Assessment of Extent of Apoptosis and DNA Defragmentation in Chilled Semen of Stallions During the Breeding Season

The objective of the study was to assess apoptosis and DNA defragmentation in equine semen diluted and chilled to +4°C. Semen was collected from nine fertile stallions, including four Arabian thoroughbreds and five coldbloods. Examinations were carried out immediately after semen collection (0) and at five storage times (24, 48, 72, 96 and 120 h). The basic semen evaluation was performed in terms of volume, sperm concentration, viable sperm percentage, progressive motility and morphology. Using flow cytometry, DNA defragmentation and cell membrane integrity of spermatozoa were determined. The results of basic tests did not demonstrate significant differences amongst stallions, except for progressive sperm motility, which was significantly higher (p < 0.05) in the semen of Arabian stallions. In the semen of the same stallions, a significant decrease in the percentage of alive spermatozoa was observed at 72, 96 and 120 h of storage, whereas a significant increase in the number of spermatozoa with DNA defragmentation was found after 24 h storage. In the semen of coldblood stallions, significantly reduced live spermatozoa percentage was observed at 96 and 120 h, while increased DNA defragmentation was observed at 48 h. These findings demonstrated that the semen of Arabian stallions chilled to +4°C retained original characteristics until 24 h of storage, whereas in coldbloods, these were preserved up to 48 h of storage.     

Does the Microbial Flora in the Ejaculate Affect the Freezeability of Stallion Sperm?

In an attempt to evaluate the possible relationship between the microbial flora in the stallion ejaculate and its ability to freeze, three ejaculates from five stallions were frozen using a standard protocol. Before freezing, an aliquot was removed for bacteriological analysis. Bacterial growth was observed in all the ejaculates studied. The isolated microorganisms were: Staphylococcus spp. and Micrococcus spp. (in all the stallions), β-haemolytic Streptococcus (in stallions 3 and 4), Corynebacterium spp. (in stallions 1, 3–5), Rhodococcus spp. (in stallion number 2), Pseudomonas spp. (in stallion number 1) and Klebsiella spp. (in stallions 1, 3 and 5). The presence and richness of Klebsiella and β-haemolytic Streptococcus in the ejaculate were related to two sperm variables post-thaw, namely the proportion of dead spermatozoa (ethidium+ cells; r = 0.55, p < 0.05) and the amplitude of lateral displacement of the sperm head (ALH, μm; r = −0.56, p < 0.05), respectively. The degree of growth of Corynebacterium spp. in the ejaculate was positively correlated with the percentage of spermatozoa showing high caspase activity post-thaw (r = 0.62, p < 0.05). The presence and number of colonies of β-haemolytic Streptococcus were negatively correlated (r = −0.55, p < 0.05) with low sperm caspase activity. It is concluded that the microbial flora of the equine ejaculate may be responsible for some of the sublethal damage experimented by the spermatozoa during cryopreservation.     

Characterisation of movement pattern and velocities of stallion spermatozoa depending on donor, season and cryopreservation

The aim of the study was to compare different types of movement pattern and velocities of stallion spermatozoa depending on cryopreservation during breeding and non-breeding season. Ejaculates were collected from four stallions during May (n = 24) and December (n = 24). Parameters of sperm movement were evaluated by computer-aided sperm analysis (CASA) system, and included percentages of motile spermatozoa, different patterns of motility, the velocity, linearity (LIN), amplitude of lateral head displacement (ALH) and beat-cross frequency (BCF). In winter the average percentages of motility were slightly higher compared to the breeding season in May (70.8 +/- 12.7% vs. 66.8 +/- 12.2%, respectively). Cryopreservation and thawing led to a significant decrease in the number of motile sperm to 11.3 +/- 5.8% in May and 15.6 +/- 7.0% in December. The pattern of motility was also changed. Detailed analysis by CASA demonstrated that cryopreservation resulted in a shift from the proportions of linear to more non-linear motile spermatozoa and to a significant increase of local motile and hyperactivated spermatozoa. Mean velocity of fresh motile spermatozoa differed between May and December (119.1 +/- 43.9 vs. 164.4 +/- 66.4 microm/sec, respectively; P < 0.05). Cryopreservation and thawing led to a slight increase of curvilinear velocity (VCL) and straight line velocity (VSL). The motility analysis has shown that the parameters BCF and ALH were highly correlated in stallion spermatozoa (r = -0.67; P < 0.001). The BCF of stallion spermatozoa was slightly reduced in the non-breeding season. Altogether, the influence of factors on the motility of stallion spermatozoa has the following rank order: cryopreservation (P < 0.0001) > stallion (P < 0.001) > season (P < 0.05).     

