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Pregnancies from vitrified equine oocytes collected from super-stimulated and non-stimulated mares
Theriogenology 58 (2002) 911±919
Pregnancies from vitri®ed equine oocytes collected from super-stimulated and non-stimulated mares L.J. Maclellan, E.M. Carnevale, M.A. Coutinho da Silva, C.F. Scoggin, J.E. Bruemmer, E.L. Squires* Department of Physiology, Animal Reproduction and Biotechnology Laboratory, Colorado State University, Foothills Campus, Fort Collins, CO 80523-1683, USA Received 20 July 2001; accepted 29 October 2001
Abstract The objectives were to compare embryo development rates after transfer into inseminated recipients, vitri®ed thawed oocytes collected from super-stimulated versus non-stimulated mares. In vivo matured oocytes were collected by transvaginal, ultrasound guided follicular aspiration from super-stimulated and non-stimulated mares 24±26 h after administration of hCG. Oocytes were cultured for 2±4 h prior to vitri®cation. Cryoprotectants were loaded in three steps before oocytes were placed onto a 0.5±0.7 mm diameter nylon cryoloop and plunged directly into liquid nitrogen. Oocytes were thawed and the cryoprotectant was removed in three steps. After thawing, oocytes were cultured 10±12 h before transfer into inseminated recipients. Non-vitri®ed oocytes, cultured 14±16 h before transfer, were used as controls. More oocytes were collected from 23 non-stimulated mares (20 of 29 follicles), than 10 superstimulated mares (18 of 88 follicles; P 0:001). Of the 20 oocytes collected from non-stimulated mares, 12 were vitri®ed and 8 were transferred as controls. After thawing, 10 of the 12 oocytes were morphologically intact and transferred into recipients resulting in one embryonic vesicle on Day 16 (1 of 12 8%). Fourteen oocytes from super-stimulated mares were vitri®ed, and 4 were transferred as controls. After thawing, 9 of the 14 oocytes were morphologically intact and transferred into recipients resulting in two embryonic vesicles on Day 16 (2 of 14 14%). In control transfers, 7 of 8 oocytes from non-stimulated mares and 3 of 4 oocytes from super-stimulated mares resulted in embryonic vesicles on Day 16. The two pregnancies from vitri®ed oocytes resulted in healthy foals. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Equine; Oocyte vitri®cation; Super-stimulation; Oocyte transfer; Cryopreservation
* Corresponding author. Tel.: 1-970-491-8409; fax: 1-970-491-3557. E-mail address: [email protected] (E.L. Squires).
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1. Introduction Oocytes are dif®cult to cryopreserve because of the high surface to volume ratio, which limits rates of movement of cryoprotectants and water into and out of oocytes [1]. Low temperature sensitivity [2,3], alterations that occur to the zona pellucida [4], and the required removal of cumulus cells [1,5] pose dif®culties in successful oocyte cryopreservation. Mammalian oocytes of many species, particularly immature oocytes, appear to be more sensitive than zygotes and embryos to cooling [3] or cryopreservation [1]. Immature equine oocytes at the germinal vesicle stage are dif®cult to mature in vitro [6]; therefore, many studies on cryopreservation of oocytes have focused on oocytes at Metaphase II (MII; [7±10]). Cryopreservation of oocytes at later meiotic stages is problematic because of spindle disorganization in ovulated oocytes at MII [11] and increased polyploidy at fertilization [12,13]. The ®rst reports of successful oocyte cryopreservation with production of blastocysts and offspring were with slow-rate freezing of bovine oocytes [14]. Recently, vitri®cation using a novel containerless technique has been used for the cryopreservation of oocytes [9] and embryos [15] as an alternative method to traditional slow-rate freezing. Improved vitri®cation protocols have resulted in advances in oocyte cryopreservation, particularly with bovine oocytes. Recently, blastocyst production rates after in vitro fertilization and culture have been similar to non-cryopreserved oocytes [3,16,17]. Decreased fertilization rates for cryopreserved germinal vesicle and MII stage oocytes [4,18,19] have been reported; however fertilization rates of bovine oocytes vitri®ed before the GV stage, the Metaphase I to Metaphase II transition, or at MII were not signi®cantly lower than controls [4]. Fertilization rates of equine oocytes vitri®ed 12 or 24 h after onset of maturation were not signi®cantly different from control oocytes after intracytoplasmic sperm injection (ICSI) [20]. In the mare, a limited number of oocytes can be collected from preovulatory follicles. Super-stimulation protocols aimed at increasing the number of preovulatory follicles per cycle would bene®t assisted reproduction technologies by providing larger numbers of oocytes for experimental purposes. Previous attempts to stimulate follicular growth in mares resulted in increased numbers of follicles 25 mm [21±24]. Initiation of treatment with equine pituitary extract (EPE) on Day 5 rather than Day 12 after ovulation has resulted in signi®cantly more follicles 25 mm [21]. Most equine oocytes matured in vivo and collected just before ovulation are at Metaphase II [25]. Oocytes have been recovered from donor mares at 24±26 h after hCG, or approximately 12 h prior to the expected time of ovulation. When such oocytes were cultured in vitro for the completion of maturation before surgical transfer to recipients, pregnancy rates of 62±92% resulted [26,27]. Collection of oocytes from mares 24±26 h after hCG for vitri®cation studies would eliminate some of the problems associated with the maturation in vitro of immature equine oocytes. Objectives of the present study were to compare embryo development rates after transfer of oocytes into inseminated recipient mares using oocytes collected from super-stimulated versus non-stimulated mares, and cultured with or without prior vitri®cation.
