248 OPTIMAL IN VITRO MATURATION TIME DEPENDS ON MORPHOLOGICAL CHARACTERISTICS OF BOVINE CUMULUS–OOCYTE COMPLEXES

Abstract

Oocytes with compact cumulus cells are usually chosen for bovine in vitro maturation and IVF experiments, while oocytes that do not have compact cumulus cells are often discarded. In this experiment, we studied the interaction of maturation time and cumulus characteristics. Five cumulus classifications (compact, partially expanded, expanded, denuded, dark-coagulated) were studied using 3 maturation times. A total of 1923 oocytes were aspirated from 3- to 8-mm follicles; matured for 1, 4, or 23 h; and fertilized with sperm from 3 bulls (2 replicates per bull with one replicate using ovaries of cull cows and the other, ovaries of fattened heifers). IVF was done with sperm centrifuged through a 45/90% Percoll gradient. All media were chemically defined (CDM) plus 0.5% fatty acid-free BSA. Sperm were co-incubated with oocytes for 16–18 h; presumptive zygotes were then vortexed to remove cumulus cells and cultured for 7 days (De La Torre-Sanchez et al. 2006 Mol. Reprod. Dev. 18, 585–596). Overall blastocyst production was low, partly due to differences among bulls (P < 0.01) (2.8, 4.5, and 11.3% blastocysts per oocyte for semen of bulls A, B, and C), and partly because some ovaries were from fattened heifers fed a progestin. There was a significant interaction between type of cumulus and maturation time for development to the 8-cell stage (P < 0.01) and blastocysts (P < 0.07), but not for cleavage (data not presented). From Table 1, it is clear that 1 or 4 h is insufficient maturation time for oocytes from all cumulus classifications except for expanded cumulus. For that category, 1 h maturation resulted in blastocyst production equivalent to oocytes with compact cumulus matured 23 h. Standard timing of 23 h maturation was suboptimal for oocytes with expanded cumulus. For oocytes with compact cumulus, 23 h maturation was superior to shorter times (P < 0.05); oocytes with partially expanded cumulus cells responded similarly to those with compact cumulus except for a higher percent of 8-cell embryos with 23 h maturation. While 8-cell embryo production was above 40% per oocyte for all groups with 23 h of maturation, blastocyst production was very low for oocytes with dark cumulus, and also low when oocytes were denuded or had expanded cumulus. In conclusion, while 23 h maturation was optimal for oocytes with compact or partially expanded cumulus, similar blastocyst rates were obtained with oocytes with expanded cumulus if maturation time was shortened to 1 h.

Table 1. Mean percentage development of oocytes to 8-cell and blastocyst stages