Flow cytometric cell cycle analysis of somatic cells primary cultures established for bovine cloning.

An important factor governing developmental rates of somatic cloned embryos is the phase of the cell cycle of donor nuclei. The aim of this experiment was to investigate the distribution of cell cycle phases in bovine cumulus and fibroblast cells cultured using routine treatment, and under cell cycle-arresting treatments. The highest percentages of cumulus cells in the G0 + G1 stage were observed in uncultured, frozen/thawed cells originating from immature oocytes (79.8 +/- 2.2%), fresh and frozen/thawed cells from in vitro matured oocytes (84.1 +/- 6.2 and 77.8 +/- 5.7%, respectively), and in cycling cells (72.7 +/- 16.3 and 78.4 +/- 11.2%, respectively for cumulus cells from immature and in vitro matured oocytes). Serum starvation of cumulus cultures markedly decreased percentages of cells in G0 + G1, and prolonged starvation significantly increased (P < 0.05) percentages of cells in G2 + M phase. Culture of cumulus cells to confluency did not increase percentages of cells in G0 + G1. Contrary to findings in cumulus cells, significantly higher percentages of cells in G0 + G1 were apparent when fibroblast cells were cultured to confluency or serum starved, and significantly increased (P < 0.01) as the starvation period was prolonged. It is concluded that for particular cell types specific strategies should be used to attain improvements in the efficiency of cloning procedures.

The effect of Ureaplasma diversum activated mononuclear leukocytes on the development and interferon-tau production by bovine IVF-derived embryos.

Ureaplasma diversum is an opportunistic pathogen of the bovine genital tract causing herd outbreaks of granular vulvitis, abortion and infertility. Early embryonic death probably contributes to reduction of the reproductive performance in cows, however, pathogenesis of the disease remains obscure. The aim of the study was to examine whether activation of mononuclear leukocytes by U. diversum may affect embryo development and IFN-tau production. Bovine peripheral blood mononuclear leukocytes were cultured with U.diversum antigen for 24 h. The levels of IL-1, TNF-alpha, NO and GM-CSF in the cell culture supernatants were measured. IVF-derived embryos were cultured in the presence of supernatants from activated leukocytes. The development of embryos until day 6 postinsemination and the rate of morulae/blastocysts were determined. IFN-tau production in supernatants of cultured embryos was examined by inhibition of a virally-induced cytopathic effect. The results showed that U. diversum stimulated mononuclear leukocyte production of IL-1, TNF-alpha and NO. Supernatants from U. diversum-activated cells did not impair the rates of the embryo development and blastocyst formation. The products of activated leukocytes increased the IFN-tau production by cultured blastocysts. This suggest that U. diversum infection provides leukocyte-mediated signals for developing embryos for generation of additional production of cytokine - an important component of innate immunity.

Nuclear configuration of bovine oocytes derived from fresh and in vitro-cultured preantral and early antral ovarian follicles.

The aim of this experiment was to characterize the growth and nuclear configuration of oocytes isolated from late preantral and early antral bovine ovarian follicles immediately after recovery and after the in vitro culture. Individual follicles were isolated by microdissection from slices of the ovarian cortex. Follicles were sorted by diameter into 175 to 224, 225 to 274 and 275 to 325 microm-size classes. The follicles selected for in vitro culture were placed singly into 40 microL droplets of medium (TCM 199 enriched with FCS, insulin, transferrin, sodium selenite, sodium pyruvate, 1-glutamine, hypoxanthine, FSH and estradiol-17beta) and cultured for 6, 8, 11, 14 or 17 d. The sizes of follicles and oocytes were related to the duration of culture and gradually increased as culture duration was prolonged. The analysis of the relationship between mean diameters of oocytes at the time of recovery and after the in vitro culture, has shown significant differences after culture lasting 8 d (76.9+/-9.9 vs. 86.1+/-11.1 microm; P < 0.05), 11 d (77.0+/-9.9 vs. 91.9+/-17.5 microm; P < 0.01), 14 d (80.0+/-9.5 vs. 97.9+/-16.5 microm; P < 0.01) and 17 d (82.6+/-6.6 vs. 97.2+/-11.5 microm; P< 0.01). No statistical differences were shown among oocytes in the 5 pre-culture groups (79.5+/-8.8; 76.9+/-9.9; 77.1+/-9.9; 80.1+/-9.5 and 82.6+/-6.6 microm). Meiotic arrest was preserved in 71.9% of oocytes in our culture system up to 14 d. Frequency of the germinal vesicle (GV) stage did not significantly differ among oocytes evaluated "fresh" or cultured for 6, 8, 11 or 14 d. No relationship was observed between the size class of follicles and the frequency of the GV-stage. Prolonging the culture period to 17 d drastically decreased the percentage of oocytes in the GV-stage (18.7%) and increased the percentage of oocytes having premature initiation of meiosis (GVBD; 46.3%) and degeneration (25.0%). These results suggest that out of all culture periods used in our experiment, Day 14 was found to be the longest culture time allowing for both oocyte growth and maintenance of nuclear configuration at the GV-stage.

The isolation and in vitro culture of bovine preantral and early antral follicles of different size classes.

