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In cattle, mating to intact, but not vasectomised, bulls has been shown to modify the endometrial transcriptome, suggesting an important role of sperm in the modulation of the uterine environment in this species. However, it is not clear whether these changes are driven by intrinsic sperm factors, or by factors of accessory gland (AG) origin that bind to sperm at ejaculation. Therefore, the aim of the present study was to determine whether ejaculated sperm, which are suspended in the secretions of the AGs, elicit a different endometrial transcriptomic response than epididymal sperm, which have never been exposed to AG factors. To this end, bovine endometrial explants collected from heifers in oestrus were (co-)incubated for 6 h alone (control), or with epididymal sperm or ejaculated sperm, following which transcriptomic changes in the endometrium were evaluated. Epididymal sperm elicited a more dramatic endometrial response than ejaculated sperm, in terms of the number of differentially expressed genes (DEGs). Indeed, RNA-sequencing data analysis revealed 1912 DEGs in endometrial explants exposed to epididymal sperm compared with control explants, whereas 115 DEGs were detected between endometrial explants exposed to ejaculated sperm in comparison to control explants. The top pathways associated with genes upregulated by epididymal sperm included T cell regulation and TNF, NF-KB and IL17 signalling. Interestingly, ejaculated sperm induced downregulation of genes associated with T cell immunity and Th17 differentiation, and upregulation of genes involved in NF-KB signalling, in comparison to epididymal sperm. These data indicate that factors of AG origin modulate the interaction between sperm and the endometrium in cattle.

Beca predoctoral de Pequeño financiada por la AEI (PRE2018-085637), Context: In the epididymis, epithelial cells manage changes in the luminal environment for proper sperm maturation. Moreover, aquaglyceroporins, a subgroup of aquaporins (AQP), modulate the transport of water, glycerol and other small molecules in epithelial cells. Aims: We aim to characterise the lining epithelium, quantify its cell composition and immunolocalise the aquaglyceroporins AQP3, AQP7, AQP9 and AQP10 alongside the epididymal ductus of three wild ruminant species, and to determine if species-specific differences could be associated with cauda sperm cryoresistance variations. Methods: Epididymides from Iberian ibex (n =5), mouflon (n =5) and chamois (n =6) were obtained. Cauda spermatozoa were collected and sperm parameters were analysed before and after freezing. Histology and immunohistochemistry of AQP3, 7, 9, 10 and T-CD3 were performed in the caput, corpus and cauda epididymal regions. Key results: This work first describes the lining epithelium in Iberian ibex, mouflon and chamois epididymis along the three anatomical regions, consisting of principal, basal, apical, clear and halo cells. However, the percentage of each cell type differed in ibex compared to mouflon and chamois. The positive T-CD3 immunolabeling of all the halo cells confirmed their T-lymphocyte nature. Aquaglyceroporin expression patterns were similar among species, except for differences in AQP7 and AQP10 immunolocalisation in ibex. Species-specific differences in epididymal sperm cryoresistance were confirmed. Conclusions: The epididymal epithelium of the three wild ruminants differ in their relative number of cell types and AQP immunolocalisation, which ultimately appears to affect cauda epidydimal spermatozoa cryoresistance. Implications: Our study provides information on the relevance of the quantitative composition and AQP pattern expression in epididymal lining epithelium on sperm cryoresistance., Agencia Estatal de Investigación (AEI), Depto. de Medicina y Cirugía Animal, Fac. de Veterinaria, TRUE, pub

Reports that mouse sperm gain small RNAs from the epididymosomes secreted by epididymal epithelial cells and that these "foreign" small RNAs act as an epigenetic information carrier mediating the transmission of acquired paternal traits have drawn great attention because the findings suggest that heritable information can flow from soma to germ line, thus invalidating the long-standing Weismann's barrier theory on heritable information flow. Using small RNA sequencing (sRNA-seq), northern blots, sRNA in situ hybridization, and immunofluorescence, we detected substantial changes in the small RNA profile in murine caput epididymal sperm (sperm in the head of the epididymis), and we further determined that the changes resulted from sperm exchanging small RNAs, mainly tsRNAs and rsRNAs, with cytoplasmic droplets rather than the epididymosomes. Moreover, the murine sperm-borne small RNAs were mainly derived from the nuclear small RNAs in late spermatids. Thus, caution is needed regarding sperm gaining foreign small RNAs as an underlying mechanism of epigenetic inheritance.

Single-cell RNA sequencing has recently led to the identification of a flurry of rare, new cell types, such as the CFTR-high ionocytes in the airway epithelium. Ionocytes appear to be specifically responsible for fluid osmolarity and pH regulation. Similar cells exist in multiple other organs and have received various names, including intercalated cell in the kidney, mitochondria-rich cell in the inner ear, clear cell in the epididymis, and ionocyte in the salivary gland. Here, we compare the previously published transcriptomic profile of cells expressing FOXI1, the signature transcription factor expressed in airway ionocytes. Such FOXI1+ cells were found in datasets representing human and/or murine kidney, airway, epididymis, thymus, skin, inner ear, salivary gland, and prostate. This allowed us to assess the similarities between these cells and identify the core transcriptomic signature of this ionocyte 'family'. Our results demonstrate that, across all these organs, ionocytes maintain the expression of a characteristic set of genes, including FOXI1, KRT7, and ATP6V1B1. We conclude that the ionocyte signature defines a class of closely related cell types across multiple mammalian organs.

Background: During epididymal transit spermatozoa acquire specific morphological features which enhance their ability to swim in a progressive manner and interact with the oocytes. At the same time, sperm cells undergo specific molecular rearrangements essential for the fertilizing sperm to drive a correct embryo development. To assess epigenetic sperm changes during epididymal maturation, the caput, corpus and cauda epididymis sperm tracts were isolated from eight bulls and characterized for different sperm quality parameters and for CpG DNA methylation using Reduced Representation Bisulfite Sequencing (RRBS) able to identify differentially methylated regions (DMRs) in higher CpG density regions. Results: Caput sperm showed significant variation in motility and sperm kinetics variables, whereas spermatozoa collected from the corpus presented morphology variation and significant alterations in variables related to acrosome integrity. A total of 57,583 methylated regions were identified across the eight bulls, showing a significantly diverse distribution for sperm collected in the three epididymal regions. Differential methylation was observed between caput vs corpus (n = 11,434), corpus vs cauda (n = 12,372) and caput vs cauda (n = 2790). During epididymal transit a high proportion of the epigenome was remodeled, showing several regions in which methylation decreases from caput to corpus and increases from corpus to cauda. Conclusions: Specific CpG DNA methylation changes in sperm isolated from the caput, corpus, and cauda epididymis tracts are likely to refine the sperm epigenome during sperm maturation, potentially impacting sperm fertilization ability and spatial organization of the genome during early embryo development.

The adhesion receptor ADGRA3 (GPR125) is a known spermatogonial stem cell marker, but its impact on male reproduction and fertility has not been examined. Using a mouse model lacking Adgra3 (Adgra3(-/-)), we show that 55% of the male mice are infertile from puberty despite having normal spermatogenesis and epididymal sperm count. Instead, male mice lacking Adgra3 exhibited decreased estrogen receptor alpha expression and transient dilation of the epididymis. Combined with an increased estradiol production, this indicates a post-pubertal hormonal imbalance and fluid retention. Dye injection revealed a blockage between the ejaculatory duct and the urethra, which is rare in mice suffering from infertility, thereby mimicking the etiologies of obstructive azoospermia found in human male infertility. To summarize, male reproductive tract development is dependent on ADGRA3 function that in concert with estrogen signaling may influence fluid handling during sperm maturation and storage.

The adhesion receptor ADGRA3 (GPR125) is a known spermatogonial stem cell marker, but its impact on male reproduction and fertility has not been examined. Using a mouse model lacking Adgra3 (Adgra3(-/-)), we show that 55% of the male mice are infertile from puberty despite having normal spermatogenesis and epididymal sperm count. Instead, male mice lacking Adgra3 exhibited decreased estrogen receptor alpha expression and transient dilation of the epididymis. Combined with an increased estradiol production, this indicates a post-pubertal hormonal imbalance and fluid retention. Dye injection revealed a blockage between the ejaculatory duct and the urethra, which is rare in mice suffering from infertility, thereby mimicking the etiologies of obstructive azoospermia found in human male infertility. To summarize, male reproductive tract development is dependent on ADGRA3 function that in concert with estrogen signaling may influence fluid handling during sperm maturation and storage.

In the epididymis, epithelial cells manage changes in the luminal environment for proper sperm maturation. Moreover, aquaglyceroporins, a subgroup of aquaporins (AQP), modulate the transport of water, glycerol and other small molecules in epithelial cells.

