Embryo collection and transfer in small ruminants

EMBRYO COLLECTION AND TRANSFER IN SMALL RUMINANTS D.C. Kraemer Departments of Veterinary Physiology and Pharmacology and Animal Science, Texas Agricultural Experiment Station, Texas A&M University, College Station, Texas 77843 ABSTRACT Recent studies of embryo transfer in small ruminants have yielded laparoscopic and nonsurgical procedures for both embryo collection and transfer. These relatively atraumatic methods for embryo collection produce results that are competitive with surgical methods for collecting uterine stage embryos. Embryos have been collected nonsurgically from sheep, goats, deer, suni antelope and the yellow-backed duiker. Laparoscopic embryo transfer is both rapid and effective when compared with surgical transfer. Although nonsurgical transfers have been achieved in goats, more data are required before this approach can be recommended for widespread application to small ruminants. Key words: Embryo transfer, Small ruminants, Nonsurgical INTRODUCTION The first successful embryo transfers in livestock species were performed on sheep and goats (1). Nevertheless, until very recently, there has been relatively little commercial embryo transfer activity with the small ruminants. Two factors that have probably contributed most to this lack of utilization of embryo transfer in these species are: a) the relative cost-benefit ratio and b) the damage caused to genetically valuable animals by surgical embryo collection procedures. The interest in international exchange of germ plasm and in preservation of endangered species has altered the cost-benefit ratio in favor of the use of embryo transfer. Also, methods for nonsurgical collection of embryos from several species of small ruminants have been developed recently. Therefore, there is markedly increased interest in the use of embryo transfer in these species. The major objective of this paper is to present information on embryo collection and transfer procedures that have been developed since the publication of the most recent reviews on the subject of embryo transfer in small ruminants (3,4). For purposes of this presentation, small ruminants are defined as those too small for manipulation of the reproductive tract by a hand inserted into the rectum. Acknowledgements: The author is grateful to N.M. Loskutoff and S.A. Workman for preparation of the manuscript and S.M. Bowen for the illustrations.

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DISCUSSION Collection of Embryos Several recent reports have shown the detrimental effects of repeated surgical embryo collections on the fertility of the donor females (5). Clearly, embryo transfer is limited in its usefulness for genetic improvement or preservation of endangered species as long as the embryos must be collected by conventional surgical methods. Fortunately, several effective alternatives are available, such as laparoscopic and nonsurgical embryo collection and in vitro fertilization. COD‘c collection of embryos. An early approach to the use of laparoscopy to colle’ct uterine stage embryos from sheep utilized the endoscope to visualize the ovarian response and to exteriorize the tips of the uterine horns (6). The collection fluid was then injected into the tips of the two uterine horns simultaneously and collected via a glass vaginal speculum placed around the external OSof the cervix. This procedure yielded a 54% (7 of 13) rate of recovery of embryos from eight naturally-ovulating ewes. Adhesions were observed following the use of this procedure; however, they were limited to the uterotubal junction area, thereby being less damaging than those formed between the ovary and oviduct during conventional surgical embryo collection. More recently, a laparoscopic procedure was developed for collecting uterine stage sheep embryos without exteriorizing the reproductive tract (7,8). The donor ewes are placed in dorsal recumbency under general anesthesia with the head end of the cradle lowered at a 30 to 45” angle. The abdomen is insufflated with CO, using a Verres needle. Two stab wounds are made in the abdominal wall, each 2 to 3 cm from the midline and approximately 10 cm anterior to the udder, for insertion of the laparoscope and a pair of laparoscopic grasping forceps. After the ovaries and uterus are located, a trocar and cannula are inserted through the abdomen on the midline, about 5 cm anterior to the other instruments. This cannula is used for insertion of an instrument (either a Verres needle, or a blunted bovine paravertebral needle) for making a small (0.25 cm) incision through the uterine wall. This is done by grasping the uterine horn caudal to the external bifurcation of the uterus and inserting the needle adjacent to, and on the ovarian side of the grasping forceps. After removing the needle, a two-way, 10 French, pediatric Foley catheter is inserted through the cammla and into the uterine lumen with the aid of a metal stylet. The 3-ml balloon is inflated and the stylet is removed. The grasping forceps are then used to hold the ipsilateral uterotubal junction for insertion of an intravenous catheter and stylet into the tip of the uterine horn. After the stylet is removed, collection medium (approximately 60 ml) is introduced through the intravenous catheter and out the Foley catheter. The procedure is repeated on the opposite horn the instruments are removed and the abdominal incisions are closed. McKelvey and Robinson (8) achieved a 50% recovery rate (56 of 111) from 21 ewes using this procedure. There were no adhesions present when the ewes were examined by laparoscopy 3 months later; however, two of the ewes had developed endometrial outgrowths. Although a pregnancy was obtained in one of the ewes with the endometrial