The ovary of cattle contains thousands of oocytes which are enclosed primarily in the preantral follicles. Methods of culturing preantral follicles are now being developed. The aim of this study was to investigate the effect of the size of bovine preantral and early antral follicles and culture media on their in vitro growth. Individual follicles isolated by microdissection of the ovarian slices were sorted into the following size classes: 75 to 124, 125 to 174, 175 to 224, 225 to 274, 275 to 324 and > or = 325 microns. The follicles were cultured individually in TCM 199 + fetal calf serum (FCS) + supplements (FSH, estradiol-17 beta, insulin, transferrin, sodium selenite, sodium pyruvate, 1-glutamine and hypoxanthine) or in Menezo B2 + FCS + supplements (Experiment 1) and in TCM 199 + steer serum (SS) with or without additional supplements (Experiment 2). The total number of isolated follicles of different size classes was similar in heifers and cows. No significant difference in the growth rate of follicles of different sizes was seen in the 2 media (TC 199 and B2). However, the culture of follicles in the TCM 199 that was supplemented only with SS and contained no other additives significantly reduced follicular survival and growth in comparison with follicles cultured in the supplemented medium. The survival time of follicles was related to their initial size at the beginning of culture. The longest period of growth was for follicles 275 to 324 microns in diameter (i.e., 10.7 +/- 5.7; 12.1 +/- 6.2 and 12.2 +/- 2.7 d, respectively, for culture in supplemented Menezo B2, TCM 199 + FCS and TCM 199 + SS). Survival and growth of some follicles was maintained for 23 d.

Demi-embryo production from hatching of zona-drilled bovine and rabbit blastocysts.

It is known that the pregnancy rate resulting after transfer of bisected embryos is lower than after transfer of whole embryos. The main reason is the reduced cell number in the demi-embryo which is less than 1 2 of that in the intact embryo, since a number of blastomeres is damaged as a result of the procedure used in conventional embryo splitting. The aim of our experiment was to develop a non-invasive procedure which would limit cell losses during microsurgery. The experiment was carried out on bovine IVM-IVF embryos at middle, late and expanded blastocyst stage and rabbit embryos at late blastocyst stage cultured in vitro from in vivo produced zygotes. The zona pellucida of these embryos was drilled on the line between the inner cell mass and the trophoblast using a glass microneedle (

The effect of co-culture system on developmental capacity of bovine IVM/IVF oocytes.

Two experiments were conducted to compare the influence of different culture systems and the oviduct donor's cycle phase on the developmental potential of co-cultured bovine embryos derived from IVM/IVF oocytes and to establish an efficient freezing method for oviduct epithelial cells. In the first experiment, the effects of media (Menezo B2, synthetic oviduct fluid SOF); sera (no serum, fetal calf serum FCS, human serum HS); and the presence or absence of monolayer of bovine oviduct epithelial cells (BOEC) on developmental capacity of bovine embryos were investigated. In the second experiment, the influence of oviduct donor's hormonal status (superovulated versus unstimulated) and the cryopreservation of oviductal tissue on the support of developmental competence of bovine IVM/IVF-derived zygotes were examined. Oviduct epithelial cells were cryopreserved according to the modified two-step method previously applied to rabbit embryos. For zygotes co-cultured with a monolayer of BOEC the following blastocyst development rates were obtained: 40.1% (63/157); 34.5% (60/174); 13.0% (7/54); and 19.2% (14/73), respectively, in B2 serum-free medium, B2 plus 20% HS, SOF plus 20% HS, and SOF plus 20% FCS medium. In the absence of BOEC the rates were 12.3% (10/81); 41.4% (36/87); and 8.9% (6/67), respectively, in B2 plus 20% HS, SOF plus 20% HS, and SOF plus 20% FCS. It was shown that the source of oviduct epithelial cells and previous freezing had no influence on the proportion of cleaved zygotes (approximately 70%) or on the percentage of blastocysts (approximately 20%).

Influence of hardening of the zona pellucida on in vitro fertilization of bovine oocytes.

The influence of hardening of the zona pellucida of in vivo matured bovine oocytes on fertilizability was investigated. For the study, 163 preovulatory and 73 postovulatory oocytes recovered from superovulated heifers were used. The preovulatory oocytes, before they were used for in vitro fertilization, consisted of: 1) those cultured in vitro for 4 to 6 h to permit final maturation and 2) those incubated in the rabbit oviduct for 4 to 5 h to permit final maturation and induce hardening of the zona pellucida. A few oocytes served as a control of nuclear maturity and the zona pellucida solubility. Preovulatory and postovulatory oocytes were both inseminated in vitro using frozen-thawed, heparin treated and swim-up separated spermatozoa. Significant differences (P<0.01) were established between fertilization rates of cultured preovulatory oocytes (68.8%) and those incubated in the rabbit oviducts (42.9%), or those recovered from bovine oviducts (40.7%). It can be concluded that hardening of the zona pellucida distinctly influences the fertilizability of oocytes. This factor should be taken into account when considering the source of oocytes or the kind of treatment to be used for in vitro fertilization.

Reversible changes in dissolution of the zona pellucida of immature bovine oocytes.

The influence of holding immature bovine oocytes in in vitro bovine oviducts on the dissolution of zona pellucida in 0.1% pronase, the role of cumulus cells in this process and the possibility of reversing the process were examined. For the study, 1,045 oocytes were obtained from 2 to 6 mm ovarian follicles. Cumulus-free oocytes were placed in isolated bovine oviducts at 37 degrees C. The average dissolution time of the zona pellucida increased in proportion to the holding time of oocytes in oviducts: 9.9, 13.8, 48.3, 239.3 and 788.3 min after 5, 20, 40, 80 and 120 min in the oviduct, respectively. For the control group, only 4.6 min were required for dissolution of the zona. When cumulus-free and cumulus enclosed oocytes were held for 120 min, no differences were seen in the average lytic time of the zona pellucida of cumulus enclosed oocytes compared with the control group. When cumulus-free oocytes were held in vitro for 120 min and then immersed in follicular fluid from 30 min to 18 h, there was a significant reversal in the sensitivity of the zona pellucida to proteolysis.