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- Author or Editor: Barry A. Ball x
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Objective—To determine glutathione peroxidase (GPX) and superoxide dismutase (SOD)-like activities in spermatozoa, seminal plasma, and reproductive tissues (ie, testis, epididymis, bulbourethral gland, prostate, vesicular gland, and ampulla) in horses.
Sample Population—Seminal plasma from 17 stallions, spermatozoa from 5 stallions, and reproductive tissues from 3 stallions.
Procedure—Activity of GPX was determined by use of assays measuring oxidation of NADPH in the presence of exogenous glutathione, cumene hydroperoxide, and glutathione reductase. Activity of SOD-like enzymes was determined by use of the nitroblue tetrazolium assay.
Results—Mean GPX and SOD-like activities in seminal plasma were 1.3 ± 0.1 nmol of NADPH oxidized/ min/mg of protein and 29.2 ± 6.6 U/mg of protein, respectively. Mean GPX activities in spermatozoa separated from seminal plasma by centrifugation and via Percoll gradient were 2.2 ± 0.3 nmol and 6.1 ± 1.3 nmol of NADPH oxidized/min/mg of protein, respectively. Mean SOD-like activity of spermatozoa separated by centrifugation was 58.6 ± 22.3 U/mg of protein; SOD-like activity was not detected in Percollseparated spermatozoa. Among reproductive tissues, the ampulla and prostate had the highest SOD-like activity, although this was not significantly different from activity in other tissues. Testes and spermatozoa from the cauda epididymis contained significantly more GPX activity than other tissues.
Conclusions and Clinical Relevance—Results suggest that although equine seminal plasma contains high SOD-like enzyme activity, spermatozoa have limited GPX and SOD-like activity. Enzymatic antioxidant activity in equine spermatozoa appears to be predominantly derived from seminal plasma adsorbed onto the plasma membrane. Removal of seminal plasma during semen processing may increase oxidative stress in equine spermatozoa. (Am J Vet Res 2005;66:1415–1419)
Objective—To identify the generation of the superoxide anion by equine spermatozoa.
Sample Population—Multiple ejaculates collected from 3 Thoroughbred stallions.
Procedures—Induced superoxide production by reduced nicotinamide adenine dinucleotides (NAD[P]H; ie, reduced nicotinamide adenine dinucleotide [NADH] and reduced nicotinamide adenine dinucleotide phosphate [NADPH]) was measured by use of a nitroblue tetrazolium (NBT) reduction assay on whole spermatozoa and a cytochrome c reduction assay on isolated membrane fractions of spermatozoa. Localization of superoxide generation was determined by use of NBT cytochemistry.
Results—A dose-dependent increase in NBT reduction was found in the presence of NADPH, which was inhibited by superoxide dismutase (SOD). The flavoprotein inhibitor, diphenyleneiodonium (DPI; 5 or 15μM), significantly decreased NBT reduction. Cytochrome c reduction by plasma membranes of spermatozoa was significantly higher in the presence of NADPH than in its absence. Cytochemical staining of equine spermatozoa in the presence of NADPH and NADH revealed diaphorase labeling in the spermatozoon midpiece and head. This staining was inhibited by DPI and SOD.
Conclusions and Clinical Relevance—Results of our study indicate that superoxide generation is associated with a membrane-associated NAD(P)H oxidase present in equine spermatozoa, although mitochondrial generation of superoxide is also detected. This oxidase may play a role in cell signaling or may also contribute to cytopathic effects associated with oxidative stress in equine spermatozoa.
Objective—To evaluate the effect of the addition of enzyme scavengers and antioxidants to the cryopreservation extender on characteristics of equine spermatozoa after freezing and thawing.
Sample Population—2 ejaculates collected from each of 5 stallions.
Procedure—Equine spermatozoa were cryopreserved in freezing extender alone (control samples) or with the addition of catalase (200 U/mL), superoxide dismutase (200 U/mL), reduced glutathione (10mM), ascorbic acid (10mM), α-tocopherol (25, 50, 100, or 500µM or 1mM), or the vehicle for α-tocopherol (0.5% ethanol). After thawing, spermatozoal motility was assessed via computer-assisted analysis and DNA fragmentation was assessed via the comet assay. Spermatozoal mitochondrial membrane potential, acrosomal integrity, and viability were determined by use of various specific staining techniques and flow cytometry.
