Small Ruminant Research 119 (2014) 100–106

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Small Ruminant Research

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Comparison of different cryoprotectant regimes forvitrification of ovine embryos produced in vivo

P.F.B. de Araújo-Lemosa, L.M. de Freitas Netob, J.V. de Meloc, M.T. Mourab,P.F. Limab, M.A.L. Oliveirab,∗

a Paraíba State Agriculture Research Company (EMEPA), Brazilb Department of Veterinary Medicine, Federal Rural University of Pernambuco (UFRPE), Brazilc Center for Strategic Technologies of the Northeast (CETENE), Federal University of Pernambuco (UFPE), Brazil

a r t i c l e i n f o

Article history:Received 24 September 2013Received in revised form 2 February 2014Accepted 25 February 2014Available online 6 March 2014

Keywords:CryobiologySmall ruminantsLambing rateDimethyl sulfoxide

a b s t r a c t

This study was aimed to compare different cryoprotectants for vitrification of sheepembryos produced in vivo. Blastocysts were obtained from superovulated Santa Inesewes and randomized into three groups: conventional freezing using ethylene glycol (EG)(control group), vitrification with EG and dimethyl sulfoxide (DMSO vitrification), or vitri-fication with EG and dimethylformamide (DMF vitrification). All groups were analyzedfor embryonic viability (propidium iodide staining), re-expansion rate after thawing (atmorphological and ultrastructural levels) and pregnancy rate after embryo transfer (ET).Embryos of DMSO vitrification group showed lower cell viability (44.44%), compared toDMF group and control embryos (77.77% and 100%, respectively). The ultrastructural studyshowed similar cryopreservation damage among control and DMF embryos, and these wereless damaged than DMSO vitrified embryos. Embryos vitrified with DMF had higher rates of

Dimethylformamide re-expansion in vitro (53.33%) than DMSO (26.66%), and control (33.33%). After ET, similarpregnancy rates were obtained from all groups (DMF: 45%, DMSO: 30%, control: 40%). Col-lectively, DMF vitrification is more efficient than DMSO vitrification and is indistinguishablefrom conventional freezing of sheep embryos.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Embryo cryopreservation was first described fourdecades ago (Whittingham et al., 1972; Wilmut, 1972).Currently, cryopreservation is a key procedure for embryotechnologies for both commercial and research scenarios,

allowing the conservation of biological material for longperiods of time, while preserving cellular, genetic, and bio-chemical properties (Whittingham, 1980).

∗ Corresponding author at: Laboratory for Applied Biotechnologies inAnimal Reproduction, Department of Veterinary Medicine, Federal RuralUniversity of Pernambuco (UFRPE), Av. Dom Manoel de Medeiros s/n, DoisIrmãos, 52171-900 Recife, PE, Brazil. Tel.: +55 81 3320 6415.

E-mail address: maloufrpe@uol.com.br (M.A.L. Oliveira).

http://dx.doi.org/10.1016/j.smallrumres.2014.02.0130921-4488/© 2014 Elsevier B.V. All rights reserved.

Two main cryopreservation methodologies have beendescribed, namely slow conventional freezing and vitrifica-tion (Massip, 2001). Conventional freezing has been widelyused for embryo cryopreservation of various species,including sheep (Bilton and Moore, 1976; Cognié et al.,2003). However, conventional freezing holds several lim-itations, such as need for sophisticated and expensiveequipment, and time consuming protocols using slowfreezing curves (Loutradi et al., 2008). In contrast, vitrifica-tion is characterized by simple and fast procedures, whiledispensing expensive equipment (Vajta and Kuwayama,

2006; Dike, 2009). These advantages have made cryopres-ervation by vitrification an attractive approach to cell andtissue cryobanking under commercial and research sett-ings (Szell et al., 1990; Vajta, 2000; Kuleshova et al., 2001).