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2. Materials and methods 2.1. Oocyte collection Light horse mares (n 33) evaluated for reproductive soundness and between 3 and 15 years of age were used for the study. Beginning 5 days after a natural ovulation, 10 mares were administered 25 mg of EPE twice daily, prepared using a modi®cation of the method of Guillou and Combarnous [28]; the protein was reconstituted with sterile 0.15 M sodium chloride solution after lyophilization, and ®ltered through a 0.22 micron ®lter without further puri®cation; gel ®ltration and ion exchange chromatography were not used. A single dose of prostaglandin, 250 mg i.m. cloprostenol (Bayer, Shawnee Mission, KS, USA) was administered i.m. 7 days after ovulation. EPE treatment was discontinued when half of the developing follicles reached a diameter 35 mm. At this time, human chorionic gonadotropin (hCG, Steris Laboratories Inc., Phoenix, AZ, USA; 2500 IU) was administered i.v. to initiate synchronous follicular and oocyte maturation. For control mares (n 23), ovarian follicular activity was monitored by transrectal ultrasonography. When relaxed uterine and cervical tone, uterine edema, and a follicle 35 mm in diameter were observed, hCG (Steris Laboratories Inc.; 2500 IU) was administered i.v. to initiate follicular and oocyte maturation. Oocytes were collected by transvaginal, ultrasound guided follicular aspirations using a linear transducer and a double lumen needle (Cook Veterinary Products Inc., New Buffalo, MI, USA). For aspirations, donor mares were sedated (Xylazine HCl, Vedco, Inc., St. Joseph, MO, USA; 0.33 mg/kg, i.v. and Butorphanol tartrate, Fort Dodge Animal Health, Fort Dodge, IA, USA; 0.01 mg/kg, i.v.) and propantheline bromide (Sigma Chemical Co., St. Louis, MO, USA; 0.05 mg/kg i.v.) was administered to promote relaxation of the rectum. Contents of the follicle were gently aspirated (150 mg Hg) using a vacuum pump (Cook Veterinary Products Inc.), and the follicle was lavaged with ¯ush medium (Emcare1 complete ¯ush solution, ICP, New Zealand). Upon collection, cumulus oocyte complexes (COC) were evaluated for cumulus expansion (graded from compact to fully expanded, Table 1). Oocytes for control transfer were cultured 14±16 h in Equine Maturation Medium I (EMMI) [20] before transfer. Oocytes for cryopreservation were placed in culture medium (EMMI) for approximately 2±4 h before vitri®cation. 2.2. Recipient mares Only mares from which an oocyte was collected from all follicles >35 mm were used as recipients. Seven control mares with an oocyte collected from a single preovulatory follicle and one super-stimulated mare with oocytes collected from two follicles were used as recipients. Recipient mares were inseminated with 2 109 progressively motile sperm from 1 of 2 stallions of known fertility, into the uterine body 15±18 h before oocyte transfer. Semen was extended in a skim-milk and glucose extender (E-Z Mixin CST; Animal Reproduction Systems, Chino, CA, USA).
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Table 1 Cumulus expansion of oocytes from super-stimulated and non-stimulated mares Treatment
Super-stimulated (n 18) Non-stimulated (n 20)
Cumulus expansion at collection None
Slight
Moderate
Good
Complete
0 0
0 0
7 11
6 9
5a 0b
Values within columns with different superscripts differ (P < 0:05).
