Use of antioxidants reduce lipid peroxidation and improve quality of crossbred ram sperm during its cryopreservation

Ram sperm are subjected to extreme oxidative stress during their preservation at −196 °C resulting in reduced quality at post thaw. Therefore, the main objective of this study was to evaluate the effect of antioxidants taurine, quercetin and reduced glutathione on the post thaw quality of crossbred ram sperm. A total of twenty four ejaculates from six crossbred rams were collected and extended with tris-based extender with no antioxidant (Control), with taurine (40 mM), quercetin (5 μg/ml) and reduced glutathione (5 mM). The post thaw sperm quality was determined by percent sperm motility, live sperm count, intact acrosome and hypo-osmotic swelling test (HOST) reacted spermatozoa and lipid peroxidation was measured in terms of malondialdehyde (MDA) level both in seminal plasma and sperm cell. At post thaw, percent sperm motility and live sperm count were significantly (p < 0.05) higher for taurine than control and reduced glutathione but did not differ significantly (p > 0.05) from quercetin. The percent HOST reacted spermatozoa were significantly higher for taurine than control, quercetin and reduced glutathione. Seminal plasma MDA level was significantly (p < 0.05) lower for taurine than control and non-significantly lower than quercetin and reduced glutathione. However, spermatic MDA level did not differ significantly (p > 0.05) among the control and antioxidants. In conclusion, taurine at 40 mM reduced lipid peroxidation and improved post thaw sperm quality of cryopreserved crossbred ram semen. Further, transportation time of semen samples in an ice chest at 4–5 °C may be included as a part of equilibration period, when collection shed and frozen semen unit are located at a distance.     

Activity, substrate detection and immunolocalization of glutathione peroxidase (GPx) in bovine reproductive organs and semen

The purpose of the current study was to further investigate the role of the antioxidant selenium-dependent enzyme glutathione peroxidase (GPx) in reproductive organs and semen from bulls. To this end a fast and convenient combined method for immune detection and substrate localization was adapted, which allows the assessment of both molecular weight and peroxidase activity of proteins on one and the same SDS-PAGE gel plate. After routine semen analysis of ejaculates, a spectrophotometrical assay of GPx activity in bovine semen was performed. For the immunological analyses performed, a rabbit polyclonal monospecific antibody against GPx was raised. Substrate detection and immunolocalization of GPx in lysates from bovine testis, epididymis, spermatozoa, and seminal plasma was performed. In order to determine the localization of GPx in spermatozoa, immunofluorescence analysis was performed. A positive correlation was established between GPx activity in semen and the number of motile spermatozoa. A negative correlation was observed between GPx activity and the number of immotile spermatozoa. The combined method for immunodetection and substrate localization was tested and proved reliable. Both tetramer and monomer forms of GPx were detected in lysates from testis, epididymis, and spermatozoa. We found no GPx activity in seminal plasma. Immunofluorescence shows the presence of GPx chiefly in the mitochondrial and in the acrosome regions of spermatozoa. GPx activity remained stable under the extreme experimental conditions.     

Effect of season on some characteristics of stallion semen.

Season had a pronounced effect upon seminal pH and refractometer 'protein', total carbohydrate, dry weight, total N2 and lactic acid in seminal plasma of first and second ejaculates. In addition, total seminal volume, spermatozoa per ml and per ejaculate, non-protein sulphhydryl and glycerylphosphorylcholine of second ejaculates were also influenced. There was a season difference in the concentrations of lactic acid in spermatozoa from first and in total N2 from spermatozoa in second ejaculates. The effects of season on seminal plasma were greater than those on spermatozoa. Spermatozoa in first ejaculates were less affected by season than those in secons ejaculates. This differential effect on first and second ejaculates was generally true of all seminal characteristics.     

Effects of dead spermatozoa on motion characteristics and membrane integrity of live spermatozoa in fresh and cooled-stored equine semen