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Vitrification of ovine embryos is as efficient as conven-ional freezing (Baril et al., 2001; Bettencourt et al., 2009),nd using direct transfer (Isachenko et al., 2003), vitrifi-ation is advantageous to generate offspring (Green et al.,009). Despite this, it remains controversial if vitrifica-ion affects the viability of sheep embryos, since differenteports have found lower or similar pregnancy rates whenompared to fresh embryos (Martinez and Matkovic, 1998;attena et al., 2000; Zhu et al., 2001; Papadopoulos et al.,002; Green et al., 2009).

In order to improve sheep embryo survival and preg-ancy rates after vitrification, different combinations ofryoprotectants and other protocol variations were testedLeoni et al., 2002; Dattena et al., 2004; Shirazi et al., 2010).owever, to our knowledge, dimethylformamide (DMF)as not been tested as a cryoprotectant for sheep embryoitrification. Despite the poor results from initial attempto use DMF for embryo cryopreservation (Kasai et al., 1981;hen and Tian, 2005), the lower molecular weight of DMFompared to glycerol motivated recent investigations usingMF for semen cryopreservation aiming to reduce osmotic

tress (Squires et al., 2004; Moustacas et al., 2011).The objective of the present research was to compare

wo different vitrification protocols to conventional freez-ng using sheep embryos produced in vivo. Experimentalroups were compared by embryo viability at morphologi-al and ultra-structural levels, re-expansion rate of thawedmbryos and pregnancy outcome after transfer of cryopres-rved embryos to synchronized recipients.

. Materials and methods

.1. Chemicals

Chemicals were obtained from Sigma–Aldrich Chemical CompanySaint Louis, USA) unless otherwise indicated.

.2. Experimental location

The experiment was performed at Research Station of Pendência, andt Research Station of Benjamin Maranhão, both research units are partf Paraíba State Agriculture Research Company (EMEPA), Brazil. Researchas also conducted at Federal University of Pernambuco (UFPE), at Federalural University of Pernambuco (UFRPE), and at the Center for Strategicechnologies of the Northeast (CETENE), all located in Recife, Brazil.

.3. Donor selection

All experimental procedures were conducted in accordance with localthics review board on animal research. Thirty Santa Ines ewes, with aver-ge age of 3.2 years, with no reported reproductive problems, and withdequate nutritional (minimum body score of 2.0) and sanitary conditionsere used. Animals were housed in a covered shed, fed with ray ad libi-

um, and concentrate supplement with 18% of crude protein, containingorn, soybean, wheat, and limestone. Animals had free access to waternd mineral supplementation.

.4. Estrous synchronization and FSH-treatment

Embryo donors had their estrous cycles synchronized by insertionf vaginal devices impregnated with 0.33 g of natural progesterone, con-rolled internal drug release (CIDR, Pfizer, Auckland, New Zealand), andonsidered it day 0 on protocol. On day 9, all CIDR were replaced by

ew devices, and were used until day 13. FSH-treatment was initiatedn day 11 until day 15, using 252 mg of follicle stimulating hormone –FSH (Folltropin-V, Bioniche, Ontario, Canada), divided in eight decreasingoses (four days), administered in 12 h intervals. Concomitant withemoval of vaginal dispositive on day 13, 200 IU de equine chorionic

ant Research 119 (2014) 100–106 101

gonadotropin (eCG) was administered (Folligon, Intervet, Boxmeer, Hol-land). Controlled natural mating was performed on day 14 with rams ofproven fertility.

2.5. Embryo collection and evaluation

Embryos were collected on days 5.5 and 6.0 after estrus onset, aimingto recover embryos at developmental stages from morulae to expandedblastocyst. Animals were not fed 24 h before collection, and were anes-thetized with 0.2 mg kg−1 xylazine chloride (Rompun, Bayer, São Paulo,Brazil) and 7.5 mg kg−1 ketamine chloride (Ketalar, Parke-Davis, BuenosAires, Argentina). Embryo collections were performed by laparotomy, andboth uterine horns were flushed with embryo collection medium Dul-becco’s modified phosphate buffered saline (DPBS, Embriocare, Cultilab,Campinas, Brazil), supplemented with 1% fetal bovine serum (FBS) at 37 ◦C.Embryos were immediately identified and placed in holding medium (TQCHolding Plus, Nutricell, Bioniche, Athens, USA).

