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ABSTRACT

Equine embryos can remain viable for 12 to 24 hourswhen cooled and stored at 5°C.1 Cryopreservation ofembryos would allow for long-term preservation of ge-netic material and more efficient management of em-bryo recipients. This study compared pregnancy ratesafter transfer of equine embryos vitrified within 1 hourof collection or cooled for 12 to 19 hours before vitrifi-cation. Mares (N = 40) were superovulated usingequine follicle-stimulating hormone (eFSH). Embryoswere recovered 6.5 days after ovulation or 8 days afterhuman chorionic gonadotropin. Forty morulae or earlyblastocysts with a grade of 1 to 2 and <300 mm in diam-eter were randomly assigned to 1 of 2 treatments:Group 1 (n = 20), washed 4 times in a commercial hold-ing medium and then vitrified; Group 2 (n = 20),washed 3 times and then stored in the same holdingmedium at 5°C to 8°C in a passive cooling device for 12to 19 hours before being vitrified. To thaw, embryoswere warmed by holding the straw in air at room tem-perature for 10 seconds and then submerged in a waterbath (20°C to 22°C) for an additional 10 seconds. Thecontents of the straw were transferred directly into arecipient that had ovulated 4 to 6 days previously.There were no differences (P > .05) in embryo diame-ter, grade, or morphology score between treatmentgroups before vitrification. Pregnancy rates (day 16)were not different (P > .05) between embryos vitrifiedimmediately after collection (15 of 20; 75%) and em-bryos cooled for 12 to 19 hours before vitrification (13of 20; 65%). Based on these results, small equine em-bryos (<300 mm) can be stored at 5°C to 8°C for 12 to

19 hours before vitrification without a significant lossof viability.Keywords: equine; embryo transfer; cooling; vitrification

INTRODUCTION

Advantages of embryo cryopreservation include theability to import and export equine embryos through-out the world, banking of genetic material from valu-able mares, and decreasing the cost of embryo transferby reducing the number of recipient mares that must bemaintained. With fresh embryos, a synchronized recipi-ent is needed; however, when embryos are cryopre-served, the embryo can be thawed and transferred atthe appropriate time in the recipient mare’s cycle.

The majority of previous studies have used a slowcool method for freezing equine embryos. Acceptablepregnancy rates were obtained with a slow cool freezingof equine embryos <300 mm in size.1-3 Recent work inour laboratory4 has shown that small equine embryoscan be placed in increasing concentrations of cryopro-tectant and vitrified. Transfer of morulae or early blasto-cysts <300 mm that had been vitrified resulted in 4 out of6 embryonic vesicles in a preliminary trial. Subsequently,embryos <300 mm were vitrified in the same ethyleneglycol-glycerol solution and transferred into synchro-nized recipients. Twenty-six of 48 embryos (54%) re-sulted in an embryonic vesicle at 14 to 16 days.4 Embryosin this study were from single-ovulating mares.Apparently, there are no studies on pregnancy rates aftertransfer of vitrified embryos from superovulated mares.

The vitrification process is quicker than slow coolcryopreservation and does not require specializedequipment. In addition, dilution of the cryoprotectantscan occur in the straw, and the embryo can be trans-ferred directly into the mare without removal from thestraw.4

Cooling and storage of equine embryos for 12 to 24hours at 5°C before transfer is a standard practice in theequine industry. Pregnancy rates are similar for embryoscooled and stored for 24 hours compared with embryostransferred immediately upon collection.1 Perhaps em-

SCIENTIFIC PAPER

The Effects of Cooling and Vitrification ofEmbryos From Mares Treated With Equine

Follicle-Stimulating Hormone on PregnancyRates After Nonsurgical Transfer

Jason Hudson, DVM, Patrick M. McCue, DVM, PhD, Elaine M. Carnevale, DVM, PhD,Susan Welch, MS, and Edward L. Squires, PhD

From the Animal Reproduction and Biotechnology Laboratory,Colorado State University, Fort Collins.Reprint requests: Edward L. Squires, Animal Reproduction andBiotechnology Laboratory, Foothills Campus, Colorado StateUniversity, Fort Collins, CO 80523-1683.0737-0806/$ - see front matter©2006 Elsevier Inc. All rights reserved.doi:10.1016/j.jevs.2005.11.012

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52 Journal of Equine Veterinary Science February 2006

bryos can be collected on the farm, cooled and stored,and then sent to a special facility for vitrification.

