australian society for reproductive · the australian society for reproductive biology inc ......

AUSTRALIAN SOCIETY

FOR

REPRODUCTIVE

UNIVERSITY ·OF •• OTAGO,DUNEDIN, .NEW .ZEALAND

AUGUST 23 .. 25.·.1993

Twenty Fifth Annual Conference

August 23 - 25 1993

University of Otago

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SA1-SA3

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CONTENTS

Acknowledgements

Copyright Australian Society for Reproductive Biology Inc., 1993ISSN 0705-6044

Personnel

PROGRAMME AND ABSTRACTS OF PAPERS

The Australian Society for Reproductive Biology me.

Campus map

Programme

Author index

Abstracts

State-of-the-Art Lectures

THE AUSTRALIAN SOCIETY FOR REPRODUCTIVE BIOLOGY INC.

August 1993

THE AUSTRALIAN SOCIETY FOR REPRODUCTIVE BIOLOGY INC

wishes to thank the following for their supportof the 1993 conference

OFFICE BEARERS

ChairmanSecretaryTreasurerCommittee Members

Postgraduate Representative

PROGRAMME COMMITTEE

ChairmanCommittee Members

Dr Rex ScaramuzziDr Lois SalamonsenDr Con TsonisDr Jim CumminsDr Graham JenkinDr Peter KayeDr Simon MaddocksDr Alan TilbrookMr David Millar

Dr Simon MaddocksDr Bill BreedDr Rob NormanDr Bob SeamarkProfessor Brian Setchell

Bayer DiagnosticsBoehringer Mannheim

Chelsea Sugar (NZ Sugar)Combined Trade Exhibitors

Dairy Advisory BureauEli Lilly & CompanyFarmitalia Carlo Erba

HoechstJohnson & Johnson

Kabi PharmaciaKellion Bequest

Meat Research Corporation of AustraliaNovo Nordisk Pharmaceuticals

Parke DavisRoche Products

ScheringSerono

Servier LaboratoriesSterling Winthrop

TemmoWyeth Pharmaceuticals

LOCAL ORGANISING COMMITTEE (Combined Societies)

ConvenorCommittee Members

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Prof David StewartMs Kerry BuchanDr Mark FisherDr Peter HurstDr Nick Lewis-BarnedDr Derek MaviusDr Bernie McLeodDr Grant MontgomeryDr Sheila SkeaffDr Peter RennieDr Don Wilson

The cont~nts of these I!<?C.eedings have not been edited by th~ S~iety and are reproduced as.sublllltted. Respon~lbl1ity for the ac~uracy of the cOI?mumcatIons and for carrying out

expenmental work on ammals of humans ill accordance WIth the approval of the appropriate ethicscommittee, or with the appropriate legislation, rests with the authors.

Material in these Proceedings may not be reproduced without permission of the Society.

Price of the Proceedings to Non-members: $25 plus postage.

iii

Details of the scientific programme for theassociated meetings of the

Endocrine Society of Australia,and the

New Zealand Society of Endocrinologycan be found in the

Combined Societies Conference Handbookavailable at Registration.

This also contains information onprogrammes of other Societies meeting

in Dunedin this week.

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AUSTRALIAN SOCIETY FOR REPRODUCTIVE BIOLOGY INC.

PROGRAMME 1993

Registration: The registration desk will be located in the Student U~ion Building (ground floor). OnSunday, August 22nd, it will be open from 1400h, on Monday - Fnday between 0830 and l700h.

The Goding, Harrison, MRC and ASRB Lectures will be presented in the :'Cast~e 2'.' Lec~r~ Theatre; theAnnual General Meeting oftlle Society, the State-of-the-Art Lectures, JUnIor SClenttst, Mimposter and Oralsessions will be held in th.e "Archway" Lecture Theatres, all as indicated in the detailed programme. Posterswill remain up for the duration of the meeting, and will be displayed in the Gazebo Lounge of the StudentUnion building or in the Marama Hall. Trade displays will be found in the Main Common Room and theUnion Hall in the Student Union, as will morning and afternoon teas.

On Sunday, August 22nd, there will be an informal social n:nction in The Otago Museum (acrossCumberland Street North, opposite the Student Union) starting at 1900h.

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MONDAY AUGUST 23 (cont.)

Boukhliq, R. ,Miller D.W. and Martin, G.B.Dietaryfatty acids stimulate gonadotrophin secretion in the ram

Smith. J.F., Cruickshank, G.J., Parr, J. and Konlechner, J.A.Effect ofdietary protein intake on the changes in levels ofFSH produced by injections offollicularfluid in ovariectomised ewes

Fit?;patrick, L.A., D'Occhio, M.l, Rhodes, F.M., Entwistle, KE. and Kinder, J.E.Effects ofovariectomy and/or weaning on plasma LH in postpartum Bos indicus cows

Curlewis, J.D., MacGregor, D., Gale, J., Loudon, A.S.l. and Brinklow, B.R.Effect ofphase two lactation on prolactin in marsupials

Cummins I.M., Meloni, B.P. and Jequier, A.M.Human sperm and testis mitochondrial DNA

MONDAY AUGUST 23

Session 1 (Oral • concurrent) : GAMETOGENESIS AND GONADAL CELLINTERACTIONS

Chairman: Dr. Gail Risbridger

Time: 0845 - 1000

)1Venue: Archway 3 Lecture Theatre

Session 3 : STATE OF THE ART LECTURE

SAl Dr. John M. Hutson

Hormonal control oftesticular descent and the cause ofcryptorchidismChairman: Assoc. Prof. David Handelsman

Time: 1030 - 1100 Venue: Archway 4 Lecture Theatre

Session 4 (Oral • concurrent) : SPERM MOTILITY AND IN-VITROFERTILIZING ABILITY

Session 2 (Oral • concurrent) : ENDOCRINE, NUTRITIONAL AND LACTATIONALINTERACTIONS

Chairman: Dr. John Smith

Time: 1100 - 1200

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3

4

5

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Swan M.A. and Gill, J. .Electron microscopy ofavian spermatogenesis and diagnosis ofmale infertility: use of laparoscoplCbiopsy

Hayes T.M., de Kretser, D.M. and McFarlane, J.R.Microtubule associatedproteins in the rat testis

Kim Y.H., Temple Smith, P.D., de Kretser, D.M., Stanton, P.G. and McFarlane, J.R.Isolation and partial characterisation ofrat sperm tai/fibrous sheath proteins

lolly P.D., Tisdall, DJ., Heath, D.A., Lun, S. and McNatty, KP.Evidence ofapotosis in bovine granulosa cells

Kern. S. and Maddocks, S.Indomethacin blocks the immunosuppressive activity ofrat testicular macrophages in-vitro

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14

15

16

Venue: Archway 4 Lecture Theatre

AI-Somai N., Vishwanath, R., Shannon, P. and Molan, P.C.Isolation and characterisation ofanionic proteins and peptidesfrom bovine seminal plasma andtheir effects on sperm motility

Prendergast E., Vishwanath, R., Shannon, P. and Molan, P.Assessing the changes in translational arid rotational velocities ofbull sperm under aerobic andanaerobic conditions after cold shock using the ITLV laser machine

Upreti G.C., Brown, K, Oliver, J.E. and Smith, J.F.Modification ofram semen diluent (RSD-l): organic nutrient(s) relJ!!irementfor ram spermatozoalmotility

Stojanov T., Maxwell, W.M.C., Rhodes, S.L. and Evans, G.Insemination ofin vitro matured sheep oocytes with low numbers offresh sperm

7 Fisher, J., Martin, G., Hughes, P., Boukhliq, R. and Gray, S.Nutritional effects ofluteinizing hormone (Ul) secretion in rams after exposure to oestrous ewes

ChaiIman: Dr. Michael D'Occhio

Time: 0845 - 1000 Venue: Archway 4 Lecture TheatreSession 5 (Poster • concurrent) IMPLANTATION AND PLACENTATIONChairman: Dr. Brendan Waddell

Time: 1100 - 1200 Venue: Gazebo Lounge, Student Union

Walkden-Brown S.W., Boukhliq, R., Fisher, J.S. and Martin, G.B.Does the nutrition ofthe ram influence its behavioural and endocrine response to oestrous ewes? 17 Walton C.E., Chen, Q. and Nancarrow, C.D.

DNA amplification ofan oestrogen-associated oviducal glycoprotein sequence from primates

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18

19

20

21

22

MONDAY AUGUST 23 (cont,)

McMillan, W.H., Macmillan, K.L, and Peterson, AJ.Is uterine function compromised in heifers synchronised with a long-duration progesteronetreatment ?

Sakoff, J.A, Murdoch, R.N, and Dunstan, R.H.Changes in the polyunsaturatedfatty acid composition ofthe uterus during induction ofthedecidual cell reaction in pseudopregnant mice

Herrmann, B., O'Neill, C. and Battye, K.M,The activity ofPAF:acetylhydrolase within the uterus during the periovulatory and preimplantati(Jnphases

Lee R.S.F., Tervit, H,R, and Peterson, AJ,Pregnancy-specific proteins in the uterinejLuid ofsheep around the time ofimplantation

Lee, C.S., Gogolin-Ewens, K. and Brandon, M.RLocalisation ofa pregnancy-associated molecule (SBU-59) in sheep

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29

32

MONDAY AUGUST 23 (cont.)

Bonello, N., Norman, R. and Briinnstrom, M.Cytokines andplasminogen activator activity in the rat ovary at ovulation

Hill, J.L., Walker, S.K. and Nancarrow, C.D.The role ofan oestrus-associated oviducal glycoprotein in the early development of the sheepembryo

Spindler R, Renfree, M.R and Gardner, D.K.Nutrient uptake during embryonic diapause and reactivation in Tammar wallaby blastocysts

Tatham, B.G., Dowsing, AT. and Trounson, AO,Enucleation by centrifugationfor bovine nuclear transplantation

Lane M. and Gardner, D.K. l.r,;

In situ removal ofembryo-toxic ammonium generated by the metabolism and breakdown ofamilwacids in culture media

23 O'Leary, P.C., Sunley, A,K. and Waddell, RJ.Endocrine interaction ofthe human trophoblast and decidua: effects ofdecidual secretions ontrophoblast steroidogenesis

24 Re~nault, T.R.H., Scaramuzzi, R.I" Sriskandarajah, N. and Oddy, V.H.Depression of insulin response to a glucose load in pregnant ewes with high circulating placentallactogen levels


NUTRITION AND FERTILITY

Wri~ht P J and Clarke, LJ,The frequency ofrelease ofIE in anoestrus Corriedale ewes is influenced by nutrient status butnot by post-partum status

Walkden-Brown, S.W., Restall, RJ., Norton, B.W" Scaramuzzi, R.J. and Martin, G.B,Nutrition affects testicular size in male goats without changing IE, FSH or testosteroneconcentrations

37

Brown B.W.Gonadotrophin and testosterone concentrations and testicular growth in rams supplemented withlupins from birth to puberty

Rhodes, F.M., Fitzpatrick, L.A" Kinder, J.E. and Entwistle, K.E.Changes in ovarian morphology associated with dietary intake in Bos indicua heifers

36 Smith, J.F., Parr, J. and Konlechner, J.A,Effect oflevel ofdietary protein intake on the plasma levels ojprogesterone in ovariectomised ewestreated with CIDR@ devices

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Session 8: (Oral. concurrent) :

Chairman: Dr. Graeme Martin

Time: 1630 - 1800


Venue: Castle 2 Lecture Theatre

GODING LECTUREProfessor G.C. Liggins

Cortisol: ajeta! hormonefor all seasons

NO.ASRBJUNIOR SCIENTIST AWARD

Chairman: Prof. Rex Scararnuzzi

Time: 1200 - 1300

Session 6

O'Connor, A.E., de Kretser, D.M. and Wreford, N.G.Human recombinant inhibin partially reverses the hemicastration induced rise in serum FSH in therat

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Godfrey S.L, Walkden-Brown, S.W., Martin, G.B., Gherardi, S.G., Lindsey, M.J.and Porter, B.L.Immuno-castration ofadult cashmere bucks with Vaxstrate (!J

Markey C.M., Jequier, AM. and Meyer, G.T.Morphological damage to the ram testis and epididymisfollowing ischaemia: possible implicationsin male infertility

Barker-Gibb, M.L. and Clarke, I.J,Chronic oestradiol treatment ofovariectomized ewes in the non-breeding season does not affect thedensity ojNPY terminals in the hypothalamus

Session 9 (Oral • concurrent) :

Chairman: Dr, Simon Maddocks

Time: 1630 - 1800

ENDOCRINE REGULATION OFREPRODUCTION IN THE MALE


viii ix

TUESDAY AUGUST 24 (cant,)

50 Shaw J.M. and MacFarlane, D.R.Rapidfreezing ofmouse expanded and hatched blastocysts

49 Maclellan. L.J.. Trounson, A. and Gardner, D.K.;~ffe~ts ofoxygen and amino acids on cattle oocyte maturation,fertilization & embryo developmentIn VItro


ASRB SPECIAL LECTURESession 12Dr Paul Wasserman

Ifr Regulation ofmammalianfertilization by gamete receptors and binding proteins

'I Chainnan: Dr. Jim CumminsTime: 0900 - 1000

Jongman. E.C., Hemsworth, P.H. and Gallow~y, D.B.. . .The effect ofsexual stimulation on testosterone zn boars In &0 different matzng systems

MJ., Garozzo, S.M., O'Connor, A. and Wreford, N.G.~~=~:;tgy and sperm production in bulls receiving the gonadotrophin releasing hormone

lore

MONDAY AUGUST 23 (cont,)

Tilbrook, A.J., de Kretser, D.M. and Clarke, I.J.Effect ofstage ofbreeding season on the pituitary actions of inhibin and testosterone to controlFSH secretion in rams

TUESDAY AUGUST 24

Session 10 (Oral - concurrent) : OVARIAN STEROIDOGENESIS

Chairman: Dr. Robert NormanTime: 0800 - 0900 Venue: Archway 3 Lecture Theatre

Session 11 (Oral - concurrent) :

51 de Kretser D.M., Foulds, L.M., Hancock, M., Miller, S., McFarlane, J., Goss, N.and Jenkin, G.Isolation ofactivin Afrom ovine amnioticfluid and persistence ofinhibin in amnioticfluid afterfoetal orchidectomy

ESA Cameron, V.A., Sawchenko, P.E. and Vale, W.W.Effects ofsteroids on activin receptor expression in rat brain andpituitary

52 Farnworth. P.G., Robertson, D.M. and Schwartz, J.ActivinA regulates production offollistatin (FS) by ovine anteriorpituitary cells in vitro

53 Kleemann. D.O., Grosser, T.I., Bindon, B.M., Russell, D. and Findlay, J.K.Effect ofthe FECB gene and age on plasma inhibin concentrations in merino rams

ESA Flemming. J.S., Galloway, S.M., Greenwood, P.J., Penty, J.M., Tisdall, D.J. andMontgomery, G.W.Genetic and structural analysis of the f3A - inhibin gene in sheep carrying the Booroola FECB

mutation

ESA Keelan, J., Song, L. and France, J.T.An immunoblotting study ofinhibin and activin production by the human placenta in vivoand in vitro

Session 13 (Oral • concurrent): ASRBIESA COMBINED SESSION :INHIBIN, ACTIVIN AND FOLLISTATIN

Chairman: Assoc. Prof. Jock Findlay (ASRB) and Dr. John France (ESA)



CRYOPRESERVATION ANDOOCYTE DEVELOPMENT

Lopes, T. and Waddell. BJ.Influence ofprolactin andLH on ovarian progestin secretion during late pregnancy in the rat

Bruce N.W.Corpora lutea ofprevious cycles are a source ofprogestins in the rat

Rabiee. A.R., Gooden, J.M., Miller, B.G. and Lean, IJ.;Evaluation oftransovarian uptake ofmetabolites and progesterone secretion

Downing, J.A., Scaramuzzi R.I. and Joss, J.Steroid secretion in ewes following an ovarian arterial infusion ofsheep growth hormone

Chairman: Dr. Leeanda Wilton

Time: 0800 - 0900

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44

43

46

47 Payne. S.R., Oliver, J.E. and Upreti, G.C.The role ofantifreeze proteins in the freezing ofram spermatozoa

48 Valiotis, M.C., Lacham-Kaplan, O. and Trounson,A.O,Pronucleiformation and embryo cleavage following electrofusion ofround spermatids withoocytes from the mouse

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TUESDAY AUGUST 24 (coot.) TUESDAY AUGUST 24 (cont.)

Session 14 : (Oral • concurrent) :

Chainnan: Dr. Ken McNatty

Time: 1030 - 1200

Session 16

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FOLLICULOGENESIS AND OVULATION

Venue: Archway 2 Lecnrre Theatre

Downing. I.A., Scaram.uzzi, RJ..and Jos~, J. .. .The direct effect of insulm on ovanan sterOId secretzon zn ewes wlth an autotransplanted ovary

Peterson, A.J., Ledgard, AM., Hodgkinson, S.C. and McDougall,. S. ..Ovarianfollicular insulin-like growthfactor binding proteins andfolllcular status In dazry cattle

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68

Marshall, J T.A, Pruss, C.A. and Nancarrow, C.D.Use ofin vitro matured andfertilized ovine oocytesfor production of embryo's with visiblepronucleifor microinjection

Berg D.K., Thompson, J.G. and Asher, G.W.Time course ofoocyte maturation,fertilisation and sperm transport events in red deer (Cervuselaphus )

HARRISON LECTURE

Session 15 (Miniposter • concurrent) : GAMETES AND EMBRYOS IN CULTURE

Chainnan: Dr. Jeremy Thompson

Time: 1030 - 1200 Venue: Archway 4 Lecnrre Theatre

60 Tervit H.R., Smith, J.F. McGowan, L.T. Wells, RW. and Pugh, P.A.Laparoscopic recovery ofovarian oocytesfrom slaughtered or living sheep

61 Fry, RC., Simpson, T.L., Squires, T.J., Miles, M.A and Niall, E.Oocyte recovery is influenced by needle type and aspiration pressure

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ANNUAL GENERAL MEETING

SA2

STATE OF THE ART LECTURE

Dr. John F. Cockrem

Seasonal breeding - are New Zealand birds different?

Chainnan: Dr. Terry Fletcher

Time: 1400 - 1430

Professor Eberhard Nieschlag

Hormonal male contraception: a challenge to endocrine research

Chairman: Assoc. Prof. David Handelsman

Time: 1200 - 1300

Session 18 :

Chairman: Prof. Rex Scaramuzzi

Time: 1430 - 1600

Session 17

Robens R.D ..Insulin-like growth factor binding proteins in the ovary of the domestlc hen (Gallus domestzcus )

O'Shea T.O., Baxter, G. and Webb, R.Factors infollicularfluid that delay or hasten oestrus

Brannstrom, M., Seamark, R, Robenson, S. and Norman. R.The rat ovary produces cytokines during ovulation

Sernia, C., Bathgate, R. and Gemmell, R.T, .,Mesotocin and arginine vasopressin in the corpus luteum ofthe brushtazl possum (Tnchosurusvulpecula)

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Chainnan: Dr. David Robertson

Time: 1630 - 1730

62 Shannon. P. and Vishwanath, RThe effect ofoptimal and sub optimal concentrations ofsperm on the fertility offresh andfrozenbovine semen

Sanchez-Partida L.G., Zupp, IL., Maxwell, W.M.C. and Setchell, B.P.Effect oflevels ofegg yolk and compatible solutes on the post-thaw motility ofram spermatozoa

Sanchez-Panida, L.G., Sowerbutts, S.F., Maxwell, W.M.C. and Setchell, B.P.Effect oflevels oftaurine and levels ofglycerol on the post-thaw motility ofram spermatozoa

Session 19 : MRC LECTURE

Prof. Yves Combarnous

Structurejunction relationships and mechanisms ofaction ofpituitaryandplacernalgonadotropms

Venue: Castle 2 Lecnrre Theatre

65 Marshall, J.T.A. and Nancarrow, C.D.Changes in saccharide-binding sites on the surface ofram spermatozoafollowing freeze-thawing

66 McGowan, L.T., Wells, R.W., Pugh, P.A., Bell, A.C.S. and Tervit, H.RCulture conditions affect thefreezability ofin vitro produced cattle embryos

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WEDNESDAY AUGUST 25 WEpNESDAy AUGUST 25 (coPt.)

Session 20 (Miniposter - concurrent) : SPERM MOTILITY AND ARTIFICIALINSEMINATION

Chainnan: Dr. R. Vishwanath

Time: 0830 - 1000

Session 23 (Miniposter - concurrent) : EMBRYO DEVELOPMENT ANDLACTOGENESIS

Chainnan: Dr. Colin Nancarrow


82 Spindler R., Renfree, M.B. and Gardner, D.K.Nutrient uptake during embryonic diapause and subsequent reactivation in mouse blastocysts

83 Bowen. J.M., Pugh, P.A and Thompson, J.G.Development of i~-vivo and.in-vitro derived ovine embryos in media supplemented with humanserum orfatty aczd-jree bovzne serum albumin

84 Rhodes S.L., Stojanov, T., Evans, G. and Maxwell, W.M.C.Effect oftype and concentration ofserum on the development of in vitro matured andfertilisedsheep embryos

85 Szell, A.Z. and Newton, p.r.The effects ofco.-cU!ture with oviduct cells and the addition ofglutamine on the development ofsheep embryos zn vztro


STATE OF THE ART LECTURE

Dr. Leeanda J. Wilton

Diagnosis ofgenetic diseases in preimplantation embryosChainnan: Dr. Peter Rogers

Time: 1030 - 1100

Session 22

SA3


Macmillan K.L. and Taufa, V.K.Pregnancy rates to first insemination in dairy cattle previously treated with progesterone during latedioestrus and with or without oestradiol benzoate

Windsor. D.P., Szell, AZ., Buschbeck, C., Edward, AY. and Milton, r.T.B.Transcervical insemination ofmerino ewes

AI-SomaL N., Vishwanath, R., Shannon, P. and Molan, P.C.High nwlecular weightprotein aggregates in bovine seminal plasma produce cationic peptides thataffect bull sperm nwtility

Upreti. G.C., Oliver, J.E., Munday, R and Smith, J.F.Effect ofdiluent type on nwtility ofram spermatozoa storedfor varying periods at 15 OC

Upreti, G.c., Board K.R, Oliver, J.E. and Smith, J.F. ",,"~"~-~"\Modification ofram semen diluent (RSD-l): effect oflipids on~~p:Spennatozo~1 motility

• 5S h A., Sanchez-Partida, L.G., Maddocks, S. l}n(ilSetchell, B.P. )uail yolk and coconut extract in diluentsfor storage ofram semen at 30 and/OC

\ /Hill, J.L. and Nancarrow C.D. \ /Absence ofbinding ofan ovine oestrus-associated oviducal glycopI.QJetf1 to ram spermatozoa

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70

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76

Session 21 (Oral - concurrent) : UTERINE REMODELLING AND IMPLANTATION

Chairman: Dr. Mike Legge


Harper. G.M., Gooneratne, A.D., Molloy, C. and Bullock, D.W.Purification ofa stage-specific lectin receptorfrom the sheep uterus

77 Hurst. P.R., Gibbs, RD., Clark, D.E. and Myers, D.B.Decrease in fibrillar collagen types I, III and V during rat implantation

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87

88

Pug~, P.A., Thompson, J.G., McGowan, L.T., McMillan, W.H. and Tervit, H.R.Surv!val after transfer offresh orfrozen bovine embryos produced in vitro in a cell- and serum-freemedzum

78

79

I Clark D.E. and Hurst, P.RDecidualisation and the remodelling oflaminin during natural embryo implantation in the rat

Macpherson, A.M. and Rogers P.A.W.Hzunan endometrial endothelial cell proliferation during the menstrual cycle

80 Marsh M.M., Hampton, AL., Findlay, J.K. and Salamonsen, L.AEndothelin secretion in vitro by cultured human endometrial epithelial cells: effect oftransforminggrowthfactor-/3J and interleuJdn-la

89

90

Knight, T.W. and Scott, I.Time and incidence offoetal nwrtality in Alpacas

Kent. J.C. and Hartman, P.E.Calcium, phosphate and citrate in sows milk during lactogenesis II

81 Abberton. K.M., Rogers, P.A.W., Taylor, N. and Critchley, H.O.D.Endometrial oestrogen andprogesterone receptor expression in women with menorrhagia

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WEDNESDAY AUGUST 25 (cont.)

Session 24 (Poster • concurrent) : FOLLICULAR DYNAMICS ANDOESTROUS SYNCHRONY

AUTHOR INpEX

Chairman: Dr. Peter Jolly

Time: 1100 -1230 Venue: Mamma Hall

91 McMillan, W.H., Hall D.R.H., Oakley, A.P. and Morris, C.A.Follicular dynamics in cows from a herd with a history of twin-calving

92 McMillan, W.H., Oakley. A.P. and Hall, D.R.H.Follicular dynamics, ovulation and oestrus in beefcows suckling either one or two calves

93 McMillan W.H., Hall, D.R.H. and Oakley, A.P.Follicle and ovulatory patterns in romney ewes teased by rams during late anoestrus

94 Macmillan KL., Taufa, V.K and Burke, C.R.Effects ofoestradiol on synchrony patterns in cattle treated with an intravaginal progesterone device

95 McMillan, W.H., Hall D.R.H. and Evans, P.H.Oestrous synchrony, ovulation rate and embryo survival in recipient ewes commencing synchronytreatment in either mid or late cycle

96 McDougall, S., Macmillan, KL. and Williamson, N.B.Effect of treatment of non-cycling, lactating dairy cows with progesterone and/or oestradiol

