Origin of a sperm motility inhibitor from boar seminal plasma

We have investigated the origin of the sperm motility inhibitor (SPMI) from boar seminal plasma. SPMI was measured by its capacity to inhibit the motility of demembranated spermatozoa and by an enzyme-linked immunosorbant assay (ELISA). Among the various reproductive and now reproductive tissues and fluids tested, only the seminal vesicle fluid and seminal plasma contained significant amounts of SPMI biological activity and SPMI antigen. Like other seminal vesicle fluid proteins, SPMI is diluted 6- to 8-fold upon ejaculation. By immunohistochemical detection at the light microscope with antibodies obtained from rabbits immunized with SPMI purified from boar seminal plasma, SPMI was found in the cytosol and/or on the plasma membrane bordering the lumen of the seminal vesicles. At the electron microscope level, SPMI appeared to be present only on the surface of the secretory cells. The data indicate that SPMI originates from a single tissue, the seminal vesicle, and suggest that only the mature form present on the luminal surface of the gland can react with the antibody generated from rabbits immunized with the secreted form of SPMI. © 1993 Wiley-Liss, Inc.     

Effect of heterologous and homologous seminal plasma on stallion sperm quality

Removing most of the seminal plasma (SP) from stallion semen has been shown to improve survival during cooled storage, yet adding small quantities of SP may improve pregnancy rates or cryosurvival. Furthermore, there is considerable controversy about whether the stallion's own SP or heterologous SP produces the best effect, possibly because of the variation between stallions in SP proteins or because some homologous SP remained in the sperm preparation. The SP is removed completely from stallion spermatozoa prepared by colloid centrifugation. Thus, the aim of the present study was (1) to investigate the effect of adding back SP to colloid centrifuged spermatozoa to determine its effect on spermatozoa; and (2) to investigate whether the stallion's own SP had a greater or lesser effect than heterologous SP. Conventional semen doses were sent from a stud overnight to the laboratory using standard transport conditions. Once at the laboratory, the semen samples were used for single layer centrifugation with Androcoll-E, and the resulting sperm preparations were treated with heterologous SP. Adding SP had a small but significant effect on sperm motility but no effect on the proportion of spermatozoa that had acrosome reacted. There were significant increases in hydrogen peroxide production and chromatin damage (P < 0.001). When homologous and heterologous SP were compared, considerable variation was observed between stallions, so that it was not possible to predict whether homologous or heterologous SP, or no SP, will produce the best motility for spermatozoa from any given stallion. Therefore, it is necessary to test different combinations of spermatozoa and SP to find the optimal effect on motility. The SP from most stallions increased reactive oxygen species and chromatin damage. In conclusion, the interaction between SP and spermatozoa depends on the origin of both SP and spermatozoa. If it is desirable to add SP to stallion sperm samples, it should be done directly before insemination rather than before storage, because of increased hydrogen peroxide production and sperm chromatin damage.     

The effect of seminal plasma on alpaca sperm function

In order to advance the development of assisted reproductive technologies in alpacas and other Camelids, the objective of this study was to explore the role of seminal plasma concentration on motility and functional integrity of alpaca sperm. Sixteen male alpacas > 3 y of age were used. In Experiment 1, epididymal sperm were incubated for 0 to 6 h in 0, 10, 25, 50, or 100% seminal plasma and motility was assessed. In Experiment 2, epididymal sperm were incubated in 0, 10, or 100% seminal plasma for 3 h and motility, acrosome integrity and DNA integrity were assessed. In Experiment 3, ejaculated sperm were incubated in 10, 25, 50, or 100% seminal plasma for 0 to 6 h and motility assessed. In Experiment 4, ejaculated sperm were incubated in 10 or 100% seminal plasma for 3 h and motility, acrosome integrity, DNA integrity, and viability were assessed. Epididymal and ejaculated sperm maintained motility longer when incubated in the presence of 10% seminal plasma compared to 0, 25, 50, or 100% seminal plasma (P < 0.001). The mean ± SEM percentage of epididymal sperm with intact acrosomes was less (P < 0.001) in samples incubated in 0% seminal plasma (39.4 ± 3.73) compared to 10% (75.3 ± 1.20) or 100% (77.4 ± 0.90) within 1 h after incubation. However, DNA integrity of ejaculated and epididymal sperm was not significantly affected by seminal plasma concentration. The mean viability of ejaculated sperm was reduced in the presence of 100 (12.7 ± 2.33) compared to 10% (36.2 ± 4.68) seminal plasma (P < 0.001) within 1 h of incubation. We concluded that alpaca semen should be diluted to a final concentration of 10% seminal plasma to prolong motility, preserve acrosome integrity, and maintain viability of sperm.     

