Morphology of spermatozoa in semen from stallions of normal fertility.

Semen samples were collected from 3 fertile stallions by means of an 'open' artificial vagina and examined under scanning and transmission electron microscopy. The stallion spermatozoon has many features in common with that of other mammals but differs specifically in that it has an asymmetric head, an abaxial position of the tail and an acrosome of small volume. The presence of microtubules in the neck is also a characteristic of stallion spermatozoa.     

Seasonal changes of semen quality and freezability in Franches-Montagnes stallions

The objective of this study was to investigate seasonal changes of semen quality parameters in Franches-Montagnes stallions and to compare the freezability of ejaculates collected in autumn and winter. Experiments were performed using 15 stallions from the National Stud Farm in Avenches (Switzerland). Ejaculates were collected and evaluated every month during 1 year as well as cryopreserved in autumn and winter (September to February). In fresh semen the gel-free volume, concentration, motility and morphology (normal sperm, major defects, vacuoles and acrosome defects) were evaluated and in frozen-thawed semen the motility as well as the viability (SYBR-14/PI) were performed. To analyse seasonal differences four periods of 3 months each were defined as autumn (September, October, November), winter (December, January, February), spring (March, April, May) and summer (June, July, August). During the 1-year experiment all fresh semen quality parameters demonstrated a clear seasonal and individual pattern. The gel-free volume was significantly (P<0.05) higher in spring and summer compared to autumn and winter while sperm concentration was significantly (P<0.05) lower in spring than at any other time of the year. Total sperm number was significantly (P<0.05) higher and sperm motility significantly (P<0.05) lower in summer than in other seasons. Regarding sperm morphology, normal sperm was significantly (P<0.05) higher in autumn than in winter and summer and major defects were lowest (P<0.05) in autumn. In frozen-thawed semen motility was significantly (P<0.05) improved in the ejaculates collected in autumn compared to winter, while viability showed no obvious differences. Our results clearly demonstrate that individual and seasonal differences occurred in semen quality of Franches-Montagnes stallions. Ejaculates collected in autumn (September, October, November) demonstrated good quality, especially regarding sperm morphology, and were more suitable for cryopreservation because of better motility in frozen-thawed semen collected during autumn than in winter.     

Effect of controlled exercise on semen characteristics in two-year-old stallions

A two-phase study was conducted to evaluate the effect of controlled daily exercise on semen characteristics of 2-yr-old stallions. Eight nonexercised, sexually inexperienced 2-year-old Morgan stallions were randomly assigned to one of two groups. In Phase 1, four stallions were randomly assigned to receive daily controlled exercise for 16 wk. In Phase 2, these stallions were confined to box stalls for 16 wk. The remaining four stallions were confined to box stalls for 16 wk during Phase 1 and received daily controlled exercise for 16 wk during Phase 2. Individual semen collections were obtained from all stallions every 14 days. Daily sperm output (DSO) was determined by daily semen collections for seven consecutive days on Wk 0, 8, 16, 24, and 32. Mean DSO (10(9)) in Phase 1 was 3.52+/-.62 and 2.79+/-.75 for Wk O and 3.12+/-.39 and 2.61+/-.68 for Wk 16 for exercised and nonexercised stallions, respectively. Mean DSO (10(9)) in Phase 2 was 2.61+/-.68 and 3.12+/-.39 for Wk 16 and 2.48+/-.69 and 3.95+/-.51 for Wk 32 for exercised and nonexercised stallions, respectively. No effect (P>.05) of treatment was observed in either phase when parameters of spermatozoa concentration, total spermatozoa per ejaculate, DSO, total volume, gel-free volume, gel volume, percentage motility, percentage live, pH, and percentage normal were measured and analyzed.     