The recipient's ovary and oviduct were exposed through a standing ¯ank laparotomy. Oocytes were placed in modi®ed G2 medium [29] containing 20 mM hepes (Sigma Chemical Co.), 5 mM sodium bicarbonate (Sigma Chemical Co.) and 8 mg/ml BSA (Pentex, Kankakee, MI, USA) with no human serum albumin or phenol red. Oocytes were pulled into a ®re-polished, glass pipette with <0.3 ml of medium. The pipette was threaded 2±3 cm through the recipient's infundibulum, and oocytes were slowly deposited within the oviduct. Only oocytes that appeared morphologically normal were transferred. Two to ®ve oocytes were transferred into each recipient. Before surgery and during the two subsequent days, the ovaries and uteri of the recipients were scanned to ensure that no recipient ovulated an additional follicle, and for the presence of intrauterine ¯uid. No ovulations were detected, but ¯uid was observed in 6 of 8 recipients. Recipients with intrauterine ¯uid received 1 ml of oxytocin (Vedco, Inc.; 20 unit/ml) before surgery and on the two subsequent days if ¯uid was still observed. The day after surgery, all recipients were given daily injections of progesterone (Sigma Chemical Co.) in cottonseed oil (Sigma Chemical Co.; 150 mg, i.m.) until diagnosis of pregnancy. Uteri of recipients were scanned with ultrasound on Days 11±14, 16, 20, and 25 to determine the presence and number of embryonic vesicles. The embryo development rate was de®ned as the number of embryonic vesicles per number of oocytes assigned to each treatment that developed into embryonic vesicles that were imaged with ultrasound by Day 16. If pregnant after transfer of cryopreserved oocytes, recipients were maintained on a synthetic progestin (altrenogest, Intervet Inc., Millsboro, DE, USA; 0.044 mg/kg) given orally until Day 120. 2.3. Vitri®cation and warming Procedures for vitri®cation and thawing oocytes were performed in a heated room (28± 30 8C), and on a heated microscope stage (37±38 8C). COC were gently pipetted with a large-bore pipette in modi®ed G2 until 8±10 cumulus cell layers remained. Cryoprotectants were loaded in three steps: 5% dimethyl sulfoxide (DMSO; Sigma Chemical Co.; 0.7 M) and 5% ethylene glycol (EG; Sigma Chemical Co.; 0.9 M) in modi®ed G2 for 30 s; 10% DMSO (1.4 M) and 10% EG (1.8 M) in modi®ed G2 for 30 s; and 20% DMSO (2.8 M), 20% EG (3.6 M), 10 mg/ml ®coll (Sigma Chemical Co.) with 0.65 M sucrose in modi®ed G2 for 20 s, before being loaded onto a 0.5±0.7 mm diameter nylon loop, mounted on a stainless steel pipe inserted into the lid of a 2 ml cryovial (Hampton Research, Laguna Niguel, CA, USA) and directly plunged into liquid nitrogen. Oocytes were stored for up to 2 months before transfer. Oocytes were thawed and diluted in three steps by immersing the
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nylon cryoloop in 0.250 M, 0.188 M and 0.125 M sucrose in modi®ed G2 at 38 8C for 30 s per step. Oocytes were cultured for 10±12 h in EMMI before transfer into inseminated recipients. 2.4. Statistical analyses Collection rates and cumulus expansion of oocytes from super-stimulated and nonstimulated mares and embryonic development rates per vitri®ed or non-vitri®ed oocyte were compared by Fisher's Exact Test. 3. Results More oocytes (P 0:001) were collected from the aspiration of follicles of 23 nonstimulated mares (20 of 29 oocytes/follicle) than 10 super-stimulated mares (18 of 88 oocytes/follicle). At collection, more (P < 0:05) of the oocytes from super-stimulated mares had complete expansion of the cumulus cell layer yet other morphological grades appeared similar (P > 0:05, Table 1). There was no relationship between degree of cumulus expansion and survival of oocytes after vitri®cation or in embryonic vesicle formation. Of the 20 oocytes collected from non-stimulated mares, 12 were vitri®ed and 8 were transferred as controls. After thawing and dilution of cryoprotectant, 10 of the 12 oocytes were morphologically normal in appearance with intact plasma membranes and were transferred into recipients. One embryonic vesicle resulted from transfer of the 10 oocytes into inseminated recipients. Oocytes from super-stimulated mares were vitri®ed (n 14), or transferred as controls (n 4). After thawing, 9 of 14 vitri®ed oocytes were considered morphologically normal in appearance with intact membranes and were transferred into inseminated recipients. Two embryonic vesicles were established in one recipient. Percentages of oocytes considered morphologically normal after vitri®cation were not different (P > 0:05) between non-stimulated and stimulated mares (10 of 12, 83% and 9 of 14, 64%, respectively). All oocytes for control transfers were considered morphologically normal and were transferred into recipients. Seven of 8 oocytes from non-stimulated and 3 Table 2 Effect of treatment on the number of oocytes transferred into recipient mares and rate of embryonic development per total number of oocytes assigned to vitrification or control Treatment
Total oocytes (n)
Non-stimulated vitrified Super-stimulated vitrified
12 14
Total control
3/26 (12%)a 7/8 (88%)b 3/4 (75%)b
8 4 12
Embryonic development 1/12 (8%)a 2/14 (1%)a
19/26 (73%)a
Total vitrified Non-stimulated control Super-stimulated control
Morphologically normal oocytes transferred into recipients
12/12 (100%)b
Values within columns with different superscripts differ (P < 0:05).
