The Influence of Developmental Stage and Morphological Quality of Frozen-Thawed Bovine Embryos on Pregnancy Rate in Bovine Embryo Transfer

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1 Journal of Reproduction and Development, Vol. 45, No. 4, 1999 Technical Note The Influence of Developmental Stage and Morphological Quality of Frozen-Thawed Bovine Embryos on Pregnancy Rate in Bovine Embryo Transfer Masahiko NISHIGAI 1,2), Hideo KAMOMAE 2), Tomomi TANAKA 2), and Yoshihiro KANEDA 2) 1) Nasu ET Institute, 7 10, Shimakata, Kuroiso, Tochigi , and 2) Laboratory of Veterinary Reproduction, Tokyo University of Agriculture and Technology, Fuchu, Tokyo , Japan Abstract. To investigate the influence of post-thawed embryonic developmental stage and post - thawed morphological quality on pregnancy rate, frozen embryos, which had been collected from Japanese Black beef cattle and qualified as morphologically excellent before freezing, were thawed by the stepwise method and transferred non-surgically to Holstein heifers on day 7 (day 0=onset of estrus). The transferred embryos ranged in development from compacted morula to blastocyst stage and were evaluated as either morphologically excellent or good quality just after thawing. Pregnancy rates of 62.0% (49/79), 66.7% (26/39), and 81.0% (34/42) were achieved in the transfers of the compacted morula, early blastocyst, and blastocyst stages, respectively, for the transfer of the excellent quality embryos. The pregnancy rate obtained with blastocyst stage embryos was significantly (P<0.05) higher than that obtained with compacted morula embryos. The pregnancy rate of morphologically excellent quality embryos of the compact morula stage was 62.0% (49/79), and that of the good quality embryos of the compacted morula stage was 58.3% (7/12) revealing no significant difference between the two morphological qualities. These results indicate that the pregnancy rate increases as the embryonic developmental stage advances from compacted morula to early blastocyst, and that post-thawed morphological qualities, excellent and good quality, have no influence on pregnancy rates for frozen-thawed embryos that are transferred to bovine recipients on day 7 of estrus. Key words: Bovine frozen-thawed embryo, Embryonic developmental stage, Non-surgical transfer, Pregnancy rate (J. Reprod. Dev. 45: , 1999) Accepted for publication: April 26, 1999 Correspondence: M. Nishigai ecipient-donor synchrony of estrous cycles is R necessary to obtain a high pregnancy rate in bovine embryo transfer. The permissible range of asynchrony in the recipient is ±1 day in estrous cycle with that of the donor. Two days disparity between the recipient and the donor decreased the pregnancy rate markedly, and three days asynchrony resulted in very low pregnancy rates [1 4]. The pregnancy rate in embryo transfer is influenced by the embryonic developmental stage. In the surgical transfer of bovine fresh embryos to bovine recipients, where the synchronized estrous cycle stage was within ± 12 hours of the donors, 6 to 8 days after estrus, the pregnancy rate was significantly lower in the early morula stage than in the compacted morula, early blastocyst, and blastocyst stages [5]. Some reports on the transfer of

2 302 NISHIGAI et al. fresh embryos by the non-surgical method have shown that the pregnancy rate improved as the embryonic developmental stage advanced [6 8]. In previous studies of the non-surgical transfer of cryopreserved bovine embryos, Leibo [9] has shown that the pre-freezing developmental stage and morphological quality of embryos are definitely related to the subsequent pregnancy rate. However, few investigations have been undertaken regarding the influence of these embryonic parameters evaluated after the thawing of frozen embryos by the stepwise method on the pregnancy rate due to the fact that there is a morphological differences in bovine embryos between before freezing, and after thawing and removal of cryoprotectants [10]. Thus, the present study investigated the influence of the post-thawed developmental stage and morphological quality of bovine frozen-thawed embryos on the pregnancy rate in embryo transfer by the stepwise method. This was accomplished by transferring frozen-thawed embryos in good or excellent morphological condition, in compacted morula, early blastocyst, and blastocyst stages, to bovine recipients 7 days after estrus, with estrous cycle stages synchronized to the donors. Materials and Methods Recipients The recipients were 172 Holstein heifers which had been bred at twelve different dairy farms. They were aged 13 to 15 months old and in good nutritional condition. Prior to embryo transfer, they were confirmed to have neither abnormal development of corpus luteum nor abnormality of genital organs by vaginal and rectal examinations on day 6 and 7 (day 0 = onset of estrus). Frozenthawed embryos were transferred to them on day 7, i.e., the day when the embryos had been collected from the donors. Collection and freezing-thawing of embryos Sixty-seven Japanese Black beef cattle, which had been bred at 34 breeding farms and were in good nutritional condition, were used