New protocol to separate llama sperm without enzymatic treatment using Androcoll-E™

The objective of this study was to design a protocol to separate spermatozoa from seminal plasma of raw llama semen without prior enzymatic treatment using a single-layer centrifugation with Androcoll-E^™ (AE). Two experiments were performed: (a) samples were divided into three aliquots (1 ml) that were deposited on the top of 4, 5 or 6 ml of AE and were centrifuged at 800g for 20 min and (b) samples were divided into two aliquots (1 ml) that were deposited on the top of 4 ml of AE and were centrifuged at 600g or 1,000g for 20 min. Columns of 5 and 6 ml of AE showed a total sperm motility (TM) significantly lower, while in the 4 ml column, this parameter was not different from the TM of samples before the AE treatment. The percentage of spermatozoa with intact and functional membranes, normal morphology and intact acrosomes, as well as the percentages of sperm with highly condensed chromatin, was conserved (p ˃ .05) in the three column heights and in the two centrifugation speeds evaluated. In conclusion, the different column heights of AE (4, 5 and 6 ml) and the different centrifugation speeds used (600, 800 and 1,000g) allow separating spermatozoa of raw llama semen without enzymatic treatment, preserving the evaluated sperm characteristics. However, of all the studied treatments, centrifugation in the 4 ml column of AE at 800g would be the method of choice to process raw llama semen samples destined for reproductive biotechnologies.     

Effect of Removing Seminal Plasma Using a Sperm Filter on the Viability of Refrigerated Stallion Semen

Cooling of equine semen obtained from some stallions results in lower seminal quality and viability when the seminal plasma (SP) is present. The objective of this study was to evaluate the effect of the removal of SP using a Sperm Filter on the viability of cooled stallion semen. For this purpose, 31 stallions were used. Their ejaculates were divided into three groups: CN, semen was diluted with an extender; FLT, SP was removed by filtration; and CT, SP was removed by centrifugation and cooled to 15°C for 24 hours. Sperm kinetics and plasma membrane integrity were evaluated immediately after collection (T0) and after 24 hours of refrigeration (T1). No difference (P > .05) was noted at T1 for total sperm motility (TM), progressive sperm motility, or plasma membrane integrity when semen samples from all the stallions were analyzed. However, when samples from stallions termed “bad coolers” were analyzed (TM = <30% at T1), a difference was observed in TM and progressive sperm motility for CN compared with FLT and CT at T1. Sperm recovery was greater when SP was removed using the filter (FLT) to that when the SP was removed by centrifugation (CN) (89% vs. 81%). Thus, we concluded that filtering with a Sperm Filter is an efficient and practical method for removal of SP from stallion ejaculates, with lower sperm loss than centrifugation. We also found that the presence of SP reduces the quality and viability of cooled semen from stallions whose semen is sensitive to the process of refrigeration.     

A Comparison of the Ability of Three Commercially Available Diluents to Maintain the Motility of Cold Stored Stallion Semen

The objective of this study was to compare the ability of three commercially available extenders to promote poststorage motility of stallion spermatozoa stored at 5°C with and without centrifugation to remove the seminal plasma. Diluents tested included skim milk glucose (SKMG), INRA 96, and VMD-Z. All diluents were tested with (-SP) and without (+SP) centrifugation to remove most of the seminal plasma. In experiment I, after 48 and 72 hours of storage, total (TM) and progressive (PM) motility values were higher (P ≤.05) for those aliquots subjected to the INRA 96-SP as compared with either SKMG treatment. After 72 hours of storage, PM of spermatozoa stored in VMD-Z-SP was superior to that of spermatozoa stored in SKMG regardless of the presence of seminal plasma (P ≤.05). In the second experiment, after 48 hours of storage, PM of spermatozoa subjected to the INRA 96-SP and VMD-Z-SP treatments were superior (P ≤.05) to those for all treatments that had been stored without removal of seminal plasma. Removal of the seminal plasma and resuspension of the sperm pellet with either INRA 96 or VMD-Z resulted in TM after 48 hours of storage that were similar to those obtained after 24 hours of storage.     