10/12 (83%)b
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of 4 oocytes from super-stimulated mares resulted in embryonic vesicles. There was no difference (P < 0:05) in embryonic development rates of vitri®ed or control oocytes from non-stimulated versus super-stimulated mares; therefore, results were pooled for vitri®ed and control oocytes. Of the 26 vitri®ed oocytes, 7 were determined to have severe morphological abnormalities after thawing and were not transferred. More (P 0:01) control oocytes than vitri®ed oocytes resulted in embryo development (10 of 12 83% vs. 3 of 26 12%, respectively; Table 2). After reduction of one of the twin vesicles, two recipients produced healthy foals from vitri®ed oocytes. 4. Discussion This study represents the ®rst pregnancies after transfer of vitri®ed and thawed equine oocytes. Although viability of vitri®ed oocytes was lower than control oocytes, the establishment of pregnancies from cryopreserved oocytes is a signi®cant advance in developing methods to preserve equine gametes. Few studies have described cryopreservation of equine oocytes [20,30±33]. Survival rates after thawing of equine oocytes, after conventional slow freezing [30] or vitri®cation [31±33] were lower than for non-cryopreserved oocytes. Successful fertilization after cryopreservation of equine oocytes has been reported twice [20,30]. In the present study, stepwise equilibration for short periods with cryoprotectant before vitri®cation resulted in successful rates of cryopreservation based on subsequent pregnancy rates. Stepwise exposure of oocytes to cryoprotectants has previously been demonstrated to be bene®cial to the vitri®cation of immature equine oocytes [32]. With immature and mature human oocytes, a short equilibration process of 20 s in cryoprotectant resulted in 56±100% morphologically normal oocytes after vitri®cation [34]. The short equilibration period in this study may have helped prevent severe osmotic damage from the high concentration of cryoprotectant in the vitri®cation solution. Vitri®cation of immature [35] and MII stage bovine oocytes [6,35] has resulted in fertilization rates similar to untreated controls [6]. Previously reported rates of fertilization of slaughterhouse immature equine oocytes following vitri®cation were signi®cantly lower than those of non-vitri®ed oocytes [31]. In the present study, embryonic development rates of vitri®ed-thawed oocytes collected from mares were signi®cantly lower than controls; however two foals were produced. Success of in vitro matured equine oocytes from small follicles remains low, indicating that conditions for oocyte maturation are not optimized [5,36]. Oocytes collected from slaughterhouse material are of unknown age, reproductive history, and stage of nuclear maturation, which can contribute to variability in results [37± 39]. Until maturation protocols are optimized for equine oocytes from small follicles, slaughterhouse oocytes are not the optimal source of material for experimentation. Embryonic development rates for super-stimulated and non-stimulated oocytes were similar in both control and vitri®ed oocyte transfers. Findings are similar to previous studies in which the administration of EPE to mares for embryo collection did not adversely in¯uence pregnancy rates [24] and oocytes collected from super-stimulated or non-stimulated mares matured at similar rates [40]. In the present study, a range of
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morphologic grades of COC was observed at collection, but morphology grades were not related to subsequent embryonic development after transfer. The pregnancy rates for nonvitri®ed controls were similar to those previously reported for oocyte transfer [26,41±43]. Super-stimulation of follicular growth with EPE increased the number of preovulatory follicles per mare but not the number of oocytes collected. The recovery rate from preovulatory follicles was 69% in control mares and 20% in super-stimulated mares. The large number of follicles present in the ovaries of super-stimulated mares presented a problem during the oocyte collection. The ovaries were large and dif®cult to manipulate during aspiration. The less successful collection of oocytes from super-stimulated than control mares may have been caused in part by the immature or atretic state of some of the follicles. A high incidence of low steroid concentrations was found in follicles in which oocytes were not recovered, indicating that the follicles were immature [44]. Collection of oocytes from immature follicles has been reported to be lower because the compact cumulus was highly attached to the follicular wall [43,45,46]. The optimal point in time from the resumption of maturation for cryopreservation of equine oocytes has not been established, and data from the present study did not establish an optimum stage for oocyte vitri®cation. Further studies focusing on the stage of maturation of equine oocytes before cryopreservation could lead to increased pregnancy rates after transfer to recipients. The results of this study were encouraging in that viable pregnancies were established after transfer of vitri®ed equine oocytes. Super-stimulation of mares with EPE did not increase the number of oocytes recovered per mare or affect the viability of oocytes after transfer to recipients. Further studies are required to improve techniques for equine oocyte vitri®cation. Acknowledgements We thank Heather Reger and Kimberly Preis for assistance with collection of oocytes and George Seidel Jr., Gabor Vatja, and Michelle Lane for scienti®c input. This research was funded by the Lucy Whittier Foundation and benefactors of the Preservation of Equine Genetics program. References [1] Vatja G. Vitri®cation of oocytes and embryos of domestic animals. Anim Reprod Sci 2000;60/61:357±64. [2] Aman R, Parkes J. Effects of cooling and rewarming on meiotic spindle and chromosomes of in vitro matured bovine oocytes. Biol Reprod 1994;50:103±10. [3] Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996;54:1059±69. [4] Fuku E, Xia L, Downey BR. Ultrastructural changes in bovine oocytes cryopreserved by vitri®cation. Cryobiology 1995;32:139±56. [5] Le Gal F, Massip A. Cryopreservation of cattle oocytes: effects of meiotic stage, cycloheximide treatment, and vitri®cation procedure. Cryobiology 1999;38:290±300. [6] Scott TJ, Carnevale EM, Maclellan LJ, Scoggin CF, Squires EL. Embryo development rates after transfer of oocytes matured in vivo, in vitro or within oviducts of mares. Theriogenology 2001;55:705±15.