97 Scott. I.e. and McLeod, BJ.The effects ofexogenous progesterone on ovulation and pregnancy in Alpacas

98 Fisher M.W., Meikle, L.M., McLeod, BJ. and Shackell, G.H.GnRH-induced accessory corpora lutea in red deer and sheep

xvi

de Kretser, D.M 11, 40Downing J A · 3,4, 38 39 51

xvii

Marshall, J.T.A. . ,. 7 8 9 25 33

~~~!t;.c.///<E.///···:···/.·E.:/E«L2>t~~\Iixviii

McLeod, B.J , 97,98McMillan, W.H. . 18,86, 91, 92, 93, 95McNatty, K.P. . 5Meikle, L.M. . 98Meloni, B.P 1Meyer, G.T ~ 26Miles, M.A 61Miller, B.G. . 44Miller, D.W 9Miller, S 51Milton, J.T.B 74Molan, P.C. . 13, 14,69Molloy, C. . 76Morris, C.A 91Munday, R. . 70Murdoch, R.N 19Myers, D.B 77Nancarrow, C.D. .. 17, 29, 65,67, 73Newton, P.J 85Niall, E 61Norman, R. . 28, 58Norton, B.W 33O'Connor, A.E. . 38, 40O'Leary, P.C. . 23O'Neill, C. . 20O'Shea, T.O. . 57Oakley, A.P 91,92,93Oddy, V.H 24Oliver, J.E. . 15, 47, 70, 71Parr, J. . 10,36Payne, S.R 47Peterson, A.J 18,21,55Porter, B.L 25Prendergast, E 14Pruss, C.A. . 67Pugh, P.A 60, 66, 83, 86Rabiee, A.R 44Regnault, T.R.H. . 24Renfree, M.B 30,82Restall, B.J. .. 33Rhodes, F.M. . 11,35Rhodes, S.L 16,84Roberts, R.D. .. 56Robertson, D.M 52Robertson, S 58Rogers, P.A.W 79, 81Russell, D 53Sakoff, J.A. . 19Salamonsen, L.A 80Sanchez-Partida, L.G 63, 64, 72Scaramuzzi, R.I 24, 33, 43, 54Schwartz, J. . 52Scott, I.C 89, 97Seamark, R.F 58, 88Semia, C 59Setchell, B.P 41, 63,64, 72,88Shackell, G.H. .. 98Shannon, P. . ; 13, 14,62,69Shaw, J.M 50

xix

· TL 61SImpson, . . 10 15 36 60 70, 71

Temple SmIth, P.D. 21 60 66 86

!~~~~;/:.G;::::.~~~::::::;;;::.:.:::~:::;.;::::::::::~:·:·:·:::::::::::.::\/>:(/L ..r>:::::~,: ~iTrounson, A.a. .. 15 47 70 71Upr~ti! G.e :.. :::::::::::: :::: ::::::: : : :.48Vahotls, M.e. .. 13 14 62 69

Zupp, J.L. ,

xx

SA 1

HORMONAL CONTROL OF TESTICULAR DESCENT AND THECAUSE OF CRYPTORCHIDISM

JM HutsonRoyal Children's Hospital, Surgical Research Unit,Flemington Road, Parkville, Victoria, 3052,Melbourne, Australia.

Testicular descent occurs in two separatemorphological and hormonal stages. In the firststage the gubernaculum enlarges to anchor thetestis near the inguinal region as the embryogrows. In the second phase the gubernacularmigrates from the groin to the scrotum. Nonandrogenic hormones control the first phase withconflicting evidence suggesting that mullerianinhibiting substance (MIS) may be the activehormone. The second phase is controlled bytestosterone, but it has been proposed recentlythat androgens act indirectly via the nervoussystem. Calcitonin gene-related peptide (CGRP) isa neuropeptide that has been found in thegenitofemoral nerve spinal nucleus, and it hasbeen proposed that CGRP may act as a secondmessenger for androgens to stimulate gubernacularmigration. The evidence for this proposal will bepresented.

The cause of undescended testes is multifactorial.True hormonal deficiencies such as in androgensynthesis or action and in the persistentmullerian duct syndrome do cause undescendedtestes. However, these hormonal anomalies arerare, and most children have no recognisablehormonal cause for their undescended testes. Thehypothesis that the genitofemoral nerve and CGRPmay mediate the action of androgens ongubernacular migration suggests that anomalies ofthe genitofemoral nerve or its content of CGRP maybe a common cause of undescended testes.

SA 1

HORMONAL CONTROL OF TESTICULAR DESCENT AND THECAUSE OF CRYPTORCHIDISM

JM HutsonRoyal Children's Hospital, Surgical Research unit,Flemington Road, Parkville, Victoria, 3052,Melbourne, Australia.

r

Testicular descent occurs in two separatemorphological and hormonal stages. In the firststage the gubernaculum enlarges to anchor thetestis near the inguinal region as the embryogrows. In the second phase the gubernacularmigrates from the groin to the scrotum. Nonandrogenic hormones control ... the first phase withconflicting evidence suggesting that mullerianinhibiting substance (1liIS) may be. the activehormone. The second phase is controlled bytestosterone, but it has be'en proposed recentlythat androgens act indirectly via the nervoussystem. Calcitonin gene-related peptide (CGRP) isa neuropeptide that has been found in thegenitofemoral nerve spinal nucleus, and it hasbeen proposed that CGRP may act as a s,econdmessenger for androgens to stimulate gUbernacularmigration. The evidence for this proposal will bepresented.

The cause of undescended testes is multifactorial.True hormonal deficiencies such as in androgensynthesis or action and in the persistentmullerian duct syndrome do cause undescendedtestes. However, these hormonal anomalies arerare, and most children have no recognisablehormonal cause for their undescended testes. Thehypothesis that the genitofemoral nerve and CGRPmay mediate the action of androgens ongubernacular migration suggests that anomalies ofthe genitofemoral nerve or its content of CGRP maybe a common cause of undescended testes.

SA 2

SEASONAL BREEDING - ARE NEW ZEALAND BIRDS DIFFERENT?

JF. Cockrem

Department of Physiology and Anatomy, Massey University, Palmerston North, NewZealand.

T?e New Zea:r~d avifauna includes many unique species, including such distinctivebIrds as th.e kiWIS~ sp.) and the kakapo~ habrgptilus). Do NewZealand bIrds also show unique features in their patterns of seasonal breeding? Inorder to answer this question the breeding seasons of three species and then of theentire New Ze~~d avifauna~ 70nsidered, and a model to explain the physiologicalcontrol of the tnmng of breeding ill New Zealand birds is presented.

!he main.egg-laying season ofkiwis extend from early winter until mid-summer, andIS longer ill the North Island brown kiwi (July-December) than the little spotted kiwi(September-December). Both male and female North Island brown kiwis have annualcycles of plasma levels of estradiol, and males have an annual cycle of testosteronelevels. ~lasI?a ste~id levels rise.in ;April and May, indicating that the reproductivesystem IS bemg s,wttched on at thIS ume. In contrast to kiwis, the yellow-eyed penguinCMegadyptes anhpodes) has a short period of egg-laying in early spring (Septemberand ea:ly October), with a correspondingly short period of elevated plasma steroidlevels m male~ an~ females. A third species, the kakapo, is a large, flightless parrotthat lays eggs m ffild-summer (January and February). Data on the months in whicheggs are laid haye been combined for all native New Zealand birds, showing a broadpeak of egg-laymg from August to February. The pattern of egg-laying for speciesthat h~v~ been introduced to ~ew Zealand (largely from the Northern Hemisphere) isvery similar to tha~ for .the nauve ~irds, suggesting that the pattern of seasonal breedingby New Zealand birds IS strongly influenced by the local conditions rather than beingdistinctive to the native species.

A speculative Jl.lodel to explain the J:lhysiological control of the timing of breeding inNew ~aland bIrds uses the suggeshon made by Nicholls ~.aI. (1) that long daylengthsthat sumulate gonadal growth in birds also initiate processes that lead tophotorefractoriness and ~onadal re~ssion. I.t is proposed that the annual reproductivec.ycles of New ~aland bIrds effecuvely start m April. Daylength is decreasing at thistlme and.the ~nod of photorefractori~essthat was initiated by the long daylengths ofsummer IS ending..This refractory penod can start before the summer solstice (e.g.yellow-eyed pengum) or ~~y not occur until well after the solstice (e.g. kakapo). In~e North I.sland br?wn kiWI gonadal growth probably commences in April, with verylittle negauve steroId feedback on the hypothalamus. In the little spotted kiwi gonadalgrowt1,l probably~~ la~r ~d steroid feedback is much stronger. The yellow-eyedp~ngum d~s n~t Im~ate sl~nifican~ gonadal growth until daylengths increase after theWInter so!su~e; I.n this ~pecles steroId feedback is strong and must be overcome byphotopenodic s?IDulau?n. The kakapo also requires photoperiodic stimulation toovercome.sterOId negauve feedback. The varied breeding seasons of other NewZealand bIrds can also be explained using this model. It is concluded therefore that thebasic phy~iological. m~chanisms that control the timing of seasonal breeding in NewZealand bIrds are similar to those of other birds.

(1) Nicholls, TJ., Goldsmith, A.R. & Dawson, A. (1988) Physiol. Rev. .Qa: 133-176.

SA 3

DIAGNOSIS OF GENETIC DISEASES IN PREIMPLANTATION EMBRYOS

L.eeanda J. Wilton, Monash IVF Pty. Ltd. and Centre for Early HumanDevelopment, Clayton Road, Clayton, VIC 3168

Genetic diseases affecting fetuses are currently diagnosed at 6-16 weeksgestation by amniocentesis or chorionic villus sampling. Diagnosis in thepreimplantation embryo has the advantage that unaffected embryos can be selectedfor transfer circumventing the need for termination of affected fetuses. Biopsy ofcells from preimplantation embryos can be done either at the blastocyst or 4-8 cellstage. Blastocysts can be collected by uterine lavage although recovery rates arelow, or by culture of embryos produced by in vitro fertilization (lVF). One or twocells can also be removed from 8-cell embryos. This does not compromise preor post-implantation development in mice or in vitro development of humanembryos. Only a small number of biopsied human embryos have been transferredbut it appears that they are unaffected by the biopsy procedure.

One of the limitations of preimplantation diagnosis is the small amount ofmaterial available for analysis. To date, preimplantation diagnosis has beenpredominantly used for sexing of embryos of patients who carry X-linked diseasessuch as muscular dystrophy, haemophilia etc. Female, and hence unaffected,embryos are selected for transfer. Initially embryo sexing was done by amplifyinghighly repeated fragments of DNA specific to the Y chromosome using thepolymerase chain reaction (PCR). Cells which did not exhibit a Y-specific bandon a gel were presumed to come from female embryos. This approach has a riskof misdiagnosis and chromosome dosage cannot be determined even whensimultaneously amplifying sequences from the X and Y chromosomes.

The use of chromosome specific probes detected by fluorescent in situhybridisation (FISH) has circumvented many of these problems. FISH isperformed on single metaphase or interphase nuclei, is rapid and efficient andchromosome dosage can be determined. To date, FISH has been usedsuccessfully for embryo sexing in patients with X-linked diseases. It is alsoapplicable to the detection of aneuploidies such as Down syndrome. In conjuctionwith chromosome painting, a technique which labels the entire chromosome, itmay enable detection of structural chromosomal anomalies such as translocations.

Detection of single gene defect diseases by embryo biopsy remains achallenge as only one or two cells are available for for analysis. The absence of ahuman B-globin transgene has been detected by PCR amplification of 1-5 cellsbiopsied from mouse blastocysts. In humans, this approach has a high failure rateparticularly when single cells are used. However diagnosis of single gene defectsis possible in the preimplantation embryo. A child free of cystic fibrosis wasrecently born to a couple who carry the mutation which causes the disease. Thechild resulted from an embryo biopsied at the 8-cell stage. As more efficient PCRprotocols are developed the prospects for preimplantation diagnosis of other singlegene defect diseases are encouraging.

---------------4Ji--------------IW::l';@t

NOTES 1

HUMAN SPERM AND TESTIS MITOCHONDRIAL DNA

J.M Cummins, B.P. Meloni & A.M. Jequier*, School of Veterinary Studies,Murdoch University, Murdoch, Western Australia 6150 and *Perth Andrology.

Many ageing and degenerative disorders are related to deletions to themitochondrial genome (mtDNA) that cause disordered oxidativephosphorylation and uncontrolled free radical release (1,2). Some forms ofhuman infertility due to spermatogenic failure may also be associated withmitochondrial disorders, resulting in accelerated testicular ageing, withprogressive spermatogenic and endocrine decline, vascular insufficiency of thetestis and free-radical-induced damage to spermatozoa (Cummins et aI,submitted). Such damage typically inhibits sperm function by lipid peroxidation,however advancing paternal (as well as maternal) age is also associated withgenetic damage in offspring (3). Human spermatozoa carry oxidative DNAbreakage products that can be decreased with ascorbate therapy (4).Nevertheless, infertile couples where the male has very severe semen defectsare increasingly being treated by sperm microinjection of oocytes in vitro,despite little understanding of aetiology or genetic background. We havetherefore started a study of male reproductive tissue mtDNA in relation to maleinfertility.

Aliquots (200~1) of frozen normal (n=3), asthenozoospermic (n=1) andteratozoospermic (n=2) semen samples were lysed and the extracted mtDNAwas subjected to 30-32 cycles of PCR using established primers and protocols(5). One additional fresh, normospermic sample was studied after Percolldensity gradient fractionation, and material was also obtained from a testicularbiopsy specimen from a 36 year old man with spermatogenic failure. Theprimers chosen amplified a small and a large sequence (398 and 5349 bprespectively). The larger of these spans a common site for age-related deletionevents in muscle and brain tissues (5).

We have successfully amplified both sequences from all samples. By serialdilution we have managed to amplify mtDNA from as few as c50 spermatozoa.On this limited series we have not yet detected any deletions fromspermatozoa. However, the testis material revealed the presence of thecommon deletion (5), consistent with the premature aging hypothesis. Weintend to increase the extent of the mtDNA that we can amplify by suitablechoice of primers, to increase resolution to single spermatozoa and to screen awide range of fertile and infertile men. If idiopathic male infertility is indeedlinked with mitochondrial disorders it may therefore be maternally inherited.This would explain some puzzling demographic and genetic aspects of thissyndrome. In addition, it may open the way for appropriate redox therapy forsome forms of male infertility (1).

1. Linnane, A.W., et al., (1992) Mutation Res. gzQ,:95-208.2. Shoffner, J.M. & Wallace, D.C. (1992) Adv. Hum. Genetics 1,a: 267-330.3 Auroux, M. (1983) J. Gynec.Obstet. BioI. Reprod.(Paris) 12: 11-17.4. Fraga, C.G. et al, (1991) PNAS USA 88: 11003-6.5. Cortopassi, G. & Arnheim, N. (1990) Nucleic Acids Res.jjl: 6927-33.

2

ELECTRON MICROSCOPY OF AVIAN SPERMATOGENESIS ANDDIAGNOSIS OF MALE INFERTILITY: USE OF LAPAROSCOPIC BIOPSY.

M. ANNE SWAN and TIM GILL

Department o~ Anatomy .and Histology, University of Sydney, N.S.W. 2006and Canley HeIghts Vetennary Clinic, 44 Harden St, Canley Heights N S W2166. ' ...

£< Successful ~aptive breeding of non-domestic species of birds is essential0: the future su~val .of m~y species. Rare and valuable parrots caged as truepaIrS are someUmes mfertIle for unknown reasons When the fi I I• £< rtil . . . ema e aysm e e eggs It IS assu~ed that the male is infertile or unable to impregnate.~or ~~ purpose ?f studymg spermatogenesis and diagnosing male infertility wee~cn t a techmque in which a sample of avian testicular tissue is bio~sied

u? er. aparoscopy and examined by light and electron microscopy. The birds~~O~S:'d, a Barbary dove (Streptopelia risoria), a rainbow lorikeet

nc gl~ssus haematodus) and a scaly-breasted lorikeet (Tricho lossusch:~rolepu!otus) v:ere all of p~ven fertility and were not rare as we wi:hed togal expenence wIth the techmque before assessing rare and valuable males.

.using 1 mm .cup forceps, the biopsy samples obtained wereapproxImat.ely 3 mm, wI~h a small piece of capsule attached. This was mo~an suffiCIent for analySIS. The Barbary dove was re-examined 10 da s aft:bIOpSy, when the testes were found to have healed. y

. When th~ biopsied testicuI~ tissue was examined by light and electronmIc:oscOPy, actIve spermatogenesIs was present in all three specimens. The.~SUCUlar: spermat?zoa of each species had elongated cylindrical sperm-heads

ondensmg nucleI were surrounded by a longitudinal manchette A .were cone-sha ed Th B b . crosomes

.th p. e ar ary dove had only a narrow subacrosomal spaceWI. a small amount of dense material adjacent to the nucleus while thelorikeets had more extensive subacrosomal space containing a dense fibrous rod.

. In the rainbow lorikeet, cytoplasmic canal formation was similar to thatm mammals: from an alignment of vacuoles along the axoneme. This occurredbefore neck attachment. The sperm-tail did not attach to the I &n k h . nuc eus to 10rm the

ec attac ments unul after considerable outgrowth from the spermatid.

In co .mpanson ~o mammals, the early stages of spermato enesis hadsome unusual featur~s m all three species examined here: sperma~o onia and~pe~atocytes h~d WIde cytoplasmic continuity and typical intercellul~ bridgeeve oped late m spermatogenesis. Initially centriole pairs were arm d. s

parallel and later assumed the more usual perpendicular orientation. nge m

3

MICROTUBULE ASSOCIATED PROTEINS IN THE RAT TESTIS.

T. M. Hayes, D.M. de Kretser, and J.R. Me Farlane. Institute of Reproduction andDevelopment, Monash University, Clayton Victoria Australia 3168.

Microtubule associated proteins (MAPs) are known to initiate and stabilisethe polymerisation of microtubuies. MAPs have been seen as decoratingmicrotubule arrays using electron microscopy, but their role as effectors ofmicrotubule function remains inconclusive. The most abundant sources of MAPsare neural tissue and the testis. Numerous MAPs from neural tissue have beenreported in the literature; these include three isotypes of MAP-1 (A, B & C, lateridentified as cytoplasmic dynein), MAP-2, MAP-4 and a heterogeneous group ofrelated proteins collectively known as Tau. The testis possesses a variety ofmicrotubular networks (eg. the axoneme, the manchette, meiotic and mitoticspindles and Sertoli cell cytoskeletal microtubules) and MAP-1A, B & C, MAP-4and Tau have all been identified in testis. In addition, Boekelheide at. al. (1)reported the presence of up to sixteen uncharacterised heat stable MAPs greaterthan 80kDa in rat testis. The aim of this study was to further characterise andidentify the MAPs present in the rat testis.

HPLC gel filtration profiles of homogenates from rat brain and testis wereassayed by ELISA using an antiserum which specifically recognises MAP-2 andTau (anti-MAPs) and a monoclonal antibody to Tau (Tau-1). The gel filtrationprofiles from the brain extract contained 2 peaks corresponding to MAP-2 (>200kDa) &Tau (60kDa) while in the testis, multiple peaks could be identified. Usingthe Tau-1 antibody a single peak corresponding to Tau could be found in thetestis extract, however, the anti-MAPaps antiserum identified peaks >200 kDa(MAP-4), 60kDa (Tau), and numerous peaks were present in the 30-50 kDa range,which may represent novel MAP-2, 4 and Tau related proteins.

Using the taxol-induced assembly method for purification of microtubules(2) we isolated MAPs from both adult rat brain and testicular tissues. Thehomogenates were made to 20IJM taxol (a plant derived alkaloid known to stabilisemicrotubules) and to 1mM GTP (necessary for in vitro polymerisation) and putthrough one cycle of temperature dependent assembly. Using the criterion of coassembly with tubulin during polymerisation and analysis on 8% 80S-PAGE, morethan twenty-five heat-stable testicular MAPs greaterthan 30kDa could be identified.Similar analysis of brain MAP extracts reveals only three to four heat stable MAPs.

These data demonstrate that the testis may contain numerous novel MAPswhich do not occur in brain tissue and that some of these putative testicular MAPsmay share the homologous microtubule binding domain found on the C-terminusof MAP 2, 4 and Tau. Further studies are underway to isolate and identify thesenovel MAPs.

(1) Boekelheide K., Neely MD., Sioussat TM., (1989). Toxicol. Appl. Pharmacol.101 :373-389.

(2) Vallee R.B., (1986). Methods In Enzymol. 134:104-115.

4

ISOLATION AND PARTIAL CHARACTERISATION OF RAT SPERM TAILFIBROUS SHEATH PROTEINS.

Y.H. Kim, P.O. Temple Smith, D.M. de Kretser, P.G. Stanton & J.R. McFarlane, Departments of Anatomy, Biochemistry and Institute of Reproductionand Development, Monash University, Clayton, Victoria, Australia 3168.

The fibrous sheath (FS) in the principle piece of the tail of most mammalianspermatozoa is composed of two structural elements: the longitudinal columnsand numerous transverse ribs which surround the outer dense fibres (ODF).The isolation and partial biochemical characterisation of rat sperm FS has beenpreviously reported (1,2) suggesting the FS to be keratin-like with extensivedisulfide linkages (3). The major protein components as revealed by SDSPAGE are reported as apprOXimately 75 kDa, 25 kDa and 14 kDa together withseveral other minor components (1,2). The aim of this stUdy was to extend thiswork, by isolating and characterising the individual protein components of theFS.

Rat sperm were obtained from the cauda epididymides of 12 adult SpragueDawley rats. The sperm filtered through a Nitex screen were decapitated bysonication and layered through a sucrose step gradient. Isolated sperm tailswere treated in several steps with Triton X-100, OTT, and urea. Finally thesolubiJised sperm tail suspensions were centrifuged through a sucrose stepgradient and the insoluble pellet fixed for electron microscopy.

The residual contamination of ODF or other sperm tail components in the FSpreparation was minimal as judged by electron microscopy. SDS-PAGE of theisolated FS resolved at least 20 different proteins of varying abundance. Therange and molecular size distribution of these 20 proteins were similar to thatreported by Oko (2), except the band at 14.5 kDa by Oko was much lE;lssabundant our preparation of rat FS. The most abundant proteins were a doubleband at 80-87 kDA, a 66 kDa, and 28.5 kDa bands. The minimal contaminationof ODF in this preparation as compared with that of Oko may explain thedifferences in abundance ofproteins. Using electrolelution following SDS-PAGEwe have isolated 10 of the most abundant proteins with a high degree of pUrity.Preliminary results of amino acid analysis on 6 of the isolated rat FS proteinsshow that these proteins have a similar composition of amino acids, abundantin aspartic acid, glutamic acid, glycine, and to a lesser extent tyrosine. Furtherwork is being conducted to obtain amino acid sequence data for these proteins,and will provide further information on the homology between these proteins.

(1) Olson, G.E., Hamilton, D.W. and Fawcett, D.W. (1976) BioI. Reprod14:517-530. .,

(2) Oko, R. (1988). BioI. Reprod., 39:169-182.

(3) Bedford J.M., and Calvin H. I. (1974) J. Exp. ZooI. , 197:181- 203

5

EVIDENCE OF APOPTOSIS IN BOVINE GRANULOSA CELLS

PD Jolly, OJ Tisdall, DA Heath, S Lun and KP McNatty

AgResearch, Wallaceville Animal Research Centre, Upper Hutt, New Zealand

Apoptosis or 'programmed cell death' is an active genetically governed process of selectivecell deletion implicated in the mechanisms of ovarian follicle atresia and luteolysis (1,2).Apoptosis is associated with the activation of an endogenous endonuclease which cleaveschromatin between nucleosomes producing DNA fragments of integer multiples of 180-200base pair subunits in size, which resolve into a characteristic ladder-like pattern on agarosegel electrophoresis (AGE). The aim of this study was to determine whether apoptotic celldeath occurs in granulosa cells (GC) from bovine follicles ~4 rom in diameter, classifiedmorphologically as healthy or atretic.

Follicles were dissected from ovaries collected postmortem from 8 cows in the luteal stageof the oestrous cycle. Individual follicles (n=30) were classified by size and as healthy oratretic according to previously defined morphological criteria (3,4). Granulosa cells werecollected and snap frozen to -7CY'C in Ca2+/Mg2+-free buffer containing EDTA (2 mM).Number of cells recovered was determined and expressed as a percentage of the maximumnumber of cells (%Gmax) previously recorded for a follicle of that size (3). Follicular fluidwas also collected and levels of oestradiol-17iS determined by RIA (4). Total DNA wasisolated from GC from each follicle and purified by treatment with ribonuclease-A(100 JLg/ml), phenol/chloroform extraction and ethanol precipitation, then quantitated byfluorescent assay. DNA"(500 ng) from each sample was labeled at 3'-ends with 50 JLCi of[d2P]dideoxy-ATP (3000 Cilmmol) using 25 U terminal transferase and 5 mM CoCl2for 60min at 37°C (1). Labeled DNA was purified by ethanol precipitation using 20 JLg glycogenas carrier and fractionated by AGE on 1.8% gels (125 ng/lane), which were then dried andexposed to Kodak X-Omat films for 15-30 min at -7CJ'C. Total DNA from some follicles wasalso fractionated by AGE (2-4 JLg/lane) for ethidium bromide staining.

Distinct ladder-like patterns of DNA fragments characteristic of apoptosis were evident byethidium bromide staining and(or) 3'-end labeling in granulosa cells from all folliclesclassified as atretic (n=24). No fragmentation was evident by either method in 2/6 folliclesclassified as healthy. Degree of fragmentation was subjectively classified from intensity ofradiolabeling of oligonucleosomes as absent, slight, moderate, or marked. Slightfragmentation only was evident by the more sensitive 3'-end labeling technique in theremaining 4/6 follicles that were classified as healthy. DNA fragmentation was slight orabsent in follicles with >5 ng/ml oestradiol-17is in follicular fluid. The degree offragmentation was related to percentage of GC that could be recovered from individualfollicles (%Gmax, mean ± SEM), being 96% ± 3 (n=2), 74% ± 6 (n= 12), 50% ± 10(n=7) and 19% ± 5 (n=9) for absent, slight, moderate and marked categories, respectively.

These results suggest that apoptosis occurs in bovine GC and that degree of internucleosomalDNA fragmentation is most evident in cells from follicles that are severely atretic. The studyof follicle atresia as an apoptotic process may offer novel insight into the intracellular andmolecular mechanisms governing foHicle regression and loss.

(1) Tilly JL et al (1991) Endocrinology 129: 2799-2801(2) Juengel JL et al (1993) Endocrinology 132: 249-254(3) McNatty KP et al (1984a) BioI Reprod 30: 159-170(4) McNatty KP et al (1984b) J Reprod Fert 72: 39-53

6INDOMETHACIN BLOCKS THE IMMUNOSUPPRESSIVE

ACTIVITY OF RAT TESTICULAR MACROPHAGES IN-VITROS. Kern and S. Maddocks

Department of Animal Sciences, The University of Adelaide,Waite Agricultural Research Institute, Glen Osmond, South Australia 5064.

2.0**

James Fisherl, Graeme Martinl,2, Paul Hughes3, Rachid Boukhliql and Stephen Grayl

The presence of oestrous ewes increases the frequency of LH pulses in rams and themagnitude of the change depends on their nutrition. The difference in responsiveness ofthe rams to oestrous ewes may be due to decreased responsiveness of the LH pulsegenerator to social stimuli when an animal is in a catabolic nutritional state as the maineffect of our diets was decreased condition and testis size of the Low rams.

High Low High Low High LowFigure 1. LH secretion in rams before (D) and after (II) the introduction of oestrous

ewes.** P < 0.01: Between treattnents, histograms ofthe same type are different.

7

1. Martin, G.B. et aI. (1989). Proc. Aust Soc. Reprod. BioI. 21, 62. Sanford, LM. et aI. (1974). Endocrino!. 95, 627-6313. Walkden-Brown, S.W. et aI. (1990). Proc. AUSl Soc. Reprod. BioI. 22, 139

NUTRITIONAL EFFECTS ON LUTEINIZING HORMONE (LH)SECRETION IN RAMS AFTER EXPOSURE TO OESTROUS EWES.

The secretion of LH pulses in rams is increased by nutritional supplements (1) andby the introduction of ewes (2). In goat bucks, the magnitude of the response to does isgreater for bucks on a 'High' plane of nutrition than for those on a 'Low' plane (3). Wetested whether rams show a similar interaction between the nutritional and social stimuli.

Twenty, 2 year old Merino rams grazing spring pasture were penned indoors andallocated to 2 nutritional groups ('High' or 'Low') following stratification by scrotalcircumference and condition score. The Low rams received 500 g hammer milled oatenhay per day while High rams received 1kg of the hay plus 750 g lupin grain per day for35 days. Mter 21 days, the rams were penned in groups of 4 and from 1800 hours bloodwas sampled every 20 minutes for 12 hours before and after the introduction of oestrousewes (4 per pen). Number of mounts per hour were recorded for each ram.

The High rams had greater condition score (2.9 vs 1.45, P < 0.001) and scrotalcircumference (31.4 vs 26.9 em, P < 0.001) than Low rams on Day 35. High ramsincreased their condition score (2.45 to 2.9, P < 0.05) but not scrotal circumference(31.0 to 31.4 cm), while both decreased in Low rams (2.55 to 1.45, P < 0.001 and 31.3to 26.9 em, P < 0.001). There was no difference between the groups in the frequency ofLH pulses before the ewes (Fig. 1). The presence of oestrous ewes increased thefrequency ofLH pulses in both groups (P < 0.001) and resulted in a higher frequency forHigh rams than Low rams (P < 0.01). Pulse amplitude was greater for High rams thanLow rams before and after ewes (P < 0.01), but there was no effect of the presence of theewes. Mean LH concentration did not differ between the groups at any stage. However,over all rams, it increased after the introduction of the ewes (P < 0.01). The averagenumber of mounts per hour was the same for both ram groups (2.6 High vs 2.5 Low).There was no relationship between mounting activity and LH secretion.

1 School of Agriculture, University of Western Australia, Nedlands,WA 6009. 2 CSIRO Animal Production,Private Bag, Wembley, WA 6014. 3 School of Agriculture & ForestrY, University of Melbourne, Vic 3023.

conditioned medium source

* P<0.05; **P<0.25; vs Control (n=5)

~oU

1.0 proliferation in the prosenee of CM120

§100.~

~ 80

£ 60

3" 40

~ 20

o

Macrophages have been recognised as a prominent cellular component of the testicular interstitial tissueof several species including man, monkey, rats and boars (1). They represent some 20% of the cellpopulation and have an intimate relationship with Leydig cells in the interstitium. We have previouslyshown that rat testicular macrophages (I'M) do not stimulate lymphocyte (L0) proliferation in culture,and infact secrete factors which inhibit the proliferation of lymphocytes stimulated in vitro by themitogen Concanavalin A (Con A) (2). These results suggest that the TM could contribute to theimmunologically privileged status afforded the testis. Subsequent studies have indicated that the poor1.0 proliferative response observed when TMs are cultured in vitro with L0S may be due partly to thelow basal production of Interleukin 1 by TMs and their inability to increase production of this cytokinein response to stimulation by factors such as Lipopolysaccharide (LPS) (3,4). However this does notexplain the suppressive nature of factors secreted by TM into culture media. The production ofprostaglandins (PO) by activated macrophages has been shown to inhibit lymphocyte function (5). POis produced by an increase in cellular cAMP levels during macrophage activation and may be blockedby indomethacin, an inhibitor of prostaglandin synthase. In the present study we have examined thepossibility that prostaglandin production by TM may be responsible for the immunosuppressionexhibited by these cells in vitro.Rat peripheral blood lymphocytes were isolated using standard protocols (2) and cultured at 107cells/mlin 96 well round bottomed culture plates. TMs were isolated by enzymatic digestion of the testis andthen cultured as in (3) but with the addition of indomethacin (ID?v1) at 10 ug/ml to some cultures inorder to inhibit PO production. Mter 48hours the TM conditioned medium (CM) was collected,centrifuged and frozen until use. Some of the CM collected was then dialysed removing molecules<14000MW, such as PO. Lymphocytes were optimally stimulated with the mitogen Concanavalin A(Con A) at IOng/ml and concurrently cultured with the CM or dialysed CM (20% of the total culturevolume). Mter48 hours of culture, 3H-thymidine incorporation was used to measure the lymphocyteproliferation.Indomethacin treated cultures and the dialysedmedia of TMs proved to be significantly lesssuppressive than untreated CM. The CMfrom TMs treated with LPS and indomethacin,and the dialysed CM from LPS stimulated TMcultures, did not significantly reducelymphocyte proliferation from that of thecontrol. That the presence of indomethacin inculture, or the removal of small molecularweight compounds from CM should removethe immune-suppression previously observedsuggests that PGs are involved in theimmunosuppression encountered in vitro.The presence ofPOs may also down-regulatecytokine production by TMs (3) and henceprovide a basis for immune privilege in thetestis. PGs may also playa role in regulatingLeydig cell steroidogenesis, and this mayprovide a basis for physiological TM-Leydigcell associations.REFERENCES1Fawcett D.W., Neaves W.B., & Flores M.N., (1973) BioI Reprod. 2: 5002.Kern S. & Maddocks S., (1991) Proc. AUSl Soc. Reprod. BioI. 23.:673.Kern S., Robertson SA, & Maddocks S., (1992) Proc. Aust. Soc. Reprod. BioI. ;M:874.Guzzardi V. & Hedger M., (1992) Proc. Aust. Soc. Reprod. BioI. ~:865.Gemsa D., Seitz M., Kramer W., Till G., & Resch K., (1978) J. ImmunoI. l2.Q: 11876.Sairam M R., (1979) Prostaglandins 17: 929

8 9

Figure 1. a) LH concentration in rams (mean ± sem, n=30) before and during exposme to oestrousewes, b) Effect of diet on LH pulse frequency (mean ± sem, n=5) in rams before (. day 35) and during(~, day 36) exposme to oestrous ewes,

The diets induced significant changes in liveweight and testicular size (3) Exposure tooestrous ewes increased LH pulse frequency (3,7 v 2,2 pulses/12h, p<0.01), pulseamplitude (5.5 v. 4,2 ng, p<0.05) and mean LH concentration (2,1 v. 1.1 ng/ml,p<O.OOl, Fig. 1a) with 21/30 rams exhibiting an increase in the number of LH pulses.There was no effect of dietary treatment on LH secretion before or during ewe contact(Fig. 1b) and no significant association between LH secretion and change in liveweightor scrotal circumference. Similarly, there was no effect of diet on any behaviouralvariable, However change in LH pulse frequency following exposure to ewes wassignificantly correlated with the frequency of investigative (r=0.66 p<O.OOl) andreproductive (r=O.56, p<0.01) but not aggressive behaviours in individual rams.

We conclude: a) full contact with oestrous ewes is not necessary to induce the femaleeffect in rams; b) supra-maintenance ram nutrition does not always enhance the endocrineresponse to oestrous ewes; c) The magnitude of the endocrine response to ewes isassociated with the frequency of investigative and reproductive behaviours expressed byrams under conditions where mating and mounting are precluded.1. Walkden-Brown, S,W. et al, (1990). Proc Aust Soc. Reprod. BioI. 22, 1392. Fisher, I.S, et al, (1993) Proc AUSl Soc, Reprod, BioI. 25 (These proceedings)3. Boukh!iq, R., Martin, G,B. and Adams, N.R. (1992). Proc Aust Soc, Reprod. BioI. 24, 100

DIETARY FATTY ACIDS STIMULATE GONADOTROPHIN SECRETIONIN THE RAM

Rachid Boukhliq1,2, David W Miller2 & Graeme B, Martin2,3

1Animal Reproduction, IAV Hassan II, Rabat-Instituts 6202, Morocco, 2School ofAgriculture, University of WA, Nedlands, WA 6009; 3CSIRO, Division of Animal

Production, Wembley, WA 6014

We previously showed ~at supple~entation,of a m~tenan~e ~et with lupins stimulatesgonadotrophin secretion m rams. This effect IS assocIated WIth mcreased ~loodconcentrations of glucose and fatty acids and could not be reproduced b~ mtravenousglucose infusion (1). This experiment tested whether fatty acIds could sttmulategonadotrophin secretion in rams.

Twenty four Merino rams were housed indoors and fed a diet consisting of 900 g ofhammer milled hay and 100 g lupins (6.17 MJ/d and 19.76 g CP/d) for 2 weeks be;forethey were randomly allocated to a control or a treated group, The control~s continuedon the same diet. The treated rams received, in their diet, a mixture of sodium ae:etate (127gld), sodium propionate (64 gld) and ~e&etable oil (60 gld) for 11 days. The estImatedmetabolisable energy intake and protem mtake were 11.83 MJ/d and 19.76 g CP/d for thetreated group. Blood was sampled every 20 min for 24 h before the start and at the end ofthe treatment period. All samples were assayed for LH and two-hourly samples :wereassayed for FSH, glucose and insulin, ~ata were log transformed before analySIS andpre-treatment values were used as covanates,

Fatty acid supplementation did not affect food intake. Mean LH pulse frequency andFSH concentrations were similar in both groups before treatment s~d. However, at theend of the treatment period, rams fed the fatty acid supplement had a higher LH pulsefrequency (p < 0.05) and ~SH concentrations (p <.0.05) than the control rams, No. ,changes in LH pulse amplitude or basal concentratlon were observed between or WIthingroups. Plasma concentrations of glucose were rel~ti~ely co~st8?t througho?t the ~ay 3;I1dbetween and within group differences were not statiStically SIgnificant MaxImum Insulinconcentrations were observed after feeding and then declined to reach a plateau 6 ~o 8hours afterwards. Neither the decline nor the plateau of insulin were statistically differentbetween the control and the supplemented rams,

The present study represe~ts the ~st ~uccess~ attempt to stimulate ~ona?otrophinsecretion in intact rams With semI-purified nutnents' Gluc?se andlor msulin do not appearto play an intermediary role as peripheral blood con~ntrati0Il;Sw~re n~t affected by thetreatment. Therefore, the stimulation of gonadotrophin secreti?n m l~~m-!ed rams may bemediated via a biological pathway associated with the synthesIS or utilisation ?f fattyacids. Future research in this area should focus on the,effect on GnRH secretion ofindividual fatty acids and their oxidative metabolites (I.e. aspartate and glutamate).

(1) Boukhliq, R., Martin, G,B, & Adams, N,R. (1991), Proc, Aust Soc, Reprod. BioI. 22:89.

Maint Glucose Casein G+C G+C+VFA Lupinsoo i Befor~ ie't'~~ iii' rfrth ie:,,~~ iii

Bam 11 5 8pm Bam 11 2 5 llpm

DOES THE NUTRITION OF THE RAM INFLUENCE ITS BEHAVIOURAL ANDENDOCRINE RESPONSE TO OESTROUS EWES?

S,W. Walkden-Brown1, R. Boukhliq1,2, I.S. Fisher1 and G.B. Martin1,3

lSchool of Agriculture, University of Western Australia, Nedlands, WA 6009, 2Anirnal Reproduction, IAVHassan n, Rabat-Instituts 6202, Morocco, 3 CSIRO Division of Animal Production, Wembley, WA 6014,

The LH response of rams and goat bucks exposed to oestrous females (the "femaleeffect") varies with the nutritional status of the male (1,2). To determine the majornutrients involved in this variation we examined the endocrine and behavioural responsesto oestrous ewes of mature Merino rams given dietary treatments designed to mimic themajor nutritional components of a 750g lupin grain supplement.

High and low control groups were fed a maintenance diet (Maint) or Maint + 750g/day of lupin grain. Three other groups were fed Maint and received abomasal infusionsof glucose (180 gld), casein (200 gld) or the same amount of both glucose and casein(GC), while the final group received GC+volatile fatty acid salts (VFA, acetate 127 g/d,propionate 64 gld) in vegetable oil (60 gld) as an oral supplement to the Maint diet.Dietary treatments were applied for 5 weeks in April-May (n=5/treatment). Liveweightand scrotal circumference were monitored weekly and blood was sampled for LH ondays 35 and 36. After the first sample on day 36, 30 oestrous ewes were penned in frontof the rams so that each ram had access to 2-3 ewes through the front of the pen whichenabled the passage of heads only, During exposure to oestrous ewes the frequency ofinvestigative (sniffs, nudges, licks), reproductive (flehmen, pawing, vocalisation) andaggressive (male and female directed) ram behaviours was recorded.

4 a) J'w E: b)=3 ~4~ llz ~ 3

3 82

:51

10

EFFECT OF DIETARY PROTEIN INTAKE ON THE CHANGES IN LEVELSOF FSH PRODUCED BY INJECTIONS OF FOLLICULAR FLUID IN

OVARIECTOMISED EWES.

11

EFFECTS OF OVARIECTOMY AND/OR WEANING ON PLASMA LHIN POSTPARTUM BOS INDICUS COWS

LA Fltzpatrlck1, MJ O'OCChlo2, FM Rhodes1, KW Entwlstle1 and JE KlnderS

1Department of Biomedical & Tropical Veterinary Sciences, James Cook University, Townsville, 48112CSIRO Division of Tropical Animal Production. Tropical Cattle Research Centre, Rockhampton, 47003Department of Animal Science, University of Nebraska, Lincoln, NE, USA

The inhibitory effects of suckling on postpartum ovarian activity are mediated by centralinhibition of LH (1) involving modulation by ovarian factors as well as mechanisms that ~reindependent of the ovaries (2,3). The aim of this study was to determine t~e relativeimportance of ovary-dependent control mechanisms and ovary-Independent suckling effectsin the suppression of LH secretion in the postpartum Bos Indlcus cow.

Twenty grade Brahman cows (%-7A1 Bos Indlcus) were allocated to groups eitherovariectomised(Ovx) and their calves weaned (OW, n=5), ovariectomised and suckled by their calves (OS,n=5), intact and their calves weaned (IW, n=5) or intact and suckled by. their calves (IS, n=5).Ovariectomy and weaning occurred at 7-10 days postpartum. At weekly Intervals, commencing7-10 days postpartum, cows were bled by coccygeal venipuncture for plasma LH determinationby RIA. The ovaries of IS and IW cows were examined weekly by ultrasonop,raphy todetermine the time of first postpartum ovulation. Bodyweight (BWT) and body conditIOn score(BCS) were recorded weekly.

There were no differences in BWT or BCS between groups i~ the first 5 weeks postP'!rtum.Mean BCS (±sem) at calving was 5±1 (1-9 scale). The mean Interval (±sem) from calving tofirst ovulation was less for weaned than for suckled cows (27±4 d vs 72±11 d, respectively;P<O.01). Plasma LH concentrations from 1-5 weeks postpartum are presented in Figure 1. At...28 days postpartum, mean plasma LH (±sem) was O.33~.03, O.56±O.12, O.94±O.03 and1.34±O.24 for IS, IW, OS and OW cows, respectively. Ovanectomy elevated plasma LH, theeffect being greater in weaned than In suckled cows (P<O.01).

J.E Smith, G.J. Cruickshank, J. Parr, 1.A. Konlechner

AgResearch, Ruakura Agricultural Centre, Private Bag 3123, Hamilton, N.Z.

Increased dietary protein results in increased ovulation rate (1). Interference withthe inhibitory feedback mechanisms on FSH release have been suggested as apossible mechanism. However, no effect of protein was found in studies onoestradiol feedback (2). The effect of protein intake on "inhibin" feedback isreported in this trial.

Twenty four ovariectomised ewes fitted with abomasal catheters and fed on a lowprotein (12%) diet were allocated to four groups. Two of these groups were givenabomasal infusion of protein (Alacen 100 gm/ewe/day) for 7 days and the other 2groups given infusions of equivalent volumes of water (l lJd). On day 3 ofinfusion, injections of charcoal stripped ovine follicular fluid (oFF-containing 14uI,n. inhibin activity) were given at 10:00 h and 22:00 h and again on day 4 at10:00 hr. One group in each of the infusion treatments received either a high(3x2.5ml) or low (3xO.5ml) dose of oFF. Blood samples for FSH assay (RIA) weretaken at 2 hr intervals between 09:00 h and 21:00 h on the day before and for days1 and 2 of infusion then for 40 h from 07:00 h on days 3 and 4 and again from09:00 h to 15:00 h on days 5 to 8.

FSH levels ranged from 6.0 to 24.0 ng/m1 for individual ewes and data has beenanalysed as variation from the mean value for the day before infusions commenced(expressed as a percentage).

There was a significant (P<0.005) effect of dose of oFF on the depression ofplasma FSH concentration with the high dose rate reducing FSH levels, to 69%of pretreated values, following the third injection. The first significant decrease inFSH was recorded 8 h after the 1st injection of oFF and pre-treatment levels wereregained on day 7. No significant depression in FSH levels was seen with the lowdose and protein infusion had no effect at either level of oFF.

:I:-J

1.8 .----.----,-----,-------r-------ri1.61.41.21.00.80.60.40.20.0 L.l----2l...-.---...l.3----4~---5""'----'