Evaluation of a portable device for assessment of motility in stallion semen

In horse breeding, quality assessment of semen before insemination is often requested. Non‐laboratory‐based techniques for objective analysis of sperm motility are thus of interest. The aim of this study was evaluating a portable device for semen analysis (Ongo sperm test) and its comparison with computer‐assisted semen analysis (CASA). Semen was collected from 10 stallions, diluted to 100, 50 and 25 × 10⁶ sperm/ml and analysed for total (TM) and progressive motility (PM). The final sperm concentration influenced total motility analysed by Ongo (p < 0.05) which was higher at 100 × 10⁶ sperm/ml when compared to 25 × 10⁶ sperm/ml (p < 0.05) but not when compared to 50 × 10⁶ sperm/ml (n.s.). Sperm concentration did not influence total motility when assessed by SpermVision (n.s.). Agreement between methods was evaluated by correlation analysis and Bland–Altman plot. Intra‐assay variation of Ongo was 5.2% ± 3.0 for TM and 6.9% ± 3.4 for PM. Correlation between Ongo and CASA was r = 0.79, 0.88 and 0.83 for 100, 50 and 25 × 10⁶ sperm/ml for TM, and r = 0.87, 0.89 and 0.87 for PM, respectively (all p < 0.001). At the 100 and 25 mio/ml dilutions, the difference between the two systems deviated significantly from 0, while no such bias existed at the 50 mio/ml dilution (TM Ongo 85.0%, CASA 82.3%; PM Ongo 64.1%, CASA 66.1%). The 95% confidence interval was 19.9%, 18.9% and 19.2% ± mean for TM and 20.7%, 17.4% and 20.3% ± mean for 100, 50 and 25 × 10⁶ sperm/ml, respectively. In conclusion, Ongo sperm test sperm motility data were strongly correlated with data obtained by CASA. In addition, at a concentration of 50 × 10⁶ sperm/ml values measured with both systems were close to identical. At this concentration, which is recommended in equine AI, Ongo and CASA can be used interchangeably.     

Effect of Post‐Thaw Addition of Seminal Plasma on Motility,Viability and Chromatin Integrity of Cryopreserved Donkey Jack (Equus asinus) Spermatozoa

Pregnancy rates in donkeys after artificial insemination with cryopreserved semen are still low, compared to the horse species. Addition of autologous seminal plasma to frozen‐thawed semen appeared to improve pregnancy rates. The aims of this study were to evaluate (1) sperm motility and plasma membrane integrity after thawing (T0) and after one and 2 h (T1 and T2) of post‐thaw incubation in either 0% (SP0) or 70% (SP70) autologous seminal plasma and (2) sperm motility, plasma membrane integrity and DNA quality (%COMP‐αt) after thawing (T0) and after 2 and 4 h (T2 and T4) of post‐thaw incubation in either 0% (SP0), 5% (SP5) or 20% (SP20) homologous seminal plasma. In experiment 1, seminal plasma decreased total and progressive sperm motility and plasma membrane intact spermatozoa immediately after dilution and at all following time points (p < 0.05). In experiment 2, total and progressive motility did not differ between treatments immediately after dilution and between SP0 and SP5 at T2, while they were lower in both SP5 and SP20 than in SP0 at T4. Plasma membrane intact sperm cells did not differ between SP0 and SP5 and were lower in SP20 at all time points. DNA quality was not affected by treatment immediately after dilution and was significantly worse for SP20 after 4 h of incubation (p < 0.05). The post‐thaw addition of seminal plasma at the tested concentrations did not improve donkey frozen semen characteristics in vitro over time.     

