69 Invitro production of ovine embryos using either fresh or frozen-thawed semen

Abstract

Invitro embryo production (IVP) is an important tool for genetic improvement in small ruminants. Semen quality is one of the most important aspects to consider for the success of this assisted reproductive technique. With ovine IVP, it is a common practice to use fresh semen for IVF, but this could be a problem because the differences between ejaculates from the same animal are well documented and a source of variation in IVP results. The objective of this research was to compare the effect of fresh and frozen–thawed domestic sheep (Ovis aries) semen on IVF for ovine IVP. The research was carried out in the reproduction laboratory at the Palominos Ranch (Jalisco, México). The IVP was performed with a continuous invitro culture system. Ovaries (n=186) were collected from a slaughterhouse (León, México) and transported to the laboratory within 2h in physiological saline solution (0.9% NaCl) supplemented with penicillin G (100IU mL−1) and streptomycin sulphate (100µg mL−1). For IVP, IVF-Bioscience™ media were used for IVM, IVF, and invitro culture (IVC). For IVM, the cumulus–oocyte complexes (COCs) were selected (only grades 1 and 2) and matured for 24h at 38.5°C in 5% CO2 in air and 100% humidity. Matured oocytes (n=1000) were invitro fertilized using either fresh or frozen–thawed semen (Triladyl™; Minitube) from the same sheep, at a concentration of 2×106 sperm mL−1, for 18h in 38.5°C, 5% CO2 in air, and 100% humidity. The presumptive zygotes were denuded by pipetting and set in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2, and 90% N2 at 100% humidity. The percentages of cleavage, embryos with more than 6 cells, and blastocysts on Day 7 of culture were evaluated, based on the initial number of oocytes entering into IVM. Statistical analyses were carried out with the GLM procedure of SAS software (version 9.3; SAS Institute Inc.) to evaluate the results of fresh versus frozen–thawed (α level=0.05). Rates of cleavage, embryos with more than 6 cells, and blastocysts on Day 7 were similar (P>0.05): fresh 52.3±3.0%, 43.6±2.6%, and 34.3±2.9%, respectively; frozen–thawed: 53.3%±3.0, 41.1±2.6%, and 33.3±2.9%, respectively. In conclusion, under the conditions of this research, the use of fresh and frozen–thawed semen had similar results for ovine IVP.