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Pregnancies from vitri®ed equine oocytes collected from super-stimulated and non-stimulated mares L.J. Maclellan, E.M. Carnevale, M.A. Coutinho da Silva, C.F. Scoggin, J.E. Bruemmer, E.L. Squires* Department of Physiology, Animal Reproduction and Biotechnology Laboratory, Colorado State University, Foothills Campus, Fort Collins, CO 80523-1683, USA Received 20 July 2001; accepted 29 October 2001
Abstract The objectives were to compare embryo development rates after transfer into inseminated recipients, vitri®ed thawed oocytes collected from super-stimulated versus non-stimulated mares. In vivo matured oocytes were collected by transvaginal, ultrasound guided follicular aspiration from super-stimulated and non-stimulated mares 24±26 h after administration of hCG. Oocytes were cultured for 2±4 h prior to vitri®cation. Cryoprotectants were loaded in three steps before oocytes were placed onto a 0.5±0.7 mm diameter nylon cryoloop and plunged directly into liquid nitrogen. Oocytes were thawed and the cryoprotectant was removed in three steps. After thawing, oocytes were cultured 10±12 h before transfer into inseminated recipients. Non-vitri®ed oocytes, cultured 14±16 h before transfer, were used as controls. More oocytes were collected from 23 non-stimulated mares (20 of 29 follicles), than 10 superstimulated mares (18 of 88 follicles; P 0:001). Of the 20 oocytes collected from non-stimulated mares, 12 were vitri®ed and 8 were transferred as controls. After thawing, 10 of the 12 oocytes were morphologically intact and transferred into recipients resulting in one embryonic vesicle on Day 16 (1 of 12 8%). Fourteen oocytes from super-stimulated mares were vitri®ed, and 4 were transferred as controls. After thawing, 9 of the 14 oocytes were morphologically intact and transferred into recipients resulting in two embryonic vesicles on Day 16 (2 of 14 14%). In control transfers, 7 of 8 oocytes from non-stimulated mares and 3 of 4 oocytes from super-stimulated mares resulted in embryonic vesicles on Day 16. The two pregnancies from vitri®ed oocytes resulted in healthy foals. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Equine; Oocyte vitri®cation; Super-stimulation; Oocyte transfer; Cryopreservation
* Corresponding author. Tel.: 1-970-491-8409; fax: 1-970-491-3557. E-mail address: [email protected] (E.L. Squires).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 9 2 0 - 2
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1. Introduction Oocytes are dif®cult to cryopreserve because of the high surface to volume ratio, which limits rates of movement of cryoprotectants and water into and out of oocytes [1]. Low temperature sensitivity [2,3], alterations that occur to the zona pellucida [4], and the required removal of cumulus cells [1,5] pose dif®culties in successful oocyte cryopreservation. Mammalian oocytes of many species, particularly immature oocytes, appear to be more sensitive than zygotes and embryos to cooling [3] or cryopreservation [1]. Immature equine oocytes at the germinal vesicle stage are dif®cult to mature in vitro [6]; therefore, many studies on cryopreservation of oocytes have focused on oocytes at Metaphase II (MII; [7±10]). Cryopreservation of oocytes at later meiotic stages is problematic because of spindle disorganization in ovulated oocytes at MII [11] and increased polyploidy at fertilization [12,13]. The ®rst reports of successful oocyte cryopreservation with production of blastocysts and offspring were with slow-rate freezing of bovine oocytes [14]. Recently, vitri®cation using a novel containerless technique has been used for the cryopreservation of oocytes [9] and embryos [15] as an alternative method to traditional slow-rate freezing. Improved vitri®cation protocols have resulted in advances in oocyte cryopreservation, particularly with bovine oocytes. Recently, blastocyst production rates after in vitro fertilization and culture have been similar to non-cryopreserved oocytes [3,16,17]. Decreased fertilization rates for cryopreserved germinal vesicle and MII stage oocytes [4,18,19] have been reported; however fertilization rates of bovine oocytes vitri®ed before the GV stage, the Metaphase I to Metaphase II transition, or at MII were not signi®cantly lower than controls [4]. Fertilization rates of equine oocytes vitri®ed 12 or 24 h after onset of maturation were not signi®cantly different from control oocytes after intracytoplasmic sperm injection (ICSI) [20]. In the mare, a limited number of oocytes can be collected from preovulatory follicles. Super-stimulation protocols aimed at increasing the number of preovulatory follicles per cycle would bene®t assisted reproduction technologies by providing larger numbers of oocytes for experimental purposes. Previous attempts to stimulate follicular growth in mares resulted in increased numbers of follicles 25 mm [21±24]. Initiation of treatment with equine pituitary extract (EPE) on Day 5 rather than Day 12 after ovulation has resulted in signi®cantly more follicles 25 mm [21]. Most equine oocytes matured in vivo and collected just before ovulation are at Metaphase II [25]. Oocytes have been recovered from donor mares at 24±26 h after hCG, or approximately 12 h prior to the expected time of ovulation. When such oocytes were cultured in vitro for the completion of maturation before surgical transfer to recipients, pregnancy rates of 62±92% resulted [26,27]. Collection of oocytes from mares 24±26 h after hCG for vitri®cation studies would eliminate some of the problems associated with the maturation in vitro of immature equine oocytes. Objectives of the present study were to compare embryo development rates after transfer of oocytes into inseminated recipient mares using oocytes collected from super-stimulated versus non-stimulated mares, and cultured with or without prior vitri®cation.
