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.     

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.     

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.     

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.     

Collection of stallion semen without a mount

Semen collection was attempted from 18 stallions with an artificial vagina (AV) and without use of a mare or dummy mount. After the stallions were sexually excited by the presence of another horse (mares, and in some instances, geldings) an AV was placed over the erect penis. Thirteen of the 18 stallions manifested thrusting and ejaculation in at least one attempt at semen collection. Semen was collected from 12 of the stallions on the first attempt with all subsequent attempts at semen collection from 11 of these 12 stallions being successful. The ejaculate from one of the stallions was judged incomplete based upon spermatozoal concentration compared with the concentration of a subsequent ejaculate obtained with a mount.     

Sexual behavior and serum concentrations of reproductive hormones in impotent stallions

In an attempt to ascertain the cause of abnormal sexual behavior, serum concentrations of hormones were examined in normal (n=7) and impotent (n=7) stallions. Normal stallions achieved an erection (63 ± 19 sec) when exposed to a teaser mare, and mounted (17 ± 6 sec) and ejaculated (38 ± 5 sec) upon subsequent exposure to an estrous mare. In general, impotent stallions did not achieve an erection, mount or ejaculate. Serum concentrations of testosterone in normal and impotent stallions were similar; however, concentrations of luteinizing hormone (LH) were lower (P < 0.05) in impotent than in normal stallions. Further, the serum concentrations of estradiol-17β in impotent stallions were also lower (P < 0.05) than those in normal stallions. Apparently, there is a physiological basis for impotence in stallions involving decreased serum concentrations of LH and estradiol, but not testosterone; however, whether the reduced concentrations of LH and estradiol occur prior to, or result from, behaviorial impotence is not clear.     

Age and seasonal effects on sexual behaviour of stallions at rest

Sexual reactions were studied in stallions kept under uniform stable conditions and having no contact with mares. The stallions were observed continuously for 7 days during the short photoperiod and for 7 days, 6 months later, when the photoperiod was long.The monotonous life and environment of the stable appear to be the main causes of the frequent occurrence of erection and masturbation.Full erection was observed on average 4 times per day per stallion and masturbation attempts on average 4.4 times per day, while the total time of erection per stallion was an average of 38 min per day during the short photoperiod and 45 min during the long photoperiod. Two periods of the day, in the morning and in the evening, were characterized by an increased amount of sexual activity. Full masturbation, ending with ejaculation was observed sporadically, and only in some of the stallions. It was found that erection time tended to be longer during the long-day period, P <0.10.     

Sexual behavior and serum concentrations of reproductive hormones in normal stallions

Recently, it has been reported that impotence in the stallion has a physiological basis that involves decreased serum concentrations of luteinizing hormone (LH) and estradiol-17beta, but not testosterone. We have found such a hormonal profile in two of nine stallions studied during an ongoing investigation of the endocrinology of the normal stallion. Nevertheless, both of these stallions possessed vigorous libido and normal seminal characteristics. We conclude that the hormonal profile of low LH, low estradiol and normal testosterone, although it may accompany impotence in the stallion, is not predictive of, or causally related to, abnormalities in sexual behavior.     

Sociosexual behavior and the ovulatory cycle of ponies (Equus caballus) observed in harem groups

Observations of sociosexual behavior of adult ponies, made on two harem groups (each comprised of one vasectomized male and three females), were correlated with follicular development and ovulation for a total of 15 cycles (minimum of 2 cycles per female). Mean cycle length (interovulatory interval) was found to be 19.7 days, with behavioral estrus lasting 7–8 days (5.5 days preovulatory; 2.3 days postovulatory). Estrous females typically showed increased frequencies of approaching and following the stallion, urinating, presenting, clitoral winking, and tail raising. Approaching and following the stallion appeared earlier and persisted longer than other estrous responses. Deviations from the modal estrous pattern included cycles with subestrus, continual estrus, behavioral estrus in the absence of ovulation, and displays of female mounting. Dominance tests revealed that a mare's status was unaffected by the phases of the estrous cycle. The presence of more than one estrous female affected the copulatory performance of both stallions, most notably in reduced latencies to first mount, intromission, and ejaculation, in spite of differences between the stallions in sexual vigor. Each stallion usually selected the dominant mare for copulation when there were multiple estrous females present, but mounts were not displayed exclusively to one female per test. The social testing situation made apparent the importance of use of space in sociosexual communication in this species, particularly in avoidance of the stallion by diestrous mares and standing alone or in proximity to him by estrous mares.     

