Effects of hormones on in vitro maturation of cattle oocytes.

The efficiency of cattle oocyte maturation in vitro was studied in protein-free MEM-α with hormones and in completely definite culture medium without hormones. Oocyte capacity to develop after fertilization to the morula/blastocyst and blastocyst stages served as a criterion of effective maturation. The increase in follicle-stimulating hormone concentration in the medium by one or two orders of magnitude in comparison with the "standard" level of 1 µg/ml deteriorated the development of embryos to the preimplantation stages. Serum gonadotropin from pregnant mares worked similarly as follicle-stimulating hormone. Oocytes that underwent maturation without hormones developed to the blastocyst stage, though the percentage of dividing embryos was significantly less and there was a trend to worse development of the embryos to the preimplantation stages.

[In vitro fertilization of bovine oocytes in in-vitro protein-free culture system].

We studied the possibility of fertilization of bovine oocyte-cumulus complexes, matured in vitro in a protein-free medium, in a protein-free culture system without preliminary capacitation of spermatozoa. The development of embryos to the morula-blastocyst and blastocyst stage was considered as a criterion of successful fertilization. It was shown that replacement of bovine serum albumin for polyvinyl alcohol or polyvinylpyrrolidone in Tyrode medium for fertilization did not affect significantly the development to the morula-blastocyst stage and the number of cells in blastocysts. It was also found that replacement bovine serum albumin for polyvinyl alcohol in all used media, Tyrode medium for washing of oocytes, medium for sperm preparation to fertilization, and Tyrode medium for fertilization, did not affect significantly the development to the morula-blastocyst and blastocyst stages, as well as on the number of cells in blastocysts. The results obtained suggest that in vitro fertilization of bovine oocyte-cumulus complexes is possible in a protein-free culture system without significant reduction in the capacity for in vitro development of the obtained embryos and number of cells in blastocysts.

[Fertilization and subsequent development of cattle oocytes after reduced incubation with spermatozoa]. / Oplodotvorenie i posleduiushchee razvitie ootsitov krupnogo rogatogo skota posle cokrashchennoi inkubatsii so spermatozoidami.

We studied the influence of the duration of joint incubation of cattle oocytes and spermatozoa (18 versus 1 h) on fertilization, cleavage, and embryonic development in vitro until the blastocyst stage. Spermatozoa of a bull of the Britanofrizskaya breed were used in the experiments. It was shown in the first experimental series that after a long-term incubation with the spermatozoa, the percentage of penetrated eggs increased: 71.7% and 56.0% (p < 0.05) after 18-hour and 1-hour incubation, respectively. However, no differences were found in the number of normally fertilized eggs: 46.5 and 39.0%, respectively. In the second experimental series, no significant differences were found in either the number of cleaving embryos (41.2 and 32.2%, respectively) or the capacity of cleaving embryos to develop in vitro until the blastocyst stage (21.7 and 15.8%, respectively). Thus, reduction in the time of joint incubation of cattle gametes upon in vitro fertilization to 1 h did not reduce the number of normally fertilized eggs and did not affect their capacity for subsequent in vitro development.

[The effect of the composition of the culture media on bovine oocyte maturation and embryo development in vitro]. / Vliianie sostava kul'turnykh sred na sozrevanie ootsitov i razvitie émbrionov krupnogo rogatogo skota in vitro.

We studied the capacity of the cattle oocyte for the resumption of meiosis and achievement of metaphase II in various protein-free culture media (DMEM, TCM-199, Ham's F-10, and Ham's F-12) and the pattern of influence of the estrous serum on in vitro development of fertilized cattle oocytes, with special reference to the time of its addition to the synthetic oviduct fluid containing BSA. In the first experimental series, it was shown that the highest number of oocytes (76.1%) resumed meiosis in DMEM medium. Meiosis was not resumed in Ham's F-12. Intermediate results were obtained for TCM-199 (55.1%), which is commonly used for maturation of cattle oocytes in vitro, and for Ham's F-10 (51.7%). The oocytes reached metaphase II in DMEM at a higher rate (45.3%) than in TCM-199 or Ham's F-10 (29.4 and 8.6%, respectively). In the second experimental series, the estrous serum was added to the culture medium within 20 h (control) or 42 h (experiment) after the beginning of fertilization. The estrous serum did not inhibit the first cleavage division (the percentage of cleaving embryos did not differ reliably: 32.7 and 37.9%, respectively). However, a later serum addition to the culture medium (within 42 h after the beginning of fertilization) reliably increased the percentage of embryos that reached the blastocyst stage (6.5% in the control and 17.8% in the experiment) and the hatched blastocyst stage (2 and 9.2%, respectively).

[The development of mouse embryos in vitro in a protein-free medium depending on the numbers of embryos in a microvolume of the medium]. / Razvitie myshinykh émbrionov in virto v srede bez belka v zavisimosti ot kolichestva zarodyshei v mikroob''eme sredy.

We studied the development of mouse embryos in vitro depending on the number of embryos in a given microvolume of the Ham's F-10 medium without protein or with the addition of serum. The absence of serum from the culture medium did not affect the development of two-cell embryos cultivate in groups of 5-6 (about 90% embryos developed until the stage of blastocyst and over 50% left zona pellucida), but the development of single embryos in the protein-free medium proceeded significantly worse. Single two-cell embryos cultivated in the serum-containing medium developed similar to embryos cultivated in groups. At the same time, no significant differences in development was found between eight-cell embryos cultivated individually or in groups of up to 10 embryos in the Ham's F-10 medium, either in the presence of serum or without it (about 95% embryos developed until the stage of blastocyst and over 70% left zona pellucida). The increase in the number of cultivated embryos over 10 had the adverse effect on development of either two-cell or eight-cell embryos. The attachment of blastocysts to the substrate after leaving zona pellucida and growth of trophectoderm was observed only in the presence of serum. These results suggest that interaction between preimplantation embryos in culture can probably be mediated by factor(s) released by embryos into the medium. Serum appears to contain such factor(s). Sensitivity of embryos to the factor(s) apparently depends on the stage of development.