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In this study, different assays were used to assess the structural and functional integrity of the sperm plasma membrane following freezing-thawing of the whole ejaculates (WEs) and sperm-rich fractions (SRFs) of boar semen. Besides sperm viability assessments (motility and mitochondrial membrane potential using a mitochondrial specific dye, JC-1 with propidium iodide, PI), sperm plasma membrane integrity (PMI) assessments were determined simultaneously using different membrane-based tests (SYBR- 14/PI and carboxyfluorescein diacetate (CFDA) with PI (CFDA/PI), and the hypo-osmotic swelling (HOS) test). ANOVA results showed that boar variability had a significant effect on the analysed parameters of post-thaw sperm characteristics. Spermatozoa harvested from the WEs exhibited a marked decline in post-thaw viability, manifested in reduced motility and mitochondrial membrane potential, than those originated from the SRFs after freezing-thawing. Cryopreservation compromised sperm PMI, as indicated in a significant decline in SYBR-stained, CFDA-stained or HOS-positive sperm cells, irrespective of the ejaculate collection procedure. It was observed that the membrane-based tests were strongly inter-correlated. Furthermore, agreement between the measurements of the membrane-based tests was confirmed by the Bland-Altman scatter plots of differences, suggesting that these tests could detect the same sperm cohorts, which were susceptible to cryo-induced membrane damage. The findings of this study indicate that dual fluorescent staining with SYBR-14/PI and CFDA/PI assays, in combination with the HOS test, provide more precise description of the sperm populations in frozen-thawed boar semen.
The aim of this study was to investigate the effect the sperm-rich fraction (F1) and the post-F1 fraction (F2) on the quality of boar spermatozoa stored in a liquid state. Ejaculates were collected from three Polish Landrace boars. Each ejaculate fraction was diluted with BTS short-term extender and Safe-Cell Plus (SCP) long-term extender and stored for seven days (D1-D7) at 17°C. Analyses included sperm motility parameters, normal apical ridge (NAR) acrosomes and plasma membrane integrity (PMI). Prior to the dilution of fractions, marked changes (p<0.05) were noted between F1 and F2 in progressive motility (PMOT), velocity average pathway (VAP) and velocity straight line (VCL). After the ejaculate was diluted, the type of fraction and type of extender significantly affected (p<0.05) PMOT, being markedly higher (p<0.05) for F1 extended in BTS. No marked changes (p<0.05) were observed between F1 and F2 extended in SCP for any of the analyzed sperm quality parameters during seven days of storage. Significantly higher (p<0.05) values of sperm quality parameters were noted in F1 compared with F2 for BTS on D7 of storage. The results of the four-way ANOVA analysis indicate that boar, fraction of ejaculate, extender type and day of storage had significant effects on the quality of boar stored spermatozoa. The F1 was characterised by higher quality of spermatozoa during storage in comparison with F2 in the short-term extender. Using the long-term extender containing the proteins allowed for a better application of F2, which could be important for the pig industry.
This study was aimed to analyze the metabolic activity and membrane integrity of boar spermatozoa following storage in long-term semen extenders. Boar semen was diluted with Androhep® EnduraGuard™ (AeG), DILU-Cell (DC), SafeCell Plus™ (SCP) and Vitasem LD (VLD) extenders and stored for 10 days at 17°C. Parameters of the analyzed sperm metabolic activity included total motility (TMOT), progressive motility (PMOT), high mitochondrial membrane potential (MMP) and ATP content, whereas those of the membrane integrity included plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome. Extender type was a significant (P < 0.05) source of variation in all the analyzed sperm parameters, except for ATP content. Furthermore, the storage time had a significant effect (P < 0.05) on the sperm metabolic activity and membrane integrity during semen storage. In all extenders the metabolic activity and membrane integrity of the stored spermatozoa decreased continuously over time. Among the four analyzed extenders, AeG and SCP showed the best performance in terms of TMOT and PMI on Days 5, 7 and 10 of storage. Marked differences in the proportions of spermatozoa with high MMP were observed between the extenders, particularly on Day 10 of storage. There were not any marked differences in sperm ATP content between the extenders, regardless of the storage time. Furthermore, the percentage of spermatozoa with NAR acrosomes decreased during prolonged storage, being markedly lower in DC-diluted semen compared with semen diluted with either AeG or SCP extender. The results of this study indicated that components of the long-term extenders have different effects on the sperm functionality and prolonged semen longevity by delaying the processes associated with sperm ageing during liquid storage.
The aim of this study was to investigate the effects of storage of semen in different commercial extenders on the pre-freezing and post-thawing quality of boar spermatozoa. Semen was diluted in BTS, Androhep (AH) and Gedil (GD), stored for 24 h at 17°C, and then frozen in accordance with the cryopreservation protocol. Analyses of the quality of spermatozoa included: motility, normal apical ridge (NAR) acrosome, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), measurements of ATP content and activity of superoxidase dismutase (SOD) and glutathione peroxidase (GPx). Prior to the freezing process, no significant effect of the extender on the sperm quality parameters was noted. After thawing the spermatozoa it was demonstrated that the type of extender used influenced PMI, MMP, ATP content and activity of GPx. In the AH extender the percentage of spermatozoa with PMI and ATP content in spermatozoa was significantly higher (P<0.05) as compared to the BTS or GD extenders. In addition, semen stored in the AH was characterised by a statistically higher (P<0.05) percentage of spermatozoa with MMP and increased activity of GPx as compared with the BTS. The results obtained indicate that for the cryopreservation process, boar spermatozoa stored for 24 hours in liquid state can be used. However, the type of extender used prior to freezing may have a significant effect on the post- thawing quality of the spermatozoa. The AH extender better secured the quality of thawed boar spermatozoa as compared with the BTS or GD.
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