Prenatal and postnatal development of Sertoli cells in rams

The morphological picture of Sertoli cells was studied in view of development. The testicular tissue of rams was studied from the 80th to the 140th day of intrauterine development and the testes of ram-lambs were studied from the 1st to the 40th day from birth; further, the testes were studied in adult rams. The development and differentiation of Sertoli cell precursors were examined, including their change into differentiated mature Sertoli cells. The electron-microscopic picture of these cells was described with special emphasis on those morphological changes in cells which are closely related with their functional activity during the development of the testes. The findings obtained so far in this field are incomplete and concern mainly laboratory animals and man. The results of the study extend the theoretical knowledge in the fields of histology, embryology, physiology, endocrinology and andrology. At the same time, they may serve as starting material for the study of the control of spermatogenesis under experimental conditions.     

Comparisons of endocrinological and testis parameters in 18-month-old Ile de France and Romanov rams

Endocrinological and testis parameters of adult 18-month-old Ile de France (IF) and Romanov (Ro) rams were compared during sexual season. Testis weights, total volumes of intertubular tissue, and of blood and lymph vessels, total seminiferous tubule length, rete testis flow rate and daily production of germ cells were significantly higher in IF than in Ro rams. These variations originated from differences in Sertoli cell numbers, which were established before puberty. When daily productions of germ cells, of ABP or of RTF were expressed per Sertoli cell, they were higher in Ro than in IF rams. Quality of spermatids, as measured by their cellular size prior to elongation, was lower in Ro than in IF. The number of FSH-binding sites per Sertoli did not differ between the two breeds but FSH plasma levels were higher in Ro than in IF rams. Total numbers of Leydig cells per testis, their individual size or their LH-binding capacity did not differ significantly between the two breeds. However, the ratio of mean testosterone upon mean LH plasma levels were greater in Ro than in IF rams while both breeds had identical LH mean plasma levels.     

Changes in spermatogenesis and endocrine function in the ram testis due to irradiation and active immunization against luteinizing hormone-releasing hormone

Spermatogonial stem cell transplantation is a technique that has potential in livestock to enhance genetic gain and generate transgenic offspring through the male germ line. A means for depletion of endogenous germ cells in a recipient's seminiferous tubules is necessary for this technology to be applied. The objectives of this study were to evaluate several methods for depletion of endogenous germ cells in the testes of adult rams and to evaluate ultrasound-guided injections into the rete testes as a means for infusing a suspension into the seminiferous tubules. Sixteen adult rams were randomly divided into 4 treatment groups (n = 4 per group). Treatments consisted of active immunization against LHRH (IMM), localized testicular irradiation (IR), LHRH immunization + irradiation (IMM+IR), and untreated control. Serial bleedings were conducted pretreatment and monthly after treatment for 4 mo, at which time all rams were castrated. Both IMM and IMM+IR rams received exogenous gonadotropin in the form of Perganol weekly for 8 wk before castration to bypass the immunization. All rams also received an ultrasound-guided injection of PBS containing 0.4% trypan blue into the rete testis of one testicle before castration. Rams receiving IMM and IMM+IR treatments had higher (P < 0.05) average percentages of seminiferous tubule cross sections with depleted germ cells compared with controls. Serum testosterone was decreased (P < 0.05) in IMM and IMM+IR rams 1 mo after treatment and throughout the remainder of the study compared with controls and IR rams, which were not different from each other. Serum inhibin concentration was unchanged in all rams following treatment indicating that Sertoli cell function was unaltered. A greater (P < 0.05) average percentage of the total testicular area could be filled with the trypan blue solution by rete testis injection in IMM and IMM+IR rams. These data demonstrate the depletion of endogenous germ cells in adult ram testes without alteration of Sertoli cell viability and function that have potential as methods for preparing recipient animals for germ cell transplantation.     