The reduction in plasma FSH levels to 70% of pretreatment levels confirms thepreviously reported effects of oFF. That this depression and subsequent recoverywas not influenced by dietary protein intake indicates that the mechanism by whichnutrition effects ovulation rate is not via the disturbance of inhibin negativefeedback mechanisms.

(1) Cruickshank, G.1., et al. (1988) Proc. N.Z. Soc. Anim. Prod. 48: 77-79.(2) Smith, J.F. et al. (1990) Proc. N.Z. Soc. Anim. Prod. 50: 145-148.

Weeks PostpartumFigure 1. Mean LH (±sem) for Intact-Suckled (IS), Intact-Weaned (IW), Ovx-Suckled (OS)

and Ovx-Weaned (OW) cows from 1-5 weeks postpartum.

In summary, both suckling and ovarian factors Inhibit secretion of LH, but ovarian factors e~ert

a much greater inhibitory influence on LH secretion than suckling during the postpartum periodof the Bos Indlcus cow.(1) Short RE st aI. (1990) J Anim Sci 68:799-816(2) Richards MW st aJ. (1991) Bioi Reprod 44:961-966(3) Jolly PD st al. (1991) Proc ASRB 22:152

This work was partially supported by the Meat Research Corporation

12 13

ISOLATION AND CHARACTERISATION OF ANIONIC PROTEINS AND PEPTIDESFROM BOVINE SEMINAL PLASMA AND THEIR EFFECTS ON SPERM MOTILITY

N. AI-Somai*, R. Vishwanath, P. Shannon and P.C. Molan*

Livestock Improvement Corporation, New Zealand Dairy Board, Hamilton and *Department ofBiological Sciences, University ofWaikato, Hamilton, New Zealand.

riots withm co umn 1 er 10<0. 5).Treatment Cone Protein cone. % Motility

(%) (ml!/m)) ohrs 18 hrs

Control 10 82 +4.0a 72 +3.0a

WFI 10 11.7 83 + 2.5a 54±3.0b

WF2 10 3.7 72 +3.0b 39±2.5c

ACNfrae 10 2.2 84 ±3.5a 73 + 3.0a

BSP 10 43.0 80 ±2.2a 65 ±5.0a

The APs in BSP are aggregates (4) which under disaggregating conditions release low MWcomponents with molecular weight <5 KDa. Both WFI and WF2 reduce motility of sperm onincubation however, WF2 was more detrimental to sperm motility compared to the ACNfrac. Theactivity of WFI and WF2 could be due to a cationic patch on the molecule similar to the APsreported earlier (4). WF2 on the other hand, possibly has the right proportion of cationic and anionicpeptides that fulfil the criteria for maximum activity. The lack of reduction of sperm motility inACNfrac could be attributed to the competitive effect of the anionic material that counteracts/reducesthe effect of the active cationic material on sperm and thus plays a part in its protection. An acidicprotein such as antiseminalplasmin reverses the effect of seminalplasmin, a cationic protein in BSP,which affects sperm motility and fertility (5). Nature may have developed this system to regulatefertility. It probably depends on which fraction the sperm meets first at the time of ejaculation.Further work is needed to determine the exact role of the anionic proteins and their counterparts, thecationic proteins in the protein aggregates in BSP. Their ultimate role in damaging the-integrity ofthe sperm membrane needs to be established.(I) Shannon, P., Curson, B., and Molan, PC. (1987). N. Z. J. Agr. Res. 30: 195-202.(2) Hameed, 1. (1987). PhD thesis, University ofWaikato, Hamilton, New Zealand.(3) ChandOIUlet, L., Roberts, KD., Chapdeline, A., and ManjlUlath, P. (1990). Mol. Reprod. Dev. 26: 313-318.

(4) Manjunath, P., and Sairam, MR. (1987). Biochem. J. 241: 685-692.(5) Rao, VN., and Bhargava, PM. (1985). Biochemical Joumal. 227:609

Proteins in bovine seminal plasma (ESP) exist in large aggregates of smaller units of molecularweight (MW) < 3.5 KDa (1). The smaller units are composed of cationic and anionic peptides ofestimated MW 1 KDa (2). Anionic Proteins (APs) in BSP have been implicated in gonadotrophinrelease, sperm capacitation and acrosome reaction (3). The aim of this work was to isolate and studythe effects of these APs and their low MW constituents on the motility of bull sperm. 10 mI BSPfrom fresh semen was loaded on a column of SP Sephadex C25, washed with 30% (vlv) acetonitrileat pH 3 (30%ACN). The unbound material was neutralised, its volume reduced to 10 mI, ammoniumsulphate (AS) added to 1.7 M and then loaded on a Phenyl Superose column. Elution of the boundmaterial was achieved by a descending gradient of 1.7 M AS followed by water and 30% ACN. Thefractions eluted with water and 30%ACN were chromatographed on a Sephadex G25 column andeluted with 30% ACN in 0.02 M HCI, pH 1.7. Fractions eluted late with water and with 30%ACNgave one peak near the bed volume (ACNfrac). The fractions eluted earlier with water were mainlyat the void volume (WF1) followed by a second peak with MW <5 KDa and>I.5 KDa (WF2) and apeak near the bed volume of <1.5 KDa. On Superose 12, at neutral pH, WFI and WF2 re-aggregatedto a higher MW while ACNfrac did not. Table I shows the effect the these fractions on motility ofwashed sperm.Table 1. Effect of the fractions from chromatography on Sephadex G·25 of the Phenyl Superosefractionation on motility of washed bull sperm at ambient temperature. Values are mean ± s.e (n=3),unlike supersc 'I d'ffi ( 0

EFFECf OF PHASE TWO LACfATION ON PROLACTIN IN MARSUPIALS

J D Curlewis, D. MacGregor, J. Gale, A. S. I. Loudon § & B. R. Brinklow §

Department of Physiology and Pharmacology, The University of Queensland,Qld 4072 and § The Institute of Zoology, Regents' Park, London NWI4RY, UK.

In marsupials, prolactin (PRL) profiles during lactation are characterized by apeak of PRL at birth, low concentrations during the first half of lactation (phase 2) andmarkedly elevated levels during the second half of lactation (phase 3). All previousstudies of phase 2 lactation have not demonstrated a difference in PRL concentrationsbetween lactating (L) and non-lactating (NL) animals. Nevertheless, it is known thatPRL is essential during this period because treatment with bl'omocriptine terminateslactation. Here we describe 2 independent studies of PRL which were undertaken tore-evaluate PRL levels during this period and a third study in which we characterizedthe molecular forms of PRL detected by the available antisera against marsupial PRL.

Study 1- Bennett's wallaby. L (n=4) and NL (n=5) wallabies were sampledeach week by venepuncture of the lateral tail vein during phase 2 lactation. PRL levelswere measured in 2 assay systems, a heterologous (As 33/1-8) and a homologouswallaby (As G3/1) assay. Irrespective of assay system, PRL fluctuated markedly inthe L group and, for the entire 4 month period, the overall effect of lactation wassignificant (P < 0.02). Nevertheless, at some time points there was no differencebetween groups. Overall, the difference between NL and L groups was greater withAs G3/1 (6.7±O.4, 2L0±2.6 ng/ml) than As 33/1-8 (3.0±0.l and 4.7±O.4 ng/ml).

Study 2 - Brushtail possum. In this experiment, a sampling method whichdid not require physical restraint of the animals was used so that frequent samplescould be collected over a relatively prolonged period without causing stress to theanimals. The jugular vein of 4 L (mean age of pouch young, 4O±12 days) and 4 NLpossums was surgically cannulated at least 1 week before sampling. On the day of theexperiment, it was necessary to disturb each animal so that aim length of tubing couldbe connected to the cannulae. Animals were then returned to their nest sites. With thisarrangement, blood could be withdrawn without physical contact with the possum and,in most cases, the animals slept during the sampling period. Blood was collected at 20min intervals for 4 h and plasma PRL levels later determined in the heterologous PRLRIA. At the onset of sampling, PRL concentrations were similar in the two groups(3.4±O.2 and 3.7±O.2 ng/ml for NL and L groups) and in the first 2 h period there wasno effect of lactation (P > 0.1). However during the sampling period, levels in thelactating animals increased gradually so that in the third and fourth hours of sampling,the effect of lactation was significant (P < 0.05). In the final sample, PRL levels were3.1±O.3 and 6.4±L8 ng/ml in NL and L groups respectively.

Study 3. The molecular forms of pituitary PRL were examined by SDSPAGE/western blot. Three antisera (As 33/1-8, As G3/1 and anti-PRL-peptide (AsS3)) were tested. Pituitaries from the red-necked wallaby, brushtail possum, koala andbandicoot were examined. In addition, for the brushtail possum, a range ofphysiological states (male/female, early/late lactation, pre-pubertal female) were alsostudied. In all cases, only one form of PRL, MW 23500, was detected. In contrastwhen pig pituitary was blotted with As S3, the expected pattern with two forms ofPRL, MWs 23000 and 26000, was observed. These correspond to the nonglycosylated and glycosylated forms of pig PRL.

In conclusion, these studies suggest that early lactation does increase PRLconcentrations in marsupials and that these differences are more evident when anhomologous assay is used. Further, frequent sampling from undisturbed animals maybe required to demonstrate these differences. Finally, the available antisera againstmarsupial PRL detect only one form of PRL.and so at present we conclude thatchanges in physiological state are unlikely to alter the processing of this hormone.

14

ASSESSING THE CHANGES IN TRANSLATIONAL AND ROTATIONAL VELOCITIES OFBULL SPERM UNDER AEROBIC AND ANAEROBIC CONDmONS AFTER COLD SHOCK

USING THE TTLV LASER MACHINE

E. Prendergast* R Vishwanath, P. Shannon and P. Molan*

* Livestock Improvement Corpomtion, New Zealand Dairy Board, Hamilton, andDepartment ofBiological Sciences, University ofWaikato, Hamilton, New Zealand.

Progressive movement in spennatozoa is an important function enabling them to reach the site offertilisation. Propulsion requires a supply of ATP which can be generated either in the presence ofox-ygen by respiration coupled with oxidative phosphorylation or anaerobically by fructolysis. When theavailable oxygen drops below a critical level, transition from one system to the other is clearly evidentusing the 1TLV laser machine (1) resulting in a sharp decrease in both Translational velocity (TV) andRotational velocity (RV). Cold shock immobilises sperm by causing plasma lemma damage, allowing itto become leaky. In this study, the 1TLV laser machine was used to monitor the differences in motilityof bull sperm stored in 3 cationic lipoprotein diluents that have been described previously (2). Thediluents were under test for their relative abilities to protect against cold shock. Measurements ofprogressive velocity (v) and rotation frequency (f) were taken before and after cold shocking the spermto DOC for 20 minutes, under both aerobic and anaerobic conditions. Semen collected from 3 bulls wasdiluted to 200 million sperm/ml in 14G citrate buffer containing 5% egg yolk (2) or one of thelipoprotein diluents at the equivalent protein concentration (8.15 mg/ml). Assessments were performedat 39°C, with the semen diluted to 20 million sperm/ml with 14G buffer and v andfwere measuredusing six-2 minute alternate runs for each. To obtain anaerobic conditions all diluents were saturatedwith nitrogen gas and semen samples were incubated anaerobically at 37°C for 45 minutes prior toassessment and cold shock.

Table 1: Translational velocities (v) in !-un/sec before (BC) and after cold shock (AC) under aerobicand anaerobic conditions. Velocities are mean :I: s.e. (n=3). Rotational (fj results not shown.

AEROBIC ANAEROBIC

TREATMENT BC AC BC AC5%EY 151.1:1:9.0 152.1:1:9.1 99.9:1:11.7 97.9:1:13.1WTM 148.8:1:10.1 150.2±9.9 97.3:1:14.2 95.5:1:14.7SLM 144.6:1:8.7 122.5:1:9.1'" 101.5:1:10.3 100.0±1O.0CTM 132.5:1:17.3 107.5:1:25.0'" 97.1:1:9.7 92.4:1:9.3

* Slgmficantly reduced after cold shock (p<0.05)

There is a marked drop in TV and RV when oxygen becomes the limiting factor. The results suggestthat 2/3 of the total velocity component is contributed by the anaerobic phase. Hence, fructolysis mustbe the regulating mechanism of sperm motility. Under aerobic conditions, 5% EY and WTM show nosignificant drop in TV after cold shock, both these extenders have high lipid:protein ratios (2) and areeffective in protecting the motile sperm. SLM and CTM on the contrary, have lesser lipid:protein ratiosand show a significant drop in TV after cold shock, however, under anaerobic conditions there are nosignificant differences either between the extenders or after cold shock. This disruption to the aerobicphase in SLM and CTM suggests that cold shock is causing mitochondrial damage. In SLM this is onlypartial, because, if there was complete respiration breakdown, the aerobic value would decline toanaerobic levels. This is almost occurring in CTM, however sufficient thermal protection is provided tomaintain fructolysis in the cytosol, as spermatozoa cold shocked in the absence of any thermalprotectant are rendered completely immotile.

(1) Schaare, P., Vishwanath, R., Shannon, P. & Curson, R (1991). Proc. Aust. Soc. Reprod. BioI. 23:8(2) Vishwanath, R., Shannon, P. & Curson, R(1992) Anim Reprod. Sci. 29: 185-194.

15

MODIFICAnON OF RAM SEMEN DILUENT (RSD-l): ORGANICNUTRIENT(S) REQUIREMENT FOR RAM SPERMATOZOAL MOTILITY

G.C. Upreti, K. Brown, J.E. Oliver and J.F. Smith

AgResearch, Ruakura Agricultural Centre, Private Bag 3123, Hamilton, N.Z.

The organic nutrients in RSD-l consist of glucose, fumarate, pyruvate andglutamate (1). RSD-l was developed from a media that was optimized for themaintenance of sheep hepatocytes (2) and the organic nutrient requirements forspermatozoal motility were not evaluated during the initial development of RSD-l.This study was undertaken to assess the requirement for these organic componentsand other tricarboxylic acid (TeA) cycle intermediates for spermatozoal motility.

Semen was collected, diluted and motility parameters were measured as previouslydescribed (1). Under incubation at 38°C, the percentage motility and velocity ofspermatozoa declined rapidly in the absence of all organic nutrients. After 1,5, and24 hours incubation at 38°C, the % motile spermatozoa and velocity score(indicated in brackets) for RSD-l incubations were 80 (3.9), 70 (3.7) and 38 (1.9)respectively compared to 70 (3.6),45 (2.8) and 4 (0.5) for incubations in deficientRSD-l (ie. RSD-l without any organic nutrient). Addition of glucose alone todeficient RSD-l, partially restored motility, increasing % motile and score to 24(1.5) after 24 h incubation at 38°C. Fumarate, pyruvate or glutamate wereineffective in restoring velocity score (0.2 to 0.4) and percentage motilespermatozoa, increased only for fumarate (19%) after 24 h storage at 38°C.

Other TCA cycle intermediates (Citrate, isocitrate, oc-ketoglutarate, oxaloacetatesuccinate, malate) alone did not improve spermatozoal motility. However acombination of glucose with oxaloacetate, or citrate or fumarate was more effectivethan glucose alone, but not different from RSD-l. Fumarate and oxaloacetate weremore effective than citrate in restoring motility in presence of glucose. The %motile and velocity score for glucose, glucose + fumarate, glucose + oxaloacetate,glucose + citrate and RSD-l were 51 (2.6),63 (3.6),60 (3.4), 54 (2.8) and 65 (3.1)respectively after 24 h incubation at 38°C. Additional improvements in motilityparameters were not obtained when either acetate or other TCA cycle intermediateswere added to the glucose + oxaloacetate combination.

The above studies have shown the involvement of glucose metabolism and TCAcycle in maintenance of sperm motility. Subsequent studies will examine the valueof alternate sugars and optimization of concentrations required under conditions ofstorage.

(1) Upreti, G.C. et al. (199la) Proc. Aust. Soc. Reprod. BioI. 23: 129.(2) Upreti, G.C. et al. (199lb) Small Ruminant Research.1: 175-187.

16 ]]

INSEMINAllON OF IN VITRO MATURED SHEEP OOCYTES WITH LOW NUMBERS OFFRESH SPERM DNA AMPLIFICATION OF AN OESTROGEN-ASSOCIATED OVIDUCAL

GLYCOPROTEIN SEQUENCE FROM PRIMATES

Proportions In columns with different superscripts differ (p<O.05)

Table 1. Effect of dose of sperm on fertilisation of oocytes.

Department of Animal Science, University of Sydney, NSW 2006

Clayton E. Walton, Qingxuan Chen! and Colin D. Nancarrow

CSIRO Division of Animal Production, P.O. Box 239, Blacktown, NSW 2148.lInstitute ofDevelopmental Biology, Chinese Academy of Sciences, Beijing.

Secretion of oviduct-specific proteins has been described for several species includingoestrus-associated glycoprotein (BOP; 90 IDa) in sheep (1) and an oestrogen-inducedglycoprotein (120 IDa) in baboons, Papio anubis (2). Recently, the cDNA sequence ofthe baboon glycoprotein was published. As these two proteins are induced by oestrogenand present during fertilization and early embryo development, we were interested intesting whether there is sufficient sequence homology to use a baboon-based probe toisolate the ovine gene.

Oligonucleotide primers of 21 base pairs (bp) length were selected by computer analysisusing the software program Oligo (version 3.4, National Biosciences) and synthesisedwith an Applied Biosystems 381A DNA synthesiser. These primers span a 537 bpregion, 77 bp at the end of the coding region and 460 bp of the subsequent 3' nontranslated sequence, DNA amplification (3) was carried out using DNA extracted fromthe blood of various species. Each reaction tube contained 1-2 units of thermostable Taqpolymerase in 50~ buffer containing 100 pmol of each primer, 0.5 ~g of the DNA to beamplified and final concentrations of 50 mM KCI, 10 mM Tris-HCI (pH 8.8), 1.5 mMMgCI2, 0.1% Triton X-1OO and 2 mM of each deoxyribonucleotide triphosphate. Thereaction mixtures were heated to 930C to denature the DNA, cooled to 530C to anneal theprimers to the target DNA and heated to 71°C for DNA replication by the Taq DNApolymerase. This cycle was repeated 40 times.

Amplification of the 537 bp DNA fragment from the Crab Eating Macaque, Macacaphilippinensis, was used to optimise the reaction. Sequences from Chimpanzee, Pantroglodytes, and Orangutan, Pongo pygmaeus, were also amplified but these were shorterthan predicted (pongo -480 bp and Pan -425 bp). We were unable to amplify sequencesfrom Spider Monkey, Ateles geoffroyi, man, salmon, several rodents, bandicoot, sheepand pigs. Restriction maps of the amplified sequences found several conserved restrictionenzyme (RE) sites in all monkey sequences amplified.

The inability to amplify a sheep sequence indicates that the baboon and sheep proteins areeither different proteins or have greater variability in the 3' non-translated region than thatseen in the primates. Whether these two proteins therefore have different functions infertilization or early embryo development remains unanswered. The primate RE mapsindicate a reasonably conserved sequence. However, the variability in RE sites andsequence length will provide a useful tool for studies on evolutionary lineage.

1) Sutton, R., Nancarrow, C.D., Wallace, A.L.C. and Rigby, N.W. (1984) J. Reprod.Fert. 72, 415-422.2) Donnelly, K.M., Fazleabas, A.T., Verhage, H.G., Mavrogianis, P.A. and Jaffe,R.C. (1991) Mol. Endocrinology 5, 356-364.3) Mullis, K.B. and Faloona, F.A. (1987) Methods in Enzymology. ISS, 335-350.

The authors wish to thank Dr D, Spielman, Taronga Zoo, Sydney, NSW for collectingand supplying the primate blood samples.

Dose of sperm No. of oocytes(million/mQ

fertilised/ polyspermic/inseminated (%) fertilised (%)

0.075 4/78 (5)8 0/4 (0)80.15 14/74 (19)b 0/14 (0)80.30 24/110 (22)b 0/24 (0)80.60 55/102 (53)C 1/55 (2)81.20 65/83 (78)d 2/65 (3t2.40 32/32 (100t 5/32(15)b

The proportion of oocytes fertilised increased with increasing dose of sperm inseminated(linear, p<0.001), as did the proportion of polyspermic oocytes (linear, p<0.001). Thisstudy confirms that 1-2 million fresh sperm/ml are required to obtain satisfactoryfertilisation of IVM oocytes, although a low level of polyspermic fertilisation is likely withdoses higher than 1 million sperm/ml. In cases where the number of sperm is limited (as insperm sexing by fluorescence activated cell sorting) some fertilised sheep oocytes may beobtained with less than 1 million sperm/ml of fertilisation medium.

T. Stojanov, W.M.C. Maxwell, S.L. Rhodes and G. Evans

('.J

Immature oocytes collected on 3 separate days, were cultured for 22hr at 39°C (5%C02 inair) in M199 + 10% FBS (foetal bovine serum), LH (10llg/ml), FSH (10Ilg/mQ andoestradiol (11lg/ml). Fresh ram semen (pooled ejaculates from 4 rams) was diluted 20-foldwith h-SOF [Hepes-buffered SOF + 3mg/ml BSA (bovine serum albumin)] and washedtwice (centrifugation at 800g, 6 min). After discarding the supernatant the semen pelletwas resuspended with b-SOF (bicarbonate buffered SOF) + 20% SS (sheep serum) to thedesired concentration of sperm. After 25 hr of culture, cumulus-expanded oocytes wererandomly allocated to 6 groups in b-SOF + 20% SS that contained 0.075, 0.15, 0.3, 0.6,1.2 or 2.4 million sperm/ml. I

'<)j'}:'

Eighteen hr after insemination presumptive Izygotes were fixed and stained with 1% acetoorcein, and examined for the presence of two pronuclei and a sperm tail as the critera forfertilisation. The results are presented in Table 1.

Satisfactory in vitro fertilisation (IVF) of in vitro matured (IVM) sheep oocytes has beenreported using 1-2 million fresh sperm/ml (1,2), although these authors did not examinelower numbers of sperm. This study examined the effect of dose of fresh sperm onfertilisation of IVM sheep oocytes.

(1) Crozet, N. et al. (1987). Gam. Res. 16, 159-170.(2) Slavik, T. et al. (1992). Theriogenology 38,749-756.

18 19

IS UTERINE FUNCTION COMPROMISED IN HEIFERSSYNCHRONISED WITH ALONG-DURATION PROGESTERONE

TREATMENT?

CHANGES IN THE POLYUNSATURATED FATTY ACID COMPOSmONOF THE UTERUS DURING INDUCTION OF THE DECIDUAL CELL

REACTION IN PSEUDOPREGNANT MICE

W H McMillan, K.L. Macmillan1 and AJ. Peterson

AgResearch and Dairying Research Corporation1, Ruakura, Private Bag 3123,Hamilton, New Zealand

J.A.SakofT, RN. Murdoch and RH. Dunstan

Department ofBiological Sciences, The University ofNewcastle, N.S.W. 2308

(1) Sirios, J. and Fortune, J.E. (1990) Endo 127: 916-925.

Day 16 Day 25 Day 40 Day 60There was no effect of synchrony treatment on pregnancy rate between day 16 and 60(Figure 1). Two thirds of the heifers with recovered material were pregnant at day 16.Based on non-return rates to day 25, the pregnancy rate had fallen to 54%. By day 40,only 45% of heifers were pregnant and this had reduced to 36% pregnant by day 60.In summary, these data show that extended-duration progesterone treatment does notcompromise either CL function or the ability of the uterus to support pregnanciesfollowing embryo transfer. The sub-fertility following AI is thus likely to be associatedwith impaired sperm transport or compromised oocyte/embryo quality.

Long-duration exogenous progesterone treatment can result in good synchrony butpoor fertility. The reason for this sub-fertility is unknown but may involvecompromise of: oocyte/embryo quality, sperm transport, corpus luteum eCL) functionor uterine function. The aim of this study was to compare uterine function (andtherefore indirectly CL function) in heifers treated to synchronise oestrus with either ashort- or a long-duration progesterone treatment. Ninety seven 18-month old Herefordx Friesian heifers were treated with a progesterone-impregnanted CrnR® device foreither 10 or 19 days (10 D and 19 D groups). In addition, the 10 D group received a 10mg oestradiol benzoate capsule at CrnR® insertion and a prostaglandin injection 6 dayslater. Each heifer received a single frozen-thawed embryo 7 days after the onset ofoestrus (onset =dO). On d 16,33 heifers were slaughtered and their reproductivetracts flushed to recover conceptus material. The remainder were monitored for returnto oestrus until d 25, and then pregnancy status assessed at d 40 (using ultrasound) andday 60 (slaughter). Two of the 46 heifers in the 10 D group had excess uterine tone attransfer, and neither were pregnant at day 60. Of the 8 heifers in the 19 D group withexcess tone, 3 were pregnant. The factors which may explain excess tone are thefailure of circulating progesterone to effectively suppress luteinising hormone secretionand therefore allow follicle persistence and excess oestrogen production (1).

Figure 1

The peri-implantation period of pregnancy is associated with a high incidence ofreproductive wastage. This may be due to either poor embryo development or failureof the uterine endometrium to provide a suitable environment for implantation. Theembryo-induced transformation of hormonally primed uterine stroma into decidualtissue in preparation for implantation is characterized by cellular proliferation anddifferentiation. Lipid metabolism and, in particular, membrane polyunsaturated fattyacid (pUFA) composition can modulate membrane fluidity which can subsequentlyinfluence carrier mediated transport systems, membrane-bound enzymes, receptorfunction, signal transduction mechanisms, prostaglandin production, and cell growth(1) all of which are associated with the decidual cell reaction (OCR) (2). In addition,intracellular fat stores provide the oxidative energy for these cellular functions.

In the present study, the PUFA composition of uterine tissue was analysed by GeMS techniques at various times (20, 40, 80, 160, 320, 640, & 1280 min [21.3h])following the induction ofthe DCR with Concanavalin A (Con A)(125ug/hom in 0.9%NaCl) (3) in the left uterine horn on day 4.5 of pseudopregnancy. The right uterinehom was not stimulated to undergo decidualization and was used as a control in allcases. The range of PUFAs extracted from whole uterine tissue included arachidonicacid C20:4(n-6), linoleic acid CI8:2(n-6), C18:2 isomers (a) and (b), C20:2(n-6]),C20:3(n-6), C20:3(n-3), C20:4 isomers (a) and (b), and C22:2(n-6), C22:4(n-6).

It was found that the PUFA relative abundance/mg of dry tissue in both the right(untreated) and left (treated) uterine horns changed significantly with time afterstimulation. This may be due to altering hormonal conditions accompanying thesestages ofpseudopregnancy. However, the levels of the PUFAs in the left horn with theexception ofthe two C18:2 isomers, did not differ significantly from those in the rightuntil 21.3h after stimulation by Con A, when levels in the left horn fell significantly(P<0.01) below those in the right hom. This decline in PUFA content in the left horncorresponded with significant increases in the weight of the horn, as opposed to theright uterine horn, due to growth changes associated with decidualization.

In conclusion, the results suggest that major changes in the PUFA composition ofwhole uterine tissue accompany the process of decidualization in pseudopregnantmice. These PUFAs may be utilized as a source of oxidative energy and/or involved incellular membrane remodelling, including altered cell function during the DCR.

(1) Spector, A.A. & Yorek, M.A. (1985) ILipid Res. 26:1015-1035.(2) Kennedy, T.G. (1983) AustJ.Bio1.Sci. 36:531-543(3) Buxton, L.E. & Murdoch, R.N. (1982) AustJ.Bio1.Sci. 35:63-72.

• 10 DAY CIDR®11I11I 19 DAY CIDR®

8070

Q) 60

!~ 50a1 §, 40~ ~ 30~c. 20

10

o

20 21

THE ACTIVITY OF PAF:ACETYLHYDROLASE WITHIN THE UTERUSDURING THE PERIOVULA1DRY AND PREIMPLANTATION PHASES.

B. Herrmann, C.O'Neill, K.M. Battye

PREGNANCY-SPECIFIC PROTEINS IN THE UTERINE FLUID OF SHEEPAROUND THE TIME OF IMPLANTATION

Human Reproduction Unit, Royal North Shore Hospital, St. Leonards, N.S.W:

AH. Specific Activity (pmoles acetate released/mg protein/min) (mean ± SEM)Day of Cycle Uterine Fluids Endometrial Tissue

Acetylhydrolase showed a significant reduction (p < 0.01) in activityfrom the beginning of the cycle to the time of ovulation (D 0), and a furthersignificant reduction (p < 0.001) from the 1st to the 4th day of pregnancy (dayof implantation). During corresponding periods the activity in endometrial tissuedid not change. The profile in the fluids and tissue of pseudopregnant animalswas similar to pregnancy. The results suggest that coincident with two keyreproductive events (fertilisation and implantation) in which PAF is claimed toplaya role, PAF metabolism may be reduced resulting in its prolonged half-lifeand hence activity within the reproductive tract.(1) Maki et al., (1988) Proc. Natl. Acad. Sci. USA. 85: 728-732.

Platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine,PAF) is produced by gametes, embryos and the reproductive tract, and has beenproposed to be involved in the establishment of pregnancy. PAF is thought tohave a short half-life in vivo due to it rapid metabolism by the essentiallyubiquitous enzyme, PAF:acetylhydrolase. This study investigated the activity ofPAF: acetylhydrolase within the uterine fluid and endometrial tissue on variousdays of the reproductive cycle.

Eight-ten week old QS female mice had ovulation synchronised by i.p.injection of 2.5 Lu. PMSG (Day-2) and hCG (D 0). In a second group, femaleswere placed with either fertile or vasectomised males until mating occurred. Thepresence of a vaginal plug represented D + 1 of pregnancy or pseudopregnancy.The uterus was removed at approximately 15:00h every day from D -2 to D +4.The contents of the uterus was flushed with Tyrode's solution. Immediately afterflushing, the uterus was opened and the endometrial tissue scrapped from theuterus into Tyrode's solution. The cells were disrupted by sonication. The proteinconcentration of both the flushings and scrapping were assayed (BioRad). Thespecific activity of PAF acetylhydrolase was assayed using a radiometric assay aspreviously described (1).

Rita S. F. Lee, H. R. Tervit and A. J. Peterson.

To determine the origin of the pregnancy-specific polypeptides, embryonictissues and explants of uterine endometrium were cultured in mediumcontaining 35S-methionine. Day 17 and 18 embryos were cultured wholeor dissected and cultured as isolated trophoblast, allantois, yolk sac orembryo proper. Secreted proteins were analyzed by using 2-D gels andautoradiography. The most abundant pregnancy-specific protein, a 39kDa polypeptide, 2 polypeptides of approximately 40 kDa and as well as apolypeptide of approximately 44 kDa were shown to be of conceptusorigin. The 39 kDa polypeptide was secreted by the trophoblast tissueonly.

Further analysis may reveal yet more proteins that are pregnancy-specific.A combination of teChniques, such as immunoblotting and peptide microsequencing will be used to identify some of these polypeptides.

1. Voris, B.P. and Young, D.A. (1980) Anal. Biochem. 104, 478-484.2. Young, D. A., eta/. (1983) Methods Enzymol. 91,190-214.3. Godkin, J.D., eta/. (1984) Endocrinology, 114,1210-130.4. Stewart, H.J., eta/. (1987) J. Endocr.115, R13-R15.

AgResearch, Ruakura Hamilton, New Zealand.

A major cause of embryonic loss in the Iifestock industry is the failure ofthe embryo to implant. In domestic animals (like sheep and cattle), theembryo is relatively developmentally advanced when implantation occurs.The process of implantation is thought to involve complex signallingbetween maternal and embryonic tissues mediated by soluble factorssecreted either by the uterine or conceptus tissues.

The aim of this study is to identify protein factors that may mediate theprocess of implantation. Pregnancy-specific proteins in uterine fluid of day15 and day 17 pregnant ewes were compared with matched, nonpregnant controls, using "giant" 2-D gel electrophoresis (1, 2). Theincreased sensitivity of detection with large-format gels allows thevisualization of several thousand polypeptide spots compared withseveral hundred spots normally seen with good, conventional-sized 2-Dgels. At least 5 polypeptides have been identified as being pregnancyspecific, apart from ovine trophoblastic proteins (OTP), which are secretedby the trophoblast of the conceptus. The properties of OTP, its interferonlike activity, and it effects on the uterine endometrium, have been wellstudied (3,4).

19.35 ±2.4918.33 + 3.2015.33 + 3.20

8.33 ± 1.529.50 ± 9.509.08 ± 1.7011.01 ± 1.50

259.50 ± 80.6052.94 ± 12.7347.03 ± 9.43

63.95 ± 11.6928.56 + 7.3814.04± 7.2718.82 ± 6.62

-2-1o

Day of Pregnancy+1+2+3+4

22

LOCALISATION OF A PREGNANCY - ASSOCIATED MOLECULE (SBU-59)IN SHEEP

C.S. Lee. K. Gogolin-Ewens and M.R. BrandonDepartment of Veterinary Science,

The University of Melbourne, Parkville, 3052

Our previous studies have shown that the monoclonal antibody,SBU-3 raised against trophoblast microvilli from the sheep placentarecognises a population of binucleate cells and the syncytia in the sheepplacenta (1). Recently a monoclonal antibody, designated SBU-59, raisedagainst purified SBU-3 glycoproteins was found to have a broader spectrumof reactivity in ovine placental tissue than SBU-3. The present studiesexamine the distribution of this glycoprotein in the placenta and otherreproductive tissues of the sheep.

Pregnant sheep uteri at 29 to 130 days of gestation were collected froman abattoir. Tissues from placentomes and interplacentomal areas werefixed in 95% cold ethanol for immunohistochemical studies. Histologicalsections of 5 to 6 ~m were stained with the indirect immunoperoxidasetechnique. In addition histological sections of foetal and cycling ovary, onecell zygote, morula and blastocyst of sheep were stained as described above.

In early pregnancy the glycoproteins recognised by SBU-59 wereconfined to the binucleate cells of the placentomal region; positive stainingalso appeared in the maternal tissue as a result of the fusion of binucleatecells with maternal uterine epithelial c~lls. In addition to staining thebinucleate cells in the placentome, SBU-59 also recognised a subpopulationof binucleate cells present in the interplacentomal areas. As transitoryerosion of the interplacentomal uterine epithelium occurred (35-40 days)SBU-59 positive binucleate cells were found interspersed or fused with theinterplacentomal uterine epithelium. SBU-59 molecules present on theapical surface of the uterine epithelial cells, the intercellular space of theuterine epithelial cells, and the lumen and epithelial cells of theendometrial glands located at the upper region of the stroma were probablysecreted by these binucleate cells. It is also interesting to note that SBU-59also stains the primary oocytes of sheep foetus, the primary oocytes of theprimordial, primary, secondary and Graafian follicles, secondary oocytes,one cell zygote and the trophoblast of early blastocyst.

It is concluded that the interplacentomal SBU-59 positive binucleatecells play an active role in the transitory erosion of the interplacentomaluterine epithelium. Furthermore, the positive SBU-59 staining of oocytesand preimplantation embryos suggests that this monoclonal antibody maybe used to study cellular differentiation in early embryogenesis.

(1) Lee, C.S., Gogolin-Ewens, K., White, T.R. and Brandon, M.R., J. Anat.140: 565-576 (1985).

23

ENDOCRINE INTERACTION OF THE HUMAN TROPHOBLAST AND DECIDUA:EFFECfS OF DECIDUAL SECRETIONS ON TROPHOBLAST STEROIDOGENESIS.

Peter C. O'Leary, Amanda K. Sunley and Brendan J. WaddeD·

Department ofBiochemistry, King Edward Memorial Hospital, Subiaco, W.A. 6008 andBDepartment ofAnatomy and Human Biology, The University ofW.A., Nedlands, W.A. 6009

Endocrine interaction between the trophoblast and adjacent decidua is likely to play animportant role in the establishment and maintenance ofmammalian pregnancy. In humans, thedecidua is a rich source ofinsulin-like growth factor binding protein-l (IGFBP-I) and prolactin(pRL), but the biological role of these decidual products has not been fully explored. In thepresent study, we investigated the possibility that decidual secretions exert a local, paracrineeffect on the steroidogenic activity ofthe villous trophoblast. Decidual IGFBP-l may beparticularly important in this regard, since IGF-I and -IT have both been shown to affecttrophoblast steroidogenesisl . The steroidogenic activity ofhuman trophoblast cells wasmeasured in the presence ofdecidual culture medium (DCM), containing secretory products ofdecidual explants. Because any action ofIGFBP-I is likely to involve interaction with IGFs, theeffects ofDCM were examined in the presence and absence ofexogenous IGF-I.

Human placentas were obtained after uncomplicated C-section at tean. Trophoblastcells were isolated from villous fragments using trypsin/DNAse dispersion and Percoll gradientpurification as previously described2. Cells (Rl5x10S) were incubated for 24h in control medium(DMEM) alone or with the addition of50% OCM (see below), 20 nglrnl IGF-I, or bothtreatments combined. All incubations were carried out on a rocking platfoan in an atmosphereof5% CO2 at 37°C. The DCM had been obtained previously from a 24-h incubation ofteandecidua parietalis fragments in DMEM. Following their initial24-h incubation, trophoblast cellswere centrifuged and the medium replaced with similar treatment medium containing 1.0 J1gImlofeither pregnenolone (Ps) or androstenedione (.6.4A). Incubation was continued for 3h, and the

400 medium was removed and stored for subsequento CONTROl.. RIA ofprogesterone (p4) and oestradiol (EV.

~ 350 l2Zl IGF-I In the absence ofadded precursors, Po(g 300 _ OCM secretion was 0.8 ± 0.1 ngl3h (mean ± SEM, n=5'0 placentas), but no~ secretion was detectable.~ 250 ~ OCM/ICF-I The figure shows rates ofP4 and E:2 synthesis in

5 200 ~:'~;'~;d.:!:t~O~:trOI) the presence oftheir respective precursors for all~ 150 groups. In control incubations, synthesis ofP4

2 was 47 ± 14 ngl3h and that of~ was 625 ± 110~ 100 pg/3h. P4 synthesis waS unaffected by IGF-I~ alone, but increased in response to DCM (11%;~ 50 P<O.05; ANOVA) and DCM/IGF-I (22%,

o P<O.05). Conversion of.6.4A~~was alsoPROGESTERONE OESTRADIOl.. unaffected by IGF-I alone, but increased

markedly in response to both DCM (3. I-fold; P<O.O1) and DCM/IGF-I (2.9-fold, P<O.OI).In conclusion, these data demonstrate that secretory products ofthe decidua dramatically

alter steroidogenic activity oftrophoblast cells in vitro. Current studies are aimed at determiningwhether either of the major decidual products, IGFBP-I and PRL, account for this stimulation.

INestIer IE (1990) Endocrinology 127:2064; 2Kliman ill et al (1986) Endocrinology 118:1567Supported by the King Edward Memorial Hospital Research Foundation.

24 25

TRH RegnaultI,2, R.J.ScaramuzziI, N.SriskandarajaJ12 and V.H.Oddy3

DEPRESSION OF INSULIN RESPONSE TO A GLUCOSE LOAD IN PREGNANTEWES WITII mGH CIRCULATING PLACENTAL LACTOGEN LEVELS

86

-----c--

2o

"'iiie 22o

en 20

4 6 8 10 0 2 4Week Week

Fig. la) Scrotal circumference and b) testosterone concentration of entire. castrated. Vax 2 and Vax 4bucks (mean ±scm)

Vaxstrate® provides effective immuno-castration following two vaccinations at either2 or 4 week intervals. The results also confirm the autumn decline in voluntary feedintake in cashmere bucks (1) and suggest that it is testosterone dependent.

Supported by the MRC. We thank Me A. Farnham and Mrs EJ. Speijers for their assistance.(1) Walkden-Brown. S.W. ct al. (1990) Prec. Aust. Soc. Anim. Prod. lB.: 564.

Susan I Godfreyl, Stephen W. Walkden-Brown2, Graeme B. Martin2,Stephen G. Gherardil, Mark J. Lindsey3 and Ben L. Porter1

IMMUNO-CASTRATION OF ADULT CASHMERE BUCKS WITHVAXSTRATE®

1. Sheep Industries Branch. Wastern Australian Dapartment of Agriculture. South Perth. W.A. 61512. Animal Science Group. School of Agriculture. University of Western Australia, Nedlands. W.A. 6009

3. Arthur Webster Pty Ltd. Castle Hill. N.S.W.• 2154

Non-surgical castration has potential applications in the live export of feral goat bucksand the control of unwanted male behaviour in growing bucks. In this study, conductedduring the breeding season in the southern hemisphere, we tested whether thecommercial anti-GnRH vaccine, Vaxstrate® (Arthur Webster Pty Ltd, Castle Hill,N.S.W., batch No 2542), would reduce testosterone concentrations and testicular size inmature cashmere bucks.

Twenty cashmere bucks (1.5 - 4 yrs) were housed and acclimatised to individualpens and ad libitum feeding of commercial goat pellets (crude protein 17.5%, ME 11.6MJ/kg) for one month. Bucks either remained as non-immunised controls (5 Entire, 5Castrated) or were immunised with Vaxstrate® (l ml sc, n=10). Imrnunised bucks weregiven booster imrnunisations after 2 (Vax 2, n=5) and 4 weeks (Vax 4, n=5).Castration and primary immunisation were performed on 21 January, 1993. Voluntaryfeed intake was measured daily while liveweight and scrotal circumference weremeasured weekly for 10 weeks following primary immunisation. Each fortnight (toweek 8) jugular blood was sampled every 30 minutes for 6 h and pooled aliquots wereassayed for testosterone.

In Entire bucks scrotal circumference and testosterone concentrations increased withthe onset of the rut (Fig. 1). Immunisation with Vaxstrate® significantly reducedtesticular size and plasma testosterone concentrations (p<O.OOl, Fig. 1). Testosteroneconcentrations fell to castrate levels within 2 weeks of booster immunisation. Entirebucks exhibited a decline in voluntary feed intake after week 3. Average intakes ofpellets (kg/day) over weeks 5 to 8 were: Entlre,'-1.36; Vax 2, 2.04; Vax 4, 1.80; andCastrate, 1.91 (sem ± 0.188). Liveweights increased throughout the experiment, but didnot differ between treatments (initial 44.5 ±2.5 kg, final 52.5 ±2.5 kg, p>0.05).

e- 32 ~18 b)

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0.0

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ICSIRO Division of Animal Production, Bladaown 2148; 2University of Western SydneyHawkesbury, Richmond 2753 and 3NSW Agriculture, Camden 2570

(1) Brelje, T.C., Scharp, D.W., Lacy, P.E. et al.(l993) Endocrinology 132: 879-887.(2) Fowden AL. (1982) J. Endocrinology 94: Supplement, 65P.

Ovine Placental Lactogen (oPL) is implicated in the repartitioning of maternal nutrients duringpregnancy (1). Its mode and site(s) of action in the proposed sparing of glu~ose for fetalrequirements, are ill-defined. One possible mechanism could be through reduced pancreaticsensitivityto glucose, as ewes carryingmore thanonefetus display adiminished insulin sensitivitywhen subjected to a glucose challenge (2).Medium woolMerinoewes, 3-4years old(45-60kgliveweight, 125 days postcoitus), ofthreelittersizes, dry (0), single (S) and twin (T) were used. A dose ofOAg glucose/kg (Lv) was injected 24hours post-feeding. Blood samples werecollected at -15,0,20,35, 55, 95, 155minutes after the dose. Data were analysed by repeated measures ANOVA.The glucose dose increased insulin concentration. Comparedto Dand Sewes,Tewes had a lower (P<0.05) insulin concentrationpeak (Figure 1). T ewes had ahigher (p<O.OOl) level of oPL, 2494 ±163 nglml (n=4), than S ewes 932 ± 94nglml (n=lI). Differences in insulin response pattern were also observed. Tewes, but not D and S ewes displayed abiphasic insulin response to the glucosedose, similar to that observed in D ewesgiven> O.5g1kg i.v. (T.R.H. Regnault,unpublished results).These data suggest that oPLmay reducepancreatic B-cell sensitivity to glucose,thereby favouring non-insulin dependent uptake of glucose by placental tissues. Moreover we speculate that, whilereducing peak insulin concentration, theglucosethres.bold for eliciting abiphasicresponse may also have beenlowered inTewes. Suchamechanismmayfacilitateglucoseuptakebythegraviduteros,whilealsoensuringtbatglucoseuptakeoccurs Figure 1: Insulin response to glucosein insulin dependent tissues.

26

MORPHOLOGICAL DAMAGE TO THE RAM TESTIS AND EPIDIDYMISFOLLOWING ISCHAEMIA: POSSffiLE IMPLICATIONS IN MALE INFERTILITY

Caroline M Markey* Anne M Jequier Geoffrey T Meyer** Department of Anatomy and Human Biology, The University of Western Australla.Nedlands 6009: Perth Andrology. Perth Surglcentre, South Perth 6151, WesternAustralla.