L’épididyme est un organe du système reproducteur masculin permettant le transport et la maturation des spermatozoïdes. Il est constitué d’une lumière, où transitent les spermatozoïdes et d’un épithélium composé majoritairement de cellules principales qui entourent la lumière mais aussi d’autres types cellulaires dont les cellules basales. Plusieurs données indiquent que des cellules souches (CS) pourraient être présentes dans l’épididyme, cependant leur existence est une controverse depuis de nombreuses années. L’hypothèse émise dans ce projet de recherche est qu’il existe une population de CS, localisée à la base de l’épithélium, au sein de l’épididyme. Les objectifs sont la démonstration de la présence de CS dans l’épididyme, la création d’un modèle d’organoïde d’épididyme de rat et l’identification d’un marqueur spécifique associé à ces cellules. La mise en culture en 3D de cellules basales d’épididyme de rats a permis d’observer la formation d’organoïdes exprimant plusieurs marqueurs de cellules basales. Ces organoïdes sont formés à partir d’une seule cellule et se maintiennent sur plusieurs générations démontrant la capacité d’auto-renouvellement des cellules basales. Après 14 jours de culture, l’apparition de cellules principales au sein des organoïdes a été observée par immunofluorescence, par microscopie électronique ainsi que par analyse de l’expression génique confirmant ainsi la capacité de différenciation des cellules basales. La présence de clusterine, une protéine impliquée dans la maturation des spermatozoïdes a également été observée au sein des organoïdes d’épididyme. Un marqueur exprimé par les CS adultes de nombreux organes, nommé LGR5, identifie une population spécifique de cellules localisées à la base de l’épithélium dans tous les segments de l’épididyme de rat adulte. La co-localisation de LGR5 et p63, un facteur de transcription spécifique aux cellules basales, nous a également permis de distinguer la présen

The concern of canine overpopulation is related to zoonoses such as rabies, which is responsible for 99% of human rabies cases, causing the death of approximately 59,000 people per year. Surgical sterilization is an effective, costly and invasive control method with limited impact. The effects of phytoestrogens on reproductive activity have been commonly studied in females and a limited number in males. Therefore, the objective of this study was to know the effect of subcutaneous administration of coumestrol on the gonadal activity of adult dogs as an alternative for their reproductive control. Basic seminal evaluation parameters, epididymal structure by ultrasonography, histological characteristics, as well as the presence of coumestrol by fluorescence and serum levels of testosterone and estrogens were determined. The administration of coumestrol for five weeks reduced sperm production, and evidenced changes in the echodensity and cellularity of the epididymis, associated with serum concentrations of estradiol and testosterone. Therefore, it is concluded that coumestrol administered subcutaneously has an estrogenic effect that can be used as a non-invasive method to help control fertility in adult dogs., La preocupación de la sobrepoblación canina se relaciona con zoonosis como la rabia, la cual es responsable del 99% de los casos de rabia humana, causante de la muerte de aproximadamente 59,000 personas al año. La esterilización quirúrgica es un método de control eficaz, costoso e invasivo siendo su impacto limitado. Los efectos de los fitoestrógenos en la actividad reproductiva se han estudiado comúnmente en hembras, y un número limitado en machos. Por lo anterior, el objetivo de este estudio fue conocer el efecto de la administración subcutánea de coumestrol en la actividad gonadal de perros adultos como alternativa para su control reproductivo. Se determinaron los parámetros de evaluación seminal básica, la estructura del epidídimo mediante ecografía, características histológicas, así como la presencia del coumestrol mediante fluorescencia y los niveles séricos de testosterona y estrógenos. La administración de coumestrol durante cinco semanas redujo la producción espermática, y evidenció cambios en la eco densidad y celularidad del epidídimo, asociados a las concentraciones séricas de estradiol y testosterona. Por lo que, se concluye que el coumestrol administrado vía subcutánea tiene un efecto estrogénico que puede utilizarse como un método no invasivo para ayudar a controlar la fertilidad de perros adultos.

The concern of canine overpopulation is related to zoonoses such as rabies, which is responsible for 99% of human rabies cases, causing the death of approximately 59,000 people per year. Surgical sterilization is an effective, costly and invasive control method with limited impact. The effects of phytoestrogens on reproductive activity have been commonly studied in females and a limited number in males. Therefore, the objective of this study was to know the effect of subcutaneous administration of coumestrol on the gonadal activity of adult dogs as an alternative for their reproductive control. Basic seminal evaluation parameters, epididymal structure by ultrasonography, histological characteristics, as well as the presence of coumestrol by fluorescence and serum levels of testosterone and estrogens were determined. The administration of coumestrol for five weeks reduced sperm production, and evidenced changes in the echodensity and cellularity of the epididymis, associated with serum concentrations of estradiol and testosterone. Therefore, it is concluded that coumestrol administered subcutaneously has an estrogenic effect that can be used as a non-invasive method to help control fertility in adult dogs., La preocupación de la sobrepoblación canina se relaciona con zoonosis como la rabia, la cual es responsable del 99% de los casos de rabia humana, causante de la muerte de aproximadamente 59,000 personas al año. La esterilización quirúrgica es un método de control eficaz, costoso e invasivo siendo su impacto limitado. Los efectos de los fitoestrógenos en la actividad reproductiva se han estudiado comúnmente en hembras, y un número limitado en machos. Por lo anterior, el objetivo de este estudio fue conocer el efecto de la administración subcutánea de coumestrol en la actividad gonadal de perros adultos como alternativa para su control reproductivo. Se determinaron los parámetros de evaluación seminal básica, la estructura del epidídimo mediante ecografía, características histológicas, así como la presencia del coumestrol mediante fluorescencia y los niveles séricos de testosterona y estrógenos. La administración de coumestrol durante cinco semanas redujo la producción espermática, y evidenció cambios en la eco densidad y celularidad del epidídimo, asociados a las concentraciones séricas de estradiol y testosterona. Por lo que, se concluye que el coumestrol administrado vía subcutánea tiene un efecto estrogénico que puede utilizarse como un método no invasivo para ayudar a controlar la fertilidad de perros adultos.

Zika virus (ZIKV) can infect developing fetuses in utero and cause severe congenital defects independent of route of maternal infection. Infected men can shed ZIKV RNA in semen for over six months. Whether prolonged viral RNA shedding in semen indicates a persistent infection in the male reproductive tract is unknown. We hypothesized that if ZIKV establishes a persistent infection in the male reproductive tract (MRT), then immunosuppressant treatment should stimulate ZIKV replication and seminal shedding. Male mice were infected with ZIKV and immunosuppressed when they shed viral RNA but not infectious virus in ejaculates. Following immunosuppression, we did not detect infectious virus in ejaculates. However, we did detect ZIKV positive and negative sense RNA in the epididymal lumens of mice treated with cyclophosphamide, suggesting that ZIKV persists in the epididymis. This study provides insight into the mechanisms behind ZIKV sexual transmission, which may inform public health decisions regarding ZIKV risks.

Zika virus (ZIKV) can infect developing fetuses in utero and cause severe congenital defects independent of route of maternal infection. Infected men can shed ZIKV RNA in semen for over six months. Whether prolonged viral RNA shedding in semen indicates a persistent infection in the male reproductive tract is unknown. We hypothesized that if ZIKV establishes a persistent infection in the male reproductive tract (MRT), then immunosuppressant treatment should stimulate ZIKV replication and seminal shedding. Male mice were infected with ZIKV and immunosuppressed when they shed viral RNA but not infectious virus in ejaculates. Following immunosuppression, we did not detect infectious virus in ejaculates. However, we did detect ZIKV positive and negative sense RNA in the epididymal lumens of mice treated with cyclophosphamide, suggesting that ZIKV persists in the epididymis. This study provides insight into the mechanisms behind ZIKV sexual transmission, which may inform public health decisions regarding ZIKV risks.

Seminal plasma (SP), the non-cellular component of semen, is a heterogeneous composite fluid built by secretions of the testis, the epididymis and the accessory sexual glands. Its composition, despite species-specific anatomical peculiarities, consistently contains inorganic ions, specific hormones, proteins and peptides, including cytokines and enzymes, cholesterol, DNA and RNA-the latter often protected within epididymis- or prostate-derived extracellular vesicles. It is beyond question that the SP participates in diverse aspects of sperm function pre-fertilization events. The SP also interacts with the various compartments of the tubular genital tract, triggering changes in gene function that prepares for an eventual successful pregnancy; thus, it ultimately modulates fertility. Despite these concepts, it is imperative to remember that SP-free spermatozoa (epididymal or washed ejaculated) are still fertile, so this review shall focus on the differences between the in vivo roles of the SP following semen deposition in the female and those regarding additions of SP on spermatozoa handled for artificial reproduction, including cryopreservation, from artificial insemination to in vitro fertilization. This review attempts, including our own results on model animal species, to critically summarize the current knowledge of the reproductive roles played by SP components, particularly in our own species, which is increasingly affected by infertility. The ultimate goal is to reconcile the delicate balance between the SP molecular concentration and their concerted effects after temporal exposure in vivo. We aim to appraise the functions of the SP components, their relevance as diagnostic biomarkers and their value as eventual additives to refine reproductive strategies, including biotechnologies, in livestock models and humans.

