Ejaculated ram spermatozoa, freed from seminal plasma by a dextran/swim-up procedure and exposed to cold shock, were incubated with ram seminal plasma proteins and analyzed by fluorescence markers and scanning electron microscopy. Seminal plasma proteins bound to the sperm plasma membrane modified the functional characteristics of damaged spermatozoa, reproducing those of live cells. Scanning electron microscopy showed that the dramatic structural damage induced by cooling reverted after incubation with seminal plasma proteins. Assessment of membrane integrity by fluorescence markers also indicated a restoration of intact-membrane cells. This protein adsorption is a concentration-dependent process that induces cell surface restoration in relation to the amount of protein in the incubation medium. Fractionation of ram seminal plasma proteins by exclusion chromatography provided three fractions able to reverse the cold shock effect. Scanning electron microscopy also confirmed the high activity of one fraction, because approximately 50% of cold-shocked sperm plasma membrane surface was restored to its original appearance after incubation. Differences in composition between the three separated fractions mainly resulted from one major band of approximately 20 kDa, which must be responsible for recovering the sperm membrane permeability characteristic of a live cell.

We recently demonstrated the presence of melatonin in ram seminal plasma and differences in its concentration in this fluid between the breeding and nonbreeding season. In this study, we investigate the hypothesis that in vitro treatment with melatonin affects ram sperm quality, and that this is reflected in the in vitro fertilization (IVF) results. Semen from nine rams was collected during the nonreproductive season and treated with 1 mum, 10 nm and 100 pm melatonin. Samples were incubated at 39 degrees C and 5% CO2, and motility, viability, capacitation status and phosphatidylserine (PS) translocation were assessed before and after melatonin addition, either 1 or 3 hr of incubation. Fertility rate of the melatonin-treated samples was determined by means of IVF. Although melatonin failed to affect both sperm kinematic parameters and viability, the exposure of ram spermatozoa to melatonin has a direct effect, decreasing capacitation and PS translocation at 1 mum, and increasing short-term capacitation at 100 pm, which caused an increased oocyte fertilization rate following IVF. Furthermore, cleavage rate of oocytes fertilized with 100 pm melatonin-treated spermatozoa was higher than that with 1 mum melatonin and control samples (P < 0.1). These results prove that melatonin has a direct effect on ram spermatozoa in the nonreproductive season, which can be explained, at least in part, by the melatonin capacity as a reactive oxygen species scavenger and antioxidant. These findings might help to select the optimal experimental conditions for IVF and to improve sperm preservation protocols.

The study evaluated the protective effect of seminal plasma (SP) added to freezing extender against cryopreservation injuries to boar spermatozoa. Pooled sperm-rich fractions collected from 9 fertile boars were frozen in 0.5-mL straws after being extended in a conventional freezing extender either alone or supplemented with 5% of SPs (SP1-SP4) collected from the sperm-rich fractions (diluted 1:1, vol/vol, in Beltsville Thawing Solution extender) from 4 boars (1-4) with known sperm cryosurvival (poor, moderate, and good sperm freezers). Cryopreservation injuries were assessed in terms of postthaw sperm motility (assessed by computer-assisted sperm analysis), viability (plasma membrane and acrosome integrity assessed simultaneously by flow cytometry), membrane lipid peroxidation (malondialdehyde [MDA] production), and the ability of thawed spermatozoa to fertilize in vitro-matured homologous oocytes. The addition of SP from good sperm freezers (SP3 and SP4) improved (P , .01) the motility and viability of thawed spermatozoa without any influence on MDA production. Moreover, SP from good sperm freezers also increased (P , .05) the percentage of penetrated (SP3) and polyspermic oocytes (SP4) with respect to the control. Neither the total amount of SP proteins, protein profiles, nor antioxidant capacity of the different SPs were related to the various cryosurvival/fertilizing capacities of the processed spermatozoa.

The role of seminal plasma (SP) in mammalian sperm function remains largely a matter of speculation as both inhibitory and stimulating effects have been found. Specific components of SP, particularly proteins, are adsorbed onto the surface of ejaculated sperm as they pass through the male and female reproductive tracts. These sperm coating components seem to have the important function of maintaining the stability of the membrane up to the process of capacitation (decapacitation factors). Therefore, they must be removed, modified or masked before the spermatozoa undergo the acrosome reaction, an essential process for successful fertilization. It is well known that low temperatures alter the function of spermatozoa. Cold shock results in the destabilization of sperm membranes and impairment of sperm function, and it is also well known that ram spermatozoa are more sensitive to cold-shock stress than those of other species. The addition of SP proteins to spermatozoa before and/or after cooling is able to minimize cryoinjury effects. The major proteins in ram SP which are able to protect and repair the cold-shock damage to sperm contain fibronectin-II domains. The significance of this domain and the role of these proteins in sperm capacitation and gamete interaction are discussed.

Background: Some breeds of sheep are highly seasonal in terms of reproductive capability, and these changes are regulated by photoperiod and melatonin secretion. These changes affect the reproductive performance of rams, impairing semen quality and modifying hormonal profiles. Also, the antioxidant defence systems seem to be modulated by melatonin secretion, and shows seasonal variations. The aim of this study was to investigate the presence of melatonin and testosterone in ram seminal plasma and their variations between the breeding and nonbreeding seasons. In addition, we analyzed the possible correlations between these hormones and the antioxidant enzyme defence system activity. Methods:Seminal plasma from nine Rasa Aragonesa rams were collected for one year, and their levels of melatonin, testosterone, superoxide dismutase (SOD), glutathione reductase (GRD), glutathione peroxidase (GPX) and catalase (CAT) were measured.Results: All samples presented measurable quantities of hormones and antioxidant enzymes. Both hormones showed monthly variations, with a decrease after the winter solstice and a rise after the summer solstice that reached the maximum levels in October-November, and a marked seasonal variation (P < 0.01) with higher levels in the breeding season. The yearly pattern of GRD and catalase was close to that of melatonin, and GRD showed a significant seasonal variation (P < 0.01) with a higher activity during the breeding season. Linear regression analysis between the studied hormones and antioxidant enzymes showed a significant correlation between melatonin and testosterone, GRD, SOD and catalase. Conclusions:These results show the presence of melatonin and testosterone in ram seminal plasma, and that both hormones have seasonal variations, and support the idea that seasonal variations of fertility in the ram involve interplay between melatonin and the antioxidant defence system. BackgroundMelatonin plays a central role in fine-tuning circadian rhythms [1] and seasonal changes [2] through its daily nocturnal increase in the blood [3]. In seasonally breeding mammals that use changes in the photoperiod to time