No abstract

A large proportion of bovine oocytes fail to develop to blastocyst stage following maturation, fertilization, and culture in vitro. While suboptimal culture conditions undoubtedly contribute to this poor development, it is recognized that immature oocytes, especially from cows with reduced reproductive performance or which are slaughtered on the end of their use, are heterogeneous in quality and developmental competence (Gordon 2003). The aim of the present study was to increase the efficiency of blastocyst production from cows after IVM/IVF by oocyte selection before maturation. Immature oocytes are known to synthesize a variety of proteins (Wassarman PM 1988, Annu. Rev. Biochem. 57, 415-442), among them, glucose-6-phosphate dehydrogenase (G6PDH). This enzyme is active in the growing oocyte, but has decreased activity in oocytes that have finished their growth phase. Brilliant cresyl blue (BCB) has been used to measure G6PDH activity. The BCB test is based on the capability of the G6PDH to convert the BCB stain from blue to colorless (Erisson et al. 1993 Theriogenology 39, 214). The ovaries were obtained from a slaughterhouse and transported to the laboratory; cumulus-oocyte complexes (COCs) were recovered by slicing the surface of the ovary. Only oocytes with a compact cumulus investment were used. Oocytes were placed into three groups: (1) control-placed immediately into culture; (2) holding control-COCs kept in PBS containing 0.4% BSA for 90 min at 38.5 • C before placement into culture; and (3) treatment-incubation with brilliant cresyl blue for 90 min at 38.5 • C before culture. Treated oocytes were then divided into BCB− (colorless cytoplasm, increased G6PDH) and BCB+ (colored cytoplasm, low G6PDH) on their ability to metabolize the stain. Activity of G6PDH was determined via measurement of NADP reduction in control, BCB−, and BCB+ groups; activity was significantly increased in BCB− COCs in comparison to the control and BCB+ COCs. After IVM, oocytes were fertilized in vitro. Embryos were cultured to Day 8. The rate of maturation to metaphase II was significantly higher for control and BCB+ oocytes (77.1 and 72.5%, respectively) than for BCB− oocytes (58.1%). The BCB+ oocytes yielded a significantly higher proportion of blastocysts (34.1%) than either control group (18.3 and 19.2%); and both controls and BCB+ oocytes had significantly higher blastocyst development than did BCB− oocytes (3.9%). The number of nuclei in the blastocysts was comparable in BCB+ and both control groups (105.5 ± 5.8 and 117.5 ± 8.5, 101.8 ± 6.2, respectively). Blastocysts in the BCB− group had a significantly lower cell number (61.0 ± 2.6) than did controls. The results show that the staining of COCs from cows before IVM may be useful in increasing the efficiency of blastocyst production during standard IVF procedures. In addition, classification of G6PDH activity on the basis of BCB staining may be used to effectively select cow oocytes with further developmental competence. To our knowledge, this is the first study to evaluate ...

The generation of hydrogen peroxide (H 2 O 2 ) by ram spermatozoa (spz) was measured using a flurometric assay with 10-acetyl-3,7-dihydroxyphenoxazine agent as a probe for H 2 O 2 detection. The kinetics of H 2 O 2 production from both live and dead spz at 1 × 10 6 , 3 × 10 6 , and 6 × 10 6 spz/well concentrations were assessed in the tyrode albumin lactate (TAL) medium every 15 min for 120 min. An increase in H 2 O 2 production from both live and dead spz was noted with a significant difference (P < 0.05) between the 1 × 10 6 and 6 × 10 6 spz/well concentrations. Although dead sperm generated higher amounts of H 2 O 2 than live ones, no significant differences (P > 0.05) were observed between the two types of sperm for the three different concentrations. The generation of H 2 O 2 by ram spz was also compared in the presence and absence of nicotinamide adenine dinucleotide phosphate (NADPH) and phenylalanine, substrates of the two specific oxidases. The supplementation with these substrates significantly (P < 0.05) increased the amounts of H 2 O 2 generated from both live and dead spz, but for the two substrates, the increase was higher with dead than with live spz especially when phenylalanine was added. Addition of the antioxidant catalase significantly (P < 0.05) decreased the generation of H 2 O 2 by live and dead spz with no significant differences (P > 0.05) between the two types of sperm before or after the antioxidant addition. This study showed the ability of live and dead ram spz to generate H 2 O 2 in TAL medium. This ability was significantly influenced by the addition of NADPH and phenylalanine and also by the supplementation of the antioxidant catalase.

Abstract. The effects of month, electro-ejaculation (EE) and copulation process on testosterone and cortisol levels were investigated in Syrian Awassi rams. Jugular blood samples were collected from 10 rams at weekly intervals for 1 year. During the breeding and non-breeding season, samples were collected 60 min before EE and copulation as well as 0 (at the time of ejaculation), 20 and 60 min after EE and copulation. Low testosterone levels were detected from October to February (4.58-5.06 nmol L −1 ), while high levels were noted from May to September (8.01-11.40 nmol L −1 ) with significant differences among months (P <0.001). In contrast, cortisol levels were low from March to October (0.63-2.27 nmol L −1 ) and the highest level was recorded in December (11.30 nmol L −1 ) with a significant month effect (P <0.001). Cortisol reached its maximum concentration in the electrically stimulated rams 20 min post electro-ejaculation with no significant difference between the two seasons at this end time point. Means of testosterone levels differed between the breeding and the non-breeding season for electro-ejaculated rams. An increase in testosterone level was observed after 60 min of copulation process in the non-breeding, while no differences were noted for this hormone over the four time periods during the breeding season. Cortisol levels rose significantly in non-breeding season at 0 and 20 min after copulation (P <0.001). In conclusion, Syrian Awassi rams displayed a clear seasonality of testosterone and cortisol. Cortisol levels indicate an acute stress response to EE treatment. Females have an effect on testosterone and cortisol levels in Syrian Awassi rams only during the non-breeding season.