Conserved ram seminal plasma proteins bind to the sperm membrane and repair cryopreservation damage

Whole seminal plasma (SP) enhances the function and fertility of frozen/thawed ram sperm. The objective of the current study was to investigate whether SP proteins capable of binding to molecules from the sperm plasma membrane were conserved among ram breeds, and whether these proteins were sufficient to overcome cryopreservation-induced reductions in sperm quality. Whole ram SP, obtained from rams of various breeds, improved progressive motility of frozen/thawed sperm at all times evaluated (P < 0.05); however, it did not improve total motility (15 min, P = 0.480; 30 min, P = 0.764; and 45 min, P = 0.795). To identify SP proteins responsible for this effect, a new method was developed to retain SP proteins that bound specifically to the sperm membrane by immobilization of sperm membrane proteins. These proteins specifically bound to the sperm surface, especially the acrosomal region. Lactotransferrin, epididymal secretory protein E1, Synaptosomal-associated protein 29, and RSVP-20 were identified (mass spectrometry) in this fraction. The retained SP proteins fraction repaired ultrastructural damage of frozen/thawed sperm and, with the addition of fructose, significantly improved motility of frozen/thawed sperm. We concluded that SP proteins that bound to the sperm membrane were conserved among ram breeds, and that when added to frozen/thawed semen (along with an energy source), they repaired ram sperm damage and enhanced sperm motility.     

Seasonal variations in seminal plasma and sperm characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua

The objective was to investigate changes, throughout the spawning season, in body size attributes and quantitative semen characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua L. Sperm velocity increased significantly throughout the spawning season of cod from both origins. Curvilinear velocity (VCL; 30 sec post-activation) increased from 78.9 ± 6.5 to 128.2 ± 6.5 μm/sec (mean ± SEM) between the beginning and end of the spawning season, respectively, for wild-caught cod, whereas for cultivated fish, it increased from 26.6 ± 2.4 to 48.9 ± 3.1 μm/sec between January and March. Spermatocrit did not undergo a significant seasonal change in wild-caught cod but did thicken for cultivated cod (24.6 ± 4.2% in January to 40.5 ± 4.4% in April; P < 0.01). Sperm head area, perimeter, length, and width declined significantly at the end of the spawning season of cod from both origins (all P values < 0.01). Seminal plasma osmolality and Na⁺ ion concentration followed a dome-shaped function through the spawning season for both wild-caught and cultivated cod (P < 0.05). For cultivated cod, seminal plasma pH was significantly lower at the start of the spawning season (P < 0.001), whereas Ca²⁺ increased then decreased (P < 0.05). Body size attributes, spermatocrit, and seminal plasma constituents had significant relationships with sperm activity variables. These relationships varied as a function of time post-activation, month, and fish origin. Our findings may be used to (i) assess spermiation stage without killing males; (ii) optimize semen collection for hatchery production; (iii) characterize the potential impact of farming on sperm quality; and (iv) improve success of sperm cryopreservation and short-term storage.     

