Comparisons of oocyte maturation times and of three methods of sperm preparation for their effects on the production of goat embryos in vitro

Various times of in vitro maturation of oocytes, and three methods of separating spermatozoa from frozen-thawed semen (Percoll density-gradient centrifugation, swim-up, and glass-wool filtration), were compared for their effects on goat embryo production in vitro. Cumulus-oocyte-complexes (COCs) from abattoir ovaries were matured in M199 supplemented with 10% fetal calf serum and hormones. In Experiment 1, COCs were fixed at 4 h intervals from 0 to 27 h of culture to assess oocyte nuclear maturation. A higher proportion cultured for 27 h than for 24 h were in Metaphase II (27/37, 73% vs. 18/33, 55%, P < 0.05). In Experiment 2, the effects of separation methods on total numbers and numbers of membrane-intact spermatozoa, and the acrosome reaction were compared. Total numbers after Percoll density-gradient centrifugation were ∼4 times higher than after swim-up and ∼2 times higher than after glass-wool filtration (P < 0.001). Progression of the acrosome reaction was not affected differentially. In Experiments 3 and 4, after 27 h of culture the COCs were inseminated with sperm isolated by the three methods. In Experiment 3, presumptive zygotes were examined for pronucleus (PN) formation at 6, 12, 18 and 24 h post-insemination. At 12 h, male PN formation rate from Percoll-treated spermatozoa was higher than from sperm subjected to swim-up and glass-wool treatments (20/37, 54% vs. 6/37, 16% and 6/38, 16%, respectively; P < 0.001). In Experiment 4, embryos were compared for cleavage at 48 h and development into blastocysts, hatching rates and cell number at 192 h. The rates of cleavage and blastocyst formation in the Percoll-treated group were higher (P < 0.05) than in the swim-up and glass-wool groups (62% and 18% vs. 50% and 11%, and 45% and 8%, respectively). Similarly, the mean cell number in the Percoll group was higher (P < 0.05) than in the swim-up and glass-wool groups (167 ± 5 vs. 149 ± 4 and 126 ± 4, respectively). We conclude that Percoll density-gradient centrifugation is superior to the other two methods for separating goat spermatozoa from frozen-thawed semen in preparation for IVF.