Catalase activity in equine semen

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 +/- 1678 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.     

Lactoferrin increases sperm membrane functionality of frozen equine semen

During cryopreservation, sperm was submitted to an increase in reactive oxygen species generation. This work aimed to improve the quality of frozen equine sperm after the addition of antioxidants lactoferrin (Lf) and catalase (Cat) to a freezing extender. Semen from six stallions was frozen with the extenders: F1) control, INRA 82 freezing extender, F2) F1 + 500 μg/ml Lf and F3) F1 + 200 IU/ml Cat. After thawing, sperm motility parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome‐reacted sperm were evaluated with a computer‐assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite, hydroperoxide and iron concentrations of frozen semen were measured with spectrophotometry. The percentage of functional membrane sperm treated with Lf was higher (50.7% ± 11.6%) compared to that of the control (37.6% ± 15.6%), while the iron (61.4 ± 11.6 vs 73.3 ± 13.8 mg/dl) and nitrite concentrations (16.3 ± 7.1 vs 25.9 ± 4.2 μM/μg protein) were lower, respectively (p < .05). Thus, it can be suggested that Lf protect stallion spermatozoon during freezing as it has increased the percentage of sperm with functional membrane and decreased the lipid oxidant agents.     

Effective removal of equine arteritis virus from stallion semen

REASONS FOR PERFORMING STUDY: A method of removing equine arteritis virus (EAV) from equine semen used for artificial insemination is urgently needed. Recent medical studies suggest that a double semen processing technique of density gradient centrifugation followed by a 'swim-up' can provide virus-free sperm preparations for assisted reproduction. OBJECTIVES: To investigate the use of the double semen processing technique to obtain virus-free sperm preparations from stallion semen containing EAV. METHODS: Aliquots of an ejaculate from an uninfected stallion were spiked with virus and processed by the double processing technique. The sperm preparations were tested by PCR for the presence of EAV. The procedure was repeated using an ejaculate from a known shedding stallion, testing processed and unprocessed aliquots by PCR and virus isolation. RESULTS: Virus-free sperm preparations were obtained using the double sperm processing technique. The 'swim-up' step is apparently required to ensure complete virus removal. CONCLUSIONS: The double semen processing technique is potentially a useful and simple tool for the removal of EAV from the semen of shedding stallions. POTENTIAL RELEVANCE: The inclusion of density gradient centrifugation and 'swim-up' in protocols for the processing of semen for artificial insemination could help prevent the transmission of viral diseases carried in semen, such as EAV.     

Freezing equine semen: the effect of combinations of semen extenders and glycerol on post-thaw motility

Objective   We evaluated combinations of two commercial semen extenders and three concentrations of glycerol to determine the combination that yielded the highest post-thaw sperm motility.
Design   A randomised 2 × 3 block design was used.
Procedure   Semen was collected from four stallions (6 collections per stallion). The sample was diluted with either a dried skim-milk glucose extender (EZ Mixin Original Formula) or a chemically defined, milk-free diluent (INRA 96), and each was used in combination with 2%, 3% or 4% glycerol in standard commercial freezing medium. Sperm motility was assessed by microscopy in fresh and post-thaw semen.
Results   There was a significant difference between the two extenders in the motility of spermatozoa after cryopreservation (48.9% for INRA 96; 38.6% for EZ Mixin OF; P < 0.0001). Glycerol at 4% in freezing medium yielded the highest post-thaw motility, significantly better than 2% ( P < 0.05). Three of four stallions had significantly higher post-thaw motility using INRA 96 relative to EZ Mixin OF ( P < 0.01), and two of four stallions had significantly higher post-thaw motility using 4% glycerol ( P < 0.05). The combination of INRA 96 and 4% glycerol in freezing medium gave the highest average post-thaw motility of 51.5%.
Conclusion   In this study, INRA 96 combined with 4% glycerol yielded an average recovery of progressively motile sperm consistently above the 35% target.     