Sperm quality during extra‐gonadal sperm reserve depletion in stallions

To estimate when, during stallions’ extra‐gonadal reserves (EGR) depletion period, sperm quality would reach its highest quality, six light breed sexually rested stallions were collected daily for 7 days to deplete EGR. On collection days 1, 3, 5, and 7, sperm output, total (TM) and progressive (PM) motility, morphology, and plasma membrane (PLM) integrity were evaluated. Sperm output decreased as EGR depletion advanced, stabilizing on days 5–7. Sperm motility (TM and PM) and morphology were not different during EGR depletion. Plasma membrane integrity improved from day 1 to 3; however, no further improvement observed on days 5 and 7. Sperm of sexually rested stallions reach the highest quality on day 3 of the EGR depletion period.     

Extender and centrifugation effects on the motility patterns of slow-cooled stallion spermatozoa

Slow-cooled stallion spermatozoa, with and without seminal plasma removed by centrifugation, were diluted in Kenney's extender (KE) containing nonfat dry skim milk with glucose and antibiotics or in KE supplemented by adding a modified high-potassium Tyrode's medium (KMT). Four ejaculates from each of four stallions were collected and divided factorially across these four treatments. Percentage of motile sperm, path velocity, and linearity immediately after treatment (0 h) and after storage at 4 degrees C for 24, 48, and 72 h were evaluated objectively by use of a HTM-2030 sperm motility analyzer. Stallions were a significant source of variation (P less than .01) throughout. After sperm had cooled, effects of stallion, extender, centrifugation, and their interactions were all found to be significant (P less than .01). The motility at 0, 24, 48, and 72 h for centrifuged KE was 74, 47, 39, and 24%; for uncentrifuged KE was 76, 56, 50, and 37%; for centrifuged KMT was 76, 75, 72, and 64%; and for uncentrifuged KMT was 80, 50, 26, and 13%, respectively. The extender x centrifugation interaction, after 24, 48, and 72 h of storage, accounted for half or more of the variation. Whereas centrifugation of semen extended in KE seemed to be harmful to sperm, motility of sperm extended in KMT after centrifugation was remarkably conserved for 72 h and was superior to all other treatments (P less than .05). This extender is promising for preserving liquid stallion semen when it must be transported before use in artificial insemination.     

Single-Layer Colloid Centrifugation as a Method to Process Urine-Contaminated Stallion Semen After Freezing-Thawing

Urospermia is a major ejaculatory dysfunction affecting stallions. It has been thought that urine-contaminated semen should not be cryopreserved; however, on select cases, urine contamination of semen cannot be avoided. A recent study suggested that urospermic semen can be cryopreserved after cushion centrifugation and extension. Thus, this study aimed to assess the use of single-layer colloid centrifugation (SLC) to process frozen-thawed urine-contaminated stallion semen. Raw ejaculates (n = 55) from eight stallions were split into three groups: no urine, low (20%), or high (50%) urine contamination. Semen was extended 1:1, cushion-centrifuged, and resuspended at 200 million sperm/mL in BotuCrio. Resuspended semen was loaded in 0.5 mL straws and cryopreserved in liquid nitrogen. Samples were thawed (37°C for 30 seconds) and processed by SLC (400 g/30 minutes). Percentages of total motility (TM) and progressive motility (PM) were assessed with computer-assisted semen analyzer. Sperm viability (%VIAB) and yield were assessed with a NucleoCounter before and after gradient centrifugation. Data were analyzed with two-way ANOVA and Tukey’s test. The motility parameters TM before SLC (control: 35 ± 2; low: 33 ± 0.7; high: 22 ± 1.8) after SLC (control: 51 ± 3.6; low: 42 ± 2.2; high: 25 ± 2.8) and PM before SLC (control: 24 ± 1.8; low: 21 ± 1.14; high: 12 ± 1.5) and after SLC (control: 40.3 ± 3.2; low: 31 ± 3.9; high: 14 ± 2) significantly decreased with increasing urine contamination. Urine contamination marginally reduced (P < .05) sperm viability after cryopreservation before SLC (control: 45 ± 0.7; low: 27 ± 0.2; high: 27 ± 0.3) and after SLC (control: 54 ± 0.5; low: 49 ± 0.7; high: 38 ± 0.6). Recovery rates of sperm after centrifugation were not significantly different between groups. In conclusion, urine contamination affects sperm motility parameters in a dose-dependent manner. Post-thaw SLC selected sperm with higher motility and viability in control and low groups but only selected sperm with higher viability in the high group.     