The aim of this study was to determine if dead spermatozoa reduced motility or membrane integrity of live spermatozoa in fresh and cooled-stored equine semen. Three ejaculates from each of three stallions were centrifuged and virtually all seminal plasma was removed. Spermatozoa were resuspended to 25 x 10(6) spermatozoa/ml with EZ-Mixin CST extender and 10% autologous seminal plasma, then divided into aliquots to which 0 (control), 10, 25, 50, or 75% (v/v) dead spermatozoa were added. Dead spermatozoa preparations contained 25 x 10(6) spermatozoa/ml and 10% seminal plasma from pooled ejaculates of the three stallions, in EZ-Mixin CST extender. Spermatozoa were killed in the pooled ejaculates by repeated freezing and thawing, then stored at -20 degrees C until warmed to 37 degrees C and mixed with aliquots of fresh spermatozoa to be cooled and stored in an Equitainer for 24h. Motion characteristics (% total motility (MOT), % progressive motility (PMOT), and mean curvilinear velocity (VCL)) for fresh and 24h cooled samples were determined using a computerized spermatozoal motion analyzer. The presence of up to 75% dead spermatozoa did not adversely affect MOT or PMOT of live spermatozoa in either fresh or cooled-stored semen. However, VCL and the percentage of membrane-intact spermatozoa were reduced compared to control samples when 75% (v/v) dead spermatozoa were added. Membrane integrity, as assessed by staining with carboxyfluoresein diacetate-propidium iodide, was highly correlated (r>0.8; P<0.001) with MOT and PMOT in both fresh and cooled-stored semen samples. Results of this study have application to the processing of both cooled and frozen equine semen.     

The effect of exogenous glutathione on the fructolysis of thawed bull Bos bovis spermatozoa

1. The effect of glutathione (5mM) addition to the diluent used for sperm preservation on fructolysis and motility of bull spermatozoa was studied. 2. Glutathione had no effect on lactate and pyruvate concentration and on the motility of spermatozoa immediately after their thawing. 3. During 3 hr incubation at 37 degrees C glutathione decreased the pyruvate formation, significantly increased the lactate production and prevented the decrease in the number of spermatozoa with maintained progressive movement.     

The effect of sperm concentration in the ejaculate on morphological traits of bull spermatozoa

Experiments were performed on 75 ejaculates obtained from 19 bulls representing different cattle breeds used at the Masovian Centre for Animal Breeding and Reproduction in ?owicz. Fresh ejaculates were measured in respect to their volume and sperm count in the ejaculates was determined. The ejaculates were classified based on the criterion of sperm concentration and divided into five groups. Sperm morphometric measurements were taken from each bull and assessment of semen morphology was done on the basis of examination under a microscope using preparations made from fresh ejaculates. For each slide, morphometric measurements were taken of 15 randomly selected spermatozoa characterised by normal morphology and well visible under the microscope. Additionally, in each preparation morphometry of 500 spermatozoa was evaluated, numbers of spermatozoa with normal morphology and morphological abnormalities were recorded and these were categorized into spermatozoa with major and minor defects. An insignificant correlation was observed between the sperm concentration in the ejaculate and morphological traits, dimensions and shapes of bull spermatozoa. The less concentrated ejaculates contained spermatozoa with a slightly larger head circumference and a more elongated head shape in comparison with the spermatozoa in the more concentrated ejaculates. The highest frequency of morphologically malformed spermatozoa, both in the case of primary and secondary alterations, was observed in ejaculates with sperm concentration of no more than 1000 x 10(3)/mm3.     

Seminal PDC-109 protein vis-à-vis cholesterol content and freezability of buffalo Spermatozoa

Advancements in reproductive technologies have shown seminal plasma (SP) as a nutritive-protective medium for spermatozoa metabolism, function and transport. At the same time quality variables and thus freezability of spermatozoa are influenced by SP proteins originating from male reproductive tract. One such protein, viz. PDC-109 is reported to influence freezability of spermatozoa in cattle. Thus the present investigation was designed to evaluate effect of seminal PDC-109 protein concentration on post-thaw cholesterol content and semen quality variables (SQP) as an indicator of membrane integrity and freezability, respectively of buffalo spermatozoa. Ejaculates (n = 42) selected on the basis of mass activity and individual motility were divided into three parts, first part for SP proteins isolation, second for cholesterol estimation and third part was cryo-preserved to evaluate freezability based on post-thaw SQP, viz. individual progressive motility, viability and acrosome integrity of spermatozoa. A total of 28 (66.7%) and 14 (33.3%) ejaculates from four bulls were found as freezable or non-freezable, respectively. Though total seminal plasma protein (TSPP) concentration was found similar in freezable and non-freezable ejaculates, the heparin binding proteins (HBP) content in non-freezable semen was greater (P < 0.01) than freezable ejaculates. There was a similar trend for the PDC-109 protein content in respective ejaculates. Cholesterol content of spermatozoa and SQP were greater (P < 0.05 and 0.01, respectively) in freezable as compared to non-freezable ejaculates of each bull at post-thaw stage. This study showed that concentrations of HBP and PDC-109 in non-freezable semen might be responsible for greater cryo-damage reflecting in poor freezability of buffalo spermatozoa.     

