PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION

Evaluation of glycerol removal techniques, cryoprotectants, and inseminationmethods for cryopreserving rooster sperm with implications

of regeneration of breed or line or both' 2,3

P. H. P ll rdv , * V. Sung. F. C. Silvericles.t and H. D. Blackburn—"I SDA-Ayrzcu1tuial Research Service-National Center for Genetic Resources Preservation, National Animal

C iinplasm Program, 1111 S. Mason St., Port Collins. CO 80521-4500; and tAgriculture and Agri-Food Canada,Aqassiz Research Centre, P0 Box 1000-6947, # 7 Highway, Agassiz, British Columbia, Canada, VOM lAO

ABSTRACT A series of experiments was designed toevaluate the quality of cryopreserved rooster sperm andits fertility so that programs needing to batik germ-piasni and recreate animals can do so utilizing a minimalmnount of cryopreserved semen. In experimentexperiment 1, roost-er semen from the National Animal Germplasm Pro-grain genebank was thawed and glycerol was removedusing a discontinuous Accudenz column or by stepwisedilution. The postthaw sperm notilities, plasma mem-brane integrity, and concentration were determinedbefore and after deglycerolization. Line differences inpostthaw sperm concentration arid progressive motil-ity were observed before deglycerolization (P < 0.05).After glycerol removal, the sperm that was centrifugedthrough Accudenz had greater total motility (37 vs.33% sperm; P < 0.05), but use of the stepwise dilutionmethod recovered more sperm per milliliter (320.4 x106) compared with the Accudenz method (239.2 x 106sperm; P < 0.05; range across 6 lines of 165.7 to 581.0X 106 sperm/mnL). In experiment 2, rooster semen wascryopreserved using Lake's diluent containing either di-methyl acetaniide (DMA) or glycerol as the cryopro-tectants. Postthaw analysis revealed that the samplescryopreserved with glycerol survived freezing better,determined by total motility (47.8 and 15.1% glycerol

and DMA samples, respectively; P < 0.05) amid annexinV analyses (1.6 and 11.3% membrane-damaged spermfor glycerol arid DMA samples. respectively; P < 0.05).Differences in sperm motilities (total and progressivemotility) and velocities (path velocity, straight-line ve-locity, curvilinear velocity) were observed between the2 cryoprotectarit treatments once the glycerol had beenremoved from those samples cryopreserved with glycer-ol, of which the glycerol samples had significantly moremotile sperm and higher velocities (P < 0.05). The fer-tility of the samples frozen using the 2 cryoprotectantswas tested using a single insemination (intravaginal orintra.magnal) of 200 x 106 sperm arid the fertility (nurri-her of live embryos) was evaluated over 18 d. Overall,the intravaginal inseminations had lower fertility thanthe intrarnagnal insemninations (P < 0.05). Jr the in-travaginal inseminations, the sperm cryopreserved us-ing DMA resulted in lower fertility, but there were nodifferences in fertility in the intrania.gnal inseminationsdue to cryoprotectant (P > 0.05). These results indi-cate that reasonable postthaw sperm quality and fertil-ity can he derived using cryopreserved rooster semen.By utilizing this information, estimations can be madefor storing sufficient material for line or breed, or both,recreation programs.

Key words: rooster, spermatozoa, crvopreservation, deglycerolization, artificial insemination

2009 Poultry Science 88:2184-2191doi: lO.3382/ps.2008-OO402

INTRODUCTION

Cryopreservation of sperm for artificial insenuna-tiori is a valuable tool for agricultural species because

©2009 Poultry Science Associationln.Received September 19, 2008.Accepted June 18, 2009.'Agaasiz Research Centre Publication 760.2 Mention of trade name, proprietary products, or specified equip-

ment does not constitute a guarantee by the USDA and does not implyapproval to the exclusion of other products that may he suitable. TheUSDA. Agricultural Research Service, Northern Plains Area, is an

it allows rapid genetic progress to occur and conser-vation of valuable genetic resources. In the instanceof poultry. however, there are certain problems associ-ated with conserving sperm; only the male genome is

equal opportunity-affirmative action employer. All agency services areavailable without discrimination.

tm All methods used were approved bythe Animal Care Commit-tees of the USDA-Agricultural Research Service Avian Disease andOncology Laboratory and the Agassiz Research Centre and followedprinciples described by the Canadian Council of Animal Care for theresearch performed at these facilities.

'Corresponding author: phil.purdynars.usda.gov

2184

E\r\1L\I I() Ok (IIHLl H\I.1) 1()I I ' H I'kI\I

21s5

captured because roosters are homoganietic, and thefertility using a single insemination of frozen-thawedrooster sperm is frequently low (Hainnierstedt and Gra-haiti, 1992: Buss, 1993: Phillips et al.. 1996). Alterna-tives that are available in other species, such as cryo-preservation of oocytes and embryos, are not availablecurrently with poultry. Therefore, because the geneticdiversity of agricultural species is shrinking (Blackburn.2006), it is necessary to preserve the existing geneticsusing the technology currently available, namely cryo-preserved sperm. In doing so, there are many unknownfactors specific to poultry sperm cryopreservation thatare paramount to programs (genebanks, breed conser-vation organizations, etc.) concerned with line regen-eration, such as the number of inseminations possiblefrom a, single insemination straw, differences in surviv-ability of cryopreserved sperm by poultry line, and fer-tility using cryopreserved samples.