[EN] This study was carried out to assess the effect of dietary levels of zinc on semen indices, growth parameters and testicular sperm reserve of heterogeneous stock of male rabbits. Twenty male rabbits aged 6 mo old were randomly allotted to four groups of five males each and fed diets containing 0, 50, 100 and 150 mg of zinc gluconate per kg diet, respectively, for eight weeks. Semen was collected from the males weekly using an artificial vagina and semen volume (mL), spermatozoa motility (%), sperm concentration (×108/mL) and live sperm cells (%) were assessed. Seminal plasma was separated from the semen by centrifugation, and its zinc concentration was determined using atomic absorption spectrometry. The weights of the males were taken weekly, and the daily feed intake was recorded. At the end of the feeding trial, the rabbits were stunned and slaughtered; the testes and epididymis were carefully removed and homogenised to determine sperm reserves using standard procedure. The result showed that semen indices were not significantly influenced by the dietary levels of zinc gluconate. Seminal zinc concentration was significantly higher in males fed 100 and 150 mg of zinc per kg compared to those in the control group. A higher body weight gain (454±50.3 g), testes weight (6.23±0.25 g) and epididymis weight (1.63±0.59 g) was recorded in males fed diet supplemented with 50 mg of Zinc gluconate per kg. No significant effect of dietary zinc supplementation was recorded in testicular sperm reserve. Epididymal sperm reserve was significantly higher in males fed the diets including 100 and 150 mg of zinc gluconate per kg. In conclusion, dietary levels of zinc gluconate did not improve semen quality and growth indices but increased seminal zinc concentration, which could result in improved prostate health in the heterogeneous stock of male rabbits in the tropics.

The objectives of this study were to assess the cooling and freezing of donkey epididymal semen harvested immediately after castration (Experiment 1, n = 4) or after the shipment (24 or 48 h) of epididymides attached to testicles (Experiment 2, n = 14) or dissected apart (Experiment 3, n = 36). In each experiment, semen was frozen immediately (Non-Centrif) in an egg yolk-based semen extender (EY) or after processing through cushion-centrifugation (Centrif) while extended in a skim milk-based extender (SC). In all three experiments, cooled, pre-freeze, and post-thaw epididymal semen was assessed for total motility (TM), progressive motility (PM), plasma membrane integrity (PMI), and high mitochondrial membrane potential (HMMP). Data were analyzed with R using mixed models and Tukey's test as posthoc. Results showed that the cooling of epididymal semen up to 24 h after harvesting did not affect motility parameters or plasma membrane integrity; furthermore, in Experiment 3, the post-thaw evaluation of both Centrif and Non-Centrif achieved similar TM and PM. Collectively, the post-thaw results revealed low motility parameters across groups; while, the PMI and HMMP did not reflect this trend, and the values remained high, suggesting that there was a lack of epididymal sperm activation with either centrifugation or extenders. In summary, freshly harvested and cooled-shipped and cooled semen had satisfactory semen parameters. Future studies need to address donkey epididymal semen fertility in mares and jennies.

[EN] This study was carried out to assess the effect of dietary levels of zinc on semen indices, growth parameters and testicular sperm reserve of heterogeneous stock of male rabbits. Twenty male rabbits aged 6 mo old were randomly allotted to four groups of five males each and fed diets containing 0, 50, 100 and 150 mg of zinc gluconate per kg diet, respectively, for eight weeks. Semen was collected from the males weekly using an artificial vagina and semen volume (mL), spermatozoa motility (%), sperm concentration (×108/mL) and live sperm cells (%) were assessed. Seminal plasma was separated from the semen by centrifugation, and its zinc concentration was determined using atomic absorption spectrometry. The weights of the males were taken weekly, and the daily feed intake was recorded. At the end of the feeding trial, the rabbits were stunned and slaughtered; the testes and epididymis were carefully removed and homogenised to determine sperm reserves using standard procedure. The result showed that semen indices were not significantly influenced by the dietary levels of zinc gluconate. Seminal zinc concentration was significantly higher in males fed 100 and 150 mg of zinc per kg compared to those in the control group. A higher body weight gain (454±50.3 g), testes weight (6.23±0.25 g) and epididymis weight (1.63±0.59 g) was recorded in males fed diet supplemented with 50 mg of Zinc gluconate per kg. No significant effect of dietary zinc supplementation was recorded in testicular sperm reserve. Epididymal sperm reserve was significantly higher in males fed the diets including 100 and 150 mg of zinc gluconate per kg. In conclusion, dietary levels of zinc gluconate did not improve semen quality and growth indices but increased seminal zinc concentration, which could result in improved prostate health in the heterogeneous stock of male rabbits in the tropics.

[EN] This study was carried out to assess the effect of dietary levels of zinc on semen indices, growth parameters and testicular sperm reserve of heterogeneous stock of male rabbits. Twenty male rabbits aged 6 mo old were randomly allotted to four groups of five males each and fed diets containing 0, 50, 100 and 150 mg of zinc gluconate per kg diet, respectively, for eight weeks. Semen was collected from the males weekly using an artificial vagina and semen volume (mL), spermatozoa motility (%), sperm concentration (×108/mL) and live sperm cells (%) were assessed. Seminal plasma was separated from the semen by centrifugation, and its zinc concentration was determined using atomic absorption spectrometry. The weights of the males were taken weekly, and the daily feed intake was recorded. At the end of the feeding trial, the rabbits were stunned and slaughtered; the testes and epididymis were carefully removed and homogenised to determine sperm reserves using standard procedure. The result showed that semen indices were not significantly influenced by the dietary levels of zinc gluconate. Seminal zinc concentration was significantly higher in males fed 100 and 150 mg of zinc per kg compared to those in the control group. A higher body weight gain (454±50.3 g), testes weight (6.23±0.25 g) and epididymis weight (1.63±0.59 g) was recorded in males fed diet supplemented with 50 mg of Zinc gluconate per kg. No significant effect of dietary zinc supplementation was recorded in testicular sperm reserve. Epididymal sperm reserve was significantly higher in males fed the diets including 100 and 150 mg of zinc gluconate per kg. In conclusion, dietary levels of zinc gluconate did not improve semen quality and growth indices but increased seminal zinc concentration, which could result in improved prostate health in the heterogeneous stock of male rabbits in the tropics.

[EN] This study was carried out to assess the effect of dietary levels of zinc on semen indices, growth parameters and testicular sperm reserve of heterogeneous stock of male rabbits. Twenty male rabbits aged 6 mo old were randomly allotted to four groups of five males each and fed diets containing 0, 50, 100 and 150 mg of zinc gluconate per kg diet, respectively, for eight weeks. Semen was collected from the males weekly using an artificial vagina and semen volume (mL), spermatozoa motility (%), sperm concentration (×108/mL) and live sperm cells (%) were assessed. Seminal plasma was separated from the semen by centrifugation, and its zinc concentration was determined using atomic absorption spectrometry. The weights of the males were taken weekly, and the daily feed intake was recorded. At the end of the feeding trial, the rabbits were stunned and slaughtered; the testes and epididymis were carefully removed and homogenised to determine sperm reserves using standard procedure. The result showed that semen indices were not significantly influenced by the dietary levels of zinc gluconate. Seminal zinc concentration was significantly higher in males fed 100 and 150 mg of zinc per kg compared to those in the control group. A higher body weight gain (454±50.3 g), testes weight (6.23±0.25 g) and epididymis weight (1.63±0.59 g) was recorded in males fed diet supplemented with 50 mg of Zinc gluconate per kg. No significant effect of dietary zinc supplementation was recorded in testicular sperm reserve. Epididymal sperm reserve was significantly higher in males fed the diets including 100 and 150 mg of zinc gluconate per kg. In conclusion, dietary levels of zinc gluconate did not improve semen quality and growth indices but increased seminal zinc concentration, which could result in improved prostate health in the heterogeneous stock of male rabbits in the tropics.

[EN] This study was carried out to assess the effect of dietary levels of zinc on semen indices, growth parameters and testicular sperm reserve of heterogeneous stock of male rabbits. Twenty male rabbits aged 6 mo old were randomly allotted to four groups of five males each and fed diets containing 0, 50, 100 and 150 mg of zinc gluconate per kg diet, respectively, for eight weeks. Semen was collected from the males weekly using an artificial vagina and semen volume (mL), spermatozoa motility (%), sperm concentration (×108/mL) and live sperm cells (%) were assessed. Seminal plasma was separated from the semen by centrifugation, and its zinc concentration was determined using atomic absorption spectrometry. The weights of the males were taken weekly, and the daily feed intake was recorded. At the end of the feeding trial, the rabbits were stunned and slaughtered; the testes and epididymis were carefully removed and homogenised to determine sperm reserves using standard procedure. The result showed that semen indices were not significantly influenced by the dietary levels of zinc gluconate. Seminal zinc concentration was significantly higher in males fed 100 and 150 mg of zinc per kg compared to those in the control group. A higher body weight gain (454±50.3 g), testes weight (6.23±0.25 g) and epididymis weight (1.63±0.59 g) was recorded in males fed diet supplemented with 50 mg of Zinc gluconate per kg. No significant effect of dietary zinc supplementation was recorded in testicular sperm reserve. Epididymal sperm reserve was significantly higher in males fed the diets including 100 and 150 mg of zinc gluconate per kg. In conclusion, dietary levels of zinc gluconate did not improve semen quality and growth indices but increased seminal zinc concentration, which could result in improved prostate health in the heterogeneous stock of male rabbits in the tropics.