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.     

Pregnancy rates of mares inseminated with semen cooled for 18 hours and then frozen

The ability to ship cooled stallion sperm for subsequent freezing at a facility specializing in cryopreservation would be beneficial to the equine industry. Stallion sperm has been centrifuged, cooled to 5 degrees C for 12 h, and frozen without a detrimental effect on motility in a previous study; however, no fertility data were available. Experiment 1 compared the post-thaw motility of sperm cooled for 18 h at 15 or 5 degrees C at either 400 or 200 x 10(6) sperm/mL and then frozen. Storage temperature, sperm concentration, or the interaction of temperature and concentration had no effect on total (TM) and progressive motility (PM) after cooling. Post-thaw TM and PM were higher for control than (P < 0.05) for treated samples. There was no difference in post-thaw TM and PM due to temperature or concentration. Experiment 2 further evaluated procedures for cooling before freezing. Ejaculates were either cooled to 5 degrees C for 18 h and centrifuged, centrifuged at room temperature and then cooled to 5 degrees C for 18 h before freezing, or centrifuged and frozen immediately (control). There was no difference among treatments on post-thaw TM or PM. In Exp. 3, mares were inseminated with semen that had been extended in skim milk-egg yolk without glycerol, centrifuged, resuspended at 200 x 10(6) sperm/mL, cooled to 5 degrees C for 18 h, and then frozen or not cooled for 18 h before freezing (control). Pregnancy rates did not differ for mares receiving semen cooled and then frozen (21 of 30, 70%) or semen frozen directly without prior cooling (16 of 30, 53%). In summary, a procedure was developed for cooling stallion sperm for 18 h before freezing without a resultant decrease in fertility.     

Level of Glycolyzable Substrates in Stallion Semen: Effect of Ejaculation Frequency on Sperm Survival after Cool Storage during the Nonbreeding Season

Although seminal characteristics are routinely evaluated in the stallion, the effect of collection schedules and seminal plasma on semen quality during cool storage is not well understood, specifically during the nonbreeding season when cryopreservation of stallion semen is preferentially performed. To address these issues, behavioral characteristics, seminal parameters, and biochemical markers (d-glucose, fructose, and citric acid) were measured in ejaculates (n = 60) obtained during the nonbreeding season. Semen was collected from three stallions, twice a day (1-hour gap between successive collections) and two times in a week. Differences between the means of first and second ejaculates were observed for erection latency (P < .001), which was higher in second ejaculates and determined a higher total breeding time (P < .1). Variations introduced by the stallion were significant for number of mounts (P < .05, in first ejaculates), erection latency (P < .001, in second ejaculates), and total breeding time (P < .001, in second ejaculates). First and second ejaculates differed significantly for sperm motility and sperm concentration (P < .001, higher in first ejaculates) and pH (P < .01, higher in second ejaculates). d-glucose was present in seminal plasma at a much higher concentration than fructose (P < .001) in both ejaculates. There were no significant stallion-associated differences in sperm vitality and pH in the first and second ejaculates as well as in sperm concentration for the second ejaculates. The effect of seminal plasma on equine sperm survival during cooled storage was analyzed by monitoring sperm motility and cell morphology after conservation in an extender medium with and without seminal plasma. When statistically considering seminal plasma and conservation time simultaneously, it was found that these variables affected acrosomal status and midpiece morphology.     

