Combined effects of resveratrol and epigallocatechin-3-gallate on post thaw boar sperm and IVF parameters.

Frozen-thawed boar semen suffer a fertility decrease that negatively affects its widespread use. In recent years supplementing frozen-thawed boar sperm with different antioxidants gave interesting and promising results; the aim of the present work was to study the effect of supplementing boar sperm thawing medium for 1 h with combination of epigallocatechin-3-gallate (EGCG, 50 µM) and Resveratrol (R, 2 mM), on boar sperm motility (assessed by CASA), viability, acrosome integrity, mitochondrial function, lipid peroxidation and DNA integrity (assessed by flow cytometry), protein tyrosine phosphorylation (assessed by immunofluorescence) and on in vitro fertilization (IVF). Our results demonstrate that sperm motility is negatively affected by R (alone or associated with EGCG, p < 0.05) in comparison to control and EGCG groups both at 1 h and 4 h; this effect is evident both in average motility parameters and in single cells kinematics, studied by cluster analysis, that showed the presence of a specific cell population with simil-hyperactivated features in R group (p < 0.01). Viability, acrosome integrity, mitochondrial functionality and lipid peroxidation are not influenced by the addition of the antioxidants; finally, DNA integrity is negatively influenced by R (both alone or associated with EGCG) both at 1 h and 4 h incubation (p < 0.05). Finally, tyrosine phosphorylated protein immunolocalization, used as capacitation parameter, is not affected by the different treatments. Penetration rate is strongly enhanced by R, both alone or associated with EGCG (p < 0.05); EGCG increases penetration rate as well but to a lower extent. Our findings demonstrate that the combination of R and EGCG could positively affect frozen-thawed boar sperm fertility in vitro; the effect is evident also in R groups, thus demonstrating that this antioxidant is predominant, and no synergic effect is present. Some insights are needed to understand if, in particular R (that showed the strongest effect) could be profitably used for artificial insemination in vivo, given the detrimental effect of this molecule on both sperm motility and DNA integrity.

Aquaporin 11 is related to cryotolerance and fertilising ability of frozen-thawed bull spermatozoa.

Aquaporins (AQPs) are channel proteins involved in the transport of water and solutes across biological membranes. In the present study we identified and localised aquaporin 11 (AQP11) in bull spermatozoa and investigated the relationship between the relative AQP11 content, sperm cryotolerance and the fertilising ability of frozen-thawed semen. Bull ejaculates were classified into two groups of good and poor freezability and assessed through immunofluorescence and immunoblotting analyses before and after cryopreservation. AQP11 was localised throughout the entire tail and along the sperm head. These findings were confirmed through immunoblotting, which showed a specific band of approximately 50 kDa corresponding to AQP11. The relative amount of AQP11 was significantly (P<0.05) higher in both fresh and frozen-thawed spermatozoa from bull ejaculates with good freezability compared with those with poorer freezability. In addition, in vitro oocyte penetration rates and non-return rates 56 days after AI were correlated with the relative AQP11 content in fresh spermatozoa. In conclusion, AQP11 is present in the head and tail of bull spermatozoa and its relative amount in fresh and frozen-thawed spermatozoa is related to the resilience of the spermatozoa to withstand cryopreservation and the fertilising ability of frozen-thawed spermatozoa. Further research is needed to elucidate the actual role of sperm AQP11 in bovine fertility.

Aquaglyceroporins 3 and 7 in bull spermatozoa: identification, localisation and their relationship with sperm cryotolerance.

The present study aimed to determine the localisation of aquaglyceroporins 3 (AQP3) and 7 (AQP7) in bull spermatozoa and their relationship with the sperm cell's resilience to withstand cryopreservation (i.e. cryotolerance). A total of 18 bull ejaculates were cryopreserved and their sperm quality analysed before and after freeze-thawing. The presence and localisation of AQP3 and AQP7 was determined through immunoblotting and immunocytochemistry. AQP3 was found in the mid-piece and AQP7 in the mid-piece and post-acrosomal region of bull spermatozoa. Immunoblotting showed specific signal bands at 30 and 60kDa for AQP3 and at 25kDa for AQP7. Neither the relative abundance of AQP3 and AQP7 nor their localisation patterns was altered by cryopreservation but individual differences between bull ejaculates were found in immunoblots. In order to determine whether these individual differences were related to sperm cryotolerance, bull ejaculates were classified as having good (GFE) or poor freezability (PFE) on the basis of their sperm quality after thawing. While the relative abundance of AQP3 before cryopreservation did not differ between ejaculates with GFE and PFE, the abundance of AQP7 was higher in GFE than in PFE ejaculates. This finding was further confirmed through principal component and linear regression analyses. In conclusion, the relative abundance of AQP7 in fresh semen may be used as a marker to predict bull sperm cryotolerance.

Aquaporins in boar spermatozoa. Part II: detection and localisation of aquaglyceroporin 3.

The proteins belonging to the aquaporin family play a fundamental role in water and solute transport across biological membranes. While the presence of these proteins has been extensively studied in somatic cells, their function in mammalian spermatozoa has been studied less. The present study was designed to identify and localise aquaglyceroporin 3 (AQP3) in boar spermatozoa. With this purpose, 29 fresh ejaculates from post-pubertal Piétrain boars were classified into two groups based upon their sperm quality and subsequently evaluated through western blot and immunofluorescence assessments. Western blotting showed the specific signal band of AQP3 at 25 kDa, whereas immunofluorescence assessments allowed us to identify two different AQP3 localisation patterns: (1) spermatozoa presenting a clear labelling located only in the mid-piece and (2) spermatozoa exhibiting a distribution pattern in the head and along the entire tail. The first staining pattern was predominant in all studied ejaculates. Despite individual differences in AQP3 content and localisation between boar ejaculates, these differences were not correlated with sperm quality. In conclusion, although AQP3 is present in boar spermatozoa in two different localisation patterns, neither the AQP3 content nor its localisation have been found to be associated with conventional sperm parameters.

