The techniques used to prepare bovine spermatozoa for in vitro fertilization, to enhance the percentage of motile sperm cells include the swim-up (SU) method, among others. The objective of the present study was to evaluate the phosphatidylserine (PS) translocation and plasma membrane integrity as the indicator of apoptosis and necrosis in post-thaw bull sperm after SU treatment using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay. A flow cytometric method was employed to measure apoptosis levels on frozen-thawed bull spermatozoa. The assay detects PS translocation across the plasma membrane using a fluorescein-labelled annexin-V and PI. By using the annexin V/PI assay four different subpopulations of sperm were observed: (i) a population of apoptotic sperm, labelled with annexin V-FITC but not with PI; (ii) a population of early necrotic spermatozoa, sperm labelled with annexin-FITC and PI; (iii) a population of necrotic sperm, labelled with PI but not with annexin-FITC; and (iv) a population of fully viable sperm cells, sperm not labelled with annexin V-FITC and without PI. Results clearly indicated that SU technique itself could have an adverse effect on the spermatozoa membrane stability. It has also been found, significant differences between bulls in the levels of apoptotic sperm, after SU treatment.

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The correlation between apoptosis and early bovine embryonic loss is still not fully elucidated. In the present study, the relationship between the arrest of bovine embryos at the different stages of development and apoptosis was evaluated. We used embryos 7 days after in vitro maturation and fertilization, and morphologic and biochemical apoptotic analyses were performed by using a phase contrast microscope and by the terminal transferase dUTP nick end-labelling respectively. For the statistic, the apoptotic cell ratio (ACR) was determined as the percentage of apoptotic cells per embryo. To evaluate the relation between ACR and fragmentation pattern, embryos were divided into five groups, groups I-V. To assess the relation between ACR and cytoplasmatic fragmentation, embryos were divided into three groups, according to the fragmentation percentage (<5%; 5-15% and >15%). Of the total 139 embryos included, 65 arrested at 2-8 cells; 14 arrested at 9-16 cells; 18 compacted morula and 42 were non-arrested blastocysts. The average number of embryonic fragmentation at different stages of the development, 2-8 cells, 9-16 cells, compacted morula and blastocyst, was 16.0 +/- 1.5, 28.7 +/- 4.4, 4.4 +/- 2.4 and 1 +/- 0.3 respectively. The embryos at the stage of arrested 9-16 cells and compacted morula had higher ACR than those at the blastocyst stage, excluding the stage of 2-8 cells (the genome is not yet active). The correlation detected between embryonic development and ACR was 0.92 (p < 0.01). It was observed that embryos possessing high fragmentation showed the higher ACR value (r = 0.98, p < 0.05). Comparing the results between fragmentation percentage and ACR, it was observed that the embryos with higher percentage of fragmentation corresponded to higher ACR (r = 0.97, p < 0.01). These results clearly demonstrated that bovine embryonic arrest at different stages of development is correlated with the apoptotic mechanisms.

In the present study, the effect of bovine oocyte quality related to ultrastructural characteristics of zona pellucida (ZP), polyspermic penetration and embryo developmental competence was evaluated. Cumulus-oocyte complexes were punctioned from 453 ovaries, classified as 1, 2, 3 and 4 according to their morphological aspect, matured for 24 h and then divided into two groups. In group A, oocytes were fixed in 2.5% glutaraldehyde and 0.1 m sodium cacodylate and examined under a scanning electron microscope. Photomicrographs were taken and ZP's pores were evaluated in squares of 6.4-microm width. In group B, oocytes were fertilized in vitro. After 48 h, non-cleaved oocytes were fixed for polyspermy evaluation. On days 7, 9 and 10, embryos were classified as developed (blastocysts and hatched blastocysts). Results showed that quality 1 oocytes revealed a ZP pore diameter of 0.50 +/- 0.07 microm, which was smaller than the observed on oocytes of quality 2 (0.83 +/- 0.10 microm), quality 3 (1.02 +/- 0.22 microm) and quality 4 (1.38 +/- 0.59 microm) (p < or = 0.05). For In Vitro Fertilization (IVF), results showed that embryos originating from oocytes classed as 3 and 4 had lower cleavage rate (68.4% and 43.8%) than those belonging to class 1 and 2 (79.5% and 69.3%) (p < or = 0.05). None oocyte classified as 3 and 4 developed to hatch blastocysts, while for oocytes belonging to quality 1 and 2, these values were, respectively, 15.2% and 12.5%. Concerning polyspermy, oocytes class 1 and 2 had lower polyspermic penetration than those belonging to class 3 and 4 (respectively 4.1%, 4.5%, 11.1% and 9.8%, for class 1, 2, 3 and 4). In conclusion, the present study demonstrated that oocytes with low qualities result in lower developmental competence and with high percentage of polyspermy after IVF, which can be the result of the ZP structure such as the number and the pore's diameter.

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