Concentrations of carnitine, glutamate and myo-inositol in epididymal fluid and spermatozoa from boars.

This study examines the concentrations of carnitine, glutamate and myo-inositol in fluid and spermatozoa from six epididymal regions. Samples were taken from six post-pubertal boars, and the sperm concentration, the protein concentration in epididymal fluid and the concentrations of carnitine, myo-inositol and glutamate in the epididymal fluid and spermatozoa were analysed. In epididymal fluid the concentration of myo-inositol decreased in a proximo-distal direction, whereas intraluminal concentrations of L-carnitine and L-glutamate increased distally. As changes in the concentration of these solutes did not parallel changes in sperm concentration, this may reflect secretion or absorption of theses solutes. The sperm content of inositol fell as they moved from the distal caput whereas glutamate content increased from the distal caput to more distal regions and carnitine content remained unchanged during epididymal transit. This is the first attempt to elucidate the changes in the content of glutamate and inositol in epididymal spermatozoa of mammals and in the fluid from different epididymal regions of boars.

Study of the polyol pathway in the porcine epididymis.

Concentrations of D-glucose, D-fructose and D-sorbitol were quantified in porcine epididymal fluid by spectrofluorimetric assays and aldose reductase (AR) and sorbitol dehydrogenase (SDH) were located immunohistochemically in the epididymal epithelium. Glucose and fructose concentrations were low (<1 mM) and decreased in the cauda whereas sorbitol concentration (4-7 mM) was rather uniform along the duct. AR was luminally located on microvilli in the caput and corpus with less presence distally and was present in the lumen. SDH was present apically and basally in epithelial cells throughout the epididymis and in the lumen. The observations are consistent with diffusion of circulating glucose into the lumen, its conversion via AR to sorbitol which accumulates in the lumen and the action of SDH on sorbitol to produce fructose. Sperm metabolism of glucose and fructose may explain their lower concentrations in the cauda and sorbitol could be a metabolic substrate or osmolyte required for volume regulation.

Development of a protocol for multiple staining with fluorochromes to assess the functional status of boar spermatozoa.

The aim of this study was to design a simple and reliable method for the simultaneous evaluation of the nucleus, the acrosome, and the mitochondrial sheath of boar spermatozoa. Sperm samples coming from healthy and sexually mature Pietrain boars were incubated with two nuclear fluorochromes--bis-benzamide specific for viable cells, and propidium iodide specific for nonviable cells--the fluorochrome Mitotracker Green FM specific for functional mitochondria, and the lectin Trypsin inhibitor from Soybean (SBTI) conjugated with the fluorochrome Alexa Fluor 488 specific for proacrosin. The results obtained from assessing the functional status of the spermatozoa using fluorochromes were compared with the conventional sperm parameters of sperm vitality using the eosin exclusion test (EE test), and sperm motility and morphology using the computer-assisted semen analyzer SCA 2002Producció. Applying the multiple staining test, it was found that the frequency of viable spermatozoa with intact acrosome and intact mitochondria was not different from the frequency of viable spermatozoa obtained with the EE test, and also correlated positively with the frequency of motile spermatozoa and the frequency of mature spermatozoa. Therefore, this technique is useful to characterize the status of boar spermatozoa by assessing the nuclear, acrosomal, and mitochondrial integrity. Moreover, it provides reliable diagnostic information about the fertility potential of boars.

Effects of a high semen-collection frequency on the quality of sperm from ejaculates and from six epididymal regions in boars.

This study examines the effect of semen-collection rhythm on the sperm maturation process in boar epididymis. Three post-pubertal boars were submitted to a high semen-collection frequency (stressed boars) during 4 days, and three males were kept as a control group (control boars). Semen samples coming from six epididymal regions and from the ejaculate of each male were evaluated for sperm concentration, sperm vitality, sperm motility and sperm morphology. In each epididymal region, either fluid resorption or fluid secretion was determined from the variation in sperm concentration. The pattern of fluid resorption-secretion along the epididymal duct differed significantly between the stressed and control boars. A high semen-collection frequency also affected the development of sperm motility and the sperm cytoplasmic droplet displacement along the epididymal duct. The incidence of some sperm abnormalities was also found to be higher in some epididymal regions and ejaculates of stressed boars. From the results of this study, it can be concluded that a high semen-collection frequency brings about an altered resorption and secretion pattern of the epididymal fluid, which results in defective sperm maturation and abnormal development of sperm motility.

In vitro culture of epithelial cells from the caput, corpus, and cauda epididymis of Sus domesticus.

This work describes a protocol to culture epididymal epithelial cells from the caput, corpus, and cauda regions of Sus domesticus. Epididymal epithelial fragments were obtained by dissection and enzymatic digestion with collagenase. About 30 epididymal fragments from each epididymal region were cultured in 24-well culture plates with supplemented RPMI-1640 medium at 37 degrees C, 5% CO2 in air, and 100% humidity. A confluent monolayer of polygonal and tightly packed epithelioid cells from the three epididymal regions was obtained after 12-16 days in culture and maintained in vitro for more than 60 days. The proportion of epididymal epithelial cells in these cultures was assessed by immunofluorescent staining for cytokeratins. Throughout the 2 months of culture, about 80% of the cells were cytokeratin-positive. Electron microscopy observations indicated that cultured cells from caput, corpus, and cauda epididymal regions were tightly adhered to each other by junctional complexes and that stereocilia were present in their apical membranes. Moreover, the presence of an extensive rough endoplasmic reticulum, Golgi apparatus and numerous vesicles in the cytoplasm suggested that cultured cells maintained secretory and absorptive activities. These results show that the epididymal epithelial cells in culture from S. domesticus retain some fundamental features that characterize the epididymal epithelium in the intact organ. This system might be a valuable tool for studying the mechanism of sperm maturation in vitro, including epididymal cell secretions and the analysis of regional differences.