Blood vessels supplying the testis and epididymis show a predisposition tosclerotic chan~es in mammals1 and these changes are also seen in both youngand aged men . This study investigated the effects of such vascular anomaliesusing the ram as a model to determine if there is a link between arteriosclerosisof the spermatic artery and testicular pathology.Spermatic arteries of 16 rams were partially occluded e;ther unilaterally orbilaterally. using a sllk ligature or lcm length of polyethylene tubing. Duringan 8 week post-operative period semen samples were assessed using aHamilton Thorn Research Motility Analyzer as an index of how testicular andepididymal function were affected by ischaemia. At the end of the 8 weeksrams were castrated and the testis and epididymis were prepared forhistological analysis. Of the epididymal regions only the caput was analysed asthis region is a common site of tubular obstruction which causes maleinfertmty3·Morphometric results indicated that a range of ischaemic insult had beeninduced in the experimental testes and epididymides (n=22) and this wasevidenced by a significant decrease (P<0.05) in the % spermatogenic epithelium,the height of the seminiferous tubule epithelium and the diameter of the tubulelumen compared to the contralateral (n=10) and control (n=5) testes. Thedecrease in seminiferous tubule diameter was accompanied by a decrease inthe tubule diameter of the caput epididymis (r=0.61; P<O.OI). The testicularbiopsy score count (TBSC)4 averaged 5.81 ± 0.72 (SEM) in the experimentaltestes, indicating a general absence of spermatozoa and spermatid, in contrastto the contralateral (8.66 ± 0.08) and control (8.88 ±. 0.16) testes whichcontained a full complement of spermatogenic cells. ThiS score highlighted thefocal damage that ensued within the testes as a result of ischaemic insult. afeature qUite typical of many human pathologies5. A histological description ofthe progression of testicular and epididymal damage due to increased degrees ofischaemia will be presented. One prominent feature of the induced OXidativedamage was the accumulation of lipofuscin within various tissue compartmentsof the testis and epididymis indicating the involvement of reactive oxygenspecies in the tissues' demise.Sperm concentration declined by a range of degrees depending upon theseverity of ischaemic damage and there was an exponential relationshipbetween this measure and the TBSC (r=0.85; P<O.OI). Testis weight alsodecreased with ischaemia and when. plotted against both % spermatogenicepithelium (r=0.73; P<O.OI) and the TBSC (r=0.73; P<O.OI) a quadrantrelationship was evident. These revealed a threshold wight below which thenormal spermatogenic epithelium is structurally impaired. Further, there wasa positive linear correlation between testicular weight and sperm output(r=0.82; P<O.OI).We hypotheSize that similar morphological damage occurs in human malesexhibiting arteriosclerosis of the spermatic artery and that this may be asignificant contributing factor to their fertility.

1. Pearson, G.R., Sllnger, W.B. (1982). Vet Pathol. 19:7102. Oshima. S.• Okayasu. I., Uchlma, H., Hatakeyama, S. (1984). Acta Pathol. Jpn.34:13273. Mitchinson, M.J., Sherman, K.P., Stainer-Smith. A.M. (1975). J. Path. 115:574. Johnsen, S.G. (1970). Hormones 1:25. Hargreave. T.B.• Jequler, AM. (1978). J. Urol. 50:415

27

CHRONIC OESTRADIOL TREATMENT OF OVARIECTOMIZED EWES IN THENON-BREEDING SEASON DOES NOT AFFECT THE DENSITY OF NPY

TERMINALS IN THE HYPOTHALAMUS

Barker-Gibb M.L., ana I.J. Clarke

Prince Henry's Institute of Medical Research, P.O. Box 152 Clayton Victoria 3168

In rats and rabbits, the effects of neuropeptide Y (NPY) on LH secretion aresteroid dependent. In contrast, recent studies on sheep (1,2) have shown that gonadalsteroids do not influence the effect of NPY on basal LH secretion, although differentialeffects have been noted around estrus (2,3). The present study aimed to investigate theeffects of oestradiol (E) on the immunocytochemical expression of NPY terminalswithin the arcuate nucleus-median eminence (ARC-ME), medial preoptic area (MPOA)and organum vasculosum of the lamina terminalis (OVLT) of the Corriedale ewe duringanoestrus when the negative feedback effect of E is maximal.

Twelve OVX ewes were divided into 2 equal groups. One group received a 3cms.c. implant of E at least 8 days prior to sacrifice, and the other group remaineduntreated. Matched pairs of sheep from each group were decapitated, and their brainsperfused with PBS and a modified Bouin's fixative and then post-fixed in the sameBouin's solution for 8 hrs. After cryoprotection in 25% sucrose, pairs of brains weresnap-frozen and sectioned at 2011m on a freezing microtome. Matched sections throughthe rostra-caudal extent of the ARC-ME, MPOA, and OVLT of each pair of sheepwere immunostained under identical treatment conditions with a polyclonal antibody toNPY. The paraventricular nucleus (PVN) was also sampled as a control area. Pairs ofsections (n=3-6, depending on nucleus/area) through each brain region were imageanalysed by sampling bilateral areas (n=4-16) of each coronal section of the area underinvestigation, via a fixed sub-area thresholding technique. The mean % area and spatialdensity of immunostained terminals within each region was obtained for each pair ofsections and totals obtained for each nucleus/area. There was no significant differencebetween E treated and OVX sheep in % immunostained area or density of NPYterminals within the ARC, ME, PVN, MPOA or OVLT. The data suggest that E doesnot influence the expression and/or release of NPY from terminals within the POAOVLT (region of GnRH cell bodies) or ME (region of GnRH terminals) which is in linewith previous evidence showing that NPY has the same effect on LH secretion in OVX,intact and OVX + E treated ewes.

(1). McShane TM, May T, MinerJL, Keisler DH. (1992) BioI Reprod. 46:1151-1157.(2). Porter DW, Naylor AM, McNeilly AS, Lincoln DW. (1993). J Neuroendocrinol.

~:163-174.

(3). Advis JP, Conover CD, McDonald JK, Kuljis RO. (1990). Ann NY Acad Sci.Ml:468-470.

28

CYTOKINES AND PLASMINOGEN ACTIVATOR ACTMTY IN THE RAT OVARY ATOVULATION

.Nigel.BQnellQ, Robert Norman, Mats Brannstrom

Department of Obstetrics and Gynaecology, The University of Adelaide, The Queen ElizabethHospital, Woodville Road, Woodville, SA 5011, Australia.

Ovulation is initiated by the luteinizing hormone (LH) surge, involves plasminogen activator(PA) directed degradation of the preovulatory follicle wall (1) and has been suggested as beingakin to an inflammatory process (2). Recent data indicates that cytokines, such as interleukin1 beta (IL-1f3) and tumour necrosis factor alpha (TNFa), derived from leukocytes infiltratingthe preovulatory ovary, can promote ovulation (3,4) and these cytokines are known to at leastpartially govern the regulation of the PA response in non-ovarian systems (5).

The aim of this study was to investigate the effects of IL-1f3 and TNFa on ovulation inducedPA activity of incubated preovulatory follicles and isolated perfused ovaries of the rat.

Preovulatory follicles of equine chorionic gonadotrophin (16IU) primed Sprague Dawley ratswere incubated with ovine LH (1 uglml) , recombinant human IL-1f3 (0.1-10nglml), recombinanthuman TNFa (1-100ng/ml), IL-1f3 (1pg/ml-10nglml) + LH or TNFa (1-100ng/ml) + LH for 14hours. Altematively, follicles were incubated with LH (1 uglml), IL-1f3 (10ng/ml) ± LH or TNFa(100ng/ml) ± LH for 6 hours. Whole rat ovaries were perfused for 8 hours with LH (O.1ug/ml),IL-1f3 (4nglml) ± LH or TNFa (40nglml) ± LH. PA activity in follicular and ovarianhomogenates was determined by a labelled fibrinolytic assay. Additionally, secreted levels ofthe ovulatory mediators progesterone (P) and prostaglandin E (PGE) were measured by RIAto determine follicular viability. The presence of leukocytes in the follicle wall was confirmedusing immunohistochemical localization.

29

THE ROLE OF AN OESTRUS-ASSOCIATED OVIDUCALGLYCOPROTEIN IN THE EARLY DEVELOPMENT OF THE SHEEP

EMBRYO

J.L. HilI!, S.K. Walker2 and C.D. Nancarrow1

lCSIRO Division of Animal Production, P.O. Box 239, Blacktown, NSW 21482Department of Agriculture, Turretfield Research Centre, Rosedale, S.A. 5350

It is well established that in vitro culture of ovine embryos using synthetic oviduct fluid(SOF) and human serum <?S) results in several developmental abnormalities, includingearly ?lastocoele. forma~IOn a~d dec:eased nu~bers of n~clei per blastocyst (1).Prelimmary expenments mvolvmg the mcorporatlon of a fractlon (PI) of oviducal fluid(OF), enriched with an C?es1;nIs-associated ov~du~al glycoprotein (BOP), into the existingembryo culture system mdicated that EOP SIgnificantly mcreased the number of nucleiper blastocyst (2). The present work examines more closely the effects of Fl on earlyembryo developmentOF was fractionated (Fl,F2) by gel filtration using a Superose 12 FPLC column(Pharmacia). Fl contained predominantly EOP while F2 consisted largely of albumin.One cell sheep embryos were incubated in synthetic oviduct fluid (SOF) containinghuman serum (HS) (10% or 20%) in the presence and absence of Fl and F2. Allembryos were incubated in 0.8 ml of the appropriate culture medium under paraffin oilat 39°C in a gas phase of 5% C02, 5% 02 and 90% N2. Cleavage of embryos wasexamined by light microscopy after 24 h and blastocyst formation was assessed every8 h from day 4 of culture. Newly formed blastocysts were removed from culturepla.c~d in fixative for a minimum of 36 h and nuclei numbers assessed by aceto-orce~stammg.

In summary, IL-1f3 dose and time-dependently inhibited the LH-induced increase in PAactivity in rat preovulatory follicles while increasing P and PGE synthesis. The PA results wereconfirmed in the perfused rat ovary. Results of the perfused rat ovary indicate TNFa may alsoplaya similar inhibitory role, but the follicle incubation results were less definitive. IL-1f3 andTNFa have been shown to promote ovulation (3,4), but this study suggests IL-1f3 (andpossibly TNFa) may also be mediators of a negative-feedback loop designed to regulate theintensity of LH activation of PA at ovulation.

LH induced significant 2.5-fold and 4-fold rises in PA activity in 6 and 14 hour follicleincubations, respectively, and enhanced PA activity in perfused ovaries 4-fold. In each caseand at each concentration, IL-1f3 and TNFa, alone, had no effect. Coincubation with LH andIL-1f3 significantly inhibited the LH-induced increase in PA activity, up to 80% in the 14 hourincubations and 95% in the perfusion studies, but not after 6 hour follicle incubations. TNFa inthe presence of LH revealed the same inhibitory trend, but was not significant in the 14 and 6hour follicle incubations, although inhibiting LH-induced PA activity by 95% in perfused ratovaries. P and PGE results were as expected for viable follicles. Monocyteslmacrophagesand neutrophils were positively identified in the thecal layer of isolated follicles.

References: (1) Beers,W. (1975) Cell 6:379-386.

(2) Espey,L (1980) Biol. Reprod. 22:73-106.

(3) Brannstrom,M., Wang,L &Norman, A. (1993) Endocrinology 132:399-404.

(4) Brannslrom,M., Bonello,N., Wang,L & Norman,R. (1993) submitted

(5) Iwamoto,T., Nakashima,Y. &Suelshi,K (1990) Kidney Int 37:1466-1476.

Table 1:

Effects of OF fractions on early embryo development

Treatment % divided % formation of Day of Mean no. Totalembryos after blastocysts from blastocyst nucIeiper no.24 h cleaved embryos (no.) formation blastocyst embryos

SOF+HS+Fl 61.0a 70.2 (33)a 5.35±0.la 50.4±1.a 78SOF+HS+F2 79.0b 55.8 (19)a 4.35±0.lb 43.6±2.b 43SOF+HS 81.4b 71.9 (41)a 4.62+0.lb 39.8+1.b 70

Means with different superscripts differed significantly p<O.05

The number of embryos cleaving in the presence ofFI was significantly lower than inother treatments (Chi-square). However, the percent of blastocysts forming fromcleaved embryos did not differ between treatments. Time taken for blastocystformation and the mean number of nuclei per blastocyst were both significantlyincreased (ANOVA) in the presence ofF!. These results suggest that Fl may delay thedevelopment of some one cell embryos when compared to the control culture system.The presence of Fl slows down the rate at which blastocyst formation occurs and thismay. b~ directly responsible for the increa~e in numbers of nuclei in blastocysts.~ehmmary dat,a su~gest !hat the effect ofF~ IS concentration dependent and this aspectIS presently bemg mvestlgated. In conclUSIOn, these results strongly imply that EOPmay play an important role in the regulation of early embryo development.

1) Walker, S.K., Heard,T.M. and Seamark, R.E (1992). Theriogenology. 31: 111-126.2) Hill, J.L., Marshall, LT. and Nancarrow, C.D. (1992). Proc. Aust. Soc. Reprod. BioI. M:I04.

30 31

ENUCLEATION BY CENTRIFUGATION FOR BOVINE NUCLEAR TRANSPLANTATION

NUTRIENT UPTAKE DURING EMBRYONIC DIAPAUSE ANDREACTIVATION IN TAMMAR WALLABY BLASTOCYSTS

1?~;~,~'~-r

R. Snindler, M.B.Renfree, and D.K.Gardner·

Brendan G. Tatham, Aneta T. Dowsing and Alan O. Trounson.

Centre for Early Human Development, Monash University, Clayton, Victoria.

Department of Zoology, University of Melbourne, Parkville and·Centre for Early Human Development, Monash University, Clayton, Victoria.

The tammar wallaby, Macropus eugenii, maintains a diapausing blastocyst in the ute~while suckling young. Blastocyst reactivation can be induced by removal of the suckingyoung. Reactivation in the wallaby blastocyst, as estimated by uridine and thymidineincorporation (1,2) and mitosis (3), occurs around 4 to 5 days after removal of pouchyoung (RPY), while blastocyst diameter does not alter significantly until da~ 8 aft~r RPY(4). Synthesis of cellular material and ion transport, necessary for cell prohfer~tIon an.dblastocyst expansion respectively, require considerable utilisation of me~ab~htes. It.IStherefore expected that substrate uptake increases over the period of reactivation. W~I~eit has been shown that glucose incorporation has increased by day 5 after RPY (5), It ISnot known which substrate(s) is used to provide the energy required during reactivation,or at what stage metabolic reactivation is initiated.

Glucose and pyruvate uptake by individual blastocysts was assessed on selecteddays after RPY, using ultramicrotluorescence (6). Blastocysts w~re incu~a~edindividually at 37°C for 3h in 1~ drops of phosphate buffered salt solution contammg0.5mM glucose and pyruvate, supplemented with 2mglml BSA. Serial nanolitre samplesof medium were taken every 30 minutes for metabolite analysis and linear rates ofnutrient uptake determined.

o 5 23.4 ± 1.0 8.0 ± 1.72 3 3.0 ± 1.0 3.2 ± 1.33 6 11.4 ± 4.3 1.1 ± 2.04 3 8.4 ± 2.1 6.0 ± 3.05 3 11.6 ± 7.3 3.0 ± 0.4

10 3 174.0 ± 28.4* 84.0 ± 27.1Uptakes are mean ±. sem.· Significantly different to day 0, P<O.05. .In contrast to previous work (5), no increase in glucose uptake before expansIOn of theblastocyst was evident. There was no significant increase in glucose uptake until day 10(P<O.05) despite the increased mitotic activity of blastocysts from day 4 after RPY \3).It is therefore conceivable that the reactivating wallaby blastocyst uses an alternativeenergy source, such as amino or fatty acids for the resumption of cell activity, whileglucose is utilised during the expansion of the blastocyst.1. Shaw, G. & Renfree, M.B. (1986) Reprod. Fert. 76, 339-347.2: Thornber, EJ. et al (1981) J. Embryoi. Exp. Morph. 62, 325-338.3. Berger, PJ.(1970) Dissertation Abstracts International, 31B, 3760-1.4. Renfree M.B & Tyndale-Biscoe C.H.(1973) Dev. BioI. 3228-40.5. Pike,I.L. (1981) J. Reprod. Fert. Supplement, 29, 203-213.6. Leese, H.J. & Barton, A,M (1984) J. Reprod. Fert. 72, 9-13.

(1) Yang, C.H. and Yanagimachi, R. (1989). Human Reproduction 4(1),63-71.(2) Gardner, D.K. et al. (1992). Proc. of Australian Society for Reproductive Biology 24, 90.

Morula (%)1/8 (13)o (0)1/11 (9)

Cleavage (%)8/15 (53)3/11 (27)6/18 (33)

Group Enucleation (%) Fusion (%)1 24/50 (48) 15/24 (63)2 68/76 (90) 11/16 (69)3 69/78 (89) 18/21 (86)

TEM studies of centrifuged oocytes show the stratification of cytoplasm into 5 phases accordingto density of its components. Lipid being least dense is found at the upper pole, followed by membranebound vesicles, smooth ER, a large organelle free region containing the MIT plate and at the lower polemitochondria are concentrated.

centrifugation results in a higher enucleation rate than micromanipulation (89% vs 48%). Thisis partially due to the emission of identifiable nucleate fragments which possess an extrusion conecontaining the M IT plate. Electrofusion rates were similar between groups (63% to 86%). Developmentof NT embryos produced in groups 1 and 3 was similar (13% vs 9%). Group 2 fragments were darkerthan the opaque group 3 fragments which contain less lipid and organelles, suggesting that successfulNT may rely on a cytoplasmic factor. Full developmental capacity is currently being tested via transferof NT embryos into recipient cows. Enucleation by centrifugation enables the rapid production of largenumbers of enucleated oocyte fragments which can be used for the successful production of bovinenuclear transplantation embryos.

Nuclear transplantation (NT) has the potential to produce large numbers of practically identicalprogeny. Before reprogramming of a donor nucleus can take place the recipient oocyte must have itsgenetic material removed or destroyed. Traditionally micromanipulation, a highly skilled, expensive andtime consuming technique was used to enucleate metaphase IT (M II) oocytes. We report the use ofcentrifugation in a Percoll gradient to rapidly enucleate large numbers of oocytes for use in successfulproduction of NT embryos. Enucleation by centrifugation relies on the separation of oocytes intofragments in such a way that the M IT plate breaks from the oocyte as it is stretched apart.

Bovine oocytes aspirated from abattoir collected ovaries were matured in TCM 199 plus FCSand hormones for 22 hours. Cumulus cells were removed by pipetting in 05% hyaluronidase andoocytes which had extruded the first polar body selected (176/278; 63%). Enucleation occurred at 24hours post maturation (hpm), group 1 oocytes were enucleated via micromanipulation. Group 2 oocyteshad the zona removed in 0.5% Pronase and 20 oocytes were centrifuged in 0.4 ml tubes in 100 j.lJ eachof 7.5, 30 and 45% iso-osmotic Percoll (1) containing 10 Ilglml cytochalasin B at 5000xg for 4 seconds.Group 3 oocytes were centrifuged at 15000xg for 2 minutes before having the zona removed andcentrifuged as in group 2.

Enucleation of micromanipulated and centrifuged oocytes was checked by staining the biopsiedkaryoplast or a sample of the fragments with Hoechst 33342 and viewing via epifluorescent microscopyat 32 hpm. Enucleated oocytes were reconstituted with blastomeres from in vivo produced frozen /thawed bovine embryos via micromanipulation (group 1) or by aggregation to a fragment using 100!J.glml phytohemagglutinin (groups 2 and 3).

Electrofusion was achieved using a BTX ECM 200 and Optimizor with Zimmerman's fusionmedia. After manual alignment an AC pulse of 8V for 5 seconds was followed by fusion via a singleDC pulse of 77V (1.75 kV/cm) for 40 !AS, fusion was assessed 15 minutes later. Fused oocytes fromgroups 2 and 3 were placed in a false alginate zona and all NT embryos were cultured in SOF plusamino acids under mineral oil with 4 embryos per 30 III drop changed every 48 hours (2). Cleavage wasassessed 40 hours post fusion and development to morula day 6 post fusion.

Pyruvate uptake(pmol!embryo!b)

Glucose uptake(pmol!embryo!h)

nDays after RPY

32 33

.9 300 b}:E.!2l

CD~ 250.!g:::l.9

~ 200

60

55

~ 50

:E 45.Ql

~ 40CD.C!:...J

35

NUTRITION AFFECTS TESTICULAR SIZE IN MALE GOATS WITHOUTCHANGING LH, FSH OR TESTOSTERONE CONCENTRATIONS

S W Walkden-Brown1,4, BJ. Restall2, B.W. Norton1, R.J. Scaramuzzi3 and G.B.Martin4,5

1Dept Agriculture, University of Qld, 4072; 2Wollongbar Agric. Inst., NSW Agriculture, WollongbarNSW 2477; 3CSIRO Animal Production, Blacktown, NSW 2148. 4School of Agriculture, University ofWestern Australia, Nedlands, WA 6009. 5CSIRO Animal Production, Wembley, WA 6014.

The endocrine and spennatogenic functions of the testis are thought to becontrolled by gonadotrophins. We describe an experiment in which differential nutritionduring the non-breeding season induced a large change in testicular size in goat buckswithout concomitant changes in peripheral concentrations of LH, FSH or testosterone.

Mature cashmere bucks were and fed ad libitum diets of High (CP 17.6%, ME8.3 MJlkg) or Maintenance (CP 6.9%, ME 6.6 MJlkg) quality for 16 weeks starting inearly July at Wollongbar (29°S 153°E) (n=6/diet). Every week liveweight was measured,testicular weight estimated from scrotal circumference and three plasma samples collectedat 40 min intervals were pooled for honnone assay. During weeks -1, 4 and 13 relative tothe onset of dietary treatments, blood was sampled every 20 minutes for 8 hours andassayed for LH and testosterone. Data was analysed by repeated measures ANOVA.

The High diet induced large increases in liveweight and testicular weight(p<O.OOI, Fig. 1). However there was no effect of diet (main effect of interaction withweek) on weekly concentrations of LH, FSH or testosterone which remained at lowconcentrations throughout the experiment (LH 2.6±O.24 ng/ml; FSH 0.86±O.68 ng/ml;testosterone 0.7±0.04 ng/ml). Data from the pulse bleeds supported the weeklymeasurements with no effects of diet on LH pulse frequency (1.0±0.16 pulses/8h), meanLH concentration (2.68±O.20 ng/ml) or testosterone pulse frequency (1.3±O.15pulses/8h). However mean testosterone concentration (overall mean 1.I±O.17) wassignificantly elevated in High bucks at week 13 (2.71±O.45 v. 1.4±O.21 ng/mI, p<0.05).

"C~

30 ,r-r-,""'T'--r""T"j..,.,.....-...-.-,-r-l'""'"'T'"""'Tj--.-....., ...., ..." ;f 150.r-.........--...-..,.....,--...-..,.....,.--...-..,....,.,--.-..,......,--.,...-"

o 4 8 12 16 0 8 12 16Week Week

Figure 1. Liveweight (a) and testicular weight (b) in bucks fed High (e) or Maintenance (0) diets.

The change in testicular weight induced by nutrition in this experiment (51%) wasmuch greater than that occurring during the seasonal cycle in bucks on a maintenance diet(17%) (1). However, in contrast with the seasonal pattern, it was not associated withclear or sustained changes in either gonadotrophin or testosterone concentrations whichremained at the low levels typical of the non-breeding season (1). These data suggest thatnutrition may affect testicular weight, and therefore spenn production, without affectingtestosterone secretion, and that this effect may be independent of changes ingonadotrophin concentrations.

1. Walkden-Brown, S.W. (1991) PhD thesis, University of Queensland, 237pp.

Michelle Lane and David K.Gardner

IN SITU REMOVAL OF EMBRYO-TOXIC AMMONIUM GENERATED BY THEMETABOUSM AND BREAKDOWN OF AMINO ACIDS IN CULTURE MEDIA

Centre for Early Human Development, Monash University, Qayton, Victoria, Australia

Mouse embryo development in culture is significantly increased by the inclusion ofspecific amino acids in the medium (1). However, amino acids are metabolised byembryos and break down at 37°C to produce ammonium in the culture medium at a rateof 100fLM every 24h. Ammonium are detrimental to mouse embryo development in vitroat a concentration as low as 75!lM. During extended culture periods it is necessary torenew the medium every 48h if amino acids are included in the formulation. However,preimplantation embryos produce an autocrine factor(s) that stimulates development (2,3).Renewing the medium may therefore deprive the embryo of the beneficial effect(s) of thisfactor(s).The aim of this study was to develop a method of enzymatically removing ammoniumfrom the culture medium. The proposed mechanism of removal was based on thefollowing reaction:

Glutamate dehydrogenase (plus ADP as an enzyme activator)NH/ + NADH + a-ketoglutarate -----------> Glutamate + NAD+ +Jr

Initially, zygotes (C57BIJ6 x CBNCa) were cultured in groups of 10 in 20111 drops ofmedium mMTF (1) under paraffin oil 5% CO2 in air at 3~C. At the 2-cell stage theindividual assay components were titrated into the medium by a 1111 injection ofax20stock. Blastocyst formation and cell number were assessed after 96h of culture. Neithera-ketoglutarate nor glutamate dehydrogenase had an effect on blastocyst formation or cellnumber at concentrations up to l.88mM and 0.375U respectively. However, NADHsignificantly stimulated cleavage (p<0.01) at concentrations from 0.03-0.2mM. Incontrast, ADP inhibited both blastocyst development (P<0.05) and cell number (p<0.01)at a concentration as low as 0.08mM. The concentration of glutamate in the culturemedium after the conversion of ammonium was determined using microfluorometry andfound to be around 0.25mM. Glutamate at this concentration significantly increasedblastocyst formation (p<0.05).Only ADP, an activator of glutamate dehydrogenase, was inhibitory to embryodevelopment. This observed inhibition is consistent with the known detrimental effects ofadenine nucleotides on embryos. In the absence of ADP the reaction still progressed,although at a slower rate, and converted O.3mM ammonium in 3h. The final enzymecocktail was therefore: NADH (0.12mM), a-ketoglutarate (0.4mM) and glutamatedehydrogenase (0.375U). Addition of this cocktail after 48h of culture in the presence ofamino acids significantly increased (P<0.05) blastocyst cell number (65.2±2.1) comparedto embryos cultured in the same drop of medium for 96h (59.2±1.9), indicating that thetoxic effects of the ammonium had been alleviated. Blastocyst cell numbers after theaddition of the cocktail were equivalent to those for which the medium was renewed after48h (67.9±2.4). The data infers that exposure to an autocrine factor(s) for 48h issufficient to elicit a response; a similar pattern of embryo stimulation has been reportedfor the sheep (4).(1) Gardner, D.K. and Lane, M. (1993) BioI. Reprod., 48, 377-385.(2) Paria, B.C. and Dey, S.K. (1990) P.N.A.S., 87,4756-4760.(3) Lane, M. and Gardner, D.K. (1992) Hum. Reprod., 7, 558-562.(4) Gardner, D.K. et aI., (1992) Proc. Aus. Soc. Rep. BioI., 24, 90.M. Lane is supported by Australian NH&MRC.

34 35

CHANGES IN OVARIAN MORPHOLOGY ASSOCIATED WITH DIETARY INTAKEIN BOS INDICUS HEIFERS

GONADOTROPHIN AND TESTOSTERONE CONCENTRATIONS AND TESTICULAR GROWTHIN RAMS SUPPLEMENTED WITH LUPINS FROM BIRTH TO PUBERTY

EM Rhodesl,LA Fitzpatrickl, JE Kinde(- and KW Entwistlel

B WBrown

CSIRO, Division of Animal Production, PO Box 239, Blacktown, NSW,2148

2ndDF10btDF

11

16

14

U

1:+o-~-~~--±--+--5~-==E>-6 101:+00-~-----:~-~--!---=--~OESTROUS CYCLE NUMBER

Fig 1. Change in mean (±8em) maximum diameter (mm) of 1st and 2nd dominant follicle (DF),ovulatory follicle (OF) and CL during tlte treatment period for CIL (-) and lRT (- - -) heifers.

It is concluded from tllis study tltat nutritional restriction results in a reduction in the maximumdiameter of dominant ovarian follicles, the ovulatory follicle and tlte ensuing corpus luteum during tlteoestrous cycle ofBos indicus heifers. The effect on steroid hormone secretion by the ovary is currentlyunder investigation.(1) Murphy MG et al (1991) J. Reprod. Fert. 62, 333-338(2) Savio JD et al (1988) J. Reprod. Fert. 83,663-671(3) Graybill FA (1976) Theory and Application of the Linear Model. DlIXbury Press

This study was partially funded by tlte Meat Research Corporation

lDepartment of Biomed. and Trap. Vet. Sciences, James Cook University, Townsville 48142Department of Animal Scie.nce, University of Nebraska, Lincoln, USA

The effect of level of dietary intake on follicular dynamics has been examined in Bos taurus heifers (1),however tlte sequential changes which occur in the ovary in tlte period preceeding the onset ofnutritional anoestrus have not been reported. The aim of tltis study was to investigate the effects ofdietary restriction on ovarian function in Bos indicus heifers.The ovaries of 17 post-pubertal maiden Brahman heifers were examined daily, using a linearultrasound scanner equipped with a 7.5 MHz tranducer (Atoka 210 DX). The growtlt and regression ofindividual follicles >5mm, and tlte diameter of the corpus luteum (CL) were recorded (2). Twelveheifers were randomly allocated to a restricted diet (TR.1) in order to produce a decrease in liveweightuntil tlte onset of anoestrus. The otlter five heifers remained on an unrestricted diet (CIL). The linearrate of change of maximum follicle and CL diameter, and initial size were compared between groups,using a random coefficients regression model (3).Mean (±sem) LWs at tlte start of tlte treatment period were 318.8±3.96 and 316.2±3.72 kg for CILand lRT heifers, respectively. A mean reduction in LW of 62.3±S.69 kg, equivalent to 19.7% of initialLW, occurred during the treatment period in the lRT heifers; during the same period LW of the CILgroup increased by 67.4±7.49 kg (21.2%). There was a significant difference between tlte groups in tlterate of change in follicle and CL size (P<O.OOl) associated witlt tlte decrease in LW in tlte TRTanimals, altltough tltere were no differences in initial diameters (Fig 1).

1 (13%)4 (40%)

LH or T at pubertyNo. rams with No. rams withpulses/12h >1 pulse/12h

Mean FSH nq/ml31-40 51-60 71-80days days days

Group

Control 0.6 0.9 0.7 8 (50%)Lupin 0.9* 0.7 0.8 10 (63%)* P<0.05 compared with mean value for controls

Mean plasma FSH levels for rams in Group B peaked 10-20 days earlierthan that for Group A and remained at an elevated level for 10-20days longer. There was no significant difference between the 2groups in the number of animals exhibiting LH or T pulses in 12h.Around puberty, mean TW for Group B was significantly greater thanthat for Group A (155.6g ± 14.4 v's 119.4 ± 9.9, t = 2.07, P<0.05).At 14 months of age, mean TW for the 2 groups did not differsignificantly but a greater proportion of rams in Group B (75%), thanin Group A (53%), had TW ~ 250g.

Data suggest that lupin supplementation of rams early in lifeadvances peak FSH secretion, increases body growth rate andtesticular weight, with likely beneficial effects on the spermproducing capacity of the animals.

(1) Kilgour et ai., (1984) Proc 10th Int Congr Anim Reprod, II, 42.(2) Tjondronegoro et ai., (1990) Proc Aust Soc Reprod BioI, 22, 7.

During early life in rams, the marked rise in plasma FSH levelsinfluences the development of Sertoli cells (1) and consequentlyspermatogenesis. Since supplementation of adult rams with lupingrain produces an increase in plasma FSH concentrations (2), it ispossible that such supplementation of ram lambs early in life mayenhance their subsequent sperm producing capacity.

Two groups of Merino rams lambs (n=16 per group) were reared inadjoining paddocks until they were 14 months of age. Group A servedas non-supplemented controls. From 2 weeks before parturition untilweaning at 12 weeks of age, the ewes in Group B were supplementedwith lupin grain at a rate of 500g per ewe per day, fed twice weekly;from weaning to puberty (28 to 30 weeks of age), the lambs in thisgroup were fed lupin grain at a rate of 10g/kg body weight per day,fed twice weekly. At frequent intervals, the lambs were weighed toassess growth rates, estimates of testicular weights (TW) were wereobtained using an orchidometer and blood (5ml) was sampled from ajugular vein for measurement of FSH. Around puberty, jugular blood(5ml) was obtained from each lamb at 20min intervals for 12h formeasurement of plasma LH and testosterone (T) concentrations.

Lambs in Group B grew at a faster rate (mean 207g/day) thancontrol lambs (197g/day) from birth to puberty, with the result thatthe animals in the former group had a significantly greater mean bodyweight than those in the latter group, both at puberty and at 14months of age (mean ± s.e.m. 37.0kg ± 0.7 v's 32.9 ± 0.9, t = 4.20;52.4 ± 0.7 v's 46.9 ± 0.8, t = 5.09, respectively, P<0.001).

36

EFFECT OF LEVEL OF DIETARY PROTEIN INTAKE ON THE PLASMA LEVELS OFPROGESTERONE IN OVARIECTOMISED EWES TREATED WITH CIDR® DEVICES

THE FREQUENCY OF RELEASE OF Ul IN ANOESTROUS CORRIEDALE EWESIS INFLUENCED BY NUTRIENT STATUS BUT NOT BY POST-PARTUM STAIDS

Plasma LH patterns in ewes (ovary-intact - OV-I, ovariectomised - OVx,OVx with oestradiol implant s.c - OVx-imp) during the anovulatory season

Plasma LH pulse frequem:y in i) ovary·intact ewes was similar in CPP and CN butlower (P<0.05) in CR ewes, in ii) ovariectomised ewes with oestradiol implants was lower inCR ewes than in CN ewes, and iii) in ovariectomised ewes without implants was similar in CNand CR ewes.

These results suggest that the increased depth of anoestrus in ewes of reducednutrient status may be associated with a greater degree of sensitivity of the hypothalamicpituitary axis to the inhibitory effects of oestradiol on the secretion of LH than in ewes ofbetter nutrient status.

The responsiveness of ewes to measures to induce ovarian cyclicity reflects their depthof anoestrus. The depth of anoestrus may be affected by nutrient and by post-partum status.We have studied the influence of these factors on the pulsatile release of LH in ewes duringthe anovulatory season as part of our investigations of the endocrine basis of the depth ofanoestrus.

4

1

4 2

2 3 4 5 6 7 8 9 10 11 12

Number of ewes with plasma LH pulse frequenCY/12 hr of

oOV-I 2 10 3 4

OV-! 5 2 1 3

OVx-imp 2 1 3 3

OVx

OV-! 3 4 1

OVx-imp 6 1 1

OVx 1 2

CR

CPP

CN

GROUP STA'IUS

Patrick J. Wright, and I.J. Clarke

Department of Veterinary Sciences, University of Melbourne, Werribee 3030and Prince Henry's Institute of Medical Research, PO Box 152, Clayton 3168,Victoria, Australia.

The frequency of release of plasma LH was determined during December in matureanoestrous Corriedale ewes that were i) 11-23 days post partum and of body conditionscore 23 (Group CPP, n=19), ii) non-post partum and of condition score :0:3 (CN, 12) and iii) ofcondition score ~2 (CR, 9). Blood samples for plasma LH determination were taken each 10min for 12 hr. CN and CR ewes were ovariectomised 2 days later and implants (O.5cm)containing oestradiol were placed s.c.. Blood samples were taken 12 days later, the implantsremoved and blood samples taken again 14 days later.

J.F. Smith, J. Parr, J.A. Konlechner

AgResearch, Ruakura Agricuttural Centre, Private Bag 3123, Hammon, N.l.

Studies into the mechanisms of nutritional influences on ovulation rate (1) have suggested thatincreased protein intake stimulates the level of liver steroid metabolism (2). These trials wereconducted to examine the effects of dietary protein intake on circulating levels of plasmaprogesterone.

Trial 1. Twenty four ovariectomised ewes fitted with abomasal catheters and fed on a lowprotein diet (100 g/d) were allocated to four groups. Two groups were given abomasalinfusions of protein (Alacen 100g/ewe/d) for 16 days while the other groups received infusionsof equivalent volume of water (1 Vd). Ewes in one group of each of the infusion treatmentswere treated with new CIDR® devices on day 4 and these were removed 11 days later. Theother groups were treated with CIDR devices that had previously been inserted into ewes for14 days (used). Daily blood samples for progesterone assay (RIA) were taken at 11.30 heach day of the trial and at 2hintervals for 8hon 2days following both insertion and removalof devices.Trial 2. Involved 10 ewes in across-over design where those infused with protein during thefirst infusion period were retreated with the same CIDR devices (5 new - 5 used) whilereceiving water infusions.

In trial 1 there were signHicant effects of CIDR type on the pattern of plasma progesteroneprofiles. Mean values 8hafter insertion were 3.7±O.28 ng/ml and 1.05±O.10 ng/ml for newand used devices respectively, while on days 4and 11 values were 1.7±O.2 and 0.90±0.1 fornew and 0.65±O.06 and 0.45±O.06 for used devices. Within 2hof device removal base levelsof 0.15 ng/ml were recorded in all groups. There was no signfficant effect of protein treatment.However, between ewe variation within groups was very high with some ewes having levels2-3 times that of others.Trial 2produced similar patterns for the different types of devices as in trial 1. However, therewas an interaction with protein treatment which had the effect of depressing the levels seenin ewes treated with new CIDRs. Mean levels at 8hwere 3.1±O.3 and 4.8±O.4 forthe proteinand water group respectively while for days 4 and 11 values were (1.6±O.3 v 2.2±0.3) and(0.9±O.2 v 1.3±O.2). These dffferences were significant in 3 of the 5 ewes. Measurment ofCIDR progesterone contents indicated no between animal or nutritional effects. Pre- and posttreatment values were 345 mg and 150 mg for new and 110 mg and 75 mg for used devices.

These resutts indicate that the absorption of progesterone from CIDR devices is virtuallyidential for all animals and that different circulating levels reflect the animal's metabolism.Within individual animals, the level of protein intake may modify the clearance rate ofprogesterone and this supports the suggestion of attered steroid metabolism being involvedin the nutritional control of ovulation rate.

(1) Smith, J.F. (1991) Proc. N.Z. Soc. Anim. Prod. 51: 15-23.(2) Payne, E. et al. (1991) Reprod. Fert. Dev.~: 725-736.

38

HUMAN RECOMBINANT INHIBIN PARTIALLY REVERSES THEHEMICASTRATION INDUCED RISE IN SERUM FSH IN THE RAT.

Anne E. O'Connor", David M. de Kretser" and Nigel G. Wreford "t

"Institute of Reproduction and Development & tDept of Anatomy, MonashUniversity, Clayton, Victoria 3168, Australia.

Testicular hypertrophy is well known to occur following hemicastration in theneonatal period. This hypertrophy is thought to be mediated by elevated FSH levelsresulting from a reduction in inhibin levels following hemicastration. It has recentlybeen shown that follicular fluid can inhibit both the hemicastration induced rise inFSH and the consequent testicular hypertrophy in prepubertal rats (1). We haverecently demonstrated that human recombinant inhibin (hRI) has little effect onserum FSH levels in young « 20 days) intact animals. We were therefore interestedto explore the effect of neonatal hemicastration on testicular development and theinfluence of hRI in the young hemicastrated animals.Newborn male rats were hemicastrated or sham operated (n=IO) under hypothermiainduced anaesthesia. Animals were killed by decapitation and trunk blood collectedat 5, 10, 15,20, 50 and 90 days. Serum FSH and inhibin were measured by specificradioimmunoassay. Elongated sperm production (ESC) was assessed at 50 and 90days. In a separate experiment, hemicastrate, sham and control groups (n=7) receiveda subcutaneous injection of IOOng hRI/g body weight or vehicle on day 10. Sixhours after injection, animals were killed and testis weight, serum FSH and inhibinwas measured.Testicular weight in hemicastrated animals was significantly increased (1.3-1.5 fold)from day 5 to 90 (p<0.05) and on day 50 and 90, there was a correspondingsignificant increase in ESC/testis. FSH levels were elevated 1.6-fold on day 5(p<0.05) and 3-fold on days 10 and 15 (p<O.OI). FSH levels remained elevated by<50% from day 20 to 90. Inhibin levels decreased by 32% on day 5, were notsignificantly different from control at days 10-20 but at days 50 and 90 weresignificantly decreased (p<O.O 1). After administration of hRI on day 10, there wasa 44% reduction in serum FSH which approached control values. The inhibin levelsremaining unchanged.This data confirms the hemicastration induced testicular hypertrophy and concomitantincrease in FSH in prepubertal animals. Interestingly, there was not a consistentdecrease in serum inhibin associated with this rise in FSH except in very younganimals. Administration of hRI resulted in a decrease in serum FSH .levels inprepubertal animals to near control values thus partially reversing the effects ofhemicastration. Whilst it appears that inhibin is involved in regulating FSH in thenewborn, the question of whether inhibin is the major factor remains open. The nearnormal levels of immunoactive inhibin observed at 10, 15 and 20 days in theseanimals may reflect the presence of pro u-C which cross-reacts in the assay.

(1) Brown, lL., Dahl, K.D. and Chakaborty, P.K. (1991) J Endocrinol 130: 207-212.

39

EFFECT OF STAGE OF BREEDING SEASON ON THE PITUITARY ACTIONSOF INHIBIN AND TESTOSTERONE TO CONTROL FSH SECRETION IN RAMS

A.J. Tilbrookl, D.M. de Kretser2 and 1.J. Clarke3

Department of Physiologyl and Institute of Reproduction and Developmenf, MonashUniversity and Prince Henry's Institute of Medical Research3

, Clayton, Vic. 3168.

Both inhibin and testosterone have negative feedback actions directly on the pituitaryto suppress the plasma concentrations of follicle stimulating hormone (FS~) in ~msduring the non-breeding season (NBS; 1). It also appears that the suppressIve actionsof inhibin may be enhanced by testosterone (1). These effects have not been studiedduring the breeding season (BS), therefore this experiment tested the hypothesis thatthe pituitary actions of inhibin and testosterone change with season.

The following procedure was conducted during the BS and NBS using 2 groups (n =5)of castrated rams (wethers) that had undergone hypothalamo-pituitary disconnection2 weeks before use in the experiment and were pulsed with 125ng of GnRH every 2h.Different wethers were used in each season. Jugular samples were collected over 4hthen 2x50p.g i.v. injections of human recombinant inhibin A (hr-inhibin) or vehiclewere given 6h apart commencing 6h after the start of sampling. Jugular samples werethen collected for 6h commencing 6h after the second injection. Each wether was thentreated for 7 days with 8mg/day of testosterone propionate (TP) given as 4mg Lm.injections every 12h and the sampling and treatment regimen was repeated. Thetreatment and sampling regimen was then repeated after 7 days of treatment of eachwether with 16mg TP/day given as 8mg i.m. injections every 12h then 32mg TP/daygiven as 16mg i.m. injections every 12h.

Treatment with hr-inhibin and TP significantly (P<O.OI) suppressed plasmaconcentrations of FSH while vehicle had no effect. The stage of the breeding seasondid not influence the direct effects of either hormone. During the BS the suppressiveeffects of hr-inhibin were not affected by treatment with TP (47% suppression beforeTP and 43% suppression with all doses of TP). In contrast, during the NBS, thedegree of suppression of plasma FSH due to treatment with hr-inhibin wassignificantly (P<O.Ol) greater when the wethers were treated with 16 or .32mgTP/day (75.4% and 84.7% suppression respectively) than when not treated WIth TPor when receiving 8mg TP/day (51.8% and 55.9% suppression respectively). Overall,the suppression in plasma concentrations of FSH due to treatment with TP alone was27%, 46% and 51 % for 8, 16 and 32mg TP/day respectively.

Our data confirm that both inhibin and testosterone have negative feedback actions onthe pituitary to influence FSH secretion in rams. While the stage of the BS did notinfluence the direct effects of either hormone, the suppressive actions of inhibin wereenhanced by testosterone during the NBS but not the BS.

1. Tilbrook, A.J., de Kretser, D.M. and Clarke, IJ. (1993). J. Endocr. In press.

40

TESTIS MORPHOLOGY AND SPERM PRODUCTION IN BULLSRECEIVING THE GONADOTROPHIN RELEASING

HORMONE AGONIST DESLORELIN

M.J. D'Occhio~, S.M. Garozzo~, A. O'Connor§ and N.G. Wreford§

'CSIRO Division of Tropical Animal Production, Rockhampton, Old.,and§Department of Anatomy, Monash University, Clayton, Vic.

/'1This study examined if accelerated testis growth in bulls treated withDeslorelin (D-Trp6-Pr09-des-Gly10-GnRH ethylamide) (1) is associated withchanges in testis morphology and sperm production. T~-month-old bullsreceived 0, 4 or 8 implants formulated to release 50 Jlg Deslorelin/implant/day(n = 6 /group). After 120 days, bulls were castrated and the right testis wasperfused with heparinised saline, followed by 5% glutaraldehyde. Testis tissuewas postfixed in osmium tetroxide/potassium ferrocyanide and processed intoEpon-Araldite. Sections (1 Ilm) were stained with 1% toluidine blue in boratebuffer and mounted under DPX. Stereological estimates were made using anOlympus BH2 microscope fitted with a Panasonic F15 camera; the videoimage was genlocked to suitable stereological probes generated by the GRID1.2 package (Graffitidata, Silkeborg, Denmark). Volume fraction ofseminiferous epithelium, lumen and interstitium were assessed by pointcounting at a magnification of 169x. Areal density of round spermatids wasassessed at a magnification of 1000x using an unbiased counting framesuperimposed on a video image. Numerical density of round spermatids wasestimated from the areal density and diameter data using the Rhines de Hoffequation. There were no differences between bulls treated with 4 or 8implants so the data for these two groups was pooled for ANOVA. Data arereported as means ± SEM. Bulls treated with Deslorelin showed a greaterincrease in testis diameter compared with control bulls (5.3 ±0.8 vs 2.5 ± 0.5mm, P ( 0.01) and at the end of treatment the weight of the right testis washeavier in the former bulls (238 ± 18 vs 172 ± 7 g, pc 0.01). Seminiferoustubule diameter was not affected by treatment but the total length of tubuleswas greater in treated bulls (5054 ± 337 vs 4009 ± 278 m, P ( 0.01). Thevolume fractions of epithelium, lumen and interstitium were not affected bytreatment nor were the absolute volumes of the tubular lumen and interstitium.However, absolute volume of the seminiferous epithelium was greater intreated bulls (168 ± 43 vs 120 ± 14, cm3 P ( 0.01). Numerical density ofround spermatids in the testis was not altered but the absolute number ofround spermatids per testis was increased in treated bulls (23.5 ± 2.5 x 109

