title: Comparisons of oocyte maturation times and of three methods of sperm preparation for their effects on the production of goat embryos in vitro.
abstract: 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 approximately 4 times higher than after swim-up and approximately 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.
authors.affiliation: College of Veterinary Medicine, Gyeongsang National University, Chinju, Republic of Korea.
authors.forename: G J
authors.initials: GJ
authors.lastname: Rho
authors.forename: A C
authors.initials: AC
authors.lastname: Hahnel
authors.forename: K J
authors.initials: KJ
authors.lastname: Betteridge
mesh_terms: D000177:Acrosome / Q000502:physiology; D000818:Animals; D002499:Centrifugation, Density Gradient / Q000662:veterinary; D018920:Coculture Techniques / Q000662:veterinary; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods / Q000662:veterinary*; D005374:Filtration; D006041:Goats / Q000502:physiology*; D007150:Immunohistochemistry / Q000662:veterinary; D008297:Male; D018613:Microscopy, Confocal / Q000662:veterinary; D008856:Microscopy, Fluorescence / Q000662:veterinary; D009865:Oocytes / Q000502:physiology*; D011247:Pregnancy; D013094:Spermatozoa / Q000502:physiology*; D013997:Time Factors