201 ENDOSCOPE-GUIDED TRANSFER OF SPERM-INJECTED OOCYTES INTO THE OVIDUCTS OF ELAND AND BONGO ANTELOPES

Abstract

We have previously established methods of gamete collection in Eland and Bongo antelopes; however, blastocyst development following IVF, intracytoplasmic sperm injection (ICSI), or somatic cell nuclear transfer (SCNT) has been sporadic, thus few developmentally competent embryos have been produced for uterine transfer. In the present study, we evaluated the possibility of oviductal transfer of presumptive zygotes. Estrous cycle synchronization and ovarian superstimulation were done as follows. Females were treated with altrenogest (0.11 g/os daily) for 7 days. The FSH Folltropin^® was dissolved in 30% polyvinylpyrrolidone and administered, i.m. on Days 5 and 7 of altrenogest treatment at 266 mg and 134 mg, respectively. Prostaglandin F_2α (Lutalyse^®, 25 mg, i.m.) was administered on Day 7. Transvaginal ultrasound-guided oocyte retrieval was conducted on Day 9 as described previously (Wirtu G et al. 2009 Anim. Reprod Sci. 111, 160-172). Eleven oocytes were recovered from 2 Elands and 4 from 1 Bongo. One oocyte of each species was degenerate at the time of recovery. Oocytes were subjected to IVM for approximately 22 h, when Piezo drill-assisted ICSI was done using frozen-thawed Eland or Bongo spermatozoa. Oocytes were activated (5% ethanol, 5 min) and cultured overnight in CR1aa medium supplemented with BSA. Presumptive zygotes were subjected to endoscopic oviductal transfer at 21 to 24 h after ICSI. The oviductal transfer was adapted from the technique developed in domestic cattle (Wetscher F et al. 2005 Theriogenology 64, 30-40). Animals were sedated by i.m. administration of xylazine HCl and butorphanol tartrate and restrained in a hydraulic chute (Tamer^®) that was used to squeeze and lift the females. An epidural block (5 mL of lidocaine) was induced after cleaning the rectum, perineal region, and the injection site. A uterine relaxant, isoxsuprine HCl (10 mg, i.v), was then administered. The Brem/Besenfelder set for laparoscopic bovine embryo transfer (Karl Storz Endoscope, Karl Storz GmbH & Co. KG, Tuttlingen, Germany) was used for the oviductal transfer. Briefly, a cannula (12.5 mm in diameter, 49.5 cm long) fitted with a blunt-tip obturator was introduced into the vagina and positioned, during transrectal manipulation, at the mid-dorsal aspect of the vaginal vault. The blunt-tip obturator was replaced with a sharp-tip obturator, which was used to puncture through the vaginal wall for entry into the pelvic cavity. The trocar was subsequently replaced with an inner sheath containing a telescope (5.5 mm in diameter, 54 cm long) and a pre-loaded transfer tubing. Visualization of the ovaries and oviducts required insufflation of the peritoneal cavity. Transfer was done into the oviduct ipsilateral to the recently ovulating ovary. Ten Eland oocytes had extruded the first polar body and 8 that survived ICSI were transferred into the right oviduct of an Eland female. Two Bongo oocytes had extruded the first polar body; both survived ICSI and were transferred to the right oviduct of a Bongo female. Pregnancy diagnosis is pending. This minimally invasive method of accessing the oviduct has the potential to advance the application of assisted reproductive technologies in large nondomestic ungulates.