All embryos were scored by development stage and qualityas described by the International Embryo Transfer Society – IETS(Stringfellow and Seidel, 1998): grade I (excellent), II (good), III (poor), andIV (dead or degenerated). Embryos scored as grade I and II were selectedfor cryopreservation.

2.6. Embryo cryopreservation

2.6.1. Conventional freezing – control groupBefore freezing, embryos remained for 5 min in TqC Ethylene Gly-

col Freezer Plus solution on a heated stage at 39 ◦C (Nutricell, Bioniche,Athens, USA) and were loaded in 0.25 mL straws. Embryos were frozenusing an automatic embryo freezer (TK 3000, Uberaba, Brazil). Placedin the embryo freezer, embryos were submitted to a freezing curve of−1.0 ◦C/min until −6 ◦C, starting from room temperature. When the tem-perature of −6 ◦C was reached, the process of freezing was stopped for5 min to induce crystallization (seeding). Moreover, after waiting 10 minto reinitiate freezing, programming was reset to −0.5 ◦C/min until −32 ◦C.After 5 min stabilizing at final freezing temperature, embryo-containingstraws were immersed in liquid nitrogen.

2.6.2. Vitrification in OPS (Open Pulled Straw) – DMSO and DMF groupsAll vitrification solutions were prepared using a basal solution of

Hepes containing-TCM-199 (M7653) (Nutricell, Bioniche, Athens, USA)supplemented with 20% FBS (Nutricell, Bioniche, Athens, USA) (holdingmedium). Embryos were initially kept in H-TCM for 5 min (Vajta, 2000).Immediately after, embryos of the dimethyl sulfoxide (DMSO) group weretransferred to holding medium containing 10% ethylene glycol (EG) and10% DMSO and transferred to a 20% EG + 20% DMSO + 0.5 M sucrose solu-tion for 1 min each. Embryos of dimethylformamide (DMF) group weretransferred to a 10% EG and 10% DMF solution for 1 min and moved to a20% EG + 20% DMF + 0.5 M sucrose solution for an additional minute. Afterthis, embryos from both groups were aspirated in 2 �L of their respectivevitrification solution containing 0.5 M sucrose, containing 1 or 2 embryosand transferred by capillarity to OPS and identified properly. Immediatelyafter, straws were transferred to liquid nitrogen and kept until further use.

2.7. Thawing of frozen embryos

Embryos were thawed by exposure of straws to room temperaturefor 10 s and immersion in water bath at 37 ◦C for 20 s. Straw contentwas deposited in a well of a four well dish containing holding medium,embryos were remained for 5 min, and were subsequently evaluated formorphology and quality.

2.8. Warming of vitrified embryos

Immediately after removal from liquid nitrogen, embryo-containingstraws were held in air for 3 s, and the thinner tip was immersed in holding

medium supplemented with 0.33 M sucrose. The cryoprotectant removalwas performed in a four well dish containing holding medium supple-mented with 0.33 M sucrose (well 1 and 2). Embryos were kept in wells(1 and 2) for 1 min each, transferred to well 3 containing H-TCM + 0.2 Msucrose for 1 min, and finally for 5 min, in H-TCM.

102 P.F.B. de Araújo-Lemos et al. / Small Ruminant Research 119 (2014) 100–106

l cells v

Fig. 1. Ovine blastocysts stained for cell viability. Embryo grade 1 with al

2.9. Evaluation of embryo viability

Cellular viability was scored by propidium iodide staining, wheremembrane-lysed cells are selectively stained and considered unviable.All blastocysts (n = 9 embryos per group) were cultured after thaw-ing/warming for 10 min and incubated for 5 min in DPBS containing 1%BSA and 125 mg mL−1 of propidium iodide, and were further transferredto slides containing drops of DPBS with 100 mg mL−1 Hoechst 33342 solu-tion. Cells that stained red or pink were considered unviable (propidiumiodide staining). The total cell number in each embryo was determinedby Hoechst 33342 staining (blue cells). Embryos with less than 50% ofred/pink cells were considered viable. Stained embryos were examinedon an inverted fluorescence microscope (Leica DM 4000). Experiment wasperformed in triplicate.

