Detection of cooling-induced membrane changes in the response of boar sperm to capacitating conditions

There is a need for methods of rapid and sensitive sperm function assessment. As spermatozoa are not able to fertilize an oocyte before having undergone a series of complex physiological changes collectively called capacitation, it is logical to assess sperm function under fertilizing conditions in vitro. In this study, the responsiveness of sperm to capacitating conditions in vitro was monitored by changes in sperm response to ionophore and by changes in the amount of intracellular calcium ions in stored boar semen. Boar semen was diluted at 32 and 20 degrees C and stored for 24 and 72 h at 16 and 10 degrees C. Ionophore-induced changes and increased intracellular calcium ion content in boar spermatozoa were recorded by flow cytometry and found to progress as a function of time during incubation under capacitating conditions. All responsiveness parameters (increases in proportions of membrane-defective spermatozoa, acrosome-reacted spermatozoa, and cells with high intracellular calcium levels) were shown to be sensitive to subtle physiological changes occurring at low storage temperatures. The initial levels of sperm with a high calcium content were higher in semen stored at 10 degrees C, but the accumulation of internal calcium was lower than in semen stored at 16 degrees C. The loss of membrane integrity and increase in the proportion of acrosome-reacted cells were higher in semen stored at 10 degrees C. Dilution at 20 degrees C had no negative effect on membrane integrity or responsiveness to capacitating conditions. There was no significant difference between semen stored for 24 and 72 h in terms of membrane integrity, acrosome reaction, and intracellular calcium after capacitation treatment. However, dynamics of cell death and acrosome reaction in response to capacitating conditions were somewhat accelerated after 72 h storage, especially in semen stored at 10 degrees C. It can be concluded that the simultaneous use of the sperm membrane responsiveness and kinetic parameters is a sensitive tool for the detection of storage-related membrane changes in boar semen.     

Differences in the ability of spermatozoa from individual boar ejaculates to withstand different semen-processing techniques

The present study aimed to evaluate the ability of spermatozoa from individual boar ejaculates to withstand different semen-processing techniques. Eighteen sperm-rich ejaculate samples from six boars (three per boar) were diluted in Beltsville Thawing Solution and split into three aliquots. The aliquots were (1) further diluted to 3×10(7) sperm/mL and stored as a liquid at 17°C for 72 h, (2) frozen-thawed (FT) at 1×10(9) sperm/mL using standard 0.5-mL straw protocols, or (3) sex-sorted with subsequent liquid storage (at 17°C for 6 h) or FT (2×10(7) sperm/mL using a standard 0.25-mL straw protocol). The sperm quality was evaluated based on total sperm motility (the CASA system), viability (plasma membrane integrity assessed using flow cytometry and the LIVE/DEAD Sperm Viability Kit), lipid peroxidation (assessed via indirect measurement of the generation of malondialdehyde (MDA) using the BIOXYTECH MDA-586 Assay Kit) and DNA fragmentation (sperm chromatin dispersion assessed using the Sperm-Sus-Halomax(?) test). Data were normalized to the values assessed for the fresh (for liquid-stored and FT samples) or the sorted semen samples (for liquid stored and the FT sorted spermatozoa). All of the four sperm-processing techniques affected sperm quality (P<0.01), regardless of the semen donor, with reduced percentages of motile and viable sperm and increased MDA generation and percentages of sperm with fragmented DNA. Significant (P<0.05) inter-boar (effect of boars within each semen-processing technique) and intra-boar (effect of semen-processing techniques within each boar) differences were evident for all of the sperm quality parameters assessed, indicating differences in the ability of spermatozoa from individual boars to withstand the semen-processing techniques. These results are the first evidence that ejaculate spermatozoa from individual boars can respond in a boar-dependent manner to different semen-processing techniques.     