Effects of extender, incubation temperature, and added seminal plasma on capacitation of cryopreserved, thawed boar sperm as determined by chlortetracycline staining

The effect on apparent capacitation status of frozen-thawed (FT), washed boar sperm was examined in capacitation-supporting medium at 39 °C without added seminal plasma (SP) or supplemented with either 10 or 20% (v/v) boar SP. The thawed sperm from three boars were washed to remove the egg yolk-based freezing medium (EY) and then incubated for 1–8 h after addition of SP. Capacitation status of the sperm was determined microscopically using chlortetracycline staining patterns. At 1 h after the addition of 10 or 20% (v/v) SP, capacitated sperm decreased from 59.7 to 30.3% and from 59.5 to 26.8%, respectively (P < 0.001). Subsequent studies examined the effect of 10% SP on capacitation status of FT sperm extended in either phosphate buffered saline or commercial thawing extender with or without prior washing of sperm to remove EY and incubated at 17 or 39 °C. No effect of SP resulted from addition to sperm when EY remained or when the temperature was maintained at 17 °C (P > 0.1). These results indicate that SP appears able to reverse capacitation of FT boar sperm, but that this effect is dependent on both temperature and composition of the thawing extender.     

Polymorphism of transferrin of carp seminal plasma: relationship to blood transferrin and sperm motility characteristics

Transferrin (Tf) is a major protein of carp (Cyprinus carpio) seminal plasma. Its relationship with milt quality is unknown. In this study, we sought to determine if Tf is polymorphic in carp seminal plasma and if this polymorphism is related to sperm motility characteristics. We screened males of purebred common carp line (Polish line R6) for Tf polymorphism in blood plasma. The majority of Tf genotypes represented only DD and DG variants. We then collected milt from preselected DD and DG genotypes and tested their sperm motility characteristics using computer-aided sperm analysis (CASA). Tf polymorphism in seminal plasma was found to be identical with that of blood. However, the relationships between Tf polymorphism and iron metabolic parameters were different for blood and semen. These data suggest different regulation of Tf in liver and testis. We found substantial differences in sperm motility characteristics between both genotypes. Spermatozoa of DG males were characterized by lower curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), higher linearity (LIN) and straightness (STR) of movement as compared to DD males. No differences were found in other sperm characteristics such as sperm concentration and percentage of sperm motility. Our results suggest that sperm motility parameters are related to Tf polymorphism and therefore this polymorphism may be related to sperm competitive ability.     

Biomarkers of in vivo fertility in sperm and seminal plasma of fertile stallions

The global proteome of sperm and seminal plasma of fertile stallions was investigated to determine whether associations with relative in vivo fertility exist. Seven stallions at stud in a commercial breeding station were collected throughout the breeding season and bred to a total of 164 mares to determine conception rates. On three occasions during the breeding season, raw semen was obtained from a regular collection for proteomic analysis using two-dimensional electrophoresis and also assessed for routine semen quality end points. First cycle conception rate was negatively related to ejaculate volume (r = −0.43, P = 0.05) and total IGF1 content (ng) per ejaculate (r = −0.58, P = 0.006), whereas overall pregnancy rate was positively related to sperm concentration (r = 0.56, P = 0.01). The abundance of three proteins known to be involved in carbohydrate metabolism in sperm was positively related to fertility. Furthermore, the abundance of four seminal plasma proteins were identified as being negatively related to fertility; these were identified as kallikrein-1E2 (KLK2), clusterin, and seminal plasma proteins 1 (SP1) and 2 (SP2). Abundance of cysteine-rich secretory protein 3 (CRISP3) was positively related to first cycle conception rate (r = 0.495, P = 0.027) and may provide a good marker of fertility. Based on stepwise regression analysis, clusterin and SP1 in seminal plasma together with sperm citrate synthase were predictive of fertility (r = 0.77, P < 0.0001). This study identified proteins within sperm and seminal plasma that could serve as biomarkers of semen quality and fertility in stallions.     

Determination of glutation peroxidase and superoxide dismutase activities in canine seminal plasma and its relation with sperm quality and lipid peroxidation post thaw

Lipid peroxidation (LPO) of dog spermatozoa was assessed in fresh semen and in samples of the same ejaculates after freezing and thawing. Particular attention was paid to individual differences in the susceptibility to LPO and its possible relationship with freezeability. Innate levels of LPO were low in fresh spermatozoa but increased after thawing in one of the dogs included in our study. The level of lipid peroxidation in fresh spermatozoa was not correlated with that of thawed spermatozoa. Negative correlations were detected between the activity in seminal plasma of GPx and sperm velocities post thaw (P < 0.01), however SOD activity was positively correlated with the percentage of linear motile sperm post thaw (P < 0.05).     

