Survival of bovine fibroblasts and cumulus cells after vitrification.

The aim of the experiment was to investigate the effect of vitrification on viability and the cell cycle of bovine cumulus cells and fibroblasts after culture with or without serum starvation. In all vitrified-thawed bovine somatic cells, the number of samples that reached the confluence stage was high (50 to 100%). The viability of vitrified somatic cells depended on the concentration of the cells. The viability was higher for cells vitrified at the concentration of 10 x 10(6) per ml than for cells vitrified at a concentration of 1 x 10(6) per ml (p < 0.05; for cumulus cell, and fibroblast). Time of cell starving has had no impact on their susceptibility to vitrification in case of vitrified cumulus cells. Starving time caused shifts in proportions of subsequent cell cycle phases of vitrified fibroblasts and cumulus cells. In conclusion, the bovine cumulus and fibroblast cells can be cryopreserved successfully by vitrification procedure.

Genetical and biotechnological methods of utilization of female reproductive potential in mammals.

The investigations included: 1/ Establishment of culture systems that would maintain the three-dimensional structure of bovine intact early antral follicles (EAF) or isolated cumulus-oocyte-granulosa complexes (COCGs) and increase the resulting portion of cumulus-oocyte complexes (COCs) with normal morphology for subsequent in vitro maturation (IVM) and in vitro fertilization (IVF), 2/ Quality assessment of IVM bovine oocytes and resulting day-8 blastocysts produced in TCM199 medium supplemented with fetal bovine serum (FBS), fatty acid free bovine serum albumin (fafBSA) or polyvinylpyrrolidone (PVP40), 3/ Testing the polymorphism of the genes: retinol binding protein (RBP4), epidermal growth factor (EGF), prostaglandin-endoperoxide synthase 2 (PTGS2), insulin-like growth factor 2 (IGF2), amphiregulin (AREG) and prolactin (PRL), and their effects on reproductive traits in swine. Isolated COCGs created in culture follicle-like structures and their oocytes achieved meiotic competence and matured to metaphase II at a higher rate than did oocytes from smaller diameter follicles which were cultured intact. The proportion of COCs with normal morphology significantly increased when isolated COCGs were embedded in microdrops of collagen gel or cultured on inserts covered with gel rather than in large gel volumes. No significant effect of maturation media composition on meiotic spindle morphology and the rate of apoptotic bovine oocytes was observed. Among day-8 embryos derived from oocytes matured with PVP40 a reduced blastocyst rate and elevated apoptotic index were found, whereas total cell count was not affected. Gene expression study also revealed a decrease in relative abundance for IGF2 and its receptor (IGF2R), and heat shock protein 70 (Hsp70) genes in PVP40 group and a significant elevation in fafBSA derived embryos. The significant effect of reproduction traits of swine (litter size and litter weight) was found for RBP4, EGF, IGF2 and AREG genes. A new polymorphism was also revealed within a promoter region of PRL gene.

Effect of donor stimulation, frozen semen and heparin treatment on the efficiency of in vitro embryo production in goats.

Investigations on in vitro embryo production in goats in comparison with other domestic species, especially cattle, have been the subject of few reports despite their usefulness for both basic research and commercial application. The objectives of this study were to compare the efficiency of IVP in goats using immature follicular oocytes recovered from FSH-primed and control goats. After IVM, oocytes were fertilized with fresh or frozen-thawed semen capacitated with or without heparin. Mature oocytes were fertilized in vitro with fresh and frozen-thawed sperm of a single buck. Sperm preparation included swim-up separation and heparin treatment (50 micrograms/ml of sperm suspension for 45 min) before spermatozoa were added to oocytes in TALP-IVF. After IVF, the zygotes were cultured for 24h and cleaved embryos were further cultured with goat oviduct epithelial cells or transferred to synchronized recipients. Mean number of oocytes recovered from FSH-primed goats (24.5 +/- 8.6) was significantly higher (P < 0.01; t test) in comparison to control does (14.7 +/- 4.7). Irrespective of fresh or frozen semen, no differences were observed in blastocyst yield when sperm was treated with heparin. However, the highest cleavage rate (99/126; 79.4%) as well as blastocyst yield (47/126; 37.3%) was obtained after IVF with fresh sperm capacitated without heparin. Contrary to fresh sperm, heparin treatment of frozen-thawed sperm significantly improved (P < 0.01) embryo cleavage. No differences between in vivo developmental competence of embryos related to sperm origin were found after transferring into recipients. Overall, more than 60% of the recipients became pregnant and 20% of all transferred embryos survived delivering 13 healthy kids. Our caprine IVP system allows obtaining embryos with developmental competence comparable to bovine IVP.