Effect of hyaluronan supplementation on boar sperm motility and membrane lipid architecture status after cryopreservation

We investigated the effect of supplementing extended boar semen with different amounts of hyaluronan (HA) prior to freezing on post-thaw sperm characteristics. Using a split sample design, the effect of HA at a final concentration of 500 or 1000 microg/ml semen on post-thaw motility parameters, and membrane lipid architecture status assessed by merocyanine-540/YOPRO-1 and flow cytometry were evaluated. HA-supplementation improved motility parameters (P < 0.05 to P < 0.001) and decreased the percentage of hyperactivated spermatozoa (P < 0.05). HA-supplemented samples had more spermatozoa showing high lipid membrane stability as assessed with merocyanine-540. In conclusion, HA appeared to preserve post-thaw spermatozoa viability in vitro and maintained membrane stability after cryopreservation.     

Assessment of sperm viability,mitochondrial activity,capacitation and acrosome intactness in extended boar semen during long-term storage

The purpose of this study was to assess sperm quality in extended boar semen during in vitro storage in order to determine which extender should be used and how long boar semen can be stored. Freshly ejaculated boar semen was diluted with equal volumes of Beltsville thaw solution (BTS), Androhep, KIEV or Zorlesco extenders and stored at 17 degrees C for up to 15 days. Sperm quality was evaluated by examining viability using SYBR-14/PI and Hoechst 33258 staining, mitochondrial activity using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) staining, acrosome intactness by Coomassie blue staining, and capacitation status by chlortetracycline (CTC) staining. There were over 50% viable spermatozoa in boar semen extended with Zorlesco and Androhep extenders on Day 13 of storage. The percentage of JC-1-stained spermatozoa was 53.8 +/- 2.1% for Zorlesco and 57.7 +/- 1.60% for Androhep extenders on Day 13 of storage. The percentage of acrosome-intact spermatozoa detected by Coomassie blue staining was higher than that in the SYBR-14PI-, Hoechst 33258-, and JC-1-stained samples in our study. The results from SYBR-14/PI, Hoechst 33258, JC-1, and Coomassie blue staining were highly correlated (r > or = 0.9461). There were less than 15% capacitated spermatozoa in the semen extended with BTS, Androhep and Zorlesco extenders during 9 days of storage. However, most viable boar spermatozoa became capacitated by Day 13 of storage. The rank order of four extenders for maintaining sperm viability and mitochondrial activity was as follows: Androhep, Zorlesco, BTS, KIEV.     

Motility and fertility of alginate encapsulated boar spermatozoa

Ejaculated boar spermatozoa are vulnerable to cold shock. Prolonged storage of boar spermatozoa at low temperatures reduces survival rate, resulting in a bottleneck for the extension of artificial insemination in pig husbandry. This study evaluated whether alginate microencapsulization processing can improve the longevity of boar spermatozoa stored at 5 degrees C and the fertility of microencapsulated spermatozoa in vivo. Sperm-rich fraction semen from three purebred boars were concentrated and microencapsulated using alginate at 16-18 degrees C, and then were stored at 5 degrees C. Following storage for 1, 3 and 7 days, the microcapsule was taken out to assess sperm release under 37 degrees C incubation with or without 110 rpm stirring. The percentage of sperm released from microcapsules with 110 rpm stirring was higher than without stirring (81 versus 60%) after 24h of incubation. In another experiment, semen was also microencapsulated to evaluate the sperm motility. The motility of spermatozoa was assessed at 10 min, 8, 24, 32, 48, 56 and 72 h following incubation at 37 degrees C for nine consecutive days. The fertility of the free and microencapsulated semen was assessed by inseminating sows, and the reproductive traits (conception rate, farrowing rate, and litter size) were recorded. The motility of encapsulated spermatozoa was significantly higher than that of free semen after 8h incubation at 37 degrees C after storing for over three days (P<0.05). No significant difference existed in conception rate, farrowing rate, and litter size between the microencapsulated and non-encapsulated semen after four days of storage. In conclusion, microencapsulation can increase the longevity of boar spermatozoa and may sustain in vivo ova fertilization ability.     