Rooster sperm typically survive cryopreser vat ion verywell, as judged by motility evaluated iinn ftiediately afterthawing, but unlike other species, the crvoprotectanttypically used (glycerol) must be removed before intra-vaginal insemination because it is contraceptive (Polge.1951; Tajima et al., 1989: Hammerstedt and Graham,1992). A common method of glycerol removal involvesstepwise dilution with a glycerol-free medium, centrifu-gation, and resuspension of the sperm. These methodsare time-consuming and decrease the fertility of thesperm due to osmotic and physical damage associatedwith processing (Harnmncrstedt and Graham. 1992).Consequently, the sample that survived cryopreserva-tion well has been damaged due to this processing andthe fertilizing potential is greatly decreased.

Recently, alternatives to traditional glycerol removalhave been investigated by Long and Kulkarni (2004),who reported a method for removing glycerol from freshrooster sperm and cryopreserved rooster sperni by cen-trifugation through a discontinuous Acciidenz column(Accurate Chemical & Scientific Corp.. Westbury, NY)without the stepwise dilution process. An alternativecryoprotectant, dimethyl acetamide (DMA), has alsobeen investigated as a means of circumventing the useof glycerol for cryopreservation of rooster sperm (Baconet al., 1986; Chalah et al., 1999: Woelders et al.. 2006;Blesbois et al., 2007). The reported benefit of usingDMA is that it is not contraceptive and therefore doesnot require removal from frozen-thawed sperm. Thus.damage to the sperm normally caused by glycerol re-moval before insemination is minimized and thereforemay result in greater fertility.

The purpose of this study was to 1) evaluate meth-ods for removing glycerol from frozen-thawed sperm,2) compare the postthaw quality of rooster sperm thatwas cryopreserved using glycerol or DMA. and 3) com-pare the fertility of frozen-thawed rooster sperm thatwas cryopreserved with either glycerol or DMA and in-seminated either intravaginally or intramnagnally. Thecryoprotectants, DMA and glycerol, were selected foranalysis because these are those most commonly used

by other genehanks such as the Netherlands (Woelderset al., 2006) and France (Bleshois et al., 2007). Thedata from these experiments will he used to determinequantities of samples required for line regeneration fornational conservation progmns.

MATERIALS AND METHODS

Experimental Design

Experiment 1 was performed to compare Accudenzor stepwise dilution methods of glycerol removal fromfrozen-thawed rooster sperm. Samples for this analysiswere from 6 different lines as described in Table 1 andwere frozen individually (not pooled) in this experi-ment. Immediately after thawing, 2 straws per roosterejaculate were analyzed separately for sperni motil-ity, concentration, and plasma membrane integrity.Aliquots from each sample were processed to removethe glycerol from the rooster sperm b y either Accu-denz centrifugatiomi or stepwise dilution and centrifuga-tion. After glycerol removal, the resulting samples wereanalyzed to determine the volume of recovered sperm,sperm concentration, motility, and plasma membraneintegrity. Semen straws were processed separately toreplicate repoptilatiomi conditions.

Experiment 2 compared the postthaw quality andfertility of rooster sperm when glycerol or DMA wereused as cryoprotectants and intramagnal and intrav-aginal insemination was used for 2 breeds. Rhode Is-laud Red and White Leghorn. The control treatmentfor each breed was a group of birds that was insemi-nated using fresh semen via intravaginal insemination.Rooster semen samples front the Rhode Island Red line50 (n = 20) and White Leghorn blue line (Table 1; n= 26) roosters were collected and semen was pooledwithin each breed. The pooled samples were split andcryopreserved in diluents containing 1 of the 2 cryopro-tectants. The postthaw quality [sperm motility, plasmamembrane integrity, and plasma membrane phiosphati-dylserme (PS) exposure] was then evaluated beforeand after glycerol removal or immediately after thawingfor the DMA samples using 4 semen straws from eachof the breed and cryoprotectant combinations. Glyc-erol removal was performed using the stepwise dilutionmethod used in experiment 1.

Semen Collection, Processing,Cryopreservation, and Thawing

The samples were collected from roosters by a.bdomiu-ina.l massage (Burrows and Quinn. 1937). After col-lection. samples that were to be frozen using glycerolas the cryoprotectant were diluted 1:1 (vol/vol) withglycerol-free Lake's diluent (Lake and Stewart, 1978),which was precooled to 5°C. The samples were thenplaced in a rack on ice and transported to the laborato-ry (<15 mi. Samples were diluted 1:2 (vol/vol; sam-ple to cryopreservation medium) with Lake's diluent

2186

PURDY ET AL.

Table 1. Description of the rooster lines acquired from the USDA Avian Disease and Oncology Laboratory (ADOL) and from theAgriculture and Agri-Food Canada Agassiz Research Centre used in this study

Description'

1) Cornell University line P: selected for tumor development after inoculation with the Jozef Marek (JM) strain of MarcUs disease virus (ii = 1).Used in experiment 1.

2) Reaseheath line C; developed by full-sibling matings; selection for large famil y size; utilized for research on avian leukosis viruses and immuneresponse traits (n = 4). Used in experiment 1.

