Development of Ovine Embryos in Synthetic Oviductal Fluid Containing Amino Acids at Oviductal Fluid Concentrations

Abstract

The effects of supplementing synthetic oviductal fluid (SOF) with amino acids, at oviductal fluid concentrations, on the development of ovine in vitro-matured/in vitro-fertilized embryos was examined in three experiments. In the first, embryo development in SOF, SOF + 2% human serum (HS), SOF + 20% HS, and SOF + BSA, with and without amino acid supplementation, was examined. Development of zygotes to the blastocyst and hatching blastocyst stages was highest in medium containing 20% HS (64.8% and 54.4%, respectively) irrespective of amino acid supplementation. However, supplementation was significantly beneficial in all other media, with up to 42.1% of zygotes developing into hatching blastocysts. In these media, supplementation also significantly increased the mean number of nuclei per newly formed blastocyst (up to a mean of 70.8) and reduced the time during which blastocysts formed. Experiment 2 was an examination of the effect on embryo development of three amino acid preparations (oviduct amino acid concentrations vs. Eagle's Basal Medium (BME) essential + Minimum Essential Medium (MEM) nonessential vs. MEM essential + MEM nonessential concentrations) and the presence or absence of BSA. Both the amino acid and BSA treatments significantly influenced the percentage of zygotes that developed to the hatching blastocyst stage but not to the blastocyst stage. The preferred medium contained amino acids at oviductal fluid concentrations and BSA (54.5% hatching rate). The amino acid treatments did not significantly influence the mean number of nuclei per newly formed blastocyst, but the addition of BSA had a significant effect (70.7 +/- 1.14 vs. 75.7 +/- 1.13). In experiment 3, embryo development to Day 13 was examined after culture in SOF containing amino acids at oviductal fluid concentrations. Embryos were cultured in the presence of either BSA, polyvinyl alcohol (PVA), or no additional supplement and were transferred to recipient ewes on either Day 0 (after in vitro fertilization), 3, or 5. The addition of BSA or PVA had no significant effect, but significantly more embryos developed to Day 13 following transfer on Day 0 (60.0%) than on either Day 3 or 5 (overall 45.4%). It is concluded that SOF containing oviductal fluid concentrations of amino acids 1) facilitates the development of a high percentage (57.5%) of blastocysts, 2) improves embryo morphology compared with that observed in medium containing HS, 3) significantly improves hatching rates compared with those obtained in SOF containing commercially available preparations of amino acids, and 4) produces embryos with relatively high levels of viability to Day 13 of pregnancy.

The effects of melatonin on in vitro oocyte competence and embryo development in sheep

“…Half of the embryos in each group were cultured in a media that contained melatonin at either a 10 -5 M concentration (embryos from the M5 and C5 oocytes) or a 10 -6 M concentration (embryos from the M6 and C6 oocytes), and the other half remained as untreated controls. The embryos from the eight groups (M5M, M5C, C5M, C5C, M6M, M6C, C6M, and C6C) were placed in a culture medium that contained SOF supplemented with essential and nonessential amino acids at oviductal concentrations (Walker et al, 1996), 0.4% BSA (w/v), 1 mM of L-glutamine, 100 IU mL -1 of penicillin G, and 100 µg mL -1 of streptomycin sulphate, covered with mineral oil, and kept at 39ºC in a maximally humidified atmosphere, with 5% CO 2 , 5% O 2 , and 90% N 2 until the blastocyst stage (day 8). The number and developmental stage of blastocysts (young, expanded, hatching and hatched blastocysts) were recorded at 6, 7, and 8 days after fertilization.…”

Superoxide dismutase affects the viability of thawed European mouflon (Ovis g. musimon) semen and the heterologous fertilization using both IVF and intracytoplasmatic sperm injection

“…The matured oocytes were divided into two groups and fertilized in vitro, as described by Walker et al (1996) with some modifications. In vitro fertilization was performed in four-well Petri dishes (Nunclon; Nalge Nunc International, Kamstrup, Denmark) by depositing 20-30 oocytes into 500 µL of semen suspension (1 × 10 6 spermatozoa mL −1 ) derived from the two experimental culture systems (SOF and 0.6% (w/v) BSA), with or without SOD, as described earlier.…”

Optimisation of in vitro culture conditions in B6CBF1 mouse embryos

“…Paraffin oil [10][11][12], mineral oil [13][14][15] and silicone oil [16,17] have been broadly used to protect culture medium from evaporation and contamination when early embryos are cultured. Further studies have indicated that the application of oil affects the embryo development either in a positive or a negative way [18,19].…”