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The efficiency of in vitro embryo production is highly variable amongst individual sires in cattle. To eliminate that this variability is not caused by sperm chromatin damage caused by separation or capacitacion, chromatin integrity was evaluated. Seventeen of AI bulls with good NRRs but variable embryo production efficiency were used. For each bull, motile spermatozoa were separated on a Percoll gradient, resuspended in IVF-TALP medium and capacitated with or incubated without heparin for 6 h. Samples before and after separation and after 3-h and 6-h capacitacion or incubation were evaluated by the Sperm Chromatin Structure Assay (SCSA) and the proportion of sperm with intact chromatin structure was calculated. Based on changes in the non-DFI-sperm proportion, the sires were categorized as DNA-unstable (DNA-us), DNA-stable (DNA-s) and DNA-most stable (DNA-ms) bulls (n=3, n=5 and n=9, respectively). In DNA-us bulls, separation produced a significant increase of the mean non-DFI-sperm proportion (p

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...

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.

The aim of this work was to characterize oocyte fertilization and embryo cleavage in nine AI bulls to find parameters suitable for prediction of in vitro fertility. According to the d8 blastocysts rate, they were categorized as high, medium and low productive (HP, MP and LP, mean: 25.4, 21.0 and 13.6% respectively) bulls. For these categories, oocyte penetration and fertilization efficiency were assessed at 6 and 18 hours post insemination (hpi), respectively. Some presumptive zygotes were cultured and cleaved and fast-cleaved embryo rates were checked at 44 hpi. The penetration rate was significantly higher for HP bulls than for MP and LP bulls (67.9 versus 50.3 and 33.1%; p<0.01). The syngamy rate was significantly higher for HP bulls than for MP and LP bulls (21.4 versus 10.2 and 5.7%; p<0.05). Conversely, no significant differences in fertilization rates were found among HP, MP and LP bulls. The cleavage rate was significantly higher for HP than LP bulls (82.4 versus 74.4%; p<0.01). The fast cleavage rate was significantly higher for both HP and MP bulls, as compared with LP bulls (82.1 and 84.7 versus 73.5%; p<0.01). A strong correlation was found between blastocyst production and penetration (r=0.803), syngamy (r=0.826), cleavage (r=0.635) and fast cleavage (r=0.709). In conclusion, all the evaluated parameters showed a predictive value, the most significant being early penetration and syngamy.

Robertsonian translocation rob(16;20) in the heterozygous state was discovered in a subfertile bull of the Czech Siemmental breed. A chromosomal analysis of its family has shown that this dicentric fusion is formed de novo. The present experiments were designed to detect rob(16;20) and determine its incidence for in vitro produced embryos, using fluorescence in situ hybridization (FISH) and rob(1;29) as a detection control. To characterize semen of both bulls with the rob translocations, their sperm was examined for DNA integrity by the sperm chromatin structure assay (SCSA). For in vitro fertilization of oocytes, spermatozoa from a rob(16;20) bull carrier (Czech Siemmental breed) and those from a rob(1;29) bull carrier (Charolais breed) were used. Embryos at the 6- to 8-cell stage were cultured in a vinblastine-supplemented medium for 17 h, and embryos at the blastocyst stage were cultured in a colcemide-supplemented medium for 4 h. The embryos were fixed in methanol and acetic acid with Tween-20. Painting probes for chromosomes 16 (Spectrum Green) and 20 (Spectrum Orange) and chromosomes 1 (Spectrum Orange) and 29 (Spectrum Green) were simultaneously hybridized. In the embryos derived from the rob(16;20) bull, the presence of this translocation was not detected. On the other hand, 52.5% of the embryos derived from the rob(1;29) bull were translocation carriers. There was no significant difference in the frequency of this translocation between early and advanced embryos.