3) ADOL line 15131; closed line; 95% inbred; selected for susceptibility to avian leukosis virus (n = 4). Used in experiment 1.4) ADOL line 0: closed line: selected for absence of endogenous avian leukosis virus proviral genesgenes and for susceptibility to avian leukosis virus

(n = 4). Used in experiment 1.5) ADOL congenic line 15.151 5; closed line: 99% inbred; selected for susceptibility to lymphoid leucosis and Marek's disease (n = 4). Used in

experiment 1.6) ADOL recombinant congenic line 6C.7W: selected for tumor resistance or susceptibilit y (n = 3). Used in exjx'riixierit 1.7) Rhode Island Red line 50 (n = 20). Used in experiment 2.8) White Leghorn blue line (ii = 26). Used in experiment 2.

'Lines 1 through 6 are described in Bacon et al. (2000), and lines 7 and 8 are descriherl in Silvcrsides et, al. (21)07).

containing glycerol (11% final concentration, 5°C) in a5°C room and loaded into 0.5-mL CBS straws (IMV,Minneapolis, MN).

Samples frozen using DMA (experiment 2) were col-lected and diluted with Lake's diluent immediatelyafter collection as described previously. Samples werethen diluted 1:2 (vol/vol) with Lake's diluent contain-ing DMA (6% final concentration: Blesbois et al., 2007)and loaded into 0.5-mL CBS straws.

Samples diluted with glycerol cryoprotectant werefrozen in an extruded polystyrene foam box containingliquid nitrogen vapor (6.4 cm above liquid nitrogen) for10 mm (10°C/mm) and plunged into the liquid nitrogenfor storage (Phillips et al., 1996). Samples containingDMA were placed on a rack, frozen in liquid nitrogenvapor (1 cm above liquid for 7 mm), and plunged intothe liquid for storage, which resulted in a 59°C/nunfreeze rate, an attempt to approximate the 50°C/mmrate reported by Blesbois et al. (2007).

Immediately before analysis, samples cryopreservedusing glycerol were thawed in a 5CC water bath for 5mm (Phillips et al., 1996). The samples frozen usingDMA were thawed in a 50°C water bath for 20 s (Bles-bois et al., 2007).

Evaluation of Sperm Motilityand Concentration

Spernr motility was determined using a computer-as-sisted sperm analyzer (Hamilton Thorne Motility Ana-lyzer; IVOS, Beverly, MA). An aliquot of each sample(5 i.tL) was diluted with 100 nL of Lake's diluent con-taining glycerol for postthaw analysis or with glycerol-free Lake's diluent for postglycerol removal analysis.A subsample of this solution (5 tL) was placed on aStandard Count Analysis Chamber (Spectrum Tech-nologies, Healdsburg, CA) and analyzed for motility.The following settings were used for computer-assistedsperni analyzer analysis: 30 frames acquired, framerate of 60 Hz, minimum contrast of 25, minimum cellsize of 4 pixels, path velocity (VAP) cutoff of 5 rim/s,progressive minimum VAP cutoff of 50 [mm/s, progres-sive velocity cutoff of 10 Rm/s, static head size of 0.27

to 4.24, magnification of 1.95, and a minimuni of 500sperm from at least 5 fields were observed for motilityanalysis.

Samples from each straw were analyzed for spermconcentration using a SpermaCue spectrophotometer(Minitube, Verona, WI) that was calibrated for roostersperm. Samples were diluted 1:1 or 1:2 (vol/vol) withLakes diluent and the optical density (sperm concen-tration) was determined.

Glycerol Removal

Solutions of Accudenz. 30 and 12%, were preparedaccording to McLean et al. (1998) and maintained at5°C. Discontinuous columns of Accudenz were preparedin 1.5-rnL flat-top microcentrifuge tubes (Fisher Sci-entific, Pittsburgh, PA) by layering I mL of 12% Ac-cudenz over 0.1 mL of 30% Accudenz. An aliquot fromeach sample (150 jiL) was placed on top of the columnand centrifuged at 300 x g for 25 minin a 5°C room.The volumes of Accudenz and the frozen-thawed semenare one-fifth of the volumes used by others (McLean etal., 1998; Long and Kulkarni, 2004: Peláez and Long,2007), but initial evaluation of decreasing the centrifu-gation speed to 250 x g (one-fifth of the speed reportedby the same authors) left a significant amount of spermin the 12% Accudcnz fraction (P. H. Purdy, unpub-lished data). Consequently, the speed was adjusted to300 x g, which resulted in the sperm being presentat the interface between the 12 and 30% layers aftercentrifugation (P. H. Purdy, unpublished data). The 12and 30% Accudenz layers were then removed accordingto McLean et al. (1998) and the resulting quantity ofsperm was measured to determine volume and spermconcentration, as described previously.

The stepwise dilution method was carried out in a5°C room using glycerol-free Lake's diluent at W. Thediluent was added every minute in the following incre-ments: 10 x 10 jtL (10 dilutions of 10 jiL per dilution),10 x 20 p1, 10 x 50 p1, and 5 x 100 p1 (Tajima et al.,1989; Phillips et al., 1996) and then centrifuged 300 xg for 25 mm. After centrifugation, the supernatant wasdiscarded and the pellets were measured to determine

EVALUATION OF CHYO11{ESERVED ROOSTER SPERM

2187

volume and sperm concentration, as described previ-ously.