There are toxic agents that can interfere with the germ cell differentiation process in man and animals. The research aimed to evaluate testicular damage induced by 0,5 % iodine alcohol (2,5 g / k.p.v). 9 prepubescent male guinea pigs from the Peru Genotype were selected and divided into 3 groups, the control group and those that were evaluated at 28- and 56-days post-injection with iodized alcohol. The macroscopic (weight and length of the testis, consistency) and microscopic anatomy of the guinea pig testis were performed using hematoxylin-eosin staining. In treated guinea pigs, there was a significant reduction (p<0,05) in testis weight and length. Likewise, histopathological analysis showed a decrease in the number and atrophy of the seminiferous tubules, the alteration in the formation of spermatogenic cells, especially secondary spermatocytes, spermatids and sperm. At 56 days post-treatment, vacuoles and multinucleated giant cells were observed. On the other hand, after 28 days, alteration of the structure and cell height of the epididymis and vas deferens, increased intertubular space of the epididymis, and azoospermia were evident. Therefore, it is concluded that intratesticular injection with 0,5 % iodine alcohol causes severe and irreversible histopathological alterations in the testicles of male prepubescent guinea pigs., Hay agentes tóxicos que pueden interferir en el proceso de diferenciación de las células germinales en el hombre y en los animales. La investigación tuvo como objetivo evaluar el daño testicular inducido por el alcohol yodado 0,5 % (2,5 g / k.p.v). 9 cuyes machos prepúberes del Genotipo Perú fueron seleccionados y divididos en 3 grupos, el control y los que se evaluaron a los 28 y 56 días posinyección con alcohol yodado. Se realizó la evaluación de la anatomía macroscópica (peso y longitud del testículo, consistencia) y microscópica del testículo de los cuyes utilizando la tinción de hematoxilinaeosina. En los cuyes tratados, hubo una reducción significativa (p<0,05) en el peso y la longitud de los testículos. Asimismo, el análisis histopatológico mostró la disminución en el número y atrofia de los túbulos seminíferos, la alteración en la formación de las células espermatogénicas, especialmente de los espermatocitos secundarios, espermátidas y espermatozoides. A los 56 días postratamiento, se observó la presencia de vacuolas y células gigantes multinucleadas. Por otro lado, a partir de los 28 días, se evidenció alteración de la estructura y la altura celular del epidídimo y el conducto deferente, aumento del espacio intertubular del epidídimo y azoospermia. Por lo tanto, se concluye que la inyección intratesticular con alcohol yodado 0,5 % causa alteraciones histopatológicas severas e irreversibles en los testículos de los cuyes prepúberes machos.

The objectives of this study were to assess the cooling and freezing of donkey epididymal semen harvested immediately after castration (Experiment 1, n = 4) or after the shipment (24 or 48 h) of epididymides attached to testicles (Experiment 2, n = 14) or dissected apart (Experiment 3, n = 36). In each experiment, semen was frozen immediately (Non-Centrif) in an egg yolk-based semen extender (EY) or after processing through cushion-centrifugation (Centrif) while extended in a skim milk-based extender (SC). In all three experiments, cooled, pre-freeze, and post-thaw epididymal semen was assessed for total motility (TM), progressive motility (PM), plasma membrane integrity (PMI), and high mitochondrial membrane potential (HMMP). Data were analyzed with R using mixed models and Tukey's test as posthoc. Results showed that the cooling of epididymal semen up to 24 h after harvesting did not affect motility parameters or plasma membrane integrity; furthermore, in Experiment 3, the post-thaw evaluation of both Centrif and Non-Centrif achieved similar TM and PM. Collectively, the post-thaw results revealed low motility parameters across groups; while, the PMI and HMMP did not reflect this trend, and the values remained high, suggesting that there was a lack of epididymal sperm activation with either centrifugation or extenders. In summary, freshly harvested and cooled-shipped and cooled semen had satisfactory semen parameters. Future studies need to address donkey epididymal semen fertility in mares and jennies.

Elevated levels of branched-chain amino acids (BCAAs) and their metabolites are strongly positively associated with obesity, insulin resistance, and type 2 diabetes. Bariatric surgery is among the best treatments for weight loss and associated morbidities. Clinical studies have reported that bariatric surgery decreases the circulating levels of BCAAs. The objective of this study was to test the hypothesis that reduced BCAA levels contribute to the metabolic improvements of sustained weight loss and improved glucose tolerance after vertical sleeve gastrectomy (VSG). We find that, as in humans, circulating BCAAs are significantly lower in VSG rats and mice. To increase circulating BCAAs, we tested mice with either increased dietary intake of BCAAs or impaired BCAA catabolism by total body deletion of mitochondrial phosphatase 2C (Pp2cm). Our results show that a decrease in circulating BCAAs is not necessary for sustained body weight loss and improved glucose tolerance after VSG.

There are toxic agents that can interfere with the germ cell differentiation process in man and animals. The research aimed to evaluate testicular damage induced by 0,5 % iodine alcohol (2,5 g / k.p.v). 9 prepubescent male guinea pigs from the Peru Genotype were selected and divided into 3 groups, the control group and those that were evaluated at 28- and 56-days post-injection with iodized alcohol. The macroscopic (weight and length of the testis, consistency) and microscopic anatomy of the guinea pig testis were performed using hematoxylin-eosin staining. In treated guinea pigs, there was a significant reduction (p<0,05) in testis weight and length. Likewise, histopathological analysis showed a decrease in the number and atrophy of the seminiferous tubules, the alteration in the formation of spermatogenic cells, especially secondary spermatocytes, spermatids and sperm. At 56 days post-treatment, vacuoles and multinucleated giant cells were observed. On the other hand, after 28 days, alteration of the structure and cell height of the epididymis and vas deferens, increased intertubular space of the epididymis, and azoospermia were evident. Therefore, it is concluded that intratesticular injection with 0,5 % iodine alcohol causes severe and irreversible histopathological alterations in the testicles of male prepubescent guinea pigs., Hay agentes tóxicos que pueden interferir en el proceso de diferenciación de las células germinales en el hombre y en los animales. La investigación tuvo como objetivo evaluar el daño testicular inducido por el alcohol yodado 0,5 % (2,5 g / k.p.v). 9 cuyes machos prepúberes del Genotipo Perú fueron seleccionados y divididos en 3 grupos, el control y los que se evaluaron a los 28 y 56 días posinyección con alcohol yodado. Se realizó la evaluación de la anatomía macroscópica (peso y longitud del testículo, consistencia) y microscópica del testículo de los cuyes utilizando la tinción de hematoxilinaeosina. En los cuyes tratados, hubo una reducción significativa (p<0,05) en el peso y la longitud de los testículos. Asimismo, el análisis histopatológico mostró la disminución en el número y atrofia de los túbulos seminíferos, la alteración en la formación de las células espermatogénicas, especialmente de los espermatocitos secundarios, espermátidas y espermatozoides. A los 56 días postratamiento, se observó la presencia de vacuolas y células gigantes multinucleadas. Por otro lado, a partir de los 28 días, se evidenció alteración de la estructura y la altura celular del epidídimo y el conducto deferente, aumento del espacio intertubular del epidídimo y azoospermia. Por lo tanto, se concluye que la inyección intratesticular con alcohol yodado 0,5 % causa alteraciones histopatológicas severas e irreversibles en los testículos de los cuyes prepúberes machos.

There are toxic agents that can interfere with the germ cell differentiation process in man and animals. The research aimed to evaluate testicular damage induced by 0,5 % iodine alcohol (2,5 g / k.p.v). 9 prepubescent male guinea pigs from the Peru Genotype were selected and divided into 3 groups, the control group and those that were evaluated at 28- and 56-days post-injection with iodized alcohol. The macroscopic (weight and length of the testis, consistency) and microscopic anatomy of the guinea pig testis were performed using hematoxylin-eosin staining. In treated guinea pigs, there was a significant reduction (p<0,05) in testis weight and length. Likewise, histopathological analysis showed a decrease in the number and atrophy of the seminiferous tubules, the alteration in the formation of spermatogenic cells, especially secondary spermatocytes, spermatids and sperm. At 56 days post-treatment, vacuoles and multinucleated giant cells were observed. On the other hand, after 28 days, alteration of the structure and cell height of the epididymis and vas deferens, increased intertubular space of the epididymis, and azoospermia were evident. Therefore, it is concluded that intratesticular injection with 0,5 % iodine alcohol causes severe and irreversible histopathological alterations in the testicles of male prepubescent guinea pigs., Hay agentes tóxicos que pueden interferir en el proceso de diferenciación de las células germinales en el hombre y en los animales. La investigación tuvo como objetivo evaluar el daño testicular inducido por el alcohol yodado 0,5 % (2,5 g / k.p.v). 9 cuyes machos prepúberes del Genotipo Perú fueron seleccionados y divididos en 3 grupos, el control y los que se evaluaron a los 28 y 56 días posinyección con alcohol yodado. Se realizó la evaluación de la anatomía macroscópica (peso y longitud del testículo, consistencia) y microscópica del testículo de los cuyes utilizando la tinción de hematoxilinaeosina. En los cuyes tratados, hubo una reducción significativa (p<0,05) en el peso y la longitud de los testículos. Asimismo, el análisis histopatológico mostró la disminución en el número y atrofia de los túbulos seminíferos, la alteración en la formación de las células espermatogénicas, especialmente de los espermatocitos secundarios, espermátidas y espermatozoides. A los 56 días postratamiento, se observó la presencia de vacuolas y células gigantes multinucleadas. Por otro lado, a partir de los 28 días, se evidenció alteración de la estructura y la altura celular del epidídimo y el conducto deferente, aumento del espacio intertubular del epidídimo y azoospermia. Por lo tanto, se concluye que la inyección intratesticular con alcohol yodado 0,5 % causa alteraciones histopatológicas severas e irreversibles en los testículos de los cuyes prepúberes machos.

