Post-thaw quality of boar spermatozoa is affected by ejaculate fractions and extenders.

The aim of this study was to investigate the effect of different extenders on the post-thaw (PT) quality of sperm originating from the sperm-rich fraction (SRF) and post-sperm-rich fraction (PSRF) of boar ejaculate. Motility and velocity parameters, analyzed using a computer-assisted semen analysis (CASA) system, and membrane integrity parameters were markedly higher in frozen-thawed (FT) spermatozoa of the SRF in both the Belstville Thawing Solution (BTS) and Androhep Plus (AHP) extenders, irrespective of the post-thaw (PT) storage time. Furthermore, reduced cryo-survival was more marked in FT spermatozoa of the PSRF in both extenders following storage for 60 min. It was found that the SRF-stored samples in the AHP extender for 60 min exhibited significantly higher percentages of spermatozoa with total motility, mitochondrial function and acrosome integrity than those stored in the BTS extender. The findings of this study confirm that components of the ejaculate fractions and extender have varying effects on the cryo-survival of boar spermatozoa.

Interactions of ostrich egg yolk lipoproteins with seminal plasma of fractionated ejaculates and their effects on post-thaw boar semen quality.

Electrophoretic methods were used to identify protein complexes formed between ostrich egg yolk lipoprotein fractions (LPFo) with seminal plasma (SP) of fractionated ejaculates, and to investigate the effect of these complexes on boar semen quality after cryopreservation. Chromatographic SP fractions (F1, F2 and F3), with or without LPFo solution, were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Comparative electrophoretic analyses of the SP revealed marked differences in the SDS-PAGE protein profiles among boars. Electrophoretic analyses showed that the interactions of LPFo with SP resulted in the appearance of high-intensity protein bands. Spermatozoa were exposed to SP chromatographic fractions originating from F1, F2 and F3, and the whole SP (wSP) before being frozen. Spermatozoa exposed to F1 and F2 exhibited significantly higher post-thaw motility compared to those treated with either F3 or wSP. In most of the boars the proportions of membrane- -intact frozen-thawed spermatozoa differed among the treatments, being significantly lower in the wSP-treated samples. The incidence of frozen-thawed spermatozoa with DNA fragmentation was less prevalent in samples exposed to F3 or the wSP. The results of this study confirmed that the interactions of LPFo with fractionated SP during the cooling period contributed to alterations in the sperm membranes, rendering them less susceptible to temperature-related injury.

Effect of post-thaw supplementation of fractionated seminal plasma on survival of boar spermatozoa.

In our recent study we demonstrated that the holding of fresh semen in fractionated seminal plasma (SP1, >40 kDa; SP2, ⟨40 kDa), obtained by gel filtration chromatography, significantly improved the sperm quality characteristics following cryopreservation (Wasilewska-Sakowska et al. 2019). In this study we investigated the effect of post-thaw (PT) supplementation of fractionated SP (SP1 and SP2) on the survival of spermatozoa from boars with good and poor semen freezability, GSF and PSF, respectively. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis showed distinct differences in the protein profiles of SP1 and SP2 from boars with GSF or PSF regarding the number of protein spots. Sperm motility characteristics and the motion patterns, assessed using the computer-assisted sperm analysis (CASA) system, were markedly higher in PT semen supplemented with SP1 and SP2 from boars with GSF. Post-thaw supplementation of either SP1 or SP2 from boars with GSF significantly improved mitochondrial function, plasma membrane and acrosome integrity, and viability during storage. The findings of this study have confirmed that the presence of protective protein components in varying abundance in either fractionated SP from boars with good freezability ejaculates significantly improved the sperm survival following PT storage.

Freezability and fertility of frozen-thawed boar semen supplemented with ostrich egg yolk lipoproteins.