Flow Cytome tryPlasma membrane integrity (Donoghue et al., 1995)

was determined via flow cytometry using a minimumof 5,000 sperm per treatment sample. Lake's diluent,with glycerol for immediate postthaw and glycerol-freefor postglycerol removal analysis, was used to dilute5-1iL samples of semen. The fluorescent stains SYBR.-14 (Molecular Probes, Eugene. OR.; 5 1iL of a 20-.iMsolution in climethyl sulfoxide) and propidiumn iodide(PT; 10 iL of a 2.4 niM solution in water) were addedto the diluted semen (10 x 10 6 sperm/mL) and incu-bated at 23°C for 10 min in the dark. The samples wereanalyzed using a CyAN ADP flow cytometer (DakoCy-tomation, Fort Collins, CO) equipped with an argonlaser (488 nm) at 20 mW of power, the FL-1 detector(530-nm band pass filter) to detect SYBR-14 positive-PT-negative sperm (plasma membrane intact), and theFL3 detector (613-nm band pass filter) to detect PT-positive-SYBR- 14-negative (plasma membrane com-promised) sperm.

Phosphatidylserine is normally present on the innerleaflet of the plasma membrane, but when sperm are al-tered due to such things as capacitation. the acrosomereaction, or cryopreservation damage, PS will be ex-posed (Glander and Schaller, 1999). Phosphatidylserineexposure was determined using the staining combina-tion of Annexin V (Molecular Probes; PS exposure)and plasma membrane integrity (PT). Semen sampleswere thawed as described previously and maintained at41°C. Aliquots (1 x i0( sperm per sample) were dilutedinto 100 1rL of annexin-binding buffer (10 mM HEPES,140 mM NaCl, 2.5 mM CaCl2 . pH 7.4) containing 5 ItLof Annexin V and 5 iL of P1 according to the instruc-tions of the manufacturer. The samples were incubatedwith the stains for 15 minat 23°C. diluted with 900 1mLof annexin-binding buffer, and analyzed immediatelyafter thawing for both DMA and glycerol samples andafter glycerol removal (glycerol samples only). The flowcytometer was set up iii the same manner as for theSYBR-14-PI analyses, but in this instance, the 530-nnriband pass filter will detect PS-positive sperm. The au-tocompensation feature of the flow cytometer was usedto assist in separation of the sperm populations.

Insemination and FertilityOne hundred fifty-seven White Leghorn and Rhode

Island Red hens, aged 52 wk, were randomly assignedto a cryoprotectant protocol arid insemination method.The hens were housed in cages in groups of 2 to 3 birdsper cage, by breed, and all birds in a cage were insemi-nated only once with the same treatment. Intravaginalinseminations were performed using 200 x 106 spermof fresh pooled sperm, or frozen-thawed sperm fromeither the DMA samples or the glycerol samples. The

DMA samples were thawed and inseminated withoutfurther processing, whereas the glycerol samples werethawed and processed to remove the cryoprotectant. Nopostthaw processing was performed for the intramagnalinseminations. All samples were inseminated within 10minof the completion of glycerol removal or thawingin the case of DMA samples and imitramagnal glycerolsamples.

Intramagnal uisemrnnat.ion was performed as describedby Song and Silversides (2007). The liens were anaes-thetized by injection of 0.15 mL (White Leghorn) or0.20 mnL (Rhode Island Red) of xylazine (20 mg/mL)into the brachial vein. The left leg of the lien was drawnup while the hen was lying on its right side. Feathersoverlying the left abdominal wall were removed and thearea around the site of the surgery was wetted with70% ethanol. An incision of approximately 2.5 cm wasmade in the skin between the thigh and the breast closeto the last rib and all of approximately 1 cmwas made in the underlying muscle where it is thinnest.A retractor was used to expose the magnum, which liesalongside the body wall in this area of the abdomen.A section of the magnum was held with large forcepswhile the sperm suspension (200 x 106 sperm) was in-jected using a 1-mL syringe equipped with a 20-gaugeneedle. The exposed loop of the magnum was then re-turned to the peritoneal cavity and the skin was closedby continuous sutures.

Eggs were collected daily, stored at 18°C, and settwice a week. Fertility was determined after 6 d of incu-bation by breaking the eggs. Live embryos were clearlydistinguishable from dead embryos and other materialof embryonic origin (blood and membranes). Eggs wereconsidered fertile but dead early in incubation if bloodor membranes were present.

StatisticsIn experiment 1, differences in sperm inotilities, con-

centration, and plasma membrane integrity before andafter glycerol removal were analyzed using a mixedmodel ANOVA (PROC MIXED ANOVA) and type 3sums of squares (SAS Institute, 1985). The model in-cluded as fixed effects glycerol removal method, andline; random effects included rooster within line, andsemen straw within rooster and within line.

In experiment 2, differences in postthaw niotilities,plasma membrane integrity, and the percentage ofsperni displaying phosphatidyl serine were analyzed us-ing a mixed model ANOVA (PROC MIXED ANOVA)for the main effects of rooster, breed (fixed). cryopro-tectamit (fixed), and straw (random), which was repli-cated (random effect within line; SAS Institute, 1985).The fertility measures were anal yzed using a GLMmodel that consisted of breed, treatment, and breed xtreatment interaction, all of which were fixed effects.Cage was the experimental unit for this portion of theexperiment. The experiment was an incomplete facto-rial; therefore, 5 treatments were formed as: 1) control

2188

PURDY ET AL.