Disappearance of spermatozoa from the ejaculates of geldings.

Twenty-three geldings were used to determine changes in seminal characteristics following castration and the effect of frequency of ejaculation on these seminal characteristics. In Exp. 1, semen was collected from 8 geldings every other day after castration until the number of spermatozoa per ejaculate was below 1% of the precastration value. An average of 3 ejaculates was required to reduce the number of spermatozoa below this level. In Exp. 2, 15 stallions were castrated and each stallion was assigned to 1 of 3 groups for seminal collection at 7, 14 or 21 days post-castration. The ejaculates collected on these days contained an average of 23, 14 and 2 X 10(6) spermatozoa/ejaculate, respectively. In both experiments, all spermatozoa in ejaculates collected 7 or 8 days after castration were non-motile. Frequency of ejaculation did not appear to hasten the disappearance of spermatozoa from the ejaculates. It is considered that after castration several months may be required before the ampulla and vas deferens become devoid of spermatozoa and the ejaculates azoospermic, and that pregnancy is unlikely to result from mating or insemination 1 week after castration.     

Sperm morphology of cattle and domestic pigs

Sperm morphology was evaluated (using Blom classification) in 44 ejaculates of 11 bulls and 44 ejaculates of 11 boars. Significant differences in sperm morphology were found between bulls and boars. In addition, the correlations between frequency of morphological changes and morphometrical traits of boar spermatozoa were demonstrated. More morphological anomalies of spermatozoa were recorded in ejaculates containing longer spermatozoa.     

Total and specific activities of superoxide dismutase (SOD) in seminal plasma are related with the cryotolerance of jackass spermatozoa

This study investigated whether the activities of four antioxidant enzymes present in jackass seminal plasma (SP), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) and glutathione reductase (GSR), are related to the sperm ability to withstand cryopreservation. Eighteen ejaculates from 16 healthy jackasses were collected and split into two aliquots. The first one was centrifuged (3,000×g, 4 °C for 10 min) and used to determine the activities of these four enzymes in SP, whereas the other was diluted in a skim-milk extender and then cryopreserved. Assessment of sperm motility and membrane integrity was performed before and after cryopreservation. Based on the percentages of total motile and viable spermatozoa at post-thaw, samples were classified as good (GFE) or poor (PFE) freezability ejaculates through cluster analyses. Total and specific activities of SOD in seminal plasma were higher (P < 0.05) in GFE than in PFE, whereas no significant differences between GFE and PFE were observed regarding total and specific activities of CAT, GPX and GSR. However, post-thaw sperm parameters were positively correlated with total and specific activities of CAT and negatively correlated with those of GSR. In conclusion, determination of total and specific activities of SOD in the seminal plasma of a given jackass ejaculate may predict the sperm ability to withstand cryopreservation. In addition, our results warrant further research on addressing whether SOD activity in seminal plasma does not only allow predicting the sperm cryotolerance of a given ejaculate but also that of all ejaculates from a given jackass.     

Sequestration of PDC-109 protein improves freezability of crossbred bull spermatozoa

A study was carried out to assess the effect of sequestration of PDC-109 protein, a majority constituent of heparin binding proteins (HBP) of seminal plasma, on freezability and in vitro fertilizing ability of crossbred bull spermatozoa after cryopreservation. The study consisted of isolation and characterization of PDC-109 protein to raise anti-sera against it in rabbits. Following which, raised antibodies against PDC-109 protein was quantitated and coated in tubes used for collection of ejaculates. Semen ejaculates thus collected were cryopreserved using EYTG extender. Physico-morphological characteristics, viz. motility, viability, acrosomal integrity and HOS response as an indicator of freezability of cryopreserved spermatozoa were determined at pre freeze as well as post thaw stage. At pre freeze stage, a significant (p<0.05) improvement in viability (83.83 ± 2.18 vs 75.17 ± 2.42) and acrosome integrity (81.33 ± 2.38 vs 72.83 ± 2.39) in antibodies treated group than control was observed. Similarly, increase in HOS responsive spermatozoa was highly significant (p<0.01) than control (78.83 ± 1.69 vs 67.5 ± 1.75). At post thaw stage, significant (p<0.05) improvement in viability (69.50 ± 2.16 vs 60.33 ± 2.19) and HOS responsive spermatozoa (68.67 ± 1.62 vs 58.50 ± 1.32) and highly significant (p<0.01) increase in individual motility (56.17 ± 1.83 vs 47.00 ± 1.86) and acrosome integrity (75.17 ± 2.38 vs 61.83 ± 2.1) was observed in antibodies treated group when compared to control was observed. The results from the study revealed that sequestration of PDC-109 protein from semen samples leads to significant improvement in pre-freeze and post-thaw values of above parameters in cryopreserved spermatozoa. It is thus concluded that sequestration of PDC-109 protein from ejaculates improves freezability of crossbred bull spermatozoa.     

