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The effect of D-penicillamine on the fertile life span of frozen-thawed bull spermatozoa was studied in Experiment 1. After thawing, the washed spermatozoa were incubated for 8 h in fertilization medium with 1 mg/mL polyvinyl alcohol (PVA) and 0.5 mg/mL D-penicillamine. The addition of cumulus-free oocytes together with 10% of bovine serum (BOS) + 4 IU/mL heparin to the 8-h incubated spermatozoa resulted in high fertilization and polyspermy rates (97/103, 94.2% and 49/97, 50.5%, respectively). When BOS was substituted with 3 mg/mL of BSA, the fertilization and polyspermy rates decreased to 33/91 (36.3%) and 3/33 (9.1%), respectively. The total absence of fertilization was observed after substitution of proteins with PVA. The 8-h sperm incubation in fertilization medium without D-penicillamine and following fertilization in medium + different capacitation supplements resulted in the total absence of or a very low fertilization rate. In Experiment 2, the first set of cumulus-oocyte complexes (COC) and cumulus-free oocytes were fertilized for 8 h with nonincubated spermatozoa. The second set of COC and cumulus-free oocytes were fertilized in the same wells with spermatozoa after removal of the first set. The fertilization rate for the first set of COC and cumulus-free oocytes was 65/67 (97%) and 73/73 (100%), respectively, with 21/65 (32.3%) and 32/73 (43.8%) polyspermy, respectively. In the second set, the high penetration rate (67/73, 91.8%) was observed only for COC, while that for cumulus-free oocytes (19/76, 25%) was significantly lower (P < 0.01). In Experiment 3, the fertilization rate of COC in medium + PVA + 10 or 100 IU/mL heparin was 69/92 (75%) and 52/74 (70.2%), respectively, with no polyspermy. In medium + BSA + 10 or 100 IU/mL heparin, the fertilization rate of COC was 64/72 (88.9%) and 70/79 (88.6%), respectively, with polyspermy at 2/64 (3.1%) and 7/70 (10%), respectively. In medium + BOS + 10 or 100 IU/mL heparin, the fertilization rate was 79/82 (96.3%) and 79/80 (98.7%), respectively, with a significantly (P < 0.05) higher polyspermy rate (34/79, 43% and 30/79, 38%, respectively). The vitality and capacitation changes of frozen-thawed and 8-h incubated spermatozoa were assessed by Hoechst and chlortetracycline (CTC) staining.

Two principal kinases, p34cdc2 kinase and MAP kinase play a pivotal role in maturation of mammalian oocytes. In the porcine and bovine oocytes both kinases are activated around the time of germinal vesicle breakdown (GVBD). Butyrolactone I (BL I), a specific inhibitor of cdk kinases, prevents effectively and reversibly resumption of meiosis in the porcine and bovine oocytes. Neither p34cdc2 kinase nor MAP kinase are activated in oocytes inhibited in the GV stage. The bovine oocytes maintained for 48 h in the medium supplemented with BL I, progress subsequently to metaphase II in 91%, their cumuli expand optimally and after in vitro fertilization they possess two pronuclei. When the cdc2 kinase is blocked in the porcine oocytes by BL I, MAP kinase, activated by okadaic acid treatment, is able to substitute cdc2 kinase and induce GVBD. The histone H1 kinase activity sharply decreases in the metaphase II oocytes treated by BL I and one or two female pronuclei are formed. These data indicate that BL I is a useful tool either for the two step in vitro culture of mammalian oocytes or for their activation in nuclear transfer experiments.

The effect of exogenous bovine recombinant somatotropin (bST) on the recruitment of follicles and quality of oocytes was investigated in the bovine. Single injections of recombinant depot form of bST were given to nine heifers and seven cows at the time of synchronized estrus (day 0). Nine heifers and six cows served as controls. Three days after bST injection the animals were slaughtered, their ovaries dissected and follicles separated into two categories according to diameter (2–5 and 5–10 mm). A total of 196 (53.2%) healthy oocyte-cumulus complexes (OCC) was isolated and selected from 369 follicles (diameter 2–5 mm) of bST treated heifers and 137 (49.8%) OCC from 275 follicles of bST treated cows. After 25–28 h of culture, 164 (44.4%) and 124 (45.1%) oocytes from follicles of heifers and cows, respectively, had developed to the metaphase of the second meiotic division (MII). From control heifers, 122 (35.5%) OCC derived from 344 follicles (2–5 mm) and from control cows 87 (39.7%) OCC derived from 219 follicles were similarly selected for maturation in vitro and 109 (31.7%) and 78 (35.6%) oocytes in control heifers and cows, respectively, developed to the MII stage. The numbers of follicles developed in the experimental and control groups were similar (P > 0.05) but differences were evident in the healthy OCC and oocytes maturing in the treated versus control groups of heifers and cows (χ2 detected at 1% and 5% levels, respectively). The injection of bST resulted in significantly increased levels of growth hormone (GH) in peripheral blood and follicular fluid (FF), the GH levels in blood plasma correlating with the levels in FF. The level of insulin-like growth factor I (IGF-I) in sera from bST treated animals was significantly higher than in sera from control animals (353.6 vs. 155.5 ng ml−1). In conclusion, recombinant bST positively affected (at day 3) the proportion of OCC and oocytes maturing to MII.

Pig zona-free and zona-intact ova were successfully penetrated in vitro by ejaculated boar spermatozoa. The oocytes were obtained from antral follicles (2–5 mm in diameter) of slaughtered gilts and matured in M-199 with porcine follicular fluid (50%) and follicle stimulating hormone (FSH) (0.25 IU ml−1) for 44 h at 38.5°C. In vitro fertilisation (IVF) was carried out with a spermatozoa-rich fraction of a fresh boar ejaculate which had been incubated in M-199 supplemented by 10% inactivated bovine serum. The penetration rate of zona-free oocytes was dependent on the duration of sperm incubation. IVF of zona-intact ova was designed to estimate the optimal spermatozoa concentration for insemination. The concentration of (1–1.5) × 106 spermatozoa ml−1 gave the best fertilisation results (87%), whereas the concentration below 1 × 106 spermatozoa ml−1 resulted in a lower fertilisation rate (62%) but in a higher number of monospermic eggs (36%) and male pronucleus formation (80%).