Nicotinic Acid (Niacin) Supplementation in Cooling and Freezing Extenders Enhances Stallion Semen Characteristics

In the present study, we aimed to evaluate the possible protective effects of the nicotinic acid (NA) at three concentrations (10, 20, and 40 mM) on the equine cooled and frozen-thawed spermatozoa quality markers including viability, plasma membrane or acrosome integrity, DNA fragmentation, lipid peroxidation, and total oxidant levels. We also evaluated the effects of NA on preservation of the post-thaw sperm quality after 6 hours of cold storage before freezing. Five stallions were used for semen collections. The current experiment was repeated six times using pooled semen samples from two stallions, each time. We showed that NA at 20 and 40 mM concentrations could significantly improve the stallion sperm quality markers during cold storage. However, the protective effects were not different between 20 mM and 40 mM concentrations in most measures. Nicotinic acid could also improve the post-thaw stallion sperm quality at 10, 20, and 40 mM concentrations. However, the 40 mM concentration showed a negative impact on some post-thaw kinematic sperm parameters. Nicotinic acid at 10 and 20 mM concentrations could preserve the sperm cryo-tolerance to be frozen up to 8 hours after collection without a significant decline in most of the post-thaw sperm quality measures. Nicotinic acid could also decrease the level of the lipid peroxidation and total reactive oxygen/nitrogen species in the cooled and frozen-thawed spermatozoa, in a dose-dependent manner. Therefore, NA at 20 mM concentration could preserve most of the stallion sperm quality measures during cold storage (42 hours, 5°C) and enabled storage of cooled stallion semen for 6 hours before freezing without significant deterioration of the post-thaw sperm quality.     

Epigallocatechin‐3‐Gallate (EGCG) Reduces Rotenone Effect on Stallion Sperm–Zona Pellucida Heterologous Binding

Stallion spermatozoa are highly dependent on oxidative phosphorylation for ATP production to achieve normal sperm function and to fuel the motility. The aim of this study was to evaluate the response of equine sperm under capacitating conditions to the inhibition of mitochondrial complex I by rotenone and to test whether epigallocatechin‐3‐gallate (EGCG), a natural polyphenol component of green tea, could counteract this effect. After 2‐h incubation of stallion spermatozoa in modified Tyrode's medium, rotenone (100 nm , 500 nm and 5 μm ) and EGCG (10, 20 and 60 μm ), alone or in combination, did not induce any significant difference on the percentage of viable cells, live sperm with active mitochondria and spermatozoa with intact acrosome. The inhibition of complex I of mitochondrial respiratory chain of stallion sperm with rotenone exerted a negative effect on heterologous ZP binding ability. EGCG at the concentrations of 10 and 20 μm (but not of 60 μm ) induced a significant increase in the number of sperm bound to the ZP compared with that for control. Moreover, when stallion sperm were treated with rotenone 100 nm , the presence of EGCG at all the concentrations tested (10, 20 and 60 μm ) significantly increased the number of sperm bound to the ZP up to control levels, suggesting that this green tea polyphenol is able to reduce the toxicity of rotenone.     