as donors. Each donor was used only once. Superovulation was induced by intramuscular injection of 20 mg of follicle-stimulating hormone (FSH) (Antrin, Denka Pharmaceuticals, Co., Ltd., Kanagawa) in decreasing doses, and PGF 2α (Veterinary Pronalgon F Injection; Upjohn Pharmaceuticals, Ltd., Tokyo) injections given in a total dose of 30 mg of dinoprost (20 mg in the morning and 10 mg in the evening), 3 days after the start of FSH administration. Artificial insemination was carried out using Japanese Black frozen semen 12 and 24 hours after the appearance of standing estrus. The embryos were collected on day 7 by non-surgical uterine flushing using a Foley catheter [11]. The embryos collected were classified into several developmental stages and morphological qualities according to the classification proposed by Lindner and Wright [12]. The 556 embryos which were in the morula to blastocyst stages and evaluated as being morphologically excellent or good qualities, were frozen and stored by the two-step glycerol equilibrium method according to Elsden and Seidel [13]. Sequentially, of the 556 frozen embryos collected 180 embryos, which were found to be morphologically excellent before freezing and approximately evenly constituted from the developmental stage of morula, early blastocyst and blastocyst, were used for this experiment. However, 8 of the 180 thawed embryos were not transferred for the following reasons: one was lost during thawing, two were fissured into the blastomeres after thawing, and five were considerably damaged in the cell membrane and were accompanied by many extruded blastomeres after thawing. The frozen embryos were thawed in warm water at 37 C after exposure to air at 25 C for 15 seconds according to the method of Seidel and Seidel [14]. The thawed embryos in straws were poured into small Petri dishes and then transferred into two kinds of solutions which consisted of 6% and 3% glycerol-containing Dulbecco s phosphatebuffered saline (PBS), respectively, with 10.3% sucrose solution at 5 minute intervals in a stepwise method. Then, the embryos were finally transferred to PBS solution without either sucrose or glycerol. All solutions used in this experiment contained 0.4% bovine serum albumin. The embryos were reevaluated in the postthawed condition for the morphological quality and the embryonic developmental stage in a phosphate buffer solution containing 0.4% bovine serum albumin according to the classification proposed by Elsden [15]. The reevaluated embryos of excellent or good quality were divided into three developmental stages: compacted morula, early blastocyst,

3 EMBRYO DEVELOPMENTAL STAGE AND PREGNANCY RATE IN ET 303 and blastocyst. The embryos which showed cellular demarcation with favorable color and density and were essentially free from extruded blastomeres or vesicles were evaluated as being morphologically excellent in quality. The embryos which had favorable color and density of the cellular demarcation, but were partly associated with blastomeres or vesicles and showed 10~20% cellular degeneration were evaluated as being of morphologically good quality. Each thawed embryo was aspirated into a 0.25 ml straw. It took 16~17 minutes from the initiation of thawing to the aspiration of the embryo into the straw. The straw was attached to an embryo transfer gun (Cassou, France), wrapped with a thermal paper towel which was kept at 37 C previously and transported in a container to the scene of transfer. Embryo transfer method In the transfer of the embryos, the recipient s vulva was flushed with a disinfectant for external use (Propodyne Scrub, Santen Pharmaceuticals, Co., Ltd., Osaka). A vaginal speculum, first immersed in a disinfectant (Osvan solution, Takeda Pharmaceutical Industries, Co., Ltd., Osaka), was inserted into the vagina to dilate it for insertion of the embryo transfer gun. The transfer gun was deeply inserted into the vagina to the external uterine orifice avoiding bacterial contamination from contact with the vestibule wall of the vagina [16]. Immediately after insertion of the embryo transfer gun, the vaginal speculum was removed and the embryo was then transferred into the uterus. The site of transfer was at the central portion of the uterine horn ipsilateral to the ovary with corpora lutea. One embryo was transferred by means of the same equipment by the person performing the transfer procedure. The time required for the completion of transfer after the aspiration of a thawed embryo into the straw was 60 minutes at most. To ensure restraint of recipients at the time of embryo transfer and to facilitate the transfer, each recipient received an intramuscular injection of 20 mg of xylazine (2% Celactal Injection, Bayer, Tokyo) and epidural anesthesia with 3 ml of 2% lidocaine hydrochloride (2% Xylocaine Injection, Fujisawa Pharmaceuticals, Co., Ltd., Osaka) 5 15 minutes before the start of transfer, i.e., the vaginal speculum insertion [17]. Table 1. The effect of embryonic developmental stage and morphological quality on pregnancy rate obtained with non-surgical transfer of frozen-thawed embryos Embryo quality Developmental stage Excellent Good Compact morula 62.0 (49/79) a 58.3 (7/12) Early blastocyst 66.7 (26/39) Blastocyst 81.0 (34/42) b Total 68.1 (109/160) 58.3 (7/12) Values are pregnancy rates (No. of pregnant heifers/no.of recipient heifers). a,b) Values with different superscripts are significantly different (P<0.05). Diagnosis of pregnancy The recipients, in which estrus did not recur, were diagnosed for pregnancy by rectal examination 40~50 days after the embryo transfer. Statistical analysis Significant differences in pregnancy rates were examined by the chi-square test for independence [18]. A P value below 5% (P<0.05) was considered to be significantly different. Results The correlation of the developmental stages and morphological quality of frozen-thawed embryos with the pregnancy rate are shown in Table 1. The pregnancy rate increased as the embryonic development advanced from compacted morula to the blastocyst stage. The pregnancy rate of the blastocyst stage embryo was significantly higher than that of the compacted morula embryos. Discussion With regard to the relationship between embryonic developmental stage and pregnancy rate in non-surgical fresh embryo transfer, some reports have shown that the transfer of blastocyst stage embryos resulted in a significantly higher pregnancy rate than that of morula stage embryos [6 8]. However, others have found that there was no significant difference in pregnancy rates between

4 304 NISHIGAI et al. blastocyst and morula embryo transfers [12, 19]. The present results of transferring frozen-thawed bovine embryos on day 7 demonstrate that the pregnancy rate with blastocysts was significantly higher than that with compacted morula, and that the pregnancy rate tended to rise as the developmental stage advanced. Concerning with these findings, embryos are known to be impaired by the formation and growth of intracellular cryohydrate and osmotic swelling, which occur in the process of freezing and thawing, and by the toxicity of cryoprotectants [20, 21]. Leibo [9] has reported that the transfer of blastocyst stage embryos which had been evaluated before freezing resulted in a higher pregnancy rate by the non-surgical transfer than that of the morula stage,and this is similar to the results of the present study. Moreover, Leibo [9] demonstrated that blastocysts cultured in vitro after thawing by the one step straw method were more cryo-resistant than the morula stage. Furthermore, Iwasaki et al. [22] have found that the total number of dead inner cell mass (ICM) in the frozen IVF morula stage was greater than that of dead ICM in the IVF blastocyst stage,when the frozen IVF morula stage embryos were thawed and glycerol was removed by the stepwise method and subsequently cultured in vitro for 48 hours. The present and previous study suggest that the significant difference in the pregnancy rate between the transfer of morulae and blastocysts is attributable to the embryo cryoresistance. Moreover, it has been shown in rabbits that the pregnancy rate was high when frozen rabbit embryos were transferred into the oviduct which was at an earlier stage than the embryonic developmental stage, because frozen embryos were retarded in their development by the impairment resulting from the freezing and thawing process [23]. The relationship between the development of transferred embryos and recipients uterine environment is well known [24]. The uterine environmental cycle does not synchronize to the developmental stage of the transferred embryos, while the embryos grow fast or slowly depending on the uterine developmental stage. It has been reported that ovine embryos grew faster than those at a stage synchronized with the recipients estrous cycle, when they were transferred to ewe recipients having a more advanced stage in the estrous cycle. In contrast, the transferred embryos, which were at a more advanced developmental stage than the recipients estrous cycle stage, grew more slowly [25, 26]. On the other hand, Albihn et al. [1] reported that the light and electron microscopic examinations of bovine embryos, which had been collected on day 4 and transferred to heifer recipients on day 7, and were again collected from the recipients on day 15. The examinations showed that the collected embryos showed faster development, but all were degenerated. In the present experiment, compacted morula and blastocyst stages of embryos were considered as being on day 6 and 8 after the beginning of standing heat respectively [13], and both were transferred to the recipients on day 7. However, considering the possible developmental retardation due to freezing and thawing [23], the post-thawed compacted morulae of embryos could have been transferred to the recipients with an almost two-day advanced estrous cycle, while the blastocyst stage could have been transferred to recipients with an estrous cycle synchronized with the embryonic developmental stage [27]. Transferred compacted morulae develop rapidly to synchronize with recipients uterine environment according to Wilmut s study [26], and some of them may degenerate. Consequently, only a minor part of the compacted morulae may have survived, compared with the blastocyst