Exposure to the seminal plasma of different portions of the boar ejaculate modulates the survival of spermatozoa cryopreserved in MiniFlatPacks

Spermatozoa present in the first collectable 10 mL of the sperm-rich fraction (SRF) of the boar ejaculate (portion 1, P1) have higher documented viability during and after cryopreservation than spermatozoa in the rest of the ejaculate (portion 2, P2), probably in relation to different features of the surrounding seminal plasma (SP). In the present study, we investigated whether the SP from these ejaculate portions (SP1 or SP2) was able to differently influence sperm viability and chromatin structure of the P1- or P2-contained spermatozoa from individual boars primarily or secondarily exposed (e.g., following cleansing and re-exposure) to pooled SP1 or SP2 from the same males during 60 min. Spermatozoa were subjected to controlled cooling and thawing in MiniFlatPacks (MFPs) and examined for motility (using computer-assisted sperm analysis, CASA) at selected stages of processing. Moreover, sperm plasma membrane intactness (investigated using SYBR-14/propidium iodide, PI), plasma membrane architecture (examined using Annexin-V-PI staining), and chromatin (deoxyribonucleic acid, DNA) integrity (tested using sperm chromatin structure assay, SCSA) were assessed post-thaw (PT). A higher proportion of P1 spermatozoa than of P2 spermatozoa incubated in their native SP portion were confirmed to be motile from collection to PT. When P1 spermatozoa were cleansed from their original SP and re-exposed to pooled P2-SP, sperm kinematics deteriorated from extension to PT. By contrast, cleansed P2 spermatozoa increased motility to P1 levels, especially PT when re-exposed to pooled P1-SP. Such differential effects on motility were not clearly accompanied by biologically related modifications of sperm membrane or chromatin structure. This influence of the SP on sperm kinematics was not sire-dependent and it was presumably related to different concentrations or either SP proteins or bicarbonate in the different ejaculate portions.     

Lysozyme activities and immunoglobulin concentrations in seminal plasma and spermatozoa of different teleost species and indications on its significance for sperm function

The occurrence of lysozyme and immunoglobulin (Ig) in semen of different teleost species (brown trout—Salmo trutta, perch—Perca fluviatilis, burbot—Lota lota) was studied. In all investigated species lysozyme activities (1.13-1.45 U ml⁻¹) and Ig concentrations (T-Ig: 1.11-1.61 μg ml⁻¹, IgG [measured only in brown trout]: 1.49 μg ml⁻¹) were detected in seminal plasma. Ig was also found in spermatozoa (T-Ig: 0.234-0.357 μg/g protein, IgG: 0.198 μg ml⁻¹) while spermatozoal lysozyme activities were low and fluctuating (0.093-0.164 U/g protein). In Salmo trutta lysozyme activities and immunoglobulin levels were compared between semen samples with high and low sperm motility as motility is an indicator for sperm fertility. Lysozyme activities were higher in seminal plasma of samples with high motility than in those with low motility while seminal plasma and spermatozoal immunoglobulin concentrations (T-Ig, IgG) were increased in samples with low motility in comparison to samples with high motility. Seminal plasma and spermatozoal IgG concentrations and seminal plasma lysozyme activities showed significant correlations with the sperm motility rate and swimming velocity. Moreover, lysozyme improved the viability of spermatozoa in in vitro experiments. Possible physiological meanings of these results are discussed.     