2.10. Ultra-structural study by transmission electronic microscopy

Embryos from control (n = 9), DMSO (n = 10), and DMF groups(n = 10) were randomly selected for ultra-structural analysis without anymorphological evaluation. Embryos were fixed in Karnovsky solution (2 hat 4 ◦C), were washed in 0.1 M sodium cacodylate buffer (pH 7.4), and

Fig. 2. Ultrastructure of ovine blastocyst produced in vivo and cryopreserved bmitochondria (m), (B) lyzed mitochondria, (C) mitochondria and vacuoles (v) and

iable (left) and a grade 2 embryo showing damaged cells (red/pink).

were post-fixed in 1% osmium tetroxide. After dehydration in increasingethanol concentrations, embryos were embedded in epon resin. Ultrathinsections were obtained with a diatome knife. Sections were mountedon copper grade and stained with uranyl acetate and lead citrate.Samples were examined and photographed with a transmission electronmicroscope (FEI Morgani 268D, Eindhoven, Netherlands) at CETENE.

2.11. Analysis of re-expansion rates after embryo cryopreservation

Cryopreserved embryos were thawed or warmed as described aboveand morphologically evaluated before in vitro culture (IVC). Embryos(n = 15) were cultured in 400 �L of Synthetic Oviduct Fluid (SOF) medium(Nutricell, Bioniche, Athens, USA) under mineral oil in four well dishes,with 5% of CO2 in air at 38.5 ◦C. Embryo survival was scored at 6, 12,and 24 h after IVC onset. Embryo viability was determined by blastocoelexpansion. Experiment was performed in triplicate.

2.12. Embryo transfer and pregnancy diagnosis

Sixty adult crossbred recipient ewes were synchronized using vaginalsponges with 60 mg of medroxyprogesterone acetate (Progespon, Syntex,

y conventional freezing using ethylene glycol. (A) Microvilli (mv) and (D) Cellular junctions (arrow).

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ig. 3. Ultrastructure of ovine blastocyst produced in vivo and cryopreseromplex (CG) and mitochondria (m), (B) nucleus (n) and nuclear membra

uenos Aires, Argentina) for 14 days followed by 400 IU eCG concomitantith sponge removal. Twenty-four hours after sponge removal, vasec-

omized males were used for estrus detection. Embryo transfer (ET) waserformed by semi-laparoscopy on day 7 after estrous detection.

Recipient ewes were anesthetized with 0.05 mg kg−1 xylosine chlo-ide (Rompun, Bayer, São Paulo, Brazil). The uterine horn ipsilateral to theorpus luteum was exposed and a single blastocyst was transferred with

tom-cat catheter (Nutricell, Bioniche, Athens, USA). Pregnancy diagno-is was performed at day 35 after ET by rectal ultrasonography (Mindray)ith a 5 MHz linear transducer.

.13. Statistical analysis

Descriptive analysis was used for embryo morphology and ultrastruc-ural studies. Statistical analysis for re-expansion, viability and pregnancyates were performed with chi-square test for multiple comparisons. Dif-erences with less than 5% probability were considered significant.

. Results

A total of 186 embryos (2 morulae and 184 blastocysts)ere recovered from 30 embryo donors (average of 6.2

able 1e-expansion rates of ovine embryos cryopreserved by conventional freezing and

Group Embryos

Freezing (control) 15

DMSO vitrification 15

DMF vitrification 15

a Percentages with different superscripts differ significantly (p < 0.05). DMSO: d

ng dimethylformamide and ethylene glycol (DMF vitrification). (A) Golgiw), (C) mitochondria (m) and (D) mitochondria (m) and microvilli (mv).

embryos per donor), of which 159 embryos (115 blasto-cysts and 44 expanded blastocysts/average of 5.5 embryosper donor) were selected and randomly divided within thethree experimental groups.

