Secretion of equine chorionic gonadotropin and its association with supplementary corpus luteum formation and progesterone concentration in Hokkaido native pony recipient mares.

The objectives of this study were to determine the plasma profile of equine chorionic gonadotropin (eCG) and its association with the formation of supplementary corpus luteum (CL) and plasma progesterone concentrations in embryo transfer Hokkaido native pony recipient mares. Blood samples and transrectal ultrasound examination of the reproductive tract were carried out weekly from the day of ovulation until week 32 of gestation (n = 4). Plasma concentrations of eCG and progesterone were measured by enzyme immunoassays. The eCG concentration was first detectable at week 5 for 2 mares and at week 6 for another 2 mares. Immediately after detection, the mean plasma eCG concentrations were observed to rise sharply and reach a peak at week 8. The concentrations then declined dramatically to a baseline (<0.5 IU/mL) by week 21. Plasma progesterone p=p concentrations increased in 2 phases. First, a sharp increase from 0.18 ± 0.05 ng/mL at ovulation to 15.9 ± 4.6 ng/mL at week 1 was observed, then a decrease to 9.69 ± 2.27 ng/mL by week 2, and maintained at this level until week 5 of gestation. The onset of the second rise occurred at week 6 and was observed to peak to 58.3 ± 21.8 ng/mL at week 10, then gradually declined to <10 ng/mL by week 26. The supplementary CLs were first detectable by pregnancy week 6 and 7 for 2 mares each. All supplementary and primary CLs regressed by week 26 for 3 mares and by week 30 for the remaining mare. The mean number of supplementary CL was 4.5 ± 0.8 and their formation in the right ovary (66.7%, 12/18) was higher (P < 0.05) than that in the left ovary (33.3%, 6/18). Among the mares, 1 mare that developed only 2 supplementary CL had 35% lower level of peak eCG and 65% lower concentration of peak progesterone compared with other 3 mares that had 5 or 6 supplementary CL. In conclusion, development of supplementary CL and blood concentrations of progesterone from around day 40 of gestation were associated with eCG concentration. The total number of supplementary CL formation in the present study in embryo transfer Hokkaido native pony recipient mares seemed higher than previously reported supplementary CL number in pregnant mares, with a greater rate in the right ovary than in left.

Evidence for the effect of serotonin receptor 1A gene (HTR1A) polymorphism on tractability in Thoroughbred horses.

Tractability, or how easily animals can be trained and controlled, is an important behavioural trait for the management and training of domestic animals, but its genetic basis remains unclear. Polymorphisms in the serotonin receptor 1A gene (HTR1A) have been associated with individual variability in anxiety-related traits in several species. In this study, we examined the association between HTR1A polymorphisms and tractability in Thoroughbred horses. We assessed the tractability of 167 one-year-old horses reared at a training centre for racehorses using a questionnaire consisting of 17 items. A principal components analysis of answers contracted the data to five principal component (PC) scores. We genotyped two non-synonymous single nucleotide polymorphisms (SNPs) in the horse HTR1A coding region. We found that one of the two SNPs, c.709G>A, which causes an amino acid change at the intracellular region of the receptor, was significantly associated with scores of four of five PCs in fillies (all Ps < 0.05) and one PC in colts (P < 0.01). Horses carrying an A allele at c.709G>A showed lower tractability. This result provides the first evidence that a polymorphism in a serotonin-related gene may affect tractability in horses with the effect partially different depending on sex.

The cross-sectional area changes in digital flexor tendons and suspensory ligament in foals by ultrasonographic examination.

REASONS FOR PERFORMING STUDY: Flexural deformities are common conditions of growing horses and are suggested to have a relationship with the contraction of musculotendinous units. However, limited studies have documented the changes in each tendon and ligament in the metacarpal region with age. OBJECTIVES: To investigate the changes in the cross-sectional area (CSA) of each tendon and ligament in the metacarpal region with age by ultrasonographic examination. STUDY DESIGN: Longitudinal study of foals from Day 1 to age 24 months. METHODS: The CSA of the superficial digital flexor tendon, deep digital flexor tendon, accessory ligament of the deep digital flexor tendon and suspensory ligament was measured by ultrasonographic examination at monthly intervals from Day 1 to age 24 months in 7 Thoroughbred foals. RESULTS: Changes in superficial digital flexor CSA in all regions were larger than those of other structures from 10 months to 15 months. The suspensory ligament CSA was significantly larger than those of other structures on Day 1 in both the region of suspensory origin (RSO) and region of suspensory body (RSBO). This condition continued until 2 months in the RSO and until 5 months in the RSBO. The changes in deep digital flexor CSA were larger than those of other structures from 2 to 5 months in both the RSO and RSBO. CONCLUSIONS: The rate of change in each structure varies with age. Thus, the functional adaptation with age that takes place may differ among structures because the primary function of each structure differs.

