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.

Influence of splicing mutation within the lysophosphatidic acid receptor 1 gene (LPAR1) on semen quality in Holstein-Friesian bulls.

Effect of single nucleotide polymorphism (SNP) in splicing site of the LPAR1 (lysophosphatidic acid receptor 1) gene on selected quality traits was investigated in frozen-thawed semen of Holstein-Friesian bulls. Splicing mutation A/G in the LPAR1 gene (rs43581860) was identified in 120 Holstein-Friesian bulls using PCR-RFLP technique (Hph I). Heterozygotes AG were the most frequent (37.5%) compared with AA (30.8%) and GG (31.7%) homozygotes. Observed differences in total motility (TM), sperm membrane integrity (SYBR-14/PI) and ATP content were significant between homozygotes AA or GG and heterozygotes AG. For all three traits disadvantageous effect of heterozygotes AG was detected. This means that LPAR1 splicing mutation has significant effect on semen quality and should be considered as a new marker of semen quality in Holstein-Friesian bulls.

Identification and changes in the seasonal concentrations of heat shock proteins in roe deer (Capreolus capreolus) epididymides.

Heat shock proteins (HSPs) act as molecular chaperones with important regulatory functions. HSPs are considered to be essential factors in animal reproduction. In view of seasonal variations in the secretory activity of the reproductive tract of mature roe deer (Capreolus capreolus), the aims of this study were to identify HSPs in the epididymides and compare the expression of the identified proteins in three periods of the reproductive season. Two-dimensional polyacrylamide gel electrophoresis revealed the highest number of polypeptides in homogenates of epididymal tissues and in caput, corpus and cauda epididymal fluids throughout the reproductive season. Epididymal tissue homogenates and epididymal fluids were analysed by tandem mass spectrometry (MS/MS) to reveal 31 polypeptides with enzymatic activity, including polypeptides with antioxidant properties, structural and cell signalling functions. Moreover, among the identified polypeptides, five of them were similar to heat shock proteins: endoplasmin (Grp94); heat shock protein 90 kDa (HSP90); 78-kDa glucose-regulated protein (Grp78); chain A, the crystal structure of the human HSP70 ATPase domain and heat shock protein beta-1 isoform X. The concentrations of the analysed polypeptides, expressed in optical density units (ODU), differed significantly (p ≤ .05) across the examined periods of the reproductive season. The highest ODU values for almost all analysed proteins were observed during the rutting period. The presence of HSPs in the epididymal tissues and fluids of roe deer in different periods of the reproductive season could indicate that those proteins play an important role in sperm maturation in the epididymis.

Effects of the platelet-activating factor (PAF) supplementation on ATP content of cryopreserved bull spermatozoa (AI).

The aim of this study was to investigate the effect of PAF supplementation in semen extender on ATP content in cryopreserved bull spermatozoa used for artificial insemination at different time intervals. Cryopreserved semen was treated with different concentrations of PAF: 1×10-5M, 1×10-6M, 1×10-7M, 1×10-8M and 1×10-9M at 37°C. In the present work we showed that content of ATP in cryopreserved semen supplemented with 1×10-9M PAF was statistically significantly higher at 90 and 120 minutes of incubation in comparison to the control group (p≤0.05). Present study indicates the potential influence of PAF on ATP content in male spermatozoa via it's protective role towards mitochondria metabolic activity.

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.

Characteristics of Antioxidant Systems of Yellow Fraction of Red Deer's (Cervus elaphus L.) Semen During the Rutting Period.