Triosephosphate isomerase (TPI) and epididymal secretory glutathione peroxidase (GPX5) are markers for boar sperm quality.

The present study sought to determine the relationship of three sperm proteins (acrosin binding protein, ACRBP; outer dense fibre protein 1, ODF1; and triosephosphate isomerase, TPI), and two seminal plasma proteins (fibronectin, FN1; and epididymal secretory glutathione peroxidase, GPX5) to conventional sperm quality parameters (sperm membrane integrity, morphology and motility) in pigs. With this purpose, 22 boar ejaculates were split into two groups according to their sperm quality (mean±standard error of the mean, % viable sperm: 95.25±0.53 vs. 78.22±1.93; % morphologically normal sperm: 96.30±0.66 vs. 80.81±2.28). The amounts of these five proteins were evaluated through Western blot analysis and subsequently compared between these two groups through a t-test for independent samples. Normalised levels of TPI in sperm were significantly higher in the low than in the high sperm quality group. In addition, TPI was found to be negatively correlated with sperm membrane integrity, morphology and several parameters describing sperm motility. On the other hand, amounts of GPX5 in seminal plasma were also significantly higher in the low than in the high quality group, and this protein was also found to be negatively correlated with sperm membrane integrity and total and progressive sperm motility. By contrast, sperm content in ACRBP and ODF1 amounts of seminal plasma protein FN1 did not significantly differ between the two groups of sperm quality. Thus, we can conclude that sperm TPI content and amounts of GPX5 in seminal plasma may be used as quality markers of boar sperm.

Aquaporins 7 and 11 in boar spermatozoa: detection, localisation and relationship with sperm quality.

Aquaporins (AQPs) are integral membrane water channels that allow transport of water and small solutes across cell membranes. Although water permeability is known to play a critical role in mammalian cells, including spermatozoa, little is known about their localisation in boar spermatozoa. Two aquaporins, AQP7 and AQP11, in boar spermatozoa were identified by western blotting and localised through immunocytochemistry analyses. Western blot results showed that boar spermatozoa expressed AQP7 (25kDa) and AQP11 (50kDa). Immunocytochemistry analyses demonstrated that AQP7 was localised in the connecting piece of boar spermatozoa, while AQP11 was found in the head and mid-piece and diffuse labelling was also seen along the tail. Despite differences in AQP7 and AQP11 content between boar ejaculates, these differences were not found to be correlated with sperm quality in the case of AQP7. Conversely, AQP11 content showed a significant correlation (P<0.05) with sperm membrane integrity and fluidity and sperm motility. In conclusion, boar spermatozoa express AQP7 and AQP11, and the amounts of AQP11 but not those of AQP7 are correlated with sperm motility and membrane integrity.

Sperm quality and fertility of boar seminal doses after 2 days of storage: does the type of extender really matter?

The present approach was designed to evaluate the extender effects on sperm quality and fertility of short-term refrigerated seminal doses from Landrace boars lodged in husbandry-controlled conditions. For this purpose, we analyzed the sperm quality of seminal doses diluted in short-term (Beltsville Thawing Solution) and extra-long-term (Duragen) extenders from Days 0 to 2 of storage at 17 °C during an 8-month period. Pregnancy rates and litter size were evaluated from double inseminations within an interval of 12 hours (36 and 48 hours of refrigeration) of multiparous females using seminal doses diluted in each extender type. Sperm quality was assessed from the analyses of sperm motility and kinetics, sperm viability, expressed as plasma and acrosome membrane integrity, membrane lipid disorder, intracellular calcium levels, and acrosin activity. Results indicated significant differences between the extenders in the sperm quality of seminal doses. Therefore, the seminal doses diluted in Duragen had higher percentages of progressive motile spermatozoa and membrane-intact spermatozoa than those diluted in Beltsville Thawing Solution throughout all the experimental months. Nevertheless, despite these differences in preserving the sperm quality, pregnancy rates (>90%) and litter sizes (>10 piglets born per litter) were similar between the extenders. Our results had great relevance from a practical point of view because they reported lack of an extender effect on the reproductive performance of seminal doses during short-tem storage.

Effects of Enterobacter cloacae on boar sperm quality during liquid storage at 17°C.

Contamination of fresh and extended boar sperm often occurs in farms and artificial insemination (AI) centres during semen collection, processing and storage. The presence of bacteria produces detrimental effects on boar sperm quality, which may cause economic losses in reproductive centres. The present study has evaluated for the first time how the presence of Enterobacter cloacae affects the preservation of boar spermatozoa in liquid storage at 15-17 °C for an 11-day period. With this purpose, extended semen samples from seven healthy post-pubertal boars were artificially contaminated with different sperm:bacterium ratios (2:1; 1:1; 1:5 and 1:10) of E. cloacae. The 1:0 ratio (non-inoculated) served as a negative control. The most infective ratios (i.e. 1:5 and 1:10) significantly damaged sperm motility and membrane integrity, increased sperm agglutination, and decreased the osmotic resistance of spermatozoa. In contrast, the negative impact that the lowest bacterial concentration (2:1) had on boar sperm quality was clearly lower. In addition, other parameters such as pH were also more affected at the highest infective ratios (i.e. 1:5 and 1:10), despite no damage being observed on sperm morphology. In conclusion, the present work shows that damage inflicted by the presence of E. cloacae in boar sperm during liquid storage at 15-17 °C compromises the longevity and fertilising ability of seminal doses when bacterial concentration is higher than a 1:1 ratio. Further research is warranted to address by which mechanism E. cloacae impairs boar sperm quality.