Changes in sperm membrane and ROS following cryopreservation of liquid boar semen stored at 15 °C

Boar semen is occasionally transferred to different locations in liquid form at 15 °C for cryopreservation. However, the use of frozen boar semen is limited due to the high susceptibility of boar sperm to cold shock. The aim of this study was to help improve the quality of frozen boar semen by determining the changes in sperm membrane and ROS during the cryopreservation processes of 15 °C-stored boar semen. Semen was collected from ten Duroc boars and transferred to our laboratory in liquid form stored at 15 °C. After cooling to 5 °C and freezing-thawing, conventional sperm parameters (total motility, progressive motility, and normal morphology), plasma membrane integrity, acrosomal membrane status, and intracellular ROS were evaluated. Sperm function, as assessed by conventional parameters, was unaffected by cooling but was decreased by freezing-thawing (P<0.05). However, the cooling and freezing-thawing processes led to damages in the sperm plasma membrane, and the cooling process caused increase in mean PNA (peanut agglutinin)-fluorescence intensity in viable acrosome-intact sperm (P<0.05). In ROS evaluation, the cooling process decreased intracellular (·)O(2) and H(2)O(2) in viable sperm (P<0.05), while the freezing-thawing process increased intracellular H(2)O(2) (P<0.05) without change in intracellular (·)O(2) in viable sperm. Our results suggest that, in liquid boar semen stored at 15 °C, cooling may be primarily responsible for the destabilization of sperm membranes in viable sperm, while freezing-thawing may induce reductions in sperm function with increase in membrane damage and H(2)O(2).     

Effect of dialysis on quality characteristics of turkey semen during liquid storage

Low molecular weight substances such as zinc and peroxides are present in seminal plasma and are responsible for deleterious effects in stored semen. On the contrary, molecules larger than 50 kDa are beneficial to in-vitro storage of spermatozoa. Since the effects of different seminal plasma fractions in turkey semen are not completely known, the purpose of the study was to determine the effects of turkey semen dialysis with a 12-14 kDa cut-off on viability, hypo-osmotic membrane integrity, or sperm motility of turkey spermatozoa stored up to 48 h at 5 degrees C. Twelve pools of semen, each pool originating from four toms, were used. Each pool was divided into two aliquots, one of which was dialyzed while the other represented the control. Each semen aliquot was evaluated for sperm viability, membrane integrity and motility after 6, 24 and 48 h of in-vitro storage. Cold storage of turkey semen for 48 h significantly worsened (P<0.01) sperm viability, hypo-osmotic membrane integrity, and sperm motility index of both control and dialyzed samples. After 24 and 48 h sperm viability, membrane integrity and sperm motility index were better (P<0.01) in dialyzed semen compared to the control.     

Influence of storage time on functional capacity of flow cytometrically sex-sorted boar spermatozoa

Sex-sorting of boar spermatozoa is an emerging biotechnology, still considered suboptimal owing to the slowness of the process, which requires long sorting periods to obtain an adequate number of spermatozoa to perform a non-surgical insemination. This period involves storage of sorted cells that could impair their functional capacity. Here, we have studied how the storage of sex-sorted boar spermatozoa affects their functional capacity. Sorted spermatozoa were assessed at various times (0, 2, 5h or 10h) during storage after sorting and compared with diluted and unsorted spermatozoa for sperm motility patterns, plasma membrane and acrosomal integrity and their ability to penetrate homologous IVM oocytes. Sex-sorted sperm motility and membrane integrity only decreased significantly (p<0.05) by the end of the storage period (10h) compared to unsorted spermatozoa. Sperm velocity, ALH and Dance increased significantly (p<0.05), immediately post-sorting, returning to unsorted sperm values during storage. Acrosome integrity was not seriously affected by the sorting process, but decreased (p<0.05) during storage after sorting. Sorted spermatozoa stored 2h after sorting did not differ from unsorted in penetration rates and numbers of spermatozoa per oocyte, reaching the highest (p<0.05) penetration rates and sperm numbers per oocyte, when co-cultured for 6 or more hours. Non-storage or storage for 5h or 10h negatively (p<0.05) affected sperm penetration ability. In conclusion, although flow cytometrically sex-sorted spermatozoa are able to maintain motility, viability and acrosomal integrity at optimal levels until 10h of storage after sorting, fertilizing ability is maintained only over shorter storage times (<5h).     