Lipid and protein oxidation levels in spermatozoa and seminal plasma of Asian Elephants (Elephas maximus) and their relationship with semen parameters

Peroxidation damage to spermatozoa and seminal plasma has an important role in sperm quality. Thus, the objective of this study was to determine the levels of lipid and protein oxidation in spermatozoa and seminal plasma of Asian elephants (Elephas maximus) with varying percentage of progressive motility. Lipid and protein oxidation was measured by the thiobarbituric acid‐reactive species (TBARS) assay and the 2, 4‐dinitrophenylhydrazine (DNPH) carbonyl groups assay, respectively. Fresh semen samples were collected from Asian elephants and classified according to the percentage of motile spermatozoa into good (>60%) and poor (≤20%) motility. Results revealed that seminal plasma malondialdehyde (MDA) and seminal plasma protein carbonyls (PCs) were significantly higher in poor motility than in good motility (p < .05). The MDA and PC levels in seminal plasma were negatively correlated with the percentages of progressive motility (p < .05). In addition, the negative correlation between sperm concentration and seminal plasma MDA level was investigated (p < .05). The sperm viability was also negatively correlated with sperm PC level (p < .05). This study indicated that lipid and protein oxidation has deleterious effect on semen quality of Asian elephants.     

Preservation of Epididymal Stallion Sperm in Liquid and Frozen States: Effects of Seminal Plasma on Sperm Function and Fertility

Three separate experiments were conducted to improve preservation of stallion epididymal sperm. In the first one, two different cooling extenders (Kenney and Gent) were compared. Sperm viability and motility patterns were assessed in 10 different epididymal sperm samples after 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours of preservation at 4°C. No significant differences were observed in any of the evaluated parameters either between extenders or throughout the storage period. The second set of experiments was designed to determine whether supplementing thawing medium (INRA Freeze) with seminal plasma had any impact on the quality of frozen-thawed epididymal sperm. Ten epididymal frozen-thawed sperm samples coming from separate stallions were used and different functional parameters (sperm membrane integrity and lipid disorder, motility, intracellular Ca²⁺ levels, and intracellular concentrations of peroxides and superoxides) were evaluated after incubation with or without 50% seminal plasma. Supplementing thawing medium with seminal plasma had no impact on sperm function and survival. The third experiment was an in vivo study. Twenty-five mares were inseminated with epididymal frozen-thawed sperm and seminal plasma, and 21 were bred with epididymal frozen-thawed sperm only. Pregnancy rates obtained for mares artificially inseminated with epididymal frozen-thawed sperm and seminal plasma were significantly (P < .05) higher than those observed when seminal plasma was not infused (64% vs. 19%). Taken together, our data indicate that the quality of epididymal stallion sperm can be maintained at 4°C for up to 96 hours. In addition, not only does supplementing frozen-thawed epididymal sperm with seminal plasma have any damaging effect on their quality but it may also improve pregnancy rates after artificial insemination.     

Effect of Seminal Plasma Components on the Quality of Fresh and Cryopreserved Stallion Semen

The importance of seminal plasma (SP) components for stallion semen quality and freezability is little known. This study aimed to evaluate the relationship between SP components and fresh/cryopreserved stallion semen quality. Semen of 30 stallions was collected, and then, SP was recovered and lyophilized. Total protein (TP), vitamin C (CVIT), vitamin E (EVIT), vitamin A (AVIT), iron (Fe), copper (Cu), magnesium, and zinc (Zn) in SP were assessed. Sperm was frozen in an extender supplemented with lyophilized SP. In fresh semen motility, abnormal morphology (AM), sperm vitality (SV), and plasma membrane integrity (PMI) were evaluated. In post-thaw semen, additionally, total motility (TM), progressive motility (PM), straight line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), amplitude of lateral head displacement (ALH), and beat cross-frequency (BCF) were assessed. Levels of component of SP were established by a distribution analysis. Generalized linear models were fitted. Comparisons of means were done with Tukey's test. Correlation and regression analyses were performed. Vitamins and ions were found to be related to fresh semen quality. For post-thaw sperm, medium TP showed higher semen quality. Negative regression and correlation coefficients between CVIT and all post-thaw semen parameters were found. Low EVIT yielded the lowest PM, VSL, and VAP values, while a high level of AVIT yielded the best results for sperm quality. A high level of Cu yielded higher results for TM, PM, VCL, and ALH. Moreover, a negative correlation was found between Zn, SV, and PMI. In conclusion, SP composition influences fresh and post-thaw stallion semen quality.     

