Ultrasonographic measurements of accessory sex glands,ampullae, and urethra of normal stallions of various size types

For the purpose of establishing clinical reference values, this paper reports results of ultrasonographic examination and measurement of accessory sex glands, ampullae, and the pelvic urethra of 102 mature, healthy breeding stallions (2-29 years of age) of various size types (7 Miniature Horses, 27 small ponies, 53 light horses and 15 heavy horses). Examinations were done per rectum in mostly unsedated stallions using an Aloka 210 scanner with a 7.5 MHz linear veterinary transrectal transducer (Corometrics Medical Systems, Inc., North Wallingford, CT, USA). Most measures of accessory sex glands, ampullae and the urethra were larger in horses of larger sizes. Except for vesicular glands, the majority of the measures for all glands were smaller for Miniature Horses and ponies than for light horses and heavy horses (P < 0.05). For vesicular glands, measures for heavy horses were greater than for those of other groups (P < 0.05). Measures were similar for Miniature Horses and ponies, and for light horses and heavy horses. For all measures, differences between left and right paired glands were not different (P > 0.10). The lumen diameter of vesicular glands and ampullae as well as prostate lobe thickness showed the greatest asymmetry. Although there were too few representatives of various breeds for statistical comparison, among the light horse breeds Arabian stallions had the smallest mean values for the majority of the measures. Among stallions, echogenic characteristics of accessory sex glands, particularly vesicular glands, varied widely, possibly related to variation in recent sexual activity. For some stallions, echogenic character, particularly that of vesicular glands, varied remarkably from left to right gland within stallions. For ampullae, there was also wide variation in lumen contents between stallions. These data are generally consistent with previous reports with smaller numbers of stallions, as well as consistent with in vitro measures in previous studies. The results provide useful clinical guidelines for size measures of accessory sex glands in horses.     

B-mode ultrasonographic examination of the accessory sex glands of boars

Thorough examinations of the reproductive system of boars are generally not performed on normal boars to be used for breeding; only boars with problems undergo a form of a breeding soundness examination. In order for veterinarians to identify pathological conditions, the normal architecture of the accessory sex glands needs to be described. The purpose of this study was to use B-mode ultrasonography to describe the accessory sex glands in the boar and to see if transrectal ultrasonography would be a viable option in which to obtain this data. Initially, cross-sectional saline bath examinations of accessory sex glands were performed on crossbred boar reproductive tracts (n = 4) using B-mode ultrasonography equipped with a 5 MHz dual frequency linear array transducer. In situ examinations were also performed on terminal line crossbred boars (n = 16) ranging in age from 10 to 23 months old using the same ultrasound methodology; four boars were under general anesthesia and the remaining 12 were standing in crates. Eight boars were abstinent for 2 days and the other eight had ejaculates collected 2 h prior to examination. The paired bulbourethral glands are best described as a long oval gland with a uniformly echogenic appearance with a large anechoic space in the center of the gland extending most of its length. The walls of the vesicular glands were found to be thin, with the parenchyma having multiple small echolucent areas that appeared to merge and form a central canal. The prostate gland was best identified as a pecan-sized gland with a uniform echogenic appearance. Visualization of the prostate gland was accomplished with more proficiency using the saline bath ultrasonography as compared to in situ examinations. All of the accessory sex glands could be examined using both methodologies of ultrasonographic examination with a 5 MHz frequency linear array transducer. It was determined that each accessory sex gland could be recognized, and differences between ejaculated and nonejaculated boars could be identified. The results of this study demonstrate that transrectal ultrasonography can be used as a diagnostic aid in assessing the accessory sex glands of boars.     

Components of stallion seminal plasma and the effects of seminal plasma on sperm longevity

Seminal plasma is a mixture of secretions produced in the testes, epididymides and accessory sex glands, and ejaculated as several consecutive fluid fractions. The composition of seminal plasma and the effects on sperm longevity vary between fractions and individual stallions. This review focuses on the sequence of ejaculation, constituents of seminal plasma and their potential use as fertility markers as well as the influence of seminal plasma on spermatozoa during storage.     

