Comparison of spermatozoal movement and semen characteristics with fertility in stallions: 64 cases (1987-1988).

Information pertaining to evaluation of single ejaculates of semen and records for 2 consecutive breeding seasons were obtained. In all, data for 99 individual breeding seasons (n = 43 Standardbreds and 56 Thoroughbreds) were evaluated. Included in each semen evaluation was examination of semen characteristics and computer-aided analysis of spermatozoal movement characteristics. On the basis of the analysis of breeding records for 4,175 mares (7,017 estrous cycles), a per-estrous cycle fertility rate was calculated from data for 96 of the breeding seasons. Stallions with lower fertility than the mean overall season fertility had significantly (P less than 0.01) lower mean values for subjective appraisal of the percentage of motile and progressively motile spermatozoa and for percentage of morphologically normal spermatozoa. Lower mean values were obtained for computer-aided movement analysis of the percentage of motile and progressively motile spermatozoa, and for mean velocity of motile spermatozoa. Semen characteristics, including spermatozoal movement characteristics, and fertility were significantly (P less than 0.05) correlated for Thoroughbred and Standardbred stallions when analyzed individually and when data for both breeds were combined. Characteristics most highly correlated (P less than 0.01) with fertility data for both breeds combined were: subjective appraisal of the percentage of motile (r = 0.40) and progressively motile (r = 0.46) spermatozoa; percentage of morphologically normal spermatozoa (r = 0.36); and computer-aided analysis of percentage of motile spermatozoa (r = 0.34). However, on the basis of evaluation of a single ejaculate for each stallion, the variation in these characteristics only accounted for approximately 20% of the observed variation in fertility rate.     

Effect of different processing techniques on motility and acrosomal integrity of cold-stored stallion spermatozoa

Two experiments were conducted to test whether stallionand/or semen processing techniques influenced spermatozoal motility and acrosomal status following cold storage. Ejaculates from each of 18 stallions (N=54) were collected and split. In Experiment I, a skim milk-glucose extender (SKMG) was added to the semen following a 5, 15 or 30 minute delay post-collection. Following each delay, sperm were packaged at a final concentration of 25 million progressively motile sperm per ml (PMS/ml) in a commercially available skim milk-glucose extender (SKMG). In Experiment II, sperm were packaged at concentrations of 25, 50, and 75 million PMS/ml both in the presence and absence of seminal plasma (SP) utilizing SKMG and SKMG plus PBS, respectively. In both experiments, aliquots were cooled, stored, and the percentage of progressively motile and acrosome intact spermatozoa were determined at 24 and 48 hours post-collection. In Experiment 1, delayed dilution resulted in a lower recovery of PMS. In Experiment II, removal of SP resulted in higher percentages of PMS following cold storage. Increasing the concentration of spermatozoa during packaging decreased the percentage of PMS; however, removal of SP reduced the harmful effects on spermatozoa motility. These data suggest that reducing the time that spermatozoa remain in an undiluted state and removal of SP maximize recovery of progressively motile, acrosome-intact spermatozoa. In addition, individualizing the processing techniques for each stallion may enhance spermatozoal survival following cold storage.     

Effect of pyruvate on the function of stallion spermatozoa stored for up to 48 hours