Effect of long-term cryopreservation on physiological characteristics, antioxidant activities and lipid peroxidation of red seabream (Pagrus major) sperm

The aim of this study was to determine the effect of long-term cryopreservation on physiological characteristics, the antioxidant activities and lipid peroxidation of red seabream sperm which were respectively cryopreserved with 15% dimethylsulfoxide (Me₂SO) for 1 month, 13 months, 26 months, 48 months and 73 months. The motility and fertility of post-thaw sperm decreased with the storage time going on. The highest motility (87.67 ± 2.52%) was obtained in sperm cryopreserved for 1 month and the lowest (50.67 ± 5.31%) was in sperm for 73 months. There were no significant differences (p < 0.05) in fertilization rates of sperm cryopreserved for 1 month (71.33 ± 8.84%), 13 months (69.22 ± 1.02%) and 26 months (60.33 ± 2.33%); however, the sperm fertility decreased significantly for 48 months (47.22 ± 3.89%) and 73 months (39.56 ± 0.69%) storage. In addition, superoxide dismutase (SOD) activities of sperm were at a stable level for less than 26 months storage, then, decreased significantly after 48 months storage. Catalase (CAT) activities of sperm cryopreserved for 13 months, 26 months, 48 months and 73 months were significantly lower than that for 1 month. There were no significant differences in the malondialdehyde (MDA) level of sperm for less than 13 months storage. After 26 months storage, the concentration of MDA increased significantly, and the highest concentration (3.22 ± 0.05 nmol/mgprot) was obtained in 73 months storage sperm.     

The cold but not hard fats in ectotherms: consequences of lipid restructuring on susceptibility of biological membranes to peroxidation,a review

The production of reactive oxygen species is a regular feature of life in the presence of oxygen. Some reactive oxygen species possess sufficient energy to initiate lipid peroxidation in biological membranes, self-propagating reactions with the potential to damage membranes by altering their physical properties and ultimately their function. Two of the most prominent patterns of lipid restructuring in membranes of ectotherms involve contents of polyunsaturated fatty acids and ratios of the abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine. Since polyunsaturated fatty acids and phosphatidylethanolamine are particularly vulnerable to oxidation, it is likely that higher contents of these lipids at low body temperature elevate the inherent susceptibility of membranes to lipid peroxidation. Although membranes from animals living at low body temperatures may be more prone to oxidation, the generation of reactive oxygen species and lipid peroxidation are sensitive to temperature. These scenarios raise the possibility that membrane susceptibility to lipid peroxidation is conserved at physiological temperatures. Reduced levels of polyunsaturated fatty acids and phosphatidylethanolamine may protect membranes at warm temperatures from deleterious oxidations when rates of reactive oxygen species production and lipid peroxidation are relatively high. At low temperatures, enhanced susceptibility may ensure sufficient lipid peroxidation for cellular processes that require lipid oxidation products.     

Characterization of capacitation, cryoinjury, and the role of seminal plasma in porcine sperm

Capacitation is a biochemical pathway sperm must undergo to be able to fertilize an oocyte, whereas cryoinjury is cryopreservation-induced biophysical damage which renders sperm immediately capable of fertilization. Similarities between capacitation and cryoinjury have not been fully elucidated. The present study attempted to characterize both processes, including the role of seminal plasma (SP). Merocyanine-540 staining detected an increase (P < 0.01) in plasma membrane disorder from 60.5% in in vitro capacitated sperm to 91.4% in cryopreserved sperm, with no effect of SP. After cryopreservation, 42.8% of sperm displayed phosphatidylserine on the outer leaflet compared to 13.6% of in vitro capacitated sperm (P < 0.01), as assessed by annexin-V staining (SP decreased phosphatidylserine inversion in both populations). Lipid raft-associated glycolipid GM₁ movement increased throughout the entire sperm membrane in cryopreserved sperm, although SP did not affect lipid raft movement in these sperm. Cryopreserved and in vitro capacitated sperm had a similar intensity of tyrosine phosphorylation (although SP reduced this intensity). In in vitro capacitated sperm, 67.5% underwent an ionophore induced acrosome reaction with 91.3% reacting in cryopreserved sperm. In both cases, SP reduced (P < 0.01) the percentage of acrosome-reacted sperm to 1.0 and 7.8%, respectively. Cryopreservation appeared to damage sperm, resulting in marked increases in membrane disorder, cholesterol efflux, and percent of capacitated sperm. In both capacitated and cryoinjured sperm, the addition of SP appeared to attenuate some of these events.     

