Small Ruminant Embryo Transfer - Opportunities and Challenges A Practitioner s perspective Brian W. McOnie Benefits of embryo transfer Embryo transfer (ET) technology gained commercial prominence in the international movement of cattle genetics in the 1970's and the rapid domestic increase in the North American population of European breeds of cattle. Similar though less dramatic events have been occurred in sheep and goat breeds with the recent importation to Canada of many cryopreserved small ruminant embryos especially Boer goat and Texel, East Friesian, Dorper and Charollais sheep. Small ruminant ET is a well described and yet underexploited animal breeding technology. The size of sheep and goats, aspects of their anatomy and seasonal reproductive behaviour, present the ET practitioner with challenges not faced by those working with cattle. Those considerations have not deterred serious breeders and ET practitioners in sheep and goat producing countries. Transrectal palpation and manipulation of the reproductive tract is not possible. The cervix of sheep and goats is tortuous, fibrous and difficult to penetrate other than during estrus. It is possible to assess ovarian and follicular status with ultrasound but this may not always be logistically practical, nor necessary. Non-surgical embryo recovery is possible in goats, and embryo recovery rates can be comparable with surgical embryo recovery. Non-surgical embryo transfer remains undeveloped. While it is possible to identify the presence of a CL by transrectal ultrasonography, transcervical penetration for embryo delivery into the appropriate uterine horn is difficult. Laparoscopic embryo recovery is technically challenging. It has most application in the recovery of embryos from very valuable (eg. transgenic founders and endangered species) donors. Most practitioners rely upon laparoscope-assisted surgical embryo recovery, which is invasive and but limits the number of times a donor can be flushed. Laparoscope-assisted transfer of embryos into recipient sheep and goats minimizes potentially traumatic handling of the reproductive tract and may be performed rapidly. The investment in developing technique, performing anesthesia and surgical procedures, and the requisite equipment, all contribute to the overall cost of the procedure. Super-ovulation As with other domestic species, super-ovulation can be initiated by the administration of gonadotrophins. Inconsistent results were obtained with ecg used in sheep and goats. The more reliable results obtained with FSH-p were further improved upon through the use of purified FSH preparations with restricted LH content (Folltropin Bioniche, CAN, Ovagen ICP, NZ). Published studies are often confounded and document little difference in outcomes (number of transferable embryos) when superovulatiing small ruminants with either Ovagen (o-fsh) or Folltropin (p-fsh). The slightly different molecular structure of o-fsh should potentially, at least, confer it some advantage over p-fsh when used to superovulate sheep and goats. 1
Table 1. Comparison of transferable embryos (mean +/- sem) resulting of from superstimulation of goats by injection with Ovagen (o-fsh) or Folltropin (p-fsh) where a progestagen was supplied by intra-vaginal sponge or CIDR-G. N.Z. Vet. J. 37:27-29 Sponge CIDR-G Ovagen * 8.7 +/- 3.1 14.7 +/- 2.2 Folltropin ** 9.1 +/- 3.4 10.8 +/- 1.8 *Ovagen given in 8 equal doses; **Folltropin in 8 declining doses The mean ovulation rate and mean number of recovered and viable embryos tend to be higher with decreasing than with constant doses of purified FSH administered over 6-8 treatments. Table 2. Superovulatory response (mean +/- sem) in sheep treated with a constant (Group 1) or decreasing (Group 2) dose of a low LH, o-fsh (Ovagen, ICP, Aukland, N.Z.) Reproduction, Fertility and Development 16:421-435. Group (n) Responding females Ovulations per female Embryos per female Transf. embryos per female Group 1 (31) 29 (93.6%) 13.6 +/- 1.4 10.4 +/- 1.5 5.3 +/- 1.1 Group 2 (31) 30 (96.8%) 15.9 +/- 1.9 13.5 +/- 2.4 7.0 +/- 1.7 Super stimulation is generally superimposed on a progestagen treatment, in most cases represented by progesterone impregnated sponges (medroxyactate progesterone 60mg sponges Veramix - Pharmacia & Upjohn) or CIDR-G (InterAg Research). Historically, progestagens were administered for as long as 14 days (sheep) or 19 days (goats) with FSH administered over the last 3-4 days of progestagen treatment. Early New Zealand goat protocols relied upon CIDRs