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The characteristics of energy status in porcine oocytes as related to their meiotic competence and in vitro maturation were studied. Cycling pubertal gilts in the early luteal to early follicular phases of the ovarian cycle were used as oocyte donors. The oocytes recovered from medium (MF) or small follicles (SF) were considered meiotically more or less competent, respectively. A half of oocytes from each category was matured by the standard protocol. The oocytes were examined before or after maturation by confocal microscopy, a bioluminescent cell assay and Western blotting. Four experiments, each in triplicate, were performed to assess both SF and MF oocytes in terms of metabolic units formed by mitochondria and lipids, ATP and lipid consumption and lipid droplets with adipose differentiation-related protein (ADRP) expression. The proportion of oocytes with metabolic units, the mean ATP content and the number of lipid droplets per oocyte, and the relative number of lipid droplets with ADRP expression were significantly higher in the MF compared to SF oocytes before maturation. On the other hand, after maturation, there was an increase in the proportion of oocytes with metabolic units and the relative number of lipid droplets with ADRP expression in the SF compared to MF oocytes. In conclusion, specific differences in energy characteristics between porcine oocytes with different meiotic competence were found. Meiotically more competent oocytes are more advanced in terms of energy reserves before maturation, while meiotically less competent oocytes are more active in replenishing energy stores during maturation.

Bovine embryos are now routinely used in agricultural systems as a means of disseminating superior genetics worldwide, ultimately with the aim of feeding an ever-growing population. Further investigations, common for human IVF embryos, thus have priority to improve cattle IVF, as has screening for aneuploidy (abnormal chromosome number). Although the incidence and consequences of aneuploidy are well documented in human preimplantation embryos, they are less well known for the embryos of other animals. To address this, we assessed aneuploidy levels in thirty-one 2-cell bovine embryos derived from early- and late-cleaving zygotes. Contemporary approaches ( Whole Genome Amplification and next-generation sequencing) allowed aneuploidy assessment for all chromosomes in oocytes from donors aged 4-7 years. We also quantified mitochondrial DNA (mtDNA) levels in all blastomeres assessed, thereby testing the hypothesis that they are related to levels of aneuploidy. The overall incidence of aneuploidy in this cohort of bovine embryos was 41.1% and correlated significantly with the timing of cleavage (77.8% in late-cleaving vs. 31.8% in early-cleaving embryos). Moreover, based on mtDNA sequence read counts, we observed that the median mtDNA quantity is significantly lower in late-cleaving embryos. These findings further reinforce the use of the bovine system as a model for human IVF studies.

AbstrAct:In this field study, embryos were derived from genetically highly valuable cows excluded from breeding due to reproductive disorders. Cows, 5 to 10 years old, of Czech Siemmental, Holstein Dairy and Beef Cattle breeds were used as oocyte donors. Oocytes were obtained either in the growth phase of the first follicular wave from cows with synchronized oestrus or in any other phases of follicular development from cows without oestrus synchronization. The embryos were prepared by a standard protocol described previously. The mean number of usable oocytes, transferable and freezable embryos per donor, and the mean percentage of usable, transferable and freezable embryos were assessed. The results were analyzed by Student's-t and Chi-squared tests. The embryos were frozen according to a slow freezing protocol. After thawing, they were transferred to recipients on Day 7 after oestrus. Irrespective of the breed, the mean numbers of usable oocytes and transferable and freezable embryos collected per donor were significantly higher (P < 0.01) for the synchronized than for the nonsynchronized donors (20.4 vs 11.7, 4.3 vs 1.0 and 3.2 vs 0.8, respectively). Similarly, the mean percentages of usable oocytes, transferable and freezable embryos were significantly higher (P < 0.01) for the synchronized than for the nonsynchronized donors (28.5% vs 20.5%, 20.9% vs 9.0% and 15.8% vs 6.5%, respectively). On comparison of the synchronized and nonsynchronized donors of each breed, the difference in the mean percentage of usable oocytes was significant (P < 0.01) in cows of all three breeds, the difference in the mean percentage of transferable embryos was significant in Czech Siemmental and Holstein Dairy cows (P < 0.01) and the difference in the mean percentage of freezable embryos was significant only in Holstein Dairy cows (P < 0.01). After the transfer of 41 frozen-thawed embryos and 43 fresh embryos, 20 heifers and 24 heifers became pregnant, respectively. In conclusion: (a) higher number of oocytes from infertile, genetically valuable cows was recovered in the growth phase compared with the other phases of follicular development; (b) greater development of these oocytes resulted in more embryos for transfer and cryopreservation; (c) the transfer of frozen-thawed and fresh embryos resulted in pregnancy rates of 48.8% and 55.8% , respectively.