Detection of Sex Chromosome Aneuploidy in Equine Spermatozoa Using Fluorescence In Situ Hybridization

The aim of our study was to diagnose aneuploidy in equine spermatozoa by multicolour fluorescence in situ hybridization (FISH) technique using specific molecular probes for equine sex chromosomes and autosome pair four (EGFR probe) labeled by different fluorochromes. These were applied on decondensed spermatozoa of four stallions. In total, more than 8800 sperm cells were examined. The total frequency of aberrant cells was 0.496%: aneuploidy of XX (0.135%), YY (0.023%), XY (0.102%), diploidy (0.057%), lack of sex chromosome (0.18%). In one stallion the ratio of normal X‐ and Y‐bearing cells was different from the expected 1 : 1 ratio (p = 0.0002), in all three other stallions this ratio was close to 1 : 1. The present study demonstrated that the FISH technique is a powerful method to identify sex chromosome aberrations in equine spermatozoa and allows for the determination of the ratio between X–Y‐spermatozoa.     

Variation among stallions in sperm quality after single layer centrifugation

Although single layer centrifugation (SLC) selects robust spermatozoa from stallion semen, the effect of individual variation has not been studied in detail. The objective of this study was to determine the variation among stallions in the effects of SLC on sperm quality during cooled storage for up to 48 hr. Semen samples from seven stallions (18 ejaculates) were split, with one portion being used for SLC and the other serving as a control (CON). Sperm quality (kinematics, reactive oxygen species (ROS) production, membrane integrity (MI) and chromatin integrity) were analysed at 0, 24 and 48 hr using computer-assisted sperm analysis and flow cytometry. Sperm quality was better in SLC than in CON at all timepoints, especially chromatin integrity and MI (p < .0001 for both), and some categories of ROS production (e.g. proportion of live hydrogen peroxide negative spermatozoa, p < .0001), but the degree of improvement varied among stallions and type of ROS (p < .05–p < .0001). Total and progressive motility were also better in SLC samples than in CON at 24 and 48 hr (p < .0001), although the effect on sperm kinematics varied. The interaction of treatment, time and stallion was not significant. In conclusion, sperm quality was better in SLC samples than in CON, although there was considerable individual variation among stallions. The improvement in sperm quality, particularly in chromatin integrity, was clearly beneficial, and therefore the use of this technique would be warranted for all stallion semen samples.     

Redox cycling induces spermptosis and necrosis in stallion spermatozoa while the hydroxyl radical (OH•) only induces spermptosis

Oxidative stress is a major factor explaining sperm dysfunction of spermatozoa surviving freezing and thawing and is also considered a major inducer of a special form of apoptosis, visible after thawing, in cryopreserved spermatozoa. To obtain further insights into the link between oxidative stress and the induction of apoptotic changes, stallion spermatozoa were induced to oxidative stress through redox cycling after exposure to 2‐methyl‐1,4‐naphthoquinone (menadione), or hydroxyl radical formation after FeSO₄ exposure. Either exposure induced significant increases (p < 0.05) in two markers of lipid peroxidation: 8‐iso‐PGF_2α and 4‐hydroxynonenal (4‐HNE). While both treatments induced changes indicative of spermptosis (caspase‐3 activation and decreased mitochondrial membrane potential) (p < 0.01), menadione induced sperm necrosis and a dramatic reduction in motility and thiol content in stallion spermatozoa. Thus, we provided evidence that oxidative stress underlies spermptosis, and thiol content is a key factor for stallion sperm function.     