Randomly selected embryos were stained for cell via-bility (Fig. 1). Embryos from the control group had 70% ofviable cells after thawing, allowing to consider them allviable (9/9) (see material and methods). From DMSO vitri-fied embryos analyzed, 33.33% embryos had all cells lyzed(3/9 embryos), 22.22% embryos (2/9 embryos) had less thanhalf of cells damaged, and 44.44% embryos (4/9 embryos)had few or no damaged cells. Based on these data, 66.66% ofDMSO treated-embryos had more than 50% of viable cellsand were considered viable after thawing (6/9 embryos).The DMF group had 22.22% embryos (2/9 embryos) clas-

sified as unviable, with all cells scored as unviable. Theremaining samples were considered viable (77.77%; 7/9embryos), which 57.14% had a small number of damagedcells (4/7 viable embryos).

vitrification.

Re-expanded embryos after cryopreservation (%)

6 h 12 h 24 h

1 (6.66) 4 (26.66) 5 (33.33)a

2 (13.33) 3 (20.00) 4 (26.66)a

4 (26.66) 8 (53.33) 8 (53.33)

imethyl sulfoxide, DMF: dimethylformamide.

104 P.F.B. de Araújo-Lemos et al. / Small Ruminant Research 119 (2014) 100–106

ved by vbrane (

Fig. 4. Ultrastructure of ovine blastocyst produced in vivo and cryopresertion). (A) Severe cellular damage, (B) Nucleus (n) with lyzed nuclear mem(arrow).

In order to better estimate the effect of embryocryopreservation at the cellular level, blastocysts fromall groups were analyzed by transmission electronmicroscopy. Conventional freezing (Fig. 2) and DMFvitrification (Fig. 3) displayed conserved cytoskeletonstructure with viable organelles such as mitochondria andGolgi complexes, although lysed mitochondria were alsoobserved (Fig. 2). Smaller structures such as microvilliand gap junctions maintained their normal dispositionand structure after DMF vitrification and conventionalfreezing (Figs. 2 and 3). In contrast, DMSO vitrificationshowed severe ultrastructural abnormalities, such as lossof cell shape by extensive cytoskeleton damage and lysedmitochondria (Fig. 4). Nuclear structure was also affectedby DMSO vitrification, concomitant with nuclear mem-brane rupture (Fig. 4). Collectively, control and DMFembryos were similar at the ultrastructure level, displayingbetter organelle integrity and cell structure preservationthan DMSO vitrified embryos.

The results concerning re-expansion rates of cryopres-erved embryos by conventional freezing and vitrificationare described below (Table 1). No difference was observedbetween re-expansion rates of control and DMSO groups.

However, embryos from the DMF group had a higher rateof survival and growth after thawing (Table 1).

The pregnancy rate after embryo transfer of controlgroup was 40% (8/20), while DMSO and DMF groups were

itrification using dimethyl sulfoxide and ethylene glycol (DMSO vitrifica-arrow), (C) Mitochondria (m) and vacuoles (v) and (D) cellular junctions

30% (6/20) and 45% (9/20), respectively. No difference wasobserved between pregnancy rates of all experimentalgroups.

4. Discussion

The cryopreservation of mammalian embryos is anestablished technology (Willadsen et al., 1976), but cur-rent procedures based on conventional freezing requireexpensive equipment and time-consuming protocols (Barilet al., 2001). Embryo cryopreservation by vitrification couldreduce costs, and should be used when conventional freez-ing generates unsatisfactory results (e.g. embryos at earlystages of development or in vitro produced embryos)(Massip, 2001). Although dimethylformamide has beeninvestigated for semen and embryo freezing (Kasai et al.,1981; Chalah et al., 1999; Chen and Tian, 2005; Mota Filhoet al., 2011; Moustacas et al., 2011; Malo et al., 2012), itspotential for mammalian embryo vitrification remainedunknown.

A standard functional assay for embryo viability aftercryopreservation is to measure re-expansion rates afterin vitro culture of thawed embryos. Although re-expansion

rates were initially similar at 6 h and 12 h, DMF group dis-played higher embryo recovery at 24 h of in vitro culture,functionally demonstrating that DMF-vitrified embryosdisplay better embryo viability after cryopreservation.