Table 2. Characteristics of frozen-thawed rooster sperm samples, by line, before glycerol removal'

Progressive motilit y (X)

28"'If13'210 I

iSto32"

Line (110.)2

1 (4)2 (4)3 (4)4 (4)5 (4)6 (3)SEhI

Concentration (10")

165.7'482.7"502.9"456.9"482.4'581.0"

25.9

Motility (%)

503732404357

2.1

I'm ' (V.)

565636393849

3.2

'Means within a colunin lacking a common superscript differ (P < 0.1)5).'Two straws per rooster ejaculate were analyzed.'Individual lines are described in Table 1, and the number iii parentheses indicates the number of males used

from that line.'Pl.siria membrane integrity.

(fresh semen inseminated intravaginally), 2) DMA andintramagnal insemination, 3) glycerol and intrarziagnalinsemination, 4) DMA and intravagirial insemination,and 5) glycerol and intravaginal insemination. Eachcage had multiple hens; therefore, actual fertility mea-surements were constructed as fertility per egg laid percage per day, fertility per egg per hen per day, and totalfertile eggs per hen.

RESULTS

Experiment 1: Accudenz and StepwiseDilution Analyses

Significant line differences in sperm concentrationand progressive motility before glycerol removal wereobserved (Table 2). From the results presented in Ta-ble 2, the number of insemination doses per straw wasalso determined by line. If 100 x iO t' total sperm perdose are to be inseminated (Long and Kulkarni, 2004),then from the lines analyzed, 0.84 to 2.91 insemninationscan he performed from a single 0.5-mL straw, whereasmore conservative methods of insemination (200 x 10sperm; Phillips et al.. 1996) would result in half of theinsemination doses.

The method of glycerol removal affected the resultingsperm concentration. Use of the dilution method re-covered significantly more sperm that had more intactplasma membranes than the Accudenz method (P <0.05; Table 3). The difference in postglycerol removalsperm concentration (68.7 x 10 sperm) results in asignificant loss (0.68 to 0.34 doses based on a 100 or 200x 10e sperm insemination dose, respectively) of insemi-nation doses due to processing with the Accudenz cen-trifugation method. The Accudenz method did producesamples with significantly greater motility, increasedelongation, and increased straightness compared withthe dilution method, but the (lilutioli method producedsamples with greater plasma membrane integrity (Ta-ble 3).

Experiment 2: Cryoprotectant Comparison

Postthaw semen characteristics for gl ycerol and DMAcomparison are presented in Table 4. No significantbreed differences in postthaw sperm quality were ob-served for Rhode Island Red and White Leghorn roost-ers; therefore, only the differences in cryoprotectantare reported. Samples cryopreserved using glycerol hadgreater motility and quality of motion (VAP, progres-

Table 3. Means of the motility characteristics of frozen-thawed rooster sperm samples after dilutionand centrifugation (Dilution) or centrifugation through a discontinuous Accudenz column' (Accu-denz) for removal of gl ycerol (n = 23 roosters: 2 semen straws per rooster)

Sperm attribute'

Concentration (10' spermn/mL)Total motility ('/(.)Progressive motility (%)Plasma membrane integrity ()Elongation (%)Area (pm 2)

Path velocit y (yAP, pm/s)Progressive velocity (VSL: p10/5)

Track speed (VCL; pm/s)Straightness (ratio of VSL:VAP)Linearit y (ratio of VSL:VCL)Lateral head amplitude (pm)Beat, cross frequency (freqimency/s)

Accudenz

239.2'37'1355030.8"

6.839.631.776.368.5'35.3

3.435.0

Dilution

320.1'33"1257"29.9"

6.538.329.771.564.7"33.8

3.333.9

Significance

0.020.020.050.041)040.30.40.30.20.030.3(1.70.9

SEM

17.2lU0.60.60.20.060.70.61.00.30.40.0.111:1

""Means within a row lacking a common superscript differ (I' < 0.05).'Accurate Chemical & Scientific Corp.. Westbury, NY.2VAP = path velocity: VSL = straight-line velocity; VCL = curvilinear velocity.

EVALUATION OF CRYOPRESERVED ROOSTEII SPERM

Table 4. Postthaw results of motility and flow cytometric analysis of rooster sperin from 2 breeds(Rhode Island Bed and White Leghorn) of poultry cryopreserved using the cryoprotectants (CPA: n= 3 straws per CPA treatment) dimethyl acetamnide (DMA) or glycerol (GLY-i-) and analyzed hal-mediately after thawing or after thawing, stepwise dilution, and centriftigation to remove glycerol(C LY-)'

CPA

Concentration (10' spurn i/in!.)Motility (%)Progressive motilit y ('A)VA VSLVCLA LHT3CFStraightnessLinearityElongationAreaPS positive (V.)PMI ((4)

Means withinin a row lacking a colitnion superscript differ (Panalyses were performed on the data combined from the 2 breeds because no statistical differences

were observed due to breed.2 VAP = path velocity : VSL = straight-line velocit y : V( 'L = curvilinear velocity : AI.1-1 = lateral head amplitude;

IIC'l" beat cross frequency: PS = phosploil idvlseri,,e: l'\ll = plasma niembrane intact

DMA CLY±

CLY- SEM

804'

808'

529"

28.1

15.1'

47.8'

28"

1.1

.8

20.3

8.6'

0.95

36.8'

61.0"

52.9"

1.3

26.8

SOft'

42.3"

12

78.9'

108.9"

96.3"

2.1

5.7'

49

4.8"

0.8

27.9"

30.8"

31.2"