Single‐Layer Centrifugation Reduces Equine Arteritis Virus Titre in the Semen of Shedding Stallions

Several countries have adopted strategies for preventing and/or controlling equine viral arteritis based on vaccination and restricting the breeding activities of carrier stallions. However, in some cases, carrier stallions are only identified after they have transmitted virus to a mare. Therefore, a mechanism for separating virus from spermatozoa in the semen of carrier stallions would facilitate control measures for preventing disease transmission. In this study, the use of several modifications of single‐layer centrifugation (SLC, SLC with an inner tube and double SLC) through Androcoll‐E, a species‐specific colloid were evaluated for their ability to separate spermatozoa from virus in ejaculates from carrier stallions. The three types of SLC significantly reduced the virus titre in fresh semen at 0 h and in stored semen at 24 h (p < 0.001) but did not completely eliminate the virus. Sperm motility parameters such as total motility and progressive motility were significantly increased after colloid centrifugation, whereas curvilinear velocity and amplitude of lateral head deviation were decreased, and the remainder (straight line velocity, average path velocity, straightness, linearity, wobble and beat cross‐frequency) were not significantly affected by the processing. Although virus titres were reduced in the SLC samples, significant levels of infectivity still remained, especially in stallions shedding large amounts of virus. It remains to be determined whether SLC‐processed sperm samples from stallions shedding low virus titres retain sufficient equine arteritis virus to cause infection in mares through artificial insemination.     

Single layer centrifugation of stallion spermatozoa consistently selects the most robust spermatozoa from the rest of the ejaculate in a large sample size

Reasons for performing study: An improvement in sperm quality after single layer centrifugation (SLC) has been seen in previous studies using small sample sizes (for example, n = 10 stallions). There is a need to investigate whether this improvement is repeatable over several breeding seasons with a larger number of stallions (n ≥ 30 stallions). Objective: To make a retrospective analysis of the results of SLC performed on more than 250 sperm samples (176 ejaculates) from 31 stallions in 3 consecutive breeding seasons. Methods: Sperm quality (motility, proportion of morphologically normal spermatozoa and the proportion of spermatozoa with undamaged chromatin) was assessed before and after SLC. Results: All parameters of sperm quality examined were significantly better in sperm samples after SLC than in their unselected counterparts (P<0.001 for each parameter). The yield of spermatozoa obtained after SLC was influenced by the type of extender used and also by the concentration of spermatozoa in the original ejaculate, with fewer spermatozoa being recovered when the loading dose contained a high concentration of spermatozoa. The optimal concentration was approximately 100 × 10⁶/ml. Sperm concentration in the samples loaded on to the colloid influenced the sperm yield while the type of semen extender affected sperm quality and survival. Furthermore, the scaled‐up SLC method was found to be suitable for use with a range of ejaculates, with similar sperm kinematics being observed for standard and scaled‐up preparations. Conclusions: SLC consistently improved the quality of stallion sperm samples from a large number of ejaculates. The method could be scaled‐up, allowing larger volumes of ejaculate to be processed easily from a wide range of stallions.     

Genetic Parameters and Breeding Values for Semen Characteristics in Hanoverian Stallions

The objectives of this study were to show whether semen traits of 30 Hanoverian stallions regularly used in AI may be useful for breeding purposes. Semen characteristics were studied using 15 149 ejaculates from 30 Hanoverian stallions of the State Stud Celle of Lower Saxony. Semen samples were collected between 2005 and 2009. Traits analysed were gel‐free volume, sperm concentration, total and motile sperm number and progressive motility. A linear multivariate animal model was employed to estimate heritabilities and permanent environmental variances for stallions. The same model was used to predict breeding values for all traits simultaneously. Heritabilities were high for gel‐free volume (h² = 0.43) and moderate for total number of sperm (h² = 0.29) and progressive motility (h² = 0.20). Gel‐free volume, sperm concentration and total number of sperm were genetically negatively correlated with progressive motility. The effect of the permanent environment for stallions accounted for 9–55% of the trait variance. The total variance among stallions explained 37–69% of the trait variance. The average reliabilities of the breeding values were 0.43–0.76 for the 30 Hanoverian stallions. In conclusion, the study could demonstrate large effects of stallions, routinely employed in a breeding programme, on semen characteristics analysed here. We could demonstrate that estimated breeding values (EBV) with sufficient high reliabilities can be predicted using data from these stallions and these EBV are useful in horse breeding programmes to achieve genetic improvement in semen quality.     