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The ultrastructural study also revealed that DMSO vitri-cation was more detrimental to cellular componentshan DMF and control groups, possibly due to excessiveehydration (Fahy, 2010). An important advantage of vitri-cation is to avoid intracellular and extracellular ice crystal

ormation, a process associated with most cellular damagey cryopreservation (Massip, 1989; Vajta and Kuwayama,006), but dehydration needs to be monitored in ordero avoid osmotic damage during freezing (Fahy, 2010).he ultrastructural analysis demonstrated a considerablereservation of cellular structure, irrespectively of cryo-reservation method. This is in agreement with reportshat ovine embryos are more resistant to cryodamage thanther species, such as bovine (Rizos et al., 2002, 2004;ettencourt et al., 2009).

In order to better estimate the viability of vitrifiedvine embryos using different cryoprotectants, thawed orarmed embryos were transferred to surrogate mothers.ere, pregnancy rates were similar among experimen-

al groups. Substantial variation is observed in pregnancynd lambing rates after vitrification of sheep embryosMcginnis et al., 1989; Fahning and Garcia, 1992; Songsasent al., 1995; Cocero et al., 2002; Dattena et al., 2004).ull-term development after embryo cryopreservation cane influenced by many factors, such as embryo develop-ental stage, species, cryoprotectants, freezing solution

omposition, among others (Massip, 2001; Dinnyes et al.,006; Bettencourt et al., 2009), but pregnancy rateesults described here fall into the range of previouseports.

The DMF group and conventional freezing were morefficient to maintain cell viability than DMSO group, asemonstrated by propidium iodide staining. Embryos withigher cell numbers have higher viability, increased poten-ial to establish pregnancies and are in accordance with thee-expansion rates.

The results described here demonstrated the potentialf using the combination of DMF with EG for vitrifica-ion of sheep embryos. These results pave the way foruture research on vitrification using DMF on other mam-

alian species and efforts to apply DMF vitrification ofvine embryos under commercial settings.

. Conclusions

The study describes the first successful use of DMF as aryoprotectant for ovine embryo cryopreservation. More-ver, it was demonstrated that the cryoprotectant regimenonsisting of EG and DMF (DMF vitrification) is more effi-ient than DMSO vitrification and similar to conventionalreezing of sheep embryos. Moreover, DMF embryos wereble to establish pregnancies at a similar rate then DMSOitrification and conventional freezing.

onflict of interest statement

The author declares that there is no conflict of interest.

ant Research 119 (2014) 100–106 105

Acknowledgments

We would like to acknowledge EMEPA, FACEPE andCNPq for financial support of the study. M.T. Moura holdsa CNPq fellowship.

References

Baril, F., Traldi, A.-S., Cognié, Y., Leboeuf, B., Bechers, J.F., Mermillod, P.,2001. The repeatability of superovulatory response and embryo recov-ery in sheep. Theriogenology 56, 147–155.

Bettencourt, E.M.V., Bettencourt, C.M., Silva, J.N.C.E., Ferreira, P., Matos,C.P., Oliveira, E., Romão, R.J., Rocha, A., Sousa, M., 2009. Ultrastructuralcharacterization of fresh and cryopreserved in vivo produced ovineembryos. Theriogenology 71, 947–958.

Bilton, R.J., Moore, N.W., 1976. In vitro culture, storage and transfer of goatembryos. Aust. J. Biol. Sci. 29, 125–129.

Chalah, T., Seigneurin, F., Blesbois, E., Brillard, J.P., 1999. In vitro compar-ison of fowl sperm viability in ejaculates frozen by three differenttechniques and relationship with subsequent fertility in vivo. Cryobi-ology 39, 185–191.

Chen, S.L., Tian, Y.S., 2005. Cryopreservation of flounder (Paralichthys oli-vaceus) embryos by vitrification. Theriogenology 63, 1207–1219.

Cocero, M.J., Moreno Díaz De La Espina, S., Aguilar, B., 2002. Ultrastructuralcharacteristics of fresh and frozen-thawed ovine embryos using twocryoprotectants. Biol. Reprod. 66, 1244–1258.