vs 16.2 ± 1.4 x 109, P (0.05). These findings indicated that testis growth and

sperm production are increased in bulls treated with Deslorelin. The apparentabsence of changes in testis morphology would suggest that the response toDeslorelin involves normal growth and maturation of the testes, albeit at anaccelerated rate.

(1) D'Occhio MJ et al 1991 Proc Aust Soc Reprod Bioi 23, 93.

41

ACTIVE IMMUNIZATION AGAINST OESTRADIOL-176 INDEVELOPING RAM LAMBS CAN HAVE MARKED EFFECTS ON THE

STRUCTURES AND FUNCTIONS OF THE TESTIS.

Dyane Auclair, S.F. Sowerbutts, B.P. Setchell.Department of Animal Sciences, Waite Agricultural Research Institute,

University of Adelaide, Glen Osmond, South Australia, 5064.

We have studied the effects of active immunization against oestradiol-17B (E2) ontesticular development, testosterone (T) production, testicular blood plasma flow(TBPF) and daily sperm production (DSP).Eight Merino ram lambs received a primary injection of 17B-oestradiol-6 (o-carboxymethyl) oxime-bovine serum albumin conjugate (E2-6-BSA) in Freund's completeadjuvant (PCA) at 14 weeks of age (E2-immunized). Nine control ram lambs weregiven bovine serum albumin (BSA) in FCA. Each lamb received a booster injectionfour weeks later with Freund's incomplete adjuvant (FIA) instead of FCA. Testicularbiopsies were taken from 4 controls (CB) and 4 E2-immunized (EB) ram lambs at 22wks of age (left testis) and at 26 wks of age (right testis). The other lambs (5 CN and 4EN) were not biopsied. The lambs were kept in a room with controlled light (12 hourslight: 12 hours dark). All ram lambs were castrated at 30 wks of age (except for the twolightest lambs, 1 CN and 1 EN, which were castrated at 34 wks of age).E2 binding in diluted (1:5000) plasma from the E2-immunized lambs varied between 40and 60 % after the booster injection whereas binding of E2 remained negligible (lessthan 4 %) in the controls during the whole experiment. Testicular volumes (TV) wereslightly increased between 22 and 26 wks of age in the E2-immunized lambs howeverthe differences between means were not statistically significant at any age. We havenoticed that TV was decreasing towards the end of the experiment in two lambs in eachgroup. Interestingly, one lamb (#15, EN) exhibited a very important increase in TVbetween 23 and 25 wks of age followed by a very steep decline. Immunizationtreatment did not significantly affect testis+epididymis weight at castration but tended toincrease TV (p < 0.08). TV was significantly smaller (p < 0.05) in CB lambs comparedwith CN and EN lambs (TV cm3± sem: CN: 163.50± 18.39; CB: 110.13 ± 14.49; EN:188.08 ± 23.37, EB: 153.07 ± 11.58). Immunization or biopsy treatment did notsignificantly affect DSP. The minimum DSP value (0.7 x 1()6 sperm per gram testis)was found in the lamb #15 (EN) which had exhibited the most pronounced decline intesticular volume. Biopsy sampling reduced circulating T level and increased PRL levelat 30 wks of age. TPBF and the production of T have been calculated for 5 testes from3 CN lambs and 5 testes from 3 EN lambs. TBPF per testis tended to be higher incontrols but the difference between the two groups was not statistically significant at anytime. However, TBPF per unit weight of testis was significantly higher (p < 0.05) incontrols than in E2-immunized lambs. Before hCG injection, mean T production pertestis tended to be higher in the EN lambs but the difference between EN and CN lambswas not statistically significant After hCG challenge, mean T production per testis wassignificantly higher (p < 0.05) in the EN lambs compared with the CN lambs (CN: 5.06± 2.09, EN: 15.14 ± 3.27 Ilglmin/testis). At 22 and 26 wks of age, large vacuoles wereobserved in the epithelium of the seminiferous tubules of one E2-immunized lamb (#3,EB). At 30 wks of age, nearly complete absence of germ cells was observed in Erimmunized lamb #15 with other apparent signs of degeneration in the seminiferoustubules such as those observed in lamb #3 at 22, 26 and 30 wks of age.We conclude that the presence of oestrogen antibodies in circulation does not result in anacceleration in testicular maturation.

42THE EFFECT OF SEXUAL STIMULATION ON TESTOSTERONE IN BOARS INTWO DIFFERENT MATING SYSTEMS.

43

STEROID SECRETION IN EWES FOLLOWING AN OVARIAN ARTERIALINFUSION OF SHEEP GROWTH HORMONE

E.C. Jongman·, P.H. Hemsworth· and D.B. Galloway·

'Department of Agriculture, Victorian Institute of Animal Science, Wembee. Vic. 3030. 'Department of Veterinary Clinical Science,Univenily of Melbourne. Werribee, Vic. 3030.

lA. Downingl •l , R J SCaramuzzil & J, Joss21 CSIRO Division ofAnimal Production, PO Box 239, Blacktown 2148 and 2Schoolof Biological Sciences, Macquarie University 2109, NSW, Australia.

GnRH

4 ~2 0 2 4 6 8 10 12 14Time relative to start of infusion (h)

coI 4Co)

a 2

Figure: The secretion of oestradiol following infusion ofovine Growth Hormone (hatched band is mean ± sem)

Growth hormone (GH) plays a role in the control of amino acid uptake by tissues and theregulation of IGF 1 production in hepatic and peripheral tissues. The effect of GH on theovary remains equivocal although it is thought to be involved in nutritional influences onovarian function. Wereport theresults ofan experiment inwhich the direct effect ofgrowthhormone on the secretion of follicular steroids was determined.

Ten sheep with ovarian autotransplants (Goding et aI., 1967) were fed apelleted dietof60 %lucerne hay and40% oats (600 g per day). Following oestrus synchronisation, theywere allocated to GH-treated. (n=5) and untreated. (n=5) groupS. Treatment was administered by direct infusion into the ovarian artery for 12 hours on day 10 (luteal phase) oftheoestrous cycle (Murrayet al. 1993). Ovine GH (immuno-potency of1.16 X Nlll oGH 87)was infused at arate of80-90 J.1g/hour, a rate thatproduced an ovarian venous concentration

of 3.9±0.2 ng/mlwithout any change inthe jugular venousconcentration(1.2±O.2 ng/ml). Allewes were givenGnRH (150ngi/v) at2.5hours and 10.5hours relative to thestart of treatment.Ovarian venous andjugular venous bloodwascollectedevery 15min from -30 to 150min relative to eachGnRHinjection andat

4 and 8 hours after the start of the infusions. Oestradiol and androstenedione levels wereassayed in ovarian venous plasma and LH, FSH and GH levels in jugular venous plasma.

Growth hormone had no effect on androstenedione secretion. Oestradiol secretion inresponse to a GnRH-induced ill-pulse was increased following the infusion of GH(Figure), an effect that was associated with a reduced ill pulse" amplitude.

These data show that short term GH infusions can increase oestradiol secretionfollowing a GnRH induced ill pulse and therefore suggest a role for GH in mediatingsteroid secretion from the follicle. Unequivocal effects ofGHon androstenedione secretionwere not observed and further work is in progress using higher infusions rates of oGH.(1) Goding, J.R., McCracken, J.A. & Baird, D.T. (1967).1. Endocrinol., 39, 37-52.(2) Murray, J.F., Downing, J.A., Evans, G., et al. (1993). 1. Endocrinol. (In Press).

(l'----'----'---'---L----'_..L----'-_'---'----'-aD -30 110 110 120 150 110 210 2AQ

'T1nIe (mIne)

10

70

80

O'--....I---L_i..---'----'_..L-.....L----J'---'-----'-110 -:010 :010 eo 110 120 HIli 110 210 24(J

'T1nIe (mint)

10

70

80

Fig. 1 Testosterone levels after sexual stimulation Fig. 2 Testosterone levels after matingLit tv(!\:> ,. i rt . blll.1.M. ~ ih!i

The magnitude of the testosterone response of boars in the DMA indicates that this mating systemprovide boars with high levels of sexual stimulation, which may have consequence for their reproductiveperformance. Blood samples are currently being analysed for LH and these data may provide furtherinformation on the level of sexual stimulation in the two mating systems.

Although base concentrations of testosterone tended to be higher for boars in the DMA, this difference wasnot statistically significant (p > 0.05). In contast to boars in the traditional system, boars in the DMAexperienced a distinct rise in testosterone concentrations following sexual stimulation (figure 1). Thistestosterone response of boars in the DMA may be due to increased sexual stimulation. All boars respondedto mating with a rise in testosterone concentrations, although this response appears to be less than thatfollowing sexual stimulation. As with sexual stimulation, the increase in testosterone concentrations followingmating was greater for boars in the DMA than those in the traditional system (figure 2).

REFERENCES(l) Hemsworth P.R., Hansen C., Coleman G.J. and Jongman E (1991). The influence of conditions at thetime of mating on reproduction of commercial pigs. J.Appl. Anim. Beh. 30; 273-285.

A new mating system for pigs has been developed at Werribee to improve the physical and sexualenvironment during oestrus detection and mating (l). In this system six boars are housed in stallssurrounding an oestrus detection and mating arena (DMA), in which females are detected for oestrus with theback pressure test and adjacent boars are introduced for mating. In the traditional system, females areintroduced to the boar's accommodation pen for oestrus detection and mating.

Six boars in the DMA and six boars in the traditional system were fitted with permanent cannulas to studythe levels of sexual stimulation provided to boars in the two systems. To avoid disturbance to the animalsduring sampling, these cannulas were extended during blood. sampling so that the boars could be bled fromoutside the accommodation shed. Concentrations of testosterone from 0900 to 1700 h. and after sexualstimulation and mating were compared in the two systems. Sexual stimulation was provided by allowingboars to observe a mating from their pen or stall for 15 minutes.

44 45

INFLUENCE OF PROLACTIN AND LH ON OVARIAN PROGESTINSECRETION DURING LATE PREGNANCY IN THE RAT

EVALUATION OF TRANSOVARIAN UPTAKE OF METABOLITES AND PROGESTERONESECRETION

A.R.Rabiee, J.M.Gooden, B.G.Miller, and I.J.Lean

Tricia Lopes and Brendan J. WaddellDepartment ofAnatomy and Human Biology,

The University ofWestern Australia, NedIands, W.A. 6009

lWaddell BJ et al (1989) BioI Reprod 41:990; 2Taya K, Greenwald GS (1981) BioI Reprod25:692Supported by an ARC Small Grant (UWA 0311259).

021

!i:o~ 50

Ovarian secretion ofprogesterone (P4) fulls gradually during late pregnancy in the rat,and there is a subsequent, rapid increase in the secretion of20a.-hydroxypregn-4-en-3-ooe(20a.-OHP) during day 22 (tenn=day 23)1. The pre-partum fall in P4 is generally thought toreflect withdrawal ofplacental luteotrophic support, but the stimulus for the increase in20a.-OHP secretion is not known. It is likely to involve the pituitary, since the rise in 20a.-OHPdoes not occur in rats hypophysectomised at midpregnancy2. In the present study, therefore, wetested the possibility that either prolactin or LH, both ofwhich increase rapidly in peripheralblood during day 22, is responsible for the rise in ovarian 20a.-OHP secretion.

Rats were anaesthetized with halothane/nitrous oxide on day 17 ofpregnancy, and acannula positioned in the dorsal aorta for subsequent collection ofblood samples. At 16.00h ondays 21, 22 and 23, rats were treated with either LH antiserum (LHAS; 0.5 ml ip; n=5), nonnalrabbit serum (NRS; 0.5 ml ip; n=4), bromocryptine (BROMO; 4 mg/kg, 0.4 ml sc; n=5) or.saline vehicle (SAL; 0.4 ml sc; n=3). Blood samples were obtained from all rats justprior tothe initial treatment, and then at 2-h intervals from 8.00h to 20.00h on day 22, the period duringwhich the major increase in 20a-OHP secretion occurs. The time ofparturition was recordedfor each rat (during day 23), and ovaries were collected on the morning ofday 24.Concentrations ofP4 and 20a-OHP were measured by RIA in all samples, and ovaries wereexamined histologically to detennine whether a post-partum ovulation had occurred.

Concentration profiles for both P4 and 20a.-OHP were similar in the two control groups(NRS and SAL) from day 21 to 23, and so these groups were combined for subsequentanalyses. As shown in Fig 1, 20a.-OHP levels increased rapidly in control rats dllling day 22

Figure 1: (P<O.O I, two-way ANOVA), but in rats treated with2SO 0 Conlrol BROMO this increase was attenuated. Thus,

concentrations of20a.-OHP in BROMo-treated ratswere lower (p<O.05) than those in controls at 16.00,18.00 and 20.00h on day 22. BROMO had noapparent effect on P4 secretion. In contrast, UIASdid not prevent the rise 20a.-OHP; indeed, the initialincrease in 20a.-OHP was evident earlier in thisgroup (by 8.00h on day 22), and this was associatedwith a slight full (p<O.05) in P4. In rats treated with

D22 UIAS, ovarian weight was reduced by more thenL1-'6.'-OO~ /''-'~.O'-0--'---12'''.OO---'--1-'6.-00-'-2-0''''.OO--' 30% (p<O.05; ANOVA) and there was no evidence

TOlE (h) ofa post-partum ovulation, which occurred in 4ncontrol rats and 3/5 BROMO rats. The timing ofpar1urition was advanced (p<O.05; MannWhitney test) in the LHAS group, but not in BROMO group.

In conclusion, these data indicate that prolactin stimulates ovarian 20a.-OHP secretionduring late pregnancy in the rat, apparently independent ofchanges in P4. LH also influencesprogestin secretion at this time, but does not appear to directly stimulate 20a.-OHP secretion.

OBF CHOL GLUe ACET PROG(ml/mln) (mmol/L) (mmol/L) (mmol/L) (ng/mI)

IF 3.0' 0.2 0.6 2.2(2.9-3.1)·· {-a. 1-0.5) (0.1-1.2) (0.9-3.4)

2F 2.8 0.5 0.9 0.1 -12.3(2.5-3.2) (-0.17-1.1) (-0.6-2.3) . (-0.1·0.3) (-16-{·8))

3L 3.3 2.5 3.A 0.04 ·20(3.0·3.5) (-0.2-5.2) (1.7-5.8) (-0.3-0.4) (-23-{-16))

4L 4.2 -0.9 -1.2 -0.1 -27(3.8-4.6) (-1.2-0.2) (-2.6-0.2) (·0.4-0.2) (-31-{-24))

5Q 1.8 0.3 1.1 0.1 3.3(1.4-2.1) (.02-0.6) (0.4-1.7) (.01-0.2) (2.1·4.5)

• Mean .. 95% Confidence lntervlil

Department of Animal science, University of Sydney, Camden NSW 2570

Nutrition influences hypothalamo-pituitary-gonadal function. However, Iitlle Information Isavailable on the effects of nutrition on steroid hormone producllon except thatundernutrition tends to produce increased progesterone concentration(1). Our study wasconducted to determine the uptake of metabolites, and progesterone secretion by ovaries ofanaesthetised ewes. The ovarian vein in five cross-bred anaesthetised ewes wascannulated, and a blood flow transducer (4R Transonic) placed around the ovarian arteryfor measuring ovarian blood flow (OBF). Sheep 1 and 2 had dominant follicularstructures(F), sheep 3 and 4 had dominant luteal structures{l), and sheep 5 had nosignificant structure{O). Dala (mean and 95% confidence interval for the mean) for OBF,glucose, acetate, cholesterol and progesterone are presented in the table. Blood flow to theL ovaries appears to be higher than F or Q ovaries. Ovarian uptake of cholesterol wassignificant only in one sheep. Glucose uptake was significant in 3 sheep with differentovarian structures. Only one sheep (0) had significant uptake of acetate. Progesteronesecretion was significant in 3 sheep (L and F ovaries).

Table: Uptake per minute of metabolites for ovaries with dUfercnt dominant structures

(1) Smith, J.F. (1988) Aus!. J. BioI. Sci. 41:27-36(2) Redmer.D.A., Grazul.A.T., Kirsch. D.J. & Reynolds, P.L.(1988)

J.Reprod.Fert.82:627-634(3) Niswender,G.D., Reimers, T.J., Diekman, M.A. & Nett, T.M.(1976)

BioI. Reprod.14:64-81.(4) Gwynne, J.T. & Strauss J.F.(1982) Endocrine Review.,2.:299-329

Higher blood flow to the L ovaries Is related to the large luteal vascular bed (2) and Isconsistent with previous studies (3). Despite previous studies showing that luteal tissueprimarily utilises cholesterol for progesterone synthesis (4), our study found that uptake ofcholesterol was not significant in most sheep. It appears that acetate was not significantlyutilised as a substrate for ovarian steroidogenesis by most sheep. This is consistent withprevious studies which found that de novo synthesis of cholesterol from acetate· wasunlikely (4). The data show considerable variation in glucose uptake. Glucose suppliesenergy used in hormone synthesis. The reasons for progesterone uptake in F and Qovaries are unclear. Progesterone secretion in sheep 2 may be due to residual luteal tissue,luteinization of the follicle or normal follicular function. Our findings Indicate the need forfurther research using these methods.

46

(1) Freeman, ME (1988) in The Physiology ofReproduction. Knobil and Meill eds RavenPress New York, PP 1893-1928. (2) Bruce, N. W. et al., (1984) J Reprod Fert 71,445452 (3) Swann, R T and Bruce N W (1986) J Reprod Fert 77,655-664.

CORPORA LUTEA OF PREVIOUS CYCLES ARE A SOURCE OF PROGESTINS INTHE RAT

Neville W Bruce

Anatomy and Human Biology, The University of Western Australia, Nedlands, WA 6009

Corpora lutea (CL) of most mammals regress functionally and structurally near the end of anon-fertile oestrous cycle. CL of the rat, however, survive for two or three cycles but arereported to have minimal steroidogenic activity unless 'rescued' by prolactin surges inducedby mating at the start of the cycle (1). We have found, however, that during pregnancy CLof previous cycles grow, have a classic steroidogenic morphology, a high rate of blood flow(2) and accumulate and convert cholesterol into precursors of progestins (3).

This study focusses on the role of CL of previous cycles on progestin secretion during thecycle and early pregnancy (Day 3). Vaginal smears were taken from 19 rats to assess stageof the cycle. To assess potential secretion rates, mass of tissue and progestinconcentrations (progesterone and 20a.-hydroxypregn-4-en-3-one) were determined from oneovary which was dissected into components of new CL (NCL) formed since the latestovulation, old CL of the previous generation (OCLl), old CL of generations earlier thanthat (OCL2) and remaining stroma which also contained luteal remnants (STRO). Ratswere injected with Evans blue dye (1 mI, 1% solution) before the latest ovulation todistinguish NCL (red) from OCL (blue): OCLl were distinguished from OCL2 by theirapproximately 50% lighter mass. The remaining ovary was examined histologically forsteroidogenic characteristics.

NCL were not observable at oestrus and were grouped with STRO at post-oestrus. TheOCLl, OCL2 and STRO collectively contained around 50% of the available progesteronein the ovary (see below) and a similar fraction of 20a.-aydroxypregn-4-en-3-one, at allstages of the cycle and on Day 3 of pregnancy. All CL examined were highly vascular and,at II light microscope level, retained an active steroidogenic appearance. Pregnancy wasassociated with a 30% (not sig) increase in ovarian progesterone content compared to theequivalent stage of the oestrous cycle. Total progestin concentrations were generally higherin NCL than in OCLl, the latter were higher than in OCL2 but overall differences at eachstage were less than 40% suggesting that all CL were active progestin secretors.

These findings together with our previous work suggest that CL do not need the prolactinstimulation consequent to earlier mating to be 'rescued', they do not functionally deteriorateby the next oestrus and they are capable of further stimulation by pregnancy.

60

50

40

30

20

10

opro-oestrus oestrus post-oestrus dl-oestrus

Progesterone content,'l(, of total +SEM

pregnant

THE ROLE OF ANTIFREEZE PROTEINS IN THE FREEZING OF RAMSPERMATOZOA

S R Paynel, J.E. Oliver and G.C. Uprete

I Meat Industry Research Institute of New Zealand, P.O.Box 617, Hamilton, New Zealand

2 AgResearch, Ruakura Agricultural Research Centre, Hamilton, New Zealand

Antifreeze proteins from polar fish have the ability to lower the freezing point ofwater by a non-colligative mechanism termed adsorption-inhibition[ll. In addition,these proteins can prevent ice recrystallisation by binding directly to the faces of icecrystalsDl.

Addition of antifreeze glycoprotein from Antarctic Cod to freezing mediaincreased the survival of pig oocytes by 25% after freezing[2,3l. The presence of 20mg/ml of antifreeze proteins from a variety of polar fish, in incubation media,increased by threefold the number of bovine oocytes able to undergo in vitromaturation, after exposure to 4°C[4l. Antifreeze proteins from all species of fishgave comparable results[4l.

The effects of the antifreeze peptide from Winter Flounder (AFP) and anantifreeze glycoprotein from Antarctic Cod (AFGP) were assessed on the motility ofram spermatozoa (Dorset and Dorset x Romney) after chilling (5°C) and afterfreeze-thaw. Concentrations of 0,0.1, 1 and 10 pg/ml were used for both AFP andAFGP. Semen was diluted and cooled to 5°C, then loaded into 0.25ml straws andfrozen in a liquid N2 plunge freezer.

After chilling and after freeze-thawing, the motility of the diluted semen wasassessed on a scale of 0 to 5 and percent motile[Sl.

After chilling, spermatozoal motility decreased significantly at an AFP or AFGPconcentration of O.lpg/ml and at concentrations above 10pg/ml (P<O.05). Theseproteins can have a mild cytotoxic effect at certain concentrations at 5°C.

However, addition of AFP or AFGP to the freezing medium at concentration of0.1 to 10 pg/ml significantly reduced the loss of spermatozoal motility that occursdue to the freeze-thaw process (P<O.OOl). The effect was not concentrationdependant, nor did it depend on which antifreeze protein was added. However, dueto the cytotoxicity during the chilling stage, only AFP at a concentration of 10pg/ml increased the percentage of motile spermatozoa significantly followingfreezing and thawing over that of the control (P<0.05).

We propose that prevention of recrystallisation of ice by antifreeze proteins duringthe brief thawing stage may have a protective effect on ram spermatozoa,particularly reducing damage to cellular structures such as membranes.

[1] DeVries, A.L., (1988), Comp.Biochem.Physiol. 90b, 3:611-621[2] Rubinsky, B. et al., (1991), Cryo-Letters 12:93-106[3] Rubinsky, B. et al., (1992), Cryobiology 29:69-79[4] Rubinsky, B. et al., (1991), Biochem.Biophys.Res.Comm. 180,2:566-571[5] Rodger, J.e. et al., (1991), ReprodFertil.Dev. 3:119-125

48 49

Lisa J. Maclellan Alan Trounson and David .K. Gardner

Centre for Early Human Development, Monash University, Clayton, Victoria, 3168

EFFECTS OF OXYGEN AND AMINO ACIDS ON CATILE OOCYTEMATURATION, FERTILIZATION & EMBRYO DEVELOPMENT IN VITRO

150±2127

151±1

125±3112±6

127±2128±3

87±579±6

104±498±4

3717**

3414**

7260**

7953**

-aa+aa

3. NCSame drop 76 15 99±7 123±3 132±10Moved @ 48h 80 39** 96±3 123±2 141±3

EB, early blastocyst; B, blastocyst; ExB, expanded blastocyst; Cell, cell number ± sem; oa, without amino acids; +oa, with amino acids; ** significant P<O.Ol.The beneficial effect of high oxygen on oocyte maturation, as determined by increases inblastocyst formation is similar to the findings of DeAzambuja et at (3). We have furtherdemonstrated a stimulatory effect of high oxygen on cleavage. In contrast to their abilityto enhance embryo development, amino acids in the fertilisation medium significantlyreduced the number of resultant embryos. This may be attributed to their action on eithersperm and/or oocytes and warrants further investigation. Similar to the data on sheepembryos in vitro (2), the build up of ammonium in the culture medium reduced thenumber of cattle embryos reaching the blastocyst stage, although there was no apparenteffect on resultant blastocyst cell number.(1) Thompson, J.G.E. et aI., (1990) J. Reprod. Fert., 89, 573-578.(2) Gardner, D..K. et aI., (1992) Proc. Aust. Soc. Reprod. BioI., 24, 90.(3) Bavister,B.D. and Yanagamachi,R. (1977) BioI. Reprod., 16,228-237.(4) DeAzambuja, R.M. et aI., (1993) Theriogeneology, 39, 184.

Reduced oxygen tension significantly increases the development of ruminant embryos invitro (1). The supplementation of culture media with amino acids significantly reduces thepercentage of sheep embryos which arrest in culture, provided ammonium produced fromthe breakdown of amino acids is avoided by renewing the media every 48 hours (2). Inthe present study we have examined these factors on the in vitro maturation (IVM) ofcumulus enclosed cattle oocytes (Experiment 1), in vitro fertilization (IVF) (Experiment2) and on subsequent in vitro culture (IVC) (Experiment 3). Immature oocyte-cumuluscomplexes were aspirated from the follicles of ovaries collected from a local abattoir.Oocytes surrounded by compact corona radiata were cultured in medium 199 + 10 %fetal calf serum and 705IU FSH, 7.5IU lB, 50m hCG and l(lg/ml oestradiol 17-13 at39"C for 24h. In Experiment 1, 422 oocytes were matured in a gas phase of either 7%0z,5%CO:z, 88%N2 or 20%02> 5%C02, 75%N2. Oocytes were then inseminated with frozenthawed bull semen at 2xHf/ml in Fert Talp medium (3) (modified to contain O.25mMpyruvate and no glucose) and cultured in SOF medium + amino acids (2) + BSA(8mg/ml). In Experiment 2, 489 oocytes were matured in 20%02, 5%CO:z, 75%N2 andinseminated in Fert Talp with or without amino acids. Subsequent embryo culture was in7%02 in SOF + amino acids + BSA In Experiment 3, 486 oocytes matured in 20%02>inseminated in the absence of amino acids and then cultured in SOF + amino acids +BSA, for 6 days or placed in fresh medium every 48h.Exveriment No Qeavage(%) Blastocyst(%) EB....Qill1. NM

20% O27% O2

2. IVF

PRONUCLEI FORMATION AND EMBRYO CLEAVAGE FOlLOWINGELECfROFUSION OF ROUND SPERMATIDS WITH OOCYTES

FROM THE MOUSE

(1) Lacham-Kaplan, 0. and Trounson, 0. (1991) Mol. Reprod. Dev. 29,85-93.(2) Ogura, A and Yanagimachi, R. (1993) BioI. Reprod. 48, 219-25.(3) Meistrich, M., Bruce, W. and Clermont, Y. (1973) Exp. Cell Res. 79, 213-27.

Centre for Early Human Development, Monash University, Qayton, Australia.

Mary C Valiotis. Orly Lacham-Kaplan and Alan 0 Trounson

The union of immature male gametes with oocytes in vitro could possibly lead toan improvement in fertilization rates for sterile men. The immature mouse sperm cellsfrom the testes are unable to fertilize oocytes with intact ZOlUle, zona-free oocytes or aftermicroinjection into the perivitelline space (1). Recently, mouse round spermatid nucleihave been injected into electrically activated hamster oocytes, resulting in the formationof male pronuclei (2). For the present studies, we have selected mouse round spermatids,by micromanipulation, and then inserted them into the perivitelline space of maturemouse oocytes; With the use of electrical stimulation the round spermatids areincorporated into the oocyte. The combination of selection, micromanipulation andelectrical fusion of the mouse round spermatid with the mature mouse oocyte has led tothe development of pronuclei and embryonic cleavage.

A single mouse testis was removed from two mature F1 hybrid males and placedin 2ml of modified T6 media. An incision was made in each testes and segments of theseminiferous tubule were removed. The cells were expressed with the aid of two fineforceps running along the segmented length of the tubules. One ml of trypsin (0.025%),was added to this solution for 15min to disaggregate the cell junctions. Addition of 1mlof fetal calf serum (FCS) stopped this reaction. The disaggregated cells were centrifugedat 1000rpm for 5min to complete the separation. The round spermatids were selectedusing a micromanipulator under interference-contrast microscopy. The typicalmorphology of round spermatids was confirmed by the Periodic Acid-Schiff staining(PAS) technique (3). A homogeneous round spermatid population was selected at about90% accuracy and the viability of the original cellular population was determined at beingabout 95% by use of the trypan blue exclusion test.

The oocytes were collected from the oviducts of superovulated female mice, at13-15h post-fusion after administration of hCG. Oocytes were freed from the cumuluscells with 4OIU/ml of hyaluronidase. A single round spermatid was then microinjectedinto the perivitelline space of the mature mouse oocyte.

The microinjected oocytes were then manually aligned in a cell fusion chambercontaining Zimmerman's fusion media. A BTX electro cell manipulator 200 "andOptimizor were used to electrofuse the two cells together. An AC pulse of 8V for 5secwas followed by a single DC pulse of 1.5kv/cm for 99llsec.

In our preliminary studies 22% of the round spermatids fused into oocytes and allfused oocytes formed two, apparently normal pronuclei, as assessed by epifluorescentmicroscopy. Embryos developed from these pronuclear oocytes at apparently normal ratesin vitro.

We have demonstrated the fon:n.ation of pronuclei from the electrical fusion of theround spermatid into the mouse oocyte and that pre-implantation cleavage anddevelopment is possible.

RAPID FREEZING OF MOUSE EXPANDED AND HATCHED BLASTOCYSTS

J.M. Shaw and D.R. MacFarlane

CEHD, IRD, Monash University., Clayton 3168, Vic. Australia.

In 1991 we rapidly froze expanded and hatched mouse blastocysts after a 3 min equilibrationperiod at room temperature (R1) in dimethyl sulfoxide and sucrose (1). Of the 20% ofembryos that reexpanded only half "plated" ie attached to the petri dish. More recentlyFujikawa et al (2) obtained 95% survival of mouse blastocysts frozen after 30 secondsexposure to a solution containing 20% ethylene glycol (EG), 20% dimethyl sulfoxide (DMSO)& 10% 1,3 butanediol. We are reluctant to use butanediol as our toxicity tests indicate thatboth 1,3 and 2,3 butanediol are very toxic to mouse embryos (unpublished data). Solutionswithout butanediol are known to be highly effective for the cryopreservation of mouse morula(3). This paper therefore investigates whether late blastocyst stage embryos can be frozen insolutions without butanediol.

Mature, superovulated, C57BL x CBA F1 mice were mated with C57BL x CBA F1 males.Embryos were collected early on D 4 at the morula early blastocyst stage and cultured inDMEM with 10% FCS. Embryos were frozen on D 5 at the expanded and hatched blastocyststage. The cryoprotectant solution developed by Kasai et al (3) (10 ml= 4 ml ethylene glycol,1.8 g Ficoll 70 000, 0.3 M sucrose, PBS) gave variable survival of late blastocysts.Modifications to Kasai's formulation resulted in higher survival rates. Here we present theresults for solutions in which the sucrose was replaced by glucose, and the PBS with water.The cryoprotectant was either DMSO or EG. In 3 replicate experiments, embryos for freezingwere randomly divided into 6 groups (9-10/group). Group 1 embryos were inserted directlyinto straws containing the cryoprotectant solution (CP). The other groups were pre-equilibratedin M2 containing dilute CPo Group 2 were in 10% CP for 5 min. Group 3 were in 10% CPfor 2.5 min followed by 20% CP for 2.5 min. After 1 minute in the concentrated CP at RTall straws were plunged into liquid nitrogen. Straws were warmed in a 35 to 37°C waterbathfor 3 to 6 sec, and the contents expelled into M2 with 0.3 M sucrose at RT. After 10 min theembryos were washed and placed in culture. The proportion of embryos which plated within3 days was 98% (61/62) in control and 100% in both DMSO (18/18) and EG (35/35) solutioncontrol groups. The proportion of embryos which plated was significantly (* P<0.01) reducedcompared to controls in only 1 of the 6 frozen-thawed groups (below).

Group 1 Group 2 Group 3Cryoprotectant % SID % STD % SID

Dimethylsulfoxide 86 4.4 93 9.4 85 13.9

Ethylene glycol 72* 18.0 90 4.7 96 5.2

There were no significant differences between solutions containing DMSO and EG, orbetween groups pre-equilibrated in dilute CPo We conclude that the use of butanediol is notnecessary to obtain high survival rates of late blastocyst stage mouse embryos.

References:1. Shaw, J.M., Diotallevi, L. & Trounson, A.O. (1991) Reprod. Fertil. DeveL, 3:621-626.2. Fujikawa S. Valdez C.A. Kanagawa H. (1992) Cryobiology 29:755.3. Kasai M. Nishimori M. Zhu S.E. Sakurai T. Machida T. (1992) Bio); Reprod. 47: 1134-1139

51

ISOLATION OF ACTIVIN A FROM OVINE AMNIOTIC FLUID ANDPERSISTENCE OF INHffiIN IN AMNIOTIC FLUID AFTER FOETALORCHIDECTOMYD.M. de Kretser, L.M. Foulds, M. Hancock, S. Miller, 1. McFarlane, N. Goss, G.Jenkin. Institute of Reproduction & Development & Department of Physiology,Monash University, Clayton, Victoria, Australia.

Our previous studies have demonstrated that immunoactive inhibin wasdetectable in foetal gonads, foetal plasma and amniotic fluid. The addition of foetaltestis extract and foetal plasma, to rat pituitary cell cultures resulted in inhibition ofFSH content. Addition of amniotic fluid however caused a stimulation of FSHcontent. We have demonstrated by two specific immunoassaysl that this FSHstimulating activity was likely to be due to activin. To characterize the nature ofactivin present in amniotic fluid, pools of amniotic fluid (2-8 litres) were collectedfrom ovine foetuses between 110-113 days gestation, and purified by dye-affinitychromatography, hydrophobic interaction chromatography, gel filtration usingSephadex-G100 in 4M acetic acid and a series of reverse-phase HPLC steps. Thepurification of activin was monitored by immunoassay. A progressive increase inactivin specific immunoactivity was obtained at each step. Polyacrylamide gelelectrophoresis of the active fractions after the final step revealed a single band witha molecular weight of 24.3 kDa which on reduction produced a single band of 16.4kDa. NH2-terminal amino acid sequences of several fractions in the immunoactiveactivin peak were identical and were GLEXDGKVNIXXKKQFYVSSGDI (X indicatesunknown). This sequence is identical to the known sequence of ovine activin A.

In attempts to determine the source of inhibin and activin in amniotic fluid,gonadectomy was performed in 4 male foetal sheep between 113 and 119 days ofgestation. Foetal and maternal carotid artery and jugular vein catheters and amnioticfluid catheters were inserted into experimental and control (n = 5) pregnant ewes andtheir foetuses. Post castration plasma and amniotic fluid samples were obtained forinhibin and activin measurements by radioimmunoassay. Gonadectomy caused aprecipitous decrease in foetal plasma inhibin concentrations from 6.3 ± 1.3 ng/ml (x± sem) to basal concentrations of 0.16 ± 0.01 ng/ml within 48 hrs. Amniotic fluidinhibin concentrations did not alter after gonadectomy, and were not different fromthose observed in amniotic fluid from control foetuses throughout the remainder ofgestation (5.15 ± 0.19 ng/ml vs. 7.63 ± 0.59 ng/ml : gonadectomy vs control).

In conclusion, the results of these studies demonstrate that activin A representsthe major component of the immunoactive activin in ovine amniotic fluid.Furthermore, the foetal gonads do not to appear be the major source of inhibin inamniotic fluid. Further studies will determine whether the changes in activin levelsin amniotic fluid parallel those of inhibin post-gonadectomy. The possibility thatactivin and inhibin are locally produced by the ovine foetal membranes is currentlybeing explored.

1. Robertson DM et al (1992) Endocrinology, 130: 1680-1687

52

ACTIVIN A REGULATES PRODUCTION OF FOLLlSTATIN (FS) BY OVINEANTERIOR PITUITARY CELLS IN VITRO.

P.G. Farnworth, D.M. Robertson, and J. SchwartzPrince Henry's Institute of Medical Research, P.O. Box 152, Clayton, Victoria 3168,

Australia

Rat pituitary cells express mRNA for the activin-binding protein, FS, and bovinepituitary folliculo-stellate cell cultures secrete FS into the medium. In the presentstudy, we have employed an heterologous RIA for FS to determine whether ovineanterior pituitary cells synthesize and secrete FS in a regUlated manner in vitro.

Primary cultures of ovine pituitary cells were prepared by standard proceduresfrom tissue obtained from a local abattoir. Cells were plated at 75,000 cells/0.3 mlper well in DMEM:F12 medium containing 10% fetal bovine serum, and treatmentswere usually added 2 days later to washed cultures in the absence of serum butpresence of insulin (1 Ilg!ml), transferrin (10 Ilg/ml). and bovine serum albumin (6mg/ml). The medium and cell lysate samples from each well were saved forsubsequent RIA for FS and FSH.

Medium conditioned for several days by ovine pituitary cell cultures containedFS-Iike immunoreactivity which diluted in parallel with a bovine FS standard in theRIA. The immunoreactive material co-eluted with authentic bovine FS through aroutine chromatographic isolation of FS, and the purified material selectivelysuppressed FSH cell content in the rat pituitary cell culture bioassay for inhibin/FS.

Under serum-free conditions, dispersed ovine pituitary cells secretedimmunoactive FS at 2-5 ng/106 cells per day. Secretion of FS was lower in culturesof closely packed cells. Dispersed cells that had been cultured for 2 to 13 days inthe presence of serum secreted up to 3-fold more FS during the subsequent 2days under serum-free conditions than did cells cultured in the absence of serumprior to the test period.

Continuous exposure of cultures to recombinant human activin A (1 - 10 nM)for 3 days increased FS levels in both the medium and cell lysate fractions by up to3-fold, and concomitantly enhanced FSH secretion and cell content. Under similarcircumstances, purified bovine inhibin (0.003 - 3.2 nM), dihydrotesterone (1-320nM). retinoic acid (0.03-30 IlM). and the synthetic glucocorticoid, RU28362 (1-100nM), suppressed FSH secretion and total FSH, but did not affect FS synthesis orsecretion, which were likewise unaffected by GnRH, GRF, somatostatin, CRF orAVP.

From these results, we conclude that 1) ovine anterior pituitary cells synthesizeand secrete FS for many days in vitro, and 2) FS production is enhanced byprolonged exposure of the cells to activin, or undefined serum factors. Activin mayregulate the production of its own binding protein in the pituitary.

Supported by the NHMRC of Australia.

53

EFFECT OF THE FECS GENE AND AGE ON PlASMA INHIBINCONCENTRATIONS IN MERINO RAMS

D.O. Kleemannl, T.I. Grosserl, RM. Bindon2, D. Russe1l3 and J.K. Findlay3

ITurretfield Research Centre, Rosedale, S.A, 5350, 2Division of AnimalProduction, CSIRO, Armidale, N.S.W., 2350, and 3prince Henry's Institute,Monash Medical Centre, Melbourne, Vic., 3168.

Utilisation of the Booroola high fecundity gene (FeeB) in sheep populations islimited by an inability to identify gene carriers easily. A recent ~tudy indicatedthat the concentration of immunoreactive inhibin was lower III a group ofprepubertal rams of high FecB gene frequency than ~ ~ .group of lo~ ge~efrequency (1). The present study determined plasma mhibm concentrations mMerino rams, either heterozygous or non-carriers of the gene, between 2 and18 months of age. B +Ten ram progeny from SA Merino rams and heterozygous (Fee Fee ) SAMerino ewes were bled at 2, 3, 4, .6, 9, 1~ and 18 months ~f age, and plasmainhibin concentrations were determmed usmg RIA (2). Fec gene status of therams was assessed by a progeny test.

Table 1. Least-squares means (SEM) of plasma inhibin concentrations (ng/l)

Ram Age (months)genotype

n 2 3 4 6 9 12 18

Fec+Fec+ 5 1694 1490 1200 871 739 610 763(163) (146) (131) (149) (107) (72) (87)

FecBFec+ 5 1698 1297 1202 827 508 529 831(133) (119) (107) (122) (88) (59) (71)

Overall inhibin concentrations did not vary significantly with FecB status. Thisis in c~ntrast to an earlier report (1) that indicated a transient chan~e ~ninhlbin concentrations due to genotype at 3 and 4 months of age. A decline minhibin concentrations with increasing age (P<0.001) confirmed previous results(1, 3). The apparent increase at 18 months may have ~ee~ due .to a seasonaleffect (4). We conclude that plasma inhibin concentratIOn IS unlikely to be ofpredictive value for the selection of FecB rams.

(1) Earl, C.R, Male, RH., Bindon, RM., Piper, L.R., Russell, D. & Findlay,J.K. (1989) Proc. Aust. Soc. Reprod. BioI. 21:5. .

(2) Findlay, J.K., Clarke, I.J & Robertson, D.M. (1990) Endocnnol. 126:528.(3) Earl, C.R, Male, RH., Rowe, J.P., Russell, D. & Findlay, J.K. (1991)

Proc. Aust. Soc. Reprod. BioI. 23:81.(4) Clarke, 1.J., Tilbrook, AJ., Galloway, D.R, Earl, C.R, Findlay, J.K. &

deKretser, D.M. (1991) J. Reprod. Fert. 43:163.

54 55

Figure: The secretion of androstenedione following infusion of insulin and glucose

THE DIRECT EFFECT OF INSULIN ON OVARIAN STEROID SECRETION INEWES WITH AN AUTOTRANSPLANTED OVARY

A.J. Peterson1, A.M. Ledgard1, S.C. Hodgkinson1and S. McDougall2

1AgResearch and 2Dairying Research Corporation, Ruakura, Hamitton, New Zealand.

OVARIAN FOLLICULAR INSULIN-LIKE GROWTH FACTOR BINDING PROTEINS ANDFOLLICULAR STATUS IN DAIRY CADLE

The insulin-like growth factors (IGF), IGF-1 and IGF-II, implicated in ovarian folliculogenesisin anumber of mammalian species, are bound with high affinity to afamily of binding proteins(IGF BPs) that modulate their action in many tissues. In bovine follicles, total binding of theIGFs declines with increasing diameter1l although the concentrations of the IGFs themselvesremain relatively constant and appear not to be influenced by the level of oestradiol in thefollicular f1uid(21. The relationship between the various IGF BPs -in follicular fluid wasdetermined in dairy cows ovariectomised following sequential uttrasonogrphy to determinefollicular status.

The ovaries of twelve lactating dairy cows were examined daily by transrectal uttrasound.After dissection, the diameter of individual follicles were measured and the follicular fluidaspirated and stored at -20°C until assay. The status of each follicle was categorised as rising(ie. diameter increasing by ~ 1 mmlday), plateau (± 1 mm diameter for 3 days), declining(diameter decreasing by ~ 1 mmlday) or dominant (the largest diameter follicle that was inrising or plateau phase) based on the individual follicle growth profile before ovariectomy.

After Westem ligand blotting using iodinated IGF-II, the various IGF BPs were quantified bydensitometry after autoradiography and standardized against ovine CSF. Their identity wasdetermined by appropriate immunoblotting. Total binding activity was determined using adot·blotting procedure.

There was little difference between total binding activity in rising and plateau follicles (878±91cpm vs 812±85 cpm) but an almost tenfold difference between declining and dominant follicles(2776±1013 cpm vs 328±13 cpm). While IGF BP3 showed little association with follicularstatus, IGF BP2 levels were lower in dominant follicules. The greatest variation inconcentration was shown by the IGF BPs with molecular weights of 24 and 30 kDa (Table).

Concentrations of IGF BPs (OD units ±S.E.)Status (n) BP3 BP2 BP 24 kDa BP 30 kDa diameter(mm)rising (14) 769±106 318±71 175±32 221±49 6.6±OAplateau (10) 837±94 472169 183±28 344±63 12.6±O.9decline (8) 912±211 328±71 355±86 553±157 7.8±1.0dominant (10) 644±89 176±30 96±12 87±13 11.3±O.9

The increased levels of the 24 and 30 kDa IGF BPs in declining bovine follicles suggest anintraovarian .role for one or both of these IGF BPs in preventing the development of granulosacell aromatase activity. Very low or neglible levels of these IGF BPs occur in bovine follicleswith high (>100 ng'ml) oestradiol concentrations(3l. The factor(s) which regulate theconcentration of IGF BPs in the follicle remain to be elucidated.

(1) McDougall, S. et al. (1992) Aust. Soc. Rep. BioI. 24: 31(2) Spicer, L.J. et al. (1988) BioI. Reprod. 39: 573-580(3) Echtemkemp, S.E. and Howard, H.J. (1992) J. Anim. Sci. (Suppl. 1) 490

Insulin +Glucose

6

12

18

Untreated

o r---+-,.----r--..,....---,.-...30 60 90 120 150 -30 0 30 60 90 120 150

Time relative to the GnRH injection (min)

o-30

The infusion of insulin or glucose did not alter the secretion of androstenedione oroestradiol when compared to untreated ewes or when compared to pre-treatmentresponsesfor each ewe. However, treatment with a combination of insulin and glucose resulted in amarkedly supressed secretion of androstenedione (Figure) and oestradiol (not illustrated).

These data suggest that insulin increases the availability of glucose to follicles andthat this is associated with decreased steroidogenic responses to UI. We propose thatnutritionaleffects onovulationmay bemediatedby the insulin-regulateduptake ofglucose.