Empirical growth curve estimation considering multiple seasonal compensatory growths of body weights in Japanese Thoroughbred colts and fillies.

Thoroughbred horses are seasonal mating animals, and their foals are born yearly in spring seasons. In northern regions or countries, the foals generally show a typical seasonal compensatory growth pattern, where their growth rate declines in winter and increases in the next spring. In this study, a new empirical approach is proposed to adjust for this compensatory growth when growth curve equations are estimated, by using BW of Japanese Thoroughbred colts and fillies raised in Hidaka, Hokkaido. Based on the traditional Richards growth curve equation, new growth curve equations were developed and fit to the weight-age data. The foals generally experience 2 major winter seasons before their debut in horseracing. The new equations had sigmoid subfunctions that can empirically adjust the first and second year compensatory growths, combined with the Richards biological parameter responsible for the maturity of animals. The unknown parameters included in the equations were estimated by SAS NLMIXED procedure. The goodness-of-fit was examined by using several indices of goodness-of-fit (i.e., Akaike's information criterion, Bayesian information criterion, -2 log likelihood, and residual sum of squares) for the multiple applications of the subfunctions. The indices indicated the best fit of the new equations including both subfunctions for the first and second compensatory growths to the weight-age data. The shapes of the growth curves were improved during the periods of compensatory growth. The proposed method is one of the useful approaches for adjusting multiple seasonal compensatory growths in growth curve estimations of Thoroughbreds and for the management of young horses during the compensatory periods.

Expression of inhibin/activin subunits in the equine uteri during the early pregnancy.

The establishment of equine pregnancy is a unique and long process during which a series of physical and possibly biochemical interactions are required between the conceptus and uterus. In this study, we investigated the expression pattern of inhibin/activin subunits in the uterus during early pregnancy. The uteri from four adult mares on cyclic day 13 or pregnancy day 25 were obtained. Immunohistochemical experiments suggested that inhibin/activin subunits were immunolocalized in the luminal and glandular epithelium on pregnancy day 25. In addition, the inhibin α and inhibin/activin ßB subunits were not detected, and inhibin/activin ßA subunit was detected, in the luminal and glandular epithelium on cyclic day 13. Real-time polymerase chain reaction and Western blotting results for the inhibin/activin subunits suggested a significant increase in the expression of inhibin/activin subunit ßB and a significant decrease in the expression of inhibin/activin subunit ßA on pregnancy day 25 compared with those on cyclic day 13. Enzyme-linked immunosorbent assays suggested a significant decrease in the concentration of activin A in endometrium extracts from cyclic day 13 to pregnancy day 25. These results suggest that inhibins or activins synthesized in the uterus, as endocrine factors and necessary nutriments, have different expression patterns and may play different, important roles during early embryonic development of the equine.

Three-dimensional ultrasound imaging of the equine fetus.

The objective was to assess the optimal procedure for real-time, three-dimensional (3D) ultrasound (US) imaging for assessing the equine fetus during the first half of gestation and the possibility of using 3D US imaging of the equine fetus in clinical applications. Seventeen pregnant mares were examined by 3D US between Days 35 and 180 of gestation. Abdominal and endo-vaginal real-time 3D transducers used in human medicine were used for transrectal and transvaginal examinations, respectively. Images were recorded by both 3D stationary and real-time movies. In a comparison of four methods, transrectal examination with a bulb-shaped abdominal 3D transducer enabled the equine fetus to be clearly visualized, and did not require sedation of the mare. Therefore, this approach was the most suitable procedure for examining equine fetuses during the first half of gestation. Each scan required only a few seconds and an entire examination took <10 min in total. The 3D volume image was easy to restore after the examination and could be rotated to any angle the examiner desired. Fetal surface structures, including the head, body, limbs, and genital tubercle, were observed as 3D images which enabled fetal development to be characterized. For early (Days 60-70), but not later (Days 90-150) periods, 3D ultrasonography was not able to evaluate fetal structure in detail as well as conventional 2D ultrasonography. In conclusion, 3D ultrasonography of the equine fetus was a valuable adjunct to 2D ultrasonography and a convenient modality for more detailed assessment of fetal structures.