Prostaglandins in stallion semen

The purpose of the experiment was to obtain preparatory information about the presence of prostaglandins in semen collected from various types of horses after different periods of sexual rest. Semen was collected with an artificial vagina. Prostaglandin-like activity was estimated by the bioassay procedure described by Vane (1). Results are expressed in ng/ml PGE(2) of seminal plasma. The total concentration of prostaglandins in the full ejaculate averaged 43.73 +/- 4.93 ng/ml of plasma while the total amount of prostaglandins in the ejaculate was 1076 ng. Taking into consideration the period of sexual rest in the stallion, statistically significant differences were found in the prostaglandin level in the semen of all the stallions.     

Insulin-like growth factor-I and insulin-like growth factor binding protein-2 and -5 in equine seminal plasma: association with sperm characteristics and fertility

The objectives of this study were 1) to determine whether insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBPs) were present in seminal plasma of stallions; 2) to compare semen parameters (IGF proteins, sperm numbers, morphology, and motility) from stallions at sexual rest (SR) and when sexually active (SA); 3) to compare semen parameters between stallions with high and low seminal plasma IGF-I concentrations; and 4) to examine the relationship between seminal plasma IGF-I concentrations and fertility parameters of stallions. Ejaculates were collected from stallions at SR (n = 51) and SA (n = 46). Concentrations of IGF-I and IGFBP-2 in seminal plasma samples were determined by radioimmunoassay. Presence of IGFBPs in equine seminal plasma was verified using immunoprecipitation and Western ligand blot procedures. IGF-I, IGFBP-2, and IGFBP-5 were present in equine seminal plasma. Concentrations of IGF-I, IGF-I/protein, total IGF-I, IGFBP-2, IGFBP-2/protein, and total IGFBP-2 were not significantly different (P > or = 0.13) in seminal plasma between stallions at either SR or SA. At SR, stallions with higher seminal plasma IGF-I had more total IGFBP-2 per ejaculate (P < 0.01), more morphologically normal sperm (P = 0.05), and higher first-cycle pregnancy rates (P = 0.02). At SA, stallions with higher seminal plasma IGF-I had fewer cycles per pregnancy (P = 0.02). An association of seminal plasma IGF-I concentration with sperm motility, sperm morphology, and pregnancy rates in bred mares suggests that IGF-I may play a role in sperm function.     

Sexual behavior and ejaculate characteristics in Pêga donkeys (Equus asinus) mounting estrous horse mares (Equus caballus)