Effect of heparin on in vitro capacitation of boar sperm

Chlortetracycline (CTC) fluorescent pattern, the ability to undergo acrosome reaction (AR) upon exposure to 10 microM calcium ionophore A23187 and vitality estimation were used to investigate the effect of the sulfated glycosaminoglycan heparin on the in vitro capacitation of porcine spermatozoa. Sperm incubation in capacitating medium (CM) supplemented with 10 mM heparin for up to 120 min, showed an increase in the number of capacitated sperm (B pattern) and acrosome reacted sperm (AR pattern), without affecting their viability. In this condition, spermatozoa were incubated in CM depleted of albumin, calcium, bicarbonate or combinations, in the presence of heparin. In either calcium or bicarbonate-free media, capacitation was only basal and did not show variations in the presence of heparin. In absence of albumin the presence of calcium and bicarbonate stimulated capacitation, which was further increased by the addition of heparin. These results suggest that heparin enhances in vitro capacitation of porcine sperm only under capacitating conditions. Additionally, when sperm were incubated with 100 microg/ml biotinylated heparin in the presence or absence of unlabeled heparin, we observed that heparin binding sites were located mostly on the acrosomal region of boar sperm in an specific and saturable manner. The in vitro effect of heparin described in this work indicates that sulfated glycosaminoglycans, which are normally present in the female reproductive tract, might play an important role in the fertilization process in porcines.     

Assessment of sperm characteristics post-thaw and response to calcium ionophore in relation to fertility in Swedish dairy AI bulls

The present study examined the relationship between bull sperm characteristics post-thawing, after swim-up, and after challenge to calcium ionophore in relation to fertility (56-d nonreturn rates) after artificial insemination (AI). Spermatozoa from 25 semen batches derived from 15 Swedish Red and White AI bulls were evaluated with regard to post-thaw motility, membrane integrity, and migration through a swim-up procedure. The swim-up separated spermatozoa were assessed in terms of sperm concentration, viability and capacitation status as well as their response to exogenous calcium ionophore (A23187). Acrosome reactions were evaluated by fluorescence microscopy and flow cytometry. Sperm motility and viability post-thawing were significantly correlated with fertility. For the swim-up separated semen, significant correlations to nonreturn rates were found for concentration, viability, number of viable spermatozoa and sperm capacitation status (Pattern F and Pattern B). The only parameter significantly correlated to fertility after the ionophore challenge was the percentage of acrosome-reacted spermatozoa with remaining equatorial fluorescence, as assessed by fluorescence microscopy, but not by flow cytometry. The regression analysis showed that combining the results of sperm membrane integrity assessment post-thawing with those of capacitation status after swim-up provided the best prediction of fertility. The accuracy of prediction did not improve when these parameters were combined with the percentage of spermatozoa in which acrosome reaction was induced by ionophore challenge.     