Effect of L-carnitine administration on the seminal characteristics of oligoasthenospermic stallions

The effect of orally administered l-carnitine on the quality of semen obtained from stallions with different semen qualities was investigated. Four stallions with proven fertility (high motility group, HM) and with normal seminal characteristics (>50% progressive motility and > 80 x 10(6) spermatozoa/ml), and four questionable breeders (low motility group, LM) with <50% of sperm progressive motility and < 80 x 10(6) spermatozoa/ml, received p.o. 20 g of l-carnitine for 60 days. Blood and semen samples were collected before treatment (T0) and after 30 (T1) and 60 days (T2). Semen evaluation were performed on five consecutive daily ejaculates (n = 120 ejaculates) and conventional semen analysis was carried out on each ejaculate, both at collection and after refrigeration for 24, 48, and 72 h. Furthermore l-carnitine, acetylcarnitine, pyruvate, and lactate concentrations, and carnitine acetyltransferase activity (CAT) were determined both in raw semen and seminal plasma. There were an increase in progressive motile spermatozoa only in the LM group (26.8 +/- 12.9, 39.1 +/- 15.5, and 48.8 +/- 8.6 for T0, T1, and T2, respectively). Free seminal plasma carnitine concentration was higher in the LM group compared to the HM one. Both pyruvate and lactate were higher in the LM group. Raw semen and seminal plasma carnitine and acetylcarnitine levels correlate positively with both sperm concentration and progressive motility; moreover, acetylcarnitine content was positively correlated with total motile morphologically normal spermatozoa. In conclusion, oral administration of l-carnitine to stallions with questionable seminal characteristics may improve spermatozoa kinetics and morphological characteristics; whereas, it seem to be ineffective in normospermic animals.     

Lipid peroxides, spermatozoa quality and activity of glutathione peroxidase in bull semen

In bull semen spontaneous lipid peroxidation measured by the level of endogenous lipid peroxides and the consequences of this process for morphological and biochemical changes was studied. Glutathione peroxidase activity as protective enzyme against peroxidative damage was also determined. Obtained results showed that approximately two thirds of GSH-Px activity in bull semen was non Se-dependent glutathione peroxidase activity. Malonaldehyde (MDA) level was negative correlated with selenium-dependent GSH-Px activity (r = = -0.38, P less than 0.01). Spermatozoa with acrosome entirely lost appeared to increase as the MDA level increased (r = 0.18, P less than 0.05). The negative correlation between Se GSH-Px activity and spermatozoa with acrosome separation from head (r = -0.28, P less than 0.01) and entirely lost (r = -0.21, P less than 0.05) suggest that selenium-dependent GSH-Px plays role in protecting the acrosome against disruption of the acrosomal membrane. The total glutathione peroxidase activity was unrelated to studied variables of bull semen.     

The effect of season on the properties of wild boar (Sus scrofa L.) semen

The objective of the study was to determine the properties of wild boar semen and their changes in annual cycle. During a 14-month study period, 167 ejaculates were sampled from 3 mature boars. In each ejaculate the volume of liquid fraction, percentage of spermatozoa motility, spermatozoa concentration and the total number of spermatozoa were determined. The activity of acid and alkaline phosphatase, and aspartate aminotransferase in the fresh semen plasma was also measured. It was shown that wild boar ejaculates did not differ from those of domestic boars, and the semen of the highest volume, concentration and number of spermatozoa was produced in late autumn. The spermatozoa motility was the lowest in summer. The activity of aspartate aminotransferase and alkaline phosphatase in the semen plasma increased with shortening of the light period.     