The objectives of this study were to determine ifcooling before vitrification would result in similar preg-nancy rates to embryos vitrified immediately upon col-lection and to determine the viability of vitrifiedembryos from superovulated mares.

MATERIALS AND METHODS

Embryos were provided from a companion study inour laboratory.5 Briefly, mares were administered ei-ther 12.5 mg of eFSH twice daily or 25 mg once per day.Treatments began 7 days after ovulation and continueddaily until the majority of follicles reached either 32mm or 35 mm in diameter, depending upon the exacttreatment. Prostaglandin (10 mg; PGF2�, Lutalyse) wasadministered to all mares on the second day of eFSHtreatment; human chorionic gonadotropin (2500 IU;Chorulon) was given to induce ovulation. Mares wereartificially inseminated with fresh semen at the time ofadministration with human chorionic gonadotropin.The following day, mares were inseminated with cooledsemen if all ovulations had not occurred. Ovulation wasconfirmed with daily transrectal ultrasonography.

Embryos were flushed from the uterus 6.5 days afterovulation if ovulations were asynchronous or 8 daysafter administration of human chorionic gonadotropinfor synchronous ovulations.5 A total of 4 L of Vigroflush medium was infused in the uterus: 1 to 2 L at atime was infused into the mare’s uterus and allowed todrain out of the uterus into a filter cup.1 The fluid re-maining in the filter cup was poured into a search dishand examined under 7� magnification for the presenceof an embryo. The catheter remained in the mare dur-ing the initial search for the embryo. If the total numberof embryos recovered matched the total number ofovulations, the catheter was removed and the mare wasadministered PGF2�. However, if the total number ofembryos recovered was less than the total number ofovulations, an extra flush was administered.6 The extraflush consisted of infusing an additional 1 to 2 L ofmedia into the uterus, and 20 IU of oxytocin was givenintravenously. The flush medium was allowed to sit inthe mare’s uterus for 3 minutes before retrieval.Filtered medium from the extra flush was searched forthe presence of an embryo.

Upon identification, each embryo was measuredusing a linear eyepiece micrometer and graded.7 Aftersizing and grading, each embryo was rinsed 4 times inVigro holding solution in a 35-mm dish. The embryowas then placed in holding medium in a separate 35-mm dish until the vitrification process was per-formed, or they were packaged and placed in a passivecooling device1 to be cooled for 12 to 19 hours beforevitrification. Embryos (N = 40) were randomly assigned

to 1 of 2 groups: Group 1, cooled to 5°C in a passivecooling device for 12 to 19 hours before vitrification;and Group 2, vitrified immediately upon collection.Embryos in Group 1 were placed in a 5-mL plastic tubecontaining Vigro holding solution. This tube was placedinside a 50-mL Falcon tube containing Vigro flushmedium, then surrounded by ballast bags and placed in-side the cooling device.

Embryos were vitrified as previously described fordirect transfer.4 Briefly, after the embryos were rinsedin Vigro holding medium, they were transferred into200-mL droplets of the first vitrification solution (VS-1) containing 1.4 M glycerol. The embryo was held inVS-1 for 5 minutes and then transferred into another200-�L drop (VS-2), which contained 1.4 M glycerolplus 3.6 M ethylene glycol. The embryo also remainedin this solution for 5 minutes. Last, the embryo wasplaced in a 30-�L drop of VS-3 solution for 50 to 60 sec-onds. VS-3 consisted of 3.4 M glycerol plus 4.6 M ethy-lene glycol. The allotted time of 1 minute includedplacing the embryo in VS-3 solution, then loading theembryo into a 0.25-mL non-irradiated polyvinylchlo-ride straw and sealing the straw.