The objective of this study was to make the preliminary characterization of the antioxidant defence systems of the yellow fraction (YF) of red deer's (Cervus elaphus L.) semen during the rutting period. The semen was collected using artificial vagina (AV). The studies included spectrophotometric determination of antioxidant enzymes activities such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). We also analysed the contents of low-molecular antioxidants such as L-glutathione (GSH + GSSG), L-ascorbate (ASC) and total antioxidant status (TAS). Additionally, the samples were subjected to PAGE and stained for SOD and GPx activities. It was demonstrated that the yellow fraction exhibited activities of SOD and GPx, with the highest activities in September and October. CAT activity was not detected. Staining for the SOD and GPx activities confirmed three protein bands with SOD activity and one protein band with GPx activity. The content of GSH + GSSG was similar in trials dating from October to December contrary to the content of ASC which was high in samples from September and October. The stable rate of TAS was observed during the whole rutting period. The results of this study showed that the YF of red deer semen is equipped with basic battery of antioxidant enzymes comprising SOD and GPx, with the supporting role of GSH + GSSG and ASC. Moreover, the samples obtained at the peak of the rutting period occurring from September to October had the highest enzymatic activity in comparison with remaining months of the rutting period, which contributed to the high quality of the semen by preventing it from the formation of oxidative stress during the short period of intense sexual activity of male red deer. The better understanding of the mechanisms of antioxidant defence systems in the YF of deer's semen may contribute to the potential use of this fraction in technology of wild ruminant semen preservation.

Effects of the platelet-activating factor (PAF) on selected quality parameters of cryopreserved bull semen (AI) with reduced sperm motility.

The aim of the study was to determine the effects of platelet-activating factor (PAF) on selected quality parameters of cryopreserved bull semen with reduced sperm motility used for artificial insemination. The aim of experiment 1 was to identify the optimal concentration of the phospholipid able to preserve sperm viability. Cryopreserved semen was treated with different PAF concentrations: 1×10(-5) M, 1×10(-6) M, 1×10(-7) M, 1×10(-8) M and 1×10(-9) M. The experiment demonstrated that PAF at concentration 1×10(-9) M increased most the sperm viability parameters (motility parameters, plasma membrane integrity and mitochondrial function) after 120 min of incubation of thawed semen at 37°C. Cryopreserved bull semen with reduced sperm motility (below 70%) was supplemented with PAF in a concentration of 1×10(-9) M. A statistically significant increase in sperm motility, percentage of linear motile spermatozoa and VSL value was observed after 120 min incubation of sperm with 1×10(-9) M PAF. Sperm supplementation with PAF also had positive effects on plasma membrane integrity and percentage of spermatozoa with preserved mitochondrial transmembrane potential, but the differences were not statistically significant. The results indicated positive effects of PAF supplementation at a concentration of 1×10(-9) M on the selected sperm quality parameters in cryopreserved bull semen with reduced motility.

Association between ETFA genotype and activity of superoxide dismutase, catalase and glutathione peroxidase in cryopreserved sperm of Holstein-Friesian bulls.

The aim of this study was to determine whether C/T missense mutation within the ETFA gene is associated with sperm antioxidant enzymatic activity. One hundred and twenty Holstein-Friesian bulls were genotyped by the PCR-RFLP technique (MwoI). Commercial straws of frozen-thawed semen were used to evaluate the activity of three antioxidant enzymes: superoxide dismutase, catalase and glutathione peroxidase. Among all bulls investigated, genotype CT was the most frequent (44.2%), in comparison with CC (42.5%) and TT (13.3%). Significant differences in glutathione peroxidase activity were observed between homozygous individuals (CC vs TT) with heterozygous CT having intermediate values. Dismutase activity was significantly associated with ETFA genotype, although only bulls with the CT genotype were significantly different from bulls carrying the CC genotype. The activity of catalase showed a similar trend (but was not statistically significant). In conclusion, we found that bulls with the ETFA TT genotype produce sperm with the highest glutathione peroxidase activity and can therefore be more efficiently protected from reactive oxygen. The mechanism of this interaction needs to be elucidated in future research.

Proteomic Characterization of Zinc-Binding Proteins of Canine Seminal Plasma.