0.5

7-1"

77.9's

75.4"

0.4

38.3'

44,9"

42.5"

0.4

32. 1"

34.4" 3330I (1.3

7.9

7,3"

0.2

11.3 .0

14.7

0.6

5 9. 7

5(1.6

1.9

2189

sive velocity, curvilinear velocity) as well as greatermembranequality, as indicated by lower PS exposure,immediately after thawing compared with the DMAtreatment (Table 4). Glycerol removal by stepwise dilu-tion (Table 4) resulted in significant alterations of themotility, quality of motion characteristics, and plasmamembrane PS exposure compared with the same sam-ples before undergoing the process of gl ycerol removal.However, the samples that underwent glycerol removalstill had more robust motion metrics than the samplescryopreserved using DMA (Table 4). Sperm treatedby all 3 approaches had similar percentages of intactplasma membranes (P > 0.05). However, the cell areawas significantly larger for those cells cryopreserved inglycerol than the DMA and the glycerol + stepwisedilution.

Fertility of Cryopreserved TreatmentsAn ANOVA using GLM was performed in winch

semen treatment, breech and the semen treatmentx breed interaction were included in the model. Themodel yielded consistent results for all response vari-ables analyzed. Semen treatment effect was found to besignificant for all measures of fertility, whereas breedand treatment x breed were found to be nonsignificantsources of variation. Linear contrasts were performed tocompare the 2 methods of insemination and the 2 cryo-protectants for the response variables. The intramagnalmethod had significantly higher fertility levels then theintravaginal approach (Table 5). The results also indi-cate no significant difference between the types of cryo-protectant used for all measures of fertility (Table 5).Confounding does exist because samples cryopreservedin glycerol were handled differently (the glycerol + in-

t.ravaginal insemination saiiiples underwent glycerol re-moval via the stepwise dilution method).

DISCUSSIONAchieving fertility using frozen-thawed rooster sperm

relies on many factors related to sperm quality, post-thaw processing. and method of insemination. Experi-ment 1 was an evaluation of postthaw glycerol removalmethods involving multiple poultry lines. The line dif-ferences in postthaw motility observed in this research(experiment. 1) are similar to those reported elsewherefor motility (Yousif et al. 1984) and mobility (Fromanand Kirby. 2005). The stepwise dilution method willsuccessfully remove glycerol from rooster sperm but itrequires attention to detail during the multiple dilutionsand is more time-consuming (60 ilium total) comparedwith the Accuclenz method (30 min total), althoughthe latter requires preparation of the column and layer-big of the sample before centrifugation. Potentially, thenumber of dilutions in the stepwise method could bedecreased, which would minimize the total processingtime (Lake et al., 1981).

The Accudenz itmet.bod y ielded greater postprocess-ing motility, elongation, and straightness of movement,but the stepwise dilution method resulted in 34% moresperm, with more intact plasma membranes, availablefor insetninatioii when compared with the Accudenzmethodology. In trying to maximize the use of storedsemen, the additional number of sperni available forinsemination with the stepwise approach comparedwith the Accudenz method is important. The addition-al number of motile sperm available with the stepwisemethod may be more important than an increase in thepercentage of motile cells obtained with the Accudenz

2190

PURDY El AL.

Table 5. Least squares means for total fertile eggs laid per cage, fertility per egg per cage per da y, fertility per egg laid per hen perday, and total fertile eggs per lien from d 2 to 8 and 2 to 18 after insemination by treatment

Days 2 to 8 after insemination

Fertility per Fertility perSeiiiuii- Nunili,'r Total fertile egg laid per egg laid perinsemination of cages eggs per cage day per cage hen per daytreatment' used lIens (SEM) (SEM) (SEM)

Days 2 to 18 after insemination

Fertility per Fertilit,v arTotal fertile egg laid per egg laid per

eggs per cage day per cage hen per day(SEM) (SEM) (SEM)

Control 12 31 10.8 (0.77)Glycerol IV' 15 35 2.2 (0.69)Glycerol tM 12 29 2.5 (0.77)DMA IV 13 32 ((.7 (0.74)DMA l\l 12 301 3.6 (0.77)

0.83 (0.08) 0.36 (0.08)

0.23 (0.07) 0.14 (0.07)

0.49 (0.09) 0.23 (0.09)

0.13 (0.08) 0.07 (0.08)

0.57 (0.08) 0.25 (0.08)

18.2 (1.45) 0.60 (0.06) ((.25 (0.03)

2.4 (133) 0.09 (0.05) 0.05 (0.03)

4.3 (1.18) 0.30 (0.06) 0.13 (0.03)

1.1 (1.43) 0.10 (0.06) 0.05 (0.03)

7.7 (1.48) 0.41 (0.06) 0.18 (0.03),.b Linear contrasts l,etweeri methods of artificial insemination were significant for all traits anal yzed within the 2 to 8- and 2 to 18-il period (P <

0.05).

'Control semen-insemination treatments represent fresh semen and an intravaginal insemination method. Crvoprotectants [dinmetliylacetamide(DMA), glycerol] were not significantly different. Use of the glycerol cryoprotectant required posttliaw stepwise dilution to renlove glycerol for theintravaginal inseminations (IV) but not for the intrainagnal (TM) inseminations, whereas the DMA samples (lid not require removal of the eryopro-tectant before insemination with either method.

method. This result underscores the need to better un-derstand the relationship between motility and fertilitylevels. Because maximization of stored semen is a con-cern for genebanks, therefore, at present, the stepwisedilution method would better meet reconstitution pur-poses and therefore it was used it in experiment 2. Still,because no fertility trials comparing frozen-thawedrooster sperm, which have had the glycerol removed byeither the Accudenz or stepwise dilution method, havebeen performed, it may be prudent to perform inin thefuture.

