86 IN VITRO-MATURED GILT OOCYTES CAN HAVE EQUAL OR BETTER DEVELOPMENTAL COMPETENCE THAN SOW OOCYTES WITH NEW MATURATION MEDIA

Abstract

It has long been thought that oocytes obtained from sows yielded a higher level of developmental competence compared with oocytes obtained from prepubertal gilts. Because gilt-derived oocytes are more readily available to our laboratory and they are less developmentally competent, we hypothesised that by making alterations to our maturation system we could improve the developmental competence of the gilt-derived oocytes to that of their sow-derived counterparts. We performed 2 experiments that evaluated the ability of each source of oocyte to develop to the blastocyst stage, using altered maturation media. The first experiment focused on the developmental ability of each source of oocytes, through IVF and culture. The second experiment again focused on the developmental competence of each oocyte source but through somatic cell NT. For both experiments, the sow-derived oocytes were obtained from Desoto Biosciences and the gilt ovaries were collected from Smithfield Inc. in Milan, Missouri. Both sets of oocytes were in vitro matured in M199 supplemented with 0.57 mM cysteine, 5 μg mL⁻¹ LH and FSH, and 10 ng mL⁻¹ epidermal growth factor; however, the gilt derived media was altered to contain 40 ng mL⁻¹ fibroblast growth factor 2 and 20 ng mL⁻¹ insulin-like growth factor and leukemia inhibitory factor. Additionally, the maturation media for the sow-derived oocytes contained the addition of 5 μg mL⁻¹ insulin and 10% follicular fluid. In the first experiment we performed IVF on oocytes from the 2 sources as per our laboratory standard IVF procedure, co-incubating the oocytes with 0.25 × 106 porcine semen for 4 h, followed by washing and moving the oocytes to MU2 culture media at 38.50°C in 5% CO₂, humidified air overnight. After overnight culture the presumptive zygotes were transferred to the same conditions with 5% CO₂, 5% O₂, and 90% N₂. After an additional 5 days, blastocyst development was assessed. The gilt oocytes yielded 39.3^a ± 7.2% blastocyst, and the sow oocytes had a blastocyst rate of 24.9^b ± 6.9%, with an n of 389 and 313, respectfully. Statistical analysis was performed by using Genmod in SAS 9.4. In the second experiment, using standard laboratory protocol for somatic cell NT, we activated both sets of oocytes with 200 μM thimerosal for 10 min followed by 30-min incubation with 4 mM dithiothreitol. The embryos were co-incubated for 15 h with 500 nM Scriptaid in the MU2 culture media in 5% CO₂, humidified air; then these embryos were also moved to 5% CO₂, 5% O₂, and 90% N₂ and cultured to Day 6. The sow oocytes produced a blastocyst percentage of 38.6%, and the gilt oocyte group had a blastocyst percentage of 43.5%, with an n of 290 and 285, respectfully. There was no difference statistically between these treatments. Both gilt and the sow oocyte sources have yielded live piglets at this time. We concluded that the maturation system used for our gilt-derived oocytes resulted in equal or better development in vitro compared with the sow-derived oocytes. Follow-up experiments evaluating in vivo development are needed for a complete comparison.

This work was funded by Food for the 21st Century University of MO, and the NIH U42OD011140.