The zinc-binding proteins (ZnBPs) of the seminal plasma are implicated in different processes related to sperm-egg fusion. The aim of this study was to characterize the ZnBPs of canine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry. The ZnBPs were isolated from the ejaculates of five dogs by affinity chromatography and subjected to 2D-PAGE analysis. The acquired spots, detected across the gels, were analysed by mass spectrometry. Using 2D-PAGE analysis, it was shown that canine seminal plasma comprised about 46-57 zinc-binding polypeptides, with molecular mass ranging from 9.3 to 138.7 kDa and pI at pH 5.2-10.0. It was found that zinc-binding polypeptides of low molecular masses (9.3-19.0 kDa and pI at pH 6.1-10.0) were predominant in the seminal plasma, and seven polypeptides, with molecular masses ranging from 11.7 to 15.4 kDa and pI at pH 6.8-8.7, were characterized by high optical density values. In addition, analysis with mass spectrometry (LC-MS-MS/MS) revealed that the identified seven polypeptides are canine prostate-specific esterase (CPSE), which is the main proteolytic enzyme of the seminal plasma. The findings of this study indicate an important regulatory role of seminal plasma zinc ions in the functional activity of CPSE, which is of great significance for maintaining the normal function of canine prostate and the spermatozoa functions.

Prostasomes of canine seminal plasma - zinc-binding ability and effects on motility characteristics and plasma membrane integrity of spermatozoa.

Prostasomes are small lipid membrane-confined vesicles that are involved in various fertilization-related processes. The aim of this study was to demonstrate canine seminal plasma prostasomes' ability to bind zinc ions, as well as examining their effects on sperm motility characteristics and plasma membrane integrity during cold storage. Ejaculates, collected from five cross-bred dogs (n = 50), were subjected to ultracentrifugation followed by gel filtration (GF) on a Superose 6 column. Prostasomes appeared as a single fraction in the elution profile. Transmission electron microscopy (TEM) analysis of canine prostasomes revealed the presence of membrane vesicles with diameters ranging from 20.3 to 301 nm. The zinc-affinity chromatography on a Chelating Sepharose Fast Flow - Zn(2 +) showed that from 93 to 100% of the prostasome proteins bind zinc ions (P(+) Zn). SDS-PAGE revealed that canine P(+) Zn comprised four protein bands, with low molecular weights (10.2-12 kDa). We have also shown a positive effect of prostasomes (p < 0.05), especially variant B (2% of total seminal plasma protein) on canine sperm motility parameters after 2 h storage at 5°C (TMOT%, 44.75 ± 5.18) and PMOT%, 12.42 ± 1.59) and VAP, VSL, VCL, when compared with Control (TMOT%, 7.30 ± 1.41 and PMOT%, 1.70 ± 0.42). Higher percentage of spermatozoa with intact plasma membrane (SYBR/PI dual staining) and intact acrosome (Giemsa stained), after 2 h storage at 5°C, was showed, in variant A (1.5% of total seminal plasma protein) and B, when compared with Control and variant C (2.5% of total seminal plasma protein). The prostasomes' effect on motility and plasma membrane integrity of canine cold-stored spermatozoa may be related to their ability to bind zinc ions and regulate their availability to the sperm.

The effect of two packaging systems on the post-thaw characteristics of canine sperm.

The aim of this study was to compare the effect of different packaging systems on some parameters of cryopreserved canine spermatozoa. The experimental material consisted of the sperm-rich fractions of ejaculates collected from four Beagle dogs. Semen samples for cryopreservation were stored in 0.25 ml plastic straws and two aluminum tubes with a total volume of 5.0 ml. Semen was frozen in static nitrogen vapor for 10 minutes (0.25 ml straws) or 15 and 20 minutes (aluminum tubes). Post-thaw assessments involved the determination of sperm motility parameters using a computer assisted sperm analyzer (CASA), sperm plasma membrane integrity (SPMI), mitochondrial membrane potential (MMP) and acrosome integrity (normal apical ridge, NAR). Regardless of the packaging system applied, no significant differences in total sperm motility (TMOT) or selected kinematic parameters were observed after freezing-thawing. However, spermatozoa frozen in 0.25 mL straws were characterized by improved functionality, in particular mitochondrial function, after thawing. The results indicate that large quantities of canine semen can be frozen in aluminum tubes. Further studies are required, however, to evaluate different freezing and thawing rates of aluminum tubes.