The impact of cushioned centrifugation protocols on semen quality of stallions

The objective was to determine if decreased cushion-fluid volume and increased sperm number during centrifugation, or if sperm concentration of extended semen following centrifugation, affected stallion sperm quality. Three ejaculates from each of three stallions were subjected to cushioned centrifugation (1,000g for 20 min). Cushion-fluid volume was set at 1 or 3.5 ml, and sperm number per centrifuge tube was set 1 billion or 3 billion. Following centrifugation, sperm pellets were resuspended in semen extender containing 20% seminal plasma (v/v) with sperm concentrations of 25 or 250 million/mL. Sperm recovery rate among centrifugation treatment groups was compared. Motion characteristics, plasma membrane intactness (SMI), and DNA quality (COMPαt) of sperm were compared among treatment groups and uncentrifuged controls immediately following centrifugation (Time 0 h) and following 24 h of cooled storage (Time 24 h). Centrifugation treatment did not affect sperm recovery rate (P > 0.05). At Time 0 h, no differences in experimental end points were detected between cushion-fluid volumes tested (P > 0.05). Values for percent total sperm motility, percent progressive sperm motility, and track straightness were similar between sperm-number treatments subjected to centrifugation (P > 0.05). At Time 24 h, values for all experimental endpoints were similar between centrifugation treatments for cushion volume per tube, and between centrifugation treatments for sperm number per tube (P > 0.05). Centrifugation treatments and control treatments were similar for five of six variables tested (P > 0.05). Sperm storage concentrations of 25 × 10⁶ and 250 × 10⁶/mL yielded similar values for percent total sperm motility, percent progressive sperm motility, percent SMI, and percent COMPαt (P > 0.05). A storage concentration of 250 × 10⁶ sperm/ml yielded higher values for curvilinear velocity, and lower values for straightness, than all other groups (P < 0.05). In conclusion, centrifugation with as little as 1 ml of cushion fluid and a sperm number of up to 3 × 10⁹ sperm in 50-ml conical-bottom centrifuge tubes had no detrimental effect on initial or cool-stored sperm quality. Additionally, storage of centrifuged sperm at a concentration of 250 × 10⁶/mL with 20% seminal plasma (v/v) did not have a detrimental effect on percentages of motile or progressively motile sperm, or sperm DNA quality.     

Possible negative effects of inbreeding on semen quality in Shetland pony stallions

Inbreeding is widely believed to negatively affect reproductive performance. Indeed, in some species, high levels of inbreeding are thought to be the major cause of poor semen quality. It is, however, not clear whether inbreeding affects fertility in horses. In this study, the relationship between inbreeding and semen quality was examined in 285 immature Shetland pony stallions submitted for breeding soundness examination in March-April of the years 1992-1997. The majority of stallions examined were 3 years old (85%) and their coefficients of inbreeding ranged from 0 to 25% (mean+/-S.D.: 3+/-4.6%). For the purpose of analysis, stallions were divided into six inbreeding classes (0-1, 1-2, 2-5, 5-8, 8-12 and >12%) containing 132, 40, 42, 27, 25 and 19 animals, respectively. The degree of inbreeding significantly affected many aspects of sperm production and quality, based on a standard examination of two ejaculates collected at a 1.5-3h interval. In particular, coefficients of inbreeding above 2% were associated with lower percentages of motile (p<0.01) and morphologically normal sperm (p<0.001). When the data set was used to estimate heritability of semen characteristics, the high values calculated for sperm progressive motility (0.46) and concentration (0.24) suggested that these traits could be improved by phenotypic selection. These findings support the hypothesis that inbreeding has a detrimental effect on semen quality in Shetland ponies, although examination of multiple ejaculates after repeated semen collection to bring the animals to daily sperm output is needed to confirm this conclusion. Nevertheless, the results support previous suggestions that inbreeding is an important cause of reduced semen quality.     