The objectives were to (i) characterize sexual behavior of donkey stallions (jacks; Equus asinus) during on-farm semen collection using estrous horse mares (mares; Equus caballus); (ii) compare behavior of young (less experienced) versus older (more experienced) jacks; (iii) determine whether semen suitable for artificial insemination (AI) could be collected using mares; and (iv) determine the suitability of using mares in field collection of semen from jacks. Six Pêga jacks (3.5 to 16 yr old), previously conditioned to breed mares, were used. Mount mares were confirmed in estrus by a teaser horse stallion (stallion) and a jack. Semen was collected with an artificial vagina, at intervals of 48 to 72 h (180 collections). The mean ± SD (young [3.5 yr] vs. old [14 to 16 yr]) were Flehmen response frequency, 7.4 ± 5.8 (8.1 ± 3.0 vs. 7.0 ± 2.0); number of mounts without erection, 1.1 ± 1.3 (2.1 ± 1.4 vs. 1.2 ± 0.4, P < 0.05); latency from first exposure to mare to full erection on the ejaculatory mount, 18.3 ± 17.7 min (25.3 ± 21.3 vs. 12.2 ± 6.2, P < 0.05); latency from erection to insertion, 5.1 ± 3.5 sec (5.3 ± 3.8 vs. 4.8 ± 3.2); and duration of copulation from insertion to dismount after ejaculation, 25.4 ± 7.8 sec (22.1 ± 2.9 vs. 28.1 ± 9.3). In all jacks, sexual behavior was generally normal, with the notable absence of open mouth behavior. Mare estrous behavior was markedly less intense than that in the presence of a stallion and usually absent. Semen characteristics were gel free volume, 47.3 ± 28.7 mL; gel volume, 71.8 ± 54.8 mL; total motility, 84.3 ± 6.0%; progressive motility, 74.3 ± 74.5%; sperm vigor, 3.9 ± 0.5 (scale 1 to 5); sperm concentration, 253 × 10⁶ cells/mL; and total number of sperm, 10.3 × 10⁹ cells. Copulation duration was significantly correlated with gel free volume (r = 0.9) and gel volume (r = 0.7). We concluded that (i) the sexual behavior of jacks during semen collection using mares was similar to that reported for natural mating to jennies, (ii) precopulatory and copulatory behavior for the young (less experienced) jacks and older (more experienced) jacks were generally similar (except number of mounts without erection and latency to full erection); (iii) semen obtained using mares as stimulus and mount females was similar to that reported with estrous jennies; and (iv) semen collection from previously conditioned jacks, using estrous mares, was appropriate for field collection of semen.     

The effect of vesiculectomy on seminal characteristics in the stallion.

Successful unilateral extirpation of an inflamed seminal vesicle in a stallion led to systematic trials of the influence of a reduction and absence of the secretion of this gland upon semen characteristics. Operations were performed by the method described for the bull. The volume of ejaculates dropped and sperm concentration per ml increased in each of 2 stallions from which the seminal vesicles had been uni- or bi-laterally removed. Total sperm number and motility remained uninfluenced, but the percentage of eosin-stained spermatozoa increased in the unilaterally operated stallion and the percentage of abnormal spermatozoa increased significantly in both. Concentration of citric acid per ml and per ejaculate was significantly reduced after bilateral vesiculectomy. Ergothioneine concentration per ml increased in the unilaterally and in the bilaterally operated stallion.     

Disappearance of spermatozoa from the ejaculates of geldings.

Twenty-three geldings were used to determine changes in seminal characteristics following castration and the effect of frequency of ejaculation on these seminal characteristics. In Exp. 1, semen was collected from 8 geldings every other day after castration until the number of spermatozoa per ejaculate was below 1% of the precastration value. An average of 3 ejaculates was required to reduce the number of spermatozoa below this level. In Exp. 2, 15 stallions were castrated and each stallion was assigned to 1 of 3 groups for seminal collection at 7, 14 or 21 days post-castration. The ejaculates collected on these days contained an average of 23, 14 and 2 X 10(6) spermatozoa/ejaculate, respectively. In both experiments, all spermatozoa in ejaculates collected 7 or 8 days after castration were non-motile. Frequency of ejaculation did not appear to hasten the disappearance of spermatozoa from the ejaculates. It is considered that after castration several months may be required before the ampulla and vas deferens become devoid of spermatozoa and the ejaculates azoospermic, and that pregnancy is unlikely to result from mating or insemination 1 week after castration.     

Modulating reproductive activity in stallions: a review

Situations in which suppression or stimulation of reproductive activity in stallions has been attempted, or is desired, include resolution of the equine arteritis virus 'shedding' state, induction of testicular descent in inguinal cryptorchids, and the improvement of sperm production capacity and/or semen quality in sub-fertile stallions. However, the most common reason for wanting to modulate reproductive activity in a stallion is to alter the expression of sexual behaviour. In the case of intact stallions used for competitive or recreational purposes, the overt expression of sexual or aggressive behaviour can be distracting for both animal and owner and, in some cases, dangerous to all concerned. By the same token, a breeding stallion that displays little interest in mounting a mare/phantom, or is slow to achieve erection and/or ejaculation, can be extremely frustrating. This paper reviews the major pharmacological agents reported to usefully modify reproductive activity in stallions, and outlines their pros and cons when compared to training, management or surgical alternatives.     