Stallion spermatozoa maintain high fertilizing capacity if cooled to 5 degrees C and inseminated within 24 h. However, if spermatozoa are stored for 48 h, fertilizing capacity declines. Therefore, multiple shipments of semen are often required to inseminate mares that remain in estrus for days. Therefore, experiments were designed to determine if adding antioxidants to stallion spermatozoa stored at 5 degrees C for 48 h could maintain motility and fertilizing ability. In the first experiment stallion spermatozoa were incubated in a skim milk (SM) or a skim milk-egg yolk medium in combination with 10 mM pyruvate, 5 mM xanthurenic acid separately or in combination for up to 48 h at 5 degrees C. Spermatozoa incubated in SM for 48 h exhibited higher percentages of motile sperm (57%) than did sperm incubated in skim milk-egg yolk (34%); antioxidant treatment had little effect. In the second experiment, spermatozoa were incubated in SM containing 0, 1, 2, or 5 mM pyruvate. After 24 h of incubation at 5 degrees C, sperm incubated with 1, 2, or 5 mM pyruvate exhibited higher percentages of progressively motile spermatozoa (45%) than control exhibited (26%; P < 0.05). After 48 h, percentages of progressively motile spermatozoa were similar (27, 19, and 30 vs 14, respectively; P > 0.05). However, when incubated at 5 degrees C for 48 h and then incubated an additional 4 h at 25 degrees C, samples containing pyruvate exhibited higher percentages of motile (63 to 80%) and progressively motile (36 to 42%) sperm than did sperm in SM alone (28 and 5%, respectively; P < 0.05). The third experiment attempted to determine the optimal pyruvate concentration to maintain spermatozoal motility. Spermatozoa incubated with 0, 2, 3.5, or 5 mM pyruvate for 48 h at 5 degrees C and then an additional 4 h at 25 degrees C, exhibited similar percentages of progressively motile cells (31, 35, and 28%, respectively) that were higher than control (11%, P < 0.05). The last experiment evaluated the fertilizing potential of cooled spermatozoa. Embryos were recovered from 35, 20, and 30% of mares inseminated with spermatozoa that had been incubated at 5 degrees C, for 24 h in SM, or for 48 h in SM or SM + 2 mM pyruvate, respectively (P > 0.05). These studies indicate that 2 mM pyruvate in SM was beneficial in maintaining spermatozoal motility in 48 h-stored sperm and, although not significant, seemed to help maintain the fertility of 48 h-cooled spermatozoa.     

Anti-oxidative status and semen quality during cooled storage in stallions

Activity of the anti-oxidative enzymes glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT), content of thiobarbituric acid reactive substances (TBARS) and SH-groups were determined in native stallion semen (n = 8 stallions). Semen was then diluted in Kenney extender, EquiPro((R)) extender either with or without addition of N-acetyl cysteine or phosphate-buffered saline (PBS) and stored for 72 h at 5 degrees C. Correlations between initial activity of enzymes and development of semen motility and membrane integrity were calculated. Activities of GSH-Px, SOD and CAT immediately after semen collections were 10.0 +/- 0.6 picokatals, 0.40 +/- 0.03 SOD units and 0.70 +/- 0.05 nanokatals/10(6) spermatozoa respectively. TBARS content was 0.06 +/- 0.01 nmol and SH-group content 1.7 +/- 0.5 mmol/10(6) spermatozoa. The loss of motile spermatozoa during storage did not differ between extenders. N-acetyl cysteine had no effect on semen motility and membrane integrity. The loss in membrane-intact spermatozoa was highest (P < 0.05) in semen diluted in PBS. Motility and membrane integrity after addition of extender were positively correlated with GSH-Px and CAT, indicating that anti-oxidative mechanisms contribute to the initial high percentage of motile and membrane-intact spermatozoa. However, in these samples the decrease in semen quality was most pronounced. No correlations existed between initial activity of anti-oxidative enzymes, peroxidation products and semen quality during storage. This indicates that once extender has been added, peroxidative damage to sperm membranes is not the predominant cause of losses in semen quality.     

Fertility of stallion spermatozoa isolated on albumin gradients

Fertility of stallion semen extended with bovine serum albumin (BSA) sucrose extender or cream-gelatin extender was compared. Pregnancy rates were 95% of 47 mares and 86% of 46 mares inseminated with BSA-sucrose and creamgelatin extended semen, respectively. Foaling rates and cycles per conception were not significantly different between treatment groups.Semen from 5 mature stallions was used in an attempt to isolate a population of highly motile spermatozoa. Immediately following collection, samples were evaluated for motility and forward movement. Seven to 10 ml of semen, extended 1:1 with BSA-sucrose extender, were pipetted onto BSA medium separation columns. After 1 hour incubation at 37°C, the top, middle and bottom layers were separately withdrawn from each separation column and pooled, respectively. A marked decrease (P<.001) was noted in the mean motility of spermatozoa in the top layer (35%) as compared to the mean pre-incubation motility (59%). Spermatozoa from middle and bottom layers were significantly (P<.001) more motile (70.6 and 87%) than those from the top layer and pre-incubation samples. Rate of forward movement (RFM) of spermatozoa in lower fractions was higher (P<.001) than RFM of spermatozoa in the top layer. Concentration of spermatozoa decreased (P<.001) as the concentration of BSA in the medium increased.     