Scrotal enlargement in rams and bucks in Qassim region,central of Saudi Arabia: clinical and ultrasonographic findings and seroprevalence of brucellosis

The aim of this study was to clarify the causes of scrotal enlargement in rams and bucks in Qassim region of Saudi Arabia. Enlarged scrotal contents of rams and bucks (n = 153) were examined by visual inspection, palpation, and ultrasonography. Blood samples were obtained and tested for Brucella sp. infection. Clinical and ultrasonographic findings showed that scrotal enlargement was mainly associated with orchitis, peri-orchitis, and epididymitis. Miscellaneous findings were scrotal hernia, scrotal hematoma, and hydrocele. The frequencies of orchitis, peri-orchitis, and epididymitis were 47.4, 21.1, and 14.1% in Awassi rams; 54.5, 21.7, and 8.7% in Najdi rams; 52.3, 20.5, and 9.1% in Ardi bucks; and 50, 16.7, and 16.7% in Damascus bucks, respectively. Orchitis was associated with no-abscess formation (23%), single-abscess formation (15.4%), and multiple-abscesses formation (61.6%). Peri-orchitis was characterized by hard consistency, atrophy of the testes, and extensive connective tissue formation. Epididymitis was observed mainly at the tail of the epididymis (82.4%) but rarely at the head (17.6%). Epididymitis was associated in many cases with abscessation (70.6%). Males with orchitis, peri-orchitis, and epididymitis were positive for Brucella melitensis and Brucella ovis in the frequency of 21.3% and 48.8%, respectively. In conclusion, scrotal enlargement in rams and bucks in Qassim region is caused mainly by inflammation of the testis and/or epididymis and associated tremendously with brucellosis seropositivity.

     

The epidemiology of bacterial infection of the genitalia in rams

The interrelationship between the various bacteria isolated from the genital tract of rams and their host animals was studied. The pathogenicity of the different isolates varied. Several of these bacteria could be cultured in a medium consisting of a suspension of pen floor debris solidified with agar, while many organisms survived in the suspension for 10 days. Epidemiological investigations showed that rams kept under intensive systems were subjected to large-scale invasion of their genitalia by bacteria which led to infection of the accessory glands and orchitis and epididymitis. Apart from the preputial cavity, some rams kept on open range were entirely free of bacterial infection of their genitalia, and those that did have bacteria in the deeper parts of their genitalia had a very significantly lower incidence of pathological lesions of their genitalia. Finding bacteria and neutrophils in semen is consistent with the epidemiological findings.     

Seasonal changes in spermatogenesis and immunolocalization of inhibin/activin subunits in the wild male ground squirrel (Citellus dauricus Brandt)

The objective of this study was to investigate the seasonal changes in spermatogenesis and the immunolocalization of the inhibin alpha and inhibin/activin (betaA and betaB) subunits during the breeding and non-breeding seasons in the wild male ground squirrel. The testicular weight and size and seminiferous tubule diameter were measured, and histological observations of testes were performed. The sections of the testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin alpha, inhibin/activin betaA and inhibin/activin betaB during the breeding and non-breeding seasons. There were marked variations in testicular weight and size and seminiferous tubule diameter between the breeding and non-breeding seasons, and all types of spermatogenic cells, including spermatozoa, were found in the breeding season. In addition, immunoreactivity was also detected for the inhibin alpha, betaA and betaB subunits in Sertoli and Leydig cells during the breeding season, but immunostaining was only present for the inhibin alpha and inhibin/activin betaB subunits in Sertoli cells during the non-breeding season. These results suggest that seasonal changes in testicular weight and size and seminiferous tubule diameter of wild ground squirrels are correlated with changes in spermatogenesis, and the cellular localization of the inhibin/activin subunits showed season related changes in the breeding and non-breeding seasons.     

Isolation of Actinobacillus seminis from rams in the United Kingdom.

