In vitro effects of nonylphenol on motility, mitochondrial, acrosomal and chromatin integrity of ram and boar spermatozoa.

The objective of this study was to determine the effects of nonylphenol (NP) on viability of ram and boar sperm in vitro. Ram or boar spermatozoa were exposed to 1, 10, 100, 250 and 500 µg NP ml(-1) for 1, 2, 3 or 4 h. Computer-assisted sperm motility analysis (CASA) system was used to evaluate sperm motility characteristics. Flow cytometry was used to determine mitochondrial membrane potential (MMP) and chromatin integrity, while epifluorescent microscopy was used to determine sperm acrosomal status. Exposure of both species spermatozoa to 250 and 500 µg NP ml(-1) was detrimental to progressive motility (P < 0.05), and its adverse effect was significant at lower (100 µg NP ml(-1) ) concentration (P < 0.05). The percentages of ram and boar spermatozoa with high MMP declined drastically after exposures to ≥250 µg ml(-1) NP (P < 0.05). Unlike chromatin integrity, which did not appear to be altered by NP exposure, there were dose-dependent NP effects (P < 0.05) on acrosomal integrity of both species at as low as 1 µg ml(-1) NP for boar spermatozoa and 10 µg ml(-1) NP for ram spermatozoa. These data show adverse effects of NP on ram and boar spermatozoa and thus its potential harmful effects on male reproduction as NP is found in fruits, vegetables, human milk, fish and livestock products.

Gamete cryobanks for laboratory research: developing a rapid and easy-to-perform protocol for the cryopreservation of the sea urchin Paracentrotus lividus (Lmk, 1816) spermatozoa.

Gamete cryopreservation is a biotechnology that can guarantee a continuous supply of gametes, regardless of the seasonal reproductive cycle. In this study we developed a protocol for the cryopreservation of the sea urchin Paracentrotuslividus spermatozoa, with a view to the creation of cryobanks of semen to be used as a model system in laboratory research and ecotoxicological tests. All the key phases of the procedure were separately considered and the effect on sperm motility was evaluated by means of computer assisted analysis. The best results were obtained using 7% dimethylsulfoxide in 1% NaCl plus 0.04 M trehalose as the extender, at a freezing rate of -20 °C/min. On thawing, in semen samples cryopreserved in accordance with this protocol the velocity parameters of the sub-population of rapid sperm (best performing spermatozoa) did not significantly differ from semen on collection; in addition also the fertilization ability was restored, and about 50% of normal developed plutei larvae were obtained by thawed semen. The developed protocol is rapid and easy-to-perform; moreover, the use of gametes from reared urchins makes it unnecessary to continuously collect specimens from natural populations, making this procedure a promising starting point for the creation of alternative and more sustainable methodologies in laboratory research on sea urchin gametes and embryos.

Development of alternative and sustainable methodologies in laboratory research on sea urchin gametes.

The aim of the present work is to develop a laboratory-scaled methodology for an on-demand supply of semen from the sea urchin Paracentrotus lividus. Firstly, sea urchin specimens were acclimatized to the long-term rearing in a recirculating aquaculture system and gonad maturation was obtained under controlled conditions. Semen samples were then collected from mature sea urchins and cryopreserved. Finally, post-thawing motility was evaluated, to verify whether the cryopreserved semen had maintained enough viability to be used in laboratory activities. The post-thawing motility parameters remained quite unchanged for up to 60 min after activation; moreover, the semen even retained the ability of motility activation for 60 min after thawing. This motility pattern makes the use of cryopreserved semen a feasible option in spermiotoxicity bioassays. The preliminary ecotoxicity test, carried out using motility parameters as endpoints, showed sensitivity levels to cadmium falling in the same order of magnitude as those recorded for fresh sea urchin semen and for cryopreserved sea bream semen. . Therefore, semen samples produced and stored according to the developed methodology described in this paper, can be considered a promising and sustainable alternative to those collected from mature sea urchins harvested in the field.

Evaluation of sperm subpopulation structure in relation to in vitro sperm-oocyte interaction of frozen-thawed semen from Holstein bulls.

The present study examined the relationship between the relative amount of high motile sperm and sperm-oocyte interactions obtained from Holstein bull ejaculates. Post-thaw sperm motility was analyzed using a computer-assisted sperm analyzer system and evaluated to determine the sperm motility subpopulations. Adhesion and penetration of zona pellucida (ZP) and pronucleus formation using post-thawed samples (15 ejaculates form 5 different bulls) with different percentages of sperm in the subpopulation with the fastest and most progressive subpopulation (subpopulation 4 [SP4]) were analyzed. The correlation between the proportion of sperm in SP4 and the number of spermatozoa bound to the zona pellucida (ZBA), the penetration rate, and the rate of pronucleus formation were calculated. A significant (P < 0.05) and positive correlation was found between the number of spermatozoa bound to the zona pellucida, the penetration rate, and the rate of pronucleus formation with the proportion of sperm in SP4 (r = 0.79, r = 0.66, and r = 0.63, respectively). Our results suggest that this specific high motile and progressive subpopulation is positively and significantly correlated with the ability of a thawed bull semen sample to interact properly with the oocyte and its extracellular vestments. These findings emphasize the relevance of analyzing semen subpopulation composition to predict bull sperm fertilizing ability and to select Holstein bulls for breeding purposes.