ABSTRACT:The objective of the study was to characterize the effect of the oestrous cycle stage on the yield, morphology and meiotic competence of porcine oocytes. A total of 46 cycling gilts, at 8.5-9 months of age, were used as oocyte donors. Their oestrous cycle was synchronized by Regumate and the onset of oestrus was checked (Day 0). The gilts were slaughtered at the early (Days 1-5), middle (6-10) and late (11-14) luteal or early and late (20-21) follicular phase. Oocytes were isolated separately from medium (5-9 mm) and small (≤ 4 mm) follicles. Cumulus-oocyte complexes with dark, evenly granulated cytoplasm and at least two compact layers of cumulus cells were selected as useful for maturation. They were matured by a standard protocol, denuded from cumulus cells, fixed in glutaraldehyde, stained with 33258-Hoechst and examined by epifluorescence. The oocytes collected from small and medium follicles differed in their yield, morphology and meiotic competence regardless of the phase. The mean number (± S.E.M.) of oocytes isolated per donor was higher (187.7 ± 48.4 vs. 16.9 ± 6.1) but the mean percentage (± S.E.M.) of useful oocytes was lower (22.4 ± 7.5% vs. 80.2 ± 6.8%; P < 0.01) for small than for medium follicles. The mean number (± S.E.M.) of useful oocytes per donor was significantly (P < 0.01) higher (42.1 ± 16.8 vs. 11.9 ± 4.3) but the mean percentage of matured oocytes was significantly (P < 0.01) lower (48.4 ± 17.8% vs. 79.9 ± 7.9%) for small than for medium follicles. The oocyte population collected from small follicles varied during the oestrous cycle. The mean number (± S.E.M.) of oocytes isolated per donor from small follicles increased during the luteal and decreased during the follicular phase, except for the late follicular phase when it increased again. The mean percentage (± S.E.M.) of useful oocytes did not differ too much during this period, except for the late follicular phase when it decreased significantly (P < 0.01). The mean number (± S.E.M.) of useful oocytes per donor increased during the luteal and decreased during the follicular phase, but the differences were not significant except for the late follicular phase (P < 0.01). Similarly, the mean percentage (± S.E.M.) of matured oocytes increased during the luteal and decreased during the follicular phase, and the differences were significant. Compared with the oocyte population from small follicles, the oocyte population from medium follicles was less variable in the period from the middle luteal to middle follicular phase, when these follicles were present on the ovaries. It can be concluded that the porcine oocyte population changes in terms of quantity, morphological quality and meiotic competence according to the stage of follicular development. The late luteal and early follicular phases appeared to be most productive for oocyte recovery, because more morphologically normal oocytes with greater meiotic competence were collected, as compared with the other stages of the oestrous cycle.

The present experiments were designed to study the effects of Robertsonian translocations on the efficiency and kinetics of in vitro fertilization and early and advanced embryo development. Spermatozoa from bulls with rob(16;20), rob(1;29) and normal karyotype (A, B and C, respectively) were used. Oocytes were matured, fertilized and cultured by the standard protocol described previously. Twenty-four hours after fertilization, adequate numbers of oocytes were fixed, stained and examined. The development of embryos was evaluated on days 2 (D2), 7 (D7) and 8 (D8) after fertilization. The rate of normally fertilized oocytes was significantly lower ( p ≤ 0.01) for bull A than for bulls B and C. However, no significant differences in the kinetics of fertilization were found between bulls A, B and C. The D2 cleavage rate of embryos was significantly lower ( p ≤ 0.01) for bull A than for bulls B and C. Both D7 and D8 blastocyst rates for bull A or bull B were significantly lower ( p ≤ 0.01 or p ≤ 0.05) than those for bull C. The percentages of both D7 advanced blastocysts and D8 expanded blastocysts were significantly lower ( p ≤ 0.01) for bulls A and B than for bull C. In conclusion, for rob(16;20), the efficiency of fertilization was strongly reduced; it resulted in low early and advanced embryo development. On the other hand, for the rob(1;29), neither fertilization nor early embryo development were affected and only advanced embryo development was decreased. But for both translocations, blastocyst formation was significantly delayed.