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.     

Oral imipramine and intravenous xylazine for pharmacologically-induced ex copula ejaculation in stallions.

This study is part of ongoing work toward developing pharmacological methods for enhancing and inducing ejaculation in stallions with ejaculatory dysfunction or disabilities that interfere with normal breeding behavior. The objective was to evaluate a treatment regimen involving oral imipramine followed by intravenous xylazine that, in uncontrolled field clinical trials, had shown promise for a higher rate of ejaculation and fewer side effects using a more easily obtained and administered form of imipramine. Eight stallions each underwent eight trials in which treatment consisted of imipramine hydrochloride (3mg/kg, orally in a small portion sweet feed) followed 2h later by xylazine hydrochloride (0.66 mg/kg, intravenously). Trials were conducted with the stallion in a stall. Semen was collected using a collection bag secured over the prepuce with a girth band. Overall, 44 of the 64 attempts (68%) resulted in ejaculation. Within-stallion ejaculation rate ranged from 3 of 8 to 7 of 8 attempts. Interval from xylazine treatment to ejaculation ranged from 1.2 to 14 min. As is typical for induced ejaculations in which imipramine is included in the treatment regimen, ejaculates were of low volume, high sperm concentration, and with a higher total number of sperm than for in copula ejaculates of these stallions. These results represent a modest improvement in rate of ejaculation over previous treatment regimens.     

Seasonal timing of sperm production in roe deer: interrelationship among changes in ejaculate parameters,morphology and function of testis and accessory glands

Roe deer are seasonal breeders with a short rutting season from mid-July to mid-August. The seasonality of reproductive activity in males is associated with cyclic changes between growth and involution of both testes and the accessory sex glands. This study characterizes morphological and functional parameters of these organs prior to, during and after breeding season in live adult roe deer bucks. Size and morphology of the reproductive tract was monitored monthly by transcutaneous (testes, epididymis) and transrectal (accessory glands) ultrasonography. Semen was collected by electroejaculation. Concentration, motility and morphological integrity of spermatozoa as well as the content of proteins and testosterone in semen plasma were evaluated. Proportions of haploid, diploid and tetraploid cells were estimated by flow cytometry in testicular tissue biopsies. Serum testosterone was measured by enzyme immunoassay. Most parts of the male reproductive tract showed distinct circannual changes in size and texture. These changes were most pronounced in the testes, seminal vesicles, and prostate. All reproductive organs were highly developed during the rut only. The volume of ejaculates, total sperm number and percentages of motile and intact spermatozoa also showed a maximum during this period and corresponded with high proportions of haploid cells in the testis. The highest percentages of tetraploid cells were found in the prerutting period. The production of motile and intact spermatozoa correlated with both the protein content of semen plasma and the concentration of testosterone in semen plasma and blood serum. These results suggest the importance of combined actions of the testes and accessory sex glands and the crucial role of testosterone in facilitating the optimal timing of intensified semen production to ensure sufficient numbers of normal spermatozoa in seasonal breeders.     

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.     

Influence of exogenous testosterone on sperm production, seminal quality and libido of stallions.

The effect of exogenous testosterone on sperm production, seminal quality and libido was studied in 24 stallions. Based on pretreatment data, a stallion was assigned to 1 of 3 groups each containing 8 animals. One member of each group received 0 (Group 1), 50 (Group 2), or 200 micrograms (Group 3) testosterone propionate per kg body weight every 2 days for 88 days. The lower dose of testosterone had no significant effect on most of the parameters studied: the higher dose depressed total scrotal width at Day 90 post-treatment (P less than 0.01), total spermatozoa ejaculated between Days 60 and 90 (P less than 0.01) and 96 progressively motile spermatozoa between Days 60 and 90 (P less than 0.10). One half of the stallions from each treatment were castrated on Day 90. In the operated stallions, the mean number of spermatids per g testicular parenchyma in the controls (Group 1) was significantly (P less than 0.05) higher than that in Group 3 whereas the difference between the number of spermatids/testis in the same stallions of these two groups was significant only at P less than 0.1. Testosterone propionate treatment did not influence time to erection, interval from first mount to ejaculation or number of mounts per ejaculation. The treatment of normal, intact stallions with testosterone propionate did not enhance libido and caused a severe depression of reproductive capacity.     