We evaluated the effects of unilateral obstruction of the vas deferens on testicular endocrine and exocrine function in rats. The left vas deferens was ligated and divided in 1-week-old Wistar rats. Additional rats of the same age underwent a sham operation. Bilateral testicular weight and epididymal weight were compared between the vasectomized and sham-operated animals at 4, 8 and 12 weeks postoperation. Intratesticular versus intraabdominal temperature difference, epididymal caudal sperm content (ECSC), motility (ECSM) and in vitro fertilizing capacity (IVF) were compared between the vasectomized and sham-operated animals at 12 weeks postoperation. Serum testosterone response to human chorionic gonadotropin and bilateral testicular weight were significantly smaller in the vasectomized animals than in the sham-operated animals at 8 and 12 weeks postoperation. Bilateral ECSC, ECSM and IVF outcomes were significantly smaller in vasectomized rats than in sham-treated rats 12 weeks postoperatively. Bilateral intratesticular versus intraabdominal temperature differences were not significantly different between the vasectomized and control rats at 12 weeks postoperation. Unilateral obstruction of the vas deferens results in a bilateral defect in spermatogenesis and in the epididymal sperm maturation process.

Sexual transmission of Ebola virus (EBOV) has been demonstrated more than a year after recovery from the acute phase of Ebola virus disease (EVD). The mechanisms underlying EBOV persistence and sexual transmission are not currently understood. Using the acute macaque model of EVD, we hypothesized EBOV would infect the reproductive tissues and sought to localize the infection in these tissues using immunohistochemistry and transmission electron microscopy. In four female and eight male macaques that succumbed to EVD between 6 and 9 days after EBOV challenge, we demonstrate widespread EBOV infection of the interstitial tissues and endothelium in the ovary, uterus, testis, seminal vesicle, epididymis, and prostate gland, with minimal associated tissue immune response or organ pathology. Given the widespread involvement of EBOV in the reproductive tracts of both male and female macaques, it is reasonable to surmise that our understanding of the mechanisms underlying sexual transmission of EVD and persistence of EBOV in immune-privileged sites would be facilitated by the development of a nonhuman primate model in which the macaques survived past the acute stage into convalescence.

Activin A is an important regulator of testicular and epididymal development and function, as well as inflammation and immunity. In the adult murine reproductive tract, activin A mRNA (Inhba) expression levels are highest in the caput epididymis and decrease progressively towards the distal vas deferens. The activin-binding protein, follistatin (FST), shows the opposite expression pattern, with exceptionally high levels of the Fst288 mRNA variant in the vas deferens. This unique pattern of expression suggests that activin A and follistatin, in particular FST288, play region-specific roles in regulating the epididymis and vas deferens. The cellular distribution of activin and follistatin and structural organization of the male reproductive tract was examined in wild-type and transgenic (TghFST315) mice lacking FST288. Compared to wild-type littermates, TghFST315 mice showed a 50% reduction in serum follistatin and a significant elevation of both activin A and B. Testicular, epididymal and seminal vesicle weights were reduced, but intra-testicular testosterone was normal. A decrease in the epididymal duct diameter in the corpus and thickening of the peritubular smooth muscle in the cauda, together with increased coiling of the proximal vas deferens, were observed in TghFST315 mice. No immune cell infiltrates were detected. Immunohistochemistry indicated that epithelial cells are the main source of activins and follistatin in the epididymis and vas deferens. Activin A, but not activin B, was also localized to sperm heads in the lumen of the epididymis and vas deferens. Expression of Inhba and another immunoregulatory gene, indoleamine-2,3-dioxygenase (Ido-1), was increased approximately twofold in the TghFST315 caput epididymis, but several other genes associated with immunoregulation, inflammation or fibrosis were unaffected. Our novel data indicate that disruption of follistatin expression has significant effects on the testis and epididymis, and suggest an associati

peer-reviewed, β-defensins are small cationic proteins with potent immunoregulatory and antimicrobial activity. The number of genes encoding these peptides varies significantly between and within species but they have not been extensively characterised in the horse. Here, we describe a systematic search of the Equus caballus genome that identified a cluster of novel β-defensin genes on Chromosome 22, which is homologous to a cluster on bovine Chromosome 13. Close genomic matches were found for orthologs of 13 of the bovine genes, which were named equine β-defensins (eBD) 115, eBD116, eBD117, eBD119, eBD120, eBD122a, eBD123, eBD124, eBD125, eBD126, eBD127, eBD129 and eBD132. As expression of the homologous cluster in cattle was limited to the reproductive tract, tissue sections were obtained from the testis, caput, corpus and cauda epididymis and the vas deferens of three stallions and from the ovary, oviduct, uterine horn, uterus, cervix and vagina of three mares. Using a quantitative real-time polymerase chain reaction approach, each of the novel β-defensin genes showed distinct region-specific patterns of expression. Preferential expression in the caput epididymis of these novel defensins in the stallion and in the oviduct in the mare suggests a possible role in immunoprotection of the equine reproductive tract or in fertility., ACCEPTED

Activin A is an important regulator of testicular and epididymal development and function, as well as inflammation and immunity. In the adult murine reproductive tract, activin A mRNA (Inhba) expression levels are highest in the caput epididymis and decrease progressively towards the distal vas deferens. The activin-binding protein, follistatin (FST), shows the opposite expression pattern, with exceptionally high levels of the Fst288 mRNA variant in the vas deferens. This unique pattern of expression suggests that activin A and follistatin, in particular FST288, play region-specific roles in regulating the epididymis and vas deferens. The cellular distribution of activin and follistatin and structural organization of the male reproductive tract was examined in wild-type and transgenic (TghFST315) mice lacking FST288. Compared to wild-type littermates, TghFST315 mice showed a 50% reduction in serum follistatin and a significant elevation of both activin A and B. Testicular, epididymal and seminal vesicle weights were reduced, but intra-testicular testosterone was normal. A decrease in the epididymal duct diameter in the corpus and thickening of the peritubular smooth muscle in the cauda, together with increased coiling of the proximal vas deferens, were observed in TghFST315 mice. No immune cell infiltrates were detected. Immunohistochemistry indicated that epithelial cells are the main source of activins and follistatin in the epididymis and vas deferens. Activin A, but not activin B, was also localized to sperm heads in the lumen of the epididymis and vas deferens. Expression of Inhba and another immunoregulatory gene, indoleamine-2,3-dioxygenase (Ido-1), was increased approximately twofold in the TghFST315 caput epididymis, but several other genes associated with immunoregulation, inflammation or fibrosis were unaffected. Our novel data indicate that disruption of follistatin expression has significant effects on the testis and epididymis, and suggest an associati

peer-reviewed, Background: beta-defensins are small, cationic, antimicrobial peptides found in species across the plant and animal kingdoms. In addition to microbiocidal activity, roles in immunity as well as reproduction have more recently been documented. beta-defensin genes in Ovis aries (domestic sheep) have been poorly annotated, having been identified only by automatic gene prediction algorithms. The objective of this study was to use a comparative genomics approach to identify and characterise the beta-defensin gene repertoire in sheep using the bovine genome as the primary reference.Results: All 57 currently predicted bovine beta-defensin genes were used to find orthologous sequences in the most recent version of the sheep genome (OAR v4.0). Forty three genes were found to have close genomic matches (> 70% similarity) between sheep and cattle. The orthologous genes were located in four clusters across the genome, with 4 genes on chromosome 2, 19 genes on chromosome 13, 5 genes on chromosome 20 and 15 genes on chromosome 26. Conserved gene order for the beta-defensin genes was apparent in the two smaller clusters, although gene order was reversed on chromosome 2, suggesting an inversion between sheep and cattle. Complete conservation of gene order was also observed for chromosome 13 beta-defensin orthologs. More structural differences were apparent between chromosome 26 genes and the orthologous region in the bovine reference genome, which is known to be copy-number variable. In this cluster, the Defensin-beta 1 (DEFB1) gene matched to eleven Bovine Neutrophil beta-Defensin (BNBD) genes on chromosome 27 with almost uniform similarity, as well as to tracheal, enteric and lingual anti-microbial peptides (TAP, EAP and LAP), suggesting that annotation of the bovine reference sequence is still incomplete. qPCR was used to profile the expression of 34 beta-defensin genes, representing each of the four clusters, in the ram reproductive tract. Distinct site-specific and differentia, PUBLISHED, peer-reviewed