PGF(2α), LH, testosterone, oestrone sulphate, and cortisol plasma concentrations around sexual stimulation in jackass

Many hormones are involved in the regulation of male reproductive functions, controlling sexual behavior, and influencing sexual arousal, the onset of erection and ejaculation, and the post-ejaculatory detumescence. The aims of this study were to analyze the plasma concentrations of 15-ketodihydro-PGF_2α (PGFM), LH, testosterone (T), oestrone sulphate (OS), and cortisol (C) in relation to sexual stimulation and to evaluate the possible correlations among circulating hormones and between hormones and semen characteristics in the donkey stallion. Thirteen sexually experienced Martina Franca jackass of proven fertility were enrolled and semen was collected through an artificial vagina. Plasma samples were collected at 12, 9, 6 and 3 min before oestrous jenny exposure, at the first erection in the mating arena in the presence of an oestrous jenny, during ejaculation, at dismounting, 3, 6, 9 and 12 min after ejaculation in box, and then every 10 min during the following 50 min. PGFM showed an increasing trend with significant differences between the pre-ejaculatory and post-ejaculatory period, suggesting a role of this hormone in the control of ejaculation. LH showed a significantly higher concentration at ejaculation compared to last samples, while T showed significantly higher levels at erection, ejaculation and dismounting, probably for its influence on these processes and on sexual behavior. Finally, OS did not show any difference in the period of observation, while C presented a significant increase only 22 minutes after erection. The only hormonal correlation found was a positive one between LH and T at erection and dismounting, while T and OS were positively correlated with total and progressive motility, respectively.     

Influence of prostaglandin F2 alpha on sperm production and seminal characteristics of the stallion

Six mature stallions were used to test the effect of prostaglandin F2 alpha (PGF2 alpha ) on sperm production and seminal characteristics. Semen was collected from each stallion twice weekly 1 hr following a 10 mg intramuscular injection of PGF2 alpha or a sham injection. A switchback design was used so that three stallions received PGF2 alpha and three served as controls during the first 9 weeks (period 1). Treatment regimens were reversed during the second 9 weeks (period 2). Treatment of stallions with PGF2 alpha resulted in an increase (P less than .05) in gel free seminal volume and a decrease in sperm cell concentration. Total spermatozoa, sperm cell motility, and percentage of primary and secondary sperm abnormalities of ejaculates were not significantly affected by treatment of stallions with PGF2 alpha before semen collection. All treated stallions exhibited a pronounced sweating response to the drug. During the experiment, two of the six stallions masturbated within 20 to 30 minutes after PGF2 alpha treatment without achieving an erection.     

Imipramine and xylazine-induced ex copula ejaculation in stallions

This study is a part of our ongoing work toward developing pharmacological methods for enhancing and inducing ejaculation in stallions with ejaculatory dysfunction. We evaluated a combination treatment of imipramine hydrochloride followed 10 minutes later by xylazine hydrochloride for the induction of ex copula ejaculation. Eight pony stallions each underwent 6 treatment trials conducted at 4-day intervals. The trials were conducted in the animals' stalls, where they were observed for 90 minutes following treatment. To evaluate the effect of pretreatment sexual stimulation on the rate of ejaculation for each of the 8 stallions, 3 of the 6 trials were preceded by exposure to a restrained ovariectomized pony mare. For 7 of the 8 stallions, 1 to 4 of the 6 trials resulted in induced ejaculation, for a total of 16 ejaculations in the 48 trials. Six of the ejaculations occurred with imipramine treatment alone, before the administration of xylazine. All ejaculations were associated with erection and masturbation. Six of 24 trials (25%) preceded by sexual stimulation resulted in ejaculation, while 10 of 24 trials (42%) without sexual prestimulation resulted in ejaculation. These proportions were not different (P = 0.11). Induced ejaculates were collected into a plastic bag positioned over the prepuce by a girth strap for the comparison of semen characteristics with 2 base line ejaculates obtained in copula from these stallions during the week preceding the series of induced ejaculation trials (with similar 4-day intervals from previous ejaculation). The induced ejaculates were of lower total volume, higher concentration, lower gel volume, higher total numbers of spermatozoa, and lower pH (P < 0.05) than the base line in copula ejaculates. Together, these semen characteristics suggest increased emission of the sperm-rich fraction and reduced emission of accessory gland fluids, probably resulting from imipramine treatment.     

