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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%).

The vitality of bovine oocytes stored in isolated follicles was examined. The aim of this work was to prolong the time of in vitro manipulation of oocytes before their maturation and develop a new alternative of oocyte "capacitation" to improve the quality of in vitro produced embryos. Follicles were dissected from the ovaries of slaughtered cows; subsequently, follicles were divided according to their diameter into three categories (2-3, 3-4 and 4-6 mm), and stored at 17-18 degrees C for 24 or 48 h in a modified tissue culture medium-199 (TCM-199) with reduced pH. After that time, the cumulus-oocyte complexes (COCs) were isolated, matured, fertilized, and embryos cultured in vitro for a total of 9 days. The percentage of total blastocysts, and hatched blastocysts developed from oocytes, initially kept ("capacitated") for 24h at 17-18 degrees C, within follicles of 3-6mm size categories, were significantly higher than that oocytes of the control [of control oocytes] (44.9 and 30.3% versus 36.2 and 20.4%, respectively). The oocytes of follicles stored for 48 h at 17-18 degrees C already had decreased developmental capacity. Interesting data were obtained when COCs of the 3-4 and 4-6 categories were additionally divided into two subgroups according to their presumed developmental history (originating from the supposed growing "fit" in contrast to the supposed regressing "unfit" follicles). The higher improvement in the rate of hatched blastocysts from 24h stored oocytes was observed only in the subgroup originated from "fit" COCs (15.3 versus 25.0%, and 20.0 versus 34.4%, in the 3-4 and 4-6mm categories, respectively). The transfer of 26 blastocysts (developed of follicles kept for 24h at 17-18 degrees C) to 26 recipient heifers resulted in 18 pregnancies. Storage of follicles at 17-18 degrees C in vitro resulted not only in recovery of higher numbers of blastocysts of better quality but also facilitated the safe transport of follicles for a long distance. The extended, time of follicle storage before the proper oocyte maturation allowed also the synchronization of an appropriate number of recipient animals according to the number of isolated follicles.

Bovine oocytes originated from follicles of two different size categories (medium (M), 3-6 mm and small (S), 1-2 mm) were cultured for 24 and 70 h, respectively, in a meiosis-inhibiting medium (MIM) supplemented with 100 microM butyrolactone I (BL I). At the end of culture, cumulus oocyte complexes (COC) from M and S follicles were labeled by 3H-uridine for 30 min. The autoradiography (ARG) of semi-thin sections of COC showed the labeling of the germinal vesicles (GV) as: (1) The COC of the M category labeled immediately after isolation from follicles showed only weak labeling (+) of the GV. The COC of the S category labeled immediately after their isolation showed mostly intensive labeling (4+,3+) of the GV. (2) When the COC were labeled after 24 and 70 h of culture in MIM, no labeling was observed in the M category. The S category of oocytes showed the slightly decreased labeling (3+,2+) after 24 h and negligible labeling after 70 h of culture. The pattern of very intensive labeling of granulosa cell nuclei of all mentioned groups was practically not changed during the whole 70 h period of culture in both categories. The nucleolar ultrastructure of S category oocytes revealed time dependent changes from the reticular fibrillogranular structure present in freshly isolated oocytes. The several fibrillar centers before the culture changed to the fibrillogranular appearance with few large and a number of small vacuoles and an exclusively fibrillar area after 24 h of culture. Finally, nucleoli acquired a mostly exclusively fibrillar structure with one large fibrillar center after 70 h of culture. In the second experiment, the meiotic maturation of COC of S category was inhibited in MIM for 48 h. The subsequent 24 h culture in a medium with BOS and gonadotropins resulted in 81.0% oocytes matured to metaphase II (M II). Only 27.1 and 11.3% of the control S oocytes cultured in a medium, with BOS and gonadotropins directly after isolation, reached M II after 48 or 72 h of culture, respectively. The two-step culture increased significantly the meiotic competence of cattle oocytes isolated from small antral follicles.