Increasing storage time of extended boar semen reduces sperm DNA integrity

There is an extensive use of artificial insemination (AI) in the pig industry. Extended liquid boar semen may be used for insemination for up to 5 days after collection. The objective of this study was to determine the changes in sperm quality, when boar semen was extended and stored at 18 degrees C for up to 72 h post-collection. The study included three ejaculates from five boars, for each of the four breeds: Duroc, Hampshire, Landrace and Danish Large White (n=60 ejaculates). The sperm chromatin structure assay (SCSA) showed an increase in DNA fragmentation index (DFI) after 72 h of incubation (P<0.001), with no differences between breeds (P=0.07). For two Hampshire boars, all ejaculates had a large increase in DFI after 24 h of incubation. The standard deviation of DFI (SD-DFI) differed between breeds, with the SD-DFI for Hampshire being significantly greater than for the other breeds. The SD-DFI did not change during the 72 h of storage. Sperm viability was determined using SYBR-14 and propidium iodide in combination with flow cytometry. The sperm viability did not differ between breeds (P=0.21), but a difference in viability during storage (P<0.001) was detected. In conclusion, the SCSA cytogram patterns were consistent for different ejaculates within boars and storage of extended boar semen at 18 degrees C for 72 h significantly decreased the integrity of sperm DNA.     

Comparison of assessment of pigeon sperm viability by contrast-phase microscope (eosin-nigrosin staining) and flow cytometry (SYBR-14/propidium iodide (PI) staining) [evaluation of pigeon sperm viability

The aim of these experiments was to compare the conventional, microscopic method of evaluating pigeon sperm viability to sperm assessed by flow cytometry. Semen was collected twice a week from two groups of pigeons. In every group were 20 males (Group I: meat-type breed; Group II: fancy pigeon breed). Semen was collected using the lumbosacral and cloacal region massage method. Ejaculates collected from each group were pooled and diluted to 10 × 10⁶ sperm/ml in BPSE solution. Samples were divided into three equal parts and estimated after collection as well as after in vitro storage for 3, 6 and 24 h. The first part was using for semen motility evaluation. The proportion of motile spermatozoa (MOT) and progressive movement (PMOT) of fresh and stored semen were evaluated using the CASA-system. The second part was examined subjectively by microscope (eosin-nigrosin (EN), eosin-nigrosin staining), the third one was assessed using dual fluorescence SYBR-14/propidium iodide (PI) and flow cytometry (FC). There were not any significant differences in sperm viability and motility between the groups at 0, 3, 6, and 24 h post collection. The percentage of viable spermatozoa in fresh semen determined by EN and FC was not different in Groups I and II (I - 88.71 ± 5.42 and 84.01 ± 3.19, respectively; II-90.87 ± 6.01 and 87.38 ± 5.57, respectively). Significantly lower percentages of viable spermatozoa were detected by FC compared to the EN method in both groups after 6 h (P ≤ 0.05) as well as 24 h (P ≤ 0.01) of storage. Moreover, the dual fluorescent SYBR-14/PI staining allowed for the identification a third population of double stained, moribund spermatozoa. High positive correlations in percentage of live spermatozoa were noted between EN and FC methods in both groups of birds. Evaluation of sperm viability by FC is a rapid, accurate, sensitive, and objective method for the assessment of pigeon sperm viability in fresh as well as stored semen.     

Sperm treatment affects capacitation parameters and penetration ability of ejaculated and epididymal boar spermatozoa

This work was designed to study how this ability is affected by different sperm treatments routinely used for in vitro fertilization (IVF) assay. In this study, boar sperm samples from epididymal or ejaculated origin were processed by three different methods: left unwashed (NW group), washed in Dulbecco's phosphate-buffered saline supplemented with 0.1% BSA (BSA group), and washed on a Percoll^® gradient (PERCOLL group). After preparation of semen samples, changes in motility patterns were studied by CASA, calcium uptake by spectrofluorimetry, and ROS generation, spontaneous acrosome reaction, and lipid disorder by means of flow cytometry. Finally IVF assays were also performed with the different semen samples and penetrability results evaluated at 2 and 4 h post insemination (hpi). Independently of the sperm treatment, epididymal spermatozoa showed higher values of progressive motility, percentage of live cells with low lipid disorder, and penetration ability at 4 hpi than the corresponding ejaculated spermatozoa. Ejaculated spermatozoa showed higher levels of calcium uptake, ROS generation and percentage of spontaneous acrosome reaction than epididymal sperm. Regarding sperm treatments, PERCOLL group showed the highest values for some motility parameters (linearity of the curvilinear trajectory, straightness, and average path velocity/curvilinear velocity), ROS generation and penetration ability at 2 and 4 hpi; however this same group showed the lowest values for sperm curvilinear velocity and lateral head displacement. From all experimental groups, ejaculated-PERCOLL-treated spermatozoa showed the highest fertilization ability after 2 hpi. Results suggest that capacitation pathways can be regulated by suitable treatments making the ejaculated sperm able to reach capacitation and fertilize oocytes in similar levels than epididymal spermatozoa, although most of the studied capacitation-associated changes do not correlate with this ability.     