for 19 days and FSH administerd twice daily on days 17-20 inclusively. No hormones other than CIDRs and FSH were used as progestagens were supplied for longer than the normal duration of a CL. In Canada, only Veramix sponges are approved for use in sheep; no product is approved for use in goats. Most of our superovulatory programs are based on Veramix sponges for 12 days. P-FSH is administered every 12 hours for 4 days in a decreasing dose, generally beginning on day 9. Cloprostenol is given close to the time of the first injection of p-fsh. Current research suggests that this duration of P4 exposure is unnecessarily long, although the impact on fertility is equivocal. As with cattle, there is evidence that the ovarian response to super-stimulation is dampened in the presence of a dominant follicle. Strategies which may militate against the effect of a dominant follicle, and attempt to recruit follicles 4-6 mm., in diameter are documented. Follicles smaller than 4 mm. at the beginning of superovulation are presumed to lack the competence to be recruited and subsequently ovulated and fertilised. Donors are usually in standing estrus 24-36 hours after sponge withdrawal. We recommend that breeding should begin no earlier than 24 hours after sponge withdrawal (assuming the donor is in standing estrus), and continue every 8 hours until the donor is out of heat. Laparoscopic AI can be performed at approximately 48 hours after progestagen withdrawal. Most breeding is by natural service although we have experienced good results with transcervical AI in sheep (fresh extended semen) and goats with frozen semen. If AI is being considered, we recommend laparoscopic AI where logistically possible. Embryo recovery occurs 8 days after the withdrawal of the progestagen. 2
Table 3. Ova fertilisation (%) as a function of superovulatory response and means of breeding (goat). All semen doses contained 200 x 10 9 motile sperm. (Therio 59:171-188) Superovulatory response <15 CL >15 CL Total Hand mating 82 72* 75 Cervical AI (x2) 66 47* 55 Laparoscopic AI (frozen) 63 52* 55 Laparoscopic AI (fresh) 68 65 66 ( * Differ significantly from < 15 CL within a row P <0.01) Embryo recovery Success in non-surgical embryo recovery is limited to goats and has not been part of our practice. Procedure Sedation and local anesthesia of the donor may provide adequate restraint but we prefer general anesthesia for optimal control and safety. The anesthetized donor is placed in dorsal recumbency and the ventral abdomen cranial to the udder prepared routinely for surgery. Response to super-stimulation is assessed laparoscopically. Poor responders (<=3 CL) are usually not flushed unless at the request of the owner. Typically 15-20% of sheep and 10% of goats fail to stimulate adequately. The uterus and uterine horns are exteriorized through a ventral (either midline or para-median) abdominal incision. A Foley catheter is introduced through a stab incision in the uterine horn, threaded distally and the cuff of the catheter inflated. Embryo flush fluid (usually a 20mL aliquot) is introduced through a blunted 20g needle at the utero-tubule junction and directed down the uterine horn, towards the Foley and collected in a gridded search dish. The procedure is repeated with the contra-lateral horn. The uterine stab incisions are closed (3-0 PDS). The serosal surfaces of the reproductive tract are rinsed free of blood clots or foreign material with saline. Abdominal closure is routine. Careful technique is required to minimise the formation of adhesions which can impair future fertility. While donors may be flushed 2-3 times in a season, allowing them to become pregnant after a few flushes lets the reproductive tract become stretched, and breaks down some of those adhesions. Identification and classification of embryos Examination of the recovered flush fluid is routine. The size, morphology and developmental stages of small ruminant embryos are typical of bovine embryos. Small ruminant embryos may be held in proprietary embryo holding solutions formulated for bovine embryos. 3