L’épididyme est l’organe responsable de la maturation des spermatozoïdes, son rôle est essentiel dans l’acquisition de la fertilité masculine. Ce processus crucial est dépendant des interactions entre les spermatozoïdes et le microenvironnement luminal de l’épididyme. La composition de ce microenvironnement spécifique est régulée par les différents types cellulaires présents au sein de l’épithélium de l’épididyme et évolue tout au long de cet organe. Afin de synchroniser leurs actions, les cellules épithéliales communiquent entre elles par l’intermédiaire des jonctions lacunaires. Les jonctions lacunaires sont composées de canaux transmembranaires entre deux cellules adjacentes, ce qui leur permet de communiquer par la diffusion directe d’ions et de petites molécules. Les connexines (Cxs) sont les protéines qui composent ces canaux transmembranaires. Dans l’épididyme du rat, Gjb5 (Cx30.3), Gjb4 (Cx31.1), Gjb1 (Cx32) et Gja1 (Cx43) sont présentes au sein de l’épithélium adulte différencié alors que Gjb2 (Cx26) est uniquement détectable chez les jeunes animaux, lorsque l’épithélium est indifférencié. Ces changements d’expression des Cxs modifient la sélectivité des jonctions lacunaires et permettent aux cellules de l’épithélium de modifier leurs échanges. On observe un changement de l’expression des Cxs pendant la différenciation du tissu, ce qui suggère un rôle clé des Cxs dans la régulation de la différentiation de l’épithélium épididymaire. Il n’existe aucune information sur la régulation transcriptionnelle des Cxs dans l’épididyme durant la différenciation.L’objectif principal de ce projet était d’identifier les mécanismes impliqués dans la régulation de l’expression des Cxs lors de la différentiation de l’épididyme. Afin d’élucider de tels mécanismes, le promoteur de Gjb2 a été caractérisé. Les résultats ont montré que Gjb2 possède un site unique d’initiation de la transcription au sein de l’épididyme. L’analyse de la séquence du promoteur de Gjb2 a révélé la pré

Understanding the physiological processes involved in the sperm maturation process is critical to elucidating mechanisms underlying male infertility. Maturation of the spermatozoa during epididymal transit is associated with the integration of specific proteins and acquisition of functionality. Many underlying processes are unclear thereby making it challenging to develop applications for assisted reproductive technologies. The goal of this research was to understand key phases in the sperm maturation process associated with functional effects of protein incorporation via epididymosome secretion on sperm cell motility and fertilization. Specific objectives were to (1) identify essential proteins that contribute to sperm centrosome maturation throughout epididymal transit, (2) characterize the transfer of key factors via epididymosomes between the epididymis and maturing sperm cells and (3) demonstrate the impact of such mechanisms on the acquisition of motility and fertilizing ability by the spermatozoa. Using the domestic cat model (Felis catus), we demonstrated that the secreted epididymal vesicles termed epididymosomes supply critical proteins to the developing spermatozoa and examined the effects of exposure of these vesicles on motility and ability to successfully fertilize an oocyte. Exposure of immature sperm cells to epididymosomes in vitro resulted in the significant incorporation of cenexin, critical for the maturation of the sperm centrosome, which then enables proper pronuclear syngamy and formation of the first mitotic spindle following oocyte penetration. Furthermore, this research focused on assessing the impact of malformed spermatozoa at the centrosome on fertilization success in the domestic cat. Findings revealed that improper maturation of the centrosome may be a source of infertility in this species as well as in wild felids. In summary, this research has provided insights into the sperm cell maturation process which will enhance the use of assi

Quassia amara is a Brazilian medicinal plant traditionally used as an herbal remedy for a variety of diseases. Quassin is the major secondary chemical compound in it, which is under investigation as an anti-malarial compound. There are preliminary studies implicating Q. amara in male reproductive toxicity. Hence, this elaborate study was undertaken to find the male reproductive toxic effects of Q. amara and quassin. Methanolic extract of Q. amara bark and quassin were administered to male mice for 35 days. The cauda epididymidal sperm, testis and epididymis were subjected to analysis adopting light and transmission electron microscopy. Sperm counts, motility and viability decreased. The incidence of sperm with abnormal morphologies increased. There was premature loss of germ cells from the seminiferous tubules, which correlated with Sertoli cell pathology. The Leydig cells were vacuolated, suggesting a hypo-androgen status. The epididymis indicated severe histopathological changes, including reflection of a mitogenic / tumorigenic effect of Q. amara. Thus, this study indicates that Q. amara, when applied as a therapeutic, could be severely toxic to male reproductive mechanisms and the principal mechanism of action appears to be through Leydig cells. Quassin, the major secondary metabolite responsible for the therapeutic potential of Q. amara, is also the one causative of the male reproductive toxic effects. Thus, this study substantiates the claim that Q. amara is toxic to male reproductive system.

Doria P.B.A., Rodrigues A.L.R., Leite C.R. & Botteon P.T.L. [Surgical esterilization of rams (Ovis aries) with the use of cryosurgery.] Esterilização cirúrgica de carneiros (Ovis aries) com o emprego da criocirurgia. Revista Brasileira de Medicina Veterinária, 38(Supl.2):86-92, 2016. Programa de Pós-Graduação em Medicina Veterinária (Patologia e Ciências Clínicas), Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, BR 465 Km 7, Seropédica, RJ 23851- 790, Brasil. E-mail: phillipebauer@vm.uff.br; phillipebauer@hotmail.com The current study aims to develop a minimally invasive surgical technique with the use of cryosurgery that provides sterilization of rams. It was used nine rams, pubescent. The animals were weighed, clinical examination, and samples of blood and feces were collected for hematological and feces tests. After this the animals were submitted to an andrologic evaluation and libido test. The animals were submitted to cryosurgery sterilization and observed for a period of 32 days performed before surgery. All of the animals look good, health, with no pain or discomfort, maintenance the intake of water and food, with no significant changes in body weight. No signs of post-operative complications were seen in surgical site. It was observed decrease on scrotal circumference of six animals. The exams showed no changes in the scrotal and testicular parenchyma, however demonstrating changes in the shape and increased epididymis tail dimensions, irregular form and firm texture. Semen analysis performed at 32 days revealed azoospermia in six of the nine rams. There were no changes in the libido test. It concludes that the proposed technique is safe, fast, and easy to perform, with low cost, minimally invasive and free of the occurrence of postoperative complications., O presente estudo visa ao desenvolvimento de técnica de esterilização cirúrgica minimamente invasiva com o emprego da criocirurgia em carneiros. Foram utilizados nove ovinos, machos, inteiros, púberes. Os animais foram submetidos à pesagem, avaliação clínica, hematológica, coproparasitológica, exame andrológico e teste de libido. Os animais foram submetidos à técnica de esterilização por criocirurgia e acompanhados por um período de 32 dias, repetindo-se ao final desta etapa todos os exames realizados antes da intervenção cirúrgica. Os animais apresentaram-se bem clinicamente, sem sinais de dor ou desconforto ou alteração do escore corporal. Não foram observados sinais de complicações pós-operatórias no sítio cirúrgico. Durante o exame andrológico constatou- -se aumento de volume da cauda dos epidídimos, com formato irregular e consistência firme. Seis animais apresentaram discreta redução na circunferência escrotal. O espermograma realizado aos 32 dias revelou azoospermia em seis dos nove carneiros. Não houve alterações referentes ao teste de libido. Concluímos que a técnica cirúrgica proposta mostrou-se segura, rápida, de fácil execução, baixo custo, minimamente invasiva e isenta da ocorrência de complicações pós-operatórias.

Quassia amara is a Brazilian medicinal plant traditionally used as an herbal remedy for a variety of diseases. Quassin is the major secondary chemical compound in it, which is under investigation as an anti-malarial compound. There are preliminary studies implicating Q. amara in male reproductive toxicity. Hence, this elaborate study was undertaken to find the male reproductive toxic effects of Q. amara and quassin. Methanolic extract of Q. amara bark and quassin were administered to male mice for 35 days. The cauda epididymidal sperm, testis and epididymis were subjected to analysis adopting light and transmission electron microscopy. Sperm counts, motility and viability decreased. The incidence of sperm with abnormal morphologies increased. There was premature loss of germ cells from the seminiferous tubules, which correlated with Sertoli cell pathology. The Leydig cells were vacuolated, suggesting a hypo-androgen status. The epididymis indicated severe histopathological changes, including reflection of a mitogenic / tumorigenic effect of Q. amara. Thus, this study indicates that Q. amara, when applied as a therapeutic, could be severely toxic to male reproductive mechanisms and the principal mechanism of action appears to be through Leydig cells. Quassin, the major secondary metabolite responsible for the therapeutic potential of Q. amara, is also the one causative of the male reproductive toxic effects. Thus, this study substantiates the claim that Q. amara is toxic to male reproductive system.