Lipoproteins, isolated from ostrich egg yolk (LPFo), provide excellent protection for boar spermatozoa against cryo-induced damage. The present study was performed to investigate the effects of LPFo on the freezability and fertilizing capacity of frozen-thawed (FT) boar semen after post-cervical artificial inseminations (post-CAIs). Semen, collected from 7 Polish Large White (PLW) and 4 Polish Landrace (PLR), was frozen in an extender containing LPFo. Post-CAIs were performed in 38 multiparous sows, using a catheter-cannula kit. Sows were inseminated 2× within one oestrus, and fertility parameters were recorded after farrowing. Neither boar (within breed) nor breed affected the quality of the pre-freeze (PF) semen, such as total motility (TMOT), mitochondria membrane potential (MMP), plasma membrane integrity (PMI), osmotic resistance test (ORT) and DNA fragmentation. Differences in the freezability of boar semen were observed among the boars, whereas there were no marked breed effects. Post-thaw TMOT markedly declined over storage time in most of the boars, particularly at 60 min after thawing. Inseminations of post-weaned oestrus sows resulted in pregnancy and farrowing rates of 84.2% and 81.6%, respectively. Neither the mean number of piglets born (NB) nor the mean number of piglets born alive (NBA) was affected by boar or breed. The total number of piglets born was 365, resulting in 11.8 NB piglets, whereas the total number of piglets born alive was 353, with 11.4 NBA piglets per litter. The findings of this study reaffirm the variations in the freezability of boar semen. In this study the supplementation of ostrich egg yolk lipoproteins to the freezing extender of boar semen produced high proportions of functionally viable FT spermatozoa that were capable of providing acceptable fertility results after post-CAIs in multiparous sows.

Relation between nitric oxide (NO) level in semen and certain properties of boar spermatozoa stored at 17°C.

The aim of this study was to measure the NO level in boar semen held in a liquid state and to determine its putative relation to spermatozoa motility, plasma membrane integrity, mitochondrial membrane potential and ATP content. Generally, the percentage of spermatozoa which generated nitric oxide gradually increased, while NO level in the surrounding medium declined during the liquid preservation. NO generation in semen preserved in BTS was higher as compared to those in Androhep®Plus. We demonstrated the positive correlation between the NO level in fresh spermatozoa and their quality. We also showed negative correlation between nitric oxide level in spermatozoa preserved in BTS and sperm cells motility as well as plasma membrane integrity. Results obtained in this study confirm that NO may affect sperm physiology in a dualistic manner.

Effect of boar ejaculate fraction, extender type and time of storage on quality of spermatozoa.

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.

The benefits of cooling boar semen in long-term extenders prior to cryopreservation on sperm quality characteristics.

This study investigated the effects of long-term extenders on post-thaw sperm quality characteristics following different holding times (HT) of boar semen at 17 and 10°C. Sperm-rich fractions, collected from five boars, were diluted in Androhep(®) Plus (AHP), Androstar(®) Plus (ASP), Safecell(®) Plus and TRIXcell(®) Plus (TCP) extenders. The extended semen samples were held for 2 hr at 17°C (HT 1) and additionally for 24 hr at 10°C (HT 2), after they were evaluated and frozen. CASA sperm motility and motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome integrity were assessed in the pre-freeze and frozen-thawed semen. The Vybrant Apoptosis Assay Kit was used to analyse the proportions of viable and plasma membrane apoptotic-like changes in spermatozoa. Results indicated that boar variability, extender and HT significantly affected the sperm quality characteristics, particularly after freezing-thawing. Differences in the pre-freeze semen were more marked in the sperm motion patterns between the HTs. Pre-freeze semen in HT 2 showed significantly higher VCL and VAP, whereas no marked effects were observed in the sperm membrane integrity and viability (YO-PRO-1(-) /PI(-) ) among the extenders. Post-thaw sperm TMOT and PMOT were significantly higher in the AHP and ASP extenders of HT 2 group, whereas VSL, VCL and VAP were markedly lower in the TCP extender. Furthermore, spermatozoa from the AHP- and ASP-extended semen of HT 2 group were characterized by higher MMP, PMI and NAR acrosome integrity following freezing-thawing. In most of the extenders, the incidence of frozen-thawed spermatozoa with apoptotic-like changes was greater in HT 1. The findings of this study indicate that holding of boar semen at 10°C for 24 hr in long-term preservation extenders modulates post-thaw sperm quality characteristics in an extender-dependent manner. These results will further contribute to the improvement in the cryopreservation technology of boar semen.

Age and seasonal-dependent variations in the biochemical composition of boar semen.

