Non-Surgical Transfer of Fresh or Frozen-Thawed Ovine Embryos by Laparoscopy

Transcription

1 Journal of Reproduction and Development, Vol. 45, No. 4, 1999 Non-Surgical Transfer of Fresh or Frozen-Thawed Ovine Embryos by Laparoscopy Naohisa ISHIDA, Yeon-Gil JUNG, Ryoko ITAGAKI, Midori OKADA, Tomoe OGISO, Daisuke ISHIKAWA and Yutaka FUKUI Laboratory of Animal Genetics and Reproduction, Obihiro University of Agriculture and Veterinary Medicine, Obihiro , Japan Abstract. The present study was conducted to examine survival of fresh and frozen-thawed ovine embryos after embryo transfer (ET) using laparoscopy or in vitro culture. Embryos were collected on Day 7.5 (Day 0 =sponge removal) from donor ewes superovulated by pre-treatment of progestogen-impregnated intravaginal sponges (FGA) for 12 days and a single injection of follicle stimulating hormone (FSH) and equine chorionic gonadotrophin (ecg), at 2 days and 1 day before sponge removal. The donor ewes showed estrus between 12 h and 30 h (the mean time: 15.6 h) after sponge removal. Embryo transfer of fresh or frozen-thawed embryos was carried out on Day 7.5 in 37 recipient ewes using laparoscopic technique. Frozen-thawed morulae or blastocysts were cultured to examine their viability in a modified synthetic oviduct fluid medium (SOFM) for 24 h. The lambing rates after ET with fresh and frozen-thawed embryos were 33.3% (6/18) and 26.3% (5/19), respectively. The developmental stage (morula or blastocyst) affected the survival rate of frozenthawed embryos transferred (54.5% and 0%, respectively). The survival rate (development to blastocyst or hatched blastocyst) of frozen-thawed embryos after 24 h in culture was 40.9% (9/22). The present results indicate that ovine ET using laparoscopy is a useful technique. But, the lambing and survival rates of both fresh and frozen-thawed embryos were low, and remained to be improved. Key words: Embryo transfer, Frozen embryo, Laparoscopy, Sheep. (J. Reprod. Dev. 45: , 1999) E mbryo transfer (ET) in sheep was mainly carried out by surgically exteriorizing the reproductive tract which involved some degree of surgical trauma and the formation of postoperative adhesions among the oviduct, the uterus and ovaries. The development of non-surgical transfer procedures have improved the potential of ET technology to beef and dairy industries. However, in small ruminants such as sheep, trans-cervical transfer of embryos is not feasible. Accepted for publication: May 18, 1999 Correspondence: Y. Fukui Recently, however, several groups have employed a laparoscopic technique to transfer ovine embryos to recipient ewes which has resulted in the establishment of pregnancies [1 4]. But, to date, in Japan with mainly Suffolk sheep, there is no report on non-surgical transfer of ovine embryos by laparoscopy, and no offspring has been reported after transfer of frozen-thawed ovine embryos in Japan. Furthermore, development of laparoscopic technique of ovine embryo transfer could reduce the adhesions than the ordinary surgical transfer, especially in fatty, heavy type of ewes such as Suffolk breed. Cryopreservation of embryos is a key factor in commercial embryo transfer technology.

2 290 ISHIDA et al. Frozen embryos can be easily transported from one location to another, and can be safely maintained in storage while their progenitors are being tested for disease [5]. In addition, this technique allows for the long-term storage of embryos when the number of recipients is limited or when the production of live offspring is needed to be postponed to a later date [6]. The objective of the present study was to examine survival of fresh and frozen-thawed ovine embryos (morula and blastocyst) after ET using laparoscopy or in vitro culture. Materials and Methods Donor ewes A total of 23 mature and healthy ewes aged 2 to 8 years old with 3 different breeds (six Suffolk, twelve Merino-Dorset and five Southdown ewes) were used for donor during the breeding season in The ewes were brought into the Laboratory Farm one month before commencing the study and kept under the same condition. All ewes were pre-treated for synchronization of estrus using progestogen-impregnated intravaginal sponges (FGA: Chrono-gest 40; Intervet, The Netherland). Superovulation was induced with a single injection of 15 or 20 mg follicle stimulating hormone (FSH: Antorin; Denka Chemical Co., Japan) two days before sponge removal and a single injection of 250 or 500 IU equine chorionic gonadotrophin (ecg: Serotropin; Teikoku-zoki Co., Japan) one day before sponge removal. The ewes were then examined for estrous incidence from 12 to 36 h (0 h: the time of sponge removal) using 2 3 entire rams