Results—The addition of enzyme scavengers or antioxidants to cryopreservation extender did not improve spermatozoal motility, DNA fragmentation, acrosomal integrity, viability, or mitochondrial membrane potential after thawing. Superoxide dismutase increased DNA fragmentation, likely because of the additional oxidative stress caused by the generation of hydrogen peroxide by this enzyme. Interestingly, the addition of the vehicle for α-tocopherol resulted in a significant decrease in live acrosome-intact spermatozoa.
Conclusions and Clinical Relevance—The addition of antioxidants to the cryopreservation extender did not improve the quality of equine spermatozoa after thawing, which suggests that the role of oxidative stress in cryopreservation-induced damage of equine spermatozoa requires further investigation. Our data suggest that solubilizing α-tocopherol in ethanol may affect spermatozoal viability; consequently, water-soluble analogues of α-tocopherol may be preferred for future investigations. (Am J Vet Res 2005;66:772–779)
Objective—To evaluate Coomassie blue staining of the acrosome of equine and canine spermatozoa.
Sample Population—Spermatozoa of 5 mixed-breed male dogs and 3 Thoroughbred stallions.
Procedure—Various proportions of intact and acrosome-damaged spermatozoa were fixed in 2% phosphate-buffered formaldehyde or 4% paraformaldehyde, smeared onto glass slides, and stained with Coomassie blue stain. Acrosomal status (damaged vs intact) was also assessed by use of flow cytometry after staining with fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and propidium iodide. Comparisons were made between percentages of expected and observed acrosome-intact spermatozoa in different proportions of live and flash-frozen samples; the percentages of acrosome-intact spermatozoa as determined by use of Coomassie blue staining and flow cytometry were also compared.
Results—Strong correlations were found between the expected and observed distributions of acrosome-intact spermatozoa when fixed in 4% paraformaldehyde (r = 0.93 and 0.89 for canine and equine spermatozoa, respectively) as well as between Coomassie blue-stained cells and those stained with FITC-PSA and assessed by use of flow cytometry (r = 0.96 and 0.97 for canine and equine spermatozoa, respectively). However, in canine samples that were fixed in 2% phosphate-buffered formaldehyde, these correlations were weak.
Conclusions and Clinical Relevance—Staining with Coomassie blue stain was a simple and accurate method to evaluate the acrosome in equine and canine spermatozoa after fixation in 4% paraformaldehyde. This assay should be useful in routine evaluation of semen samples from these species.
Objective—To characterize generation of reactive oxygen species (ROS) by equine spermatozoa.
Sample Population—Multiple semen samples collected from 9 stallions.
Procedure—Equine spermatozoa were separated from seminal plasma on a discontinuous polyvinylpyrrolidone (PVP)-coated silica gradient and resuspended in a modified Tyrode albumin-lactate-pyruvate medium. Amount of hydrogen peroxide (H2O2) generated was assayed by use of a 1-step fluorometric assay, using 10-acetyl-3,7-dihydroxyphenoxazine as a probe for detection of H2O2 in a microplate assay format. Concentration of H2O2 was determined by use of a fluorescence microplate reader.
Results—Amount of H2O2 generated increased significantly with time and spermatozoa concentration for live and flash-frozen spermatozoa, and amount of H2O2 generated was significantly greater for flash-frozen than for live spermatozoa. Addition of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) significantly increased generation of H2O2 by live and flash-frozen spermatozoa. Addition of a calcium ionophore also significantly increased the amount of H2O2 generated by live spermatozoa but did not have an effect on amount of H2O2 generated by flash-frozen spermatozoa. Abnormal equine spermatozoa generated significantly greater amounts of H2O2 than did normal spermatozoa.
Conclusion and Clinical Relevance—Equine spermatozoa generate ROS in vitro, possibly via a NADPH-oxidase reaction. Spermatozoa damaged during flash-freezing or morphologically abnormal spermatozoa generated significantly greater amounts of ROS than did live or morphologically normal spermatozoa. Damaged and abnormal spermatozoa generate greater amounts of ROS that may contribute to reduced fertility or problems related to semen preservation. (Am J Vet Res 2001;62:508–515)
Objective—To identify risk factors for rectal tears in horses; assess the effect of initiating cause on tear location, size, and distance from anus; and determine short-term survival rate among horses with various grades of rectal tears.
