68 Comparison of two Percoll gradients for selection of frozen semen for in vitro production of ovine embryos

Abstract

Sperm selection methods are routinely applied to prepare semen for IVF in animal species. These procedures are used to improve sperm quality characteristics as well as to remove other background material and debris. Percoll gradient is widely used in animal IVF laboratories. Different Percoll gradient concentrations and volumes can be used to improve the sperm sample motility percentage. The objective of this research was to evaluate the effect of 2 different Percoll concentrations for ovine IVF sperm selection and effects, if any, on in vitro embryo production (IVP). Specifically, Percoll gradients consisted of Group 1 (G1) 40–80% and Group 2 (G2) 45–90%, 400 µL each. The research was carried out in the reproduction laboratory at Palominos Ranch (Jalisco, México). The IVP was performed with a continuous in vitro culture system. Ovaries (n = 157) were collected from a slaughterhouse (León, México) and transported to the laboratory within 2 h in physiological saline solution (0.9% NaCl) supplemented with penicillin G (100 IU mL⁻¹) and streptomycin sulphate (100 µg mL⁻¹). For IVP, IVF Bioscience™ media were used for IVM, IVF, and in vitro culture (IVC). For IVM, the cumulus–oocyte complexes (COCs) were selected (only grades 1 and 2) and matured for 24 h at 38.5°C in 5% CO₂ in air and 100% humidity. Matured oocytes (n = 800, divided equally into 4 replicates) were divided into 2 groups, G1 and G2. The IVF process was conducted with semen selected through a mini-Percoll technique with gradients at a concentration of 45% (top layer) and 90% (bottom layer) or 40% (top layer) and 80% (bottom layer) for G1 and G2, respectively, using frozen–thawed semen from the same ram, at a concentration of 2 × 10⁶ sperm mL⁻¹, for 18 h 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. The statistical analyses were carried out with the GLM procedure of SAS software (version 9.3; SAS Institute Inc.) to evaluate the results of G1 versus G2 (α level = 0.05). Cleavage rate was 47.8% ± 2.5 and 55.9% ± 2.5, respectively, in G1 and G2. The percentages of embryos with more than 6 cells were 38.1% ± 2.2 and 43.5% ± 2.2, respectively, in G1 and G2. The percentage of blastocysts on Day 7 was 27.4% ± 1.1 and 37.3% ± 1.1, respectively, in G1 and G2. There were no significant differences between groups (P > 0.05) for percentage of cleavage and embryos with more than 6 cells, although the percentage of cleavage tended to be greater for G2 (P = 0.06). Additionally, G2 had a larger percentage of blastocysts on Day 7 compared with G1 (P < 0.05). In conclusion, under the conditions of this research, the use of a Percoll gradient at a concentration of 40–80% increased the percentage of blastocysts for ovine IVP.