Comparison of acetamide and dimethylsulfoxide as cryoprotectants of European brown hare (Lepus europaeus) spermatozoa

The aim of the present study was to assess the effect of dimethylsulfoxide (DMSO) and acetamide on the post-thaw properties of hare semen and to perform an AI trial with frozen-thawed semen. Semen was collected under general anaesthesia by the electroejaculation method from 6 males. Immediately after collection, the semen was diluted with an extender containing the following components: 250 mM Tris, 80 mM citric acid, 70 mM glucose, 1.0 M DMSO, egg yolk (17% v/v) and kanamycin (80 mg/l); this extender was used for Protocol I (n=17). In Protocol II (n=15), the DMSO was replaced with 1.0 M acetamide. Immediately after thawing and after incubation for 90 and 180 min at 37 C, the properties of semen were evaluated by computer-assisted semen analysis, and the percentage of viable, acrosome intact spermatozoa was evaluated using flow cytometry. During the 3-h incubation, the percentages of motile spermatozoa and spermatozoa with progressive motility were significantly higher in Protocol I (P<0.01). Immediately after thawing, path and straight velocity were significantly higher in Protocol I (P<0.01), as was the curvilinear velocity (P<0.05). The amplitude of lateral head displacement was higher after 3-h incubation in Protocol I (P<0.05), and no differences in beat cross frequency were found between Protocol I and II at any incubation time. The percentage of viable, acrosome intact spermatozoa determined with flow cytometry was higher in Protocol I (P<0.01) at all incubation times. As a result of artificial insemination with the semen frozen with DMSO as a cryoprotectant, two out of three inseminated females delivered two healthy young each. Following artificial insemination with the semen frozen with acetamide as a cryoprotectant, two out of three inseminated females delivered one healthy young each. On the basis of the results, it should be stated that DMSO ensures better post-thaw properties of hare spermatozoa than acetamide.     

Use of a computerized system for evaluation of equine spermatozoal motility

Three ejaculates from each of 3 stallions were used to evaluate a computerized system (Hamilton-Thorn motility analyzer; HTMA) for measuring equine spermatozoal motility. Variance components (ejaculate-within-stallion, chamber-within-ejaculate, and microscopic field-within-chamber) were determined for each stallion after diluting ejaculates to 25 x 10(6) spermatozoa/ml with a skim milk-glucose seminal extender. The HTMA was compared with frame-by-frame playback videomicrography (VIDEO) for determining: percentage of spermatozoal motility and spermatozoal number in microscopic fields; curvilinear velocity and straight-line velocity of individual spermatozoa for 5 track types; and repeatability of those velocity measurements. The effect of spermatozoal number per microscopic field on incidence of intersecting spermatozoa and the outcome of intersecting spermatozoa also were evaluated. Greatest variability in motility measures was generally attributed to the microscopic field-within-chamber component. The HTMA was highly correlated with VIDEO for estimation of spermatozoal numbers per microscopic field (r = 0.99; P less than 0.001) and motility (r = 0.97; P less than 0.001); however over the entire range of spermatozoal numbers, the HTMA yielded higher spermatozoal numbers per microscopic field (P less than 0.05) and higher motility (P less than 0.05) than did VIDEO. The HTMA- and VIDEO-derived measurements of curvilinear and straight-line velocities were highly correlated for all spermatozoal track types, but both measures were higher (P less than 0.05) by use of the HTMA than by use of VIDEO for most track types. For 3 of 5 track types, measurements of curvilinear and straight-line velocities were less variable (P less than 0.05), using the HTMA, rather than VIDEO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of degenerative germ cells in semen from a quarter horse stallion

A 3-year-old Quarter Horse stallion was presented for evaluation of potential fertility. An impairment in spermatogenesis was suspected as the stallion produced semen with reduced percentages of progressively motile and morphologically normal spermatozoa. In addition, when semen was collected daily from the stallion, daily spermatozoal output (DSO) was found to be below normal. Semen from this stallion also contained spherical cells of unknown origin. The following report describes the identification of these cells as degenerating spermatids by the use of light and electron microscopy.     