Design—Retrospective case series.
Procedures—Medical records for horses with a rectal tear were reviewed, and data including age; sex; breed; cause, location, and size of the tear and its distance from the anus; tear grade; treatment; and outcome (short-term survival [ie, survival to discharge from the hospital] vs nonsurvival) were recorded. Data for age, sex, and breed of horses with rectal tears were compared with data for all horses evaluated at the hospital during the same interval to determine risk factors for rectal tears.
Results—Arabians, American Miniature Horses, mares, and horses > 9 years of age were more likely to develop a rectal tear than other breeds, males, or younger horses. Dystocia had a significant influence on rectal tear size. Location of a rectal tear and its distance from the anus were not associated with cause. Applied treatments for grade 1, 2, and 3 rectal tears were effective, unlike treatments for grade 4 rectal tears. Irrespective of treatment, the overall short-term survival rate among horses with grade 1, 2, 3, and 4 rectal tears was 100%, 100%, 38%, and 2%, respectively.
Conclusions and Clinical Relevance—Accurate identification of risk factors could help practitioners and owners implement adequate measures to prevent the development of rectal tears in horses.
Objective—To characterize the activity of catalase in equine semen.
Animals—15 stallions of known and unknown reproductive history.
Procedure—Seminal plasma was collected from raw equine semen by centrifugation, and samples of seminal plasma were frozen prior to assay for catalase activity. Tissue samples (n = 3 stallions) from the bulbourethral gland, prostate gland, vesicular gland, and testis were homogenized, and cauda epididymal fluid was collected for determination of catalase activity. Catalase activity was determined as an enzyme kinetic assay by the disappearance of H2O2 as measured by ultraviolet spectrophotometry.
Results—Catalase activity in equine seminal plasma was 989.3 ± 167.8 U/ml (mean ± SEM), and the specific activity of catalase in equine seminal plasma was 98.7 ± 29.2 U/mg of protein. Specific activity of catalase in tissue homogenates was significantly higher in the prostate gland (954 ± 270 U/mg of protein) than in the ampulla (59 ± 5 U/mg of protein), bulbourethral gland (54 ± 11 U/mg of protein), vesicular gland (39 ± 3 U/mg of protein), cauda epididymal fluid (11 ± 3 U/mg protein), or testis (54 ± 6 U/mg of protein).
Conclusions and Clinical Relevance—Equine seminal plasma contains a high activity of catalase that is derived primarily from prostatic secretions. Procedures such as semen cryopreservation that remove most seminal plasma from semen may reduce the ability to scavenge H2O2 and thereby increase the susceptibility of spermatozoa to oxidative stress. (Am J Vet Res 2000;61:1026–1030)
Objective—To determine the incidence of equine herpesvirus-1 (EHV-1) infection among Thoroughbreds residing on a farm on which the virus was known to be endemic.
Design—Prospective cohort study.
Animals—10 nonpregnant mares, 8 stallions, 16 weanlings, 11 racehorses, and 30 pregnant mares and their foals born during the 2006 foaling season.
Procedures—Blood and nasopharygeal swab samples were collected every 3 to 5 weeks for 9 months, and placenta and colostrum samples were collected at foaling. All samples were submitted for testing for EHV-1 DNA with a PCR assay. A type-specific EHV-1 ELISA was used to determine antibody titers in mares and foals at birth, 12 to 24 hours after birth, and every 3 to 5 weeks thereafter.
Results—Results of the PCR assay were positive for only 4 of the 1,330 samples collected (590 blood samples, 590 nasopharyngeal swab samples, 30 placentas, and 30 colostrum samples), with EHV-1 DNA detected in nasal secretions from 3 horses (pregnant mare, stallion, and racehorse) and in the placenta from 1 mare. Seroconversion was detected in 3 of 27 foals during the first month of life.
Conclusions and Clinical Relevance—Results suggested that there was a low prevalence of EHV-1 infection among this population of Thoroughbreds even though the virus was known to be endemic on the farm and that pregnant mares could become infected without aborting. Analysis of nasopharyngeal swab samples appeared to be more sensitive than analysis of blood samples for detection of EHV-1 DNA.