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.

We have used chlortetracycline (CTC) analysis to investigate mechanisms that may play important roles during bull sperm capacitation in a culture medium (containing glucose, heparin, and caffeine) known to promote capacitation and fertilization in vitro. In initial experiments employing the Ca2+ ionophore A23187, we identified three discrete CTC patterns so similar to those described for mouse and human sperm that we have employed the same nomenclature: "F," characteristic of uncapacitated, acrosome-intact cells; "B," characteristic of capacitated, acrosome-intact cells; "AR," characteristic of capacitated, acrosome-reacted cells. Over a 60-min period, A23187 stimulated significant increases in B and AR pattern cells, with concomitant decreases in F pattern cells, suggesting a very rapid transition from the uncapacitated to the capacitated state and then on to exocytosis. Without ionophore, significant changes in the proportions of F and B pattern cells were also observed, but the maximum responses required 4 hr; the proportion of AR cells was consistently approximately 15% throughout, indicating a low incidence of spontaneous acrosome loss. Analysis of cells in media with altered composition indicated that the inclusion of either heparin or caffeine significantly promoted capacitation to about the same extent, but together, heparin plus caffeine had an even more stimulatory effect. Despite this, none of these treatments triggered acrosome loss above the levels seen in media lacking these constituents. In the presence of caffeine, with or without heparin, the inclusion of glucose had little effect on responses, but in the presence of heparin there were fewer B cells.(ABSTRACT TRUNCATED AT 250 WORDS)

In the present study, attempts were made to develop a protein-free (PF) in vitro maturation (IVM) system for pig oocytes and to examine subsequent embryo development after in vitro fertilization. In experiment 1, four IVM media were tested: 1) control: North Carolina State University (NCSU) 23+10% porcine follicular fluid; 2) PF-NCSU: NCSU 23+0.1% polyvinyl alcohol (PVA)+1% amino acids; 3) PF-TCM: Tissue culture medium (TCM) 199+PVA; and 4) PF-WM: PF-Waymouth MB 752/1 medium (WM)+PVA. Oocytes were cultured in the respective media containing eCG and hCG (10 IU/ml each) for 20-22 h and then without hormonal supplements for an additional 20-22 h. After culture, the degree of cumulus expansion and frequency of nuclear maturation were determined. Some oocytes were coincubated with frozen-thawed spermatozoa for 5-6 h in modified Tris-buffered medium containing caffeine and BSA. In experiment 2, oocytes were matured in control, PF-TCM, and PF-WM, fertilized in vitro, and cultured for 144 h in NSCU 23+BSA. Fewer (p < 0.01) oocytes reached metaphase II stage in PF-NCSU (45% vs. 80-85%) than in the other media. Oocytes matured in control medium showed the most cumulus expansion, followed by those in PF-TCM and PF-WM; those in PF-NCSU showed very slight expansion. A lower (p < 0.05) penetration rate was obtained for oocytes matured in PF-NCSU than in the control medium (59% vs. 81%). In contrast to those in control (96%) and PF-TCM (93%), oocytes in PF-WM (65%) showed a lower male pronuclear formation. Compared to that in the control, a significantly lower (p < 0.05) cleavage rate was also observed for oocytes matured in PF-WM. Similar proportions of embryos developed to the blastocyst stage when oocytes were matured in control (22%) and PF-TCM (13%). These results indicate that pig oocytes can be successfully matured in a protein-free medium with subsequent development to the blastocyst stage.

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