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2. Materials and methods 2.1. Oocyte collection Light horse mares (n 33) evaluated for reproductive soundness and between 3 and 15 years of age were used for the study. Beginning 5 days after a natural ovulation, 10 mares were administered 25 mg of EPE twice daily, prepared using a modi®cation of the method of Guillou and Combarnous [28]; the protein was reconstituted with sterile 0.15 M sodium chloride solution after lyophilization, and ®ltered through a 0.22 micron ®lter without further puri®cation; gel ®ltration and ion exchange chromatography were not used. A single dose of prostaglandin, 250 mg i.m. cloprostenol (Bayer, Shawnee Mission, KS, USA) was administered i.m. 7 days after ovulation. EPE treatment was discontinued when half of the developing follicles reached a diameter 35 mm. At this time, human chorionic gonadotropin (hCG, Steris Laboratories Inc., Phoenix, AZ, USA; 2500 IU) was administered i.v. to initiate synchronous follicular and oocyte maturation. For control mares (n 23), ovarian follicular activity was monitored by transrectal ultrasonography. When relaxed uterine and cervical tone, uterine edema, and a follicle 35 mm in diameter were observed, hCG (Steris Laboratories Inc.; 2500 IU) was administered i.v. to initiate follicular and oocyte maturation. Oocytes were collected by transvaginal, ultrasound guided follicular aspirations using a linear transducer and a double lumen needle (Cook Veterinary Products Inc., New Buffalo, MI, USA). For aspirations, donor mares were sedated (Xylazine HCl, Vedco, Inc., St. Joseph, MO, USA; 0.33 mg/kg, i.v. and Butorphanol tartrate, Fort Dodge Animal Health, Fort Dodge, IA, USA; 0.01 mg/kg, i.v.) and propantheline bromide (Sigma Chemical Co., St. Louis, MO, USA; 0.05 mg/kg i.v.) was administered to promote relaxation of the rectum. Contents of the follicle were gently aspirated (150 mg Hg) using a vacuum pump (Cook Veterinary Products Inc.), and the follicle was lavaged with ¯ush medium (Emcare1 complete ¯ush solution, ICP, New Zealand). Upon collection, cumulus oocyte complexes (COC) were evaluated for cumulus expansion (graded from compact to fully expanded, Table 1). Oocytes for control transfer were cultured 14±16 h in Equine Maturation Medium I (EMMI) [20] before transfer. Oocytes for cryopreservation were placed in culture medium (EMMI) for approximately 2±4 h before vitri®cation. 2.2. Recipient mares Only mares from which an oocyte was collected from all follicles >35 mm were used as recipients. Seven control mares with an oocyte collected from a single preovulatory follicle and one super-stimulated mare with oocytes collected from two follicles were used as recipients. Recipient mares were inseminated with 2 109 progressively motile sperm from 1 of 2 stallions of known fertility, into the uterine body 15±18 h before oocyte transfer. Semen was extended in a skim-milk and glucose extender (E-Z Mixin CST; Animal Reproduction Systems, Chino, CA, USA).
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Table 1 Cumulus expansion of oocytes from super-stimulated and non-stimulated mares Treatment
Super-stimulated (n 18) Non-stimulated (n 20)
Cumulus expansion at collection None
Slight
Moderate
Good
Complete
0 0
0 0
7 11
6 9
5a 0b
Values within columns with different superscripts differ (P < 0:05).