Epigallocatechin‐3‐gallate (EGCG) and green tea polyphenols do not improve stallion semen parameters during cooling at 4°C

Stallion semen storage for artificial insemination is mainly based on liquid cooled storage. In many stallions this technique maintains sperm quality for an extended period of time (24–72 hr) at 7°C. While this technique is commonly used in the horse industry, there can be a decline in fertility in some stallions, due to an inability of their sperm to tolerate the cool storage process. The aim of the present work was to evaluate the effect of two natural antioxidants (epigallocatechin‐3‐gallate (EGCG) at 20, 60 and 120 μm and green tea polyphenols, and p at .001, .01 and .1 mg/ml) on some sperm parameters (sperm motility, viability/acrosome integrity and DNA quality) in extended semen immediately after its collection (T0) and after 2, 6, 24 and 48 hr of cool storage. Two ejaculates from three trotter stallions were analysed after 48 hr of storage at 4°C. No beneficial effect on the analysed parameters was observed: the two antioxidants were not able to improve sperm quality after 48 hr of storage. These results are in agreement with previous findings on the effect of different antioxidants reported by other researches, who have demonstrated that stallion semen keeps good antioxidant capacity after dilution for 24 hr. In conclusion, the positive effect exerted by antioxidant molecules in other species is not confirmed in the equine one.     

Quality and Fertilizing Ability In Vivo of Sex‐Sorted Stallion Spermatozoa

Little information is available on the quality of stallion spermatozoa after sex sorting. The objectives of the present study were to assess the quality of sex‐sorted stallion spermatozoa and determine its fertilizing ability after hysteroscopic low dose insemination. Ejaculates from four stallions were collected and sorted by a MoFlo SX^® flow cytometer/sperm sorter. Before and after sorting, spermatozoa were evaluated for motility by Computer Assisted Sperm Analysis, viability (SYBR 14‐propidium iodide), mitochondrial function (JC‐1) and acrosomal status (fluorescein isothiocyanate Pisum sativum agglutinin conjugated). A fertility trial was carried out on four mares (seven oestrous cycles) by hysteroscopic insemination, depositing 5 × 10⁶ X‐bearing spermatozoa. Sex sorting resulted in a significant decrease (p < 0.001) in all motility characteristics. Sperm viability and percentage of spermatozoa with functional mitochondria were not affected by the sorting process, while the percentage of reacted spermatozoa was higher (p < 0.01) for non‐sorted than sorted spermatozoa. Pregnancy rate was 28.6% (2/7) after low dose hysteroscopic insemination. Only one pregnancy was carried to term with the birth of a healthy filly. In conclusion, despite the reduction in sperm motility, sex sorting did not impair stallion sperm viability and mitochondrial activity immediately post‐thaw; moreover, the sexed spermatozoa retained the ability to fertilize in vivo.     

Effect of BAPTA-AM on Thawed Stallion Spermatozoa Extended in INRA 96 or Tyrode's Medium

We studied the effect of 1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM) on the outcome of cryopreservation of stallion spermatozoa and whether reextension of thawed sperm in a more physiological and Ca²⁺-containing medium might improve the characteristics of thawed stallion spermatozoa. Individual ejaculates from six stallions were collected and split into three subsamples. The first two samples were supplemented with the membrane-permeable Ca²⁺ chelator BAPTA-AM at final concentrations of 5 and 10 μM, respectively, while the third subsample served as control. After 4 weeks of storage, samples were thawed in a water bath at 37°C and evaluated using flow cytometry and computer-assisted sperm analysis (CASA). In a second experiment, in order to determine whether restoring Ca²⁺ could improve sperm quality after cryopreservation, thawed semen was washed by centrifugation and resuspended in Tyrode's complete medium. BAPTA-AM supplementation did not modify the outcome of cryopreservation; however, changing the spermatozoa from INRA 96 to Tyrode's complete medium resulted in significant improvements in the percentages of live sperm and total motility post thaw.     