Quassia amara is a Brazilian medicinal plant traditionally used as an herbal remedy for a variety of diseases. Quassin is the major secondary chemical compound in it, which is under investigation as an anti-malarial compound. There are preliminary studies implicating Q. amara in male reproductive toxicity. Hence, this elaborate study was undertaken to find the male reproductive toxic effects of Q. amara and quassin. Methanolic extract of Q. amara bark and quassin were administered to male mice for 35 days. The cauda epididymidal sperm, testis and epididymis were subjected to analysis adopting light and transmission electron microscopy. Sperm counts, motility and viability decreased. The incidence of sperm with abnormal morphologies increased. There was premature loss of germ cells from the seminiferous tubules, which correlated with Sertoli cell pathology. The Leydig cells were vacuolated, suggesting a hypo-androgen status. The epididymis indicated severe histopathological changes, including reflection of a mitogenic / tumorigenic effect of Q. amara. Thus, this study indicates that Q. amara, when applied as a therapeutic, could be severely toxic to male reproductive mechanisms and the principal mechanism of action appears to be through Leydig cells. Quassin, the major secondary metabolite responsible for the therapeutic potential of Q. amara, is also the one causative of the male reproductive toxic effects. Thus, this study substantiates the claim that Q. amara is toxic to male reproductive system.

Doria P.B.A., Rodrigues A.L.R., Leite C.R. & Botteon P.T.L. [Surgical esterilization of rams (Ovis aries) with the use of cryosurgery.] Esterilização cirúrgica de carneiros (Ovis aries) com o emprego da criocirurgia. Revista Brasileira de Medicina Veterinária, 38(Supl.2):86-92, 2016. Programa de Pós-Graduação em Medicina Veterinária (Patologia e Ciências Clínicas), Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, BR 465 Km 7, Seropédica, RJ 23851- 790, Brasil. E-mail: phillipebauer@vm.uff.br; phillipebauer@hotmail.com The current study aims to develop a minimally invasive surgical technique with the use of cryosurgery that provides sterilization of rams. It was used nine rams, pubescent. The animals were weighed, clinical examination, and samples of blood and feces were collected for hematological and feces tests. After this the animals were submitted to an andrologic evaluation and libido test. The animals were submitted to cryosurgery sterilization and observed for a period of 32 days performed before surgery. All of the animals look good, health, with no pain or discomfort, maintenance the intake of water and food, with no significant changes in body weight. No signs of post-operative complications were seen in surgical site. It was observed decrease on scrotal circumference of six animals. The exams showed no changes in the scrotal and testicular parenchyma, however demonstrating changes in the shape and increased epididymis tail dimensions, irregular form and firm texture. Semen analysis performed at 32 days revealed azoospermia in six of the nine rams. There were no changes in the libido test. It concludes that the proposed technique is safe, fast, and easy to perform, with low cost, minimally invasive and free of the occurrence of postoperative complications., O presente estudo visa ao desenvolvimento de técnica de esterilização cirúrgica minimamente invasiva com o emprego da criocirurgia em carneiros. Foram utilizados nove ovinos, machos, inteiros, púberes. Os animais foram submetidos à pesagem, avaliação clínica, hematológica, coproparasitológica, exame andrológico e teste de libido. Os animais foram submetidos à técnica de esterilização por criocirurgia e acompanhados por um período de 32 dias, repetindo-se ao final desta etapa todos os exames realizados antes da intervenção cirúrgica. Os animais apresentaram-se bem clinicamente, sem sinais de dor ou desconforto ou alteração do escore corporal. Não foram observados sinais de complicações pós-operatórias no sítio cirúrgico. Durante o exame andrológico constatou- -se aumento de volume da cauda dos epidídimos, com formato irregular e consistência firme. Seis animais apresentaram discreta redução na circunferência escrotal. O espermograma realizado aos 32 dias revelou azoospermia em seis dos nove carneiros. Não houve alterações referentes ao teste de libido. Concluímos que a técnica cirúrgica proposta mostrou-se segura, rápida, de fácil execução, baixo custo, minimamente invasiva e isenta da ocorrência de complicações pós-operatórias.

Quassia amara is a Brazilian medicinal plant traditionally used as an herbal remedy for a variety of diseases. Quassin is the major secondary chemical compound in it, which is under investigation as an anti-malarial compound. There are preliminary studies implicating Q. amara in male reproductive toxicity. Hence, this elaborate study was undertaken to find the male reproductive toxic effects of Q. amara and quassin. Methanolic extract of Q. amara bark and quassin were administered to male mice for 35 days. The cauda epididymidal sperm, testis and epididymis were subjected to analysis adopting light and transmission electron microscopy. Sperm counts, motility and viability decreased. The incidence of sperm with abnormal morphologies increased. There was premature loss of germ cells from the seminiferous tubules, which correlated with Sertoli cell pathology. The Leydig cells were vacuolated, suggesting a hypo-androgen status. The epididymis indicated severe histopathological changes, including reflection of a mitogenic / tumorigenic effect of Q. amara. Thus, this study indicates that Q. amara, when applied as a therapeutic, could be severely toxic to male reproductive mechanisms and the principal mechanism of action appears to be through Leydig cells. Quassin, the major secondary metabolite responsible for the therapeutic potential of Q. amara, is also the one causative of the male reproductive toxic effects. Thus, this study substantiates the claim that Q. amara is toxic to male reproductive system.

Doria P.B.A., Rodrigues A.L.R., Leite C.R. & Botteon P.T.L. [Surgical esterilization of rams (Ovis aries) with the use of cryosurgery.] Esterilização cirúrgica de carneiros (Ovis aries) com o emprego da criocirurgia. Revista Brasileira de Medicina Veterinária, 38(Supl.2):86-92, 2016. Programa de Pós-Graduação em Medicina Veterinária (Patologia e Ciências Clínicas), Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, BR 465 Km 7, Seropédica, RJ 23851- 790, Brasil. E-mail: phillipebauer@vm.uff.br; phillipebauer@hotmail.com The current study aims to develop a minimally invasive surgical technique with the use of cryosurgery that provides sterilization of rams. It was used nine rams, pubescent. The animals were weighed, clinical examination, and samples of blood and feces were collected for hematological and feces tests. After this the animals were submitted to an andrologic evaluation and libido test. The animals were submitted to cryosurgery sterilization and observed for a period of 32 days performed before surgery. All of the animals look good, health, with no pain or discomfort, maintenance the intake of water and food, with no significant changes in body weight. No signs of post-operative complications were seen in surgical site. It was observed decrease on scrotal circumference of six animals. The exams showed no changes in the scrotal and testicular parenchyma, however demonstrating changes in the shape and increased epididymis tail dimensions, irregular form and firm texture. Semen analysis performed at 32 days revealed azoospermia in six of the nine rams. There were no changes in the libido test. It concludes that the proposed technique is safe, fast, and easy to perform, with low cost, minimally invasive and free of the occurrence of postoperative complications., O presente estudo visa ao desenvolvimento de técnica de esterilização cirúrgica minimamente invasiva com o emprego da criocirurgia em carneiros. Foram utilizados nove ovinos, machos, inteiros, púberes. Os animais foram submetidos à pesagem, avaliação clínica, hematológica, coproparasitológica, exame andrológico e teste de libido. Os animais foram submetidos à técnica de esterilização por criocirurgia e acompanhados por um período de 32 dias, repetindo-se ao final desta etapa todos os exames realizados antes da intervenção cirúrgica. Os animais apresentaram-se bem clinicamente, sem sinais de dor ou desconforto ou alteração do escore corporal. Não foram observados sinais de complicações pós-operatórias no sítio cirúrgico. Durante o exame andrológico constatou- -se aumento de volume da cauda dos epidídimos, com formato irregular e consistência firme. Seis animais apresentaram discreta redução na circunferência escrotal. O espermograma realizado aos 32 dias revelou azoospermia em seis dos nove carneiros. Não houve alterações referentes ao teste de libido. Concluímos que a técnica cirúrgica proposta mostrou-se segura, rápida, de fácil execução, baixo custo, minimamente invasiva e isenta da ocorrência de complicações pós-operatórias.

Quassia amara is a Brazilian medicinal plant traditionally used as an herbal remedy for a variety of diseases. Quassin is the major secondary chemical compound in it, which is under investigation as an anti-malarial compound. There are preliminary studies implicating Q. amara in male reproductive toxicity. Hence, this elaborate study was undertaken to find the male reproductive toxic effects of Q. amara and quassin. Methanolic extract of Q. amara bark and quassin were administered to male mice for 35 days. The cauda epididymidal sperm, testis and epididymis were subjected to analysis adopting light and transmission electron microscopy. Sperm counts, motility and viability decreased. The incidence of sperm with abnormal morphologies increased. There was premature loss of germ cells from the seminiferous tubules, which correlated with Sertoli cell pathology. The Leydig cells were vacuolated, suggesting a hypo-androgen status. The epididymis indicated severe histopathological changes, including reflection of a mitogenic / tumorigenic effect of Q. amara. Thus, this study indicates that Q. amara, when applied as a therapeutic, could be severely toxic to male reproductive mechanisms and the principal mechanism of action appears to be through Leydig cells. Quassin, the major secondary metabolite responsible for the therapeutic potential of Q. amara, is also the one causative of the male reproductive toxic effects. Thus, this study substantiates the claim that Q. amara is toxic to male reproductive system.