Effect of a dietary antioxidant supplementation on semen quality in pony stallions

Lipid peroxidation contributes to the damage of the sperm plasma membrane. In different species, dietary supplementation with antioxidants has been shown to improve semen quality. Therefore, we tested effects of dietary supplementation with antioxidants and l-carnitin on semen quality in Shetland pony stallions (n=6). Semen was collected twice a week over a time period of 16 weeks. From weeks 5 to 12, a special diet for stallions containing a variety of antioxidants (STALLION, Pavo Pferdenahrung GmbH, Goch, Germany; tocopherol 300 mg/day; ascorbic acid 300 mg/day; l-carnitin 4000 mg/day; folic acid 12 mg/day) was added to the basal diet (hay, mineral supplements, water). Ejaculates were evaluated for total sperm count, semen motility (percentage of totally and progressively motile spermatozoa, longevity for 24 h at 5 degrees C) and membrane integrity (SYBR-14/PI staining): All values given are means+/-S.E.M. No changes in motility, progressive motility and membrane integrity or semen longevity for 24 h were detected. A slight but significant reduction of morphologically abnormal spermatozoa was found (weeks 1-4: 43.7+/-7.1%; weeks 13-16: 39.4+/-7.2%, p<0.05). Results show that a supplementary diet with antioxidants in the given concentration and duration does not result in pronounced effects on semen quality of stallions. It is therefore questionable to support stallions with dietary antioxidants as long as they receive an adequately balanced basal diet.     

Influence of ejaculation frequency of stallions on characteristics of semen and output of spermatozoa.

Approximately 1 week was required to stabilize the extragonadal sperm reserves in stallions ejaculated daily for 10 weeks. The true daily sperm output of a stallion was equal to the mean daily sperm output of seven ejaculates +/- 1-35 X 10(9) spermatozoa. Mean concentrations of spermatozoa/ml and number of spermatozoa/ejaculate were higher (P less than 0-01) for X1 and X3/week ejaculation frequencies than for a X6/week frequency. Sperm output/week was nearly identical for a X6/week frequency. Sperm output/week was nearly identical for the X3 and X6 frequencies and higher (P less than 0-01) than the X1 frequency. Increase of ejaculation frequency from one to two ejaculates/day twice weekly significantly (P less than 0-01) raised the output of spermatozoa/week. Gel-free semen volume, spermatozoa/ml, and number of spermatozoa/ejaculate were higher (P less than 0-01) in the first, than in the second, ejaculate. Collection of semen on alternate days would be a practical ejaculation frequency for inseminating mares. Two ejaculates collected twice a week would be a practical ejaculation frequency for long-term storage of stallion semen.     

Estrogens and prostaglandin F2alpha in the semen and blood plasma of stallions

A study was performed to determine the levels of estrogens and prostaglandin F(2)alpha in the stallion ejaculate. Simultaneous semen and blood plasma samples were collected from 19 stallions, 2 weeks apart, during the breeding season. Although not statistically different, the total mean estrogen content tended to be higher in seminal plasma (4447 pg/ml) than in blood (2497 pg/ml). A tendency was found for higher mean estrone sulphate concentrations than for total free steroid in both seminal (4116.1 vs 330.5 pg/ml) and blood plasma (2447.1 vs 49.5 pm/ml). Mean concentrations of estrone in ejaculate and blood plasma were 257.1 +/- 267.0 (SD) and 9.5 +/- 5.4 pg/ml, respectively. Estradiol-17beta concentrations were 73.4 +/- 87.4 and 40.0 +/- 27.6 pg/ml in ejaculates and blood plasma, respectively. Mean PGF(2)alpha concentrations tended to be much higher than total estrogens (1106.8 +/- 1636.4, SD, vs approximately 260 ng/ejaculate, respectively). To our knowledge this is the first report of PGF(2)alpha and estrogen concentrations in the stallion ejaculate.     

Sexual behavior of stallions during in-hand natural service and semen collection: an observation in French studs.

The sexual behavior of 42 stallions from French national and private studs was examined in two contexts: semen collection for artificial insemination (AI) and in-hand natural service (NS). Each stallion was observed twice in the same context. Erection and ejaculation latencies, the number of mounts leading to ejaculation, dismount latency and total breeding time were measured and compared between AI and NS. Mount without erection was rare (6/83 observations). Erection latency was 89+/-11s, and was not different between NS (62+/-22s) and AI (100+/-13s, P=0.128). Stallions ejaculated after either one mount (62/83 observations), or two (11/83 observations) or three mounts (10/83 observations). Ejaculation latency was 85+/-15s (84+/-19 in AI and 86+/-28 in NS). If 1st mount did not lead to ejaculation, then ejaculation latency increased several fold following the 2nd mount during both AI and NS. The results provide reference measures for semen collection in French studs. Difference in erection latency between AI and NS, although not statistically significant, may reflect different contributions of excitatory inputs from the brain and the genital area to the activation of spinal networks controlling erection. In contrast, lack of difference in ejaculation latency between AI and NS suggests that the spinal network that controls ejaculation follows a more rigid motor pattern.     

