No abstract

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.

The present study was designed to evaluate how environmental factors in a dry-summer subtropical climate in Terceira-Azores (situated in the North Atlantic Ocean: 38° 43′ N 27° 12′ W) can affect dairy cow (Holstein) fertility, as well as seasonal influence on in vitro oocytes maturation and embryos development. Impact of heat shock (HS) effects on in vitro oocyte’s maturation and further embryo development after in vitro fertilization (IVF) was also evaluated. For such purpose the result of the first artificial insemination (AI) performed 60 to 90 days after calving of 6,300 cows were recorded for one year. In parallel, climatic data was obtained at different elevation points (n = 5) from 0 to 1,000 m and grazing points from 0 to 500 m, in Terceira island, and the temperature humidity index (THI) was calculated. For in vitro experiments, oocytes (n = 706) were collected weekly during all year, for meiotic maturation and IVF. Further, to evaluate HS effect, 891 oocytes were collected in the cold moths (December, January, February and March) and divided in three groups treated to HS for 24 h during in vitro maturation at: C (Control = 38.5°C), HS1 (39.5°C) and HS2 (40.5°C). Oocytes from each group were used for meiotic assessment and IVF. Cleavage, morula and blastocyst development were evaluated respectively on day 2, 6, and 9 after IVF. A negative correlation between cow’s conception rate (CR) and THI in grazing points (−91.3%; p<0.001) was observed. Mean THI in warmer months (June, July, August and September) was 71.7±0.7 and the CR (40.2±1.5%) while in cold months THI was 62.8±0.2 and CR was 63.8±0.4%. A similar impact was obtained with in vitro results in which nuclear maturation rate (NMR) ranged from 78.4% (±8.0) to 44.3% (±8.1), while embryos development ranged from 53.8% (±5.8) to 36.3% (±3.3) in cold and warmer months respectively. In vitro HS results showed a significant decline (p<0.05) on NMR of oocytes for every 1°C rising temperature (78.4±8.0, 21.7±3.1 and 8.9±2.2, respectively for C, HS1, and HS2). Similar results were observed in cleavage rate and embryo development, showing a clear correlation (96.9 p<0.05) between NMR and embryo development with respect to temperatures. Results clearly demonstrated that, up to a THI of 70.6, a decrease in the CR occurs in first AI after calving; this impairment was confirmed with in vitro results.

Antioxidants are compounds that protect cells against the damaging effects of reactive oxygen species (ROS) such as singlet oxygen, superoxide, peroxyl radicals, hydroxyl radicals and peroxynitrite. Oxidative damage occurs to cells in vivo and in vitro from exposure to free radicals generated by exogenous agents (e.g., radiation, chemicals, hyperoxia) and endogenous processes such as normal cellular metabolism. An imbalance between antioxidants and ROS results in oxidative stress, leading to cellular damage. Oxidative stress (OS) has been linked to cancer, ageing, atherosclerosis, ischemic injury, inflammation, and neurodegenerative diseases. Under extreme oxidative conditions, or if the antioxidant protective mechanisms of cells are compromised, cellular injury and death may occur. Early mammalian embryos are susceptible to damage from reactive oxygen species, and they increase the production of oxygen free radicals when cultured in vitro. ROS generation results from mitochondria´s oxidative phosphorylation. The electrons will leak from the inner mitochondrial membranes, being transferred by the oxygen molecule, resulting in an unpaired electron in the orbit. This leaking results in the generation of the superoxide molecules. ROS can also be generated by the cytoplasmic NADPH-oxidase, cytochrome p450 enzymes and the xanthine oxidoreductase enzymes. The excess of OS can have negative effects on the cellular environment and can result in impaired cellular growth in the embryo or apoptosis resulting in embryo fragmentation. Similarly to what happens in females, oxidative energy production in males is inevitably associated with the generation of ROS excessive concentrations of which can lead to cellular pathology. It has been established that ROS can function as signaling molecules and evidence is emerging that sperm may generate low and controlled concentrations of ROS, specifically O 2-H 2 O 2 , as well as other species such as nitric oxide (NO), which, in his turn, act to mediate the processes of capacitation, hyperactivation and acrosome reaction crucial to the acquisition of fertilizing ability. In the present review the most important antioxidants and their mechanisms of action related to animal reproduction, are discussed.