Cognié, Y., Baril, G., Poulin, N., Mermillod, P., 2003. Current status ofembryos technologies in sheep and goat. Theriogenology 59, 171–188.

Dattena, M., Ptak, G., Loi, P., Cappai, P., 2000. Survival and viability of vit-rified in vitro and in vivo produced ovine blastocysts. Theriogenology53, 1511–1519.

Dattena, M., Accardo, C., Pilichi, S., Isachenko, V., Mara, L., Chessa, B.,Cappai, P., 2004. Comparison of different vitrification protocols on via-bility after transfer of ovine blastocysts in vitro produced and in vivoderived. Theriogenology 62, 481–493.

Dike, I.P., 2009. Efficiency of intracellular cryoprotectants on the cryopres-ervation of sheep oocytes by controlled slow freezing and vitrificationtechniques. J. Cell Anim. Biol. 3, 44–49.

Dinnyes, A., Meng, O., Polgar, Z., Boonksol, D., Somfai, T., 2006.Criopreservac ão de embriões mamíferos. Acta Scientiae Veterinariae34 (Suppl. 1), 171–190.

Fahning, M.L., Garcia, M.A., 1992. Status of cryopreservation of embryosfrom domestic animals. Cryobiology 29, 1–18.

Fahy, G.M., 2010. Cryoprotectant toxicity neutralization. Cryobiology 60,S45–S53.

Green, R.E., Santos, B.F., Sicherle, C.C., Landim-Alvarenga, F.C., Bicudo, S.D.,2009. Viability of OPS vitrified sheep embryos after direct transfer.Reprod. Domest. Anim. 44, 406–410.

Isachenko, V., Alabart, J.L., Dattena, M., Nawroth, F., Cappai, P., Isachenko,E., Cocero, M.J., Oliveira, J., Roche, A., Accardo, C., Krivokharchenko, A.,Folch, J., 2003. New technology for vitrification and field (microscopefree) warming and transfer of small ruminant embryos. Theriogenol-ogy 59, 1209–1218.

Kasai, M., Niwa, K., Iritani, A., 1981. Effects of various cryoprotective agentson the survival of unfrozen and frozen mouse embryos. J. Reprod.Fertil. 63, 175–180.

Kuleshova, L.L., Shaw, J.M., Trounson, A.O., 2001. Studies on replacing mostof the penetrating cryoprotectant by polymers for embryo cryopres-ervation. Cryobiology 43, 21–31.

Leoni, G., Bogliolo, L., Berlinguer, F., Rosati, I., Pintus, P.P., Ledda, S., Naitana,S., 2002. Defined media for vitrification, warming, and rehydration:effects on post-thaw protein synthesis and viability of in vitro derivedovine embryos. Cryobiology 45, 204–212.

Loutradi, K.E., Kolibianakis, E.M., Venetis, C.A., Papanikolaou, E.G., Pados,G., Bontis, I., Tarlatzis, B.C., 2008. Cryopreservation of human embryosby vitrification or slow freezing: a systematic review and meta-analysis. Fertil. Steril. 90, 186–193.

Malo, C., Gil, L., Cano, R., Martínez, F., García, A., Jerez, R.A., 2012. Dimethyl-formamide is not better than glycerol for cryopreservation of boarsemen. Andrologia 44 (Suppl. 1), 605–610.

Martinez, A.G., Matkovic, M., 1998. Cryopreservation of ovine embryos:slow freezing and vitrification. Theriogenology 49, 1039–1049.

Massip, A., 1989. Some significant steps in the cryopreservation of mam-malian embryos with a note on a vitrification procedure. Anim.Reprod. Sci. 19, 117–129.

Massip, A., 2001. Cryopreservation of embryos of farm animals. Reprod.Domest. Anim. 36, 49–55.

ll Rumin

106 P.F.B. de Araújo-Lemos et al. / Sma

Mcginnis, L.K., Duplantis, S.C., Waller, S.L., Youngs, C.R., 1989. The use ofethyleneglycol for cryopreservation of sheep embryos. Theriogenol-ogy 31, 226 (abstract).

