Fertilization and Subsequent Development in Vitro of Pig Oocytes Inseminated in a Modified Tris-buffered Medium with Frozen-Thawed Ejaculated Spermatozoa1

Abstract

The present study examined the penetrability of pig oocytes by frozen-thawed ejaculated boar spermatozoa, prepared by the pellet method, coincubated in a modified Tris-buffered medium. Subsequent embryonic development of fertilized oocytes was also determined. Porcine oocyte-cumulus complexes were cultured in BSA-free North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml), and hormonal supplements (eCG and hCG: 10 IU/ml each) for 20-22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20-22 h. After culture, cumulus cells were removed and oocytes were coincubated for 12 h with three different (1 x 10(5), 5 x 10(5) and 1 x 10(6)/ml) sperm concentrations (experiment 1). In experiment 2, oocytes were coincubated with sperm (5 x 10(5)/ml) for 3, 6, 9, and 12 h. In experiment 3, at 6 h after coincubation with sperm at 5 x 10(5)/ml concentration, oocytes were transferred into NCSU 23 + 0.4% BSA medium. At 48 and 144 h, cleavage and blastocyst formation rates, respectively, were evaluated. Insemination with 1 x 10(5)/ml resulted in a 40% sperm penetration rate of oocytes with 16% polyspermy. Mean number of sperm (MNS) per oocyte was 1.2 +/- 0.1. At 5 x 10(5) and 1 x 10(6)/ml, penetration rate (84-87%) and polyspermy (57-64%) increased (p < 0.001), with no difference between the two concentrations. However, MNS per oocyte increased (p < 0.05) with increasing sperm concentration. Penetration rate was 31% at 3 h and increased (p < 0.001) at 6-12 h (80-88%), with no difference between these time points. Polyspermy increased (p < 0.05) in a time-dependent manner up to 9 h, with no difference between 9 and 12 h. Compared to 3 h, MNS per oocyte increased (p < 0.05) at 9 and 12 h, with no mean difference at 6 h. At 48 after culture, the cleavage rate was 40%, and at 144 h, the blastocyst rate was 19%. This study describes the cryopreservation of ejaculated boar semen by the pellet method and the successful in vitro fertilization of pig oocytes by frozen-thawed spermatozoa with subsequent development to the blastocyst stage.

Aberrant DNA methylation in porcine in vitro‐, parthenogenetic‐, and somatic cell nuclear transfer‐produced blastocysts

“…COCs were matured for 40-44 hr in 5% CO 2 in air at 398C prior to the removal of the cumulus cells by vortexing for 3 min in Hepes-buffered medium with 0.1% (w/v) hyaluronidase. Denuded oocytes were washed and held in modified Tris-buffered medium (mTBM) prior to fertilization (Abeydeera and Day, 1997).…”

Correlation of Developmental Differences of Nuclear Transfer Embryos Cells to the Methylation Profiles of Nuclear Transfer Donor Cells in Swine

“…COCs were matured for 40-44 hours in 5% CO 2 at 39°C prior to the removal of the cumulus cells by vortexing for three minutes in Hepes-buffered medium with 0.1% (w/v) hyaluronidase. Denuded oocytes were washed three times and held in modified Tris-buffered medium 22 prior to fertilization or SCNT.…”

PAWP, a Sperm-specific WW Domain-binding Protein, Promotes Meiotic Resumption and Pronuclear Development during Fertilization

“…Porcine Oocyte Collection and in Vitro Fertilization-Ovaries were collected from pre-pubertal gilts at a local slaughterhouse, and oocytes were collected as described previously (27). Oocytes were washed twice and transferred into TCM-199 medium containing 0.1% polyvinyl alcohol, 10 ng/ml epidermal growth factor, 0.5 g/ml FSH, 0.5 g/ml LH, and 0.57 mM cysteine (mTCM-199) at 39°C in atmosphere of 5% CO 2 in humidified air.…”