José Alvaro Cebrian Pérez

This work aimed to determine the contribution of the testis and epididymis and the effect of the photoperiodic regimen on ram seminal plasma (SP). Semen was collected from 15 mature rams located in an equatorial (Colombian Creole and Romney Marsh, eight intact and two vasectomized) or a temperate climate (Rasa Aragonesa, three intact and two vasectomized). SP proteins were analyzed by Bradford, SDS-PAGE and difference gel electrophoresis (DIGE). Melatonin and testosterone concentrations were quantified by ELISA, and activity of glutathione peroxidase (GPx), glutathione reductase (GRD), and catalase by enzymatic assays. Vasectomy increased protein concentration and the intensity of high molecular weight bands (p < 0.001), with no differences between breeds. DIGE revealed the absence of six proteins in vasectomized rams: angiotensin-converting enzyme, lactotransferrin, phosphoglycerate kinase, sorbitol dehydrogenase, epididymal secretory glutathione peroxidase and epididymal secretory protein E1. Vasectomy also decreased melatonin concentrations in seasonal rams, and testosterone in all of them (p < 0.001), but did not affect antioxidant enzyme activity. Equatorial rams showed lower melatonin and testosterone concentration (p < 0.01) and catalase, but higher GPx activity (p < 0.05). In conclusion, vasectomy modifies the protein profile and hormonal content of ram seminal plasma, whereas the exposure to a constant photoperiod affects hormonal concentration and antioxidant enzymes activity.

Nitric oxide (NO·), synthesized from L-arginine by nitric oxide synthase (NOS), is involved in sperm functionality. NOS isoforms have been detected in spermatozoa from different species, and an increment in NOS activity during capacitation has been reported. This work aims to determine the presence and localization of NOS isoforms in ram spermatozoa and analyse their possible changes during in vitro capacitation. Likewise, we investigated the effect of melatonin on the expression and localization of NOS and NO· levels in capacitated ram spermatozoa. Western blot analysis revealed protein bands associated with neuronal NOS (nNOS) and epithelial NOS (eNOS) but not with inducible NOS (iNOS). However, the three isoforms were detected by indirect immunofluorescence (IFI), and their immunotypes varied over in vitro capacitation with cAMP-elevating agents. NO· levels (evaluated by DAF-2-DA/PI staining) increased after in vitro capacitation, and the presence of L-arginine in the capacitating medium raised NO· production and enhanced the acrosome reaction. Incubation in capacitating conditions with a high-cAMP medium with melatonin modified the NOS distribution evaluated by IFI, but no differences in Western blotting were observed. Melatonin did not alter NO· levels in capacitating conditions, so we could infer that its role in ram sperm capacitation would not be mediated through NO· metabolism.

The melatonin catabolism is very complex and not completely understood. Melatonin can be metabolized by free radical interaction, but also pseudo‐enzymatically or by enzymatic pathways. We have previously detected the existence of melatonin‐synthesizing enzymes and melatonin receptors MT1 and MT2 in the ram reproductive tract; thus, in order to start to elucidate melatonin catabolism in these organs, we have investigated the presence of the melatonin‐catabolizing enzymes indoleamine 2,3‐dioxygenase (IDO, both IDO1 and IDO2 isoforms) and myeloperoxidase (MPO) in testis, epididymis and accessory glands. Gene expression analyses by real‐time PCR showed the presence of MPO, IDO1 and IDO2 in all the organs of the ram reproductive tract and revealed that MPO is the main melatonin‐catabolizing enzyme, which is mainly expressed in the testis and the bulbourethral glands (p < .05). These results were further corroborated by immunohistochemical staining, and by Western blot. Likewise, MPO was also evidenced in epididymal and ejaculated spermatozoa by indirect immunofluorescence and Western blot. In conclusion, melatonin‐catabolizing enzymes MPO, IDO1 and IDO2 are expressed in the ram reproductive tract, and MPO is the most expressed one, mainly in the testis and the bulbourethral glands. The presented results warrant further studies on the function of these enzymes and their melatonin‐metabolizing activity.