The embryo was loaded as follows: 90 �L of dilutionsolution consisting of 0.5 M galactose and phosphate-buffered saline; a 5- to 10-�L air bubble; 30 �L of VS-3containing the embryo; a second air bubble; and 90 �Lof dilution solution.4 Also during this 1-minute period,the straw was double heat-sealed and then placed into acooled plastic goblet (10�120 mm; IMV, Maple Grove,MN) held vertically in liquid nitrogen within an insu-lated container. This allowed liquid nitrogen to sur-round the goblet and produce a mixture of cooled airand nitrogen vapor around the straw at a temperatureof �190°C. The straw remained in the vapor for 1minute before the goblet and the straw were plungedinto liquid nitrogen. Straws were then placed in a liquidnitrogen storage tank until being warmed for transferinto recipient mares. For vitrification of cooled em-bryos, the embryos were removed from the Equitainer,placed into a 5°C holding medium, and allowed to equi-librate to room temperature before vitrification.

Embryos were transferred during the month ofAugust. Recipients were administered PGF2� to induceestrus and then examined daily with ultrasonography todetermine the day of ovulation. Embryos were trans-ferred into recipients 4 to 6 days after ovulation. Eachrecipient was administered 500 mg of flunixinmeglamine intravenously and 150 mg of xylazine intra-venously before transfer.

Embryos were warmed by removing the straw fromthe liquid nitrogen tank and holding it in air at roomtemperature for 10 seconds before plunging it into abath of water (20°C to 22°C) for an additional 10 sec-onds. The straw was removed from the water bath

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and flicked like a clinical thermometer 4 to 5 times toassure mixing of the solutions. The straw was examinedin a horizontal position under the dissecting scope forthe presence of the embryo. Straws were kept in a hori-zontal position for about 6 minutes before transfer.

For transfer, the straw was loaded into a specialCassou gun designed for cut straws. Each embryo wastransferred into a recipient within 8 minutes after re-moval of the straw from the liquid nitrogen tank. Alltransfers were performed using a nonsurgical, transcer-vical approach. Ultrasonography was used to determinepregnancy 6 to 10 days after transfer.

StatisticsData were analyzed by Fisher exact test or t test. All

data are presented as the mean ± SEM.

RESULTS

A total of 73 flushes were performed on 38 eFSH-treated mares. On average, mares had experienced 3.5ovulations per cycle for a total of 255 ovulations with148 embryos recovered (58% embryo recovery perovulation). During an untreated control cycle, 40 ovula-tions were detected (1.05 ovulations per mare), but em-bryo recovery was not attempted on control cycles. Allmares flushed 6.5 days after ovulation or 8 days afterhuman chorionic gonadotropin provided morulae orearly blastocysts that were �300 mm.

Pregnancy rates were similar, in both groups, for re-cipients that were 4, 5, or 6 days post-ovulation on theday of transfer (Table 1). There was no difference inpregnancy rates between morulae and early blastocysts(Table 2). Average embryo diameter was similar be-tween the 2 treatments (cooled/vitrified 185 ± 7.1 �m;vitrified 187 ± 5.0 �m).

Embryo grades were similar (P > .05) between the 2treatment groups before and after vitrification.Averagescores for embryos previtrification were 1.4 ± 0.1 and1.2 ± 0.1 for the cooled/vitrified and vitrified groups, re-spectively.

There was no difference (P > .05) between preg-nancy rates of embryos that were vitrified immediatelyupon collection versus those that were cooled for an av-erage of 15 hours before vitrification (Table 2).Pregnancy rates for the 2 groups were 13 out of 20

(65%) for embryos cooled before vitrification and 15out of 20 (75%) for embryos directly vitrified. Theoverall pregnancy rate was 70% (28 out of 40).