Age-related differences of semen quality,seminal plasma,and spermatozoa antioxidative and oxidative stress variables in bulls during cold and warm periods of the year

The aims of this study were to determine the presence and quantities of antioxidative status and oxidative stress (OS) variables in the seminal plasma and spermatozoa of bulls of varying age during cold and warm periods of the year, and to establish the correlation of these variables with semen quality parameters. The study was conducted on two groups each comprising nine Simmental bulls: one group contained younger animals (aged 2 to 4 years) and the second older animals (aged 5 to 10 years). Semen samples were collected using an artificial vagina for biochemical analysis. Seminal plasma and spermatozoa activities of total superoxide dismutase (TSOD), manganese superoxide dismutase (MnSOD), copper–zinc superoxide dismutase (CuZnSOD), catalase (CAT), selenium-dependent glutathione peroxidase, reduced glutathione and concentrations of total protein (TP), thiobarbituric acid reactive substances (TBARS) and protein carbonyl content (PCC) were determined. Several antioxidants in seminal plasma were also determined: total glutathione peroxidase (TGSH-Px), selenium-independent glutathione peroxidase (Non-SeGSH-Px), uric acid, albumins (ALB) and alkaline phosphatase (ALP). Significantly higher spermatozoa motility was observed during the cold v. warm period, and a significantly higher volume and total number of spermatozoa per ejaculate was observed in older than in younger bulls. Significantly higher values of ALP, TP and ALB were found in seminal plasma of older bulls than in younger bulls during the warm period. The seminal plasma of younger bulls showed significantly higher activities of TSOD, MnSOD, CuZnSOD, TGSH-Px and Non-SeGSH-Px. Younger bulls had significantly higher PCC concentration and activity of CAT in seminal plasma than older bulls during the cold period. Significantly higher concentrations of PCC and TBARS, and activities of TSOD, MnSOD and CuZnSOD were established in spermatozoa of the younger than in older bulls during the warm period. It could be concluded that antioxidative and OS variables differ significantly depending on bull age and time of year. Younger bulls were more sensitive to elevated ambient temperatures during the warm period, when the higher enzymatic antioxidative protection in seminal plasma and spermatozoa were insufficient to counteract the intensive oxidative processes in spermatozoa, which eventually resulted in decreased spermatozoa motility. The estimation of antioxidative and OS variables in seminal plasma and spermatozoa may have practical value for the assessment of bull semen quality.     

Preservation of ejaculated and epididymal stallion spermatozoa by cooling and freezing

The suitability of ejaculated and epididymal stallion spermatozoa for cooled storage (5 degrees C) and cryopreservation was examined in 5 ejaculates from each of 6 stallions and in spermatozoa recovered from the cauda epididymidis after castration of these stallions. The percentage of progressively motile spermatozoa, examined by subjective estimation (cooled samples) or by computerized analysis (frozen-thawed samples), was used as parameter. In ejaculated semen samples containing 5 and 25% seminal plasma in a skim milk glucose extender, the lower amount of seminal plasma supported spermatozoal motility significantly better throughout storage at 5 degrees C. Addition of 5 or 25% seminal plasma to perfused epididymal spermatozoa (0% seminal plasma) resulted in a significant stimulation of spermatozoal motility by 25% seminal plasma at 0 h (P<0.05) and to a lesser extent at 24 and 48 h. Post-thaw motility of ejaculated as well as epididymal spermatozoa was not influenced by slow cooling to 15 degrees or 5 degrees C with or without glycerol prior to rapid freezing in liquid nitrogen vapor. During cooled storage, seminal plasma had a stimulatory effect on epididymal spermatozoa and depressed motility in ejaculated spermatozoa. Results on cryopreservation indicate that freezability of equine spermatozoa is already determined when spermatozoa leave the tail of the epididymis.     