Effects of the Addition of Autologous Seminal Plasma to Highly Concentrated Stallion Semen After 48 Hours of Cooled Storage

Insemination with chilled transported semen has become distinctly important in the horse-breeding industry. To ensure cell survival during cooled storage, semen is diluted with an appropriate extender and the concentration of seminal plasma (SP) is reduced. Nevertheless, SP plays an important immunomodulatory role in the female genital tract and supports sperm fertility. The aim of the present study was to evaluate the effect of the addition of autologous SP after cooled storage to highly concentrated stallion semen. Therefore, SP was removed by simple centrifugation of extended semen, aspiration of the supernatant, and resuspension of the sperm pellet with semen extender. Motion characteristics were evaluated after cooled storage for 48 hours at concentrations of 333 × 10⁶ sperm/mL in comparison with stored samples at concentration of 25 × 10⁶ sperm/mL (control). The highly concentrated semen samples were diluted with an extender containing 0%, 5%, 20%, and 80% SP directly before motility analysis. Dilution of the cooled semen with a fresh semen extender without SP (0%) increased kinematic parameters (curvilinear velocity [VCL] 137.3 vs. 151.8; straight-line velocity [VSL] 49.0 vs. 57.5; average path velocity [VAP] 69.5 vs. 79.4 μm/second; amplitude of lateral head [ALH] 3.1 vs. 3.3 μm; beat cross frequency [BCF] 31.6 vs. 33.5 Hz; P < .05) but not total motility (51% vs. 43%) and progressive motility (46% vs. 36%) compared with controls. The addition of SP after storage for 48 hours decreased sperm total motility and progressive motility regardless of SP concentration: 5 (38% and 34%), 20 (37% and 33%), and 80% SP (27% and 22%; P < .05). In contrast, kinematic parameters were enhanced by extenders containing 5% and 20% SP (VCL: 148.0 and 155.6; VSL: 59.2 and 60.9; VAP: 78.7 and 81.9; BCF: 33.4 and 35.7; ALH: 3.4 and 3.4; P < .05). However, using an extender containing 80% SP was detrimental to kinematic parameters (VCL: 151.2; VSL: 52.2; VAP: 76.9; BCF: 34.8; P < .05) except for ALH, which increased (3.5; P < .05). In conclusion, cooled storage at concentrations of 333 × 10⁶ sperm/mL did not affect sperm motility. The addition of a fresh extender or an extender containing small concentrations of SP to highly concentrated ejaculated sperm increased kinematic values after storage; however, increasing concentrations of SP decreased sperm motility.     