The aim of the work was to study a potential relationship between acrosome response characteristics of bovine spermatozoa and their ability to fertilize oocytes and produce in vitro embryos. Sperm of artificial insemination bulls with a high rate (22.0 +/- 4.1%, group A, n = 7) or a low rate (10.3 +/- 4.1%, group B, n = 8) of embryos were used. For acrosome assessment, motile spermatozoa from a Percoll gradient were incubated with or without heparin and examined by the fix-vital sperm assay (FVSA). The differences between the heparin-treated (H+) and the non-treated (H-) spermatozoa were significant (p < 0.01) in all bulls at all tested intervals. According to the kinetics of the heparin response, the bulls fell into three categories: fast (FR, n = 7), moderate (MR, n = 5) or slow (SR, n = 3) acrosome responses (p < 0.01). Five MR bulls were found in group A in comparison with two MR bulls in group B (57.1 vs 12.5%; p < 0.05). Intensity of the acrosome response (response index) was significantly higher in bull group A compared with bull group B (7.0 vs 4.6, p < 0.01). A positive correlation was recorded between response index and embryo rate (r = 0.668, p < 0.01). In conclusion (a) the kinetics of spermatozoa response to heparin may be important for in vitro fertilization, bulls with a moderate response appearing to be most suitable for embryo production; (b) greater spermatozoa response to heparin was related to more effective embryo production.

232The application of biotechnology in cattle breeding has allowed us to reduce the generation interval and to increase selection pressure in animal populations on the basis of more objective evaluation of the genotype. It facilitates more effective utilization of the reproductive potential of animals, makes a broader range of parental combinations available and thus allows for modification of the animal population in a desired way.The prerequisite for achieving these goals involves efficient cryopreservation methods for long-term storage of embryos, exchange of genetic material and creation of genetic resources. Today the embryos obtained by superovulation can be fro- ABSTRACT: The present study was designed to compare the efficiency of bovine embryo production for cryopreservation between oocytes collected from donors in the growth phase of follicular development (GPFD) and those recovered from donors in the undefined phase (UPFD). Cyclic cows, Czech Siemental or Holstein dairy breeds, 4-6 years of age, slaughtered at the local abbatoir were used. They were divided into two groups based on ovarian morphology: I. GPFD donors with ovaries corresponding to the growth phase of the first follicular wave (estrus cycle days 3-4; n = 52), and II. UPFD donors with ovaries in any other phase of follicular development (undefined estrus cycle days; n = 89). A total of 3 771 oocytes were collected and 1 134 embryos were prepared as two separate populations by standard protocol. In total 352 excellent or good quality embryos at the early, advanced or expanded blastocyst stage from both donor groups were pooled and used for cryotolerance assessment. They were frozen on day 7 (D7) or day 8 (D8) after fertilization by the standard procedure. After thawing, the embryos were cultured for 72 h to the hatching stage. The percentages of both D7 embryos and advanced blastocysts were significantly higher (P ≤ 0.01) for oocytes collected from GPFD donors than for oocytes collected from UPFD donors (33.7 vs 28.6% and 43.0 vs 29.5%, respectively). The percentages of excellent or good quality embryos obtained from both D7 embryos and fertilized oocytes were significantly higher (P ≤ 0.01) for oocytes collected from GPFD donors than for oocytes collected from UPFD donors (63.6 vs 49.4% and 21.4 vs 14.1%, respectively). The post-thaw survival rates were significantly higher (P ≤ 0.01) for D7 than D8 embryos (80.4 vs 66.3%). In relation to the developmental stage, the development and hatching rates were significantly higher (P ≤ 0.01) for D7 than D8 early blastocysts (75.0 vs 41.2% and 50.0 vs 5.9%, respectively) and for D7 than D8 advanced blastocysts, (73.7 vs 57.1 and 52.6 vs 28.6%, respectively). No differences were found between D7 and D8 expanded blastocysts after freezing-thawing. In conclusion, the collection of oocytes from donors in the growth phase positively influenced the in vitro production of bovine embryos for cryopreservation. The development of embryos produced with oocytes from GPFD donor group was accelerated and more ex...