Timing of nuclear maturation of nonstored and stored domestic cat oocytes.

In this study we compared the effects of preculture storage of ovaries, IVM medium, a reduced O(2) atmosphere and duration of culture on in vitro maturation (IVM) of domestic cat oocytes. One randomly selected ovary of each pair (69 pairs) was stored in PBS at 10 degrees C for 16-24h before oocyte recovery. The second ovary from each pair was used as a nonstored control. In Experiment I, we investigated the effect of culture media (TCM 199 versus SOF) and a reduced O(2) atmosphere (a humidified gas atmosphere of either 5% CO(2) in air or 5% CO(2):5% O(2):90% N(2)) on IVM of both stored and nonstored oocytes. In the second experiment, we compared timing of nuclear maturation of both stored and nonstored oocytes cultured for 17-18, 20-21, 24-26, 28-30, 33-34 or 42-45 h before being evaluated for meiotic status. In both, Experiments I and II, the recovery rate, quality and competence for maturation of oocytes originating from stored ovaries did not differ (P>0.05) compared with nonstored. In Experiment I, neither culture medium (37.5 versus 43.2% of Metaphase II, respectively in TCM 199 versus SOF) or gas atmosphere (40.0 versus 32.5% of Metaphase II, respectively in 5% CO(2) in air versus 5% CO(2):5% O(2):90% N(2)) affected oocyte maturation. In Experiment II, the mean proportion of oocytes achieving Metaphase II within 17-18 h of culture was 36.1% and did not significantly increase (P>0.05) over time up to 28 h. The highest proportion of oocytes (67.3%) reached Metaphase II stage after 42-45 h of culture. Therefore, we conclude that two "waves" of nuclear maturation of cat oocytes can be distinguished. The first wave takes place within 26 h and it is likely that most oocytes of this wave mature by 17-18 h; the second wave occurs after 28-30 h of IVM. It can be assumed that this double wave may reflect the presence of two oocyte populations with two different degrees of "prematuration" which require different lengths of IVM.

In vitro developmental competence of domestic cat embryos after somatic cloning: a preliminary report.

This work was undertaken in order to study the developmental competence of nuclear transfer feline embryos with regard to the recipient-cytoplast's age and type of somatic cells used as donor nuclei. Oocytes were recovered by mincing the ovaries in HEPES-buffered TCM-199. Selected cumulus-oocyte complexes (COCs) with compact cumulus cell mass and a dark, homogenous ooplasm were cultured for maturation in the modified medium TC-199 for 24, 35, and 43 h, and after enucleation were used as a source of recipient cytoplasts for exogenous somatic nuclei. Two experiments were carried out. In Experiment 1, the source of recipient cytoplasts was oocytes matured in vitro for 24 h (Group 1), 35 h (Group 2), and 43 h (Group 3), while the source of donor nuclei was cycling fetal fibroblasts. Somatic cell-cytoplast complexes (SC-CCs) were fused electrically by double DC pulses of 2.0 kV/cm for 15 micros. The reconstructed embryos were cultured in B2 medium for 72 h after NT, then co-cultured with BRL cells in the same medium supplemented with 10% FBS at 38.5 degrees C under 5% CO2 in air. In Groups 1, 2, and 3, the fusion rates were 71.4 (25/35), 74.6 (47/63), and 57.5% (46/80), respectively. The cleavage rates in Groups 1, 2, and 3 were 80.0 (20/25), 55.3 (26/47), and 60.8% (28/46), respectively. The development to morula and blastocyst stages was higher in Groups 1 and 2 compared to Group 3 (morula stage 14/25 (56.0%), 16/47 (34.0%), and 13/46 (28.2%); blastocyst stage 2/20 (8.0%), 4/47, (8.5%), and 0/46, respectively). In Experiment 2, the oocytes matured for 24-35 h were used as a source of recipient cytoplasts and cycling fetal fibroblasts and cumulus cells derived from mature COCs were used as a source of donor nuclei. The fusion rates were 115/193 (59.6%) versus 65/143 (45.4%) for fetal fibroblasts and cumulus cells, respectively. The cleavage rate was 72/115 (62.6%) versus 48/65 (73.8%), and the development to blastocyst stage 6/115 (5.2%) versus 5/65 (7.7%), for fetal fibroblast and cumulus cells, respectively. In conclusion, a prolonged maturation period of cat oocytes decreases developmental competence of reconstructed embryos, especially the ability to reach the blastocyst stage. The in vitro development of reconstructed embryos with either nuclei of fetal fibroblasts or cumulus cells was at approximately the same level.