Pathology of lethal peripartum broad ligament haematoma in 31 Thoroughbred mares.

REASONS FOR PERFORMING STUDY: Broad ligament haemorrhage in peripartum mares is a life-threatening disease and there are few reports on the aetiology and pathogenesis of broad ligament haematoma. OBJECTIVES: To obtain information regarding the sites for the early diagnosis and pathogenesis of broad ligament haematoma of mares. METHODS: Thirty-one mares that died of broad ligament haematoma peripartum were examined pathologically for bleeding sites. The arterial distribution of 5 young mares with several parities served as negative controls. RESULTS: Age and/or multiparity were the predisposing factors for the disease. Arterial injuries were most commonly observed in the uterine artery (24 of 31 mares). Among these, the proximal uterine artery that lies within 15 cm of the bifurcation of the iliac artery was the most frequent site for rupture (18 mares). The lesions occurred preferentially at the bifurcations, lateral part of curvatures and abrupt flexures of the artery. The morphology of the injuries was classified into 3 types: ruptures with and without longitudinal fissures, and transections. Histologically, the arterial wall adjacent to the rupture showed atrophy of smooth muscle cells with fibrosis of the tunica media and disruption and/or calcification of the internal elastic lamina. CONCLUSIONS: Arterial injuries that led to broad ligament haematoma in peripartum mares occurred most frequently in the proximal uterine artery, and atrophy of smooth muscle cells with fibrosis of the arterial wall was as one of the predisposing factors in aged and multiparous mares. POTENTIAL RELEVANCE: Monitoring small aneurysms, mural tearing, medial fibrosis at the proximal uterine artery by transrectal echography could provide useful information for the early diagnosis and possible prevention of broad ligament haematoma of peripartum mares.

Population of follicles and luteal structures during the oestrous cycle of mares detected by three-dimensional internal structure microscopy.

The structure of the equine ovary is different from that of other mammals in its extremely large size, the presence of ovarian fossa and the inverted location of its cortex and medulla. A three-dimensional internal structure microscopy (3D-ISM), which consists of a computer-controlled slicer, a CCD camera, a laser disc recorder and a PC, is very useful for the observation of the internal structures in equine ovaries. In addition, the three-dimensional images of follicles and corpus luteum (CL) reconstructed by the segmentation technique can clarify the spatial arrangement in the equine ovary. In this study, to understand the changes in the ovarian internal structures of the mare during the oestrous cycle, the size and numbers of follicles and luteal structures were analysed by 3D-ISM in addition to the concentrations of progesterone (P(4)) and oestradiol-17beta. As a result, many small follicles (<10 mm in diameter) were detected. It was recognized that the luteal structures were distinguished into three types, such as the corpus haemorragicum (CH), which is formed by blood elements at the cavity after ovulation, CL and corpus albican (CA). There were some CHs and CL in the group, which had the concentration of P(4) > 1 ng/ml. CHs were also observed in the group, which had low level of P(4) (P(4) < 1 ng/ml). CAs were found regardless of the P(4) level. In conclusion, 3D-ISM enabled the internal observation of the ovarian structures in detail, and estimation of the stage of the ovarian cycle with complementary physiological information. The findings by 3D-ISM provide basic information for clinical applications.

Interleukin-1 receptor antagonist expression in the equine endometrium during the peri-implantation period.

To identify factors involved in the establishment of pregnancy in the mare, endometrium was collected from day 13 (day 0=day of ovulation) cyclic and day 13, 19, and 25 pregnant animals. From initial cDNA subtraction studies, interleukin-1 receptor antagonist (IL-1RN) mRNA was found as a candidate molecule expressed uniquely in the pregnant endometrium. Expression of IL-1RN mRNA was markedly increased in day 19 and 25 gravid endometrium. In situ hybridization analysis revealed that IL-1RN mRNA was localized to the glandular epithelium. Interleukin-1 receptor antagonist (IL-1RN) protein was found in the extracts of day 25 gravid endometrium and was immunochemically localized to the glandular epithelium/luminal cavity of the pregnant uterus. High concentrations of estradiol-17beta (E(2)) were detected in day 25 conceptuses. Concentrations of E(2) were higher in the gravid endometrial portion than in other endometrial regions. On the other hand, progesterone concentrations did not differ among endometrial samples analyzed. Furthermore, the expression of IL-1RN mRNA was up-regulated in endometrium culture samples treated with 10 ng/mL E(2) and 10 ng/mL progesterone. In the analysis of related gene expression, increased amounts of IL-1alpha and IL-6 mRNA were also found in the day 25 gravid endometrium; however, these expressions in endometrial culture samples were not up-regulated by the steroid treatment. These results indicate that expression of IL-1RN in the endometrium is likely regulated by E(2) and progesterone and suggest that IL-1RN regulates the degree of IL-1 signal transduction and thereby plays an important role in the establishment of equine pregnancy.