In vivo adverse effects of alpha‐tocopherol on the semen quality of male bucks

Oxidative stress has detrimental effects on semen quality during spermatogenesis and semen processing for artificial insemination. This work was conducted to study the effect of different levels of vitamin E on the semen traits, oxidative status and trace minerals in Beetal bucks. Thirty‐six bucks of similar body weight and age (1 year) were randomly divided into four groups. One group was kept as control with no supplementation (group 1), and the others were supplemented with 200 (group 2), 400 (group 3) and 800 IU (group 4) vitamin E/animal/day for 2 months. At the end of the experiment, semen samples were collected and evaluated. Seminal plasma was separated to study the concentration of superoxide dismutase (SOD), glutathione peroxidase (GPx), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and trace minerals (Zn, Cu, Mn and Fe). Group 3 showed significantly higher (p < 0.05) semen volume and per cent motility and lower dead sperm percentage compared to control group. Superoxide dismutase, GPx, Zn, Cu and Mn were higher in the same group. The level of AST decreased in group 3 without any change on the concentration of ALT. It is suggested that vitamin E at the rate of 400 IU/buck/day supported higher semen volume, per cent motility, per cent live spermatozoa, antioxidants (SOD, GPx) and trace mineral levels (Zn, Cu, Mn) in the seminal plasma. The increased supplementation from 0 to 400 showed a general increasing trend in improving semen quality. However, the dose of 800 IU/kg had no useful effect in further improving the semen quality.     

Liquid Storage of Goat Semen in Chemically Defined Extenders

The suitability of certain commercial and self‐made chemically defined extenders for liquid storage of goat semen was tested and the effects of storage temperatures, dilution rates and sperm washing and pH of extenders on the goat sperm during liquid storage were observed. Semen was collected from nine goat bucks of the Lubei White and Boer breeds using an artificial vagina. Each ejaculate after initial evaluation was diluted with a specific extender, cooled and stored at a desired temperature. Stored semen was evaluated for sperm motility and other parameters every 24 or 48 h of storage. The ranking order of the existing milk‐ and yolk‐free extenders in sustaining goat sperm motility was Androhep > Zorlesco > Beltsville thawing solution > the Tris–glucose medium. The new extender (mZA) which was formulated based on Zorlesco and Androhep was more suitable for goat sperm than Androhep. The mZAP extender with Bovine Serum Albumin (BSA) replaced with polyvinyl alcohol (PVA) worked as efficiently as the mZA in maintaining sperm motility, membrane integrity, acrosome intactness and capacitation status. Goat sperm motility was best maintained at 5°C during liquid preservation, but decreased significantly as the temperature increased. When semen was sixfold diluted, sperm motility was maintained longer (p < 0.05) after centrifugation, but sperm motility did not differ between the centrifuged and non‐centrifuged groups when semen was 11‐fold diluted. When the extender pH was adjusted from 6.6 to 6.04, the efficiency increased significantly in both Androhep and mZAP. A forward sperm motility of 34% was maintained for 9 days when buck semen was 11‐fold diluted and stored at 5°C in mZAP, with pH adjusted to 6.04. It is concluded that for liquid storage of buck semen, the mZA extender was more suitable than other extenders; BSA can be replaced with PVA in mZA; centrifugation to remove seminal plasma can be omitted by adequate dilution; and the storage temperature and pH of extenders affected sperm motility significantly.     

Relationship of Sperm Quality to Fertility after 4 Days of Cooled Storage of Equine Semen

This study evaluated measures of sperm quality in relation to fertility achieved with fresh semen or semen cooled and stored. Semen from 1 stallion was collected and processed to provide 3 treatments: group 1 received fresh semen; group 2 received cooled semen containing 50% seminal plasma (SP) stored for 4 days; and group 3 received cooled semen containing 50% SP stored for 1 day, then centrifuged and resuspended in fresh extender containing 10% SP on days 1 to 3. Inseminates were evaluated for sperm motion characteristics and the percentage of sperm with intact membranes (SMI). Mares (n = 34) in estrus were treated with an ovulation-inducing drug and inseminated with 100 million membrane-intact sperm on the following day. Pregnancy status was determined via transrectal ultrasonography 2 weeks after ovulation. The mean percentage of SMI was higher in group 1 (81%, initial) than in group 2 (74%, day 4) or group 3 (74%, day 4) (P < .05). The median percentages of total sperm motility differed among the groups (77%, 5%, 59% for groups 1, 2, and 3 respectively; P < .05). Median values for the percentages of progressively motile sperm and curvilinear velocity for group 1 (55%, 216 μm/s) and 3 (37%, 186 μm/s) were higher than for group 2 (1%, 73 μm/s) (P < .05). Pregnancy rates did not differ among groups (5 of 11, 45% in group 1; 5 of 11, 45% in group 2; and 7 of 12, 58%, in group 3; P = .77). These data suggest that, at least for this stallion, sperm membrane integrity may be a more valuable means of assessing potential fertility of cooled-stored semen than sperm motion characteristics.     