Restoration of seminal plasma to stallion spermatozoa selected by colloid centrifugation increases sperm progressive motility but is detrimental to chromatin integrity

There is controversy about whether the presence of some seminal plasma (SP) in an equine insemination dose is necessary for promoting fertility. A new technique for improving stallion sperm quality, single layer centrifugation (SLC) using a species-specific colloid, Androcoll-E, selects a sperm subpopulation that is highly motile with normal morphology, intact membranes and good chromatin integrity from the rest of the ejaculate and removes SP. The present study was designed to investigate the effect of restoring homologous SP (5% and 10%) on the progressive motility, velocity, and chromatin integrity of SLC-selected stallion spermatozoa in 44 semen samples over time. Sperm progressive motility (P < 0.01) and the proportion with class A velocity (>50 μm/sec) were increased in samples where SP was restored, whereas the proportion with class B velocity (10 to 50 μm/sec) was decreased compared with SLC samples. However, after 24 h cold storage of treated samples, progressive motility was not different for the SP-treated groups compared with SLC, whereas chromatin damage DNA fragmentation index (%DFI) was higher. In contrast, adding SP to untreated 24 h-stored SLC samples did not affect progressive motility although it did increase the proportion of spermatozoa with class A velocity. There was individual variation between stallions whether 5% or 10% SP produced a greater increase in progressive motility. In conclusion, 5% to 10% SP can be added back to SLC-selected samples if considered necessary to optimize fertility. However, it should be added immediately before insemination rather than before storage of the sperm dose, to benefit from the transient increase in sperm progressive motility and avoid increased chromatin damage.     

Analysis of lipid and protein components of ejaculated bull sperm surface and seminal plasma

Surface components of ejaculated bull sperm were radiolabeled by enzymatic iodination with lactoperoxidase and Na¹²⁵I. The sperm were lysed and the labeled components analyzed on SDS-7.5% polyacrylamide gels. Electrophoresis of solubilized radioactivity resolved six components with approximate molecular weights of 77, 61, 44, 36, 24, and 15 kilodaltons. To identify components that might be adsorbed to the bull sperm surface from seminal secretions, seminal plasma was labeled. Electrophoresis of labeled seminal plasma resolved four components with approximate molecular weights of 74, 33, 24, and 15 kilodaltons, each of which comigrated with a labeled sperm surface component. To identify the chemical composition of the radiolabeled components, labeled sperm surface and labeled seminal plasma were submitted to isopycnic density gradient centrifugation in cesium chloride. The ¹²⁵I incorporated into bull sperm surface separated into two discrete areas of radioactivity, one having a density characteristic of protein and the other, of lipid. Iodinated seminal plasma banded in one discrete area that had a density characteristic of protein. Electrophoretic analysis of each area of radioactivity recovered from the gradients demonstrated that five of the six sperm surface and all of the seminal plasma components were in the protein fractions. The 15-kilodalton sperm surface component banded as a lipid, whereas the 15-kilodalton seminal plasma componènt banded as a protein.     

Cell biology and functional dynamics of the mammalian sperm surface

Theriogenology has now a 40-year rich history on covering sperm biological aspects with a special emphasis on farm and husbandry animals. The major and most influential of these contributions will be placed into an evolutionary perspective of ongoing and intriguing progresses made in this field. Although many molecular details have been published, it is more the aim of this contribution to provide a guide through the main established aspects and concepts of sperm surface biology and refer only to major molecular players and mechanisms involved in sperm physiology. Those interested in more molecular details and in-depth knowledge can easily access the most relevant literature which is included here for reference purposes. With this approach, a logical and easy to follow buildup can be made of the general picture of sperm surface dynamics and of the ergonomics of sperm physiology and their function in mammalian fertilization. Understanding the ins and outs of sperm surface biology and the dynamics thereof, might challenge future researchers to design novel generation of better sperm-handling procedures. This could be beneficial for assisted reproductive technology and animal breeding industries.     

Purification and partial characterization of the 17 kDa sperm coating protein from boar seminal plasma.