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outgrowth, this could be a serious problem since these outgrowths are frequently accompanied by fistulas. The usefulness of this procedure would be greatly enhanced by the development of a method for closure of the puncture wounds in the uterus. ~onsur~~l collection of embrvos The first nonsurgical collection of embryos from small ruminants was repo;ed by Bondurant et al. in 19&Q(9). They obtained blastocysts from dairy goats nonsurgically with the aid of a Laminaria japonica tent for dilation of the cervix. The tents were inserted into the cervical canals following light sacrococcygeal epidural anesthesia (1.5 ml of 2% Iidocaine) using dressing forceps and Jackson rotational forceps. They were held in place by two gauze sponges that were packed into the anterior vagina and removed after 6 to 12 h. The embryos were collected with the animals standing, using epidural anesthesia. The collection device was a modified two-way 24-ga. Foley catheter. A stainless steel tube, 50 cm long by 1.5 mm I.D. (OD. approximately 3 mm) was placed through the catheter, piercing the latex of the catheter at the convergence of the two channels with the tip of the tube penetrating the tip of the catheter. A Sovereign catheter (5 French) was inserted through the stainless steel tube for in~odu~on of the collection fluid. Fluid exited via the channel in the Foley catheter. Three embryos were recovered from one doe and 10 from the other. Coonrod et al. (10,ll) developed a method for catheterizing the sheep cervix and collecting embryos nonsurgically. This procedure has been adapted for use in angora goats (131, white-tailed deer (141, suni antelope (15) and the belly-baled duiker (12). The procedure works most effectively under general anesthesia, although a few collections have been performed using tranquilizers. The cervix is located using a cylindrical plastic speculum and the cervix is grasped using laparoscopic forceps (Fig. 1). The speculum is removed and retracted onto the shaft of the forceps and the cervix is retracted into the vagina. The cervix is then visualized using a duck-billed speculum and the cervix is grasped with Allis tissue forceps (Fig. 2). The laparoscopic forceps are then removed and a catheter is placed into the cervical OS(Fig. 3). The duckbilled speculum is then removed and the index finger of a gloved hand is inserted into the vagina along side of the retracted cervix (Fig. 4). The finger is used to guide the catheter through the cervix. Several types of catheters have been used with this approach. The most useful for passage through tight cervices is the internal cammfa of a Verres needle. The tip is smooth and rounded with the opening on the side. Additional openings may be drilled on the sides of the cannula tip to increase the rate of return flow. A second type of catheter, which is often used in goats, is a 14-ga. angiocath with a blunted stylet. Neither of these catheters utilizes a balloon to seal the internal cervical OS,so only small amounts of medium (5 to 10 ml) are introduced at each flush to keep the pressure low and minimize fluid leakage. Until the amount of pressure necessary to push fluid through the uterotubal junction is known for these species, it is probably best to use a Iowpressure system to avoid loss of the embryos into the abdominal cavity. The catheters are-pulled out slightly and then reinserted periodically to induce

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PLASTIC SPECULUM

Fig. 1. The ceti is visualized using a tubular plastic speculum and grasped with Iaparoscopic forceps.