This study investigated the effect of age- and seasonal-related variations in the composition of boar semen over a 3-year period. At the onset of 8 months of age, ejaculates were collected from four boars and allocated into three groups: 8 to 18, 19 to 30, and 31 to 42 months and were divided into two seasonal periods: autumn-winter and spring-summer. Boar variability had a significant effect on most of the analyzed semen parameters. Significantly, higher ejaculate volumes were observed in the boars older than 18 months of age during the autumn-winter period. Sperm concentration was higher in boars less than the age of 18 months of age, whereas the total sperm numbers were significantly higher during the autumn-winter period, regardless of the age group. Seasonal effects in sperm motility were more marked in boars at the age of 19 to 30 months, being significantly higher during the autumn-winter period. The proportions of spermatozoa with intact, normal apical ridge acrosome, and osmotically tolerant acrosomal membranes were markedly higher in boars at the age of 19 to 30 months during the autumn-winter period. Spermatozoa harvested during the spring-summer period were more susceptible to lipid peroxidation, irrespective of the age group. Significantly, higher levels of protein content and concentrations of nonthiol-containing antioxidant components of the seminal plasma (SP) were detected in boars less than 18 months of age during the autumn-winter period. Seasonal effects on the pH and proteinase inhibitory activity in the SP were more marked in boars less than 18 months of age, whereas alkaline phosphatase activity was greater in boars at the age of 19 to 30 months during the autumn-winter period. Substantial amounts of the thiol-containing antioxidants of the SP were detected in boars older than 18 months of age during the spring-summer period. Neither osmolality nor total antioxidant status was affected by differences in the seasonal periods or age groups. The findings of this study indicate that age- and seasonal-related variations affect the reproductive tract functions in the boar, resulting in marked changes in the biochemical composition of the semen.

Individual and seasonal variations in the quality of fractionated boar ejaculates.

Reproductive seasonality has been shown to affect the quality of boar semen. In this study, effects of seasonal variations in the characteristics of spermatozoa and seminal plasma (SP) of fractioned ejaculates from individual boars have been investigated. Fractionated ejaculates, designated as fraction 1 (F1), fraction 2 (F2), and fraction 3 (F3), were collected from five mature boars during the autumn-winter (October through March) and spring-summer periods (April through September). A total of 10 fractionated ejaculates (F1, F2, and F3) were collected from each boar within each seasonal period. Assessments of the sperm quality characteristics included computer-assisted sperm analysis motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity, normal apical ridge acrosomes, and DNA fragmentation. Besides SDS-PAGE and densitometric analyses of the SP proteins, the antiperoxidant activity was monitored. There were marked differences in the sperm quality characteristics among the boars, except for sperm MMP. Distinct seasonal differences (P < 0.05) were observed in the ejaculate volume of F3 during the autumn-winter and spring-summer periods (107.78 ± 5.45 and 87.80 ± 4.75 mL, respectively). Significantly higher (P < 0.05) sperm concentration and the total number of spermatozoa in the fraction were observed during the autumn-winter period. Seasonal effects in MMP and plasma membrane integrity were manifested in significantly higher (P < 0.05) percentages of spermatozoa with functional mitochondria and intact plasma membrane during the autumn-winter period. However, the seasonal effects were less marked in either sperm normal apical ridge acrosomes or sperm DNA fragmentation. Sodium dodecyl sulfate-PAGE and densitometric analyses revealed marked variations in the protein composition of the SP profiles among the boars, regardless of the ejaculate fraction and seasonal period. Distinct seasonal variations, observed in the SDS-PAGE profiles, were associated with an abundance of protein fractions of low-molecular and high-molecular weight components, particularly during the autumn-winter period. There were wide variations in antiperoxidant activity in the SP among the boars, being significantly higher in the autumn-winter period, irrespective of the ejaculate fraction. It can be suggested that marked deterioration of the quality of fractionated ejaculates during the spring-summer period was probably caused by impaired reproductive function in the boar.

Effects of storage in different semen extenders on the pre-freezing and post-thawing quality of boar spermatozoa.

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.

Effect of commercial long-term extenders on metabolic activity and membrane integrity of boar spermatozoa stored at 17 degrees C.

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 degrees 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.