Actinobacillus seminis was isolated from the semen of five rams on four farms. Four of the rams had abnormal semen and three were also infertile. The isolates of A seminis showed similar phenotypic profiles and electrophoretic protein patterns to the type strain of A seminis but were distinct from Histophilus ovis previously isolated from rams with epididymitis in Scotland. The infection appeared to be subclinical but two of the five rams had palpable abnormalities of their testes. Three rams were treated with antibiotics but the infection persisted. No gross lesions were found in the genitalia of two of three rams examined post mortem but one had necrotic abscesses in the testes and epididymis. A seminis was isolated from the seminal vesicles and epididymis of one ram without gross lesions but not from the genitalia of the other two. On one farm the infection in a recently purchased ram led to the detection of another case as a result of the bacteriological screening of 11 stock rams not in contact with the initial case. These five subclinical cases, which included a supposedly healthy stock ram, suggest that A seminis infection may be widespread and should be considered in cases of infertility.     

Malfunction of spermatogenesis in experimental ischemic mice

An experimental ischemia (EI)-induced mouse model was used to analyze pathological and biochemical alterations in testes. Initial morphological changes were observed in Sertoli cells of EI testes at the light microscopic level. Examination of the ultrastructure using transmission electron microscopy confirmed that Sertoli cells were partially detached from the basement membrane of the seminiferous epithelium and that the cell membranes of adjacent Sertoli cells were not joined. The functional integrity of the blood-testis barrier (BTB) was assessed using the lanthanum tracer technique. Lanthanum had penetrated into the spaces between adjacent Sertoli cells in the adluminal compartment up to the lumen of the seminiferous epithelium in EI testes. Proteome analysis showed that the expression of heat shock protein (HSP) 70 was significantly upregulated in EI testes. Western blot analysis confirmed that the expression of HSP70 increased in a time-dependent manner after the EI procedure. HSP70 immunostaining was observed in spermatocytes and in round and elongated spermatids in EI testes. Our results suggest that a change in the junctions between adjacent Sertoli cells on the basal compartment is involved in the BTB disruption in EI testes. Therefore, male infertility caused by the BTB disruption could be associated with heat stress induced by ischemia.     

The Effect of Copper from Industrial Emission on the Seminiferous Epithelium in Rams

Contents: The changes in the testes of adult rams have been followed by methods of light-and electronmicroscopy after intoxication with copper emitted from a copper plant. In the toxic phase of the disease primarily the germinative epithelium was damaged. The breaking or decay of the cellular membrane with consequent devastation or stagnation of the spermiogenesis was observed. The toxic effect of the copper affects irreversibly not only all the cells of spermiogenesis in the developmental stages, but it damages also the Sertoli cells including the components of the interstitium. These changes participated in the impairment of reproduction in the flock under study.     

Gene and protein expression in the reproductive tract of Brazilian Somalis rams

Brazilian Somalis is a locally‐adapted breed of rams raised in tropical climate and native pastures. The present study was conducted to evaluate gene expression and proteome of the reproductive tract of such rams. Samples were collected from testes, epididymides, seminal vesicles and bulbourethral glands of four rams. Expression of clusterin (CLU), osteopontin (OPN) and prostaglandin D2 synthase (PGDS) genes were evaluated in all samples by real‐time PCR. Shotgun proteomic analysis was performed using samples from the head, corpus and cauda epididymides and from all other structures as well. Gene ontology terms and protein interactions were obtained from UniProtKB databases and MetaCore v.6.8 platform. CLU trasncripts were detected in the testes, epididymides, seminal vesicles and bulbourethral glands of the Somalis rams. The initial region and body of the epididymis had the greatest CLU expression. OPN mRNA was localized in all tissues of the ram reproductive tract. PGDS mRNA was detected in the testes and epididymides. Lable‐free mass spectrometry allowed the identification of 137 proteins in all samples. Proteins of the epididymis head mainly participate in cellular processes and response to stimulus, participating in catalityc activity and binding. Proteins of epididymis body acted as regulatory proteins and in cellular processes, with binding and catalytic activity. Cauda epididymis molecules were associated with cellular processes and regulation, with binding function and catalytic activity as well. Testis proteins were mainly linked to cell processes and response to stimuli, and had catalytic function. Seminal vesicle proteins were involved in regulation and mainly with binding functions. Most bulbourethral gland proteins participated in cellular processes. The present study is the first to evaluate the proteome and gene expressions in the reproductive tract of Brazilian Somalis rams. Such pieces of information bring significant cointribution for the understanding of the reproductive physiology of locally‐adapted livestock.     