Effects of Dietary Thyme (Thymus vulgaris) and Fish Oil on Semen Quality of Miniature Caspian Horse

Thyme (Thymus vulgaris) is a subshrub from the lamiaceae family with plants that are rich in essential oils and antioxidative phenolic substances. The aim of the study was to investigate the effect of dietary thyme and fish oil supplementation on the semen characteristics of miniature Caspian horse. Sixteen stallions were randomly allocated into four groups and received four different diets: unsupplemented control diet, supplemented with fish oil at 2.5% dry matter intake (DMI), supplemented with fish oil (2.5% DMI), and thyme (0.02% DMI), and supplemented with thyme (0.02% DMI). All experimental diets were formulated according to National Research Council (1998). Semen was collected at 0, 30, 60, and 90 days. The semen samples were cooled and preserved at 5°C. Cooled diluted semen samples were evaluated in vitro by microscopic assessments of chilled sperm motility, acrosomal and other abnormalities (head, midpieces, and tail), viability (evaluated by Eosin–nigrosin), and plasma membrane integrity (evaluated by hypo osmolarity swelling test), and the level of malondialdehyde (MDA) was determined during cool storage 0, 24, and 48 hours after collection. The results showed that total and progressive sperm motility and plasma membrane integrity and functionality in all groups were significantly decreased with increasing storage time. On the other hand, the level of MDA in all groups was significantly increased with increasing storage time. Also, the results showed that most sperm quality parameters in this study were significantly higher in fish oil–thyme and fish oil group compared with thyme and control groups after 24 and 48 hours of storage at 5°C. We concluded that dietary supplementation of fish oil and thyme can improve sperm quality in miniature Caspian stallions during storage in cool condition via increasing total and progressive motility and plasma membrane integrity and functionality. More advances in vitro evaluations and artificial insemination are required to reveal the exact effects of thyme on miniature Caspian stallion sperm quality and its fertilizing ability.     

The influence of centrifugation on quality and freezability of stallion semen

The aim of the present study was to investigate the influence of various centrifugation methods on sperm loss and quality of frozen-thawed semen. From at a total of 8 Warmblood stallions of the National Stud Farm in Avenches, 3 ejaculates each were collected and seminal plasma was removed using 3 different centrifugation regimes. In method I (reference method) centrifugation occurred by a speed of 600 x g during 10 minutes. In method II 1000 x g was used during 2 minutes while in method III centrifugation was performed by 2000 x g during 2 minutes. After centrifugation 90%, of the supernatant was removed and sperm loss calculated. After resuspension of the pellet with freezing medium, functional membrane integrity was evaluated by HOS-test and motility determined. In frozen-thawed semen motility, viability as well as functional membrane integrity (HOS-test) and acrosome status using chlortetracyclinassay (CTA) were assessed. Our results demonstrate that mean sperm loss (I, 1.9%; II, 8.7%; III, 3.7%) was significantly (P < 0.05) different between the three centrifugation regimes. Regarding semen quality of frozen-thawed semen, HOS in method III (52.1%) was significantly lower than in methods I (55.5%) and II (55.3%). Evaluation of the acrosome status by CTA showed that more than 70% of sperm cells were capacitated and 25% capacitated and acrosome reacted. From our results we conclude that sperm loss and functional membrane integrity (HOS-test) in frozen-thawed semen were significantly influenced by the centrifugation regime. Therefore, stallion semen should be centrifuged at 600 x g during 10 minutes before freezing in order to obtain low sperm loss and a good quality of frozen-thawed semen.