Fertility of Mares Inseminated With Frozen-Thawed Semen Processed by Single Layer Centrifugation Through a Colloid

The aim of this study was to determine whether there was an increase in pregnancy rates when frozen-thawed stallion semen was processed by single layer centrifugation (SLC) through a colloid before insemination. In addition, changes in semen parameters, including motility, were determined before and after SLC. Twenty light-horse mares (aged 3-16 years) and one Thoroughbred stallion (aged 16 years) having average fertility with fresh and cooled semen (>50% per cycle) and displaying a postthaw motility of >35% were used. Control mares were inseminated using 4- × 0.5-mL straws (200 × 10⁶/mL) of frozen-thawed semen. Treatment mares were inseminated with 4 × 0.5 mL of frozen-thawed semen after processing by SLC. Pregnancy rates were compared using Fisher exact test, and continuous parameters were evaluated by a Student t test. The pregnancy rates at day 14 were not different for the mares inseminated with control versus SLC-processed semen, despite the difference in sperm number (171 × 10⁶ ± 21, 59 × 10⁶ ± 25 progressively motile sperm). After frozen-thawed semen was processed by SLC, the percentage progressively motile sperm improved (P < .05), and SLC processing resulted in a 21.8% recovery of spermatozoa. In summary, centrifugation of frozen-thawed semen through a single layer of colloid increased the percentage of motile spermatozoa, but did not improve pregnancy rates after deep horn insemination.     

Determination of the best practical method of thawing bovine semen

An experiment was designed to determine the best practical method of thawing which could be applied to all semen processed by Canadian A.I. centers. Semen in Tris, Triladyl, whole milk, or Fresh Plus Extender packaged in 1 mL ampules or 0.5 mL French straws was used. Semen was thawed in water at 35°C, 20°C, or 5°C, or in a shirt pocket for 5, 5, 10, or 10 minutes, respectively. Post-thaw viability of sperm cells was assessed by determining the percentage of progressively motile cells, rate of progression, and percentage of intact acrosomes, at 0 hours and after 2 hours of incubation at 37°C. Post-thaw viability was significantly higher (P < 0.05) for the 35°C thaw than for any of the other thaw methods regardless of extender or packaging. Our data indicate that when AI center recommendations for thawing semen are unknown, semen in ampules and straws should be thawed in a 35°C water bath and maintained at that temperature until it can be inseminated.     

Einflüsse von Prostatasekret und Verdünner auf die Spermienmotilität und ATP-Konzentration sowie die Aktivität der sauren und alkalischen Phosphatase von Beagle-Samen*

Influences of seminal plasma and extender on sperm motility, ATP-concen-tration, and the activity of acid and alkaline phosphatases of beagle dog semen. The percentage of progressively motile spermatozoa, the time of sperm survival, ATP-concentration, and the activity of the acid and alkaline phosphatases were determined in the semen of five healthy beagle dogs immediately after collection and after storage for 24 hours at +5°C. The sperm rich fraction was examined in the native state as well as after the addition of prostatic secretion and a Tris-eggyolk-medium, resp. The percentage of progressively motile spermatozoa was 64.4% in the fresh undiluted semen, 68.4% after the addition of prostatic secretion, and 74.8% after dilution with Tris-eggyolk-medium. It decreased within 24 hours to 31.6%, 20.4%, and 59.2%, resp. After 0 und 24 hours, resp., the sperm survival time in a coverslide preparation was     

Cholesterol Loaded Cyclodextrin Supplementation Enhances the Cholesterol-to-Phospholipid Ratio and Diminishes Oxidative Stress in Jack Spermatozoa During Cryopreservation

The present study was conducted with the hypothesis that addition of cholesterol to the extender would stabilize the sperm membranes by increasing the cholesterol-to-phospholipid (C:P) ratio and would result in an improved post-thaw semen quality and reduce oxidative stress in the jack (Martina franca) semen. Forty-eight ejaculates from six jacks were collected and analyzed for the present study. The freshly collected semen sample of each jack stallion was divided into five equal fractions after addition of the primary extender without cholesterol-loaded cyclodextrin (CLC) (C) and with 1, 1.5, 2, and 3 mg/mL CLC to obtain 120 × 10⁶ sperm/mL spermatozoa concentration. The semen was cryopreserved using customized freezing protocols. Evaluation of seminal parameters, the C:P ratio, and the oxidative status of jack spermatozoa was analyzed at all stages of cryopreservation. The oxidative status in the jack semen was evaluated by measuring malondialdehyde, glutathione and total antioxidant capacity levels. The results indicated that the mean percent values for various seminal quality parameters and the oxidative parameters were found to be significantly higher (P < .05) in CLC-treated groups with the highest values for 2 mg of CLC/120 × 10⁶ spermatozoa. In conclusion, the present study revealed that the supplementation of CLC before cryopreservation has significantly reduced the oxidative stress and also increased the C:P ratio during semen cryopreservation process. Furthermore, a reduction in lipid peroxidation levels, reduced damage to the sperm plasma and acrosome membranes and improvement in the post-thaw sperm integrity as well as stability were recorded.     