Sperm morphology in Estonian and Tori breed stallions

The standard procedure for assessing the breeding potential of a stallion includes the parameter total number of spermatozoa classified as morphologically normal. This study investigated sperm morphology of fresh semen in randomly chosen Estonian (E, n = 8) and Tori (T, n = 7) breed stallions with proven fertility. Two ejaculates were examined from each stallion. An aliquot from each ejaculate was fixed in 1 mL formol-saline immediately after collection and examined with phase-contrast microscope at a magnification 1000x for all types of morphological abnormalities. Furthermore smears were prepared and stained according to Williams (carbolfuchsin-eosin) for a more detailed examination of the sperm heads with light microscope at a magnification 1000x. Analysis of variance was applied to the data, and results are presented as LSmeans (+/- SE). One T stallion that had a disturbance in the spermatogenesis and one 22-year-old E stallion were not included in the analyses. The T stallions had on average 57.5 +/- 4.1% and the E-stallions 74.4 +/- 3.8% morphologically normal spermatozoa (p = 0.012). In 4 of 7 T stallions and 7 of 8 E stallions both ejaculates had > 50% morphologically normal spermatozoa. There was a significant difference between breeds in mean percentage of proximal droplets (17.3 +/- 2.7% and 2.9 +/- 2.5% for T and E stallions, respectively; p = 0.003).     

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.     

Testicular Measurements and Daily Sperm Output of Tori and Estonian Breed Stallions

Evaluation of testicular measurements and daily sperm output (DSO) yields valuable information for predicting the reproductive capacity of stallions. The present study evaluated testicular measurements (height, length, width and circumference) and DSO of eight Tori and eight Estonian breed stallions. One ejaculate of semen was collected daily for 10 subsequent days from each stallion. The gel‐free volume of semen was measured with a graduated glass cylinder and the sperm concentration was assessed with a Chorjajev chamber. The volume of gel‐free fraction was multiplied by the sperm concentration to give the total number of spermatozoa (TSN). The DSO was calculated as mean TSN of collection on days 8–10 in Tori breed stallions and on days 4–10 in Estonian breed stallions. The DSO of Tori breed stallions was 12.9 × 10⁹ spermatozoa and of Estonian breed stallions 4.5 × 10⁹ spermatozoa (p < 0.001). Testicular measurements (in cm) 1 day after the last semen collection were as follows: left testis– height 7.3, length 10.4 and width 7.3 in Tori breed stallions, and 5.9, 8.1 and 5.9, respectively, in Estonian breed stallions; right testis– height 7.4, length 10.6 and width 7.4 in Tori breed stallions, and 5.5, 7.4 and 5.3, respectively, in Estonian breed stallions. All these testicular measurements were significantly smaller in Estonian than in Tori breed stallions (p < 0.001). Testicular circumference was 45.4 and 35.4 cm in Tori and Estonian breed stallions, respectively (p < 0.001). The testicular circumference was correlated with DSO in both Estonian (p < 0.05) and Tori breed stallions (p = 0.071). The results give us valuable information on the reproductive capacity of Tori and Estonian breed stallions.     

Review on Effects of Fescue Grass Ergot Alkaloids in the Horse and Preliminary Study on Effect of Fescue Grass Ergot Alkaloid in the Stallion

Feed with ergot alkaloids ingested by horses has a deleterious clinical and economic impact on the industry. The clinical manifestation of the effects in mares is early embryonic mortality, abortions, prolonged gestation, dystocia, thickened (edematous) or retained placental membranes, agalactia, and increased rates of newborn mortality. For the stallion, very little is known, although ergot alkaloids decrease the ejaculate volume. However, a large number of breeding stallions graze endophyte infected (E+) pastures. This is especially true in the southeastern United States, and clinically we do not perceive that any stallions have any defined problems that could be attributed to ergot alkaloids. However, the number of spermatozoa produced by any stallion might mask the effects. The effects on fertility may be more subtle and only evident with sperm cell manipulation such as chilling or freezing. A preliminary study was performed on six breeding stallions fed feed containing infected fescue seed. We were not able to determine any significant (P < .05) detrimental effects on sperm motility, number morphology, and sperm morphology when compared with controls.     