Effect of dialysis of dog semen on sperm characteristics and some biochemical components of seminal plasma.

The aim of this study was to investigate the effect of dog semen dialysis on sperm characteristics and some biochemical components of seminal plasma. Whole ejaculates were dialyzed against Tris-citrate-fructose extender for a 5 h period at room temperature (using semi-permeable cellulose tubing of 12-14 kDa molecular weight cut-off). It has been demonstrated that the long-term dialysis of dog semen causes a significant decrease in sperm quality parameters and disrupts the biochemical properties of seminal plasma. This procedure requires further improvement.

Semen characteristics and selected biochemical markers of canine seminal plasma in various seasons of the year.

The aim of this study was to evaluate the influence of season on selected qualitative semen characteristics and biochemical markers of canine seminal plasma. Whole ejaculates were collected from 5 crossbred dogs aged 2-8 years. The study covered a period of one year divided into four seasons: spring (March, April, May), summer (June, July, August), autumn (September, October, November) and winter (December, January, February). Semen samples were subjected to macroscopic and microscopic analyses to determine semen volume, total sperm counts and sperm morphology parameters. The study also involved the determination of sperm motility parameters (CASA system), sperm plasma membrane integrity (SPMI, fluorescent staining SYBR-14/PI), sperm mitochondrial membrane potential (MMP, fluorescent staining JC-1/PI) and the ATP content of sperm cells. Total protein content (TPC) and the activity of alkaline phosphatase (AP) and acid phosphatase (AcP) were determined in biochemical analyses of seminal plasma. No significant differences in ejaculate volume, SMPI or ATP content of sperm cells were observed between seasons. The highest total sperm counts were reported in ejaculates acquired in summer and autumn. The lowest MMP values were determined in summer ejaculates. No significant differences in sperm motility (MOT) were observed throughout the experiment, but ejaculates collected in autumn and winter were characterized by the highest progressive motility (PMOT). AP activity and TPC were not significantly affected by season. However, AcP activity levels were significantly lower in autumn than in the remaining seasons. Seasonal variations in the analyzed macroscopic and microscopic parameters of ejaculates and biochemical markers of seminal plasma did not exert a clear negative effect on the quality of canine semen.

Selected qualitative and biochemical parameters of cryopreserved semen of Holstein-Friesian (HF) AI bulls.

Selected qualitative and biochemical parameters were determined in cryopreserved semen used for artificial insemination, sampled from 120 bulls reared at the Animal Breeding and Insemination Center in Bydgoszcz. The total average motility of the analyzed sperm samples was determined at 62.51%. The percentage of motile spermatozoa displaying progressive forward motility was 21.65%. Analyzed samples were characterized by a high percentage of sperm cells with a intact plasma membrane (71.21%) and active mitochondria (71.32%). High efficiency of the enzymatic antioxidant system of the evaluated sperm cells was demonstrated by high activity of CAT, GPx and SOD (494.37, 2847.83 and 5.31U/1x10(9) spermatozoa, respectively) values and low values of the DNA Fragmentation Index (9.32). The results of the study, obtained with the involvement of advanced analytical methods, indicate a high fertilizing capability of the analyzed sperm samples.

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.

Supplementation of different concentrations of Orvus Es Paste (OEP) to ostrich egg yolk lipoprotein extender improves post-thaw boar semen quality.