Antimicrobial therapy for rams with Brucella ovis infection of the urogenital tract.

Rams shedding Brucella ovis in semen but without palpable abnormalities of the epididymides were treated with long-acting oxytetracycline for 15 days and dihydrostreptomycin for 7 days (n = 9) or conventional oxytetracycline and dihydrostreptomycin (n = 9) for 7 days. Nine rams were not treated. More treated rams were considered to have satisfactory breeding soundness examination results at posttreatment weeks 3, 7, 12, and 19. Nontreated rams continued to shed B ovis in semen. After treatment, B ovis was not recovered from 78% of rams given long-acting oxytetracycline and dihydrostreptomycin or from 89% of rams given conventional oxytetracycline and dihydrostreptomycin. At week 21, all rams were euthanatized, and specimens of the testes and epididymides were bacteriologically cultured for B ovis. Brucella ovis was not recovered from the testes of rams or from the epididymides from rams not shedding the organism in the semen. In one treated ram, B ovis was recovered from the semen but not from other tissues. All rams remained ELISA-positive, with the exception of 2 treated rams that ceased shedding B ovis in semen immediately after treatment was started; both these rams became ELISA-negative on the last examination at week 19.     

Early sequential findings in the genitalia of rams experimentally infected with Actinobacillus seminis

AIM: To investigate and assess the sequential pathological changes in the epididymis and testis of young rams injected intra-epididymally with Actinobacillus seminis. METHODS: Twenty yearling Suffolk and Suffolk-cross rams were randomly divided into two groups comprising 16 test and four control animals. Each test ram received 2.3 x 109 cfu/ml of A. seminis injected intra-epididymally. Every 24 h post-inoculation (p.i.), two test rams were randomly selected, euthanised, and necropsied, until the end of the experiment at 192 h p.i. One control animal was euthanised at 24 h, 48 h, 96 h and 144 h p.i., respectively. The reproductive tract of each ram was carefully examined, lesions photographed, and tissues cultured. Thin sections of tissue samples were fixed and examined by light microscopy; additionally, epididymal tissues were examined by scanning electron microscopy (ScEM). RESULTS: Gross lesions were observed in the cauda epididymis of all test rams, and ranged from swelling at 24 h p.i. through enlargement and granuloma formation from 72 h p.i., to gradual enlargement and increasing firmness by 192 h p.i. Gross testicular atrophy was observed in three rams. Histologically, spermatic granulomas were evident in the epididymis and the tunica vaginalis of 10 and four rams, respectively. Cauda epididymitis was present in all rams, and caput and corpus epididymitis in eight and four rams, respectively. Interstitial orchitis was observed in seven, testicular degeneration in 14, and localised and diffuse tunica vaginalitis in 12 rams. Epididymal vasculitis and infiltration of eosinophils were observed as early as 24 h p.i. Moderate disruption of the epididymal duct from 72 h p.i., with subsequent release of spermatozoa into the interstitium, was revealed by ScEM. Actinobacillus seminis was cultured from the granuloma of six test rams from 72 h p.i. CONCLUSIONS: Actinobacillus seminis has the ability to persist in the genitalia of young rams following experimental infection. Suppurative epididymitis is observed as early as 24 h p.i., and spermatic granuloma within 72 h p.i. Infiltration of eosinophils appears to be an early host response to the bacterium, and may play a role in the pathogenesis of the epididymitis.     