Contribution of the secretory material of caecilian (amphibia: Gymnophiona) male Mullerian gland to motility of sperm: a study in Uraeotyphlus narayani

Caecilians are a unique group of limbless burrowing amphibians with discontinuous distribution. Several caecilian species are viviparous, and all practice internal fertilization. In amniotic vertebrates the sperm undergo post-testicular physiological maturation when they are initiated into motility under the influence of an epididymal secretion. Further, during ejaculation mammalian sperm are suspended in a fluid secreted by the male accessory sex glands, viz., prostate gland and seminal vesicles. Caecilians lack comparable glands, but still practice internal fertilization. Uniquely, male caecilians retain the Mullerian ducts in the adults as a pair of functional glands. It has long been hypothesized, based on indirect evidence, that the Mullerian gland would be a male accessory sex gland, secreting a fluid in which sperm are suspended during ejaculation and which would also provide nutritional support to the ejaculated sperm. In the present study, the secretory material of the Mullerian gland of Uraeotyphlus narayani was mixed with sperm obtained from the testis, and the changes in motility were recorded. Uraeotyphlus narayani sperm possess a perforatorium of the acrosome proceeding deep into the endonuclear canal of the nucleus. The midpiece is characterized by closely applied centrioles, the anterior ends of the axoneme and axial fiber, and a mitochondrial sheath. The long tail has an undulating membrane on one side, supported by the axoneme and an axial fiber. The live sperm possess a mitochondrial vesicle, also known as the cytoplasmic droplet, anywhere along the head and the midpiece, as in anuran sperm, which is shed from sperm that have ceased motility. Uraeotyphlus narayani sperm are motile the moment they are released directly from the testis, indicating that the sperm do not require post-testicular physiological maturation. On being mixed with the secretory material of the Mullerian gland, the spermatozoa are enhanced in speed as well as duration of motility. Therefore, the caecilian male Mullerian gland is considered to be the male accessory sex gland.     

Alkaline phosphatase in stallion semen: characterization and clinical applications

Significant amounts of alkaline phosphatase (AP) activity have been found in semen plasma from numerous species. In species in which the majority of semen plasma AP (SPAP) activity originates from the epididymis and testicle, SPAP activity can be used clinically as a marker to differentiate testicular origin azoospermia or oligospermia from ejaculatory failure. Information on SPAP activity in stallions to date has been limited. In this study, a standard clinical chemistry analyzer was used to determine AP activity in pre-ejaculatory fluid and ejaculates from groups of normal stallions. Additionally, accessory glands, epididymides, testicles and other components of the urogenital tract of normal stallions were assayed to determine which tissues contain SPAP activity. The results indicated that levels of AP activity are low in pre-ejaculatory fluid, but significantly higher in ejaculatory fluid from normal stallions. Spermatozoa were not a significant source of SPAP activity. High levels of SPAP activity were found in the testes and epididymides. These findings suggest that SPAP activity is a candidate for a sperm-independent marker for ejaculation in the stallion. Finally, AP activity was determined in ejaculatory fluid from a stallion with bilaterally blocked ampullae, both before and after relief of the blockage. While the blockage was present, AP activity in ejaculatory fluid was low. However, following relief of the blockage, AP activity in ejaculatory fluid rose dramatically, thus suggesting that AP activity will be useful as an inexpensive, simple clinical assay for differentiating ejaculatory failure or excurrent duct blockages from testicular origin azoospermia and oligospermia.     