(1) Goding, J.R., McCracken, lA. & Baird, D.T. (1967). J. Endocrinol., 39, 37-52.(2) Murray, J.P., Downing, JA., Evans, G., et ai., (1993).1. Endocrinol., (In Press).

Sheep vary their reproduction in response to nutrition by mechanisms that are not wellunderstood; the role ofinsulin in mediating direct ovarian responses to nutrition is also notclear. We report the results of an experiment in which the direct effect of insulin on thesecretion of follicular steroids was detennined.

Twenty three sheep with ovarian autotransplants (Goding et ai., 1967) were fed, adlib, a diet of oaten straw supplemented with minerals. Following oestrus synchronisation,they were allocated to four treatments; glucose (n=6), insulin (n=6), insulin +glucose (n=5)anduntreated(n=6). Treatments wereadministeredbydirectinfusioninto theovarian arteryfor 13.5 hours on day 11 (luteal phase) ofthe oestrous cycle (Murray etai., 1993). Nominalrates ofinfusionwere 1.25 g/hourand20~g/hourforglucose andovineinsulinrespectively.All ewes were given GnRH (150 ng i/v) at -2.5h, +12h and +24h relative to the start oftreatment. Ovarian venous and jugular venous blood was collected every 15 min from -30min to +150 min relative to the GnRH injection. Androstenedione and oestradiol wereassayed in ovarian venous plasma and LH, insulin and glucose in jugular venous plasma.

J A Downing1.2, R.I. Scaramuzzi1& J. Joss2

lCSIRODivisionofAnimalProduction,P.O.Box239,Blacktown2148and2Schoolof Biological Sciences, Macquarie University 2109, N S W., Australia.

56

INSULIN-LIKE' GROWTH FACTOR BINDING PROTEINS IN THEOVARY OF THE DOMESTIC HEN (GALLUS DOMESTICUS)

R. D. RQberts*

A.F.R.C. RQslin Institute (Edinburgh), RQslin, MidlQthian, EH25 9PS, U.K.

• Now at CSIRO Division of Animal Production, P.O. Box 239, Blacktown, NSW 2148, Australia

Insulin-like grQwth factQr-I (IGF-I) is produced by granulQsa and thecal cellsfrQm pre-QvulatQry Qvarian follicles in the domestic laying hen and has been shQwn tQbe mitQgenic for bQth cell types in vitro (1). Binding proteins fQr IGF-I have beendemonstrated In mammals; so far, six Qf these have been identified and shown tQ havevarious effects on the actions Qf IGF-I in different tissues (2). Some of the proteinsare known to be involved in granulosa cell activity in mammals' however theexistence of similar proteins in avian Qvaries has not been previously ~vestigate~

Gr~ulos~ and thecal cells were.obt~ed from ~e largest pre-ovulatory fQlliclesof dO.n:estIc lay~g hens and cultured zn Vltro. AnalYSiS of the resulting granulosa cellcondi~Qn~d mediu~ (GCM) by yYestern ligand blQtting revealed the presence Qf fiveIGF bmding proteIns of approXimate molecular weights 32.5, 31.5, 30.5, 29.5 and24.5 kDa. Thecal cQnditiQned medium contained four proteins with approximatemQlecular .weights of 32.5, 31.5, 29.5 and 24.5 kDa. Treatment of granulosa cellcultures With IGF-I (25 ng/ml) caused an increase in the cQncentrations Qf all fiveproteins present in GCM with the greatest increase in the 29.5 kDa protein. Similartreatments Qf thecal cells hadnQ detectable effect on the protein concentrations.Treatment of granulQsa cells with luteinizing hormone (LH) had nQ effect on proteinconcentrations and treatment with LH and IGF-I produced results similar to treatmentwi~h IGF-I alone. Thus there are differences in the IGF binding proteins produced bychicken granulosa and thecal cells in vitro and in the regulation of this production byIGF-I.

~ese results a:e consisten~ with the existence of a cQmplex IGF-I intra-ovariansystem In the domestIc hen. In thIS system IGF-I modulates the production of bindingproteins from granulosa cells, these proteins may themselves modulate the establishedactiQn~ of IGF-I on both granulQsa ~d thecal cells. Further investigation is requiredtQ elUCidate the nature of these protems and thus determine whether they are similar tothose already characterised in mammals. investigatiQns of both the in vitro and in vivoactions .of these proteins may be of use in determining the intra-ovarian regulatorymechanisms controlling normal follicular grQwth in dQmestic hens.

(1) Roberts, R. D. (1992). PhD Thesis. Univ. Edinburgh.

(2) Shimasaki, S and Ling, N. (1992). Prog. Growth Factor Res. 3, 243-266.

FACTORS IN FOLLICULAR FLUID THAT DELAY OR HASTENOESTRUS

T. O'Sheal , G. Baxter2 and R. Webb2

lDepartment of PhysiQlogy, University of New England, Armidale, NSW 2351, and2AFRC Institute of Animal PhysiolQgy and Genetics Research, Roslin Institute,Midlothian EH25 9PS

We reported that inhibin-depleted bovine follicular fluid (bFF) suppressed fQlliculardevelopment and delayed onset of oestrus when injected into heifers (1). The presentstudy compared the effects of inhibin-depleted and inhibin-enriched follicular fluidfractions Qn oestrous onset in sheep. Sequential chromatography was used tocommence isolation of the active fraction(s).

CharcQal treated bFF was separated into inhibin-depleted (pool 1) and inhibin-enriched(Pool 2) fractions with a monoclonal antibody to the alpha subunit of bovine inhibin(2). Some of Pool 1 was divided into pools 3 (nQt-retained) and 4 (retained) with bluesepharose CL6B (pharmacia). Sephacryl-200 (Pharmacia) was used to isolate materialof mQlecular weights> 110 KDa (Pool 5) and 56-110 KDa (Pool 6) from Pool 3.Inhibin cQncentratiQns (ng/ml by RIA) were: - Pool 1, 50 ng; Pool 2, 558 ng; Pool 4,34 ng; POQI 5, 4 ng; PQol 6, 2 ng.

In January 1992, 100 adult ewes were treated with intravaginal progestagen spQnges.At sponge removal ewes were injected Lm. at 8 hr intervals for 2 days with pOQIvolumes equivalent to 3 ml initial bFF starting material (additional groups injected with1 ml of PQols 1 or 2). Animals were Qbserved for oestrous activity, daily bloodsamples were taken until 1 week after oestrous Qnset, and laparoscopy was carried Qut1 week after oestrus.

Onset Qf oestrus and growth of corpora lutea were delayed by Pool 1 and Pool 4 (Table1). The differences between the data for the POQI 5 and saline grQUPS approachedsignificance.

Table 1. Onset of oestrus and time to reach 0.4 ng progesterone/ml plasma (Prog) inewes injected with bFF fractions. Values are means ± se. n = 19 for the saline groupand 8 - 10 for the treatment groups.

Saline Pool 1 Pool 2 Albumin Pool 4 Pool 5 Pool 61ml 3ml Iml 3ml

Oestrus 67 92 132 69 92 69 102 53 72(hr) ±5 ±16 ±15 ±6 ±8 ±13 ±14 ±5 ±5

Prog 8.1 10.8 10.8 8.4 8.7 8.2 10.6 6.9 8.1(days) ± 0.9 ± 0.4 ± 0.7 ±0.5 ± 0.5 ± 0.9 ± 0.7 ± 0.3 ± 0.4

In cQnclusion the main factor in bFF that delays onset of oestrus in both cattle andsheep is a factQr other than inhibin. It chromatQgraphs with albumin Qn bluesepharose. A secQnd factQr in follicular fluid has Qpposing effects.

1. Law, A.S. et al. (1992) J. Reprod. Fertil. .26.: 603 - 616.2. O'Shea, T. et al. (1989) Reprod. Ferri!. Dev.l: 347 - 355.

58

THE RAT OVARY PRODUCES CYTOKINES DURING OVULATION

Mats Brannstrom, Robert Seamark, Sarah Robertson, Robert Norman

Reproductive Medicine Unit and Medical School, Department of Obstetrics and Gynaecology,University of Adelaide, The Queen Elizabeth Hospital, Woodville, SA.

The infiltration of leukocytes into the ovary at ovulation and the concomitant release ofinflammatory mediators including eicosanoids, collagen-degrading enzymes and vasoactivesubstances has prompted the comparison of mammalian ovulation to an inflammatoryprocess(1l. To examine the production of cytokines by the ovary during ovulation, ovarieswere obtained from immature rats and from eCG-hCG primed immature rats at differentstages of the ovulatory process (p'rior to hCG injection, 10h after hCG, and 20h after hCG)and perfused ill vitro for 5 hours(2). The steroid and cytoki.le contents of the recirculatingperfusates were measured by immuno- and specific bio-assays, respectively(3).

The neuropeptide hormones arginine-vasopressin (AVP) andoxytocin (OT) have been found in the ovarian follicles andcorpora lutea (CL) of many eutherian mammals. Inruminants, there is persuasive evidence that luteal OT isinvolved in luteolysis via stimulation of uterineprostaglandins. However, based on scant evidence, themarsupial ovary has been viewed as being devoid of OT-likeand AVP-like peptides. In this study, corpora lutea fromthe brushtail possum were examined for AVP, OT andmesotocin (MT) by a combination of reverse phase HPLC,radioimmunoassay (RIA) and immunohistochemistry (IRC).Peptides extracted from each of 5 CL were separated byHPLC (C-18 RP-HPLC column; 250 x 4.6 mm) and each fractionwas assayed for AVP, MT and OT. Two peaks were found,corresponding to AVP and MT standards. The amount of eachpeptide was 8.79+/- 2.25 ng MT/g (mean+/- sem) and 6.17+/- 1.08 ng AVP/g respectively. The mean AVP/MT ratio was0.69 compared to 4.2 for the pituitary. IHC(streptavidin-peroxidase method) of Bouins-fixed CL showedstaining for MT in the cytoplasm of luteal cells which wasabsent in stromal tissue and non-luteal ovarian tissue.Not all luteal cells were immunopositive and notopographical distribution of stained cells was observed.IHC localization of AVP was not attempted. It wasconcluded that the CL of the brushtail possum contains lowquantities of MT and AVP, probably synthesized by theimmunochemically staining cells of the CL.

MESOTOCIN AND ARGININE VASOPRESSIN IN THE CORPUS LUTEUM OFTHE BRUSHTAIL POSSUM (Trichosurus vulpecula) .

Conrad Sernia, Ross Bathgate and 'Robert T. Gemmell.

Departments of Physiology and Pharmacology , and'Anatomical Sciences, University of Queensland, Queensland4072.

pedi<:le

GM·CSF Ulml

200..----------------,

80

Large quantities of interleukin (IL)-6 and granulocyte-macrophage colony-stimulatingfactor (GM-CSF) bioactivity (Fig 1 and 2) and smaller amounts of tumor necrosis factor(TNF)u and IL-1 bioactivity were found in the perfusate. Interleukin 2 and IL-3 were notdetectable in the perfusion media. The GM-CSF content was significantly higher in theperfusate of ovulating ovaries (obtained 10h after hCG) compared to the earlier stages.Studies on preOVUlatory ovaries (prior to hCG injection) revealed that GM-CSF release wasnot influenced by LH, but was markedly increased when recombinant human IL-1J3 (4ng/ml)was added to the perfusion medium. IL-6 was released in similar amounts from ovaries atall stages. The identity of bioactive GM-CSF was confirmed by neutralisation with a specificpolyclonal antibody against murine GM-CSF. Size-exclusion chromatography of perfusionmedium revealed peaks of GM-CSF and IL-6 bioactivity at approximate molecular weightsof 21-23kD and 24-25kD, respectively.

lL-6 U(xl0·2)1ml100,-------------------,

Agure1Levels of IL-6 bioactivity In the perfuslon media al5h of Immature ovaries (10).preowtalory ovaries (PO). ovulating ovartes (00). Luteinized ovaries (LO). and theovarian vascular padlcle (pedicle). Each bar represenlS the mean.t SEM of valuesobtatned In six (10. PO. 00. LO) or four (pediCle) perfuslons.

Agure2Levels of GM-CSF bioactivity In the perfusion medium at 5h ofImmature ovaries (10). preoVUlatory ovartes (PO). ovulatingovaries ((00). lutelnlzed ovaries (LO).and the ovarian vascularpedicle (pedicle). Each bar represents the mean.t SEM otvalues obtatned In six (10. PO. 00. LO) or lour (padlde)perfuslons.ri =slgnltlcantly (p<O.01) higher than 10 and PO.

Cytokines have important roles in ovulatory events, including recruitment of leUkocytes,activation of prostaglandins, effects on steroid biosynthesis and regulation of collagenases.These results indicate that the ovary makes cytokines in detectable quantities.

References

1) Brannslrom M, Janson PO (1991). In: Hillier S (ed) Ovarian Endocrinology 133-166 Oxford.

2) Brannslrom M, Wang L, Norman RJ (1993) Endocrinology 132:399-404.

3) Robertson SA, Mayrhofer G, Seamark RF (1992) Bioi Reprod 46:1069-1079.

61

LAPAROSCOPIC RECOVERY OF OVARIAN OOCYTES FROM SLAUGHTERED ORLIVING SHEEP

OOCYTE RECOVERY IS INFLUENCED BY NEEDLE TYPE ANDASPIRATION PRESSURE.

H.R. Tervit, J.F. Smith, L.T. McGowan, R.W. Wells and PA Pugh

AgResearch Ruakura, Private Bag 3123, Hamilton, New Zealand

R.C. Fry, T.L. Simpson, T.J. Squires, M.A. Miles and E. Niall.

Victorian Institute of Animal Science, Werribee, Vic 3030.

SO

Vacuum (HII mm)

• n - 753... n - 723

o L-_-'--_-'

20

30

10

100

n - 1233n - 417

" - ...01" - 04.001-

1'0 70

Vaouu.... (HlI mm)

n - 15715n - 885

o 17; S Total Oocyt..• 1711 S Vl.,bl. Oocyt••C 20g STatal Oacylaa• 2011 S Vr.,bl. Oocyt••c.. 179 D Total Oooyt••A. 17 D Vlobl. 00 t ••

L --~__ 2-- - - - - I - - 80

- - - 0- 2: 50

-40

ao

gao

140!:l 30

g 20

10

021'

Oocyte pick-ups in humans and livestock use a variety of needles (160-200 singleand double lumen) and aspirate at a vacuum pressure (VP) of about 100 mmHg (1). Beforebeginning a research program on the repeated collection of immature oocytes from heifers,we evaluated the effect of various needle types and VP's on the in vitro recovery ofoocytes from bovine ovaries. To mimic the aspiration of follicles in vivo, the needle wasfixed loosely in two clamps, to be held at around 10" from horizontal, the ovary wasbought up to the tip of the needle, the candidate surface follicle was measured and itscontents aspirated into a 50 m1 Falcon tube containing 1 m1 ova maintenance media. Foreach parameter setting, 3 operators conducted a number of runs, each run consisting of theaspiration the all surface follicles >2 mm diameter from 10 ovaries. At the completion ofeach run the media was searched for oocytes and their number and quality recorded.

We conducted two experiments. The first compared 170 and 200 single lumenneedles at VP's of 25,50, 75 and 100 mmHg. The second compared 170 single and 170double lumen needles at the optimum VP of 50 mmHg. When using the double lumenneedle, the follicle was subjected to continuous aspiration and flushed twice for 2 secondswith Dulbecco's PBS at a rate of 0.75 mlIsec. Figure 1 demonstrates the effect of needletype and VP on the total recovery rate of oocytes (dotted lines) and the proportion thatwere viable (A,B,C class) for IVM/IVF procedures (solid lines).

Flg.1 Percentoge of aocytes Recovered per FollIcle AspIrated70 70

A total of 7,300 follicles were aspirated from 968 ovaries, giving a mean of 7.54follicles aspirated per ovary. Of these follicles, 92% were 2-4 mm, 6% 5-10 mm and 2%>10 mm in diameter. The total recovery rate of all oocytes was 50.2%.

These in vitro studies demonstrate that both the needle type and VP effect therecovery rate and quality of oocytes aspirated from bovine follicles 2 mm in diameter andgreater. We recommend using either a 170 single or 170 double lumen needle at a VP of50 mmHg to produce the greatest number of viable oocytes for in vivo follicle aspiration.1. Reichenbach et al. (1983) Theriogenology 39, 295.

Repeated collection of oocytes from living animals followed by in vitro maturation (IVM),fertilisation (IVF) and cutture (IVC) has the potential to produce more offspring than traditionalMOET procedures. We report here further resutts of our technique development in sheep.

Forty ewes were synchronised and superovulated with our standard double CIDR® (CarterHott Harvey, NZ) PMSG (Intervet, Holland) plus FSH (Ovagen, ICP, NZ) regimen (1).Immediately after CIDR removal the ewes were starved overnight and on the followingmorning were either slaughtered or subjected to laparoscopy (2). Laparoscopic oocyterecovery was attempted from the small (S; 3-6 mm) and large (L; > 6 mm) ovarian folliclesof both the living and slaughtered ewe ovaries by 2 operators using 2 types of aspirationneedles and modified human IVF equipment (Stortz, West Germany). Recovered oocyteswere IVM, IVF and IVC in our standard system (3) and development to blastocysts recorded.As an IVMlIVF/IVC control, oocytes were recovered from our usual source, the ovaries ofcommercially slaughtered untreated ewes.

There were no effects of needle type on any of the parameters. The mean number of Lfollicles aspirated in slaughtered ewes was 8.2±1.7 (sem) and 3.8±1.3 in laparoscoped ewes(P<0.01). Similar numbers of S follicles were aspirated from both ewe-types (4.7±1.6,4.1±1.3; respectively). Overall oocyte recovery rate from all follicles was 49.5%. Recoveryfrom L follicles was 55.3% and was affected by operator (1,69.1%; 2, 44.6%; P<0.05) andrecovery date (July 6, 45.2%; July 8,61.8%; P<0.01). Recovery from Sfollicles was 45.2%and was affected by operator (1,50.0%; 2,37.1%; P<0.05). Overall, a mean of 5.2±0.6oocytes were recovered from each ewe. Oocyte yield was affected by operator (1, 5.9±0.8;2, 4.6±O.9; P<0.05), follicle size (L, 3.4±O.6; S, 1.8±O.3; P<0.05) and ewe-type (Iaparoscopic,3.9±0.6; slaughter, 6.6±1.1; P<0.05).

Oocyte quality did not differ between slaughtered and laparoscoped ewes and betweenrecovery dates. However, a higher proportion of better quality oocytes (grade 1+2) wererecovered from Lthan Sfollicles (73.3% v48.6%; P<0.001). Overall development of oocytesto blastocysts from all 3 ewe types was 16.5% and was affected by follicle size (L, 20.6%; S,2.8%; P<0.01), recovery date (July 6, 10.0%; JUly 8, 20.6%,P<0.01) and oocyte quality (grade1, 21.9%; 2, 22.7%; 3, 3.4%; P<0.001). There was an inexplicable interaction between oocytequality and ewe type (P<0.01).

The resutts described are an improvement on our previous work (2). Further research is stillneeded to increase the numbers of large follicles available for aspiration in laparoscoped ewesand to improve oocyte recovery rates.

(1) Thompson, J.G.E. et al. (1990) Theriogenology 33: 1297-1304(2) Tervit, H.R. et al. (1992) Proc. Aust. Soc. Reprod. BioI. 24: 26(3) Pugh, PA et al. (1991) Theriogenology 36: 771·778

62

mE EFFECT OF OPTIMAL AND SUB OPTIMAL CONCENTRATIONS OF SPERM ONmE FERTILITY OF FRESH AND FROZEN BOVINE SEMEN

P. Shannon and R Vishwanath

Livestock Improvement Corporation, New Zealand DailY Board, Pvt Bag 3016, Hamilton NewZealand

The probability that a sperm will fertilise an ovum depends upon at least one sperm surviving at thesite of fertilisation until the arrival of the ovum. This obviously depends on several factors andprimary among them are; the pattern of survival of sperm in the female tract, survival time of viablesperm in the tract and the probability that an inseminated sperm will reach the site offertilisation (1).Differences between bulls or treatments that affect survival pattern of sperm will be reflected in adifference in conception rates. To a certain extent these effects can hi:: masked by high sperm numbers.If fertility is arranged so that it is well below optimum levels, by manipulating sperm concentrations,maximum differences between bulls and treatments can be observed (1). In this study, two semendilution techniques have been compared using this method. The two techniques are, rediluted deepfreeze (RDF) (2) and fresh semen diluted in Caprogen (3). Ejaculates from eleven bulls were frozen ata concentration of 400 million spermlml by the RDF method (2) subsequently thawed and rediluted inCaprogen (3) to a concentration of 40 or 10 million sperm/ml. For fresh semen, ejaculates from thesame bulls were split and diluted to contain either 5 or 1 million spermlml in Caprogen. A O.5ml dosewas used per insemination so that sperm numbers were 20 and 5 million for RDF and 2.5 and 0.5million for fresh semen. All diluted semen was stored at ambient temperature (18-21°C) and used onthe day after dilution. The results from the field trial are shown in Table 1.

Table 1. Effect of optimum and sub optimum dilution rate on fertility ofRDF and fresh semen.Fertility measured as percentage of non returns (NRR) 49 dayS after first service.

Dilution Optimum Sperm Sub optimum SpermTechnique dilution concentration % dilution concentration %

Inseminations million/dose NRR Inseminations million/dose NRRRDF 6004 20 67.6 3710 5 59.7Fresh 14792 2.5 68.1 9034 0.5 61.1

The difference between optimum and sub optimum dilution rates for the two techniques was 7.9% forRDF and 7% for fresh. The techniques did not differ significantly at optimum dose rates (difference,0.5%, t=O.7, p>O.I). An analysis of variance of the dilution rate results showed up some interestingeffects. For both the techniques, differences between bulls and dose rates were highly significant,however, bull x dose rate interaction whilst significant for RDF, was not significant for fresh semen.The features of interest in this trial are that at the optimum dose, freezing and redilution proceduresdid not affect the absolute time ofsurvival in the female tract, albeit many more sperm were required toachieve comparable fertility with fresh semen. The estimated motile sperm at the two dose rates was atleast 8 and 2 million for RDF and 2 and 0.4 million for fresh. The significant interaction between siresfor RDF indicates that the process of freezing had not altered the mean survival rate of sperm for allbulls but that individual bulls had been affected differently. It is therefore reasonable to conclude thatthe freezing procedure alters the probability of a sperm reaching the site of fertilisation or the patternof survival in the female reproductive tract. Mean survival rates in in vitro trials need to be conductedat different dose rates to establish if there is any link with field fertility results.

(1) Shannon, P. (1978). 1. Reprod. Fert. 54: 519-527(2) Shannon, P. (1972). Proc. VlIth Int. Congr. Anim. Reprod. A.I., Munich. 4: 1363-1367(3) Shannon, P. (1965). 1. Dairy Sci. 48: 1357-1361

63

EFFECf OF LEVELS OF EGG YOLK AND COMPATIBLE SOLU1ES ON THE POSTTHAW MOTILITY OF RAM SPERMATOZOA

L.G. Sanchez-Partida!, J.L. Zupp!, W.M.C. MaxwelI2& B.P. SetchelIl

Departments of Animal Sciences at ! Waite Agriculmral Research Instimte, University ofAdelaide, Glen Osmond, SA 5064, 2University of Sydney, NSW 2006.

The presence of egg yolk in diluents for short and long term storage of ram spermatozoais necessary to minimize the adverse effects of cold shock (1). The addition of compatiblesolutes are reported to enhance post-thaw motility of ram spermatozoa in the presence ofegg yolk (2). In that study only the absence and presence of egg yolk were assessed, andin order to investigate the effect of different levels of egg yolk in the presence or absence ofcompatible solutes the following study was conducted.

A 5 x 2 x 2 experiment was designed using tris-citrate diluents with four levels of eggyolk (0,5,10,15,20 % v/v), two levels of proline (0 and 54mM) and two levels of glycinebetaine (0 and 56mM) as factors. All the diluents contained 5% glycerol (v/v) at pH 7.0.Semen was collected from 3 Merino rams by artificial vagina, consecutive ejaculates fromeach ram were pooled, divided into 20 parts and diluted five-fold with the respectivediluents at 30°C, pellet-frozen on dry ice and stored in liquid nitrogen. Three pellets perdiluent per ram were individually thawed and assessed using a Hamilton-Thorn motilityanalyser after incubation in a water bath (37°C) for 0, 5 or 10 h.

The presence of compatible solutes in the diluents improved post-thaw motility in thepresence of egg yolk (table 1.). When egg yolk was not present in the diluent motility waslower (P<O.OOl) and there was no significant effect of the compatible solutes. (table 2).The inclusion of egg yolk in the diluents improved post-thaw motility of spermatozoa(compared with no egg yolk: 5% egg yolk (P<O.Ol); 10, 15 and 20 % egg yolk (p<O.Ol).

Table 1.Post-thaw motility (%) ofram spermatozoa pellet-frozen in diluents with 10, 15or 20% (v/v) egg yolk and in the presence or absence of compatible solutes.

Incubation GB absent GB presenttime (h) P absent P present P absent P present

0 45.8 62.4** 57.8** 56.6**5 25.5 36.2** 33.2 32.110 11.16 I-7.3 18.8 13.7

Each value IS the mean of 27 observations, means dIfferent by more than 8.8 WIthin thesame incubation time would be significantly different (**: P <0.01)Table 2. Post-thaw motility of ram spermatozoa pellet-frozen in diluents in the absence of

egg yolk and in the presence or absence of compatible solutes.

Incubation GB absent GB presenttime (h) P absent P present P absent Ppresent

0 33.2 34.0 43.0 29.65 12.3 14.7 10.9 11.510 5.6 3.8 3.7 6.0

Each value IS the mean of9 observatlons, means different by more than 15 WIthin the sameincubation time would be significantly different (P <0.01)

A low post-thaw motility of sperm frozen-thawed in diluents without egg yolk inthis study has already been reported (1,2). The better post-thaw motility presence ofcompatible solutes in the diluents corresponds with results previously reported (2).(1) Watson, P.P. & I.C.A. Martin. (1973) Aust. 1. bioI Sci., l8.:311-320.(2) Sanchez-Partida, L.G., Maxwell, W.M.C., Paleg, L.G. & Setchell, B.P. (1992)

Repred.. Ferri!. Dev. 1:113-118.

64

EFFECf OF CONCENTRATION OF TAURINE AND LEVELS OF GLYCEROL ONTHE POST-1HAW MOTILITY OF RAM SPERMATOZOA

L.G. Sanchez-Partida1, S.F. Sowerbutts1, W.M.C. MaxwelI2 & B.P. SetchelI1Departments of Animal Sciences at 1Waite Agricultural Research Institute, University of

Adelaide, Glen Osmond, SA 5064, 2University of Sydney, NSW 2006.Taurine is a sulfonic aminoacid compound which has been found in high

concentrations in reproductive tissues and fluids in the male (1) and it may be important forsperm motility and fertilization. The addition of taurine to a high K+ phosphate bufferprotected rabbit spermatozoa from loss of motility when incubated under aerobicconditions (2). The aim of this experiment, was to assess the effect of different levels oftaurine and glycerol on the the post-thaw motility of ram spermatozoa.

A 6 x 3 experiment was designed using six concentrations of taurine (OmM, 50mM,100mM, 150mM, 200mM and 250mM) and three levels of glycerol (0% v/v, 3%v/v and5% v/v) as factors. All the diluents were tris-citrate based, contained 15% egg yolk (v/v)at pH 7.0. Semen was collected from 3 Merino rams by artificial vagina, consecutiveejaculates from each ram were pooled, divided into 18 parts and diluted five-fold with therespective diluents at 30°C, pellet-frozen on dry ice and stored in liquid nitrogen. Threepellets per diluent per ram were individually thawed and assessed using a Hamilton-Thornmotility analyser after incubation in a water bath (37°C) for 0, 3 or 6 h.

The addition of taurine to the diluents containing glycerol increased post-thaw motility(table 1). When glycerol was absent post-thaw motility was lower (P<O.OOl) (table 2),but taurine still had a beneficial effect on motility; no difference was observed between the3% and 5% levels of glycerol.Table 1. Post-thaw motility of ram spermatozoa pellet-frozen in diluents with different

concentrations of taurine in the presence of 3 or 5 % glycerol.Time (h) Taurine (mM)

0 50 100 150 200 2500 51.0 64.0** 49.6 30.9 20.7 14.73 37.2 56.6** 36.7 20.3 12.3 16.46 21.7 35.8** 16.9 11.0 7.3 4.2

Each value IS the mean of 27 observatlOns, means different by more than 6.9 wlthm thesame incubation time are significantly different (**: P <0.01 higher than controls).Table 2. Post-thaw motility of ram spermatozoa pellet-frozen in diluents with different

concentrations of taurine in the absence of glycerol.Time (h) Taurine (mM)

0 50 100 150 200 2500 16.3 31.0** 25.0** 12.6 7.0 3.63 9.5 18.6** 12.8 5.2 2.5 2.86 4.9 8.1 8.2 3.0 3.3 1.4

Each value IS the mean of 54 observatIons, means dIfferent by more than 6.9 wlthm thesame incubation time are significantly different (**: P <0.01 higher than controls).

The addition of the lowest concentration of taurine (50mM) improved post-thawmotility (P <0.01), but motility was suppressed at higher concentrations. Furthermorethese results indicate that the percentage of glycerol in diluents for the freezing of ramspermatozoa can be reduced to as little as 3% without compromising post-thaw motilityand this may help to counteract the negative effects of glycerol reported on the fertilizationcapacity of frozen thawed sperm (3).(1) Kochakian, C.D. (1975) Am. J Physiol. 228:1231-1235.(2) Alvarez, lG. and Storey, B.T. (1983) BioI. Reprod 29:548-555.(3) Abdelhakeam, A.A., Graham, E.F., Vazquez, LA. and Chaloner K.M.(1991)

Cryobiology. 28:43-49.

65

CHANGES IN SACCHARIDE-BINDING SITES ON THE SURFACE OF RAMSPERMATOZOA FOLLOWING FREEZE-THAWING

James T.A. Marshall and Colin D Nancarrow.

CSIRO Division of Animal Production. P.O. Box 239. Blacktown, N.S.W. 2148

Artificial insemination (AI) in sheep, using semen from genetically superior nuns, has the potential tohasten genetic progress in the wool producing flocks. This potential is not being realized since frozensemen has poor fertility when used with vaginal or cervical AI. This is due to a diminished longevity in.and decreased transport through, the cervix (1). Proper interaction between the cervix and spermatozoamust exist if disruption of spermatozoan transport and consequent lower fertility is to be averted.Glycoproteins on the spermatozoan surface change as the spermatozoa transit both the male and femalereproductive tracts, including the addition of glycoproteins and saccharide residues in the epididymis andremoval of glycoproteins in the uterus. These changes might be expected have some function, possiblyrelated to longevity and transport in the cervix.

Preliminary studies were undertaken to define the terminal glycosyl reisdues on the sperm plasmamembrane of fresh and frozen-thawed spennatozoa. Semen was obtained from rams by ejaculation into anartificial vagina. Semen was frozen, stored and thawed using techniques described (2) using a trisglycerol-egg yolk medium as diluent, and frozen using the fast pellet method. Fresh or frozen-thawedspermatozoa were washed three times in PBS+ + 0.4% BSA (pH 7.2) at 500 g for 10 min. Plasmamembranes were extracted and isolated by a variation of the method described (3); dilution in ice-cold1Nbuffer (minus protease inhibitors), disruption by vortexing at high speed for 2 min, centrifugation at 500g for 15 min and centrifugation of the supernatant onto a 15%/50% w/v sucrose gradient at 100000 g for90 min. The plasma membrane band at the 15%/50% interface was diluted with an equal volume of1Nand spun at 100000 g for 60 min. The precipitate was taken up in 1N buffer, protein concentrationmeasured, extract subject to PAGE (7.5%. 12% and 15%) and then either stained for total protein orWestern blotted. Biotinylated lectins, proteins with a high saccharide-binding affinity and specificity,were used to probe the blots, which were subsequently reprobed with streptavidin-horseradish peroxidaseand colour developed.

Coomassie blue staining revealed several differences between fresh and frozen spermatozoa including boththe appearance of additional bands at >200 kDa, 150 kDa, 45-50 kDa and 25 IDa, and apparent changes inamounts of proteins between 35-45 IDa. Lectin binding studies showed that for both fresh and frozenmembrane extracts, Peanut agglutinin (PNA. ~-D-Gal) and Ricinus communis agglutinin (RCA, ~-D

GalNAc) had high affinity, Wheat germ agglutinin (WGA. ~-D-GluNAc) and Ulex europaeus agglutinin(UEA, a-L-Fucose) had moderate affinity, and BandeiIaea simplicifolia lectin (BSL, a-D-Gal) andSoybean agglutinin (SBA, a-D-GalNAc) had weak affmity, results consistent with other studies (4).SBA and UEA bound more intensely, and WGA less intensely, to frozen semen compared to fresh semen.Additional or more intense lectin binding occurred consistently at around 21 kDa, 43 IDa and 66 kDawith the exception ofWGA which had less binding at 66 kDa.

Such changes in the patterns ofsaccharide-binding sites are consistent with the hypothesis that changes inglycosylation of the spermatozoa surface affects longevity and transport in the cervix. Studies indicatethat some nun ejaculates give commercially acceptable conception rates after cervical AI (5) and that asmall proportion of spermatozoa in most nun ejaculates have good longevity and fertility (1). Aknowledge of the biochemical and biological chamcteristics offrozen semen with acceptable longevity andconception rates, will assist in development of new freeze-thawing techniques that will increase thefertility offrozen semen.

(1) Mattner, P.E., Entwistle, K.W. and Martin, LCA. (1969) Aust J. BioI. Sci. 22,181-7.(2) Evans. G. and Maxwell, W.MC. (1987) In "Salamons Artificial Insemination of Sheep and Goats."Butterworths, Sydney, Australia. pp. 122-141.(3) Srivastava, A and Olson G.E. (1991) Mol. Reprod. Dev. 29, 357-64.(4) Magargee, S.F., Kunze. E. and Hammerstedt, R.H. (1988) BioI. Reprod. 38, 667-85.(5) Windsor, D.P. (1992) Proc. ASRB 24,5.

66 67USE OF IN VITRO MATURED AND FERTILIZED OVINE OOCYTES FOR PRODUCTION OF

EMBRYOS WITH VISIBLE PRONUCLEI FOR MICROINJECTION

CULTURE CONDITIONS AFFECT THE FREEZABILITY OF IN VITRO PRODUCEDCADLE EMBRYOS

L.T. McGowan, R.W. Wells, P.A. Pugh, A.C.S. Bell and H.R. Tervit


Survival was also affected by thawing day (P<0.01) with more embryos categorised as goodor fair, and fewer as poor, on the first compared to the second thaw day (day 1; 34, 23, 43%;day 2; 21, 11, 68%). Survival was not affected by any other factor and embryo cell numberat the end of post·thaw cutture was not affected by cutture system.

The resutts show that IVMlIVF cattle embryos cuttured in SOF + HS have very poor survivalafter freezing and that cutture with oviduct cells, either during co-cutture or in the sheepoviduct, improves freezability. Recent atterations to SOF (2) have, however, enabled us toproduce a more freezable product from a no co-cutture system (3). The observation thatthawing day affected survival is intriguing as the same media were used each day. Itsuggests that small and unintentional changes in protocol can effect resutts.

(1) Fukui, Y. et al. (1991) J. Reprod. Fert. 92: 125-131(2) Gardner, OX et al. (1992) Proc. Aust. Soc. Reprod. BioI. 24: 90(3) Pugh, PA et al. (1993) Proc. Aust. Soc. Reprod. BioI. (these proceedings)

100Figure 1(8) Figure 1(b)E'2I PN visible

80

60%

40

20

012.5 14 15 16 17 19.5 15 16 17 19.5

Time (h) Time (h)

Results suggest that length of time to fertilize anel/or mte of development of NM/ IVF ova, andtherefore PNV, is variable and probably dependant upon several factors. Small differences in numbers of2-cell ova observed at each assessment after IVF suggests sub-optimal conditions for development beyond1-cell. A significantly higher mte of cleavage after 17h IVF than after 15 or I6h suggests that eitherfertilization is not completed until after I6h IVF or that a certain stage of development must be reachedprior to manipulation or IVC.

In conclusion, ova for microinjection can be produced by IVM and IVF of oocytes obtained fromovaries randomly collected from an abbatoir, but at very low mtes of PNV. Given that all fonns ofembryo manipulation and IVC seem to lead to decreased viability and increased developmentalabnormalities (1) it could be concluded that methods of IVM/ IVF embryo production need furtherimprovement before use in the production of transgenic livestock.1) Aust Assoc. Anim. Artificial Breeders. Proc. 5th AGM Adelaide (1992).2) Walker, S. et al. (1988). Proc. 11th Int Congr. Anim. Reprod. A.I. ~:483.

Marshall, James T.A., Pruss, Cathy A. and Nancarrow Colin D.

CSIRO Division of Animal Production, P.O. Box 239, Blacktown, N.S.W. 2148

Several problems exist with normal development of both embryos and offspring produced fromin vitro matured (IVM), fertilized (IVF) or cultured (IVC) ovine oocytes (1) and from manipulation ofin vivo -produced ovine embryos. Solutions for these problems are necessary if NM/ IVF ova are to beused for microinjection in the production of transgenic offspring. A further prerequisite is an adequate rateof pronuclear visibility (PNV) in embryos following IVF. We undertook studies to investigate someparameters of PNV in in vitro-produced embryos.

In six experiments during May-June 1992, ovaries were collected from abbatoir slaughtered ewesof random age and origin, and transported (1.5 hat 350C in PBS, pH 7.4) to the labomtory. Cumulusoocyte complexes (COC) were collected by slashing tlte surface of washed ovaries held under HEPESbuffered TCMI99 plus 10% heat-inactivated fetal calf serum (HI-FCS). cac with unexpanded cumulusoophorus were matured in bicarbonate-buffered TCMI99 plus 10% HI-FCS <± additives LH, FSH, E2and/or granulosa cells) for 27h at 390 C under humidified 5% CO2 in air. After IVM, COC withexpanded cumulus oophorus were partially stripped and incubated for 12.5 to 19.5h with fresh swim-uptreated spenn (1 x 106/ mL). Ova were immediately assessed for PNV and division; some were evaluatedfor cleavage after 5d culture in synthetic oviduct fluid medium supplemented with 20% HI-human serum(2) at 390C under humidified 5%C02: 5%02:90%N2. Results from all matwation groups were pooled.

In all, 98/459 ova (21.4%) were PNV, 95/459 (20.7%) were 2-cell and, following IVC, 213/338ova (63.0%) had cleaved. PNV against time of assessment, pooled from six experiments, is shown inFigure l(a). PNV, division to 2-cell and cleavage within these groups after IVC, at four times ofassessment, are shown in Figure I(b). Data were analysed statistically by Chi2_test Highest and lowestmtes occurred at, respectively, for PNV I5h (27.4%) and I9.5h (12.0%) (08), for division to 2-cell15h(15.8%) and I9.5h (8.0%) (08), and for cleavage after IVC 17h (75.6%) and 15h (57.4%) (p< 0.05). Thedifference in cleavage rates at 17h and I6h was also significant (75.6% and 63.0%, p< 0.05)

503184

Poor

62114

Proportion of embryos scoring

44482

Good Fair

494216

embryos

OviductCo-cuttureSOF + HS

In vitro matured (IVM) and fertilised (IVF) cattle embryos produced in our laboratory have, untilrecently, invariably been cuttured (IVC) in a non co-cutture system involving SOF mediumenriched with human serum (HS) under humidified 5% CO.j5% 0.j90% ~ (1). We reporthere the viability of embryos frozen in this and 2 systems involving co-cutture.

Oocytes from slaughtered cattle were IVM and IVF as previously reported (1). They were thenIVC for 24 h in SOF+HS and cleaved embryos then cuttured for a further 4-6 d in either:ligated oviducts of 02 (oestrus = 00) ewes; co-cuttured with bovine oviduct epithelial cells inTCM 199 + 10% FCS under 5% CO/air; or SOF+HS. All fair to excellent quality late morulato hatched blastocysts were deep frozen in straws with glycerol (G, 10%) as cryoprotectant.The straws were seeded at -rc and cooled to -33°C before plunging into liquid nitrogen.ThaWing was accomplished on 2 consecutive days and involved G removal via a sucrosegradient. Blastocoele development and visual quality after 24 hcutture in SOF+HS was usedto score embryos for survival (good, fair or poor chance of establishing a pregnancy).

Seventy percent of the embryos transferred to sheep oviducts were recovered and 10% ofthose recovered were freezable. The proportion of co-cuttured and SOF+HS embryosfreezable was 19 and 18%, respectively (P<0.01). Embryo survival was affected by cutturesystem (P<0.001) as follows:

No.

68 69

TIME COURSE OF OOCYTE MATURATION. FERTILISATION AND SPERM TRANSPORTEVENTS IN RED DEER (GeNus elaphus).

D.K. Berg. J.G. Thompson &G.W. Asher

AgResearch, Ruakura Agricuttural Centre, Hammon, N.Z.

An increase in deer farming and captive propagation of endangered species have stimulatedinterest in applying artificial breeding techniques to deer. However, there is no basicinformation on gamete/embryonic physiology for any deer species. This study determinedtiming of oocyte maturation, fertilisation, sperm transport and motility events in red deer(GeNUS elaphus).

Time to peak LH (PLH) from CIDR device withdrawal was determined in synchronised (12 dCIDR + 200iu PMSG) mature red hinds (N=26). Time to oestrous onset (00) from devicewithdrawal was recorded. Reproductive tracts were surgically exteriorised at various timesafter estimated PLH (range 0-36 h). Preovulatory follicles were aspirated. and ampullary (A) .and isthmic (I) regions of oviducts were flushed separately. Recovered follicular and ovulatedova were fixed and stained with 1% lacmoid. Sperm motility was recorded on video.

Mean±s.e.m for 00 and actual PLH following device withdrawal were 39±1 hand 37±1 h,respectively. Ovarian morphology. ovum recovery and sperm recovery data are presentedbelow:

HIGH MOLECULAR WEIGHT PROTEIN AGGREGATES IN BOVINE SEMINAL PLASMAPRODUCE CATIONIC PEPTIDES THAT AFFECT BULL SPERM MOTILITY

N. AI-Somai*, R. Vishwanath, P. Shannon and P.C.Molan*

Livestock Improvement Corporation, New Zealand Dairy Board, Hamilton and *Department ofBiological Sciences, University ofWaikato, Hamilton, New Zealand.

Reversible disaggregation of proteins in bovine seminal plasma (BSP) produce cationic peptides(CPs) of <5 KDa (1, 2). A group of CPs have been described including seminalplasmin, a cationicprotein which affects bovine sperm motility and acrosome reaction (3). We have recently reportedthe isolation of low molecular weight components (LMW) from the diffusate of dialysed BSP whichare detrimental to sperm motility (4). As a continuation of this work we have further investigated theeffect of cationic proteins in BSP and its constituent LMW peptides on motility ofbull sperm. 10 mIBSP obtained from fresh semen was loaded on a SP Sephadex C25 column, washed with 1.5 1 of30% acetonitrile at pH 3, and the bound material was eluted with 0.01 molll NaOH. The pH of theeluted material was adjusted to 7, and the volume reduced to 10 mI. SOS-PAGE of the eluate showeda major band with estimated molecular weight of 14-16 KDa. The eluate was adjusted to 30%acetonitrile in 0.02 molll HCl, pH 1.7 (30% v/v ACN), incubated for 2 hours then chromatographedon Sephadex G25 and eluted with 30% ACN. The loaded sample resolved into three peaks: one atthe void volume and two in the region <5 KDa. These three peaks were designated as: HMWcation,MMWcation, and LMWcation. Fractions were freeze-dried and reconstituted in the control bufferand tested on sperm. Table 1 shows the effect the these fractions on the motility ofwashed sperm.

ActualPLH (h) No.

0-6 37-12 313-18 719-24 5

alues are mean ± s.e (n:=3), unlike superscripts Within co umn eqp< .

Treatment Cone Protein cone. % Motility(%) (mg/mt) Ohrs 18 hrs

Control 10 81±2.9a 72±3.1a

HMWcation 10 5.2 77±4.0ao 37±3.0o

MMWcation 10 1.6 73±3.3° 59±3.8a

LMWcation 10 1.1 7l±2.90 34±5.6°

BSP 10 43.0 81±2.9a 65±5.7a

Table 1. Effect of the fraction from gel permeation chromatography on Sephadex G-25 of the elutedmaterial from the cation exchanger (SP Sephadex C 25) on motility of washed sperm at ambienttemperature. V .. I diffi ( 0 05).

Ovum Spermrecovery (n) recovery

Gv (2) I (non-motile)M1 (1) I (non-motile)

GV (1). Ml (1). M2 (1) I (non-motile, FP, CM). A (FP)M2 (3) I (FP. CM)A(1) A(FP)

2PN (3) I (CM)A(1) I (2) A (FP, CM, HA)

1PN (1), 2PN (1) I (CM, HA)A(1) I (1) A(CM, HA)

7-8mm F5-7mm F10mmF

7-10mm F(2)OV(3)

7mm F(1)OV(4)OV(3)

OVananmorphology (n)

3

5

31-36

25-30

F=tolhcles, Ov=ovulatlons, Gv=gerrmnal veSIcle, M1=mefaphase 1, M2=metaphase 2,FP=forward progressive, CM=circular motion, HA=hyperactivated

The majority of oocytes had meiotically matured (M2) by 24 h post-PLH (range 16.5-25.5 h).Mean time from PLH to ovulation was 20±1.5 h. with the earliest ovulation at 18.5 h.Fertilisation was first observed in an ovum recovered from the ampulla at 27.5 h post-PLH.All ovulated ova had lost their cumulus oophorus by the time of recovery. Sperm had reachedthe oviduct as early as 8 h after DO. but were all non-motile. Forward progressive motilitywas first observed in the isthmus 8.5 hafter 00. Two other types of sperm motility were alsoobserved: circular motion (CM) and hyperactivation (HA,"figure-8" motility pattern). One hindexhibited all three types of sperm motility at 28.5 h post-DO. After 31 h post-DO (and postPLH) no FP sperm motility pattern was observed in any hind.

These data demonstrate that, in general, gamete maturation and transport in female red deerreproductive tract follows similar patterns to those of other ruminant species, in that: meioticmaturation is complete by 24 h after PLH; ovulated oocytes rapidly lose their cellularvestments; fertilisation appears to occur shortly after ovulatIon; a rapid transport phase ofnon-viable sperm is followed by establishment of a population in the isthmus; hyperactivatedsperm move from an isthmic reservoir to the ampulla.