Seasonal variations in antioxidant enzyme activity in ram seminal plasma

Seminal oxidative stress status is emerging as a significant prognostic tool in assisted reproductive technology. A dynamic interplay between pro- and anti-antioxidant substances in the ejaculate is essential. In this study, we determined seasonal changes in the activity of the antioxidant enzyme defense system comprising superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx) and catalase (CAT) in seminal plasma (SP) of mature Rasa Aragonesa rams. This breed corresponds to a local Spanish genotype with a short seasonal anoestrus between May and July. In addition, the activity of these enzymes was measured in protein fractions isolated from ram SP by exclusion chromatography. Total protein content in ram SP was higher during the breeding season (October-February) with a significantly higher value in first ejaculates. Antioxidant enzyme activities were higher during the non-breeding season (March-September). Comparing first and second ejaculates, SOD and CAT activities were higher in the first of all months. However, GR and GPx activities changed throughout the year. Thus, GR activity was higher in July and August in first ejaculates, this difference being significant in July (4.53 versus 2.37 nmol substrate/minmg protein, P<0.05). Conversely, GPx activity was significantly higher in September and November in second ejaculates (21.1 versus 6.81 and 10.91 versus 5.33, respectively, P<0.05). After SP fractionation by exclusion chromatography, GR activity was located in fractions 1 and 2 being irrelevant in the following peaks, and CAT activity was not detected all along the chromatographic profile. GPx and SOD activities were spread out along all fractions with a main peak in fractions 6 and 7. Given that these two fractions showed the greatest capacity to recover and prevent cold-shock membrane injury [Barrios B, Pérez-Pé R, Gallego M, Tato A, Osada J, Muino-Blanco T, Cebrián-Pérez JA. Seminal plasma proteins revert the cold-shock damage on ram sperm membrane. Biol Reprod 2000;63:1531-7, Barrios B, Fernández-Juan M, Muino-Blanco T, Cebrián-Pérez J. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins that protect ram spermatozoa against cold shock. J Androl 2005;26:539-49], we could suggest that the protective effect might be, at least partially, due to the antioxidant enzyme activity.     

Morphology of spermatozoa in the cauda epididymidis before and after electroejaculation and a comparison with ejaculated spermatozoa in the domestic cat

When 2 ejaculates are collected by electroejaculation from the domestic cat within a period of 10 min the first ejaculate has a higher proportion of abnormal spermatozoa than the second. The reason for this difference is not known for the domestic cat, but in other species long-term epididymal storage results in a higher proportion of abnormal spermatozoa. The aims of this study were to determine the proportions of abnormal spermatozoa in the cauda epididymidis and to ascertain if electroejaculation affects this proportion. Therefore the proportions of spermatozoa in the cauda epididymidis with different morphological abnormalities were compared before and after ejaculation. In addition, the proportion of morphologically abnormal spermatozoa in the epididymis was compared with that in the ejaculate. Nine privately-owned domestic cats were anesthetized, and one testicle was surgically removed. An ejaculate was collected by electroejaculation, after which the remaining testicle was ectomized. There were no significant differences in the proportions of different sperm abnormalities between the cauda epididymidis removed before ejaculation and the one removed after ejaculation. A significantly (P = 0.009) higher proportion of spermatozoa with tail abnormalities was found in the ejaculates compared with the cauda epididymides (11.1 and 1.6%, respectively), while, as expected, there was a lower proportion of spermatozoa with distal droplets in the ejaculates than in the cauda epididymides (35.1 and 75.9%, respectively). This new information contributes to the understanding of the etiology of sperm defects in the domestic cat, and is of importance when evaluating a semen sample in this species.     

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).     

Determination of acrosin amidase activity in equine spermatozoa

Acrosin amidase activity of spermatozoa has been been associated with in vitro fertilization success in humans and has been proposed as an additional method for assessing sperm function in vitro. In this study, acrosin amidase activity was determined in equine spermatozoa by the hydrolysis of an arginine amide substrate. This assay includes a detergent to release acrosomal enzymes into a medium of basic pH to activate proacrosin to acrosin, which subsequently hydrolyses N-alpha-benzoyl-DL-arginine para-nitroanilide-HCl (BAPNA) to a chromogenic product. Spermatozoa (n = 3 ejaculates from each of 4 stallions) were washed free from seminal plasma by centrifugation through Ficoll and incubated with a detergent-substrate mixture (BAPNA in triton X-100; pH = 8.0) at room temperature for 3 h in the dark. At the end of the 3-h incubation, benzamidine was added to test samples to stop the reaction, and samples were centrifuged to remove spermatozoa. Absorbance at 410 nm was measured to determine acrosin amidase activity (microIU acrosin/10(6) sperm). Acrosin amidase activity increased with sperm concentration (P < 0.001; r(2) = 0.75), and there were significant effects (P < 0.001) of stallion and ejaculate within stallion on acrosin activity. Acrosin activity detectable in equine seminal plasma was 312 +/- 49 microU/ml (n = 3 ejaculates). Addition of a cryopreservation medium containing egg yolk, skim-milk, glycerol and sucrose to equine spermatozoa and subsequent cryopreservation significantly (P < 0.05) increased acrosin amidase activity compared with spermatozoa from raw semen. This result is in contrast to that previously reported for frozen-thawed human spermatozoa. Determination of acrosin amidase activity in equine spermatozoa may provide an alternative method for assessing sperm function in vitro; however, further studies are needed to determine the relationship between acrosin activity and fertility in the horse.