Effect of depletion tests (DT) on the composition of boar semen

We conducted two depletion tests during the summer (DT 1) and winter (DT 2) to study their effect on selected biochemical parameters of boar semen. We subjected three boars to DT for 10 consecutive days. The first 3 days (Period 1) of ejaculate collections represented the reserves of the extragonadal spermatozoa and accessory sex gland secretions, whereas the other seven days (Period 2) represented the daily spermatozoa output and the secretory capacity of the accessory sex glands. We observed noticeable changes in the quantity and quality of the semen in DT 1 and 2. There was an increase in the number of spermatozoa with morphological defects, particularly coiled tails and detached acrosomes. The secretory activity of the accessory sex glands, particularly the vesicular glands, was slightly influenced by season. Depletion tests caused disturbances in the qualitative relations of secretions of the accessory sex glands, which were related to changes in the sperm plasmalemma integrity. These tests can be used to determine the total spermatozoa output, and to assess the secretory capacity of the accessory sex glands of boars.     

Development of some male characteristics supported by oestrone but not dehydroepiandrosterone in the boar

Prepubertally castrated boars received subcutaneous injections twice weekly, from 13 to 35 weeks of age, of dehydroepiandrosterone (2 mg/5 kg) or oestrone (1 mg/5 kg). Dehydroepiandrosterone did not support the growth and secretory activity of the accessory organs, or induce copulatory behaviour. However, oestrone caused hypertrophy of the prostate, seminal vesicles and bulbourethral glands which was due to an increase in fibrous stromal tissue and not to the secretory epithelium. Oestrone also induced some male mating behaviour patterns in the presence of an oestrous gilt, although penile extrusion and ejaculation did not occur. The morphological and behavioural effects of the steroid treatments were supported by steroid profiles in blood plasma as seen in comparison with androgen and oestrogen values of intact and untreated castrated boars. It is concluded that oestrogen in the intact boar might enhance the secretion of the accessory organs by affecting the neural control of the secretory processes rather than by increasing the amount of secretory epithelium in the glands.     

Investigations on reproductive physiology in the male Eurasian lynx (Lynx lynx)

This study characterized (in vivo) morphological and functional parameters of reproductive organs of adult male lynx (n = 3) prior to, during, and after the breeding season (n = 3). Size and morphology of the reproductive tract were monitored by transcutaneous (testes) and transrectal (accessory sex glands) ultrasonography. Semen was collected by electroejaculation. Ejaculate volume, sperm number, motility, and morphology of spermatozoa as well as testosterone concentrations in blood serum and feces were evaluated. The testes and prostate had seasonal changes in size and echotexture. The mean (+/- S.D.) maximum and minimum testicular volume were 2.8 +/- 0.8 cm3 and 1.5 +/- 0.3 cm3, respectively. Fecal testosterone concentrations were highest in February (1240 +/- 393 ng/g feces), with a second increase in May (971 +/- 202 ng/g feces), but concentrations were lowest in January (481 +/- 52.9 ng/g feces). Ejaculate volume, total sperm number and percentage of motile, and intact spermatozoa were maximal in March (the middle of the breeding season). In one of the eight litters, multiple paternity was proven; however, in the remaining seven litters, all 16 cubs were sired by the same male. This particular male had the most developed and active testes and best semen quality, which may be important for sperm competition.     

Erectile function and bulbospongiosus EMG activity in estrogen-maintained castrated rats vary with behavioral context.

Electromyographic (EMG) activity in the bulbospongiosus muscles (BS) was recorded to monitor potential castration-induced alterations in muscle activity during copulation and reflexive erections. EMG recordings were made from intact male rats and from castrated rats maintained from 7 to 50 days on estradiol benzoate (300 micrograms/day) or testosterone (200 micrograms/day). Despite a 40-50% postcastration reduction in the weight of the BS and accessory sexual glands in estrogen-treated rats, the pattern of EMG activity during copulation was similar across groups. In estradiol-treated males, the EMG burst frequency during mounts and burst duration during intromissions exceeded the parameters of intact males and of castrated males maintained on testosterone. Between intromissions, and following ejaculatory patterns, estrogen-treated males displayed spontaneous muscle bursts accompanied by visually confirmed erection of the glans penis, but these males quickly lost the capacity for reflexive erections. These data demonstrate that despite castration-induced atrophy of the penile muscles and, presumably, their spinal motor nuclei, the motor output to these muscles is maintained following androgen removal. The capacity for substantial penile erection is retained during copulation long after reflexive erections have diminished.     