CPs have detrimental effects on sperm motility. Washed sperm when incubated with these fractionsshow curved tails when observed after 18 h incubation indicating damage to their membranes. TheLMWcation was significantly more detrimental to sperm motility than the other two fractions or BSP(p<O.05). Some hyper activation of sperm motility was seen soon after addition of the HMWcationfraction. Some ofthe effects of the HMWcation on sperm motility could be attributed to the presenceof seminalplasmin (4) in this fraction. The CPs in BSP are aggregates which under disaggregatingconditions release LMW components with molecular weights <5 KDa. Reduced activity in somecationic fractions is probably due to their inability to bind to the sperm membrane. CPs do bind tosperm membranes (5) and exert their damaging activity in a manner similar to that described inbacterial membranes (6, 7). Further work is being carried out to identify the mechanisms by whichthese fractions affect sperm.(I) Shannon, P., Curson, B., and Molan, PC. (1987). N. Z. J. Agr. Res. 30: 195-202.(2) Hameed, 1. (1987) PhD thesis. University ofWaikato, Hamilton, New Zealand.(3) Shivaji, S. (1988). Bioscience Report. 8: 609-618.(4) Al-Somai, N., Vishwanath, R., Shannon, P. et al (1991).Proc. Aust. Soc. repro. BioI. 23: 128.(5) Shannon, p., and Curson, B. (1983). N.Z. Agr. Res. 26:455-460.(6) Farley, MM., Shafer, WM., and Spitznagel, JK. (1989). Infection and Immunity 56: 1589-92.(7) Rosenthal, SK., and Buchanan, AM. (1974). Biochim. Biophys. Acta. 363: 141-150.

70

EFFECf OF DILUENT TYPE ON MOTILITY OF RAM SPERMATOZOASTORED FOR VARYING PERIODS AT 15°C

G C Upreti. J.E. Oliver, R. Munday, J.P. Smith

AgResearch, Ruakura Agricultural Centre, Private Bag 3123, Hamilton, N.Z

Successful use of fresh semen in sheep AI systems is dependant on the use of diluentsthat can maintain sperm viability for extended periods. We investigated the effect oftwo diluents on viability of semen stored at 15°C and during subsequent incubationat 3SOC

Ram semen, collected by artificial vagina, was diluted to 800 x 1(fi spermatozoa perml either in a defined diluent (RSD-l) or in a milk diluen,t (1). Diluted semen wascooled to 15°C (2) and stored at this temperature for up to 8 days. Total andprogressive motilities (3) of sperm stored in RSD-1 and total motility of those storedin milk diluent were assessed following incubation at 3SOC for 1, 7 and 24 hours.Samples were assessed after 0, 1, 2, 3 and 8 days of storage.

The percentage of motile spermatozoa declined linearly with time of storage in bothdiluents when assessed at 1 and 7 hrs of incubation and the decline was more rapidin case of the milk diluent Values at 1 h of incubation after 0, 3 or 8 days of storagein RSD-l were 80%, 60%,45% and the corresponding values for milk were 75%,45% and 10%.

There was a more rapid decline in motility during incubation with the milk comparedto the RSD-l diluent and this was more pronounced with increasing time of storage.No motility was found after 24 h of incubation in the milk diluent even on day 0 ofstorage whereas motilities of 65% on day 0 to 25% on day 8 were recorded after 24h incubation with RSD-l. The pattern of changes in velocity score was similar to thatof % motile sperm. Measurements on progressive motility (at 1· hr of incubation)after storage in RSD-l for up to 4 days did not show any changes in % progressivelymotile sperm or in the velocity.

RSD-1 is thus a better diluent than milk for maintaining spermatozoal survival asassessed by in vitro motility. A recent insemination trial (4) has shown, however, thatsemen diluted and stored in RSD-l gave no better conception rates than semen storedin milk diluent Thus the motility parameters, we currently use for in vitro assessmentof semen are inadequate for predicting fertilizing ability in vivo.

(1) Upreti, G.c., et al. (1991) Proc. Aust Soc. Reprod. Bioi 2.1: 129.(2) Upreti, G.c. et al. (19918) Proc. N2 Soc. Anim. Prod. ,52: 251-254.(3) Upreti, G.c., et al. (l992b) Proc. Aust Soc. Reprod. BioI. 24: 15.(4) Smith, J.F. et al. (1993) Proc. NZ. Soc. Anim. Prod.~: in press.

71

MODIFICATION OF RAM SEMEN DILUENT (RSD·1): EFFECT OF LIPIDS ON RAMSPERMATOZOAL MOTILITY

G.C. Upreti, K.R. Board. J.E. Oliver and J.F. Smith

AgResearch, Ruakura Agricuttural Centre, Private Bag 3123, Hamitton, N.Z.

Semen diluents based on egg yolk and milk contain considerably more lipid than RSD·1.Krebs-Ringer satts, organic nutrients and bovine serum albumin are the major componentsof RSD-1 (1). This study examined the effects of addition of lipids to RSD·1 on spermatozoalmotility.

Qualitative variations in lipids were obtained by using lipids extracted from various bi~logical

sources such as red blood cells (RBC) and livers of sheep and cattle, egg YOI.k, milk ~nd

foetal-ealf and can sera. The total lipids (TL) from some sources were also fractionated Intoneutral (NL) and phospholipids (PL). Lipids (total or fractionate~) were suspended in RSD-1(1 mg/ml) by heating to 50°C and sonicating for 10 min, under nitrogen atmosphere.. ?emenwas collected and diluted in RSD·1 as previously described (1). Percentage motility andvelocity score were assessed for freshly diluted spermatozoal suspensions and those storedat 15°C for 2 days after 1 and 24 h incubation at 38°C (2).

Spermatozoal motility was markedly inhibited by TL from liver and RBC after .24 h incubationfor both freshly diluted and stored semen. The motility was completely lost In the pr?s?nceof TL from sheep liver and cattle RBC for both freshly diluted and stored samples. SimilarlyTL from sheep RBC completely inhibited motility for stored samples only. The perce~tage

motile and velocity score (indicated in brackets) declined for sheep RBC - 4(0.08), cattle liver·10.5 (0.42) and for cattle liver TL treatment for stored samples -5 (0.33), compare~ to. ~SD-1

freshly diluted -50 (2.2) and stored - 40 (2.08). TL from milk and egg yolk did not Significantlyinfluence motility parameters.

The PLs from milk and sera were toxic to sperm but those from egg yolk were not toxic. NLsfrom egg yolk and milk were less toxic than NL from serum. After 24 h incubation, %motilityand velocity score values of 30 (1.5), 38 (1.5),21 (1.1), 13 (0.8) were recorded for NL fromegg yolk, milk, can·serum and foetal-calf serum respectively compared to 4~ (1.;) for ~SD.1.Serum NLs were less toxic in samples stored for 2 days. The corresponding Yo motile andvelocity score values for foetal-can-serum NL and c~n.seru?1 NL were 45 (1.8) and 42 (1.6)respectively compared to 45 (2) for RSD-1 after 24 Incubation of stored samples.

Incubations in RSD-1 under anaerobic conditions improved the velocity score to 3.5 after 24h at 38°C but did not effect the %motile. These beneficial effects of anaerobic incubationswere mai~tained in samples stored for 2 days at 15°C. However the toxicity of fractionatedPLs was retained even under anaerobic conditions.

(1) Upreti, G.C. et al. (1991) Proc. Aust. Soc. Reprod. BioI. 23: 129(2) Upreti, G.C. et al. (1992) Proc. N.l. Soc. Anim. Prod. 52: 251-254

72QUAIL YOLK AND COCONUT EXTRACT IN DILUENTS FOR

STORAGE OF RAM SEMEN AT 30 AND SoC.

73

Chairussyuhur, A, Sanchez-Partida, L.G., Maddocks, Sand Setchell, B.P.Department of Animal Sciences, Waite Agricultural Research Institute, University of

Adelaide, Glen Osmond S.A. , 5064.ABSENCE OF BINDING OF AN OVINE OESTRUS-ASSOCIATED

OVIDUCAL GLYCOPROTEIN TO RAM SPERMATOZOA

The motility of sperm stored in quail yolk diluent was comparable to those stored in henyolk diluent; t!I~ inclu~ion of coconut extract significantly (* : P<0.05, by Tukey's test)unproved motility dunng storage at both temperatures. Incorporation of coconut extractwas so effective that the use of this component may prove to be ofpractical importance.

(1) Y.annakopoulos, A.L. and Tserven~-Gousi, A.S. (1986). Br.Poultry Sci. 27:171-176(2) SlIva, I.S. de A, Nunes, J.F.& Freitas, V.J.P. (1992). Anim. Breed. Abstr. 60:486(3) Banzon, lA. and Velasco, J.R. (1982). Coconut Production and Utilization -

Phillippine Coconut Research and Development, Inc., p.215 '

lL. Hill and C.D. Nancarrow

CSIRO Division of Animal Production, P.O. Box 239, Blacktown, NSW 2148

An oestrus-associated glycoprotein (BGP) is known to be produced by the ovineoviduct for 3-6 days around oestrus (1). This coincides with the time at which gametetransport, fertilization and early embryo development occur in the oviduct Preliminarywork suggested that this glycoprotein, which binds to oocytes and embryos, may alsobind to spermatozoa, thereby implying a role in fertilization (2). The present set ofexperiments were designed to reassess the ability of EGP to bind to spermatowa.

Oviducal fluid (OF) was collected daily from catheters positioned in the funbrial end ofoviducts of cyclic ewes. Fractionation of OF was carried out using a Superose 12FPLC column (Pharmacia), Fl being the EGP-enriched fraction. Spermatozoa werewashed 3 times with PBS before being incubated in duplicate with the appropriateprotein (OF, Fl or F2) at 39°C. In experiment 1, semen was collected from 2 ramsand pooled. The spermatozoa were divided into 12 treatment groups, each incubatedwith either OF, Fl or F2 for 10, 20, 40 or 60 min. Experiment 2 involved acomparison of the abilities of capacitated and uncapacitated spermatozoa to bind EGPover periods of 60 min. or overnight. Experimen~ 3 tested whether EG? bound. tospermatozoa in vivo, and used 2 groups of 3 ewes killed 2 hand 6 h after mtrautenneA.I. Tracts were removed and divided into uterus, ampulla and isthmus which wereflushed with PBS to recover spermatozoa. Control spermatozoa were incubated withoviduct proteins as described in experiment 2. In all experiments, spermatozoa weresubsequently washed twice in PBS before being fixed onto slides using methanol.Specimens were then assessed for the presence of EGP; binding sites were blockedwith BSA before slides were incubated overnight with a monoclonal antibody againstEGP (MAC264) at a dilution of 1:1000. Slides were then washed twice in PBS beforebeing incubated with an FITC second antibody (1:64), washed in PBS and examinedunder an epifluorescent microscope. All experiments included a slide of oviductepithelial cells collected from oestrous ewes as a positive control. This was treated asfor spermatozoa except without incubation with oviduct proteins. Oocytes were used asa second positive control and were incubated with the proteiD: in exactly the same wayas sperm. A non-reactive primary antibody was used for negative controls.

In all of the experiments described, no fluorescence was associated with spermatowa atlevels higher than that obtained with negative controls. The oviducal epithelial cellsgave a consistently strong signal. These results imply that EGP does not bind tospermatozoa at any stage of capacitation and/or fertilization. However, this does notnecessarily mean that EGP plays no role in fertilization. It is widely documented thatEGP and similar glycoproteins in a variety of species become associated with the zonapellucida and plasma membranes of the oocyte and embryo. Although we haveevidence to suggest that this protein piays a role in regulating early embryodevelopment (3), it may also influence fertilization of the oocyte.

1) Sutton, R., Nancarrow, C.D., Wallace, AL.C. and Rigby, N.W. (1984). J.Reprod. Fert. 72,415422.

2) Sutton, R., Wallace, A.L.C., Engel, H. and Nancarrow C.D., (1984). In:Reproduction in Sheep.Eds: Lindsay, D.R. and Pearce, D.T. Aust. Acad. Sci., Canberra. pp.140-143.

3) Hill, J.L., Marshall, J.T. and Nancarrow, C.D. (1992). Proc. Aust Soc. Reprod. BioI. 24:104.

83.579.6*43.3*

87.139.63.2

88.939.03.0

85.3

85.285.380.878.8*71.5*

85.6

84.984.466.625.413.8

83.5

77.980.965.927.012.9

The effect of quail yolk and coconut extract on % motile sperm during storage.Temp. 5 0C 5 0C 5 0C 30 0C 30 OC 30 0CDiluent HYCG QYCG CEQY HYCG QYCG CEQYTime ofstorage (h)o244896144192240

Cryopreservation of spermatozoa is common in the sheep and cattle industries indeveloped countries. However the ingredients required for cryoprotective diluents, andthe technology for cryopreservation are not readily available in many developingcountries, such as Indonesia and there is little information on the requirement formaintaining viable spermatozoa at ambient or cool, rather than frozen, temperatures. Thepresent study investigated the suitability of two diluent components that are readilyavailable in Indonesia, and examined the motility of spermatozoa maintained in this noveldiluent at ambient (30 dc) and cool (5 DC) temperatures. Chicken egg yolk is used widelyas an important cryoprotective agent during chilling of spermatozoa. However quail eggsare more readily available in Indonesia, and have a greater yolk proportion than chickeneggs (1). Coconut water, found within the endosperm cavity, has previously been foundto be a viable "natural" diluent for sheep semen (2). However, coconut extract (pressedfrom the coconut endosperm) has a higher protein content (3), and since protein is alsoimportant in semen diluents we have used this extract in the present studies.Single ejaculates were collected from each of three Merino rams with an artificial vagina,twice a week, for a period of two months. Each ejaculate was split into 4 aliquots, each ofwhich was diluted five fold at 30 DC. Diluents used were a standard citrate-glucosemedium containing 20% hen yolk (HYCG), the same medium with 20% quail yolk(QYCG) and 15% coconut extract plus 5% quail yolk in citrate buffer (CEQY). For eachexperiment four repetitions were conducted. Assessment of semen was performed with aHamilton-Thorn Motility Analyser. Samples to be stored at 30 DC were maintained at thistemperature in a water bath, while those to be stored at 5 DC were cooled down from 30 to5 DC over 2 hours and maintained at this temperature in a water bath until assessed.

74

TRANSCERVICAL INSEMINATION OF MERINO EWES

D.P Windsor, A.Z. Szell, C. Buschbeck1, AY. Edward and IT.B. Milton2

Sheep Industries Branch, Department of Agriculture, Katanning WA 63171 Department of Population Medicine, University of Guelph, Ontario, Canada

2 Animal Science Group, School of Agriculture, University ofWA, Nedlands WA 6009

There have been a number of reports of successful transcervical artificial insemination inthe ewe, but the techniques described have not been widely adopted in Australia. Theaim of this study was to compare a recently described method (1) to laparoscopic andcervical artificial insemination (AI) for use with frozen semen in Merino ewes.

Ewes were treated for 12-14 days with 30mg FGA sponges (Ovakron, ESP [exp 1] orChronogest, Intervet [exp 2]). PMSG (350iu; Pregnecol, Horizon) was administered atsponge withdrawal. Al (51-56 hrpost-sponge withdrawal) with semen frozencommercially as pellets was by either 1.Iaparoscopic(LAl; 2), 2. cervical (CAl) or 3.transcervical (TAl; 1) methods. Thawed semen doses were: LAl: 2x30pL; CAl: 200(exp 1) or 100 (exp 2) pL; TAl: 80pL (diluted x6 in Dulbecco's PBS prior to Al).Depth of TAl by experienced (expl) or inexperienced (exp 2) operators was scored as:1: intrauterine; 2: deep intracervical; 3: shallow intracervical; 4: at the cervical os.

Pregnancy was determined by ultrasound 65-75 days post-insemination, and dataevaluated by logit analyses. Pregnancy rates (% Preg) and the proportion (%Al) of TAIewes falling into each depth of insemination category are shown in the table.

DepthofTAIExp. Datum LA! CAl 1 2 3 4 total

1 %AI - - 76% 4% 14% 3%I2251 %Preg 48% 9% 32% 7% ' 8% 0% 26%

2 %AI - - 43% 10% 20% 27%12182 %Preg 38% 1% 39% 17% 0% 0% 19%

LAl produced higher pregnancy rates than the overall TAl results, which were in turnsuperior ~o CAl, ~ both experiments (p<0.05). LAI produced higher pregnancy ratesthan utenne TAl m exp 1 (p<0.05), but not exp 2. TAl pregnancy rates were limited bylow rates of cervical penetration in exps 1 and 2, and reduced fertility in exp 1. Thebenefit o~ increased depth of intracervical Al (by TAl) was less apparent in exp 1 thanexp 2. It IS concluded that TAl is unlikely to displace LAl in commercial practice. Theresults of exp 2 indicate, however, that TAl may have practical application ifcervicalpenetration rates can be increased by operator experience or technical improvements.

(1) Halbert, G.W., Dobson, H., Walton, IS. & Buckrell, B.C. (1990) Theriogenologyn.: 993-1010.(2)Killeen, LD. & Caffery, GJ. (1982) Aust. Vet J.~: 95.

75

PREGNANCY RATES TO FIRST INSEMINATION IN DAIRY CATTLEPREVIOUSLY TREATED WITH PROGESTERONE DURING LATEDIOESTRUS AND WITH OR WITHOUT OESTRADIOL BENZOATE

K.L. Macmillan and V.K. Taufa

Dairying Research Corporation, Private Bag 3123, Hamilton, New Zealand

The tested hypothesis was that concurrently treating cows with oestradiolbenzoate (ODB) when a CIDR device (Eazibreed CIDR; Inter Ag, Hamilton)was inserted into the vagina for 7 or 12 days would affect the fertility of thepost-treatment oestrus. A preliminary study had shown that pregnancy ratesdeclined if devices were removed at increasing intervals after Day 20 (oestrus= Day 0).

Records were available for 1028 cows which had their first insemination from2 to 14 days after removal of a CIDR device on Days 20 to 24. Approximatelyhalf of the cows within each of the 12 participating herds had a treatmentperiod of 7 days and the remainder for 12 days (7 or 12-day CIDR). Half ofthe devices used within each of these treatment intervals each had a gelatincapsule containing 10 m'g ODB placed in its grooved surface before insertioninto the vagina. Devices were removed during a morning milking and eachcow was presented for insemination after it had been detected in oestrus.Most inseminations were made 48 h (57.4%), 72 h (28.5%) or 96 h (7.2%)after device removal. There were from 42 to 65 cows inseminated withineach of the 20 sub-cells produced by 2 treatment duration, with or withoutODB and with device removal at from Days 20 to 24. Date of conception wasconfirmed by rectal palpation of uterine tissues or contents from 35 to 70 daysafter first insemination.

The average pregnancy rates to first inseminations for cows treated with aCIDR device without an ODB capsule for 7 days (n=261) or 12 days (n=262)were 47.9% and 47.7%, and both of these were less than among herdmatestreated with a device for 7 days (n=252) or 12 days (n=253) but whichincluded a capsule (54.8% and 56.5%; p<0.001). This effect on pregnancyrate was associated with a significant treatment interaction involving Day ofdevice removal. The difference in pregnancy rates between the twotreatments varied from 2.1 % higher with no ODB for Day 22 to 16.3% higherwith ODB for Day 24 (p<0.01). The inclusion of ODB reduced but did noteliminate the decline in fertility associated with removing a CIDR device afterDay 20.

There were 263 cows inseminated 48 h after removal of devices which hadoriginally included an ODB capsule. Another 242 were inseminated at 72 h or96 h later. The pregnancy rates for these treatment sub-groups were 53.2%and 59.6%. The equivalent results for herdmates treated with CIDR deviceswithout ODB and inseminated at 48 h (n=327) or later (n=196) were 40.4%and 60.8% (time to insem. x treatment = p<0.05). The difference inpregnancy rate of 20.4% (60.8% - 40.4%) between inseminations made at 48h after the end of a CIDR treatment or at longer post-treatment intervals wasreduced to 6.4% (59.6% - 53.2%) by the use of an ODB capsule at deviceinsertion.

Day Type I6 1.83 ±0.137 1.48 ±0.128 0.96±0.03

76

PURIFICATION OF A STAGE-SPECIFIC LECTIN RECEPTOR FROMTHE SHEEP UTERUS

G.M. Harper, A.D. Gooneratne, C. Molloy and D W Bullock

Animal and Veterinary Sciences Group, Lincoln University, Canterbury,New Zealand

The uterine endometrial surface is receptive to attachment of the trophoblast for only ashort period in early pregnancy, with attachment in the sheep occurring after 15 - 16days of pregnancy. To investigate the biochemical changes associated with thiswindow of receptivity, frozen sections of uterus from pregnant or non-pregnant eweson Days 11, 13, 15, 16, 17 and 18 after mating (Day 0) were cut and stained with thebiotinylated lectins Dolichos biflorus (DBA), Glycine max., Arachis hypogaea, Ricinuscommunis-I, Concanavalin A, Ulex europaeus-I (DBA) and Wheat germ agglutinin.Only DBA and UEA reacted differentially with the endometrial epithelium of pregnantand non-pregnant ewes around the time of attachment. DBA stained strongly in theluminal and glandular epithelium of pregnant ewes up to Day 16, but showed nostaining in non-pregnant ewes. UEA stained strongly both luminal and glandularepithelium on all days examined in both pregnant and non-pregnant ewes, except for acomplete absence of staining on Days 15 - 16 of pregnancy. There was no differencein staining between caruncular and intercaruncular areas. The UEA receptor has beenpurified to apparent homogeneity by lectin-affinity chromatography and preparativegel electrophoresis. SDS-gel electrophoresis of the affinity column fractions revealed adiffuse lectin-stained band, suggestive of a heavily glycosylated glycoprotein. Thepurified product from the preparative gel was identified after electrotransfer by lectinstaining as a discrete band of~ approximately 100,000. Silver-staining did not detectany protein bands. Characterisation of the protein is in progress and its associationwith stage-specific lectin staining of the epithelium remains to be established.

Tl

DECREASE IN FIBRILLAR COLLAGEN TYPES I, ill AND V DURING, RAT IMPLANTATION

Peter R. Hurst, Rochelle D. Gibbs, Dawn E. Clark & Don B. Myers.

Department of Anatomy and Structural Biology, Medical School, University ofOtago, Dunedin, New Zealand.

Total collagen concentration has been shown to decline in the uterine sites ofimplantation (1). Furthermore by day 7 and 8 the decidual region had particularly lowcollagen levels (2) and by the use of immunohistochemistry the major fibrillarcollagen type I was found to be virtually absent in the decidual tissues (3). The studyreported here was designed to determine if other extracellular matrix fibrillarcollagens, namely types III and V, also decrease during rat implantation.

Implantation sites and segments of uninvolved uterine tissue were dissectedfrom 3 or 4 Wistar rats each on days 6, 7 and 8 of pregnancy. Collagen was extractedafter pepsin digestion of homogenised tissues. Portions of the extracts were (a) usedfor hydroxyproline assay in order to determine total collagen and (b) dissolved inreducing buffer before loading on SDS-PAGE gels. The running gels contained 3.8M urea to aid the separation of different collagen a chains. Gels were either stainedwith Coomassie blue for quantitation by scanning with an LKB densitometer ortransferred to Immobilon membranes for immunoblotting with antibodies to types I,III and V. The concentration of each collagen type was determined by dividing thetotal collagen concentration of the extracts by the densitometric proportions of eachcollagen band in the gels (Table I).Immunoblotting revealed distinctive and identifiable a bands of collagen types I (aI,a2), III (al) and V (aI, (2). Densitometric evaluation of the bands in relation to totalcollagen showed a significant reduction (by ANOVA) of type I concentration at eachday in both uterine regions. A similar decline was found also for type III but thisonly reached significance by day 8. Type V was found to be reduced by day 8 butonly in the implantation sites.

Table 1. Collagen concentrations as mg/l00mg wet weight tissue ± S.D.Implantation sites Uninvolved AreasType III Type V Type I Type III Type V0.29 ± 0.13 0.17 ± 0.04 3.05 ± 0.22 0.73 ± 0.15 0.43 ± 0.040.16 ± 0.07 0.11 ± 0.07 2.40 ± 0.25 0.63 ± 0.15 0.40 ± 0.130.09 ± 0.04 0.05 ± 0.02 2.05 ± 0.08 0.51 ± 0.09 0.33 ± 0.05

These results indicate that in the implantation regions (and to a lesser extent in theuninvolved regions) there is a decline in all the fibrillar collagens studied. The rate ofdecline was less for types III and V than for type 1. This may indicate slightlydifferent catabolic mechanisms involved in remodelling of the extracellular matrixproteins as important aspects of the uterine response to early pregnancy. Furtherstudies designed to determine the histological distribution of type III and V wouldascertain if, as is the case for type I, these collagens are greatly reduced withindecidualising tissues.(1) Myers, D.B., Clark, D.E. & Hurst, P.R. (1990) Reprod. Fertil. Dev. 2, 607-612.(2) Clark, D.E., Hurst, P.R. Myers, D.B. & Spears, G.F. (1992) J. Reprod. Fert. 94,169-175.(3) Clark, D.E., Hurst, P.R., Mclennan, 1.S. & Myers, D.B. (1993) Anat Rec. In press.

78 79

An unexpected finding was the pattern of cellular pr?lifer.ation seen with th~ PCNAantibody. In particular, evidence of glandular proliferation :vc:s apparen~ m 11/.15biopsies in the mid- and late-secretory phases of the cycle. ThIS IS not conSIstent WIththe commonly reported pattern of mitoses seen in human endometrium through thecycle' hence the comparasin between PCNA and BrdU staining. To date, the resultsfrom 'the second part of the study have shown that the pattern of cellular proliferationseen with PCNA, and BrdU staining are similar.

Overall the results do not support the concept of three discrete peaks ofangiogenic activity through the cycle corresponding to such events as post-menstrualrepair, endometrial proliferation, or spiral arteriole growth.

HUMAN ENDOMETRIAL ENDOTHELIAL CELL PROLIFERATIONDURING THE MENSTRUAL CYCLE

Anne M Macpherson and Peter A W RogersMonash University, Department of Obstetrics and Gynaecology,

Monash Medical Centre, Clayton 3168, Australia

Angiogenesis (new blood vessel growth) occurs rarely in normal adult tissue~,

however the female reproductive tract provides several exceptions. An early step mthis process is endothelial cell (BC) p~olifera?o? Previous stu.dies ind.icate that.theremay be three separate episodes of angIOgeneSIS m the endometrium durmg the pnmatemenstrual cycle. The aims were to quantify EC proliferation in human endometriumduring the menstrual cycle using immunohistochemistry, and to compm:e the.patternof cellular proliferation seen with two different markers: PCNA (prolIferating cellnuclear antigen - a nuclear protein whose expression increases during the late ~! phaseof cycling cells, and peaks during the S phase), and BrdU (bromodeoxyundme - athymidine analogue which is incorporated into DNA as it is syn~es~sed).. .

Endometrial biopsies were collected from women undergomg mvestigation forinfertility, and were used only if the infertility was unrelated to ute~e or.endoc~efactors. Biopsies were formalin fixed and wax embedded for routine hIstologICaldating, and immunohistochemistry. Portions of biopsies for the secondary study werecollected into medium with 10% FCS and incubated with 2mM BrdU for 3hr at 3rbefore fixation. For the first part of the study, one section from each biopsy wasstained immunohistochemica1ly using a streptavidin-biotin method, and incoJ:I?Orateda double staining technique for proliferating EC's. Proliferating cells were stained togive red nuclei using mouse-anti-rat-PCNA (clone PCI0; Novocastra) and AECchromogen. EC's were stained blue using mouse-anti-human-CD34 (cloneQBEND/lO; Serotec) and AP-Blue. An index of EC proliferation was derived fromthe number of proliferating EC's / the total number of EC's, expressed as %. F?r thesecond part of the study, consecutive sections were stained using the PCNA antibodyor mouse anti-BrdU (clone Bu20a; Dako).

Results (table) show that there was great variability in the EC proliferativeindices between biopsies throughout the menstrual cycle, and from the same stage ofthe cycle, and that there were no significant peaks of EC proliferation.

Menstrualgroup 1

2.47 2.561.43 1.23

12.535.57

Secretory789

5.34 8.452.28 3.72

6

17.43 5.79 18.726.78 4.87 9.54

Proliferative3452

14.026.4

meansem

DECIDUALISATION AND THE REMODELLING OF LAMININDURING NATURAL EMBRYO IMPLANTATION IN THE RAT.

Embryo implantation in most mammalian species is associated with thedifferentiation of uterine stromal cells into the maternal decidual cells ofpregnancy. The embryo which is located within the antimesometrial aspect of thelumen is surrounded by the large closely adhering primary decidual cells.Secondary decidual tissue is located more peripherally and contains less tightlypacked polygonal cells. By day 8 of pregnancy there is a functioningchoriovitelline (yolk sac) placenta on the mesometrial side of the implantationsites. Pericellular decidual laminin has been shown during an artificiallystimulated decidual response (1). Expression of laminin in the early stages ofnatural implantation has not however been examined. This study usedimmunohistochemical techniques to' investigate the spacio - temporal distributionof laminin over days 5 to 8 of natural pregnancy in the rat.

Rats were mated and the morning on which spermatozoan were found in thevaginal smear was called day 1 of pregnancy. A rabbit anti - mouse lamininantibody (Gibco) was used and its specificity tested by western blotting, slotblotting and immunoabsorbtion. Laminin was detected using immunofluorescenceon transverse frozen sections which passed through the embryo.

The presence of punctate laminin around the primary decidual cells was detectedon day 6 of pregnancy but not days 5 and 7. Transient, punctate laminin expressionaround mesenchymal cells has also been observed at the stromal/epithelial interfaceduring development and differentiation of both the intestine and kidney (2,3).Expression in the uterus may be associated with the differentiation program andsuggests that developmental mechanisms may be activated during decidualisation.The adhesive properties of laminin suggest that it m,ight be involved in theadhesion of the primary decidual cells. Other than the localised punctateexpression the decidual cells did not have pericellular laminin and thus are unlikelyto have a basement membrane.

Dawn E. Clark and Peter R. Hurst

Department of Anatomy and Structural Biology, University of Otago, Box 913,Dunedin, New Zealand.

(1) Glasser, S.R., Lampelo, S., Munir, M.l. & Julian, J. (1987) Differentiation,15::132-142.

(2) Simon-Assmann, P., Simo, P., Bouziges, F., Haffen, K. & Kedinger, M.(1990) Digestion, 46 (Supp!. 2):12-21.

(3) Ekblom, P. (1989) FASEB 1. .3.:2141-2150.

By day 8 the mesometrial vessels of the choriovitelline placenta have formed andlaminin was found in association with the arterial vessels but only faintly within thevenous sinusoids. Antimesometrially immunohistochemistry also revealed anetwork of small vessels. It is hypothesised here that this network of vessels isessential for the transport of the endocrine factors produced in this region. Theseresults lay the foundation for further investigations into the role of punctate lamininin the differentiation program of the uterine stromal cells and laminin expressionduring angiogenesis and placentation.

ENDOTHELIN SECRETION IN VITRO BY CULTURED HUMANENDOMETRIAL EPITHELIAL CELLS: EFFECT OF

TRANSFORMING GROWTH FACTOR-~1 AND INTERLEUKIN-1a.

Marion M. Marsh, Anne L. Hampton, Jock K. Findlay, & Lois A. Salamonsen.

Prince Henry's Institute of Medical Research, Clayton, Victoria 3168.

Endothelin (ET) is a potent vasoconstrictor (1), which has diversebiological actions. Immunohistochemically ET localizes predominantly toendometrial glands with increasing intensity in the late secretory phase (2).ET secretion by human endometrial epithelial cells (3) and passaged stromalcells (4) in vitro have been described. ET has been shown to interact with awide variety of cytokines and growth factors in different cell types.

In this study we examined ET production by isolated humanendometrial epithelial cells in culture and the secretory response totransforming growth factor-~ (TGF) and interleukin-1a (IL-1). Cells known toproduce both TGF and IL-1 are present in the endometrium and thesecytokines are potentially involved in the regulation of endometrial ETproduction.

Endometrial tissue from women without evidence of endometrialdysfunction was obtained at curettage. Epithelial cells were prepared fromindividual or pooled specimens. Cultures were established in Medium 199with antibiotics (M199) and 10% charcoal-treated (CT) foetal calf serum(FCS) for 48 hours, cells washed and further incubated in M199 and a serumfree mixture: insulin, transferrin, selenite, hydrocortisone and 0.01 % BSA(SF). TGF (2, 5, 10 ng/ml) or IL-1 (1, 10, 100 IU/ml) in triplicate wells, wasadded for a further 48 hours. SF alone and 10% FCS were negative andpositive controls respectively. Media was assayed for ET and cells for DNAcontent and ET secretion was calculated as pg/mcg DNA. Mean ETsecretion by unstimulated individual cell preparations is variable (133-353pg/mcg DNA), however, is increased 140%-192% by FCS. Comparativeanalysis was performed by expressing ET secretion as a percentage of thenegative control.

TGF at all doses stimulated ET production (P<0.05) (N=3). Mean ET%control +/- SE ranged from 122% +/- 22 to 191% +/- 36. This effect was notdose responsive and in general less than that induced by 10% FCS (MeanET% control +/- SE: 144% +/- 38 to 192% +/- 13). Preliminary data suggeststhat IL-1 also stimulates ET secretion (P=O.053). Mean ET% control +/- SE:110% +/-3 to 164% +/-10.

These results demonstrate in vitro stimulation of ET by TGF and IL-1.This is potentially important in the regulation of ET production and inunderstanding the role of ET in human endometrium.

Supported by the NH &MRC and WHO.1. Yanagisawa et aI., (1988) Nature, 332: 411-415.2. SaJamonsen et al (1992) Am J OOOtet & Gynecol,167: 163-167.3. Salamonsen et aI., (1992) Proc ASRB (Abstract).4. Economos et aI., (1992) J elin Endocrinol Metab., 74: 14-19.

81

ENDOMETRIAL OESTROGEN AND PROGESTERONE RECEPTOREXPRESSION IN WOMEN WITH MENORRHAGIA.

K.M. Abberton. P.A.W. Rogers, N. Taylor, H.O.D. Critchley.Monash University Department of Obstetrics and Gynaecology, MonashMedical Centre. 246 Clayton Rd. Victoria 3168.

Menorrhagia, defmed as menstrual blood loss in excess of 80 mls per period,affects 15% of all women, often with no obvious cause. Sex steroid profiles inmenorrhagic women are similar to those of normal women suggesting that,in mostcases menorrhagia is due to aberrations at a local level within the endometrium.The ~m of this study was to examine sex steroid receptor distribution inendometrium from women with menorrhagia compared to normal controls.

47 menorrhagic women were recruited (average age 37, range 22-50). Objectiveblood loss measurements were performed showing a mean menstrual blood loss of122 ml (range 85.5-559 ml). Endometrial biopsies were collected from eachwoman during a hysteroscopy to rule out any clinical reason for. excessivebleeding. Samples were dated using histological criteria and divided into fivemenstrual cycle stages; menstrual, proliferative, early, mid, an~ late secret?ry.Immunostaining for sex steroid receptors was performed as preVIously descnbed(1), and staining was semi quantitatively scored by two independent ?bservers.Results were compared with a control group of 58 women from a preVIOUS study(1). Analysis of variance and Fishers test for least significant differences wereperformed for pairwise comparison. . . . .

Maximal levels of oestrogen (ER) receptor lmmunostaimng occur In themenstrual and proliferative stages of the cycle, while progesterone (PR) receptorimmunostaining is maximal during the proliferative and early secretory stages.Immunostaining for both sex steroid receptors declined over the mid and latesecretory stages of the cycle.

PR immunostaining in both the stromal and glandular components wassignificantly higher in menorrhagic tissue during the proliferative phase of thecycle(stroma p=O.01, gland p=O.OOI), and in the glandular component during themenstrual stage of the cycle (p=0.05) when compared to the control group. ERimmunostaining did not significantly differ at any stage of the cycle betweenmenorrhagic and control samples. .

In summary, while endometrial receptor expression followed th~ same pattern mboth menorrhagic and control tissue, there was an increase In glandular PRimmunostaining during the menstrual and proliferative stages. PR immunostainingin the stroma was .also increased during the proliferative stage. As yet it is notknown if this reflects a functional increase in progesterone receptor activity in thesetissues.

(1) Immunohistochemical sex steroid receptor distribution in endometriumfrom longterm subdermal levonorgesterol users and du~g the normal menstru~cycle. Critchley H.O.D, Bailey D.A, Au C.L, Affandl B, Rogers P.A.W. (mpress).

82 83

SOF+HS 66 3 0 10 0 0JAM 78 3 0 12 25 0

CRlaa 40 6 0 12 0 0SOF+BSA 6 33 0

No. %Bl %HBI No. %BI %HBI

SOF+HS 42 33 10 13 23 46JAM 57 19 0 13 54 15

CRlaa 57 9 0 11 18 0SOF+BSA 6 67 0

20%(air)

7%

More embryos developed to blastocysts ill 7% O2 than ill aIr (P<O.OOl), th~sconfmning a previous report (3). Highest levels of development were observed illSOF+HS when incubated under 7% 02 (43±8% and 69±13% for in vitro and in vi~o

derived embryos, respectively). In vivo derived embryos performed equally well illJAM and SOF+HS media. However, at 7% 02, in vitro embryos had improveddevelopment in SOF+HS (43±8%) compared to JAM (19±6%). Development inCRlaa was poor for embryos of either source.

A striking morphological difference was observed between embryos,particularly blastocysts, incubated in ~edia .containing HS or FAF-BSA. , ?I?brro~incubated in FAF-BSA were much lIghter ill appearance and had fewer lipId-likeinclusions that characterised embryos incubated in SOF+HS. We believe thismorphology may be induced by low density lipids and lipid peroxides present inserum.

Performance of embryos in JAM, particularly in vivo derived embryos,demonstrate that this medium is suitable for investigation of factors affectingembryonic development.(1) Walker, S.K., Heard, T.M. & Seamark, R.F. (1992) Theriogenology 37,111-126.(2) Rosenkrans, C.F. & First, N.L. (1991) Theriogenology 35, 266.(3) Thompson, lO.E., et al. (1990) 1 Reprod. Fert. 89, 573-578.

[02] Medium In Vitro Derived In Vivo Derived

DEVELOPMENT OF IN-VIVO AND IN VITRO DERIVED OVINE EMBRYOS INMEDIA SUPPLEMENTED WITH HUMAN SERUM OR FATTY ACID-FREE

BOVINE SERUM ALBUMIN.

J M Bowen1, P.A. Pugh & lO. Thompson

AgResearch, Ruakura Agricultural Centre, Hamilton, N.Z.lAnim. & Vet Sciences Orp., Lincoln Univ. Canterbury, N.Z.

Domestic animal embryo culture systems are largely undefilled, comprising eithersomatic cells in co-culture or simple media supplemented with human (HS) or animalsera (1). Cell-free, defined media are advantageous in allowing direct examination ofmedia components on development and reducing variability in culture conditions.This study compared development of ovine embryos in two serum-free media (amodified SOF, known as JAM) and CRlaa (2), both supplemented with 5 mg/ml fattyacid-free bovine serum albumin (FAF-BSA) and SOF supplemented with 20% HS.

Embryos were derived either from IVMIF, or from day 1.5 superovulatedewes. Embryos were incubated for 6 days at 390C in either humidified 5% C02/airor 5% C0217% °2/88% N2 and in one of the 3 media: SOF+HS, JAM, CRlaa. Inaddition, some in vivo embryos were incubated in SOF+5 mg/ml FAF-BSA(SOF+BSA). Development to blastocyst (Bl) or hatched blastocyst (HBl) stageswere as follows:

R. Spindler M.B.Renfree", and D.K.Gardner

NUTRIENT UPTAKE DURING EMBRYONIC DIAPAUSE ANDSUBSEQUENT REACTIVATION IN MOUSE BLASTOCYSTS

Centre for Early Human Development, Monash University, Clayton and"Department of Zoology, University of Melbourne, Parkville, Victoria.

Many eutherians, including the mouse (Mus musculus), carry a diapausing blastocystin the uterus while suckling young. This delayed implantation is readily induced andterminated experimentally by administrating hormones to ovariectomised pregnant mice (1).It has been determined that within 6h of oestradiol-17f3 (E2) administration, there aresignificant changes in enzyme activities in the reactivating ~ouse blastocyst (2). Previousstudies on nutrient utilisation of blastocysts during delay and reactivation have employedradiolabel techniques, requiring extended incubation periods to assess metabolism (1,2). Incontrast, microfluorimetry, employed in this study, allows the accurate determination ofnutrient uptakes within minutes as opposed to hours.

eF1 mice were ovariectomised on day 4 of pregnancy before 8 a.m. and injected withprogesterone on days 6 - 9 of pregnancy, which delays implantation (diapause). Blastocystreactivation was induced by injecting E2 on day 9. In each experiment half of the mice withdiapausing blastocysts were injected with E2, so that diapausing and reactivated embryoswere examined simultaneously to control for interexperimental variation. Individualblastocysts were incubated at 37°C for 3h in 100nl drops of modified MTF medium (3)containing 0.5mM glutamate, pyruvate and glucose with 2mg/ml BSA. Serial nanolitresamples of medium were taken to determine substrate uptake by the blastocysts. Mermetabolic analysis, blastocysts were incubated in colcimed (2!lg/ml) for 2h before cellnumber and mitoses per embryo (mitotic index) were determined.

Mitotic index and cell numbers increased significantly within 8h ofE2 administration,compared to that of delayed controls (0.5 ± 0.1 vs 022 ± 0.02; P<O.Ol, and 137.9 ± 0.5 vs134.6 ± 0.8; P<0.05, respectively). There was no difference in glutamate uptake betweendelayed controls and reactivating blastocysts at any timepoint studied. In contrast, pyruvateuptake by reactivatingblastocysts (9.33 ± 1.1) was significantly higher than delayed controls(5.8 ± 1.6 pmol/e/h; P<0.05), within 4h of E2. However, significant differences in glucoseuptake between the two groups were not evident until 20h after E2 (reactivating, 12.5 ± 1.4;control, 7.3 ± 0.8 pmol/e/h; P<0.05). In contrast, at 20h after E2, pyruvate uptake byreactivating blastocysts was not significantly different to the delayed controls. Metabolicanalysis of reactivated embryos 24h after E2 was not possible as embryos could not beflushed and are presumed to have implanted.

The delayed mouse blastocyst has a similar substrate preference to the early cleavagestage embryo, ie preferential utilisation of pyruvate over glucose. The ATP/ADP ratio in bothzygotes and delayed blastocysts is high (attributed to low levels of biosynthesis), inhibitingphosphofructokinase, a key regulatory enzyme of the glycolytic pathway (1,4). Asbiosynthesis increases during reactiyation, and the ATP/ADP ratio falls as a result of ATPutilisation for cleavage and biosynthesis, glycolysis will be de-inhibited, resulting in anincrease in glucose uptake. As glucose utilisation increases, pyruvate uptake decreasesconcomitantly. The reactivated blastocyst therefore resumes a nutrient preference of a nondelayed embryo.1. Nieder, G.L & Weitlauf, H.M., (1984) J. Exp. Zool., 231, 121-129.2. Sakhuja-Talwar, D. et. al. (1984) Reprod. Fert., 70, 185-189.3. Gardner, D.K. & Leese, HJ. (1990) J. Reprod. Fert 88, 361-368.4. Barbehenn, E.K.,et. al. (1974) Proc. Natn. Acad Sci. U.S.A. 711056-1060.

84 85

EFFECT OF lYPE AND CONCENTRATION OF SERUM ON THE DEVELOPMENT OFIN VITRO MATURED AND FERTIUSED SHEEP EMBRYOS

S.L. Rhodes, T.Stojanov, G.Evans, W.M.C. Maxwell.

TIIE EFFECTS OF CO-CULTURE WITH OVIDUCT CElLS AND THE ADDmON OFGLUTAMINE ON THE DEVELOPMENT OF SHEEP EMBRYOS IN VTIRO

Table 1. Effect of Type of Serum on Embryo Development.

The overall cleavage rate (76% oocytes cleaved/inseminated) was not affected by type orconcentration of serum. The number of cleaved embryos developing to blastocysts was notaffected by concentration of serum (37/158, 10% vs 27/169, 20%) but was affected by typeof serum {Table 1).

Department of Animal Science, University of Sydney, NSW 2006

Synthetic oviduct fluid (SOF) supplemented with 20% human serum (HS) has been reportedto support development to blastocysts of in vivo produced sheep zygotes (1). We examinedfour sources of serum for their ability to support development to blastocysts of in vitro maturedand in vitro fertilised sheep zygotes.

Type of serum No. Blastocysts/ Mean Cell No.Cleaved zygotes (%) (± S.E.M.)

HS1 26/88 (30)a 83.5 <±.23.60)

HS2 20/85 (24)d 107.5 (±34.29)

SS 10/76 (13)C 80.2 <±18.88)

FBS 7/74 (9)b 73.5 <±34.94)

A.Z. Sull and p.r. Newton

Sheep Industries Branch, Department of AgricultureKatanning, WA 6317

The most successful methods for the in vitro culture of sheep embryos use oviduct epithelial cellsupport (1) or synthetic oviduct fluid (SOF; 2). We examined whether oviduct c~ll supp?rt willimprove the viability of embryos cultured in SOF. In addition, the effects of glutamme, WhICh wasshown to be beneficial for hamster embryos (3), were investigated on the development of sheepembryos.