Effect of pentoxifylline treatment on testicular perfusion and semen quality in Miniature horse stallions

The objective was to investigate the effects of pentoxifylline (PTX) on testicular perfusion and sperm production in stallions. In a preliminary study, six mature Miniature horse stallions were given 0, 8.5, or 17.0 mg/kg of PTX orally, twice daily, for 3 d. Total Arterial Blood Flow Rate (TABFR) was higher (P < 0.05) in all treated versus control stallions during and after treatment. Two months later (during the fall and winter), the same stallions received either 0 or 17 mg/kg of PTX orally, twice daily for 60 d. Resistance and pulsatility indices (RI and PI, respectively) decreased in PTX-treated stallions between Treatment 1 and Post-treatment periods. Arterial diameter, as well as Total Arterial Blood Flow (TABF), decreased in controls between Baseline and Treatment 1 (P < 0.05). A similar decrease in arterial diameter was delayed in Group TREATED, but reached significance during Post-treatment (P < 0.05), whereas TABF did not change in this group. Furthermore, TABFR had a transient tendency to increase during Treatment 1 (P < 0.1), whereas it steadily decreased in controls and reached significance in the Post-treatment period (P < 0.05). Both RI and PI were negatively correlated with end diastolic velocity (EDV) in both groups (P < 0.0001). There were positive correlations between RI and peak systolic velocity (PSV) in treated stallions during Treatment 1 (RI: r = 0.53, P = 0.021; PI: r = 0.59, P = 0.007). Also, there were negative correlations between Time Averaged Maximum Velocity (TAMAX) and Doppler indexes in treated stallions during Treatment 2 period (RI: r = −0.49, P = 0.006; PI: r = −0.47, P = 0.008), and during Post-treatment periods (RI: r = −0.40, P = 0.049; PI: r = −042, P = 0.039). Transient hydrocele occurred in all treated stallions (a potential complication of high-dose PTX). Semen end points were not significantly affected by PTX treatment. In conclusion, PTX delayed the seasonal decrease of testicular perfusion in stallions. Sperm quality and quantity were not significantly affected; perhaps they would have been enhanced by prolonged treatment.     

Aversive conditioning of periodic spontaneous erection adversely affects sexual behavior and semen in stallions

Periodic spontaneous erection and penile movements known as masturbation (SEAM) occur normally at approximately 90 min intervals in awake equids. SEAM in horses has traditionally been misunderstood by many horsemen as aberrant behavior that should be eliminated. Accordingly, it is not uncommon for trainers of performance stallions or managers of breeding stallions to punish SEAM in an attempt to eliminate the behavior. Previous clinical observations and preliminary unsystematic trials had suggested that attempts to stop stallion SEAM may lead to an increase rather than a decrease in SEAM, and at the same time may suppress sexual behavior (SB) in a breeding stallion. The present work evaluated the effects of aversive conditioning of SEAM on SEAM, SB, and semen. In Experiment 1, four mature pony stallions were subjected to aversive conditioning of SEAM in a within- and between-subjects half cross-over design. The SEAM erection interval tended to be less after aversive conditioning, suggesting an increase in SEAM frequency. Eleven other SEAM measures were each similar before and after aversive conditioning of SEAM. In standard sexual behavior trials with a stimulus mare and dummy mount, erection latency, ejaculation latency, mount readiness latency, and number of mounts to ejaculation increased after aversive conditioning of SEAM; erection rigidity score, number of ejaculatory pulses, and vocalization rate decreased. Number of thrusts to ejaculation was similar before and after aversive conditioning of SEAM. All affected SB measures indicated suppressed sexual arousal and breeding efficiency after SEAM. In Experiment 1, ejaculated semen was not evaluated. Because in Experiment 1, the number of ejaculatory urethral pulses was less after aversive conditioning, Experiment 2 was similarly designed, but included evaluation of semen, both immediately and again 1 week after aversive conditioning was completed. Experiment 2 included 12 aversively conditioned stallions, and 4 yoked controls. In Experiment 2, masturbation episode duration tended to be less after aversive conditioning, while the remaining 11 SEAM measures were unaffected by aversive conditioning of SEAM. Of SB measures, erection latency, mount readiness latency, thrusts to ejaculation, and ejaculation latency were significantly greater after aversive conditioning. Erection rigidity score and number of ejaculatory pulses were less after aversive conditioning. These differences are consistent with suppressed sexual arousal and reduced breeding efficiency. Semen volume and total number of sperm per ejaculate were significantly less after aversive conditioning. These findings are consistent with clinical anecdotes and preliminary trials indicating that aversive conditioning of SEAM in stallions suppresses sexual arousal and breeding behavior. Of considerable interest both clinically and theoretically, is the finding that aversive conditioning target behavior of SEAM was not suppressed by aversive conditioning, while SB and semen during semen collection trials were both adversely affected.     