DISCUSSION

Superovulation can be used to increase embryo pro-duction from mares. In this study, during the pretreat-ment cycles, mares ovulated approximately 1 follicleper cycle. In response to eFSH treatment, mares ovu-lated an average of 3.5 follicles per cycle and providedapproximately 2 embryos per recovery attempt.Although the mares were not flushed for embryos onthe control cycle, generally embryo recovery in un-treated mares is approximately 0.5 to 0.6 embryo perrecovery attempt.1

Only embryos <300 �m were selected for thisstudy, since previous studies have demonstrated thatembryos >300 �m either slow cooled2,3 or vitrified4

resulted in very poor pregnancy rates. It has been sug-gested that the acellular capsule that forms after ap-proximately day 7 impedes penetration of the embryoby the cryoprotectants.8 Upon entering the uterus, theembryo grows very rapidly, making it difficult to ob-tain embryos <300 �m unless the mares are examinedfrequently for detection of ovulation. However, inthis study and in a previous study,4 instead of fre-quent examinations for detection of ovulation, themare’s uterus was flushed either 8 days after adminis-tration of human chorionic gonadotropin or 6.5 daysafter the last ovulation. The average interval to ovula-tion after human chorionic gonadotropin is approxi-mately 36 hours. All the embryos recovered fromsuperovulated mares by this procedure were �300mm in size. This is similar to the success of using thisprocedure on single-ovulating mares.4

Effect of recipient synchrony on pregnancyrates (%) after nonsurgical transfer.*

Days From Cooled/ VitrifiedOvulation Vitrified Immediately

4 1/2 (50%) N/A5 11/16 (69%) 12/17 (71%)6 1/2 (50%) 3/3 (100%)

*No significant difference noted (Fisher exact test).

Table 1

Effect of embryo developmental stage on pregnancy rates (%) after nonsurgical transfer.*

Developmental Stage Cooled/Vitrified Vitrified Immediately Combined

Late morulae 5/7 (71%) 4/4 (100%) 9/11 (82%)Early blastocysts 8/13 (62%) 11/16 (69%) 19/29 (66%)Combined 13/20 (65%) 15/20 (75%) 28/40 (70%)

*There were no significant differences (P > .01); Fisher exact test.

Table 2

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opment of pregnancy in these mares. However,Eldridge-Panuska et al4 did conduct a similar studywhere embryos were vitrified, warmed, and transferredinto recipients and pregnancy exams done on day 16.Seven pregnancies were allowed to continue beyondday 16, and all developed normally to �35 days, atwhich time the pregnancies were terminated.

CONCLUSIONS

In summary, cooling of embryos for 12 to 19 hours be-fore vitrification did not reduce pregnancy rates after warm-ing and transfer. Embryos <300 �m in size can be cooled,vitrified, thawed, and directly transferred into recipientmares with high pregnancy rates. Further studies are neededto determine whether other slow-cooling methods for freez-ing or other vitrification methods can be used to freeze em-bryos >300 �m in size.

ACKNOWLEDGMENTS

Funding was provided by the benefactors of thePreservation of Equine Genetics Program. Equinefollicle-stimulating hormone and Vigro flush mediumand holding medium were provided by Bioniche AnimalHealth, Athens, Georgia, and AB Technology, Pullman,Washington, respectively. Special thanks to JoAnneStokes, Amanda Beisner, and Chris Wilson for their tech-nical assistance and Jason Abraham for providing recip-ient mares.

REFERENCES1. Squires EL, McCue PM, Vanderwall DK. The current status of

equine embryo transfer. Theriogenology 1999;51:91-104.2. Slade NP, Takeda T, Squires EL, Elsden RP, Seidel GE Jr. A new

procedure for the cryopreservation of equine embryos.Theriogenology 1985;24:45-58.

3. Squires EL, Seidel GE Jr, McKinnon AO. Transfer of cryopreservedequine embryos to progestin-treated ovariectomized mares.Equine Vet J 1989;8(Suppl):89-91.

4. Eldridge-Panuska WD, Caracciolo de Brienza V, Seidel GE Jr,Squires EL, Carnevale EM. Establishment of pregnancies after se-rial dilution or direct transfer by vitrified equine embryos.Theriogenology 2005;63:1308-19.