Vimentin expression in testes of Arabian stallions

Reasons for performing study: Specific patterns of cytoskeletal filaments reflect a functional state of the cell. In testicular cells intermediate filaments (IFs) are of the vimentin type. Since it is known that Sertoli cells regulate the spermatogenic function in the male gonad, it became important to propose a system that could quantify the state of seminiferous tubular quality. To date, a Johnsen score system has never been used to equine testes. Objectives: To demonstrate the expression pattern of vimentin in testes of mature Arabian stallions and correlate its distribution with grade of seminiferous tubule impairment as indicated by a Johnsen score. Methods: For histological examination by the Johnsen method, routine haematoxylin‐eosin staining was used. Vimentin expression and its presence in testicular sections and testicular homogenates were detected by immunohistochemistry and western blot, respectively. Both analyses were performed qualitatively and quantitatively and further validated by ANOVA tests. Results: Distinct morphology of seminiferous tubules was found in testes harvested from 3 stallions. Vimentin in IFs was immunolocalised to the cytoplasm of Sertoli, Leydig and peritubular‐myoid cells. The intensity and pattern of the IFs staining was different in individual seminiferous tubules suggesting a correlation between vimentin expression and the severity of tubule degeneration. Qualitative results by immunohistochemistry and western blot were confirmed by further quantitative analyses. Conclusions: In equine testes, differential expression of vimentin was found to be correlated with the impairment of seminiferous tubules indicated by a decrease in Johnsen score. Potential relevance: The Johnsen score system may be a useful method to facilitate the identification of tubular alterations in the stallion testes. Combined histological and immunohistochemical approach may provide a detailed phenotypic classification of stallions with decreased fertility.     

Seasonal changes in immunolocalization of inhibin/activin subunits and testicular activity in wild male raccoon dogs (Nyctereutes procyonoides)

Thirty-four pairs of testes from wild adult raccoon dogs (Nyctereutes procyonoides) were obtained between September 2000 and May 2003. The cellular localization of the inhibin alpha and inhibin/activin (betaA and betaB) subunits in wild raccoon dog testes was investigated. The testicular weight and size and seminiferous tubule diameters were measured. There were marked seasonal variations in testicular weight and size and seminiferous tubule diameters, with values relatively low in September and high in March. Spermatogonia and primary spermatocytes were observed in September, and spermatogonia, spermatocytes, and round spermatids were present in January. All types of spermatogenic cells, including mature spermatozoa, were found in March, indicating that the breeding season is around March in Japan. Thereafter, spermatogonia and degenerating spermatocytes were observed in April. The sections of testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin alpha, inhibin/activin betaA and inhibin/activin betaB. The inhibin alpha and inhibin/activin (betaA and betaB) subunits were only expressed in Leydig cells in September. On the other hand, the inhibin alpha, betaA, and betaB subunits were observed in Leydig cells and Sertoli cells, but not in germ cells, in March. These results suggest that the testes of wild raccoon dogs have the ability to synthesize inhibins, and the cellular localization of inhibin/activin subunits showed season-related changes in the breeding and non-breeding seasons.     

Lack of evidence of Brucella ovis infection in rams in Quebec

A study was conducted to estimate the seroprevalence of Brucella ovis infection in rams in the Estrie and Bas-Saint-Laurent regions (Quebec). Rams sera (n = 258) were serologically evaluated from 224 rams in 30 commercial flocks and from 34 rams at 2 slaughterhouses by using an enzyme linked immunosorbent assay. Epididymides and testes were examined by palpation on farms and microscopically for culled rams. No ram was seropositive to Brucella ovis or had lesions suggestive of brucellosis from the farm or slaughterhouse surveys.     

Influence of age and testosterone levels on masculine development in rams

Fifty wethers and 51 spring-born rams were divided into five groups and slaughtered at different seasons of the year at average ages of 271, 361, 459, 557 or 652 d to determine the age and season at which differences in secondary sex characteristics could be detected. Serum testosterone concentrations and testes weights were low in January when the rams were 271 d of age and again in April at 361 d of age. By July, at 459 d of age, testosterone concentrations and testes weights had peaked and then decreased the following November at 557 d and February at 652 d. In contrast with plasma testosterone concentrations and testes weights, buckiness scores, splenius to semimembranosus or semitendinosus muscle ratios, splenius muscle weights and neck and shoulder percentages were not seasonal. All of these measures increased significantly up to July and continued to increase slowly, but not significantly, thereafter. Muscle color and texture scores and rib eye color scores tended to increase in a linear manner for both rams and wethers as age increased. Subcutaneous fat from rams was yellower and softer than that from wethers over all age groups. Ram fat firmness did not change (P greater than .05) with age, and the only significant change in ram fat color was between the groups at 271 and 361 d of age. Overall, season of year coupled with higher levels of serum testosterone was related to initial development of secondary sex characteristics in ram lambs.     