Day 2 sheep embryos were cultured with or without oviduct cell support in SOF + BSA supplementedwith 0 or 1 mM glutamine. The embryos were collected at the 1 to 8-cell stage (2 days after theinsemination of donor ewes) and cultured in 96-well culture plates. The stage of embryonicdevelopment reached was evaluated after 4 days in vitro culture. Additionally, in the last two of fourreplications of the experiment. the viability of embryos which developed to the late morula/blastocyststage was tested in vivo by transfer into recipient ewes (lor 2 embryos/recipient).

The proportions of embryos which developed to or beyond the late morula (LM) stage w~ notaffected by treatments (Table 1). However, there was an interaction between the effects of OVIductcell support and glutamine on the proportions of embryos which reached the early blastocyst (EB),blastocyst (BL) or late blastocyst (LB) stages (p<0.05) and on the proportions of recipients pregnant(p<O.02). In the absence of oviduct cells, the addition of glutamine improved development to theearly to late blastocyst stages (p<O.Ol) and the proportion of recipients pregnant (p<O.05). In thepresence of oviduct cells, glutamine caused a non-significant reduction in the viability of embryos.

Table 1. The effect of sheep oviduct epithelial cells (SOEC) and glutamine (GIn) on the percentagesof embryos developing to or beyond different developmental stages and on the percentagesofrecipients pregnant on Day 51

SOEC GIn Development in vitro Pregnancy

(mM) n LM EB BL LB n Rate

- 0 36 53 6 3 3 8 38

1 31 48 35 26 26 5 100

+ 0 34 53 32 18 15 12 58

1 31 35 16 16 6 5 40

Total 132 48 22 15 12 33 52ac and db P<0.05ab P<0.01

Immature oocytes, collected on 4 separate days, were cultured at 39° C under low O2 tension(5%02,5%C021 90%NJ in M199 + 10% FBS(foetal bovine serum), LH(10~/ml), FSH (10j.Lg/ml)and oestradiol (1 j.Lg/ml). After 22hr, 427 oocytes were inseminated with frozen-thawed sperm(1 x 10a/ml) prepared by percoll gradient. Eighteen hours after insemination presumptivezygotes were allocated to 8 treatment groups investigating 4 types of serum at 2concentrations: HS1 (human serum pooled from 4 males and 1 female), HS2 (human serumfrom 2 females), SS (sheep serum from 1 ewe, 2 days after ovulation) and FBS each at 10 and20% v/v with SOF (+ 1mM glutamine, - glucose). Embryos were cultured for 6 days in 500).11of appropriate medium in 4-well dishes under low 02 tension at 39° C. On the 4th day afterinsemination, glucose was added to the culture media to give a final concentration of 1.5mMfor each treatment. On the 2nd and 7th day after insemination the zygotes were assessedfor cleavage and development to blastocyst respectively. Blastocysts were subsequentlyfixed, stained and the number of cells counted.

Media containing human serum were superior to those containing SS or FBS(P<0.001).There was no differences in mean cell numbers per blastocyst but overall the cell numberswere higher than those generally reported for totally in vitro produced blastocysts. Thesehigher cell numbers may indicate an increase in viability in vivo.

(1) Walker, S.K, Lampe, R.J. &Seamark, R.F. (1989) Theriogenology~: 797-804

These results demonstrated that glutamine, used as a single amino acid, can improve the viability ofsheep embryos cultured in SOF. It was also shown that the requirements for glutamine are differentfor embryos cultured with or without oviduct cells. This may be explained by differences in themetabolism ofembryonic and somatic cells.

(1) Gandolfi, F. & Moor. R.M. (1987) J. Reprod. Fert. ID.: 23-28.(2) Walker, S.K., Seamark, R.F., Quinn, P., Warnes, G.M., Ashman, R.J., Smith, D.H. & Ancel, P.

(1988) Proc. 11th Int. Congr. Anim. Reprod. & A.I.1: 483-485.

(3) Carey.E.W. & Bavister, B.D. (1987) J. In Vitro Fert. Embryo Transf.1: 162-167.

86 87

PARTITIONING OF GLUCOSE CARBON IN POST COMPACTION OVINEEMBRYOS

J G Thompson and A.C.S. Bell.

AgResearch, Ruakura Agricultural CentrePB 3123, Hamilton, New Zealand

To further characterise glucose carbon partitioning in the acid-soluble (AS)fraction, lOOuL samples were fractionated by HPLC (0.6 mllmin) into 5 fractions(ASl-5). Fraction AS2 corresponded to the retention time for glucose and AS4corresponded to lactate. Proportion (%) of radioactivity in each fraction follows:

Fracuon Not AS 1 AS2 AS3 AS4 AS5 (pNe!24h) of

Retention (min) recov'd 0-9 9-10.5 10.5-13 13-14.5 14.5-20 Pooled

Glutamme 16±6 15g 44±12 20g 5±1 2±1 89±13+ 15+1 17±4 36±9 23±4 8±2 2±1 75±14

Results demonstrate that neghgible glycogen productIon occurs m postcompaction sheep embryos, thus extending the earlier observation in 8- to 16-cellembryos (1). The presence of glutamine tended to improve morphologicaldevelopment over the 24 h culture period, but had no significant effect on glucoseincorporation. Finally, most glucose carbon was associated with the acid-solublenon glycogen fraction. HPLC revealed that this was predominantly glucose(approximately 40%), in addition to other metabolic intermediates. A smaller thanexpected portion was isolated in the lactate fraction. This does not, however, reflecttotal lactate production. Lactate would perfuse into the incubation medium as wellas serve as an oxidisable energy substrate.(1) Pike, I.L. & Wales, R.G. (1979) Proc. Aust. Soc. BioI. 11,9.(2) Ozias, C.B. & Stern, S. (1973) BioI. Reprod. 8,467-472.(3) Edirisinghe, W.R., Wales, R.G. & Pike,I.L. (1984) Aust. 1. BioI. Sci. 37, 137146.

2.3±0.4 21.l±3.61.6±0.1 14.2±2.7

Acid insolubleDesmogly. Non gly.

<1 57.5±3.5<1 57.7±6.2

Total

81.3±5.673.8±7.3

55+

Glutamine Reps

Sheep and cattle embryos appear to metabolise glucose in a different mannerto mouse embryos. Indeed, one study concluded that little glycogen productionoccurs in 8- to l6-cell sheep embryos (1) in contrast to the mouse, where glycogenproduction constitutes a significant proportion of glucose metabolism (2). Thecurrent study examined glucose partitioning in post-compaction sheep embryos.

Day 5 sheep embryos (late morulae and early blastocysts) were surgicallyrecovered from superovulated ewes. Groups of embryos (4-7) were incubated for 24hat 390 C under humidified 5% C02, 7% 02, 88% N2 in 50 uL microdrops of asubstrate-free SOF Ilfedium (12 mglml BSA, m-SOF) containing 0.9 mM coldglucose, 0.1 mM [U- 4C]-glucose (S.A. 10.6 GBq/mmol) and with or without 1.0mM glutamine. Following incubation, embryos were washed in m-SOF (0.9 mMcold glucose) and stored at -700 C until fractionation. Embryos were fractionatedinto acid-soluble (glycogen/non glycogen) and acid-insoluble (desmoglycogen/nonglycogen) fractions as described in (3). Partitioning of glucose carbon is presentedbelow: *

(pg atomslembryo/24h)Acid soluble

Glycogen Non gly.

SURVIVAL AFTER TRANSFER OF FRESH OR FROZEN BOVINE EMBRYOS PRODUCEDIN VITRO IN ACELL· AND SERUM·FREE MEDIUM

P.A. Pugh, J.G. Thompson, L.T. McGowan, W.H. McMillan and H.R.Tervit.

AgResearch Ruakura,Private Bag 3123, Hamitton, New Zealand.

Abattoir derived. oocytes were matured and fertilised using procedures similiar to thosepreviously described (1). Presumptive zygotes, free of surrounding cells, were then culturedin SOF/ANBSA (4) with media changes at 48h intervals. After 5·7 days cutture, morulae andblastocysts were removed and either transferred non-surgically to recipient heifers(2/recipient) or frozen in 1.5M ethylene glycol (EGJ at 0.50C/min to -350C before plunginginto liquid nitrogen. Embryos were thawed in a 30 C water bath and EG removed by directtransfer to enriched PBS. Following 30·60 min at room temperature embryos that werereexpanding were then transferred (2/recipient). Pregnancy was determined at slaughterbetween days 17 and 60.

Ot 1036 oocytes matured, 717 (69%) cleaved at least once. Overall development tomorula/blastocyst was 48% (500/1036) and 381 (76%) of these were judged excellent quality.These embryos were lighter in appearance and cavitated later than the embryos that werecuttured previously in our SOF +human serum system (1,2).Overall, of heifers receiving fresh embryos 20/28 (71%) were pregnant compared to 8/26(31%) which received frozen embryos (p<0.01). Embryo survival rates at day 17 were notsignificantly different between fresh and frozen embryos (11/18;61 % v 7/18;39%respectively). There was no significant embryonic loss up to day 60 for fresh embryos with9/9 (100%) recipients pregnant and 11/18 (61 %) embryos surviving at slaughter on day 60.However, for frozen embryos, there was a significant (p<0.05) reduction in embryo survivalsoon after day 17 so that 2/8 (25%) recipients were pregnant and 2/16 (13%) embryossurvived to slaughter on day 60. '

These resutts demonstrate that high survival rates can be achieved after transfer of fresh invitro produced bovine embryos and also reports, to our knowledge for the first time,pregnancies from frozen bovine embryos produced in vitro in the absence of somatic cells orserum. The increased embryo mortality between day 17 and day 60 for frozen embryosindicates that failure to hatch from the zona pellucida is not the primary cause of embryonicloss after freezing. Further research to optimise cutture conditions and freezing protocols isrequired before the transfer of frozen in vitro produced embryos would produce commerciallyacceptable resutts.

1.Fukui,Y. et al.(1991) J.Reprod. Fert.j.2:125-1312.Tervit,H.R. et al.(1991) Proc. A.S.R.B. ~:1223.McGowan,L.T. et al.(1993) Proc. A.S.R.B.these proceedings.4.Gardner,D.K. et al.(1992) Proc. A.S.R.B. 24:90

In vitro matured and fertilised bovine oocytes can develop to blastocysts when cuttured in asomatic cell· free medium (1). However the survival of these blastocysts after fresh transfer(2) or freezing (3) is low. Augmentation of SOF medium with amino acids (M) and BSAtogether with replacement of the medium at 48 hr intervals increases blastocyst cell numberand quality of cuttured sheep embryos (4). We investigated whether these changes improvebovine embryo development in vitro and affect survival in vivo after fresh or freeze/thawtransfer.

88 89

FERTILISING ABILITY OF SPERMATOZOA FROM RAMSSUBJECTED TO INTERMITTENT SCROTAL INSULATION AND

DEVELOPMENT OF THE RESULTANT EMBRYOS IN VITROTIME AND INCIDENCE OF FOETAL MORTALITY IN ALPACAS

(1) Ekpe, G. et al, (1992) Proc Aust.Soc Reprod.Biol. 24; 51.

6.1

>100

5.2

71 - 100

6.1

41 - 70

5.5

31 - 40

Foetal loss (%)

Days gestation

Low reproductive performance in alpacas is a major problem in SouthAmerica, with an estimated 50% of females failing to produce young eachyear. A high proportion of this loss is attributed to embryo and foetalmortality (1). Alpacas in New Zealand graze abundant green pasture overmost of the year and this may mean lower levels of embryo and foetalmortality than reported in South America. This paper presents informationon the incidence of foetal mortality in two alpaca flocks in New Zealand.

Over the years 1990-92, 46 autumn (Feb.-Apr.) and 33 spring (Oct.Dec.) mated alpaca at Flock House, and in 1992,34 autumn mated alpacasat Tara Hills Research Station were diagnosed as pregnant byultrasonography. Pregnancy in spring 1990 was diagnosed using a 3.5 mHztransabdominal probe with the first scan being at 49-113 days gestation. In thesubsequent years a 5 mHz transrectal probe was used with first scans of theautumn 1991 mated alpacas being at 51-60 days, while in spring 1991 andautumn 1992 most first scans were at 21-30 days of gestation. Once an alpacawas diagnosed as pregnant it was scanned every 10-14 days at Flock Houseand every 15-36 days at Tara Hills. The transrectal probe was used up to 80days gestation and thereafter the transabdominal probe was used until thefoetus became too large to scan.

Overall 23% of foetuses were lost between days 21-30 of gestation andparturition with the losses being 28% for autumn and 18% for spring matedalpacas at Flock House, and 23% for autumn mated alpaca at Tara Hills.The major foetal loss (6%) occurred between the scanningson days 21-30 anddays 31-40 gestation. The 6% foetal loss after day 100 of gestation indicatesthat losses in late pregnancy can occur.Table 1: Incidence of foetal mortality over gestation

Total embryo and foetal loss from the alpacas in this experiment waslikely to be greater than the 23% recorded since only a small proportion ofthe losses from fertilisation to day 30 of gestation would have been identifiedin this experiment. Foetal loss in the alpacas was much higher than the 2-6%loss in sheep after days 30-40 (2).(1) Fernandez-Baca, S.; et al. 1970. BioI. Reprod. 3: 243-251.(2) Wilkins, J.F.; et al 1984. Anim. Prod. Aust. 15 : 768

T.W. Knight1 and 1. Scote

1 AgResearch, Flock House Agricultural Centre, Private Bag 1900, Bulls, NZ.2 AgResearch, Invermay Agricultural Centre, Private Bag, Mosgiel, NZ.

65(91)***64(91)***

2.94(2.89)3.38(2.89)##

48(60)*31(71)***5w40(56)**3w

% ova fertilizedRam 47 58(58)2wRam 33 54(59)3wDevelopment scoresRam 47 2.00(2.33) 2.24(2.83)*** 2.16(2.54)*Ram 33 1.92(2.53)*** 2.30(2.79)***Rams 12+16 3.57(3.48)lw 2.99(3.34)**3wfrom previous expt.Bold figures are for the insulated rams, 36 ± 1.8 eggs/rarn/week; figures in parentheses are pooled valuesfor the two controls, for different corresponding periods as insulat;on of ram 33 began 1 week later thanram 47. 1w, 2w or 3w : no of weeks for which data are pooled. Development scores were calculated bycounting 1 for 2-4 cell embryos, 2 for 8-16 cell, 3 for morula and 4 for blastocyst. *, **, ***: P < 0.05,0.01 or o.cxn respectively less than controls. ##: P< 0.01 greater than control by 4x2 chi-square test.

Ekpe, G., Zupp, J.L., Seamark, R.F. & Setchell, B.P.Departments of Animal Sciences and Obstetrics and Gynaecology,

University of Adelaide, South Australia.

In 1992, we reported (1) that insulating ram scrota for 16h per day for 3 weeks resultedin the production of spermatozoa which were motile but less effective in fertilising eggsin vitro. In the present study, we have extended the duration but reduced the intensityof the treatment to 8h/day, and continued weekly observations through the recoveryperiod. The techniques used were the same as described previously (1). In a roommaintained at 21°C, 2 rams were kept as controls and insulating bags which raised thesubcutaneous scrotal temperature by about 20C were applied to 2 rams at 0900h andremoved at 1700 each day. As in the previous experiment, the percentage of eggsfertilised by the same number ofmotile, capacitated spermatozoa was less during theperiod of insulation, and furthermore, the effect continued with the semen from oneram for 7 weeks after the end of the 5-week period of insulation. Semen from thesecond ram deteriorated during insulation to such an extent that after of 3 weeks ofinsulation, it was no longer possible to obtain enough motile spermatozoa for the invitro studies. Insulation was therefore stopped after 4 weeks but the lack of motilespermatozoa continued for a further 5 weeks; then, the semen quality returned tonormal, but for the last 2 weeks of the experiment, spermatozoa from this ram alsofertilised a lower proportion of the eggs than controls. Development of the embryoswas assessed by c1assifiying them as 2 or 4 cell, 8 or 16 cell, morula or blastocyst;when the proportion of embryos in the 4 classes was compared, development of theembryos produced was significantly slower than control, for one ram during the 5week insulation period and for 5 weeks afterwards, and for the other during the preinsulation period (associated with appreciable numbers of abnormal sperm), and for the3 weeks of insulation before the number of motile spermatozoa fell. During the last 2weeks of the study, spermatozoa from both rams produced embryos which developednormally. When the same calculation was made on the data from the previousexperiment, a small but significant retardation of development could again bedemonstrated, and development was significantly different between the two studies,which were conducted in April and August-November respectively.

Pre-insulation Scrotum Post-insulation Post-insulationinsulated 8h/24. weeks 1-5 weeks 6-7

90

CALCIUM, PHOSPHATE AND CITRATE IN SOWS' MILKDURING LACfOOENESIS II

Jacqueline C Kent and Peter E. Hartmann

Department of Biochemistry, The University of Western Australia, Nedlands, WA 6009

Holt & Muir (1) proposed a physicochemical model to explain some of the the ioninteractions in milk and predicted a direct relationship between the diffusible fractionsof calcium and citrate in milk. The model has been corroborated in the milk of cowscollected in different seasons (1), in the milk of eight different species (2) and in humanmilk during lactogenesis II (3). Citrate has been called the 'harbinger of lactogenesis'(4), increasing in concentration during lactogenesis II in all species previously studied.However, Holmes & Hartmann (5) have shown that the concentration of citrate in themilk of sows decreases during lactogenesis II. Therefore, we examined therelationship between diffusible calcium (Cll(!) and diffusible citrate (Ci1:d) in sow milkduring the peripartum period to further test the model (1).

Milk and blood samples were collected from sows 4h to 1min before farrowing, duringfarrowing, and 1-2h, 4-6h, 1d, 2d, 3d, 4d, 7d and 9d after farrowing. Theconcentration of lactose was measured in the blood plasma. Ionized calcium (Ca2+)and total calcium (Cator) were measured in whole milk. The diffusible fraction of milkwas prepared by ultrafiltration, and the concentrations of lactose, Cad, Citd, anddiffusible phosphate (Pid) were measured.

The concentration of lactose in the blood plasma began to increase during farrowing,and reached a peak 4-6h after farrowing, confirming the occurrence of lactogenesis IIduring or just before farrowing. Catot increased from 20mM before farrowing to42mM by day 2 after farrowing and remained above 40mM for the remainder of thestudy period. Cact was 9mM before farrowing, increasing to 12mM on day 2 afterfarrowing, and decreasing again to 8mM on day 3. The casein concentration of sowmilk increases from the time offarrowing until2d after farrowing [calculated from (6)],and this may be responsible for binding the non-diffusible calcium. Ca2+ showed asimilar pattern of change, starting at 4mM before farrowing, increasing to 7mM on day2 after farrowing, and decreasing again to 5mM on day 3. Citct decreased from 8mMbefore farrowing to 6mM on 1-2h after farrowing, then decreased further to 4mM onday 3. Pid decreased from 11mM before farrowing to 6mM by day 2 after farrowing.Cact was significantly related to Ci1:d (r2 = 0.585, P = 0.0001), and this relationshipimproved slightly when Cact was corrected for Ca2+ (r2 =0.621, p =0.0001).

The general relationship between Cact and Ci1:d in established lactation also appliesduring the rapid increases in Cad and Ci1:d in human milk during lactogenesis II. Wehave now shown that the predicted relationship still applies during lactogenesis II in thesow which is unique in showing a decrease in Ci1:d concentration concurrent with arapid increase in Catot.

(1) Holt, C. & Muir, D.D. (1979) J Dairy Res 46: 433-439(2) Holt, C. & Jenness, R (1984) Comp Biochem Physiol 77A: 275-282(3) Kent, I.C., Arthur, P.G., Retallack, RW. & Hartmann, P.E. (1992) J Dairy

Res 59: 161-167(4) Peaker, M. & Linzell, J.L. (1975) Nature 253: 464(5) Holmes, M.H. & Hartmann, P.E. (1993) J Dairy Res (In press)(6) Klobasa, F., Werhahn, E. & Butler (1987) J Animal Science 64: 1458-1466

91

FOLLICULAR DYNAMICS IN COWS FROM A HERD WITH AHISTORY OF TWIN-CALVING

W.H. McMillan, D RH Hall, A.P. Oakley and C.A. Morris


Ultrasonic monitoring studies of ovarian follicle turnover in cows have led to thedevelopment of models highlighting follicular dominance as a major mechanism bywhich cows remain uni-ovular. Although twin-calving is relatively rare in cattle (1-3%in most breeds) studies have confirmed that repeated twin-calving invariably arises as aresult of twin ovulations. Furthermore, the incidence of twin ovulation and twincalving can be increased if sufficient selection pressure is applied. The Ruakura twincalving selection herd was established in 1982 to study genetic and physiologicalfactors associated with twinning in cattle (1). In the present study cows from theRuakura twin-calving selection herd and an unrelated control herd were used tocompare ovarian and oestrous activity using ultrasound scanning. In particular wewere interested in testing the hypothesis that twin-ovulations were the result of tworather than one dominant follicle developing during the latter part of the oestrous cycle,probably as a result of a diminishing of the dominance effect observed in singleovulating cows. Oestrus was synchronised in 14 cows from the twin-calving selectionherd (Selected) and 16 cows from an unrelated herd with no history of twin ovulationsor twin calving (Control). Beginning on the day of oestrus, ovaries were examinedonce a day using a 7.5 Mhz ultrasonic probe and the growth of each follicle greater than4 mm was monitored. Ovarian monitoring ceased once cows had shown a secondoestrus and ovulated. Data from 3 Control cows were excluded for being incomplete.Table 1 Follicle data from Selected and Control cows

No. Max. Follicle Mean Multiple Multiple CycleCows Follicle Waves Daily Follicle Ovulations Length

Diameter per Total Waves % daysmm Cycle Follicles %

Selected 14 14.4 2.7 15.1 71 43 22.8Control 13 13.4 2.9 15.2 23 0 22.2

P<0.05 P<0.05

There was no difference in the maximum diameter of the largest follicle during thecycle, the number of dominant follicle waves per cycle or the mean total number offollicles present per day (Table 1). A major difference favouring the Selected cows wasthe number of follicle waves with more than one dominant follicle and the incidence ofmultiple ovulations (Table 1). In all instances, twin ovulations were immediatelypreceded by the development of twin dominant follicles. Cycle length ranged from 1928 days in Selected cows and 20-26 days in Control cows. These data support thehypothesis that twin ovulations in cows are associated with a preceding development oftwin dominant follicles. A pair of dominant follicles is present for several days and canoccur during the 1st, 2nd or 3rd follicle wave. It is thus likely that dominance isattenuated in cows with a history of twin-calving. As a consequence, more folliclescontinue to grow and the incidence of multiple follicles/wave and therefore multipleovulations is increased in cows with a history of twin-calving. Furthermore, the twinfollicles present during the ovulatory follicle wave are more likely to twin-ovulate in theSelected group compared with Controls. Thus, a higher incidence of twin-folliclewaves as well as a higher propensity to ovulate appear to have contributed to the higherincidence of twin-ovulations and therefore twin-calving in the Selected herd.(1) Morris, C.A., Day, A.M., Amyes, N.C. & Hurford, A.P. (1992). NZ J Agric.Res. 35: 379-391.

92 93

IFigure 21Ovulator Cl

20 Non-ovulator_

SE~E1S.....cy.

~A10mill+ltI-U:~ 5

::::Ell.

N",33

NonOvulator

IFigure 11N",32 N=19

16

]iE14~~122''':'10~~ 8+I III 6"""'1ic= 4

~;2 2

o r----IL.,--._--,Single Twin

Ovulator Ovulator

in ewes responding to fIrst teasing as single ovulators, twin ovulators or non ovulators(Figure 1). Restricting the analysis to only ewes with follicles larger than 3 mm or 4.5mm confIrmed this result Re-teasing increased the incidence of ewes ovulatingcompared with re-isolation (75 vs 31 %, P<0.05), while more re-isolated ewes ovulatedcompared with contemporary continuously isolated ewes (2/45) (P<0.05). Totalfollicle numbers prior to re-teasing or re-isolation were lower in re-isolated ewes whichovulated (10.0 vs 16.6, P<O.Ol), but no such effect was observed in re-teased ewes(Figure 2). These data demonstrate that: 1. follicle numbers prior to teasing do notappear to influence the ability of individual ewes to ovulate. Instead, follicularsensitivity to gonadotrophin and/or gonadotrophin secretion patterns may be moreimportant. 2. immediately re-teased ewes will respond to novel rams. This fIndingquestions the need for long term isolation and highlights the urgency for a descriptionof 'physiological' rather than physical isolation. 3. ovarian responses to teasing can be'carried over' for several days since more re-isolated ewes than continuously isolatedewes ovulated. Current models used to describe ram-induced ovulations need to bemodifIed to explain these fmdings.(1). McMillan, W.H. (1987). Proc. N.Z. Soc. Anim. Prod. 47: 135-137.

FOLLICLE AND OVULATORY PATTERNS IN ROMNEY EWESTEASED BY RAMS DURING LATE ANOESTRUS

W.E. McMillan, D.R.H. Hall and A.P. Oakley


In British sheep breeds such as the Romney, the proportion of ewes ovulating inresponse to teasing increases linearly over the mid-January to mid-February period inNew Zealand (1). We know very little about why some ewes respond and others donot, but between-ewe differences could arise from differences anywhere along thehypothalamus-pituitary-ovary axis. The aim of this study was to describe some ovarianfactors that may explain some of the differences. Eighty four Romney ewes,continuously isolated from rams for several weeks, were examined using a laparoscopyto count surface ovarian follicle numbers (>1-2 mm diameter) during a 3 week periodbeginning in mid-January. The ewes were then continuously teased with rams and fourdays later ovaries were again examined. Non responsive (ie anovular) ewes were theneither re-teased with novel rams or re-isolated for 4 days and their ovaries re-examined.Total follicle numbers per ewe were 13.9 ± 0.65 (mean ± SEM, range 3-36) prior tofIrst teasing. About 17% of these follicles were >3 mm diameter, although 77% ofewes had at least one such follicle. Twenty fIve per cent of ewes had at least one large(> 4.5 mm) follicle. Overall, 61 % of ewes ovulated to fIrst teasing and 37% of thesewere twin ovulators. Total follicle numbers >1-2 mm prior to fIrst teasing were similar

m cows WIth SIlent ovulatIons

No. Interval to 'Silent' Interval to Interval IntervalCows First Ovulations 1st 1st to 2nd to First

Dominant (% of Ovulation Ovulation OestrusFollicle cows) (days) (days)1 (days)(days)

Single-suckled 23 10.5 64 38 10 43Twin-suckled 12 13.3 27 46 12 45

P<0.05 P<O.1 NS NS NS1 . , . ,

(1) Murphy, M.G., Boland, M.P. & Roche, J.F. (1990). J. Repr. Fert. 90: 523-533.

The fIrst dominant follicle took longer to appear in twin- compared with single-suckledcows (Table 1). There was no relationship (r2 <5%) between the interval to theappearance of the fIrst dominant follicle and either the interval to fIrst ovulation or theinterval to fIrst oestrus. There was a tendency for fewer twin-suckled cows to show a'silent' ovulation before fIrst oestrus, and for the interval from calving to fIrst ovulationto be longer (Table 1). The interval from the fIrst to the second ovulation as well as~ro~ calving to fIrst oestrus was similar in both groups of cows (Table 1). Our results~ smgle-suckled cows are very similar to those achieved in Irish studies, althoughmtervals to oestrus were about 10 days shorter in the present study (1). We could nottest our hypothesis because twin-suckling did not extend the intervals to ovulation oroestrus. The lack of a relationship between the timing of the appearance of the fIrstdominant follicle and the interval to either fIrst oestrus or ovulation indicates that themajor factors controlling the timing of these events are different. These results indicatethat twin-suckling need not necessarily be associated with extended intervals fromcalving to fIrst oestrus.

FOLLICULAR DYNAMICS, OVULATION AND OESTRUS IN BEEFCOWS SUCKLING EITHER ONE OR TWO CALVES

W.H. McMillan, A.P. Oakley and D.R.H. Hall

AgResearch Ruakura, Private bag 3123, Hamilton, New Zealand

Recent ultrasound scanning studies on post pactum activity in cows have focused ondominant follicle turnover as being the key ovarian event leading to the timing of fIrstoestrus and ovulation. In single-suckled beef cows under Irish conditions the extendedperiod of post-pactum anoestrus, when compared with dairy cows, is due to the lack ofovulation of dominant follicles rather than a failure of dominant follicles to develop (1).Twin-suckling can further increase the interval to fIrst oestrous activity, but dominantfollicle activity has not been monitored in twin-suckled cows. The aims of the currentstudy were .to describe ovarian and oestrous activity in cows rearing either one or twocalves. In particular, we wanted to test the hypothesis that the extended interval to fIrstoestrus in twin-suckled cows was a consequence of continued dominant follicleturnover rather than the absence of dominant follicle development The ovaries oftwenty three single-suckled Hereford x Friesian cows and 12 contemporary twin-calvedand twin-suckled cows were scanned daily beginning about 1 week after calving untilfIrst oestrus and follicle development described. All cows initiated a dominant folliclebut 1 cow in each group did not ovulate or show oestrus (day 77 and 120 post-partum).

Table 1

EFFECTS OF OESTRADIOL ON SYNCHRONY PATTERNS IN CATTLETREATED WITH AN INTRAVAGINAL PROGESTERONE DEVICE

K.L. Macmillan, V.K. Taufa and C.R. Burke

Dairying Research Corporation, Private Bag 3123, Hamilton, New Zealand

An experiment was designed to test the hypothesis that the concurrentadministration of oestradiol benzoate (ODB) at the initiation of a treatmentwith progesterone would alter the subsequent synchrony pattern in lactatingdairy cows. Cycling animals in 12 seasonal dairy herds each had a CIDR~evice (~azibreed C!DR; Inter Ag, Hamilton) containing 1.9 g progesteroneInserted Into t~~ vagina for 7 or 12 da~s. Half of the devices had a gelatincapsule containing 10 mg ODB plaeed In a grooved surface before insertion.The dates of insertion were arranged so that similar numbers of cows within aherd had devices removed during the morning milking on each of Days 20 to24 (Oestrus = Day 0). The herd owners recorded oestrous and inseminationdat~s for the trial cows. Those not detected in oestrus within 14 days ofdevIce removal were presented for a veterinary examination.

The analysed results were from 1028 of 1091 cows (94.2%) after excluding2.4% which lost their device, 0.5% which were anoestrus and 2.8% whichovulated without being detected in oestrus. There were 41 to 65 cows ineach of the 20 sub-cells (7 vs 12 days x .:t0DB x 5 removal days).

The addition of ODB in a 7-day CIDR treatment reduced the percentage ofcows detected in oestrus by 48 h after device removal (61 % vs 29%'p<0.001) with compensatory increases by 72 h (25% vs 48%; p<0.01) and 96h (5% vs 14%; p<0.05). These effects were most pronounced when a devicewas removed on Days 20 or 21 (day x treatment; p<0.01).

A contrasting effect occurred with a treatment period of 12 days. Theinclusion of ODB increased the 48 h synchrony rate (65% vs 75%' p=0.02)with these increases varying from 4% (Day 24) to 14% (Day 20;' p>0.10).There were 96.8% of these cows detected in oestrus by 96 h after deviceremoval if they were treated with ODB compared to 92.3% of those which didnot receive ODB (p=0.05). .

The synchrony patterns were similar for animals treated with a CIDR devicefor 7 or 12 days without ODB. The maximum synchrony rate at 48 h was 72%with device removal on Day 23. This Day coincided with the equivalentmaximums for cows also treated with ODB, but it was only 49% whenassociated with a 7-day CIDR compared to 84% for 12 days. Minimumsynchrony rates at 48 h were 16% and 17% if the use of ODB occurred with 7days of treatment and device removal on Days 20 or 21 (p<0.01).

Further studies are being completed to test a consequential hypothesis thatoestradiol from the capsule in combination with progesterone from the CIDRdevice is altering follicle wave patterns in late dioestrus as well as the time ofluteolysis.

95

OESTROUS SYNCHRONY, OVULATION RATE AND EMBRYOSURVIVAL IN RECIPIENT EWES COMMENCING SYNCHRONY

TREATMENT IN EITHER MID OR LATE CYCLE

W.H. McMillan, D.R.H. Hall and P.H. Evans


Oestrous synchrony treatment duration approximating the luteal phase can lead tonormal or extended cycle lengths depending on the stage of the oestrous cycle at thecommencement of treatment Some of the effects of extended cycle length on donorewes have been reported (1) but little is known of the effects on ewes treated forsynchrony only. The aim of this study was to describe oestrous and ovarian activity aswell as embryo survival in ewes treated for synchrony only. Two hundred and nineRomney ewes were each treated with a CIDR® device for 12 days. Beginning either inmid cycle (d 7-9) or late cycle (d 13-15), a second CIDR® was inserted for 12 days.Interval to onset of oestrus was recorded twice daily for 3 days. Ovulation rate wasrecorded in about half of the ewes and 2 fresh embryos were transferred to each.Correlations between interval from CIDR® removal to onset of oestrus were calculated.Table 1

Treatment No Ewes % Oestrous Interval to Correlation. Ovulation %over 3 Oestrus between Rate Embryodaysl h 1 Intervals Survival

Mid Cycle 99 97 36.5 0.36 1.65 71Late Cycle 110 100 32.9 0.20 1.75 73

NS P<0.05 NS NS NSISecond synchromsatIOn

No ewes were in oestrus within 8 h of CIDR® removal and 96% of cyclic ewes hadcommenced oestrus by 48 h after CIDR® removaL Other results show that: 1.oestrus can be synchronised in virtually all cyclic ewes within 48 h of CIDR® removal,2. ewes treated from late in the cycle have a 3-4 h shorter interval to oestrus (P<0.05,Table 1), mainly because more ewes are in oestrus between 8 and 24 h (30 vs 19%,NS), 3. within ewes, the interval to oestrus is inconsistent from treatment to treatment(Table 1), 4. neither ovulation rate nor embryo survival is affected by synchronytreatment (Table 1). Since none of the ewes were expected to have functional corporalutea (CL) at CIDR® removal, differences in onset pattern may reflect differences in:clearance of exogenous progesterone (unlikely); follicle parameters (turnover and/orfollicle sensitivity to gonadotrophin); or higher neural centres controlling oestrousbehaviour. Whatever the explanation, the difference (3-4 h) is unlikely to be ofpractical significance in AI or recipient synchrony programs. The low correlationsreported indicate that interval to onset in individual ewes cannot be reliably predictedfrom cycle to cycle. Extended periods of progesterone treatment (19-27 d) do notappear to impair corpus luteum function or the capacity of the uterus to supportpregnancies following embryo transfer. However, additional studies are required todetermine the implications of extended progesterone exposure on sperm transport,ovum competence for fertilisation and subsequent embryo development. We may findthat synchrony systems to maximise reproductive competence in recipients differ fromthose that maximise reproductive competence when artificial insemination or naturalmating is planned.

(1) McMillan et al., 1992. Proc. 12th ICAR, The Hague, pp 241-243.

.96

EFFECT OF TREATMENT OF NON·CYCLlNG, LACTATING DAIRY COWSWITH PROGESTERONE AND/OR OESTRADIOL

S. McDougall1, K.L. Macmillan1, and N.B.Williamson2

10airying Research Coiporation, Private Bag 3123, Hamilton, 20epartment of VeterinaryClinical Sciences, Massey University, Private Bag, Palmerston North, New Zealand

It was hypothesised that (a) progesterone (P4) pre-treatment would increasethe proportion of ovulations accompanied by oestrus and (b) oestradiol-17j3(E2) treatment following P4 pre-treatment would increase the number ofovulations in non-cycling post-partum mixed-age dairy cows. Thesehypotheses were tested with a 2 x 2 design. One hundred and sixty-nine cowswere milk ~ampled 3 times weekly from calving.for subsequent analysis of P4concentration by RIA (Coat-a-count, DPC, Calif, USA). At weekly intervalsthose cows that were 14-20 days postpartum were stratified on the basis ofage (2,3,>3 years) and palpated per rectum for the presence of a CL. Half ofthe cows then received either an intravaginal device containing 1.9 g of P4(CIDR-B, InterAg, Hamilton, NZ) and the others a blank device (Blank). Thedevice was removed after 5 days, and 2 days later (Day 0). each cow. wastreated with either 600 J.lg of E2 or 3 ml of 0.9% sodium chloride (saline) Lm..Cows were observed twice daily for oestrus. Cows were removed fromanalysis if they ~ad reproductive tract pathology upon palpation (n=11), werelate calvers or Induced to calve (29), or had ovulated (ie had a milk P4concentration >2.5 ng/ml) or were detected in oestrus on, or before Day 0(31). Oestrus was defined as having occurred if a cow was detected inoestrus within 14 days and ovulation if the P4 was >2.5 ng/ml within 19 days ofthe Day O. The response to treatment was coded as: no ovulation or oestrus(No resp), ovulation but no oestrus (Ovuln only), ovulation and oestrus(Oestrus and Ovuln) and oestrus without ovulation (Oestrus only).There were significant differences among treatments in response (x2=19.5,p<0.01; Table 1).

Table 1. Response to treatment with P4 and/or E2 of non-cycling cows.

Treatment No resp Ovuln only Oestrus+Ovuln Oestrus only Totaln (%) n (%) n (%) n . (%) n

Blank+saline 8 (36.4) 7 (31.8) 5 (22.7) 2 (9.1) 22Blank+E2 7 (29.2) 9 (37.5) 5 (20.8) 3 (12.5) 24CIOR+saline 9 (32.1) 4 (14.3) 15 (53.6) 0 (0.0) 28CIDR+E2 3 (13.6) 3 (13.6) 15 (68.2) 1 (4.6) 22

P4 pre-treatment increased the number of cows expressing behaviouraloestrus at ovulation (81 % vs 38% of ovulating cows had concurrentbehavioural oestrus in cows pre-treated with P4 or Blank; respectively). E2treatment did not alter the number of animals ovulating, eXhibiting behaviouraloestrus or both compared to saline treated cows.The ~ypothesis th~t P4 increases the prop?rtion of cow~ expressingbehaVIoural oestrus IS accepted, but E2 does not Increase ovulation rate eitheralone or following P4 pre-treatment.

97

THE EFFECTS OF EXOGENOUS PROGESTERONE ON OVULATION AND PREGNANCY INALPACAS.

I.C. Scoll and DJ. McLeodAgResearch, Invermuy Agricultural Centre, Private Bag, MosgieI.

As alpacas are induccd ovulators the stage of development of dominant follicles at the time of matingcan vary widely. However, in order for ovulation to occur a maturc follicle (7-12 mm) must bepresent at the time of copulation (1). In the absence of mating, follicles grow and regress in wavesWiUl a periodicity of about 12 days (2), and therefore on anyone day only about 40% of females willhave a mature follicle present. TIle reproductive cycles of conventional farm animals (eg sheep, cows,goats) can be controlled by administration of exogenous progesterone, the removal of which resultsin Immediate and synchronous follIcle growUl. Depressed follIcle grOWUl in Ule presence of a corpusluteum (CL) has been reported in alpacas (1). It was Ulerefore proposed to test if exogenousprogestcronc would control folliclc dcvelopmcnt in alpacas, thus ensuring Ihat mating coincided withUle presence of a mature follicle.

Twenty-one mixed age,lactating alpacas had eiUler an cmpty (Group C, 11=10) or progesteroneimpregnated (Group P, n=ll) controlled Internal drug releaslng-device (CIDR) inserted in Ule anteriorvagina for a period of 10 days. Four days after CIDR removal all females were mated to one of fourentire males. Each female was blood sampled, lested for receptivity to a male (2) and had it's ovariesobserved by ultrasound on Ule day of CIDR insertion, and every second day thereafter for twenty-eightdays.

TABLE l. Effect of 10 days exogenous progesterone treatment on receptivity, follicle size, ovulationand pregnancy.

Trealrnent Group C (n= 10) Group P (n=Il)

Days from CIDR insertion 0 2 10 14 0 2 10 14NumBer receptive 8 10 9 7 8 2 4 8Mean follicle si7.e 6.2 6.5 7.1 7.8 7.7 6.9 6.0 6.8(±SE) (0.8) (0.8) (0.8) (0.9) (0.9) (0.8) (0.7) (0.8)Number ovulated 8 10

Fewer Group P animals were receptive over the IO days of CIDR inserlion. Mean si7.e of Uledominant follicle decreased in Group P animals following CIDR ;l1sertion, hut increased in Group Canimals. Follicle size increased in both groups after CIDR removal, but two more Group P UlanGroup C alpacas ovulated following a single copulation four days later. Regression of Ule CLsubsequently formed was noled In 3/8 Group C and 1/10 Group P alpacas wiUlin twelve days ofmating. Forty-six days after mating 4 Group C and 5 Group P alpacas were pregnant, indicating lossof conceptus in 1/5 Group C and 4/9 Group P alpacas.

Treatment wilh progesterone CIDRs did not adequately synchronise follicle growth, and UIerewas no advantage in Ihe number of alpacas ovulating following a single mating four days after deviceremoval and subsequently becoming pregnant.

l. Bravo, P.W., Stabenfeldt G.H.• Lasley, D.L. and Fowler, M.E. (1991). Diol. Reprod. 45: 5532. Bravo P.W. and Sumac J. (1989). An. Reprod. ScLli: 271

98

GnRH - INDUCED ACCESSORY CORPORA LUTEA IN RED DEER AND SHEEP

M. W. Fisher. L. M. Meikle, B. J. McLeod and G. H. ShackeU

AgResearch, Invermay Agricultural Centre, Private Bag 50034, Mosgiel, NEW ZEALAND

Red deer hinds, unlike sheep. often have an accessory corpus luteum during pregnancy,although their origin and function are unknown. They may represent incompletely regressed corporalutea formed during an oestrous cycle prior to conception, or be formed following ovulation duringpregnancy. The aims of this experiment were to determine:(1) whether accessory corpora Iutea could be induced with a single bolus of GnRH at two stages

during the luteal phase of the oestrous cycle;(2) whether accessory corpora lutea influence the timing of luteolysis of the primary corpus luteum

and(3) the fate of accessory corpora lutea following luteolysis of the primary corpus luteum.

Mature hinds (n = 18) and ewes (n = 18) were synchronised with exogenous progesterone(CIDR-G, AHI Plastic Moulding Co.) and then treated with either saline or GnRH (Fertagyl, Intervet)at the rate of 250~g (ewes) or 500~g (hinds) 11 or 14 days after progesterone withdrawal. Ovarieswere examined laparoscopically under sedation (ewes) or general anaesthesia (hinds) 48 hours priorto saline or GnRH treatment, and again 72 hours and 18 (day 11 treated animals) or 15 (day 14 treatedanimals) days later. Blood sampling was undertaken to monitor plasma LH after GuRH (2 h for 6 h)and progesterone (daily).

Plasma LH concentrations reached a peak 2 hours after GnRH treatment: mean 9.86:1: 2.10 (sd)ngimi in GuRH-treated hinds compared with 0.04 :I: 0.02 ngimi in untreated hinds. Ewes averaged57.41 ± 21.97 ngimi 2 h after GnRH compared with 0.50 ± .45 ngimi without treatment.

Accessory corpora Iutea, absent in all saline-treated animals, were observed in GnRH treatedanimals as follows:

NOTES

SpeciesProportion of animals with induced corpora Iutea

day 11 day 14

Deer

Sheep

ControlGnRH

ControlGnRH

0/35/5

0/34/6

0/31/6

0/34/6

One hind had not ovulated prior to treatment and was removed from the experiment. Theremaining hinds all had a single corpus Iuteum and in 6 animals GuRH treatment induced a furthereasily identifiable single accessory corpus Iuteum. The ewes had 2 or 3 corpora present at the timeof the flISt laparoscopy, and GnRH induced the formation of a further 1 or 2 accessory corpora Iuteain 8 animals. All induced or accessory corpora lutea appeared macroscopically normal, but wereyounger-looking, and smaller than the primary corpus Iuteum.

Plasma progesterone profiles indicated that luteolysis had occurred at the same time in controland GnRH-treated animals in each species (hinds: 20.9 ± 1.30 d and ewes: 18.4 ± 1.04 dafter CIDRwithdrawal, respectively.

At laparoscopy during the subsequent luteal phase (18 or 15 days after GnRH) 3/6 hinds but0/8 ewes that had previously had accessory corpora Iutea had an abnormal or pale and partiallyregressed luteal structure in addition to a new, apparently normal primary corpus luteum.

These results demonstrate that accessory corpora Iutea can be induced in both hinds and ewesby a single bolus of GuRH during the luteal phase. Furthermore, their formation does not appear toaffect the timing of regression of the primary corpora Iutea. However, in the hinds at least, theaccessory corpora lutea may not completely regress at this time but they possibly persist into the lutealphase of the next oestrous cycle.

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