Some melatonin functions in mammals are exerted through MT₁ and MT₂ receptors. However, there are no reports of their presence in the reproductive tract of the ram, a seasonal species. Thus, we have investigated their existence in the ram testis, epididymis, accessory glands and ductus deferens. Real-time polymerase chain reaction (qPCR) revealed higher levels of m-RNA for both receptors in the testis, ampulla, seminal vesicles, and vas deferens, than in the other organs of the reproductive tract (p < 0.05). Western blot analyses showed protein bands compatible with the MT₁ in the testis and cauda epididymis, and for the MT₂ in the cauda epididymis and deferent duct. Immunohistochemistry analyses revealed the presence of MT₁ receptors in spermatogonias, spermatocytes, and spermatids, and MT₂ receptors in the newly-formed spermatozoa in the testis, whereas both receptors were located in the epithelial cells of the ampulla, seminal vesicles, and ductus deferens. Indirect immunofluorescence showed significant differences in the immunolocation of both receptors in spermatozoa during their transit in the epididymis. In conclusion, it was demonstrated that melatonin receptors are present in the ram reproductive tract. These results open the way for new studies on the molecular mechanism of melatonin and the biological significance of its receptors.

Melatonin (MLT) is present in seminal plasma (SP) of mammalian species, including pigs, and it is credited with antioxidant properties. This study aims to identify the sources of variation and the role of boar SP MLT on sperm quality and functionality and in vivo fertilizing ability of liquid-stored semen doses used in AI programs. The SP MLT was measured using an ELISA kit in a total of 219 ejaculates collected from 76 boars, and reproductive records of 5,318 AI sows were recorded. Sperm quality was assessed according to motility (computer-aided sperm analysis) and viability (cytometry evaluation). Sperm functionality was assessed according to the cytometric determination of intracellular H₂O₂ generation, total and mitochondrial O₂⁻ production, and lipid peroxidation in liquid AI semen samples stored at 17°C over 144 h. The concentration of SP MLT differed among seasons (P < 0.01) and day length periods (P < 0.001) of the year, demonstrating that the ejaculates collected during the increasing day length period (9.80 ± 1.38 pg/mL, range: 2.75–21.94) had lower SP MLT concentrations than those collected during the decreasing day length period (16.32 ± 1.67 pg/mL, range: 5.02–35.61). The SP MLT also differed (P < 0.001) among boars, among ejaculates within boar, and among portions within the ejaculate, demonstrating that SP from the first 10 mL of sperm-rich ejaculate fraction (SRF) exhibited lower MLT concentrations than post-SRF. The SP MLT was negatively related (P < 0.001) to mitochondrial O₂⁻ production in viable sperm. The SP MLT did not differ among AI boars (n = 14) hierarchically grouped according to high and low fertility outcomes. In conclusion, SP MLT concentration in AI boars varies depending on the season of ejaculate collection and differs among boars, ejaculates within boar, and portions within ejaculate. The SP MLT may act at the mitochondrial level of sperm by reducing the generation of O₂⁻. However, this antioxidant role of SP MLT was not reflected in sperm quality or in vivo fertility outcomes of AI semen doses. Copyright © 2017. American Society of Animal Science .

