Effects of components of semen extenders on the binding of stallion spermatozoa to bovine or equine zonae pellucidae

Silva, Marco A. Coutinho da; Seidel, G. E.; Squires, E.L.; Graham, James K.; Carnevale, E.M.

doi:10.1530/rep-11-0099

Cited by 22 publications

(13 citation statements)

References 35 publications

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“…These authors demonstrated that after incubation in TALP and co-incubation with heterologous bovine oocytes, an average of fewer than 30 sperm were bound to each oocyte (Clulow et al 2010). Similar results were obtained through binding assays incorporating equine sperm and bovine and equine oocytes, with averages of 29 and 36 sperm/egg being obtained respectively (Coutinho da Silva et al 2012). In comparison, the number of sperm bound to oocytes after incubation in PVA/mbcd (119.4 sperm/egg) and BSA/mbcd (88.3 sperm/egg) greatly exceeded those observed in previous studies.…”

Section: Discussionmentioning

confidence: 57%

“…This could have particular implications with regard to the use of sperm stored in milk-based extenders for artificial insemination, where long-term sperm survival is crucial for successful fertilisation (Bailey et al 2000), and for gamete intrafallopian transfer, where sperm are deposited directly into the oviduct and the components of semen extenders may be present during gamete interaction (Coutinho da Silva et al 2012). Although the mechanisms of sperm activation in the presence of milk proteins are poorly understood, it has recently been shown that incubation of stallion sperm in a skimmed milk-based extender (EZM) based on the composition of Kenney's extender (Kenney et al 1975) promotes a fourfold increase in the number of cells capable of binding to either bovine or equine ZPs (Coutinho da Silva et al 2012). Previously, these authors have demonstrated that the addition of milk proteins, casein, native phosphocaseinate, and caseinoglycopeptide, enhances the binding of stallion sperm to bovine and equine ZP in a dose-dependent manner (Coutinho da Silva et al 2005).…”

Section: Discussionmentioning

confidence: 99%

“…4D). This assay has recently been developed to address difficulties associated with the acquisition of sufficient horse oocytes and, importantly, has been used to assess stallion sperm capacitation status (Clulow et al 2010, Coutinho da Silva et al 2012. The use of this assay in our study demonstrated that media containing PVA and mbcd supported the highest levels of ZP binding with an average of 119.4 sperm bound to the ZP of each bovine oocyte, a result that significantly exceeded the number of sperm bound after incubation in NC conditions (11.6 cells/oocyte).…”

Section: Discussionmentioning

confidence: 99%

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Capacitation in the presence of methyl-β-cyclodextrin results in enhanced zona pellucida-binding ability of stallion spermatozoa

et al. 2014

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While IVF has been widely successful in many domesticated species, the development of a robust IVF system for the horse remains an elusive and highly valued goal. A major impediment to the development of equine IVF is the fact that optimised conditions for the capacitation of equine spermatozoa are yet to be developed. Conversely, it is known that stallion spermatozoa are particularly susceptible to damage arising as a consequence of capacitation-like changes induced prematurely in response to semen handling and transport conditions. To address these limitations, this study sought to develop an effective system to both suppress and promote the in vitro capacitation of stallion spermatozoa. Our data indicated that the latter could be achieved in a bicarbonate-rich medium supplemented with a phosphodiesterase inhibitor, a cyclic AMP analogue, and methyl-b-cyclodextrin, an efficient cholesterolwithdrawing agent. The populations of spermatozoa generated under these conditions displayed a number of hallmarks of capacitation, including elevated levels of tyrosine phosphorylation, a reorganisation of the plasma membrane leading to lipid raft coalescence in the peri-acrosomal region of the sperm head, and a dramatic increase in their ability to interact with heterologous bovine zona pellucida (ZP) and undergo agonist-induced acrosomal exocytosis. Furthermore, this functional transformation was effectively suppressed in media devoid of bicarbonate. Collectively, these results highlight the importance of efficient cholesterol removal in priming stallion spermatozoa for ZP binding in vitro.

