139 BOVINE AMNIOTIC FLUID FOR THE CULTURE OF TWO-CELL MURINE EMBRYOS

Abstract

The objective of the experiment was to evaluate bovine amniotic fluid as an alternative medium for culture of two-cell murine embryos. Variability in embryo development rate and high purchase prices of medium supplements such as fetal bovine serum have prompted the search for a serum alternative. Amniotic fluid was collected postmortem from first-trimester bovine fetuses, pooled, heat inactivated at 56°C for 30 min and stored at −20°C until used. Two-cell mouse embryos were collected from (Balb/C × C57BL6) F₁ females superovulated with 10 IU PMSG and 10 IU hCG. The experiment was performed to evaluate the effect of commercial fetal bovine serum (F4135, Sigma, South Africa) supplementation to amniotic fluid. Treatments consisted of (1) bovine amniotic fluid, (2) bovine amniotic fluid supplemented with 10% fetal bovine serum, (3) M16 (M7292, Sigma) supplemented with 10% fetal bovine serum, and (4) Medium 199 (M4530, Sigma) supplemented with 10% fetal bovine serum. The latter two media were controls. Twenty-four hours before use the culture media were supplemented with 1% antibiotic antimycotic solution (A5955, Sigma). Media were equilibrated for 24 hours at 37°C and 5% CO₂ before use. Embryos were cultured in 50-μL droplets with oil overlay at 37°C in 5% CO₂. A minimum of 5 and maximum of 10 embryos per droplet were allowed. Six replications per treatment were done giving a total of 292 embryos in treatment (1), 318 in treatment (2), 304 in treatment (3), and 303 in treatment (4). The embryos were monitored under an inverted microscope (Olympus model IX70) at 24-h intervals for 72 h for blastocyst formation. The differences between embryo growth in the different culture media were assessed by one-way analysis of variance. All culture media supported the development of mouse embryos to the hatched blastocyst stage. A higher (P < 0.05) number of embryos hatched in M16 (64.6%) and Medium 199 (55.0%) supplemented with 10% fetal bovine serum than in frozen bovine amniotic fluid (12.2%) and frozen bovine amniotic fluid supplemented with 10% fetal bovine serum (17.8%). M16 was superior to all other treatments in supporting embryo development up to the morula stage. More than twice the number of embryos (94.9%) reached the morula stage in M16 than in frozen bovine amniotic fluid with (37.4%) or without (29.1%) serum supplementation. Bovine amniotic fluid obtained from postmortem first trimester fetuses supported the development of two-cell mouse embryos; however, embryo development in frozen fetal fluid was lower (P < 0.05) than that obtained in the control media. Fetal bovine serum, when added to amniotic fluid, did not increase the development rate. It seems likely that freezing the amniotic fluid had an adverse effect on in vitro embryo development.