Application of Techniques for Sperm Selection in Fresh and Frozen-Thawed Stallion Semen

The objective of this research was to improve the techniques in processing chilled and frozen‐thawed horse semen. In a preliminary experiment (Exp. I), different techniques for sperm selection and preparation [Swim‐up, Glass wool (GW) filtration, Glass wool Sephadex (GWS) filtration; Percoll] were tested for their suitability for equine spermatozoa and results were compared with the routine procedure by dilution (Exp. I). In the main experiment (Exp. II), two sperm preparation techniques (GWS, Leucosorb^®) refering to the results of Exp. I and a previous study of our group (Pferdcheilkunde 1996 12, 773) were selected for processing complete ejaculates either for cooled‐storage or cryopreservation. In a third experiment (Exp. III), pregnancy rates from inseminations with semen processed according to the techniques tested in Exp. II were compared with those obtained with semen processed according to routine procedures. In Exp. I (six stallions, six ejaculates/stallion), between 48 and 92% of spermatozoa were lost following the different sperm selection procedures (p < 0.05). Preparation of sperm increased percentage of progressively motile spermatozoa (pms) [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] and decreased percentage of sperm head abnormalities [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] probably by not improving the quality of individual cells, but by elimination of spermatozoa of inferior quality. In Exp. II (eight stallions, three ejaculates/stallion) Leucosorb^® and GWS procedures allowed the filtration of large volumes (extended ejaculates) for routine laboratory practice. GWS and Leucosorb^® filtration resulted in increased motility, membrane integrity and sperm viability after storage of spermatozoa until 48 h at +5°C when compared with control (diluted) and centrifuged semen (p < 0.05). Significantly more spermatozoa were recovered after centrifugation (87.8 ± 15.4%) compared with GWS (63.5 ± 18.6%) and Leucosorb^® filtration (53.6 ± 22.3%). GWS or Leucosorb^® procedure resulted in successful cryopreservation of stallion semen without centrifugation for removal of seminal plasma. The per cycle conception rate of inseminated mares using 200 × 10⁶ pms transferred within 8 h after collection of semen was not affected by GWS filtration or Leucosorb^® separation when compared with centrifugation (n.s.; Exp. III). In conclusion, GWS and Leucosorb^® filtration results in the improvement of semen quality and should be considered as a method for stallion semen processing. Additional studies are needed for the evaluation of potentially higher fertilizing ability of stallion spermatozoa separated by techniques for sperm selection.     

Applicability of a New Cell Culture Device for Cooled‐Storage of Stallion Semen

A new device for storage and shipping of cell cultures – the Petaka G3 cell management device – was tested for its applicability for cooled‐storage of equine semen. Semen from three stallions was processed with EquiPro extender either without antibiotics (three ejaculates per stallion) or with gentamicin (250 mg / l; three ejaculates per stallion). Semen was either stored at five (anaerobic conditions) or 15°C (aerobic conditions) in syringes or cell culture devices. Total and progressive motility, as well as membrane integrity of spermatozoa, were evaluated from days 1 to 7 after collection with computer‐assisted semen analysis. In experiment 1 (extender without antibiotics), total motility, progressive motility and viability of spermatozoa significantly decreased over time (p < 0.05). The decrease was significantly faster at 15°C than at 5°C (p < 0.05). In the presence of gentamicin (experiment 2), this difference was no longer present. It can be concluded that cooled‐storage of equine semen in sophisticated devices for cell culture is not advantageous to syringes for successful maintenance of semen longevity.