The recipient's ovary and oviduct were exposed through a standing ¯ank laparotomy. Oocytes were placed in modi®ed G2 medium [29] containing 20 mM hepes (Sigma Chemical Co.), 5 mM sodium bicarbonate (Sigma Chemical Co.) and 8 mg/ml BSA (Pentex, Kankakee, MI, USA) with no human serum albumin or phenol red. Oocytes were pulled into a ®re-polished, glass pipette with <0.3 ml of medium. The pipette was threaded 2±3 cm through the recipient's infundibulum, and oocytes were slowly deposited within the oviduct. Only oocytes that appeared morphologically normal were transferred. Two to ®ve oocytes were transferred into each recipient. Before surgery and during the two subsequent days, the ovaries and uteri of the recipients were scanned to ensure that no recipient ovulated an additional follicle, and for the presence of intrauterine ¯uid. No ovulations were detected, but ¯uid was observed in 6 of 8 recipients. Recipients with intrauterine ¯uid received 1 ml of oxytocin (Vedco, Inc.; 20 unit/ml) before surgery and on the two subsequent days if ¯uid was still observed. The day after surgery, all recipients were given daily injections of progesterone (Sigma Chemical Co.) in cottonseed oil (Sigma Chemical Co.; 150 mg, i.m.) until diagnosis of pregnancy. Uteri of recipients were scanned with ultrasound on Days 11±14, 16, 20, and 25 to determine the presence and number of embryonic vesicles. The embryo development rate was de®ned as the number of embryonic vesicles per number of oocytes assigned to each treatment that developed into embryonic vesicles that were imaged with ultrasound by Day 16. If pregnant after transfer of cryopreserved oocytes, recipients were maintained on a synthetic progestin (altrenogest, Intervet Inc., Millsboro, DE, USA; 0.044 mg/kg) given orally until Day 120. 2.3. Vitri®cation and warming Procedures for vitri®cation and thawing oocytes were performed in a heated room (28± 30 8C), and on a heated microscope stage (37±38 8C). COC were gently pipetted with a large-bore pipette in modi®ed G2 until 8±10 cumulus cell layers remained. Cryoprotectants were loaded in three steps: 5% dimethyl sulfoxide (DMSO; Sigma Chemical Co.; 0.7 M) and 5% ethylene glycol (EG; Sigma Chemical Co.; 0.9 M) in modi®ed G2 for 30 s; 10% DMSO (1.4 M) and 10% EG (1.8 M) in modi®ed G2 for 30 s; and 20% DMSO (2.8 M), 20% EG (3.6 M), 10 mg/ml ®coll (Sigma Chemical Co.) with 0.65 M sucrose in modi®ed G2 for 20 s, before being loaded onto a 0.5±0.7 mm diameter nylon loop, mounted on a stainless steel pipe inserted into the lid of a 2 ml cryovial (Hampton Research, Laguna Niguel, CA, USA) and directly plunged into liquid nitrogen. Oocytes were stored for up to 2 months before transfer. Oocytes were thawed and diluted in three steps by immersing the
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nylon cryoloop in 0.250 M, 0.188 M and 0.125 M sucrose in modi®ed G2 at 38 8C for 30 s per step. Oocytes were cultured for 10±12 h in EMMI before transfer into inseminated recipients. 2.4. Statistical analyses Collection rates and cumulus expansion of oocytes from super-stimulated and nonstimulated mares and embryonic development rates per vitri®ed or non-vitri®ed oocyte were compared by Fisher's Exact Test. 3. Results More oocytes (P 0:001) were collected from the aspiration of follicles of 23 nonstimulated mares (20 of 29 oocytes/follicle) than 10 super-stimulated mares (18 of 88 oocytes/follicle). At collection, more (P < 0:05) of the oocytes from super-stimulated mares had complete expansion of the cumulus cell layer yet other morphological grades appeared similar (P > 0:05, Table 1). There was no relationship between degree of cumulus expansion and survival of oocytes after vitri®cation or in embryonic vesicle formation. Of the 20 oocytes collected from non-stimulated mares, 12 were vitri®ed and 8 were transferred as controls. After thawing and dilution of cryoprotectant, 10 of the 12 oocytes were morphologically normal in appearance with intact plasma membranes and were transferred into recipients. One embryonic vesicle resulted from transfer of the 10 oocytes into inseminated recipients. Oocytes from super-stimulated mares were vitri®ed (n 14), or transferred as controls (n 4). After thawing, 9 of 14 vitri®ed oocytes were considered morphologically normal in appearance with intact membranes and were transferred into inseminated recipients. Two embryonic vesicles were established in one recipient. Percentages of oocytes considered morphologically normal after vitri®cation were not different (P > 0:05) between non-stimulated and stimulated mares (10 of 12, 83% and 9 of 14, 64%, respectively). All oocytes for control transfers were considered morphologically normal and were transferred into recipients. Seven of 8 oocytes from non-stimulated and 3 Table 2 Effect of treatment on the number of oocytes transferred into recipient mares and rate of embryonic development per total number of oocytes assigned to vitrification or control Treatment
Total oocytes (n)
Non-stimulated vitrified Super-stimulated vitrified
12 14
Total control
3/26 (12%)a 7/8 (88%)b 3/4 (75%)b
8 4 12
Embryonic development 1/12 (8%)a 2/14 (1%)a
19/26 (73%)a
Total vitrified Non-stimulated control Super-stimulated control
Morphologically normal oocytes transferred into recipients
12/12 (100%)b
Values within columns with different superscripts differ (P < 0:05).