Cyproterone acetate diminishes sexual activity in male rabbits.

Cyproterone acetate (CA) was injected daily in eleven rabbits for 3 weeks at a dose of 20 mg/day, and for a further week at a dose of 40 mg/day. After 3 weeks of treatment, the ejaculation frequency was reduced but other measures of sexual behaviour were not significantly changed. There was no reduction in the fructose concentration of the semen, but the volume of the ejaculates decreased. The vesicular glands from the experimental animals showed histological changes typical of those occurring after castration. It was concluded that CA reduced the activity of at least one of the accessory sex glands as well as sexual behaviour. This lends support to the current hypothesis that the endocrine regulation of rabbit sexual behaviour differs from that of the rat.     

玉米螟赤眼蜂对亚洲玉米螟益它素的嗅觉反应

在实验室条件下利用四臂嗅觉仪测定了玉米螟赤眼蜂Trichogramma ostriniae Pang et Chen对来源于寄主亚洲玉米螟Ostrinia furnacalis (Guenée)成虫、卵、附腺以及性信息素的益它素的嗅觉反应。结果表明: 已交配尚未产卵亚洲玉米螟雌蛾及其附腺对玉米螟赤眼蜂有明显的吸引作用,而处女蛾、产卵前期雌蛾、产卵后期雌蛾及其附腺却没有作用;当玉米螟卵块数量为2块及卵表正己烷提取物当量为0.25块卵时,对玉米螟赤眼蜂也有明显的吸引作用,尤其以产在玻璃管内卵块吸引最为显著,达到极显著水平(P<0.01);性信息素组分之一,反-12-十四碳烯乙酸酯(E12.14: Ac)在剂量为1 μg时亦显著刺激了赤眼蜂的活动,表现为进入处理区的次数明显增加,且滞留时间明显延长。     

Differential ubiquitination of stallion sperm proteins: possible implications for infertility and reproductive seasonality

Antibodies against ubiquitin, a universal proteolytic marker, show increased cross-reactivity with defective spermatozoa in men and bulls. We investigated sperm ubiquitination in the stallion, a seasonally polyestrous mammal. Immunofluorescence and immunoelectron microscopy demonstrated that anti-ubiquitin antibodies bind to the surface of both membrane-intact and aldehyde-fixed spermatozoa. Cross-reactivity to the ubiquitin-conjugating enzyme E2 was also detected in sperm. Immunohistochemistry showed that ubiquitinated spermatozoa were first detected in the caput epididymis, coincident with a strong accumulation of ubiquitin and ubiquitin C-terminal hydrolase, protein gene product 9.5, in the apical stereocilia of the epididymal epithelium. Testicular spermatozoa did not display significant ubiquitin cross-reactivity. Similarly, lesser accumulation of ubiquitin cross-reactive substrates was identified in the accessory sex glands. Semen samples were collected from three fertile stallions and one subfertile stallion between December and February and probed for ubiquitin by flow cytometry and immunoblotting. Flow cytometric analysis showed that sperm from the subfertile stallion had higher ubiquitin levels than sperm from the other three stallions. In addition, immunoblot analysis of sperm proteins from the subfertile stallion showed two unique ubiquitin cross-reactive bands that were not present in sperm extracts from the three fertile stallions. To screen for a possible role for ubiquitin in seasonal changes in sperm production, semen samples from two fertile stallions were collected in March, June, September, and December and subjected to a flow cytometric ubiquitin assay. The lowest levels of ubiquitin-labeled sperm were found in March, approximately coincident with the onset of the natural horse breeding season. A progressive increase in sperm ubiquitin levels was found during summer and fall, with a peak in December. These data suggest that stallion sperm are differentially ubiquitinated during epididymal maturation and that this ubiquitination may reflect changes in sperm numbers and semen quality. The association between changes in sperm ubiquitination and seasonal changes in sperm production will be subjected to further studies in a larger cohort of animals.     