aproned and equipped with marking harneses and crayons, and received 100 µg gonadotrophin releasing hormone (GnRH: Conceral; Takeda Chemical Industries Co., Japan) at estrous detection. Estrus was detected between 12 and 30 h (the mean time:15.6 h) after sponge removal. An intrauterine insemination with frozen-thawed semen was carried out at 18 h after estrus by laparoscopy [7]. Semen was collected by an artificial vagina and was frozen in 0.2 ml pellets, as previously described [8], and thawed at 37 C in a water bath immediately before insemination. Postthawing motility ranged from 40 to 50%. The insemination dose was ml containing total spermatozoa per uterine horn. Embryo recovery On Day 7.5 (Day 0 = day of sponge removal) donor ewes were administered atropine sulfate (Tanabe Chemical Co., Japan) and celactal (Bayer; Germany), and laparotomy was carried out under a local anesthesia administered with 2% xylocaine. Firstly, both ovaries were examined for the number of newly formed corpora lutea (CL). Embryos were then recovered by flushing each horn according to the methods of Tervit et al. [9]. Twenty to 30 ml of phosphate-buffered saline (PBS) supplemented with 0.3% bovine serum albumin (BSA) (flushing medium) was used for flushing an uterine horn. The recovery rate, which calculated by dividing the number of ovulations estimated by the number of new CL, was 60%. The recovered embryos were frozen if they were fair, good, or excellent quality at the morula to expanded blastocyst stages, and maintained in a modified synthetic oviduct fluid medium (SOFM) supplemented with 0.8% BSA, 21.0 mm Hepes, 2% (v/v) minimal essential medium (MEM) containing essential amino acids (EAA; 50, Life Technologies Inc., Grand Island, NY, USA) and 1% (v/v) MEM containing nonessential amino acids (NEAA; 100, Life Technologies Inc.) [10]. Freezing and thawing of embryos Embryos were washed in PBS supplemented with 0.4% BSA and then frozen by the methods of Tervit et al. [11]. Embryos were equilibrated in PBS containing ethylene glycol (EG: Waco, Co., Japan) by a stepwise manner (0.5 M EG for 10 min, 1.0 M EG for 10 min and 1.5 M EG for 20 min in order) at room temperature, and then loaded into 0.25 ml straws (2 3 embryos/straw). They were then placed in a planner programmable freezer (ET- UM; Fujiya Yano Science, Sapporo, Japan) which cooled at 1 C / min to 7 C and at this temperature seeding was induced by squeezing the tip of straws with a forcep pre-cooled in liquid nitrogen. The straws were then cooled at 0.3 C / min to 35 C, 0.1 C / min to 38 C and then plunged into and stored for 1 day to 2 months in liquid nitrogen. Thawing was conducted by exposing the straws to room temperature for 3 5 sec and then to 37 C water for 20 sec. Embryos were expelled into a petri dish, and left in the flushing medium (PBS+BSA) for 7.5 min at room temperature. Em-

3 LAPAROSCOPIC EMBRYO TRANSFER IN SHEEP 291 bryos were added in PBS containing 0.5 M sucrose for 10 min and then in PBS for 10 min at room temperature. The embryos were then washed three times in SOFM supplemented with 0.8% BSA, 21.0 mm Hepes, 2% EAA and 1% NEAA. In Vitro Culture (IVC) Frozen-thawed embryos were cultured in SOFM supplemented with 0.8% BSA, 2% EAA and 1% NEAA. The embryos were placed in 30 µl drop (5 embryos / drop) in plastic culture dishes (60 15 mm; Becton-Dickinson Labware, U.S.A.) under mineral oil and cultured in a humidified atmosphere of 5% CO 2, 5% O 2 and 90% N 2. Development to expanded or hatched blastocysts were examined after 24 h in culture. The survival rate was expressed by number of developed embryos. Embryo Transfer (ET) Thirty-seven recipient ewes were synchronized the estrous cycle by treatment with FGA sponge inserted for 12 days and 500 IU ecg one day before the removal of FGA sponge. ET was carried out on Day 7.5 using a modified method of the laparoscopic technique described by Mckelvey et al. [3] and Walker et al. [4]. Recipient ewes were treated with a local anesthesia administered with 2% xylocaine and 0.5 mg/ml Utemerin (Kissei, Co., Japan), a smooth muscle relaxant, to reduce tract turgidity facilitated aseptic transfer of embryos into the upper uterine horn [12], and before transfer, the presence of newly formed CL [13] was determined by laparoscopy. The tip of an uterine horn ipsilateral to functional CL was exteriorized through a 2-cm mid-ventral incision. The uterus was then punctured with a hypodermic needle (22G 1 1 / 2 "R.B.) close to the utero-tubal junction. Two to three fresh or frozen-thawed embryos in SOFM supplemented with 0.8% BSA, 2% EAA and 1% NEAA were transferred to each recipient using a Tom Cat Catheter attached to a 1-ml syringe [14]. When a CL was presented in both ovaries, one embryo was transferred to the each uterine horn. The uterine horn was thereafter returned to the abdominal cavity and the incision was sutured. The implantation rate was expressed by number of implanted embryos at Day 60 after ET by ultrasonic scanning, and the survival rate after ET was expressed by number of new-born lambs. Statistical analysis The pregnancy, lambing and implantation rates were analyzed by the chi-square test. The survival rate was analyzed by the categorical data modeling (CADMOD). Results Eighteen and nineteen recipient ewes were used for ET of fresh and frozen-thawed embryos, respectively (Table 1). The pregnancy and lambing rates of fresh embryos were 38.9% and 33.3%, and those of frozen-thawed embryos were 26.3% and 26.3%, respectively. Both pregnancy and lambing rates between fresh and frozen-thawed embryos were not significantly different. Thirty-seven (15 morulae and 22 blastocysts) and forty-one (11 morulae and 30 blastocysts) ovine embryos were used for ET of fresh and frozenthawed embryos, respectively (Table 2). There were significant differences (P<0.01) in the implantation and survival rates of frozen thawed embryos between morula and blastocyst stage at recovery. The developmental stage (morula or blastocyst) did not affect on the survival rate of fresh embryos transfer (20.0% and 27.3%), whereas no frozenthawed blastocysts survived after ET, and all lambs were born from ET of frozen-thawed morulae. Table 1. Results of laparoscopic transfer with fresh or frozen-thawed ovine embryos Embryo No. of No. of ewes No. of ewes recipient ewes pregnant* 1 (%) lambed (%) Fersh 18 7 (38.9) 6 (33.3)* 2 Frozen-Thawed 19 5 (26.3) 5 (26.3)* 3 * 1 A pregnancy test was performed by a real-time ultrasonic scanning at Day 60 after ET. * 2 Three recipients lambed twins. * 3 One recipient lambed twins.

4 292 ISHIDA et al. Table 2. Survival of fresh or frozen-thawed ovine embryos Embryo Stage of No. of embryos No. of implantated No. of survival development* 1 transferred embryos* 2 (%) embryos* 3 (%) Fresh Morula 15 4 (26.7) 3 (20.0) Blastocyst 22 6(27.3) 6 (27.3) Total (27.0) 9 (24.3) Frozen-Thawed Morula 11 7 (63.6) a 6 (54.5) a Blastocyst 30 0 b 0 b Total 41 7 (17.1) 6 (14.6) * 1 The blastcyst stage include expanded blastcyst stage. * 2 A pregnancy test was performed by a real-time ultrasonic scanning at Day 60 after ET. * 3 The number of new-born lambs after ET. a-b: P<0.01. After IVC, 9 out of 22 frozen-thawed embryos (40.9%) developed to expanded or hatched blastocysts. There was a significant difference (P<0.01) in the survival rate between morula and blastocyst stage embryos (Table 3). Discussion This study demonstrated successful lamb production by using fresh or frozen-thawed embryos and non-surgical laparoscopic embryo transfer for the first time in Japan. This method reduces the time required for ET compared to the conventional transfer by laparotomy. In this study, embryos were collected from three different breeds of donor ewes superovulated by pre-treatment of FGA for 12 days and a single injection of 15 or 20 mg FSH and 250 or 500 IU ecg, at 2 days and 1 day before sponge removal. However, the differences of superovulation methods or donor breeds did not affect ovulation rate and number of normal embryos (unpublished data). The lambing rate following transfer of fresh embryos was lower than the results reported by other groups (50 to 80%) [3, 4]. Two considerable reasons exist to explain why the lambing rate was low. Firstly, the other researchers used only good or excellent quality embryos for ET or freezing, on the contrary, all morulae and blastocysts except poor quality embryos were used for ET or freezing in this study. Only good or excellent quality embryos should have been used for ET and IVC by more critical selection at recovery. Secondly, embryo transfer in 10 out of 18 recipient ewes received fresh embryos was carried out more than 2 Table 3. Effect of developmental stage at recovery to survival rate after IVC. Stage of No. of embryos No. of survvial development* 1 cultured emryos (%)* 2 Morula 9 7 (77.8%) a Blastocyst 13 2 (15.4%) b Total 22 9 (40.9%) * 1 Developmental stage of the embryo at recovery. * 2 The number of development to expanded or hatched blastocysts after 24 h in culture. a-b: P<0.01. h after the recovery of embryos because the collected embryos had to be transported from the University to a sheep farm where the recipient ewes were kept, and resulted in a low lambing rate (20.0%) in the farm than that of the other farm (50.0%) (data not shown). During transport, embryos in SOFM supplemented with 0.8% BSA, 21.0 mm