stage embryos which synchronized with the recipients uterine environmental cycle and are highly cryo - resistant. Thus, a high pregnancy rate in blastocyst stage of embryos was observed in comparison with that of compacted morula. In the present experiment, the pregnancy rates achieved with blastocysts was higher than that with compacted morula. This result indicates that the blastocysts transferred to the recipients bearing either just corresponded to or earlier developed uterine environments synchronized the subsequent embryonic developmental stage to the uterine environmental stage. The pregnancy rate of excellent and good quality fresh embryos transferred non-surgically was approximately 45%, showing no significant difference between excellent and good embryos [12]. Moreover, in the present experiment, there was no significant difference in pregnancy rates between the post-thawed excellent and good quality embryos using compacted morula. According to the study conducted by Schneider and Mazur [10], in the process of glycerol removal after thawing in frozen embryos by the stepwise method, the extra-

5 EMBRYO DEVELOPMENTAL STAGE AND PREGNANCY RATE IN ET 305 cellular sucrose solution becomes hypertonic gradually as glycerol diffuses to the extracellular space during the glycerol removal process and the embryos are consequently dehydrated and shrunk. In the present study, morula stage embryos increased in number after thawing, possibly due to the postthawed microscopic judgement of compacted morulae, some of which were derived from the pre-freezing early blastocyst or blastocyst stage of embryos, owing to the shrinkage and disappearance of blastocoele cavity during the process of the freezing, thawing and glycerol removal. From this experiment, for day 7 embryo transfer of bovine frozen-thawed embryos, the following points are indicated: (1) the pregnancy rate increases as the embryonic developmental stage advances from compacted morula to early blastocyst and to blastocyst; (2) the pregnancy rate is not different between the excellent and good quality embryos. Acknowledgments The authors deeply thank Dr. Yuri Kobayashi, Kobayashi ET Clinic, and Dr. Toshiyuki Kojima, Head of the 1st Division of Animal Reproduction, National Livestock Breeding Center, Ministry of Agriculture, Forestry and Fishery, for their support of this study. References 1. Albihn A, Gustafsson H, Rodriguez-Martinez H. Maternal influence on the early development of asynchronously transferred bovine embryos. Anim Reprod Sci 1991; 24: Hasler JF, MaCauley AD, Lathrop WF, Foote RH. Effect of donor-embryo-recipient interactions on pregnancy rate in large-scale bovine embryo transfer program. Theriogenology 1987; 27: Rowson LEA, Moor RM, Lawson RAS. Fertility following egg transfer in the cow: effect of method, medium and synchronization of oestrus. J Reprod Fertil 1969; 18: Rowson LEA, Lawson RAS, Moor RM, Baker AA. Egg transfer in the cow: synchronization requirements. J Reprod Fertil 1972; 28: Shneider HJ Jr, Castleberry RS, Griffin JL. Commercial aspects of bovine embryo transfer. Theriogenology 1980; 13: Donaldson LE. Matching of embryo stages and grades with recipient oestrous synchrony in bovine embryo transfer. Vet Rec 1985; 117: Halley SM, Rhodes RC III, McKellar LD, Randel RD. Successful superovulation,nonsurgical collection and transfer of embryos from Brahman cows. Theriogenology 1979; 12: Wright JM. Non-surgical embryo transfer in cattle embryo-recipient interactions. Theriogenology 1981; 15: Leibo SP. A one-step method for direct nonsurgical transfer of frozen-thawed bovine embryos. Theriogenology 1984; 21: Schneider U, Mazur P. Osmotic consequences of cryoprotectant permeability and its relation to the survival of frozen-thawed embryos. Theriogenology 1984; 21: Elsden RP, Hasler JF, Seidel GE Jr. Non-surgical recovery of bovine eggs. Theriogenology 1976; 5: Lindner GM, Wright RW Jr. Bovine embryo morphology and evaluation. Theriogenology 1983; 20: Elsden RP, Seidel GE Jr. Freezing and thawing procedures for embryos. In: Procedures for Recovery, Bisection, Freezing and Transfer of Bovine Embryos. Bulletin, Colorado University, 1985; No.2: Seidel GE Jr, Seidel SM. Cryopreservation of bovine embryos. In: Training manual for embryo transfer in cattle, FAO 1991: Elsden RP. Classification of embryos after thawing. In: 1988 Short Course Proceedings. Animal Reproduction Laboratory, Colorado State Universiy, 1988: Nishigai M, Kamomae H, Tanaka T, Kaneda Y. Pregnancy rate of non-surgical frozen embryo transfer using vaginal speculum in cows. J Reprod Dev 1996; 6: (In Japanese) 17. Nishigai M, Kamomae H, Tanaka T, Kaneda Y. Effect of Xylazine tranquilization during embryo transfer to bovine recipients on pregnancy rates. J Reprod Dev 1997; 43: (In Japanese). 18. Statistical Analysis System. SAS User s Guide: Statistics, Version 6.03 Edition, Cary, NC: SAS Inst., Inc.; Shea BF. Evaluating the bovine embryo. Theriogenology 1981; 15: Kasai M. Simple and efficient methods for vitrification of mammalian embryos. Anim Reprod Sci 1996; 42: Massip A, Mulnard J. Time-lapse cinematographic

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