Effects of pre-natal glucocorticoids on testicular development in sheep

We tested the hypothesis that acute pre-natal exposure to high levels of synthetic glucocorticoid (betamethasone) would alter fetal testicular development through actions on gonadal glucocorticoid receptors (GRs). Pregnant Merino ewes bearing singleton male fetuses (n = 24) were allocated randomly among four equal groups to be injected intramuscularly with saline or betamethasone (0.5 mg/kg) either on day 109 of gestation or on both day 109 and day 116 of gestation. Fetal testes were collected at post-mortem, 5 days after each treatment. The volume of interstitial tissue and the volume, length and diameter of the sex cords were measured, and Sertoli cells and gonocytes were counted. For cord volume and interstitial tissue volume, control testes demonstrated maturational changes as fetal age advanced from 109 to 116 days of gestation. For that period, the single injection of betamethasone significantly reduced Leydig cell proliferation (P < 0.05), but had no effect on Sertoli cell numbers. Immunohistochemistry was used to localize GR and proliferating cell nuclear antigen in testicular cells. GR immunoexpression in Leydig cells was higher in fetuses exposed to betamethasone at 109 days of gestation than in control fetuses. Sertoli cells showed low levels of GR. It was concluded that, during mid-gestation, a brief period of glucocorticoid treatment could affect testicular development in male sheep fetuses. The mechanism probably involves direct effects on Leydig cells, as these cells express extra-GR in response to the treatment. Sertoli cells seem to produce less GR than Leydig cells, perhaps explaining their lack of response to betamethasone. These outcomes may have important implications for future fertility in male offspring.     

Epididymal and testicular lesions in rams following experimental infection with Actinobacillus seminis

AIM: To investigate and assess the epididymal and testicular lesions in rams up to 44 days after inoculation with Actinobacillus seminis via various routes. METHODS: Forty-four young (18-24 months old) rams were randomly divided into nine test and two control groups (n=4 per group). The test rams were infected by installation, drenching or injection of A. seminis organisms cultured for 24 h in a brain-heart infusion (BHI) broth containing 2.3 x 10(9) cells/ml, via the following nine routes: intra-epididymal (1 ml), intravenous (3 ml), intra-urethral (3 ml), intra-preputial (3 ml), vas deferens (1 ml), intramuscular (3 ml), oral (10 ml), intranasal (3 ml), and intra-conjunctival (3 drops). All test rams were necropsied 9-44 days post-inoculation (p.i.). Control rams were subdivided into in-contact and non-contact groups and necropsied at 45 and 46 days p.i., respectively. Thin tissue sections were examined for histopathology. RESULTS: Gross lesions were evident only in rams inoculated intra-epididymally. Epididymides on the inoculated side were two to three times larger than those on the un-inoculated side, and the testes attached to the inoculated epididymides were also enlarged. Fibrinopurulent periorchitis and tunica vaginalitis were seen in three rams and atrophy in one. Microscopically, epididymitis was present in 17 (47%) rams, the highest incidence being in the cauda, followed by the caput and the corpus epididymis. Seminiferous tubular degeneration with areas of lymphocytic infiltration were seen in four rams: three inoculated via the cauda epididymis and one via the urethra. No epididymal and/or testicular lesions were seen in rams inoculated via the nasal and conjunctival routes. CONCLUSIONS: Injection of A. seminis in young rams by all routes except intra-conjunctival and intranasal resulted in epididymitis, predominantly in the cauda epididymis. Development of lesions in the reproductive tract following non-genital routes of inoculation supports earlier suggestions that non-venereal transmission of genital actinobacillosis occurs. This study confirmed the predilection of A. seminis for the epididymis, especially the cauda.