126 THE EFFECT OF FORSKOLIN ON IN VITRO PRODUCED BRAHMAN-SIRED BOVINE EMBRYOS

Abstract

Forskolin (Colforsin) is a lipolytic agent that can be used to reduce intracellular lipids within in vitro produced (IVP) porcine embryos (Men et al. 2006 Theriogenology 66, 2008–2016). Lowering lipids within bovine embryos has shown to decrease sensitivity to cryopreservation. The objective of this preliminary experiment was to determine developmental differences and lipolytic effect on bovine IVP embryos exposed to 10 or 40 μm forskolin. Bovine oocytes (obtained from an abattoir) were fertilized with frozen/thawed Brahman semen (Day 0) and cultured in G1.5/G2.5 medium supplemented with 8 mg mL^–1 BSA (Vitrolife, Englewood, CO, USA). On Day 6 post-fertilization, a total of 352 viable embryos were randomly submitted to 3 treatment groups; Control (no treatment, n = 116), 10 μm (n = 122) and 40 μm (n = 114) forskolin (Sigma, St. Louis, MO, USA) in G2.5 medium for 24 h. On Day 7, embryos were washed in holding medium (Vigro Holding Plus, Bioniche, Pullman, WA, USA). From each treatment group, a randomized half was cultured 72 h in G2.5 and assessed for hatched blastocyst rates (development), with the balance stained on Day 7 in Hoechst 33342 (2.5 μg mL^–1) under UV light for total cell counts before being fixed/stained in Nile red (1 μg mL^–1). Nile red stained embryos were mounted on glass slides with cover slips gently applied over each embryo. Embryos were photographed under UV light using the proper excitation filters. The area measurements of compact morula and blastocysts did not include the zona pellucida. Nile red units (NRU) were measured with computer software Image J (www.rbsweb.nih.gov) from 0 to 255 shades/pixel (0 = no lipids; 255 = highest lipid accrual). Each image was adjusted for variation in embryo size. Student’s t-test was used for statistical analysis. There was no difference (P > 0.05) between development rates for control, 10 μm and 40 μm forskolin (62%, 67% and 65%, respectively). Mean NRU by total cell numbers were calculated per embryo for each treatment group: control, 10 μm and 40 μm with the resulting means being 72.4 ± 5.0, 63.1 ± 5.1 and 66.6 ± 4.8 respectively. With treatment groups combined by embryo stage, compact morula tended to exhibit a higher mean NRU than blastocysts (78.6 ± 6.8 to 64.7 ± 3.1, respectively, P = 0.07). In conclusion, forskolin did not significantly affect embryo development rates or lipid content as measured by Nile Red staining. Compact morula tended to yield a higher NRU mean than blastocysts indicating an elevation in intracellular lipids. Unlike pig IVP embryos exposed to forskolin, forskolin failed to show a significant decrease in intracellular lipids within bovine IVP embryos. However, further research is in progress to test forskolin’s lipolytic effect on cryopreservation of in vitro and in vivo produced bovine embryos.

The authors acknowledge support from the American Brahman Breeders Association and Dr. Moisés Barceló Fimbres.