Lerma C. Ocampo

Effects of culture time, ovarian activity, cumulus cells and sera on the nuclear and cytoplasmic maturation of pig oocytes in vitro

Pig cumulus-oocyte complexes (COC) were used in this study to assess the effect of length of culture, ovarian activity, cumulus cells and sera on maturation and fertilization in vitro. In Experiment 1, the incidence of nuclear maturation increased (P<0.01) from 24 to 48 h of culture and the ability for male pronucleus (MPN) formation peak at 42 h (86.6%). The penetrability of immature COC and those at the metaphase 2 (M2) stage appeared to be not influenced by the stage of meiosis (75.3–89.3%). In Experiment 2, no significant difference in the nuclear maturation (84.8–88.0%) and penetration (82.1–92.0%) rates of COC collected from ovaries with or without corpora lutea (CL) or corpora albicans (CA) was observed, though MPN formation differed (P<0.05) between O1 (82.6%) and O3 (61.5%) oocytes. In Experiment 3, significant differences (P<0.01) on the nuclear maturation and MPN formation of oocytes cultured with cumulus cells versus those without or lacking cumulus cells was observed. It shows that the presence of cumulus cells is important in supporting maturation in vitro. In Experiment 4, serum supplementation of the medium improved the rate at which COC reached the M2 stage (P<0.05) and formed MPN except at the 5% level when using fetal bovine serum (FBS) and calf serum (CS). The use of porcine serum (PS) consistently supported the maturation of COC. Serum supplementation is thus necessary for maturation in vitro and the concentration and type of serum to be used is important.

Blastocyst formation of pig embryos derived from in vitro fertilization of in vitro matured pig oocytes in the amniotic fluid of a developing chick embryo

In this study, the acquisition for developmental competence of early-stage pig embryos derived from in vitro fertilization of in vitro matured pig oocytes was determined by culturing in a modified Whittens medium (mWM) or amniotic fluid of a developing chick embryo (CEAm fluid). Of 1080 oocytes inseminated, 420 were fixed after 15 h revealing a nuclear maturation rate of 95.2% (400420) with a fertilization rate of 84.3% (354420). The monospermic fertilization rate was 36.4% (129354). Of 660 oocytes that were allowed to continue development, 74.2% (490660) cleaved. Of these, 220 and 157 embryos were cultured in mWM and CEAm fluid with 19.5% and 25.7% progressing to blastocyst stage, respectively. The mean (±standard error of the mean) cell number of blastocyst stage embryos derived from CEAm fluid (52.0 ± 3.0) was significantly higher (P < 0.001) than those obtained from mWM (39.0 ± 1.7). The results suggest that CEAm fluid is a better in vitro culture system for pig embryo development than mWM.