Sperm coating proteins of 16, 17, and 19 kDa have been purified from boar seminal plasma. The 17 kDa protein has been identified as an antigen recognized by monoclonal antibody ACR.3 and is thus identical to low molecular mass zona pellucida binding protein from boar spermatozoa (Moos et al., 1990). The 17 and 19 kDa proteins are glycosylated and tend to form hetero-complexes. The 17 kDa ACR.3 antigen is sequentially released from the sperm cell surface during capacitation and, after induction of the acrosome reaction, the 16 kDa form was also observed. Immunocytochemical studies on boar reproductive tissues have suggested that the seminal vesicle epithelium may be the source of these proteins.     

Effect of daily semen centrifugation and resuspension on the longevity of equine sperm quality following cooled storage

An experiment was conducted to determine whether cooled semen quality could be maintained for a longer interval by conducting daily centrifugation of extended semen, with resuspension of the sperm pellet in fresh extender. Semen treatments included SP10NC and SP50NC which contained 10 and 50% seminal plasma, respectively, were not centrifuged (NC), and were stored at 4 to 7 °C for 96 h. Treatments SP10C and SP50C contained 10 and 50% seminal plasma, respectively, but were centrifuged (C) after 24, 48, and 72 h of cooled storage, with daily resuspension in fresh extender containing 10% seminal plasma. Percent total sperm motility (TMOT) and progressively motile (PMOT) was reduced (P < 0.05) in the SP50NC treatment after 24, 48, 72, and 96 h of storage, and TMOT did not differ (P > 0.05) in the SP10C, SP50C, SP10NC groups after the same storage periods. The % COMP-_αt did not differ (P > 0.05) among treatments at any time period. Percent membrane intact sperm (SMI) was reduced in SP50NC, as compared to SP10C at 48, 72, and 96 h (P < 0.05). Daily centrifugation and resuspension of sperm exposed to 50% seminal plasma for the first 24 h (SP50C) yielded similar TMOT, PMOT, VCL, SMI, % COMP-_αt (P > 0.05) to Groups SP10NC and SP10C after 96 h of storage. Daily centrifugation and resuspension of cool-stored equine semen in fresh extender may be a method to increase sperm longevity.     

Effect of cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activity in fowl semen

The aim of the present study was to determine the influence of chicken semen cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activities. Pooled semen from 10 Black Minorca roosters was used in the study. Semen samples were subjected to cryopreservation using the “pellet” method and dimethylacetamide (DMA) as a cryoprotectant. In the fresh and the frozen-thawed semen sperm membrane integrity (SYBR-14/propidium iodide (PI)), acrosomal damage (PNA-Alexa Fluor^®488) and mitochondrial activity (Rhodamine 123) were assessed using flow cytometry. Malondialdehyde (MDA) concentration, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in sperm cells and seminal plasma by spectrophotometry. All sperm characteristics evaluated using flow cytometry were affected by cryopreservation. After freezing-thawing, there was significant (P < 0.01) reduction in sperm membrane integrity, sperm acrosome integrity and mitochondrial activity. Following cryopreservation, MDA concentration significantly increased in chicken seminal plasma and spermatozoa (P < 0.01, P < 0.05). The CAT activity in seminal plasma significantly decreased (P < 0.05), while intracellular activity of this enzyme did not significantly change in frozen-thawed semen. In seminal plasma of frozen-thawed semen the significant increase (P < 0.01) in GPx activity was detected. Whereas GPx activity in spermatozoa remained statistically unchanged after thawing. The SOD activity significantly increased (P < 0.01) in cryopreserved seminal plasma with simultaneous decrease (P < 0.01) of its activity in cells. In conclusion, this is probably the first report describing the level of antioxidant enzymes in frozen-thawed avian semen. The present study showed that the activity of CAT, GPx and SOD in chicken semen was affected by cryopreservation, what increased the intensity of lipid peroxidation (LPO). Catalase appeared to play an important role in the sperm antioxidant defense strategy at cryopreservation since, opposite to SOD and GPx, its content was clearly reduced by the cryopreservation process. Change in the antioxidant defense status of the chicken spermatozoa and surrounding seminal plasma might affect the semen quality and sperm fertilizing ability.