Effects of different concentrations of enterotoxigenic and verotoxigenic E. coli on boar sperm quality

The presence of bacteria in boar semen causes economic losses in artificial insemination (AI) centers, as a consequence of alterations on boar sperm quality. For this reason, the effects of different concentrations of enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC) on boar sperm quality were determined in this study, by conducting two experiments. The first one consisted of assessing these effects on boar sperm quality after incubating the inoculated doses at 37°C for a 96-h period, whereas the second inoculated doses were stored at 15°C during 11 days. In both experiments, the infective concentrations ranged from 10(8)cfu mL(-1) to 10(2)cfu mL(-1); the negative control being a non-inoculated dose. Twenty-four hours after inoculation, we checked by PCR for the presence of bacteria in all tubes. Sperm quality (sperm motility, sperm viability and sperm morphology) was assessed at 24h, 48h, 72h and 96h after inoculations in the first experiment (37°C), and after 3, 5, 7, 9 and 11 days in the second (15°C). Whereas no changes were observed in sperm morphology in both experiments, the percentages of progressive motile spermatozoa dramatically diminished after 24h of incubation at 37°C, the effect being more detrimental at the highest infective concentration of microbes. Moreover, a significant decrease in the percentage of viable spermatozoa in the tube inoculated with the highest concentration (10(8)cfu mL(-1)) was detected after 24h of incubating contaminated doses at 37°C. After 48h of incubation, the presence of infective concentrations of ETEC and VTEC from 10(8)cfu mL(-1) to 10(3)cfu mL(-1) resulted in a significant diminution in the percentage of viable spermatozoa. These results suggest that ETEC and VTEC PCR analyses should be done in doses destined for AI to minimize the use of doses with diminished sperm quality due to the presence of bacteria and to avoid the potential spread of infective diseases.     

Viability, susceptibility to peroxidation and fatty acid composition of boar semen during liquid storage

The changes in viability, susceptibility to peroxidation and fatty acid composition of total phospholipid were studied in boar spermatozoa during 5 day liquid storage in a standard or alpha-tocopherol (alphaT) enriched diluent. The sperm rich fraction of the ejaculates was collected from 6-month old boars. Sperm viability progressively decreased during storage and alphaT inclusion into the diluent significantly inhibited this trend. alphaT inclusion also decreased significantly peroxidation (TBARS production of spermatozoa). Spermatozoa stored in the treatment diluent became rapidly enriched in alphaT with a concomitant decrease of alphaT content in the medium. The proportion of polyunsaturates, mainly 22:6n-3, decreased with a complementary increase in the content of the saturates, mainly 18:0. The inclusion of alphaT into the diluent was effective in totally preventing the significant decrease of 22:6n-3 observed in sperm phospholipid in the control samples during the storage period. It is concluded that the alphaT inclusion in the boar semen diluent increased cell viability through its prevention of an oxidative reduction in the levels of the major polyunsaturated fatty acids, namely 22:6n-3.     