The effect of x-radiation upon the quality and fertility of stallion semen

The exposure that stallion semen might receive during examination using an airport x-ray security screening system was found to be between 0.5 and 1.0 micro Sieverts (μSv). Ejaculates from 2 stallions were diluted 1:4 (volume:volume) using a nonfat dried milk-glucose extender. A total of 6 ejaculates from each stallion was collected, and each ejaculate was divided into 3 aliquots and these were then exposed to x-radiation at a dose of 0, 1.0, or 10.0 (μSv. Semen quamy was examined immediately post exposure, and the aliquots were then placed into a water bath at 37 °C, after which sample longevity was evaluated.In a second trial, 3 groups of 8 pony mares were inseminated with semen that had been exposed to x-radiation at doses of 0, 1.0, or 10.0 μSv. An entire ejaculate was irradiated and inseminated into each mare on one occasion during estrus, based upon ultrasonographic evaluation of the reproductive tract.After exposure to x-radiation there were no differences among the 3 treatment groups for spermatozoal motility, morphology, or longevity. The 14-d pregnancy rates for the 3 treatment groups were 0 μSv (7 mares), 1.0 μSv (8 mares), and 10.0 μSv (7 mares). One mare (0 (μSv) aborted at 65 d of pregnancy; 21 mares had a pregnancy of normal length, with each delivering a foal at term, although 1 foal died at parturition (1.0 μSv).These findings indicate that the exposure of stallion spermatozoa to x-radiation up to doses of 10 μSv does not have deleterious effects upon spermatozoal motility, morphology, longevity or fertility. The exposure received during examination using an x-ray security screening system is likely to be lower than this dose.     

Density gradient centrifugation of sperm from a subfertile stallion and effect of seminal plasma addition on fertility

Stallions are not selected for fertility but for other criteria (pedigree, conformation, performances, progeny), therefore valuable but subfertile stallions with poor semen quality are frequently used in commercial breeding programs. The object of this study was to evaluate whether sperm selection through a silane-coated silica colloid gradient centrifugation, with or without the addition of seminal plasma of a high fertile stallion, could improve the pregnancy rates of an oligospermic valuable stallion in a commercial breeding program. In 2008 breeding season (experiment 1, n=104 mares), simple centrifugation and density gradient centrifugation of the sperm were compared. In 2009 and 2010 breeding seasons (experiment 2, n=125 mares), the effect of the addition of 5% seminal plasma to the extender after sperm selection was evaluated. In all mares deep horn uterine insemination was performed with 1 ml containing 50×10(6) morphologically normal progressive motile spermatozoa, 24-30 h after induction of ovulation with hCG. Pregnancy diagnosis by ultrasonography was performed 14 days following ovulation. Results showed a higher per cycle pregnancy rate (P>0.05) when sperm selection through a density gradient was used (62% vs. 42.3%, exp 1), while the addition of 5% seminal plasma did not influence the outcome (45.9% vs. 47.6%, exp 2) (P>0.05). An age-related decrease in the fertility of the stallion was observed when comparing the results from the different breeding seasons (P<0.05). In conclusion, sperm selection through a discontinuous density gradient enabled a normal per cycle pregnancy rate to be achieved from an oligospermic-subfertile stallion in a commercial breeding program, and no differences were observed regarding the addition of seminal plasma.