Three-dimensional reconstruction of the equine ovary.

The equine ovary has a very unique structure in terms of its extreme large size, the presence of the ovulation fossa and the inverted location of its cortex and medulla. In the previous study, it was recognized that the application of three-dimensional internal structure microscopy (3D-ISM) to observe the mare ovary is very effective. Three-dimensional reconstruction of serially sliced images made by 3D-ISM was successful in this study with the aid of the sophisticated image processing technique. The rotation of the reconstructed ovary has been carried out with and without the application of the transparency technique in the ovarian stromal region. The spatial localization of follicles and corpus luteum was clearly visualized by rotating the reconstructed image of the ovary. The extraction of the images of follicles and corpus luteum was also available and gave a quantifiable understanding of their structure.

Mechanisms responsible for increase in circulating inhibin levels at the time of ovulation in mares.

In female mammals, inhibin is secreted by the granulosa cells and selectively inhibits secretion of FSH. Although circulating immunoreactive (ir)-inhibin levels decrease after ovulation as a result of the disappearance of its main source, they abruptly increase at the time of ovulation in mares. To investigate the mechanisms responsible for this increase, 50 ml of equine follicular fluid (eFF) was administered into the abdominal cavity of mares during the luteal phase (eFF, n = 4). One hour after treatment, plasma levels of ir-inhibin and inhibin pro-alphaC (but not estradiol-17beta) were significantly higher in eFF-treated mares than in control mares (n = 4). The hormone profiles in eFF-treated mares were similar to those in mares with the spontaneous or hCG induced ovulations. The present study demonstrates that the release of follicular fluid into the abdominal cavity when the follicle ruptures is responsible for the ovulatory inhibin surge in the mare. These findings also suggest that circulating inhibin pro-alphaC may be useful for determining the time of ovulation in the mare.

Equine follicle-stimulating hormone: molecular cloning of beta subunit and biological role of the asparagine-linked oligosaccharide at asparagine(56) of alpha subunit.

Equine FSH (eFSH) and eCG are members of the glycoprotein hormone family. These proteins are heterodimeric, composed of noncovalently associated alpha and beta subunits. We have previously reported that recombinant eCG has potent LH- and FSH-like activities and that the oligosaccharide at Asn(56) of the alpha subunit plays an indispensable role in expressing LH- but not FSH-like activity. In the present study, we cloned eFSH beta subunit cDNA and expressed wild-type recombinant eFSH and a partially deglycosylated mutant FSH (eFSH alpha56/beta) to investigate the biological role of the oligosaccharide at Asn(56) in FSH activity. The wild-type eFSH and eCG stimulated estradiol production in a dose-dependent manner in the primary cultures of rat granulosa cells, indicating that these equine gonadotropins have FSH activity. Partially deglycosylated eCG (eCG alpha56/beta) also stimulated estradiol production, confirming that the FSH-like activity of eCG is resistant to the removal of the N-linked oligosaccharide. Partially deglycosylated eFSH (eFSH alpha56/beta), however, did not show any FSH activity, indicating that the oligosaccharide at Asn(56) was necessary for eFSH. Thus, FSH-like activities of two gonadotropins, eCG and eFSH, are evoked through the distinct molecular mechanisms regarding the biological role of oligosaccharide at Asn(56) of the alpha subunit.

The application of three-dimensional internal structure microscopy in the observation of mare ovary.