L’épididyme joue un rôle important dans l’acquisition de la fertilité, en permettant aux spermatozoïdes d’acquérir mobilité et pouvoir fécondant lors de leur progression dans la lumière. Cette maturation ne peut se faire que grâce à un environnement spécifique qui garantit la protection des spermatozoïdes et est maintenu par la barrière hémato-épididymaire. Cette barrière est formée de jonctions serrées entre les cellules de l’épithélium, et isole le compartiment luminal du compartiment basal. Les cellules basales de l’épididyme sont localisées à la base de l’épithélium et possèdent des projections apicales minces régulées par des facteurs testiculaires, qui seraient capables de traverser la barrière hémato-épididymaire et d’atteindre la lumière du tubule, notamment au niveau des jonctions tripartites. En plus de la participation à la barrière hémato-épididymaire, il a été suggéré un rôle de détoxification, une fonction immunitaire et un rôle de régulation des fonctions d’autres cellules épithéliales. L’un des principaux obstacles à l’étude des cellules basales de l’épididyme est l’absence de protocole pour les isoler.L’objectif principal de ce projet était de caractériser la participation des cellules basales à la barrière hémato-épididymaire via la jonction tricellulaire, et d’étudier les voies de signalisation pouvant être impliquées dans cette participation. Trois sous-objectifs étaient nécessaires : (1) Etudier l’expression et la localisation de la tricelluline dans l’épididyme et déterminer si les cellules basales participent à la barrière hémato-épididymaire par le biais de cette protéine. (2) Mettre au point une méthode pour isoler les cellules basales. (3) Etablir le profil d’expression génique des cellules basales afin de pouvoir étudier les gènes et voies de signalisation spécifiques à ces cellules. Les résultats ont montré que la tricelluline était exprimée dans tous les segments de l’épididyme à des niveaux similaires, et que les niveaux de tricelluline

Microvesicles are of increasing interest in biology as part of normal function of numerous systems; from the immune system (T cell activation) to implantation of the embryo (invasion of the trophoblasts) and sperm maturation (protein transfer in the epididymis). Yet, the mechanisms involved in the appearance of apical blebbing from healthy cells as part of their normal function remain understudied. Microvesicles are produced via one of two pathways: exocytosis or apical blebbing also termed ectocytosis. This work quantifies the histological appearance of apical blebbing in the porcine epididymis during development and examines the role of endogenous estrogens in regulating this blebbing. Apical blebbing appears at puberty and increases in a linear manner into sexual maturity suggesting that this blebbing is a mature phenotype. Endogenous estrogen levels were reduced with an aromatase inhibitor but such a reduction did not affect apical blebbing in treated animals compared with their vehicle-treated littermates. Epididymal production of apical blebs is a secretion mechanism of functionally mature principal cells regulated by factors other than estradiol.

Liver growth factor induces testicular regeneration in EDS-treated rats and increases protein levels of class B scavenger receptors. Am J Physiol Endocrinol Metab 308 E111-E121, 2015. First published November 11, 2014; doi10.1152/ajpendo.00329.2014.-The aim of the present work was to determine the effects of liver growth factor (LGF) on the regeneration process of rat testes after chemical castration induced by ethane dimethanesulfonate (EDS) by analyzing some of the most relevant proteins involved in cholesterol metabolism, such as hormone sensitive lipase (HSL), 3(3-hydroxysteroid dehydrogenase (3(3-HSD), scavenger receptor SR-BI, and other components of the SR family that could contribute to the recovery of steroidogenesis and spermatogenesis in the testis. Sixty male rats were randomized to nontreated (controls) and LGF-treated, EDS-treated, and EDS + LGF-treated groups. Testes were obtained on days 10 (T1), 21 (T2), and 35 (T3) after EDS treatment, embedded in paraffin, and analyzed by immunohistochemistry and Western blot. LGF improved the recovery of the seminiferous epithelia, the appearance of the mature pattern of Leydig cell interstitial distribution, and the expression of mature SR-BI. Moreover, LGF treatment resulted in partial recovery of HSL expression in Leydig cells and spermatogonia. No changes in serum testosterone were observed in control or LGF-treated rats, but in EDS-castrated animals LGF treatment induced a progressive increase in serum testosterone levels and 3(3-HSD expression. Based on the pivotal role of SR-BI in the uptake of cholesteryl esters from HDL, it is suggested that the observed effects of LGF would facilitate the provision of cholesterol for sperm cell growth and Leydig cell recovery. © 2015 American Physiological Society. All rights reserved.

Genetic diversity is a primary component of adaptive evolution, and its loss or reduction can decrease the long-term survival probability of populations. Utilization of cryopreserved semen may be considered a perfect tool to improve genetic diversity, reduce inbreeding, and avoid animal translocation for breeding. The present study aimed at finding a reliable epididymal sperm freezing protocol for the critically endangered onager (Equus hemionus onager). Six testicles from three animals were processed postmortem. The effects of two transportation temperatures (22°C and 4°C; testicles submerged in saline), two cryopreservation techniques (conventional liquid nitrogen vapor freezing in straws and directional freezing in 8-ml HollowTubesTM), and two postthaw incubation temperatures (22°C and 37°C; evaluated after 0.5, 1, 2, and 3 hr) were tested in a 2 × 2 × 2 experimental design. Sperm samples were evaluated for motility, viability, acrosome integrity, and sperm morphology. The resulting optimal freezing protocol includes transportation of testicles at 4°C, cryopreservation by directional freezing, and, if needed, postthaw incubation at 22°C. With this combination of transportation temperature and cryopreservation technique, the authors obtained the following postthaw values normalized to prefreezing values 60.3 ± 8.8% motility, 60.7 ± 13.3% viability, 75.3 ± 9.5% acrosome integrity, and 94.7 ± 2.9% normal morphology (excluding defects due to the epididymal origin of the sperm). After incubation at 22°C, motility values for the above combination were 40 ± 5.7%, 30.3 ± 5.2%, 28.3 ± 4.4%, and 16.7 ± 4.4% for 0.5, 1, 2, and 3 hr, respectively. In conclusion, with this protocol, good quality semen can be stored for future use in artificial inseminations when and where needed. © 2015 by American Association of Zoo Veterinarians.

Objective: To determine whether dendritic cells (DCs) are present in semen and whether their abundance and activation correlate with sperm quality and inflammatory status of the male genital tract. Design: In vitro prospective study. Setting: University hospital laboratory. Patient(s): Eighty infertile men and twelve control fertile men were enrolled in this study. Intervention(s): None. Main Outcome Measure(s): The percentages of DCs, intracellular staining of cytokines, and spermatozoa DNA fragmentation index were assessed by flow cytometry. Seminal cytokines, neutral alpha-glucosidase, zinc, and fructose were measured with commercial kits. Result(s): A significant number of CD11c+HLA-DR+DCs were detected in ejaculates from patients with chronic inflammation of genital tract, which was negatively correlated with spermatozoa motility, vitality, and DNA integrity. Intracellular staining of cytokines of seminal DCs showed enhanced ability to secrete inflammatory cytokines (interleukin [IL] 23p19, tumor necrosis factor [TNF]-related apoptosis-inducing ligand [TRAIL], and TNF-alpha). Furthermore, a significant correlation was found between DCs and the seminal concentrations of IL-6, IL-17, IL-23, TRAIL, and neutral alpha-glucosidase, the marker of epididymal function, in the inflammatory group but not in the noninflammatory and fertile groups. Conclusion(s): The abundance and activation of seminal DCs of infertile men may be closely associated with poor epididymal function and sperm quality. (Fertil Steril (R) 2014; 101: 70-7. (C) 2014 by American Society for Reproductive Medicine.)

Cold transport of epididymides from genetically modified mice is an efficient alternative to the shipment of live animals between research facilities. Mouse sperm from epididymides cold-stored for short periods can maintain viability. We previously reported that cold storage of mouse epididymides in Lifor® perfusion medium prolonged sperm motility and fertilization potential and that the sperm efficiently fertilized oocytes when reduced glutathione was added to the fertilization medium. Cryopreservation usually results in decreased sperm viability; an optimized protocol for cold storage of epididymides plus sperm cryopreservation has yet to be established. Here, we examined the motility and fertilization potential of cryopreserved, thawed (frozen-thawed) sperm from previously cold-stored mouse epididymides. We also examined the protective effect of sphingosine-1-phosphate (S1P) on sperm viability when S1P was added to the preservation medium during cold storage. We assessed viability of frozen-thawed sperm from mouse epididymides that had been cold-transported domestically or internationally and investigated whether embryos fertilized in vitro with these sperm developed normally when implanted in pseudo-pregnant mice. Our results indicate that frozen-thawed sperm from epididymides cold-stored for up to 48 h maintained high fertilization potential. Fertilization potential was reduced after cold storage for 72 h, but not if S1P was included in the cold storage medium. Live pups were born normally to recipients after in vitro fertilization using frozen-thawed sperm from cold-transported epididymides. In summary, we demonstrate an improved protocol for cold-storage of epididymides that can facilitate transport of genetically engineered-mice and preserve sperm viability after cryopreservation.