5. Squires EL, Welch SA, Denniston DJ, Bruemmer JE, McCue PM.Evaluation of several eFSH treatments for superovulation ofmares. Proc Society for Theriogenology 2005 (abstr); in press.

6. Hudson JJ, McCue PM. How to increase embryo recovery ratesand transfer success. In: Proc 50th AAEP Convention; 2004. pp406-9.

7. McKinnon AO, Squires EL. Morphological assessment of theequine embryo. J Am Vet Med Assoc 1988;192:401-6.

8. Legrand E, Bencharif D, Barrier-Battut I, Delajarraud H, CorniereP, Fieni F, et al. Comparison of pregnancy rates for days 7-8 em-bryos frozen in glycerol with or without previous enzymatic treat-ment of their capsule. Theriogenology 2002;58:721-3.

9. McKinnon AO, Squires El, Voss JL, Cook VM. Equine embryotransfer. Comp Cont Educ Pract Vet 1988;10:343-55.

10. Squires EL, Carnevale EM, McCue PM, Bruemmer JE. Embryotechnologies in the horse. Theriogenology 2003;59:151-70.

11. Maclellan LK, Carnevale EM, Coutinho da Silva MA, McCue PM,Seidel GE Jr, Squires EL. Cryopreservation of small and largeequine embryos pre-treated with cytochalasin-B and/or trypsin.Theriogenology 2002;58:717-20.

The majority of transfers were made into mares thathad ovulated 5 days earlier. Only small numbers of em-bryos were transferred into mares ovulating 4 and 6days before a transfer. Pregnancy rates are similar forrecipient mares that ovulate 1 day before the donor andup to 3 days after the donor when fresh embryos aretransferred.1,9 Although the majority of recipients usedin this study were synchronized to ovulate within a1-week period to accommodate transfer of all the em-bryos in a short period of time, a major advantage offrozen embryos is to provide flexibility in not having tomaintain a large number of recipient mares. The em-bryo can remain in liquid nitrogen until the recipientmare cycles naturally and then warmed and placed intothe mare at the appropriate stage of the cycle.

Generally, if one flushes a mare’s uterus 6.5 daysafter ovulation, either late morulae or early blastocystswill be obtained. In this study, the pregnancy rates weresimilar for embryos in these 2 stages of development.However, in other studies, when morulae were col-lected from mares that had ovulated 7 or 8 days previ-ously, pregnancy rates for transfer of morulae was lessthan that for early blastocysts or blastocysts.10 This isdue primarily to the retarded development of thesemorulae flushed from the mare’s uterus 7 or 8 daysafter ovulation.

Squires et al1 reported that pregnancy rates fromgrade 1 and 2 embryos were similar and higher thanthose of grade 3 embryos. Superovulation did not ap-pear to affect the grade of embryos, since the majorityof embryos (>90%) obtained in this study were ofgrade 1 or 2.

Cooling embryos slowly to 5°C and storing embryosfor 12 to 24 hours does not affect pregnancy rates afternonsurgical transfer.1 Thus, shipping cooled embryos toa facility that maintains a large number of recipients isa common practice. Based on the results of this study, itappears that one has the flexibility to either transfer thecooled embryo upon arrival at a recipient facility or, ifrecipients are in short supply, vitrify the embryo.Apparently, this is the first study on pregnancy rate ofcooled, stored, and vitrified embryos. Maclellan et al11

froze embryos by slow cool and, after thawing, culturedembryos for 6 hours before nonsurgical transfer.Pregnancy rates were similar between embryos cul-tured for 6 hours before transfer and those transferredimmediately upon thawing.

The overall pregnancy rates for vitrified embryoswas 70%. This is similar to rates obtained for nonfrozenembryos10 and slow-cooled frozen embryos.11 The via-bility of vitrified embryos from superovulated mares inthis study was no different from that of vitrified em-bryos from single-ovulating mares (16 of 26; 62%) in aprevious study in our laboratory.4

The end point of day 16 of pregnancy limits the con-clusions that can be drawn, relative to continued devel-

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