Impotence and abnormal sexual behavior in the stallion

Clinical observations have been made on stallions exhibiting impotence and other abnormal forms of sexual behavior. Procedures for diagnosing and retraining consisted of presenting the patient to one or more estrcus mares and observing his sexual behavior. Following sufficient observations, patients were classified into one or more of the following categories: (a) failure to obtain or maintain an erection; (b) incomplete intromission, or lack of pelvic thrusts after intromission; (c) dismounting at onset of ejaculation; (d) failure to ejaculate despite a complete, prolonged erection and repeated intromissions; and (e) ejaculate normally for several ejaculates, then cannot ejaculate without sexual rest, although libido remains high.To successfully diagnose and treat patients with abnormal sexual behavior the following are needed: (a) a variety of mares in estrus; (b) other stallions to provide a competitive environment; (c) a phantom; (d) an artificial vagina and laboratory equipment for seminal evaluation; and (e) extreme patience.Selected clinical cases are presented, including history, diagnosis and treatment with recommendations in relation to reproductive management. Some of the more important observations were: (a) impotence was often diagnosed as aspermatogenesis, due to failure of the impotent stallion to ejaculate; (b) erection was not always essential for ejaculation; (c) pain from injury or disease was often the etiology of abnormal sexual behavior; (d) in excess of 125 ml of semen were collected from a stallion without the occurrence of complete ejaculation; (e) the presence of gelatinous material (gel) in the semen did not necessarily mean complete ejaculation occurred; (f) occasionally seminal emissions from some stallions contained spermatozoa, usually nonmotile, with a large proportion of morphologically abnormal forms, without complete ejaculation.It was concluded that the most common cause of abnormal sexual behavior was mismanagement, such as excessive use, abusive training procedures, injuries during breeding, etc. The majority of the stallions responded well to retraining and recovery was essentially complete without the use of drugs.     

Honey bee males and queens use glandular secretions to enhance sperm viability before and after storage

Internal fertilization requires live sperm to be transferred from male to female before egg fertilization. Both males and females assist the insemination process by providing sperm with glandular secretions, which have been inferred to contain subsets of proteins that maintain sperm viability. Here we show that in the honeybee (Apis mellifera) secretions of the male accessory glands, the major contributors towards seminal fluid, enhance sperm survival. We further demonstrate that the protein fraction of the male accessory gland secretion is indeed important for achieving the maximal effect on sperm survival. After sperm storage, the queens also provide sperm with secretions from spermathecal glands and we show that these secretions have a comparable positive effect on sperm viability. SDS gels show that the proteomic profiles of accessory gland secretion and spermathecal fluid secretion hardly overlap, which suggests that males and females use different proteins to enhance sperm viability during, respectively, ejaculation and final sperm storage.     

Effects of age and frequency of ejaculation on sperm production and extragonadal sperm reserves in stallions.

Extragonadal reserves totalled 89 X 10(9) spermatozoa for 5--16-year-old sexually rested stallions and 60 X 10(9) for 2--4-year-olds. Regardless of age, the cauda epididymidis contained 62% of the total reserves and the vas deferens, including the ampulla, contained 7% of the total reserves of spermatozoa. The caput plus corpus epididymidis from 5--16-year-old stallions (N = 41) contained 14.9 X 10(9) spermatozoa per side as compared (P less than 0.01) to 8.5 X 10(9) for 2--4-year olds (N = 30). Frequency of ejaculation did not influence the number of spermatozoa found in caput plus corpus epididymidis. Across all ages, the number of spermatozoa potentially available for ejaculation from the cauda epididymidis and vas deferens on each side totalled 54 X 10(9). Collection of 5 successive ejaculates from sexually rested stallions removed 40--60% of the available spermatozoa while ejaculation every 2nd day reduced (P less than 0.05) the number of spermatozoa potentially available for ejaculation by 27%. Nevertheless, sufficient spermatozoa are produced daily (6.4 and 4.2 X 10(9) for 5--16-and 2--4-year-olds) to permit use of an average stallion once or twice daily, during spring and summer, providing sexual behaviour is adequate.