The ovary of the mare has a unique structure which differs totally from that of other mammals. However, because of its relatively large size, conventional histological techniques were unsuitable for the observation of the internal structure of the whole ovary. Three-dimensional internal structure microscopy (3D-ISM) consists of a cryotome-CCD camera-laser disc recorder-PC-based control system coupled with a graphic workstation. The internal structure of the ovary is observed by processing over more than 1,000 stored images of serially sliced surfaces of each frozen equine ovary. The 3D reconstruction was done using the full-coloured, volume-rendering method. The relationship between the localization of medulla, cortex and ovulation fossa was clarified. The ovulation fossa is localized in the centre of the ovary and is surrounded by a broad ovarian cortex. A trace of ovulation was observed only at the ovulation fossa. Medulla are localized in narrow peripheral areas. The phenomenon of the competition to occupy the cortical area ahead of the ovulation fossa by developing secondary follicles was visualized. Spatial localization of various sized follicles was identified from 3D-reconstructed images. In this study, it has been clarified that application or this novel computerized technique can clarify the anatomical arrangements of the equine ovary and the complex mechanism of equine follicular development.

In vitro detection of equine arteritis virus from seminal plasma for identification of carrier stallions.

Equine arteritis virus (EAV) was readily isolated in RK-13 cell monolayers by plaque assay from seminal plasma of experimental carrier stallions when they contained high titers of virus regardless of the presence of non-viral cytotoxicity in the seminal plasma. The cytotoxicity interfered with virus isolation from seminal plasma which contained virus at titers less than 10 PFU/ml. However, it was possible to detect the virus in seminal plasma pretreated with PEG (#6000). EAV was consistently identified by RT-PCR from crude seminal plasma which contained virus at titers of more than 10(2.7) PFU/ml. In vitro detection of EAV by virus isolation supplemented with RT-PCR using seminal plasma was proved to be an effective alternative to the standard test mating as a diagnostic method for carrier stallions.

Study of early pregnancy factor (EPF) in equine (Equus caballus).

PROBLEM: Early pregnancy factor (EPF) is an immunosuppressive protein detected in the early pregnancy serum. We have already reported that we developed the rosette inhibition test for mare EPF and detected EPF in thoroughbreds. The aim of this study was to determine whether or not our method could be used clinically. METHODS OF STUDY: The rosette inhibition test for equine EPF was carried out on serum from six nonpregnant and six pregnant Shetland ponies, a female and a male Chinese pony, and four nonpregnant and 13 pregnant thoroughbred mares. In the thoroughbreds sera were collected during the pregnancy period. Furthermore, we measured progesterone and detected pregnant mare serum gonadotrophin (PMSG) in order to confirm pregnancy of the Chinese pony 3 and 6 months after mating. RESULTS: In the nonpregnant Shetland ponies, the rosette inhibition titre (RIT) was 6.0+/-1.0 and EPF was negative. In contrast, in the pregnant ponies, the RIT was 9.2+/-0.4 and EPF was positive. Based on these results, we diagnosed pregnancy of the Chinese pony. The RIT of the female Chinese pony (3 months after mating) was above 10 and EPF was positive. Furthermore, we detected PMSG and progesterone in the serum of this pony. EPF appeared in the maternal blood circulation at 24-72 hr after mating, it was detected until the second trimester, and after that it disappeared from the maternal serum. CONCLUSIONS: The pony's EPF was detected by using the same rosette inhibition test as in the thoroughbred and was present from 24 to 72 hr after mating until the second trimester. The results indicated that our method was useful for pregnancy diagnosis of Equine.

Testicular secretion of inhibin in stallions.

In this study, clear seasonal changes in the circulating concentrations of immunoreactive inhibin were observed in stallions throughout the year. The highest plasma concentrations of immunoreactive inhibin were observed during the breeding season and the lowest concentrations were observed during the nonbreeding season. There was positive correlation between the circulating concentrations of immunoreactive inhibin, steroid hormones and gonadotrophins. Inhibin alpha subunit positive staining was observed in Sertoli cells and more clearly in Leydig cells. Both cell types also stained positively for inhibin betaA and betaB subunits. Immunoreactivity to 3beta-hydroxysteroid dehydrogenase and aromatase was observed in the Leydig cells only. The testes contained large amounts of inhibin pro-alphaC and small amounts of inhibins A and B, and the inhibin B content was higher than the inhibin A content. These results indicate that inhibin B is the major bioactive form of inhibin in equine testes. However, only inhibin pro-alphaC was detectable in the circulation and the inhibin pro-alphaC concentrations changed with the concentrations of immunoreactive inhibin. In conclusion, both Leydig and Sertoli cells secrete testicular inhibin in stallions. The positive correlation observed between plasma immunoreactive inhibin and inhibin pro-alphaC concentrations and testicular activity during the breeding and nonbreeding seasons indicates that the plasma concentrations of these hormones are potentially useful indicators of testicular activity in stallions.

Ovarian secretion of inhibin in mares.