Assessment of fresh extended equine semen samples using spectrophotometry and resazurin

The purpose of this study was to determine if spectrophotometric assessment of resazurin dye in fresh extended equine semen samples was associated with spermatozoal parameters.This technique could be beneficial to veterinarians and horse producers for evaluating semen samples prior to artificially inseminating a mare. The reducible dye resazurin (blue color) is reduced via an oxidation-reduction reaction in the presence of metabolically active spermatozoa to resorufin (pink color), and upon further reduction to dihydroresorufin (colorless). Sixty semen samples were collected from six stallions (5 Quarter Horse and 1 Arabian) using a Missouri style artificial vagina. Sample aliquots were diluted using a 1:30 (semen: extender) ratio with a non-fat dry skim milk (NFDSM) glucose extender T. The diluted sample was then assessed microscopically at 250x to determine concentration, the number of motile, and progressively motile spermatozoa/mL. The remainder of the sample was diluted at a 1:1 (semen: extender) ratio prior to dye incubation and spectrophotometric analysis. The resazurin dye (50 μL from a 0.338 mM solution) was added to 4 (2 mL) aliquots of extended sample, thoroughly mixed, and incubated at 37°C. Butyl alcohol (4.8 mL) was added at five-minute increments (0,5, 10, and 15 minutes) to stop spermatozoal metabolism and draw the color out of the sample. Each aliquot was then vortexed prior to centrifugation at 700xg to extract the butanol color layer. Spectrophotometric absorbance values (615 nm) of the butanol color layer were recorded. Relationships between spectrophotometric absorbance values and spermatozoal parameters were assessed using correlation analyses on square root transformed data. At the 0 minute incubation time there were no associations between spermatozoal parameters and spectrophotometric absorbance values. However, at the five minute incubation time the spectrophotometric absorbance values were negatively correlated with concentration (r=−0.31; P=0.02), number of motile (r=−0.27; P=0.04) and progressively motile (r=−0.30; P=0.02) spermatozoa/mL. At the 10 minute incubation time negative correlations were observed between the spectrophotometric absorbance values and concentration (r=−0.48; P=0.0001), number of motile (r=−0.45; P=0.0004) and progressively motile (r=−0.46; P=0.0002) spermatozoa/mL. At the 15 minute incubation time negative correlations were also found between spectrophotometric absorbance values and concentration (r=−0.52; P=0.0001), number of motile (r=−0.50; P=0.0001) and progressively motile (r=−0.52; P=0.0001) spermatozoa/mL. Spectrophotometric absorbance values were associated with spermatozoal parameters at the 5, 10, and 15 minute incubation times.     

Retention of liquid within insemination equipment using various equine frozen semen insemination methods and two semen freezing extenders

The objective of this study was to examine the effect of different insemination techniques and extenders on the volume of liquid dispensed from insemination equipment. The method of insemination has a significant effect on the volume of semen deposited into the mare's uterus when low volumes are used. Insemination pipettes that allow for direct deposit of straw contents into the uterus are preferred. Aspiration of semen into a pipette is preferred over aspiration into a syringe with deposition through a pipette when direct deposit is not possible. Use of a pipette with a smaller lumen and less length of contact with liquid provides better results. Contact of semen with equipment may allow for residual liquid accumulation on the luminal surfaces and a decrease in overall semen dose. Extenders with differing amounts of egg yolk did not influence volume of liquid dispensed.     

Comparison of two different centrifugation extenders for preservation of frozen equine semen

This study compares a commercial semen extender (control group) to ultra high temperature (UHT) skimmed milk (treatment group) used during centrifugation for subsequent cryopreservation of equine semen. Following post‐thawing of semen samples parameters measured included motility, sperm motion kinetics (using computerised assisted semen analysis) as well as acrosome and plasmatic membrane integrity (using fluorescent dyes). After collection and analysis, the sperm‐rich fraction was divided and diluted with either: control (1:1 dilution in a skimmed milk‐glucose extender) or treatment (1:1 dilution in UHT skimmed milk). The milk used in this experiment was of the same source, commercial brand, of only one lot. After dilution, samples were subjected to centrifugation at 600 g for 10 min and sperm pellets were resuspended in a freezing extender to a concentration of 200 × 10⁶ cells/ml. Aliquots were packed into 0.5 ml straws placed in a stainless steel support and kept inside the refrigerator (5°C) for 20 min. Subsequently, these straws were placed at a height of 6 cm over liquid nitrogen for 20 min in an isotherm box. No significant differences were observed in total sperm motility (42.71 vs. 38.29%), progressive sperm motility (12.29 vs. 7.86%), plasma membrane integrity (53.43 vs. 60.14%) or acrosomal membrane integrity (93.29 vs. 93.71%) with a P>0.05 calculated between the control and the treatment groups, respectively. Considering that UHT skimmed milk has a lower cost than the commercial semen extender, this could be an option used during the centrifugation protocol to decrease the expense of the equine semen cryopreservation process and increase shelf life.     