Coital exanthema in stallions.

Equine coital exanthema can be produced experimentally in stallions by inoculation with an equine herpesvirus (strain 65/61) and be transmitted during coitus with an infected mare. Serological responses to this infection include the production of complement-fixing and serum-neutralizing antibodies which reach maximum levels 14 to 21 days after infection. Complement-fixing antibodies decline rapidly and are usually not detectable by 60 days after infection, whereas serum-neutralizing antibody activity is maintained for at least 1 year. This disparity provides a useful method for the diagnosis of recent infections and estimation of the temporal incidence of infection in groups of mares. It seems likely that clinically normal carrier mares spread the virus on studs where the disease has previously not occurred.     

Effect of initial freezing temperature, addition of glycerol and dilution on the survival and fertilizing ability of deep-frozen ram semen.

The influence oftemperature, addition of glycerol, initial freezing temperature, method of dilution, level of glycerol in the diluted semen, equilibration time and type of diluent on the survival and fertilizing capacity of deep-frozen according to the best conditions was compared with that of "fresh" semen. The addition of glycerol at plus30 degrees C resulted in a highly significant decrease in the mean proportion of motile spermatozoa immediately after thawing compared with the effect of addition at plus 4 degrees C. The immersion of the straws at minus55 degrees C significantly reduced the revival of the spermatozoa compared with initial freezing at lower temperatures. The exposure time to glycerol had no significant effect on the survival of spermatozoa after thawing and incubation, but fertility was significantly higher with 4% than with 2% glycerol. The I. N. R. A. diluent provided better sperm survival and a significantly higher conception rate than did lactose-egg yolk extender. The semen frozen according to the best conditions (about 50% of the samples) had a fertilizing ability similar to that of "fresh" semen when the proportion of motile spermatozoa before, and after 1 or 3 hr of incubation was equal to or above 45, 40 and 30% respectively.     

Testicular measurements and reproductive characteristics in stallions.

Factors affecting testicular measurements in situ and the relationships among the measurements and various reproductive characteristics were studied using data from 48 stallions. Mean values during the breeding season are provided for scrotal width, widths and lengths of individual testes, combined weight of testicular parenchyma, daily sperm production and daily sperm output. Testicular measurements were highly repeatable from day to day and for repeated measurements on a given day; technician provided the largest source of variation in the measurements of a given stallion. Age significantly affected all testicular measurements; testicular size for 2- to 3-year-old stallions did not differ (P greater than 0.05) from that for 4- to 6-year-olds, but was smaller (P less than 0.05) than testicular size of stallions greater than or equal to 7 years old. Scrotal width was correlated (P less than 0.01) with daily sperm production (r = 0.75) and daily sperm output (r = 0.55) and was generally the most repeatable measurement.     