Mota Filho, A.C., Teles, C.H., Jucá, R.P., Cardoso, J.F., Uchoa, D.C., Campello,C.C., Silva, A.R., Silva, L.D., 2011. Dimethylformamide as a cryoprotec-tant for canine semen diluted and frozen in ACP-106C. Theriogenology76, 1367–1372.

Moustacas, V.S., Cruz, B.C., Varago, F.C., Miranda, D.A., Lage, P.G., Henry,M., 2011. Extenders containing dimethylformamide associated ornot with glycerol are ineffective for ovine sperm cryopreservation.Reprod. Domest. Anim. 46, 924–925.

Papadopoulos, S., Rizos, D., Duffy, P., Wade, M., Quinn, K., Boland, M.P.,Lonergan, P., 2002. Embryo survival and recipient pregnancy ratesafter transfer of fresh or vitrified, in vivo or in vitro produced ovineblastocysts. Anim. Reprod. Sci. 74, 35–44.

Rizos, D., Fair, T., Papadopoulos, S., Boland, M.P., Lonergan, P., 2002. Devel-opmental, qualitative, and ultrastructural differences between ovineand bovine embryos produced in vivo or in vitro. Mol. Reprod. Dev.62, 320–327.

Rizos, D., Gutierrez-Adan, A., Moreira, P., O’Meara, C., Fair, T., Evans,A.C., Boland, M.P., Lonergan, P., 2004. Species-related differences inblastocyst quality are associated with differences in relative mRNAtranscription. Mol. Reprod. Dev. 69, 381–386.

Shirazi, A., Soleimani, M., Karimi, M., Nazari, H., Ahmadi, E., Heidari, B.,2010. Vitrification of in vitro produced ovine embryos at variousdevelopmental stages using two methods. Cryobiology 60, 204–210.

Songsasen, N., Buckrell, B.C., Plante, C., Leibo, S.P., 1995. In vitro and in vivosurvival of cryopreserved sheep embryos. Cryobiology 32, 78–91.

ant Research 119 (2014) 100–106

Squires, E.L., Keith, S.L., Graham, J.K., 2004. Evaluation of alternative cry-oprotectants for preserving stallion spermatozoa. Theriogenology 62,1056–1065.

Stringfellow, D.A., Seidel, S.M., 1998. International Embryo Transfer Man-ual: A Procedural Guide and General Information for the Use of Embryotransfer Technology Emphasizing Sanitary Procedures. IETS publish.,Savoy, USA.

Szell, A., Zhang, J., Hudson, R., 1990. Rapid cryopreservation of sheepembryos by direct transfer into liquid nitrogen vapour at −180 ◦C.Reprod. Fertil. Dev. 2, 613–618.

Vajta, G., 2000. Vitrification of oocytes and embryos of domestic animals.Anim. Reprod. Sci. 60–61, 357–364.

Vajta, G., Kuwayama, M., 2006. Improving cryopreservation systems. The-riogenology 65, 236–244.

Whittingham, D.G., Leibo, S.P., Mazur, P., 1972. Survival of mouse embryosby frozen to −196◦C and −296◦C. Science 178, 411–414.

Whittingham, D.G., 1980. Principles of embryo preservation. In:Ashwood-Smith, M.J., Farrant, J. (Eds.), Low Temperature Preserva-tion In Medicine and Biology. Pitman Medical Ltd., England, UK,pp. 65–83.

Willadsen, S.M., Polge, C., Rowson, L.E.A., Moor, R.M., 1976. Deep freezingof sheep embryos. J. Reprod. Fertil. 46, 151–154.

Wilmut, I., 1972. The effect of cooling rate, cryoprotective agent and stage

of development on survival of mouse embryos during freezing andthawing. Life Sci. II 11, 1071–1079.

Zhu, S.E., Zeng, S.M., Yu, W.L., Li, S.J., Zhang, Z.C., Chen, Y.F., 2001. Vitri-fication of in vivo and in vitro produced ovine blastocysts. Anim.Biotechnol. 12, 193–203.