A comparative study of Sephadex, glass wool and Percoll separation techniques on sperm quality and IVF results for cryopreserved bovine semen

The aim of this study was to compare the effects of spermatozoa separation techniques on sperm quality and in-vitro fertilization (IVF) results for cryopreserved bovine semen. Sephadex, glass wool and Percoll gradient separation techniques were used for sperm separation and sperm motility, morphology and membrane integrity were evaluated before and after separation. Also, cleavage and blastocyst developmental rate were investigated after IVF with sperm recovered by each separation technique. The motility of samples obtained by the three separation techniques were greater compared to the control samples (p < 0.05). The percentage of spermatozoa with intact plasma-membrane integrity, identified by 6-carboxyfluoresceindiacetate/propidium iodide fluorescent staining and the hypo-osmotic swelling test, was highest in the glass wool filtration samples (p < 0.05). The cleavage and blastocyst rate of total oocytes produced from glass wool filtration samples were also higher than the control and Sephadex filtration samples (p < 0.05), but were not significantly different from Percoll separation samples. However, a significantly greater number of cleaved embryos produced by glass wool filtration developed to blastocyst stage than those produced by Percoll separation (p < 0.05). These results indicate that spermatozoa with good quality can be achieved by these three separation techniques and can be used for bovine IVF. In particular, it suggests that glass wool filtration would be the most effective method of the three for improving sperm quality and embryo production for cryopreserved bovine spermatozoa.     

Viability of equine articular chondrocytes in alginate beads exposed to different oxygen tensions

Ischaemia and reperfusion are suspected to alter chondrocyte metabolism. Here, we studied the effects of three oxygen (O2) tensions on the viability of equine articular chondrocytes isolated from the cartilage of the distal interphalangeal joint of horses. Chondrocytes were cultured in alginate beads under 1%, 5% or 21% gas phase O2 concentration for 14 days, cellular growth kinetics were measured (n=6), and the cells were observed by light microscopy after staining for necrotic and apoptotic cell detection. For information about the metabolic status, the intracellular adenosine triphosphate (ATP) content was measured. The number of chondrocytes remained stable for the first eight days, then decreased especially at 1% and 21% O2. At 21% O2, normal cells decreased and necrotic cells increased at the end of the 14 day-period. No significant variations were found at 5% O2 except for a decrease in necrotic cells at day 14. Most apoptotic cells were found at 1% O2 from days 5 to 11, and normal cells decreased during the same period. But an unexpected increase in normal cells and decrease in apoptotic cells were observed at day 14. The intracellular ATP content remained stable. It was concluded that, in a three-dimensional culture model of equine articular chondrocytes, O2 tension affected the viability of the cells after an 11-day period, with the most important effects observed at 21% and 1% O2 conditions.     

Single step purification procedure for the rapid separation of equine leucocytes

Percoll gradients have been used to separate relatively pure populations of viable equine polymorphonuclear (PMN) and mononuclear (MN) cells. In preliminary studies, a continuous density gradient of 70% Percoll solution was used to separate two distinct leucocyte-rich bands. After measurement of the density of each band on the continuous gradient, discontinuous Percoll gradients, using 60% and 75% Percoll solutions, were used to provide a rapid means of separating PMN and MN cells. The yield of viable cells per ml of blood was 3.0×10⁶ and 3.2×10⁶ for MN and PMN cells, respectively. Corresponding values for recovery were 45% and 72%. The purity was 94% for PMNs and 99% for MNs.     

Single step purification procedure for the rapid separation of equine leucocytes

Percoll gradients have been used to separate relatively pure populations of viable equine polymorphonuclear (PMN) and mononuclear (MN) cells. In preliminary studies, a continuous density gradient of 70% Percoll solution was used to separate two distinct leucocyte-rich bands. After measurement of the density of each band on the continuous gradient, discontinuous Percoll gradients, using 60% and 75% Percoll solutions, were used to provide a rapid means of separating PMN and MN cells. The yield of viable cells per ml of blood was 3.0 X 10(6) and 3.2 X 10(6) for MN and PMN cells, respectively. Corresponding values for recovery were 45% and 72%. The purity was 94% for PMNs and 99% for MNs.