GRASPING FORCEPS

\

‘-’ .I

‘ALLIS

TISSUE FORCEPS

Fig. 2. The plastic speculum and cervix are retracted and a duck-billed speculum is used to place Allis tissue forceps on the cervix.

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AUIS

c-=-R--

a

INDEX

FINGER

TISSUE FORCEPS

MODIFIED VERRES NEEDLE

Fig. 3. The laparoscopic the cervical OS.

’

ti

.p

forceps are removed and the catheter is inserted

into

I

Fig. 4. An index finger is placed into the vagina and used to guide the catheter through the cervix.

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return flow. In the duiker, Pope et al. (12) used Hank’s dilators to expand the cervical canal and flushed the uterus with a Foley catheter (10-14 French). The balloon was inflated with 6 to 10 ml of air and was kept firmly against the internal OSof the cervix. They used gravity flow for infusion of the uterus with 260 to 910 ml of collection fluid. They recovered two embryos from one of four collection attempts. In sheep, this nonsurgical collection procedure yielded successful catheterization of the cervix in 59% of the attempts (10). From the 28 ewes that were successfully catheterized the recovery rate was 75% (195 of 319). In Angora goats, catheters can be passed in over 90% of the attempts. Bessoudo et al. (13) obtained 9.5 ova per donor nonsurgically and 113 ova per donor surgically in a commercial trial involving 252 Angora and 84 Kashmir does. The number of ovulations were not known; therefore, percentage recoveries were not calculated. Four ova were recovered nonsurgically from three collection attempts from naturally-ovulating small white-tailed deer (14). The number of ovulations was not determined. In a total of nine nonsurgical embryo collections from five superovulated sum antelope, an average of 4.4 + 4.0 ova (range O-10) were recovered while an average of 6.6 + 5.0 corpora lutea (range O-14) were observed per donor animal (15). Although these animals are very small(4 to 6 kg), catheterization of the cervix was achieved in every attempt. There is one additional report of a successful nonsurgical embryo collection from a small ruminant: the Shiba goat. Nagashima et al. (16) catheterized the cervix using a vaginal speculum and a cervical expander. The collection catheter consisted of a metallic inner tube for inflow and a plastic outer tube (2.5 mm O.D.) for outflow. There was no balloon on the collection catheter, and fluid (5 to 10 ml) was introduced and collected in syringes. Each uterine horn was washed separately. Embryo collection succeeded in 15 of 26 attempts yielding a recovery rate of 89.5%. Embryo Transfer Laoarosconic embtvo transfer. Several groups have reported successful laparoscopic transfers of embryos in small ruminants (17,18,19,20). The general approaches are quite similar in that the reproductive tract is visualized via the laparoscope and the uterus is held by grasping forceps. In each case, stab wounds were made in the uterine wall. In three of the reports the embryos were deposited using flexible tubing; in the fourth report (20) the embryos were deposited utilizing glass pipettes. In the latter case, a pregnancy rate of 75% (54% of the embryos) was achieved in 20 recipients. Mutiga and Baker (17) obtained pregnancies in two of three ewes. Walker et al. (19) obtained a 41% pregnancy rate in 54 recipient ewes and McKelvey and Robinson (18) achieved three pregnancies, two of which lambed, in six recipients.

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One offspring was obtained from a laparoscopic antelope utilizing a flexible catheter (15).

transfer in a small suni

Nonsurgical embN0 transfer. Only three reports were found of nonsurgical embryo transfer attempts in small ruminants. All were with goats. Procedural details are not available. Agrawal and Bhattacharyya (23) obtained a 40% conception rate in seven recipients that received 17 embryos.

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Warwick, B.L., Berry, R.O. and Horlacher, W.R. Result of mating rams to angora female goats. Proc. 27th Arm. Meet. Am. Sot. Anim. Prod., pp. 225-227 (1934).

2.

Warwick, B.L. and Berry, R.D. Inter-generic transfers. J. Hered. 4_Q:297-303 (1949).

and intraspecific

3.

Bondurant, R.H. Embryo transfer in sheep D.A.,(ed) Current Therapy in Theriogenology. Co., Philadelphia, 1986, pp. 63-66.

and goats. In: Morrow, 2nd ed., W.B. Saunders

4.