Effects of Thawing Temperature and Post‐thaw Dilution on the Quality of Cat Spermatozoa

The present study aimed to compare cat sperm quality after thawing using two different temperatures (37 and 70°C) and to investigate the effects of post‐thaw dilution on the sperm quality and longevity of ejaculated cat spermatozoa. Six ejaculates of each of six male cats were collected using an electroejaculator (total 36 ejaculates). The semen was frozen in 0.25‐ml straws using a Tris egg yolk extender containing Equex STM paste. Four straws prepared from each ejaculate were thawed at four different occasions; (i) at 37°C for 15 s, (ii) at 37°C for 15 s and diluted 1 : 2 with Tris buffer (v/v), (iii) at 70°C for 6 s, (iv) at 70°C for 6 s and diluted 1 : 2 with Tris buffer (v/v). The percentages of motile spermatozoa, the scores of progressive motility, the percentages of spermatozoa with intact plasma membrane (using SYBR‐14/EthD‐1 stains) and intact acrosome (using fluorescein isothiocyanate conjugated peanut agglutinin/propidium iodide stains) were evaluated in fresh semen at 0, 2, 4 and 6 h after thawing. The thawing temperature had no effect on any sperm parameters throughout the incubation period (p > 0.05). The dilution after thawing improved sperm motility, progressive motility and acrosome integrity (p < 0.05). The thawing of cat spermatozoa and subsequently diluting with Tris buffer resulted in an immediate (at 0 h) overall (combined over temperature) percentage of motile sperm of 64.8 ± 10.7 (mean ± SD), a score of progressive motility of 4.0 ± 0.5, a percentage of spermatozoa with intact plasma membrane of 64.4 ± 12.1 and intact acrosome of 44.8 ± 20.2. In conclusion, frozen cat semen can be thawed either at 37 or 70°C and post‐thaw dilution is recommended to reduce the toxic effect of some ingredients in the extender during post‐thaw incubation.     

Effects on boar semen quality after infection with porcine reproductive and respiratory syndrome virus: a case report

The effect of porcine reproductive and respiratory syndrome virus (PRRSV) on semen quality was examined in a group of 11 spontaneously infected boars in a commercial boar stud. Semen samples were collected 4 weeks prior to 4 weeks post-infection (wpi). Infection with PRRSV of the European genotype subtype 1 (EU-1) was verified by specific quantitative real-time polymerase chain reaction (RT-PCR) in 36% of the serum samples. All boars seroconverted before 4 wpi and remained in normal condition throughout the study. Comparison of the percentage of morphologically intact spermatozoa revealed an increase of acrosome-defective spermatozoa (P = 0.012) between −4 and 4 wpi. Significant deleterious effects on semen quality were detected for membrane integrity when semen had been stored for 2 days after sampling. Analysis of sperm subpopulations in a thermoresistance test on day 7 after sampling revealed alterations in the percentage of circular, progressively motile spermatozoa (P = 0.013), in the percentage of non-linear, progressively motile spermatozoa (P = 0.01), and on the amplitude of lateral sperm head displacement (P = 0.047). There was no difference in the incidence of mitochondrially active spermatozoa (P = 0.075). Investigation of routine production data between pre- and post-infection status showed no differences on ejaculate volume (P = 0.417), sperm concentration (P = 0.788), and percentage of motile spermatozoa (P = 0.321). This case report provides insights into a potential control strategy for PRRSV outbreaks in boar studs.     

Breeding soundness examination of North American bison bulls

OBJECTIVE: To evaluate the breeding soundness examination procedure in plains bison bulls. DESIGN: Multiyear (1993 through 1997) cross-sectional clinical procedure evaluation. ANIMALS: Two hundred thirty-four 28- to 30-month-old bison bulls at Custer State Park. PROCEDURE: Breeding soundness examinations were performed on all bison bulls using 1992 Society for Theriogenology guidelines for beef cattle semen evaluation and reproductive tract examination. Linear and logistic regression analyses were used to detect correlations and associations among breeding soundness examination variables. RESULTS: Scrotal circumference (SC) was significantly correlated with body weight, percentage of normal spermatozoa, percentage of primary spermatozoal defects, and percentage of motile spermatozoa. Scrotal circumference was positively associated with increased odds of semen collection, satisfactory motility (> or = 30% motility), satisfactory morphology (> or = 70% normal spermatozoa), and simultaneous satisfactory motility and morphology. Receiver-operator characteristic curve analysis selected 29 cm as the optimal SC cutoff most predictive of simultaneous satisfactory spermatozoal motility and morphology. Only 36.2% (83/229) of the bison bulls had a SC of 29 cm or greater and satisfactory spermatozoal motility and morphology. CLINICAL IMPLICATIONS: SC is a good indicator of adequate spermatozoal motility and structure in bison. We recommend use of 30% spermatozoal motility, 70% normal spermatozoal morphology, and 29-cm SC as minimal satisfactory measurements for breeding soundness examinations of 28- to 30-month-old bison bulls that have been raised on forage-based nutrition.     

Impact of Ultra‐Rapid Freezing on the Motility,Morphology, Viability and Acrosome Integrity of Epididymal Cat Sperm Diluted in Sucrose‐Based Extenders

The objective of the present study was to investigate the influence of different sucrose‐based extenders on the motility, morphology, viability and acrosomal integrity of epididymal cat spermatozoa cryopreserved by ultra‐rapid freezing method. Nine cats were castrated, and collected semen was diluted 1 : 1 with Dulbecco`s phosphate‐buffered saline‐BSA1%‐based extender supplemented with different sucrose concentrations (0, 0.25, 0.4 and 0.6 m ). After ultra‐rapid freezing, samples were thawed and sperm motility, morphology, viability and acrosome status were assessed. At thawing, the number of progressively motile (p < 0.01) and morphologically normal (p < 0.01) sperm was higher in the sucrose‐supplemented groups than in the sucrose‐free group. Viability of spermatozoa cryopreserved without sucrose was significantly reduced. In extender supplemented with 0.4 m sucrose, spermatozoa viability showed higher values (57.0 ± 4.7; p < 0.01). No significant differences were detected among groups for sperm acrosome integrity. Results support that cat sperm survive after ultra‐rapid freezing using sucrose as a cryoprotectant, and the best results were achieved when 0.4 m of sucrose was used. This is the first report on sperm ultra‐rapid freezing of cat sperm and further studies on extenders, sperm management or cryovials should be carried out to improve sperm cryosurvival.     

Effect of Semen Extender and Density Gradient Centrifugation on the Motility and Fertility of Turkey Spermatozoa

In the absence of commercially viable methods for cryopreserving turkey spermatozoa, new processing methods are required to extend the functional life of stored turkey spermatozoa for artificial insemination. The present study evaluates the efficacy of a new extender (Turkey Semen Extend) and investigates the use of density gradient centrifugation in processing turkey spermatozoa for artificial insemination. The new extender is compared with two commercially available turkey semen extenders, Beltsville Poultry Semen Extender and Ovodyl. Turkey spermatozoa in Turkey Semen Extend were still motile 20 h after collection, representing a considerable improvement over the other semen extenders (40%, 0% and 8% for Turkey Semen Extend, Beltsville Poultry Semen Extender and Ovodyl, respectively). A field trial on a commercial turkey farm showed improved fertilization rates following insemination of turkey hens with semen extended in Turkey Semen Extend (89.7%) compared with Beltsville Poultry Semen Extender (86.9%). This difference is statistically significant (p < 0.05). Processing on a density gradient, optimized for turkey spermatozoa, also increased sperm survival (50% gradient-prepared spermatozoa still motile after 18 h compared with <10% non-processed spermatozoa). Preliminary studies indicate that gradient preparation of spermatozoa may aid survival during cryopreservation.     