The range of motility values observed in experiment 2was in agreement with other studies that used glycerolas the cryoprotectant, as was the range of plasma mem-brane integrity values (Seigneurin and Blesbois, 1995;Phillips et al., 1996). Furthermore, the significant dif-ferences between the samples cryopreserved with glyc-erol (before and after glycerol removal) and DMA havealso been documented previously (Chalah et al,, 1999).A potential cause for the low levels of motility with theDMA samples could be attributed to the freeze rate.In this experiment, the samples were frozen using theavailable method, which was a box and static liquid ni-trogen vapor resulting in a 59'C/min freeze rate, whichwas similar to the 50°C rate reported by Blesbois et al.(2007). If the freeze rate was increased to 200°C/mm,as demonstrated by Woelders et al. (2006), potentiallythere could have been an increase in postthaw motilityand quality. Unfortunately, access to a programmablefreezer to create a 200'C/i-nin rate was not available atthe time but will he explored in the future.

The decrease in motility and changes in sperm qual-ity (PS exposure) before and after glycerol removal(stepwise dilution method; experiment 2) attest to thefragile nature of frozen-thawed rooster sperm. Theseresults underscore the care with which frozen-thawedrooster sperm must be treated through the dilution andcentrifugation process to minimize cellular damage. Inaddition, it has been our experience that if strict atten-tion to handling is not adhered to, the use of frozen-

thawed sperm is greatly compromised. Although therewere no differences in plasma membrane integrity, amore subtle indicator of sperin quality, PS exposure,which is indicative of poor plasma membrane qual-ity (Glander and Schaller, 1999), increased due to theglycerol removal process. Although no differences in PSexposure in the samples at the time of inseminationwere observed, these analyses once again demonstratethe damaging effects of the glycerol removal process.Due to the sensitivity of PS, this test may have a roleto play as an alternative method for evaluating roostersperm, a method that couples plasma membrane integ-rity analysis with a membrane quality analysis (An-nexin V; Glander and Schaller, 1999).

Examination of the experiment 2 fertilizing poten-tial demonstrated that sperm samples cryopreservedin glycerol and before undergoing stepwise dilutiongenerally had significantly better motility and cellu-lar performance characteristics than did the DMA andglycerol stepwise dilution treatments (Table 4). Thesemeasures did not translate into an advantage for fertil-ity measures, in which the DMA intramnagnal insemina-tions resulted in higher fertility levels (Table 5). Thissuggests that the intramnagrial insemination is perhapsmore robust and semen with a wider range of qualitycan be used successfully. This is particularly evident incomparing the fertility results of the intravaginal andiritramagnal inseminations, in which DMA was used asthe crvoprotectant.

The results indicated that intramagnal inseminationyielded the highest fertility levels regardless of cryo-protectant used, which is in agreement with results re-ported by Bacon et al. (1986). The intravaginal insemi-nations resulted in fertility that was within the rangeof other reports (Phillips et al., 1996; Blesbois et al.,2007), but it must be emphasized that in this experi-ment, hens were only inseminated once over the evalua-tion period of 18 d. Other research using cryopreservedrooster sperm resulted in fertility rates consistentlygreater than 60%, but this involved inseminations every

EVALUATION OF CRYOPRESERVED ROOSTER SPERM

2191

3 to 4 d using 300 x 106 sperm per insemination (Cha-lab et al., 1999; Tselutin et al., 1999; \Voelders et al.,2006), all of which are methodologies that attemptedto maximize fertility using multiple inseminations ofmoderate sperm numbers (Brillard, 1993). The currentresearch only used 200 x iO sperm per insemination(Buss, 1993; Phillips et al.. 1996) and single insemina-tions to maximize the number of doses attainable fromcryopreserved samples and to determine the numberof fertile eggs attainable from single inseminations. Byincreasing the number of inseminations performed, timefertility would increase.

This research was performed to replicate line-breedregeneration that may occur with national conserva-tion programs and therefore, only single inseminationswere performed because limited cryopreserved sampleswould be available frorn these programs. Because weendeavored to replicate regeneration conditions, wechose to evaluate both intravaginal and intramagnal in-seminations. It is apparent that although both of thesetechniques have limitations, the intramagnal techniqueis invasive and may not be readily usable by many in-stitutions. Still, in the event that drastic measures needto be employed for purposes of line-breed regeneration,utilizing intramagnal insenrunations may be the bestoption to ensure greater levels of fertility with fewerfrozen-thawed samples. Furthermore, the lack of sig-nificant breed differences for fertility suggests that theintramagnal insemination is a robust approach.

Froni this set of experiments, several conclusions rel-evant to genebanking poultry semen can he derived.First, intrarnagnal insemination appears more robust,suggesting that it be the insemination method of choicefor regeneration-expansion of lost or endangered lines.Given the insemination method, type of cryoprotectantused becomes less important. However, utilization ofDMA does have a slight but nonsignificant advantageover glycerol. Finall y, these results suggest that breedeffect may be a nonsignificant source of variation whenintramagnal insemination is employed, but this wouldneed to he further investigated.

