Methods for improving boar and stallion semen quality

Handling of semen is a very important tool for a successful artificial insemination in both pig and horse farming. One of the main problems during liquid and frozen semen storage is the loss of fertilizing potential mainly due to a sudden reduction of temperature, an excessive production of reactive oxygen species and alterations in the antioxidant defense systems. Supplementation of sperm preparation with different antioxidants gave interesting and promising results and, during recent years, use of plant antioxidants has been gaining the attention of several research groups. Moreover, an increase in the fertilising ability of boar sperm has been demonstrated after photo-stimulation. On these bases, the objective of the present thesis was to assess whether: 1) Resveratrol (RESV) or Epigallocatechin-3-gallate (EGCG) supplementation of thawing boar semen extender is effective in influencing sperm quality parameters and in vitro fertilization ability; 2) EGCG and green tea extract polyphenols improve stallion semen parameters during cooling at 4°C; 3) Photo-stimulation improves frozen-thawed boar semen quality. The results obtained demonstrate that the addition of RESV or EGCG, alone or in combination, to thawed boar semen positively affects in vitro penetration rate while no positive effects were registered in cooled stallion semen up to 48 h storage; as a consequence, the addition of these substances in cooling medium for stallion sperm storage is not useful. Therefore, it is evident that the effects of antioxidants may vary depending on species as well as on the way semen is processed for preservation (cooled vs frozen-thawed). Moreover, photo-stimulation may be considered as a potential tool to increase the cryotolerance of poor freezability ejaculates (PFE) but more researches on the mechanisms underlying the detrimental effects on sperm motility are required; they may allow to understand whether such increase in cryotolerance has a significant impact upon the fertilizing ability of PFE

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

Supplementing Maturation Medium With Insulin Growth Factor I and Vitrification-Warming Solutions With Reduced Glutathione Enhances Survival Rates and Development Ability of in vitro Matured Vitrified-Warmed Pig Oocytes

The present study sought to determine whether in vitro maturation (IVM) of pig oocytes in a medium supplemented with insulin growth factor-I (IGF-I) and subsequent vitrification with or without reduced glutathione (GSH) affect their quality and developmental competence, and the expression of genes involved in antioxidant, apoptotic and stress responses. In Experiment 1, cumulus-oocyte complexes were matured in the absence or presence of IGF-I (100 ng·mL−1) and then vitrified-warmed with or without 2 mM of GSH. Maturation rate was evaluated before vitrification, and oocyte viability, DNA fragmentation and relative transcript abundances of BCL-2-associated X protein (BAX), BCL2-like1 (BCL2L1), heat shock protein 70 (HSPA1A), glutathione peroxidase 1 (GPX1) and superoxide dismutase 1 (SOD1) genes were assessed in fresh and vitrified-warmed oocytes. In Experiment 2, fresh and vitrified-warmed oocytes were in vitro fertilized and their developmental competence determined. Whereas the addition of IGF-I to maturation medium had no effect on oocyte maturation, it caused an increase in the survival rate of vitrified-warmed oocytes. This effect was accompanied by a concomitant augment in the relative transcript abundance of HSPA1A and a decrease of BAX. Furthermore, the addition of GSH to vitrification-warming media increased survival rates at post-warming. Likewise, the action of GSH was concomitant with an increase in the relative abundance of GPX1 and a decrease of BAX transcript. Blastocyst rates of vitrified-warmed oocytes did not differ from their fresh counterparts when IGF-I and GSH were combined. In conclusion, supplementing maturation medium with 100 ng·mL−1 IGF-I and vitrification-warming solutions with 2 mM GSH improves the quality and cryotolerance of IVM pig oocytes, through a mechanism that involves BAX, GPX1 and HSPA1A expression

Improvement of in vitro fertilization by a tannin rich vegetal extract addition to frozen thawed boar sperm

Boar spermatozoa are very susceptible to cryopreservation injuries and, for this reason, pig remains one of the few species in which fresh semen is still preferred to thawed one for routine artificial insemination (AI). The present work evaluated the effect of supplementing boar sperm thawing medium with Silvafeed SP (SSP), a mixture of Chestnut and Quebracho wood extracts (60/40 w/w) rich in polyphenols (92.4% tannin content) on in vitro fertilization (IVF) and on the following sperm parameters: sperm motility (assessed by CASA), viability, acrosome integrity, mitochondrial function and lipid peroxidation (assessed by flow cytometry) and capacitation status (immunolocalization of tyrosine phosphorylated proteins). Thawed spermatozoa were incubated 1 h at 37\ub0C in BTS without (CTR) or with (5, 10, 20 g/mL) SSP. After incubation sperm suspension was divided in three aliquots: one was used for IVF trials, one for sperm analysis, and the last one was capacitated for 1 h at 39\ub0C 5% CO2 in IVF medium. Sperm motility parameters, viability, acrosome integrity, mitochondrial functionality, lipid peroxidation and tyrosine phosphorylated protein immunolocalization, used as capacitation parameter, were not influenced by SSP. However, oocytes inseminated with thawed spermatozoa pretreated with all the different SSP concentrations presented a significant (P < 0.01) increase in penetration rate compared to CTR. In addition, 5 g/mL SSP exerted a positive effect (P<0.05) on the total efficiency of fertilization. These results encourage the use of SSP in the thawing medium since post-thawing fertility is a limit for the large-scale use of boar frozen semen

Alkaline phosphatase added to capacitating medium enhances horse sperm-zona pellucida binding