Membrane status of boar spermatozoa after cooling or cryopreservation

This study tested the hypothesis that sperm membrane changes during cooling contribute substantially to the membrane damage observed after cryopreservation of boar spermatozoa. Flow cytometry was used to assess viability (percentages of live and dead cells) of boar sperm cells after staining with SYBR-14 and propidium iodide (PI) and acrosome status after staining with FITC-pisum sativum agglutenin and PI. Incubation (38 degrees C, 4 h), cooling (to 15 or 5 degrees C) and freezing reduced the proportion of live spermatozoa compared with those in fresh semen. There were more membrane changes in spermatozoa cooled to 5 degrees C than to 15 degrees C. The proportion of live spermatozoa decreased during processing for cryopreservation and cooling to 5 degrees C, but was unaffected by freezing and thawing if held at 15 degrees C for 3.5 h during cooling. Spermatozoa not held during cooling exhibited further loss of viability after freezing and thawing. Holding the spermatozoa also increased the proportion of acrosome-intact spermatozoa at both 15 degrees C and 5 degrees C and at thawing compared with that of the unheld controls. The results of this study suggest that a substantial proportion of the membrane changes associated with cryopreservation of boar spermatozoa may be attributed to the cooling of the cells to 5 degrees C rather than to the freezing and thawing process, and that sperm membrane changes are reduced when semen is held at 15 degrees C during cooling.     

The storage of pure ram semen at room temperature results in capacitation of a subpopulation of spermatozoa

We investigated whether storage of pure ram semen at room temperature would facilitate the sperm capacitation process, as assessed by means of the chlortetracycline method. Objective motility, membrane integrity and ability of spermatozoa to undergo acrosome reaction induced by A23187 for 15 min were simultaneously evaluated to gain further insight into this process. Storage for 4 h at room temperature had a clear capacitating effect in approximately 50% of spermatozoa and increased their ability to respond to A23187. Beyond that time, the percentage of motility and membrane integrity remained unchanged. Moreover, storage did not alter the ability of those spermatozoa that remained noncapacitated under these conditions to become capacitated in SOF-m medium. Storage for 4 h increased the percentage of spermatozoa showing swelling of the apical ridge from 3 to 13%. In conclusion, storage of ram semen at room temperature for 4 h in the dark has a marked capacitating effect on a subpopulation of spermatozoa, without changes in motility or membrane integrity, and a low effect on the appearance of the acrosome. Since semen storage is generally included in different IVF protocols, the results presented here contribute toward a clearer understanding of its role in these procedures.     

AMP-activated kinase AMPK is expressed in boar spermatozoa and regulates motility

The main functions of spermatozoa required for fertilization are dependent on the energy status and metabolism. AMP-activated kinase, AMPK, acts a sensor and regulator of cell metabolism. As AMPK studies have been focused on somatic cells, our aim was to investigate the expression of AMPK protein in spermatozoa and its possible role in regulating motility. Spermatozoa from boar ejaculates were isolated and incubated under different conditions (38,5°C or 17°C, basal medium TBM or medium with Ca(2+) and bicarbonate TCM, time from 1-24 hours) in presence or absence of AMPK inhibitor, compound C (CC, 30 μM). Western blotting reveals that AMPK is expressed in boar spermatozoa at relatively higher levels than in somatic cells. AMPK phosphorylation (activation) in spermatozoa is temperature-dependent, as it is undetectable at semen preservation temperature (17°C) and increases at 38,5°C in a time-dependent manner. AMPK phosphorylation is independent of the presence of Ca(2+) and/or bicarbonate in the medium. We confirm that CC effectively blocks AMPK phosphorylation in boar spermatozoa. Analysis of spermatozoa motility by CASA shows that CC treatment either in TBM or in TCM causes a significant reduction of any spermatozoa motility parameter in a time-dependent manner. Thus, AMPK inhibition significantly decreases the percentages of motile and rapid spermatozoa, significantly reduces spermatozoa velocities VAP, VCL and affects other motility parameters and coefficients. CC treatment does not cause additional side effects in spermatozoa that might lead to a lower viability even at 24 h incubation. Our results show that AMPK is expressed in spermatozoa at high levels and is phosphorylated under physiological conditions. Moreover, our study suggests that AMPK regulates a relevant function of spermatozoa, motility, which is essential for their ultimate role of fertilization.     