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Section: Discussionmentioning

confidence: 57%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Capacitation in the presence of methyl-β-cyclodextrin results in enhanced zona pellucida-binding ability of stallion spermatozoa

et al. 2014

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“…The following variables were evaluated: plasma membrane integrity, mitochondrial membrane potential, intracellular levels of ROS, lipid peroxidation, plasma membrane fluidity, DNA fragmentation and sperm motility. In addition, a heterologous sperm‐binding assay using bovine oocytes and stallion sperm was performed, due to the limited availability of equine oocytes and that heterologous oocyte binding assays have been developed and validated demonstrating similar binding responses of stallion sperm with both bovine and equine zona pellucida (Coutinho da Silva, Seidel, Squires, Graham, & Carnevale, ; Sessions‐Bresnahan, Graham, & Carnevale, ). The best antioxidant treatment identified in both conditions was chosen for this assay.…”

Section: Methodsmentioning

confidence: 99%

Cryopreservation of stallion semen: Effect of adding antioxidants to the freezing medium on sperm physiology

Contreras

Treulén

Arias

et al. 2020

Reprod Domestic Animals

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Cryopreservation of stallion semen has not reached the level of efficiency and positive results described in other species. This is mainly due to the greater sensitivity of stallion sperm to the freezing process, showing higher rates of oxidative stress and plasma membrane damage, which trigger the activation of several cell damage pathways that ultimately culminate in DNA fragmentation and cell death. Therefore, finding molecules that improve the efficiency of this technique in stallion by preventing oxidative stress and cell damage is required. Thus, the aim of the present study was to evaluate the effect of adding three antioxidants (MnTBAP, NAC and FeTPPS) to the freezing medium on the quality and functional parameters of stallion sperm. Semen samples from three stallions frozen with the antioxidants were evaluated in two conditions: (a) adding the antioxidants before freezing, and (b) before and after freezing. Plasma membrane integrity, mitochondrial membrane potential, lipid peroxidation, intracellular ROS levels, membrane lipid disorder, DNA damage, sperm motility and binding to the zona pellucida were assessed. The results showed that MnTBAP was the antioxidant treatment that best controlled the oxidative stress process and post‐thaw cell damage, showing higher plasma membrane integrity, mitochondrial membrane potential, sperm motility, number of spermatozoa bound to the zona pellucida of bovine oocytes and lower lipid disorder. Additionally, it was determined that a second post‐thaw application of antioxidants is detrimental since induced higher cell damage and lower sperm motility, without showing any beneficial effect on the spermatozoa.

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“…The aim of this study was to test the influence of factors utilized in the standard capacitating medium (BSA, calcium, and bicarbonate), and the effects of environmental pH, on the capacitation-related events of ROS production, cholesterol efflux, PY, and binding of capacitated equine sperm to the ZP. The latter was tested in a heterologous (bovine) system, as validated previously (Coutinho da Silva et al 2012), due to scarcity of equine oocytes. These findings may help to determine, in equine sperm, factors influencing the physiological changes canonically associated with capacitation, thus helping to illuminate mechanisms underlying the failure of equine IVF and adding to our understanding of comparative sperm biology.…”

Section: Introductionmentioning

confidence: 99%

Effect of calcium, bicarbonate, and albumin on capacitation-related events in equine sperm

Macías‐García¹,

González‐Fernández²,

Loux³

et al. 2015

Reproduction

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Repeatable methods for IVF have not been established in the horse, reflecting the failure of standard capacitating media to induce changes required for fertilization capacity in equine sperm. One important step in capacitation is membrane cholesterol efflux, which in other species is triggered by cholesterol oxidation and is typically enhanced using albumin as a sterol acceptor. We incubated equine sperm in the presence of calcium, BSA, and bicarbonate, alone or in combination. Bicarbonate induced an increase in reactive oxygen species (ROS) that was abolished by the addition of calcium or BSA. Bicarbonate induced protein tyrosine phosphorylation (PY), even in the presence of calcium or BSA. Incubation at high pH enhanced PY but did not increase ROS production. Notably, no combination of these factors was associated with significant cholesterol efflux, as assessed by fluorescent quantitative cholesterol assay and confirmed by filipin staining. By contrast, sperm treated with methyl-b-cyclodextrin showed a significant reduction in cholesterol levels, but no significant increase in PY or ROS. Presence of BSA increased sperm binding to bovine zonae pellucidae in all three stallions. These results show that presence of serum albumin is not associated with a reduction in membrane cholesterol levels in equine sperm, highlighting the failure of equine sperm to exhibit core capacitation-related changes in a standard capacitating medium. These data indicate an atypical relationship among cholesterol efflux, ROS production, and PY in equine sperm. Our findings may help to elucidate factors affecting failure of equine IVF under standard conditions.

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Effects of components of semen extenders on the binding of stallion spermatozoa to bovine or equine zonae pellucidae

Cited by 22 publications

References 35 publications

Capacitation in the presence of methyl-β-cyclodextrin results in enhanced zona pellucida-binding ability of stallion spermatozoa

Capacitation in the presence of methyl-β-cyclodextrin results in enhanced zona pellucida-binding ability of stallion spermatozoa

Cryopreservation of stallion semen: Effect of adding antioxidants to the freezing medium on sperm physiology

Effect of calcium, bicarbonate, and albumin on capacitation-related events in equine sperm

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