Alkaline phosphatase (AP) is present in equine seminal plasma and spermatozoa, but its functional role is not fully understood yet. Being that, sperm-oocyte interaction in equine species has been reported to be enhanced at a slightly basic pH, this work aimed at verifying whether exogenous alkaline phosphatase exerts any role on stallion spermatozoa and sperm-oocyte interaction at different pHs (7.4; 8.0; 9.0). Stallion spermatozoa were capacitated in Tyrode's medium at pH 7.4, 8.0, and 9.0 for 4 hours at 38 °C, 5% CO2 with 2.5-IU AP (AP group) or without AP (capacitated spermatozoa group); viability with mitochondrial activity, motility, and acrosome integrity were measured. In addition, a homologous binding assay was carried out: stallion spermatozoa were capacitated 1 hour at 38 °C, 5% CO2 with 2.5-IU AP (AP group) or without AP (capacitated spermatozoa group). Oocytes were then added to sperm suspensions and coincubated for 1 hour. Our results indicate that AP at pH 9.0 significantly increases the percentage of living cells with active mitochondria, whereas it significantly reduces the percentage of acrosome-damaged cells at pH 8.0. No significant differences were registered in motility parameters. The homologous binding assay showed a strong effect of AP, that increased the number of sperm bound to the oocyte's zona pellucida at all pHs tested. In conclusion, AP can induce some modifications on sperm membranes thus enhancing their capacity to bind to the zona pellucida of equine oocytes

Supplementing maturation medium with insulin-like growth factor I and vitrification medium with reduced glutathione enhances survival rates and development ability of 'in vitro' matured, vitrified-warmed pig oocytes

The present study sought to determine whether in vitro maturation (IVM) of pig oocytes in a medium supplemented with insulin growth factor-I (IGF-I) and subsequent vitrification with or without reduced glutathione (GSH) affect their quality and developmental competence, and the expression of genes involved in antioxidant, apoptotic and stress responses. In Experiment 1, cumulus-oocyte complexes were matured in the absence or presence of IGF-I (100 ng·mL−1) and then vitrified-warmed with or without 2mM of GSH. Maturation rate was evaluated before vitrification, and oocyte viability, DNA fragmentation and relative transcript abundances of BCL-2-associated X protein (BAX), BCL2-like1 (BCL2L1), heat shock protein 70 (HSPA1A), glutathione peroxidase 1 (GPX1) and superoxide dismutase 1 (SOD1) genes were assessed in fresh and vitrified-warmed oocytes. In Experiment 2, fresh and vitrified-warmed oocytes were in vitro fertilized and their developmental competence determined. Whereas the addition of IGF-I to maturation medium had no effect on oocyte maturation, it caused an increase in the survival rate of vitrified-warmed oocytes. This effect was accompanied by a concomitant augment in the relative transcript abundance of HSPA1A and a decrease of BAX. Furthermore, the addition of GSH to vitrification-warming media increased survival rates at post-warming. Likewise, the action of GSH was concomitant with an increase in the relative abundance of GPX1 and a decrease of BAX transcript. Blastocyst rates of vitrified-warmed oocytes did not differ from their fresh counterparts when IGF-I and GSH were combined. In conclusion, supplementing maturation medium with 100 ng·mL−1 IGF-I and vitrification-warming solutions with 2mM GSH improves the quality and cryotolerance of IVM pig oocytes, through a mechanism that involves BAX, GPX1 and HSPA1A expression

Supplementing maturation medium with insulin-like growth factor I and vitrification medium with reduced glutathione enhances survival rates and development ability of 'in vitro' matured, vitrified-warmed pig oocytes

The present study sought to determine whether in vitro maturation (IVM) of pig oocytes in a medium supplemented with insulin growth factor-I (IGF-I) and subsequent vitrification with or without reduced glutathione (GSH) affect their quality and developmental competence, and the expression of genes involved in antioxidant, apoptotic and stress responses. In Experiment 1, cumulus-oocyte complexes were matured in the absence or presence of IGF-I (100 ng·mL−1) and then vitrified-warmed with or without 2mM of GSH. Maturation rate was evaluated before vitrification, and oocyte viability, DNA fragmentation and relative transcript abundances of BCL-2-associated X protein (BAX), BCL2-like1 (BCL2L1), heat shock protein 70 (HSPA1A), glutathione peroxidase 1 (GPX1) and superoxide dismutase 1 (SOD1) genes were assessed in fresh and vitrified-warmed oocytes. In Experiment 2, fresh and vitrified-warmed oocytes were in vitro fertilized and their developmental competence determined. Whereas the addition of IGF-I to maturation medium had no effect on oocyte maturation, it caused an increase in the survival rate of vitrified-warmed oocytes. This effect was accompanied by a concomitant augment in the relative transcript abundance of HSPA1A and a decrease of BAX. Furthermore, the addition of GSH to vitrification-warming media increased survival rates at post-warming. Likewise, the action of GSH was concomitant with an increase in the relative abundance of GPX1 and a decrease of BAX transcript. Blastocyst rates of vitrified-warmed oocytes did not differ from their fresh counterparts when IGF-I and GSH were combined. In conclusion, supplementing maturation medium with 100 ng·mL−1 IGF-I and vitrification-warming solutions with 2mM GSH improves the quality and cryotolerance of IVM pig oocytes, through a mechanism that involves BAX, GPX1 and HSPA1A expression