In mares, circulating immunoreactive inhibin concentrations increase during the follicular phase and decrease at the start of the LH surge. Thereafter, sharp increases in circulating immunoreactive inhibin concentrations, the 'ovulatory increase', are observed during ovulation. In the present study, the cellular sources and molecular form of ovarian inhibin were investigated to determine the mechanism responsible for this unique ovulatory increase. Three sizes of ovarian follicles (small, < 15 mm; medium, 15-30 mm; large, > 30 mm in diameter) were selected. Inhibin alpha-subunit was localized by immunohistochemistry to the granulosa cells of follicles of all sizes and the theca cells of large follicles, whereas inhibin betaA- and betaB-subunits were detected in the granulosa and theca cells of large follicles only. High concentrations of immunoreactive inhibin, inhibin pro-alphaC and inhibin A were detected in the follicular fluid of large follicles compared with small and medium follicles, whereas there were no significant differences in the concentrations of inhibin B in the follicular fluid of medium and large follicles. These results indicate that mature large follicles secrete large amounts of inhibins pro-alphaC and A, whereas small or medium follicles secrete small amounts of inhibins A, B and pro-alphaC. These findings also indicate that the large amount of inhibin pro-alphaC produced by the ovulatory follicle is the source of the ovulatory increase in the concentrations of circulatory immunoreactive inhibin observed during ovulation in

A selective increase in circulating inhibin and inhibin pro-alphaC at the time of ovulation in the mare.

The relationship between a selective increase in circulating immunoreactive (ir)-inhibin and the time of ovulation was investigated in mares. Concentrations of plasma ir-inhibin were measured every 4 h during the periovulatory period. Inhibin pro-alphaC, a precursor protein of the inhibin alpha-subunit, was also measured. The changes in ir-inhibin and inhibin pro-alphaC in circulation were parallel. Concentrations of both ir-inhibin and inhibin pro-alphaC in the plasma increased at the same time when ovulatory follicles ruptured, and the peak levels of circulating ir-inhibin and inhibin pro-alphaC were maintained for 4-8 h. There was no selective increase in plasma concentrations of estradiol-17beta during the process of ovulation. These results suggest that the selective increase in ir-inhibin and inhibin pro-alphaC was caused by the absorption of follicular fluid after the rupture of ovulatory follicles. These results also suggest that the measuring of plasma concentrations of ir-inhibin or inhibin pro-alphaC in mares might be a useful method for detecting the time of ovulation.

Morphological study on pigmented cells in the horse testis.

One of the most attractive characteristics of a horse testis is the change of the weight during development. As the testicular weight changes and the number of Leydig cells decreases, pigments appear in interstitial tissues. In the present study, the characteristics of the pigments found in the interstitial tissues were examined histochemically and ultrastructurally. Specific stainings indicated that the pigmented granules showed almost all of the histological and histochemical characteristics of ceroid or ceroid-like pigment. The cells showed positive reaction for acid phosphatase while the pigmented cells contained a lot of lysosomes ultrastructurally. These results suggest that macrophages might phagocytize Leydig cells, and store their digested materials as ceroid-like pigment.

Effects of anabolic steroid (19-nortestosterone) on the secretion of testicular hormones in the stallion.

The aim of this study was to clarify the effect of anabolic steroids on the testicular endocrine function of mature stallions. Mature thoroughbred stallions were treated with 800 mg nandrolone decanoate every 3 weeks for 3 months. After the first treatment, plasma concentrations of LH, immunoreactive inhibin and testosterone decreased rapidly to the nadir. These hormones were maintained at significantly lower concentrations compared with concentrations in intact stallions. Histology of the testicular tissue indicated the arrest of advanced spermatogenesis in the seminiferous tubules and a severe depletion of the number of Leydig cells in the interstitial compartment as a result of treatment. Most of the immunopositive cells for the inhibin alpha-subunit and steroidogenesis enzymes in the interstitial compartment decreased below detectable amounts, whereas immunopositive reactions of inhibin alpha-subunit in the seminiferous tubules were clearly observed. In conclusion, the treatment of mature stallions with nandrolone decanoate caused a decrease in the secretion of ir-inhibin and testosterone from the testis, the depletion of the number of Leydig cells and a decrease below detectable amounts of inhibin alpha-subunit and steroidogenesis enzymes. The concentration of ir-inhibin in the peripheral blood may be a useful marker for the examination of testicular activity in stallions being treated with anabolic steroids.