Preliminary Results: The Advantages of Low-Density Lipoproteins for the Cryopreservation of Equine Semen

The aim of this study was to determine the best concentration of low-density lipoproteins (LDL) in a semen extender to improve the percentage of motile spermatozoa in equine sperm after freezing and thawing in comparison with standard extenders. Ten extenders were compared: 1 with 2% egg yolk (EY), 8 with different concentrations of LDL (0.25%, 0.50%, 0.75%, 1%, 2%, 3%, 4%, and 5%), and INRA 96; all of the extenders contained 2.5% glycerol. Fourteen ejaculates were collected from four different stallions. The first dilution was made with equal parts at +37°C, centrifuged (600 × g/10 min), and resuspended in the corresponding extenders to obtain a final concentration of 100 × 10⁶ spermatozoa/ml. The resulting mixture was cooled to 4°C over 1 hour, packed into four 0.5-ml straws, and left for a further 30 minutes at +4°C. Finally, the straws were frozen in nitrogen vapors 4 cm over liquid nitrogen for 10 minutes before being immersed in liquid nitrogen at −196°C and stored. Two straws per extender and per ejaculate were thawed in a water bath at +37°C for 30 seconds. The contents of each straw were recovered into a cryotube and placed in a water bath at +37°C for 10 minutes before being examined with an image analyzer. The best post-thaw motility results were obtained with the extenders made with 0.5%, 2%, and 3% LDL and with the control extender made with egg yolk; no significant difference was observed between these extenders. The last two straws were thawed to perform four sperm function tests. The hypo-osmotic test was used to assess the integrity of the plasma membrane; the 2% and 3% LDL treatments were the most suitable and were comparable to that with whole egg yolk for protecting stallion sperm during cryopreservation (32.3%, 32.4%, and 31.3%, respectively). The Pisum sativum agglutinin-fluorescein isothiocyanate test was used to verify the integrity of the acrosomes; the best results were obtained with the 0.5%, 0.75%, and 3% LDL and INRA96 extenders; no significant differences were observed among the 85.8%, 85.0%, 84.7%, and 84.8% extenders. The acridine orange test was used to assess DNA integrity; there were no significant differences among the various extenders: the DNA was preserved in 98% of the spermatozoa. Finally, spermatozoal morphology was examined using Spermac stain; 78% of the spermatozoa did not present any anomalies in the 0.25% and 2% LDL extenders. In conclusion, the 2% LDL extender gave the best post-thaw percentage of motile spermatozoa. The results of the sperm function test were also superior for this extender.     

Assessment of the cryopreservation of equine spermatozoa in the presence of enzyme scavengers and antioxidants

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 (10 mM), ascorbic acid (10 mM), alpha-tocopherol (25, 50, 100, or 500 microM or 1 mM), or the vehicle for alpha-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 alpha-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 alpha-tocopherol in ethanol may affect spermatozoal viability; consequently, water-soluble analogues of alpha-tocopherol may be preferred for future investigations.     

Effect of adding different levels of caffeine in the extender on some biochemical constituents,enzymatic activities and physical characteristics of chilled and frozen ram semen

Two experiments were carried out to determine the efficiency of supplementation of ram semen extender with caffeine on chilled storage and frozen capacity of spermatozoa. In the first experiment, eighty ejaculates were collected by an artificial vagina from five adult Barki rams, aged 2–3 years and weighted 45.0 ± 2.0 kg throughout the experimental period (January to February 2017). The ejaculates were pooled and diluted (1:10) with tris‐citric egg yolk extender and were split into five groups. Group 1 served as control, whereas groups 2‐5 were supplemented with 0.1, 0.2, 0.3 and 0.4 mM caffeine. All diluted semen specimens were evaluated for physical characteristics immediately after dilution (T₀) and throughout preservation period of 48 hr at 4°C. Simultaneously, oxidative stress and indices such as total antioxidant capacity (TAC), malondialdehyde concentrations (MDA) and alkaline transaminase (AKP) concentrations and value of resazurin reduction test (RRT) were determined. In the second experiment, the raw pooled ejaculates were diluted (1:10) with glycerolated tris‐citric egg yolk extender, receiving the previously mentioned caffeine levels. The post‐thaw assessment of cryopreserved spermatozoa, in all groups, was conducted by a computer‐assisted sperm analysis (CASA) system. The results revealed that adding caffeine to ram semen extender at low (0.1 mM) or medium (0.2 mM) levels had positive impact on both physical characteristics of ram sperm and the enzymatic activities compared to the other semen groups. Caffeine supplementation also enhanced post‐thaw sperm dynamics, which implies its potential as an exogenous antioxidant supplement.