Effect of storage on sperm membrane integrity and other functional characteristics of canine spermatozoa: In vitro bioassay for canine semen

The effect of storage of canine semen on sperm membrane integrity, as determined by the hypoosmotic swelling test, and on other functional characteristics of the canine spermatozoa was evaluated by established procedures. The results of this study indicated that storage of canine semen at a chilling temperature of 5 degrees C for 24 h did not significantly impair the physical and functional characteristics of the canine spermatozoa. The overall mean percentage of motility, hypo-osmotic swelling response, which assessed sperm membrane integrity, acrosome-reacted spermatozoa, acrosomal defects, and the percentage of live spermatozoa, did not significantly differ between the fresh and chilled semen samples. However, storage altered the rate of motility and acrosome reaction. The percentage of acrosome reaction in the canine capacitating medium peaked earlier in chilled than in fresh semen. It is probable that storing semen at 5 degrees C initiated/triggered the acrosome reaction. This did not amount to impairment of functional properties. Significant correlations were observed between hypo-osmotic swelling vs motility (r=0.98, P<0.002); hypo-osmotic swelling vs acrosome reaction (r=0.83, P<0.08); and acrosome reaction vs motility (R=0.89, P<0.04) in the fresh semen, and between hypo-osmotic swelling vs motility (r=0.87, P<0.05) and hypo-osmotic swelling vs acrosome reaction (r=0.56, P<0.05) in the chilled semen. It was concluded: that 1) storage of canine semen at 5 degrees C for 24 h did not significantly impair the physical and functional integrity of the spermatozoa; 2) the significant association between motility or acrosome reaction vs hypo-osmotic swelling indicates their value in assessing sperm viability; and 3) the hypo-osmotic swelling assay could have predictive value in screening out subfertile males with apparently normal spermiograms.     

Cryopreservation-induced alterations in protein tyrosine phosphorylation of spermatozoa from different portions of the boar ejaculate

Previous studies have shown that boar sperm quality after cryopreservation differs depending on the ejaculate fraction used and that spermatozoa contained in the first 10 mL (P1) of the sperm-rich fraction (SRF) show better cryosurvival than those in the SRF-P1. Since protein tyrosine phosphorylation (PTP) in spermatozoa is related with the tolerance of spermatozoa to frozen storage and cryocapacitation, we assessed the dynamics of cryopreservation-induced PTP and intracellular calcium ([Ca²⁺]i) in spermatozoa, using flow cytometry, from P1 and SRF-P1 of the boar ejaculate at different stages of cryopreservation. Sperm kinetics, assessed using a computer-assisted semen analyzer, did not differ between P1 and SRF-P1 during cryopreservation but the decrease in sperm velocity during cryopreservation was significant (P < 0.05) in SRF-P1 compared to P1. There were no significant differences in percentages of spermatozoa with high [Ca²⁺]i between P1 and SRF-P1 in fresh as well as in frozen–thawed semen. A higher (P < 0.001) proportion of spermatozoa displayed PTP during the course of cryopreservation indicating a definite effect of the cryopreservation process on sperm PTP. The proportion of spermatozoa with PTP did not differ significantly between portions of the boar ejaculate. However at any given step during cryopreservation the percentage of spermatozoa with PTP was comparatively higher in SRF-P1 than P1. A 32 kDa tyrosine phosphorylated protein, associated with capacitation, appeared after cooling suggesting that cooling induces capacitation-like changes in boar spermatozoa. In conclusion, the study has shown that the cryopreservation process induced PTP in spermatozoa and their proportions were similar between portions of SRF.