Hepes, 2% EAA and 1% NEAA were kept at 37 C. Although clear reason for the low lambing rate of these embryos is unknown, it should have been returned to the uterus as soon as possible after recovery or thawing. Széll et al. [15] and Somgsasen et al. [16] found an enhanced permeability for EG in ovine embryos than other cryoprotectant. In addition, they found an improvement in the post-thaw survival rate of embryos frozen with EG. However, in this study using EG, the survival rate of embryos during culture after freezing and thawing was low (41%), and lambing rate following ET of frozenthawed embryos was also low (26%). The reason of the low survival rate of the cultured embryos would be that almost all embryos used for IVC

5 LAPAROSCOPIC EMBRYO TRANSFER IN SHEEP 293 after freezing and thawing were at fair stage. However, the survival rates of ET and IVC of frozen-thawed molurae were higher than those of blastocysts (Tables 2 and 3). Some workers reported a higher conception rate by ET of morulae frozen with EG rather than blastocysts [5, 15]. The differences in the survival rate between treatments for morula have been explained by differences in the permeation rates associated with cryoprotectants in early developmental stage of the embryos [15]. Therefore, the present freezing and thawing method may be more suitable for morulae than blastocysts. If this hypothesis is correct, to obtain morulae, recovery of embryos should be performed one day earlier (Day 6.5) than in the present study collecting embryos on Day 7.5, mainly blastocyst stage. In conclusion, we obtained offsprings from fresh or frozen-thawed embryos collected from superovulated ewes by a single injection of FSH combined with ecg and transferred by a non-surgical laparoscopic technique in sheep. Moreover, the present freezing and thawing method was suitable for morulae, but not blastocysts. Therefore, further studies need to be improved the survival of both fresh and frozen-thawed embryos. Acknowledgments We thank Mr. Kuniyoshi Shimoda and Mr. Koji Mutou, and Japan Lamb, Otofuke-cho, Hokkaido, Japan for supply of the animals and facilities used for this study. The authors thank to Dr. H. R. Tervit and Ms. P. A. Pugh, AgResearch, New Zealand, for advising the freezing and thawing techniques of ovine embryos and Dr. M. Tetsuka for critical reading and comments on this study. This work was supported by a grant from the Hokkaido Foundation for the Promotion of Scientific and Industrial Technology (Hokscitec), Japan. References 1. Mutiga ER, Baker AA. Transfer of sheep embryos through a laparoscope. Vet Rec 1984; 114: McKelvey WAC, Robinson JJ. Normal lambs born following transfer of embryos by laparoscopy. Vet Rec 1984; 115: Mckelvey WAC, Robinson JJ, Aitken RP. A simplified technique for the transfer of ovine embryos by laparoscopy. Vet Rec 1985; 117: Walker SK, Warnes GM, Quinn P, Seamark RF. Laparoscopic technique for the transfer of embryos in sheep. Australian Vet J 1985; 62: MartÍnez AG, Matkovic M. Cryopreservation of ovine embryos: slow freezing and vitrification. Theriogenology 1998; 49: Fahning ML, Garcia MA. Status of cryopreservation of embryos from domestic animals. Cryobiology 1992; 29: Maxwell WAC, Butler LG, Wilson HR. Intra-uterine insemination of ewes with frozen semen. J Agric Sci, Camb 1984; 102: Salamon S, Visser D. Tris-based diluents and thawing solution on survival of ram spermatozoa frozen by the pellet method. Aust J Biol Sci 1972; 25: Tervit HR, Havik PG. A modified technique for flushing ova from the sheep uterus. New Zealand Vet J 1976; 24: Tervit HR, Whittingham DG, Rowson LEA. Successful culture in vitro of sheep and cattle ova. J Reprod Fert 1972; 30: Tervit HR, Gold PG. Deep-freezing sheep embryos. Theriogenology 1984; 21: 268 (abstr). 12. Morrow CJ, Asher GW, Berg DK, Tervit HR, Pugh PA, McMillan WH, Beaumont S, Hall DRH, Bell ACS. Embryo transfer in fallow deer (Dama dama): superovulation, embryo recovery and laparoscopic transfer of fresh and cryopreserved embryos. Theriogenology 1994; 42: Tetsuka M, Kobayashi M, Fukui Y, Ono H. Observations of ovarian activities using a laparoscope in ewes induced estrus and inseminated artificially during the non-breeding season. Japanese J Anim Reprod 1985; 31: Thompson JG, Bell ACS, McMillan WH, Peterson AJ, Tervit HR. Donor and recipient ewe factors affecting in vitro development post-transfer survival of cultured sheep embryos. Anim Reprod Sci 1995; 40: Széll A, Shelton JN, Széll K. Osmotic characteristics of sheep and cattle embryos. Cryobiology 1989; 26: Somgsasen N, Buckrell BC, Plante C, Leibo SP. In vitro and in vivo survival of cryopreserved sheep embryos. Cryobiology 1995; 32: