RESUMEN
Despite the vital role of seminal plasma (SP) in maintaining sperm function and aiding gamete interaction in many species, SP is usually removed before cryopreservation of stallion sperm to improve cryosurvival of sperm. The present study assessed if the vital sperm functional parameters of genetically superior stallions producing poor quality semen can be enhanced by the supplementation of heterologous SP from the stallion producing high quality semen. Spermatozoa from poor quality semen producing stallions were divided into three aliquots: two aliquots were supplemented with SP obtained from good quality semen producing stallions at the rate of 20% and 30%, respectively, whereas the third aliquot remained as control (0% SP) and incubated at 37°C for 30 minutes. Sperm membrane integrity, mitochondrial membrane potential (MMP), mitochondrial superoxide (mtROS) generation, and intracellular calcium status were assessed at different time intervals during incubation by flow cytometry. It was observed that the dead sperm population increased (p < 0.01) during incubation in both the 20% and 30% SP-supplemented groups. However, no significant changes were observed in MMP in both the control and treatment groups at different time intervals. Interestingly, it was found that sperm mtROS production increased (p < 0.01) during incubation in the SP-supplemented groups compared with the control group. The proportion of live spermatozoa with high intracellular calcium was reduced (p < 0.01) during incubation in the SP-incubated groups. Collectively, heterologous SP addition could not repair the damages caused by the cryopreservation and further resulted in deterioration of semen quality as observed in our study by reducing viability, increasing reactive oxygen species (ROS) production possibly due to high proportion of dead cells, or some factors (yet to be identified) that are inducive of oxidative stress in stallion spermatozoa.
Asunto(s)
Preservación de Semen , Semen , Masculino , Caballos , Animales , Análisis de Semen , Calcio , Preservación de Semen/veterinaria , Preservación de Semen/métodos , Espermatozoides , Criopreservación/métodosRESUMEN
Seminal plasma proteins and pathways associated with sperm motility have not been elucidated in stallions. Therefore, in the current study, using the high throughput LC/MS-MS approach, we profiled stallion seminal plasma proteins and identified the proteins and pathways associated with sperm motility. Seminal plasma from six stallions producing semen with contrasting sperm motility (n = 3 each high-and low-motile group) was utilized for proteomic analysis. We identified a total of 1687 proteins in stallion seminal plasma, of which 1627 and 1496 proteins were expressed in high- (HM) and low- motile (LM) sperm of stallions, respectively. A total number of 1436 proteins were co-expressed in both the groups; 191 (11%) and 60 (3.5%) proteins were exclusively detected in HM and LM groups, respectively. A total of 220 proteins were upregulated (>1-fold change) and 386 proteins were downregulated in SP from LM group stallions as compared to HM group stallions, while 830 proteins were neutrally expressed in both the groups. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed dysregulation of the important proteins related to mitochondrial function, acrosome, and sperm cytoskeleton in the seminal plasma of stallions producing ejaculates with low sperm motility. High abundance of peroxiredoxins and low abundance of seminal Chaperonin Containing TCP1 Complex (CCT) complex and Annexins indicate dysregulated oxidative metabolism, which might be the underlying etiology for poor sperm motility in LM group stallions. In conclusion, the current study identified the seminal plasma proteomic alterations associated with poor sperm motility in stallions; the results indicate that poor sperm motility in stallions could be associated with altered expression of seminal plasma proteins involved in oxidative metabolism.
Asunto(s)
Preservación de Semen , Semen , Animales , Caballos , Masculino , Proteínas/metabolismo , Proteómica , Semen/metabolismo , Análisis de Semen , Preservación de Semen/métodos , Proteínas de Plasma Seminal , Motilidad Espermática , Espermatozoides/metabolismoRESUMEN
The present study was conducted with the hypothesis that addition of cholesterol to the extender would stabilize the sperm membranes by increasing the cholesterol-to-phospholipid (C:P) ratio and would result in an improved post-thaw semen quality and reduce oxidative stress in the jack (Martina franca) semen. Forty-eight ejaculates from six jacks were collected and analyzed for the present study. The freshly collected semen sample of each jack stallion was divided into five equal fractions after addition of the primary extender without cholesterol-loaded cyclodextrin (CLC) (C) and with 1, 1.5, 2, and 3 mg/mL CLC to obtain 120 × 106 sperm/mL spermatozoa concentration. The semen was cryopreserved using customized freezing protocols. Evaluation of seminal parameters, the C:P ratio, and the oxidative status of jack spermatozoa was analyzed at all stages of cryopreservation. The oxidative status in the jack semen was evaluated by measuring malondialdehyde, glutathione and total antioxidant capacity levels. The results indicated that the mean percent values for various seminal quality parameters and the oxidative parameters were found to be significantly higher (P < .05) in CLC-treated groups with the highest values for 2 mg of CLC/120 × 106 spermatozoa. In conclusion, the present study revealed that the supplementation of CLC before cryopreservation has significantly reduced the oxidative stress and also increased the C:P ratio during semen cryopreservation process. Furthermore, a reduction in lipid peroxidation levels, reduced damage to the sperm plasma and acrosome membranes and improvement in the post-thaw sperm integrity as well as stability were recorded.
Asunto(s)
Ciclodextrinas , Preservación de Semen , Animales , Colesterol , Criopreservación/veterinaria , Crioprotectores/farmacología , Ciclodextrinas/farmacología , Suplementos Dietéticos , Caballos , Masculino , Estrés Oxidativo , Fosfolípidos , Análisis de Semen/veterinaria , Preservación de Semen/veterinaria , Motilidad Espermática , EspermatozoidesRESUMEN
The present study was designed to study the adverse effects of cryopreservation and evaluation of the cryoprotective effect of reduced glutathione (GSH) and ascorbic acid (AA) supplementation on exotic jack semen in combination or alone. For this, 24 semen samples from four adult and fertile jacks were collected via artificial vagina using an estrus jenny as dummy. After semen collection, the semen was evaluated for various qualitative and quantitative parameters in fresh, cooled, and frozen-thawed semen. The semen pellet was extended with the freezing extender containing either AA (0.9 g/L), GSH (2.5 mM), or combination of both (AA 0.9 g/L + GSH 2.5 mM), and another aliquot was kept as control without adding the antioxidants. The jack semen underwent cryodamage, which was evident by the observation of significant (P < .05) decline in the seminal quantitative parameters at various stages of cryopreservation process. Prefreeze and postthaw semen evaluation revealed that the values of plasma membrane, acrosome integrity, and chromatin integrity were found to be significantly higher (P < .01) in the group of samples supplemented with the combination (0.9 g/L AA +2.5 mM GSH) than AA- and GSH-alone or control groups. Supplementation of antioxidants to the freezing extender improved jack prefreeze and postthaw semen quality with the superiority of GSH over AA alone. From the present study, it was inferred that, exotic jack spermatozoa are susceptible to injuries because of cryopreservation, but these cryo-induced damage can be ameliorated significantly (P < .05) with the use of antioxidants and contribute to the improvement of semen cryopreservation procedures.