108 THE EFFECTS OF FORSKOLIN ON BRAHMAN IN VIVO-PRODUCED EMBRYOS AND SUBSEQUENT PREGNANCY RATES POST-THAW

Abstract

It has been hypothesised that lower survival rates post-thaw of Brahman (BR) embryos is due in part to higher intracellular lipids. The objective of this study was to determine whether pregnancy rates of in vivo–produced BR embryos could be enhanced by exposing them to 10 μM of forskolin (FSK; lipolytic agent) before slow cool freezing, thawing and direct transfer. A previous report (Phase II) indicated that the addition of FSK to culture 24 h before freezing 7 days in vitro–produced, BR-sired embryos can increase survival and blastocyst hatching rates (Pryor et al. 2010 Reprod. Fertil. Dev. 22, 214). For this study (Phase III), 11 BR cows were injected with 25 mg of Lutalyse^® (Pfizer Animal Health, New York, NY, USA) to synchronize oestrus. On Day 0 (7 days after synchronized oestrus), all donors were inserted with a CIDR (Controlled Intravaginal Releasing Device; Pharmacia Co., Kalamazoo, MI, USA) and injected with 50 mg of progesterone plus 2.5 mg of oestradiol 17 β (Medshop, Longview, TX, USA). On Day 4, decreasing doses of Folltropin (Bioniche, Pullman, WA, USA) ranging from 160 to 264 mg were given twice daily for 3 days along with 0.625 mg of cloprostenol (Estrumate; Merck/Schering-Plough Animal Health, Whitehouse Station, NJ, USA) given twice on Day 6 (AM and PM) and CIDR removal on Day 7 AM. Donors were artificially inseminated 12 and 24 h post-oestrus (oestrus = Day 8) with frozen/thawed BR semen. On Day 14 (embryo age 6 days), donors were nonsurgically collected, producing a total of 75 grade-one morulae, which were randomly allocated and cultured for 24 h in Evolve (Zenith Biotechnology, Canada) supplemented with 4 mg mL^–1 of Probumin BSA (Millipore, Norcross, GA, USA; control) or with FSK (Sigma, St. Louis, MO, USA) at 38.5°C under a 5% CO₂, 5% O₂ and 90% N₂ humidified atmosphere. Immediately following treatment, 7 days compact morula/blastocysts were washed in Vigro Holding Plus medium (Bioniche, Belleville, Ontario, Canada) and submitted to Vigro Ethylene Glycol Freeze Plus medium (Bioniche) for 5 to 7 min before being frozen at 0.5°C min^–1 from –6°C to –32°C and plunged in liquid nitrogen. Frozen embryos (n = 35 FSK; n = 35 control) were air thawed for 7 s and then immersed in 30°C H₂O for 10 s before being nonsurgically transferred into synchronized recipients. Pregnancy rates were assessed by ultrasonography via rectal palpation 30 to 60 days post-transfer. Contingency analysis was performed using forskolin treatment, technician and embryo stage and location as independent variables (JMP 8.0, SAS Institute Inc., Cary, NC, USA). There were no statistical differences or interactions for any of the analysed variables. Pregnancy rates between control and FSK treatments did not vary (34.3 and 31.4%, respectively; chi-square P = 0.80). In conclusion, treating in vivo–produced BR embryos with 10 μM of forskolin for 24 h did not alter pregnancy rates.

The authors acknowledge support from the American Brahman Breeders Association.