Table 4. Transferable embryos from sheep as a function of superovulatory response. Therio. 59:171-188 # CL % ova / CL % Fertilised % Transferable. 5-9 60 84 69 10-14 62 93 76 15-19 62 89 79 20-24 67 85 73 25-29 67 82 77 >30 53* 72* 47* ( * Differ significantly from other values within columns P <0.01) Embryo cryopreservation Sheep and goat embryos are generally frozen in 0.25 ml plastic straws, each straw containing 2-4 embryos of a similar developmental stage from the same flush. Conventional cryopreservation in 1.5M EG (seeding at -6C, followed by cooling at -0.5C/min to -35C) is effective for morulae and blastocysts. It is reported that blastocysts have greater post-thaw survival than morulae. Vitrification may offer a cost effective alternative to conventional freezing of small ruminant embryos by circumventing the need for controlled rate freezers. Post-thaw survival of vitrified sheep and goat embryos compare favourably with conventionally frozen embryos of the same developmental stage. Donor and recipient cycle synchrony As with other species, donor and recipient estrus and ovulations should be synchronised as closely as possible. Asynchrony of 12-24 hours results in acceptable pregnancy rates. Synchronisation usually involves intra-vaginal progestagens, inserted concurrently with the donors, and withdrawn 0-8 hours prior to the donors. In our practice, ecg (200-500 IU) is given at progestagen withdrawal. Some groups advocate the use of GnRH or LH 24h. after progestagen withdrawal to further narrow the interval during which ovulation may occur. We prefer prolific breeds as recipients. Most will have multiple CLs at the time of ET and have a greater potential for milk production. Despite hormonal manipulation, some potential recipients fail to synchronise or are otherwise found to be unfit as embryo recipients at the time of ET. Failure of recipients to become synchronized can slow ET projects both on the day of ET, and add costs of re-programming and delays in the birth of offspring. Pre-screening of recipients by measuring serum progesterone levels has been attempted in small ruminants but with inconsistent results and has a low predictive value. Transrectal ultrasonography offers a non-invasive alternative but requires technical proficiency, suitable equipment, and does not circumvent the need to scope qualifying recipients. In our practice, final selection of recipients is made at the time of ET. Assessment of CL number and quality can be performed rapidly by laparoscopy. A detailed examination of the entire reproductive tract is impractical but obvious pathology (extensive abdominal adhesions, pelvic inflammatory disease, abscesses, distended uterine horns) disqualifies potential recipients. A single CL can, and will, support pregnancy but my preference is to see 2 or more extruded, bright to deep red CLs. If the recipient is deemed satisfactory, ET can be performed immediately. 4
Transfer of embryos ET can be performed quickly so sedation (typically xylazine/ketamine), with or without local analgesia, provides adequate restraint. Once the animal is placed on the tilt table, the ventral abdomen is prepared routinely for surgery though to a lesser extent than the donors. The location of laparoscope ports and incisions for exteriorization of the uterine horn are largely a matter of personal preference. I introduce the laparoscope 5-7 cm. cranial to the udder and 5-7 cm. lateral to the midline, attempting to avoid mammary vessels. A small incision (2 cm.) to allow forceps manipulation of the reproductive tract is made paramedian or on the ventral midline. The tip of the uterine horn ipsilateral to the CL(s) is gently exteriorized and embryos transferred at the uterotubular junction. The uterine horn is allowed to return to the abdomen and the small incisions are closed routinely. As with other species, small ruminant ET presents many opportunities, to both the livestock owner / producers and veterinarian. Practitioners continue to be faced with on-going challenges in providing optimal outcomes with current techniques and in the application of advances in the field. Areas that continue to require work are consistent super-stimulatory responses, nonsurgical embryo recovery, and vitrification with direct transfer. Typical responses Sheep Goats Super-ovulatory response 12 CL (0-30) 15 CL (0-45) Efficiency of embryo collection 75% (60-100%) 75% (60-100%) Transferable embryos / donor 6 (0-30) 9 (0-45) Embryos surving to birth 70% (0-100%) 70% (0-100%) Recipients pregnant 65% (0-100%) 65% (0-100%) References upon request Brian W. McOnie Creekside Animal Clinic Ltd 5001 24 th. Street Vernon, BC V1T 8X7 250.549.3533 animal@junction.net www.creeksideanimalclinic.com 5
Expectations and caveats Average Range Super-ovulatory response to FSH 12 CLs 0-30 Efficiency of embryo collection 8 embryos 60-95% Transferable embryos 6 embryos 0-30 Embryos surviving to birth 70% 0-100% Recipients pregnant 60% 0-100% 6