Effect of semen collection practices on sperm characteristics before and after storage and on fertility of stallions

This study analyzed effects of different methods and intervals of semen collection on the quantity and quality of fresh, cool-stored, and frozen-thawed sperm and fertility of AI stallions. In Experiment 1, ejaculates were obtained from six stallions (72 ejaculates per stallion) using fractionated versus non-fractionated semen collection techniques. Initial sperm quality of the first three jets of the ejaculate was not different from that of total ejaculates. Centrifugation of sperm-rich fractions before freezing improved post-thaw motility and sperm membrane integrity when compared to non-centrifuged sperm-rich fractions or non-fractionated centrifuged ejaculates (P<0.05). In Experiment 2, semen from four stallions (60-70 ejaculates per stallion) was collected either once daily or two times 1h apart every 48 h. The first ejaculates of double collections had significantly higher sperm concentrations, percentages of progressively motile sperm (PMS) after storage for 24h at 5 degrees C and lower percentages of midpiece alterations than single daily ejaculates. Semen collected once daily showed significantly lower values of live sperm after freezing and thawing than the first ejaculate of two ejaculates collected 1h apart every 48 h. In Experiment 3, semen was collected from 36 stallions (> or =12 ejaculates per stallion) during the non-breeding season and the time to ejaculation and the number of mounts was recorded. When time to ejaculation and the number of mounts increased, volume and total sperm count (TSC) also increased (P<0.05), whereas a decrease was observed in sperm concentration, percentage of PMS after storage for 24 h at 5 degrees C, percentage of membrane-intact sperm in fresh semen (P<0.05) as well as motility and percentage of membrane-intact sperm of frozen-thawed sperm (P<0.05). In Experiment 4, AI data of 71 stallions were retrospectively analyzed for the effect of number of mounts per ejaculation and frequency, time interval of semen collections on pregnancy, and foaling rates (FRs) of mares. Semen volume increased, but sperm concentration and percentage of PMS after 24-h cool-storage decreased with increasing number of mounts on the phantom (P<0.05). A statistically significant inter-relationship was demonstrated between frequency and interval of semen collection and FR. Mares inseminated with stallions from which semen was collected frequently (> or =1 on an average per day) showed significantly higher FRs than mares inseminated with semen from stallions with a daily collection frequency of 0.5-1 or <0.5. FR of mares inseminated with stallions having 0.5-1 days between semen collections was significantly better than FR of mares that were inseminated with stallions having semen collection intervals of 1-1.5 days or >2.5 days.     

Optimal concentrations of cryoprotective agents for semen from stallions that are classified 'good' or 'poor' for freezing

Cryopreserved stallion sperm displays a high degree of male-to-male variability with respect to cell viability after thawing. Animals that have semen with low viability after cryopreservation are classified as 'poor' freezers, and when post-thaw viability is high they are designated as 'good' freezers. Cryoprotective agents that are used for cryopreserving stallion sperm include glycerol, ethylene glycol, methyl formamide, and dimethylformamide, and are typically used in concentrations ranging from 1% to 4%. The aim of this study was to evaluate the osmotic stresses that stallion sperm is exposed to during cryopreservation, and to determine if sperm from 'good' and 'poor' freezers show differences in osmotic tolerance limits and in the suitability of cryoprotective agents. Concentrations of 2-3% of the above mentioned cryoprotectants with freezing extender osmolalities ranging from 580 to 895 mOsm kg(-1) showed the highest motility rates after freeze-thaw, both for 'good' and 'poor' freezers, for all cryoprotectants tested with slightly higher values for glycerol. Freeze-thawed semen from 'poor' freezers was found to have a lower percentage of progressively motile sperm compared to that of 'good' freezers. Assessment of plasma and acrosomal membrane integrity after return to isosmotic conditions revealed that cryopreserved sperm from 'poor' freezers showed lower osmotic tolerance limits as compared to sperm from 'good' freezers. Semen from 'poor' freezers that was frozen using freezing extenders supplemented with more then 2% cryoprotectant showed decreased viability and increased acrosome reaction upon return to isoosmotic conditions, whereas 'good' freezers could withstand cryoprotectant concentrations up to 3% before a decline in viability was observed.