Armstrong, D.T. and Evans, G. Factors influencing transfer in sheep and goats. Theriogenology a:3142

5.

Torres, S. and Sevellec, C. Repeated superovulation and surgical recovery of embryos in the ewe. Reprod. Nutr. Develop. 22:859-863 (1987).

6.

Capehart, J.S., Bowen, M.J., Bassett, J.W., Shelton, J.M. and Kraemer, D.C. A modified technique for the collection of uterine stage ovine embryos. Theriogenology a:227 (1984).

7.

McKelvey, WAC. and Robinson, by laparoscopy. Vet. Rec. m:158

a.

McKelvey, WAC. and Robinson, JJ. Repeated by laparoscopy. Theriogenology 25:171 (1986).

9.

Bondurant, R.H., Skirrow, S., Anderson, G.B., Hanson, F. and Rogers, W.H. Nonsurgical collection of blastocysts from dairy goats. Theriogenology 22:423431 (1984).

10.

Coonrod, S.A., Bowen, J. and Kraemer, D.C. Non-surgical collection of ovine embryos. Proceedings of the 5th Ann. Convention of the American Embryo Transfer Assoc., pp. 83-87 (1984).

11.

Coonrod, S.A., Coren, B.R., McBride, B.L., Bowen, M.J. and Kraemer, DC. Successful non-surgical collection of ovine embryos. Theriogenology 25: 149 (1986).

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Pope, C.E., Dresser, B.L., Kramer, L.W. and Gillespie, D. Nonsurgical embryo recovery in the yellow-backed duiker ($&&xlophuz @icuitor) a preliminary study. Proc. 11th Int. Cong. Anim. Reprod. AI., pp. 184185 (1988).

13.

Bessoudo, E., Davies, L, Coonrod, S., Gamex, J. and Kraemer, D.C. Nonsurgical collection of caprine embryos under commercial quarantine conditions. Theriogenology 293221 (1988).

14. Magyar,

S.J., Hodges, C., Seager, S.W.J. and D.C. Kraemer. Successful nonsurgical embryo collection with surgical transfer in captive white-tailed deer. Theriogenology a:273 (1988).

15.

Raphael, B.L., Loskutoff, N.M., Howard, J.G., Wolfe, B.A., Nemec, LA., Schiewe, MC. and Kraemer, DC. Embryo transfer and artificial insemination in suni (Neotrams moschatus zuluensis). Theriogenology (this issue, 1989).

16.

Nagashima, I., Matsui, K, Sawasaki, T. and Kano, Y. Non-surgical collection of embryos in Shiba goats. Exp. Anim. %:51-56 (1987).

17.

Mutiga, E.R. and Baker, AA. Transfer of sheep embryos through a laparoscope. Vet. Rec. U&401-402 (1984).

18.

McKelvey, W-AC. and Robinson, J.J. Normal lambs born following transfer of embryos by laparoscopy. Vet. Rec. U&230 (1984).

19.

Walker, SK, Wames, G. M., Quinn, P., Seamark, R.F. and Smith, D.H. Laparoscopic technique for the transfer of embryos in sheep. Aust. Vet. J. @:105-106 (1985).

20.

McKelvey, W-AC., Robinson, JJ., and Aitken, R.P. A simplified technique for the transfer of ovine embryos by laparoscopy. Vet. Rec. m:492-494 (1985).

21.

Otuski, K. and Soma, T. Transfer of the fertilized ova through the cervix of goats. Bull. Natl. Inst. Anim. Ind. 6:27 (1964).

22.

Lin, AC. Studies on the embryo transfer in goats and pigs. Bull. Nippon Vet. Zootech. College 2:215-216 (1981).

23.

Agrawal, K.P. and N.K.. Bhattacharyya, N.K. Nonsurgical transplantation of embryos in goats. Proc. 3rd Int. Cong. on Goat Prod. and Disease. Dairy Goat Journal Publishing Co., p. 340 (1982).

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