This study aimed to compare post-thaw quality of boar semen following freezing in an ostrich egg yolk lipoprotein (LPFo) extender supplemented with 0%, 0.25% and 0.50% Orvus Es Paste (OEP). Sperm assessments included total motility (TMOT), mitochondrial function (MF), plasma membrane integrity (PMI) and acrosome integrity (normal apical ridge, NAR). Considerable variations among boars and OEP treatments had a significant effect (P < 0.001) on post-thaw sperm characteristics. It was observed that post-thaw sperm characteristics were significantly compromised in semen samples frozen in the absence of OEP. By contrast, lactose-LPFo-glycerol extender supplemented with either 0.25% OEP or 0.50% OEP markedly enhanced post-thaw sperm characteristics. In all boars, there were no marked differences in post-thaw sperm TMOT between the freezing extenders supplemented with 0.25% and 0.50% OEP. However, a decline in the percentage of post-thaw motile spermatozoa was more pronounced in the extender supplemented with 0.50% OEP following a 120-min incubation period. Furthermore, the proportions of frozen-thawed spermatozoa with MF, PMI and NAR acrosomes varied significantly among the boars in the OEP-supplemented extenders. The findings of this study indicate that different OEP concentrations, in the presence of ostrich egg yolk lipoproteins, could have varying effects on post-thaw sperm survival.

Single nucleotide polymorphism within arylsulfatase D gene (ARSD) is associated with selected kinematic parameters of sperm motility in Holstein-Friesian bulls.

The aim of the study was to find out whether the single nucleotide polymorphism (SNP) within arylsulfatase D (ARSD) gene is associated with kinematic parameters of sperm motility in Holstein-Friesian bulls. 367 Holstein-Friesian bulls kept in one AI center were included in the study. Point mutation C/T at position 139037255 on chromosome X (rs42207167) was identified by PCR-RFLP method (Pflm I). Significant associations were found between ARSD genotypes and CASA-derived sperm motility parameters: average TM (Total Motility), average VSL (Straight Velocity), average VCL (Curvilinear Velocity) and for fraction of sperms showing progressive motility (a) of sperms (VSLa, VCLa and BCFa -Beat Cross Frequency). Most significant differences were observed between alternative homozygotes (CC vs TT). Our results suggest new role of arylsulfatase D gene as being involved in sperm motility.

Seasonal-dependent variations in metabolic status of spermatozoa and antioxidant enzyme activity in the reproductive tract fluids of wild boar/domestic pig hybrids.

This study investigated seasonal changes in the metabolic performance of spermatozoa and activity of the antioxidant enzymes in the seminal plasma of three wild boar/domestic pigs (aged 1.5 to 2.5 years) and the activity of the antioxidant enzymes in fluids of the cauda epididymidis and vesicular glands from 16 wild boar/domestic pig hybrids (aged 1 to 3 years). Parameters of the sperm metabolic activity, such as total motility, mitochondrial functions, and measurements of oxygen uptake, ATP content and L-lactate production, were analyzed during the spring-summer and autumn-winter periods. Besides these sperm metabolic parameters, the sperm membrane integrity was also assessed. Total protein content and activity of the antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), were measured in the reproductive tract fluids. There were no marked significant differences (P > 0.05) between the seasonal periods in terms of sperm motility, mitochondrial function and oxygen uptake; however, spermatozoa collected during the autumn-winter period exhibited higher (P < 0.05) ATP content and L-lactate production than those harvested during the spring-summer period. It was found that the vesicular gland fluid exhibited a higher level of SOD activity during the spring-summer period compared with the autumn-winter period. Furthermore, CAT activity in the seminal plasma and vesicular gland fluid was greater during the autumn-winter. Total protein content was significantly higher in the vesicular gland fluid, whereas the cauda epididymidal fluid exhibited greater SOD and GPx activities, irrespective of the seasonal period. The findings of this study confirmed seasonal-related differences in the metabolic performance of spermatozoa and activity of antioxidant enzymes of the reproductive tract of the boar/domestic pig hybrids.

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.