REFERENCESBacon, L. D.. H. D. Hunt. and 11. H. Clang. 2000 A review of the

development of chicken Imes to resolve genes determining resis-tance to diseases. Poult. Sci. 79:1082--1093.

Bacon, L. B., D. W. Salter, J. V. Motta, and L. B. Crittenden.1986. Cryopreservation of chicken semen of inbred or specializedstrains. Poult. Sd. 65:1965 -1971.

Blackburn. H. D. 2006. The National Animal Germplasin Program:Challenges and opportunities for poultry genetic resources.Poult. Sci. 85:210-215.

ljleshois. E.. F. Seigneurin. I. Grasseau. C. Liinonzin..J. Besnard.B. Gourichon, C. Coquerehle. P. Rault. and M. Tixier-Boichard.2007. Semen cryopreservation for ex situ management of genet-ic diversity in chicken: Creation of the French avian crobank.Poiilt. Sci. 86:555-564.

Brillard, J. P. 1993. Sperm storage and transport following naturalmating and artificial insemination. Poult. Sci. 72:923 928.

Burrows. W. H.. and J. P. Quinn. 1937. Collection of spermatozoafrom the domestic fowl and turkey. Poult. Sci. 16:19-24.

Buss. E. G. 1993. Cryopreservation of rooster sperm. Poult. Sci.72:944-951.

Chalali. T., F. Seigneurin. E. Bleshois, and J. P. Brillard. 1999. Invitro comparison of fowl sperm viability in ejaculates frozen bythree different techniques and relationship with subsequent fertil-ity in vivo. Cryobiology 39:185 191.

Donoghue, A. NI., D. L. Garner. D. J. Donoghue, and L. A john-son. 1995. Viabilit y assessment of turkey sperm using fluorescentstaining and flow cytometry. Poult. Sci. 74:1191-1200.

Froman. D. P.. and J. D. Kirby. 2005. Sperm mobility: Phenot ype inroosters (Callus dornesticu.$) determined by mnitochondrial func-tion. Biol. Reprod. 72:562----567.

Glander, H.....and J. Schaller. 1999. Bionding of annexin V toplasma membranes of human spermatozoa: A rapid assay fordetection of membrane changes after cryostorage. Mol. Hum.Reprod. 5:109-115.

Hamnmerstedt, R. H.. and J. K. Graham. 1992. Cryopreservation ofpoultry sperm: The enigma of glycerol. Cryobiology 29:26-38.

Lake, P. E., 0. Ravie. and J. McAdam. 1981. Preservation of fowlsemen in liquid nitrogen: Application to breeding programmes.Br. Poult. Sci. 22:71-77.

Lake, P. E., and J. M. Stewart. 1978. Preservation of fowl semen inliquid nitrogen An improved method. Br. Poult. Sci. 19:187-194.

Long, J. A., and G. Kulkarni. 2004. An effective met-hod for improv-ing the fertility of glycerol-exposed poultry semen. Poult. Sci.83:1594-1601.

McLean. D. A. J. Felt mann. and B. P. Fromnan. 1998. Transferof sperm into a- chemically defined environment by centrifugationthrough 12% (wt/vol) Accudenz . Poult. Sd. 77:163-168.

Pehiez, J.. and J. A. Long. 2007. Characterizing the glycocalyx ofpoultry spermatozoa: I. Identification and distribution of carbo-hydrate residues using flow cytomnetry'ancl epiflumorescence mi-croscopy. J. Androl. 28:342 352.

Phillips, .1.....R.. K. Bramwell, and .1. K. Graham. 1996. Cryo-preservation of rooster sperm using meth yl cellulose. Poult. Sci.75:915-923.

Polge, C. 1951. Functional survival of fowl spermatozoa after freez-ing at -79°C. Nature 167:949- 950.

SAS Institute. 1985. User's Guide: Statistics. Version 5 edition. SASInstitute Inc., Cary, NC.

Seigneurin, F., and E. Blesbois. 1995. Effects of the freezing rate onviability and fertility of frozen-thawed fowl spermatozoa. The-riogenology 43:1351-1358.

Silversicles, F. G., B. M. Shaver, and Y. Song. 2007. Pure line lay-ing chickens at the Agassiz Research Centre. Animal GeneticResources Information 40. FAO. Rome, italy.

Song. Y., and F. C. Silversides. 2007. Heterotopic transplantation oftestes in newly hatched chicken and production of viable offspringvia intramagnal insemination. Biol. Reprod. 76:598-603.

Tajima, A.. E. F. Graham. and D. M. Hawkins. 1989. Estimationof the relative fertilizing ability of frozen chicken spermatozoausing a lieterospermic competition method. J. Reprod. Fertil.85:1-5.

Tselutin, K.. F. Seigneurin. and E. Blesbois. 1999. Comparison ofcryoprotectants and methods of cryopreservation of fowl spermna-tozoa. Poult. Sci. 78:586-590.

Woelders, H., C. A. Zuidberg, and S. J. Hieumstra. 2006. Animalgenetic resources conservation in the Netherlands and Europe:Poultry perspective. Poult. Sd. 85:216-222.

Yousif, Y. F., G. A. Ansah, and R. B. Bumckland. 1984. Effect ofselection for fertility of frozen-thawed semen in chickens on thefertility of fresh and stored semen. Poult. Sci. 63:1475 1480.