10/12 (83%)b
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of 4 oocytes from super-stimulated mares resulted in embryonic vesicles. There was no difference (P < 0:05) in embryonic development rates of vitri®ed or control oocytes from non-stimulated versus super-stimulated mares; therefore, results were pooled for vitri®ed and control oocytes. Of the 26 vitri®ed oocytes, 7 were determined to have severe morphological abnormalities after thawing and were not transferred. More (P 0:01) control oocytes than vitri®ed oocytes resulted in embryo development (10 of 12 83% vs. 3 of 26 12%, respectively; Table 2). After reduction of one of the twin vesicles, two recipients produced healthy foals from vitri®ed oocytes. 4. Discussion This study represents the ®rst pregnancies after transfer of vitri®ed and thawed equine oocytes. Although viability of vitri®ed oocytes was lower than control oocytes, the establishment of pregnancies from cryopreserved oocytes is a signi®cant advance in developing methods to preserve equine gametes. Few studies have described cryopreservation of equine oocytes [20,30±33]. Survival rates after thawing of equine oocytes, after conventional slow freezing [30] or vitri®cation [31±33] were lower than for non-cryopreserved oocytes. Successful fertilization after cryopreservation of equine oocytes has been reported twice [20,30]. In the present study, stepwise equilibration for short periods with cryoprotectant before vitri®cation resulted in successful rates of cryopreservation based on subsequent pregnancy rates. Stepwise exposure of oocytes to cryoprotectants has previously been demonstrated to be bene®cial to the vitri®cation of immature equine oocytes [32]. With immature and mature human oocytes, a short equilibration process of 20 s in cryoprotectant resulted in 56±100% morphologically normal oocytes after vitri®cation [34]. The short equilibration period in this study may have helped prevent severe osmotic damage from the high concentration of cryoprotectant in the vitri®cation solution. Vitri®cation of immature [35] and MII stage bovine oocytes [6,35] has resulted in fertilization rates similar to untreated controls [6]. Previously reported rates of fertilization of slaughterhouse immature equine oocytes following vitri®cation were signi®cantly lower than those of non-vitri®ed oocytes [31]. In the present study, embryonic development rates of vitri®ed-thawed oocytes collected from mares were signi®cantly lower than controls; however two foals were produced. Success of in vitro matured equine oocytes from small follicles remains low, indicating that conditions for oocyte maturation are not optimized [5,36]. Oocytes collected from slaughterhouse material are of unknown age, reproductive history, and stage of nuclear maturation, which can contribute to variability in results [37± 39]. Until maturation protocols are optimized for equine oocytes from small follicles, slaughterhouse oocytes are not the optimal source of material for experimentation. Embryonic development rates for super-stimulated and non-stimulated oocytes were similar in both control and vitri®ed oocyte transfers. Findings are similar to previous studies in which the administration of EPE to mares for embryo collection did not adversely in¯uence pregnancy rates [24] and oocytes collected from super-stimulated or non-stimulated mares matured at similar rates [40]. In the present study, a range of
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morphologic grades of COC was observed at collection, but morphology grades were not related to subsequent embryonic development after transfer. The pregnancy rates for nonvitri®ed controls were similar to those previously reported for oocyte transfer [26,41±43]. Super-stimulation of follicular growth with EPE increased the number of preovulatory follicles per mare but not the number of oocytes collected. The recovery rate from preovulatory follicles was 69% in control mares and 20% in super-stimulated mares. The large number of follicles present in the ovaries of super-stimulated mares presented a problem during the oocyte collection. The ovaries were large and dif®cult to manipulate during aspiration. The less successful collection of oocytes from super-stimulated than control mares may have been caused in part by the immature or atretic state of some of the follicles. A high incidence of low steroid concentrations was found in follicles in which oocytes were not recovered, indicating that the follicles were immature [44]. Collection of oocytes from immature follicles has been reported to be lower because the compact cumulus was highly attached to the follicular wall [43,45,46]. The optimal point in time from the resumption of maturation for cryopreservation of equine oocytes has not been established, and data from the present study did not establish an optimum stage for oocyte vitri®cation. Further studies focusing on the stage of maturation of equine oocytes before cryopreservation could lead to increased pregnancy rates after transfer to recipients. The results of this study were encouraging in that viable pregnancies were established after transfer of vitri®ed equine oocytes. Super-stimulation of mares with EPE did not increase the number of oocytes recovered per mare or affect the viability of oocytes after transfer to recipients. Further studies are required to improve techniques for equine oocyte vitri®cation. Acknowledgements We thank Heather Reger and Kimberly Preis for assistance with collection of oocytes and George Seidel Jr., Gabor Vatja, and Michelle Lane for scienti®c input. This research was funded by the Lucy Whittier Foundation and benefactors of the Preservation of Equine Genetics program. References [1] Vatja G. Vitri®cation of oocytes and embryos of domestic animals. Anim Reprod Sci 2000;60/61:357±64. [2] Aman R, Parkes J. Effects of cooling and rewarming on meiotic spindle and chromosomes of in vitro matured bovine oocytes. Biol Reprod 1994;50:103±10. [3] Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996;54:1059±69. [4] Fuku E, Xia L, Downey BR. Ultrastructural changes in bovine oocytes cryopreserved by vitri®cation. Cryobiology 1995;32:139±56. [5] Le Gal F, Massip A. Cryopreservation of cattle oocytes: effects of meiotic stage, cycloheximide treatment, and vitri®cation procedure. Cryobiology 1999;38:290±300. [6] Scott TJ, Carnevale EM, Maclellan LJ, Scoggin CF, Squires EL. Embryo development rates after transfer of oocytes matured in vivo, in vitro or within oviducts of mares. Theriogenology 2001;55:705±15.
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