Action of reactive oxygen species, protamination failures and apoptosis are considered the most important etiologies of sperm DNA fragmentation. This study evaluated the effects of induced lipid peroxidation susceptibility on native semen profile and identified the mechanisms involved in sperm DNA fragmentation and testicular antioxidant defense on Santa Ines ram sperm samples. Semen was collected from 12 adult rams (Ovis aries) performed weekly over a 9-week period. Sperm analysis (motility, mass motility, abnormalities, membrane and acrosome status, mitochondrial potential, DNA fragmentation, lipid peroxidation and intracellular free radicals production); protamine deficiency; PRM1, TNP1 and TNP2 gene expression; and determination of glutathione peroxidase (GPx), glutathione reductase, catalase (CAT) and superoxide dismutase activity and immunodetection in seminal plasma were performed. Samples were distributed into four groups according to the sperm susceptibility to lipid peroxidation after induction with ascorbate and ferrous sulfate (low, medium, high and very high). The results were analyzed by GLM test and post hoc least significant difference. We observed an increase in native GPx activity and CAT immunodetection in groups with high susceptibility to induced lipid peroxidation. We also found an increase in total sperm defects, acrosome and membrane damages in the group with the highest susceptibility to induced lipid peroxidation. Additionally, the low mitochondrial membrane potential, susceptible to chromatin fragmentation and the PRM1 mRNA were increased in the group showing higher susceptibility to lipid peroxidation. Ram sperm susceptibility to lipid peroxidation may compromise sperm quality and interfere with the oxidative homeostasis by oxidative stress, which may be the main cause of chromatin damage in ram sperm. Abstract Action of reactive oxygen species, protamination failures and apoptosis are considered the most important etiologies of sperm DNA fragmentation. This study evaluated the effects of induced lipid peroxidation susceptibility on native semen profile and identified the mechanisms involved in sperm DNA fragmentation and testicular antioxidant defense on Santa Ines ram sperm samples. Semen was collected from 12 adult rams (Ovis aries) performed weekly over a 9-week period. Sperm analysis (motility, mass motility, abnormalities, membrane and acrosome status, mitochondrial potential, DNA fragmentation, lipid peroxidation and intracellular free radicals production); protamine deficiency; PRM1, TNP1 and TNP2 gene expression; and determination of glutathione peroxidase (GPx), glutathione reductase, catalase (CAT) and superoxide dismutase activity and immunodetection in seminal plasma were performed. Samples were distributed into four groups according to the sperm susceptibility to lipid peroxidation after induction with ascorbate and ferrous sulfate (low, medium, high and very high). The results were analyzed by GLM test and post hoc least significant difference. We observed an increase in native GPx activity and CAT immunodetection in groups with high susceptibility to induced lipid peroxidation. We also found an increase in total sperm defects, acrosome and membrane damages in the group with the highest susceptibility to induced lipid peroxidation. Additionally, the low mitochondrial membrane potential, susceptible to chromatin fragmentation and the PRM1 mRNA were increased in the group showing higher susceptibility to lipid peroxidation. Ram sperm susceptibility to lipid peroxidation may compromise sperm quality and interfere with the oxidative homeostasis by oxidative stress, which may be the main cause of chromatin damage in ram sperm.

Summary: Melatonin is a ubiquitous molecule found in a wide range of fluids, one of them being ram seminal plasma, in which it can reach higher concentrations than those found in blood, suggesting an extrapineal secretion by the reproductive tract. In order to identify the source of the melatonin found in ram seminal plasma, we first tried to determine whether the melatonin levels were maintained during the day. For this purpose, melatonin concentrations were measured in seminal plasma obtained from first ejaculates of six rams at 6:00 a.m. in total darkness, at 10:00 a.m. and at 14:00 p.m. The melatonin concentration was higher (p < 0.05) in ejaculates collected at 6:00 a.m. than at 10:00 and 14:00. There was no statistical difference between the latter. To further corroborate an extrapineal secretion of melatonin, the presence of the two key enzymes involved in melatonin synthesis, arylalkylamine‐N‐acetyltransferase (AANAT) and N‐acetylserotonin‐O‐methyltransferase (ASMT) was analyzed by RT‐PCR, q‐PCR and Western‐blot in ram testes, epididymis, and accessory glands. The RT‐PCR showed the presence of the m‐RNA codifying both AANAT and ASTM in all the tissues under study, but the q‐PCR and Western‐blot revealed that gene expression of these enzymes was significantly higher in the testis (p < 0.05). Immunohistochemistry confirmed the presence of AANAT and ASMT in the testis and revealed that they were found in the Leydig cells, spermatocytes, and spermatids. Also, measurable levels of melatonin were found in testicular tissue and the tail of the epididymis. In conclusion, our study indicates that the testes are one of the likely sources of the high levels of melatonin found in ram seminal plasma, at least during the day.

Higher temperatures lead to an increase of testicular metabolism that results in spermatic damage. Oxidative stress is the main factor responsible for testicular damage caused by heat stress. The aim of this study was to evaluate lasting effects of heat stress on ejaculated sperm and immediate or long‐term effects of heat stress on epididymal sperm. We observed decrease in motility and mass motility of ejaculated sperm, as well as an increase in the percentages of sperm showing major and minor defects, damaged plasma and acrosome membranes, and a decrease in the percentage of sperm with high mitochondrial membrane potential in the treated group until one spermatic cycle. An increased enzymatic activity of glutathione peroxidase and an increase of stressed cells were observed in ejaculated sperm of the treated group. A decrease in the percentage of epididymal sperm with high mitochondrial membrane potential was observed in the treated group. However, when comparing immediate and long‐term effects, we observed an increase in the percentage of sperm with low mitochondrial membrane potential. In conclusion, testicular heat stress induced oxidative stress that led to rescuable alterations after one spermatic cycle in ejaculated sperm and also after 30 days in epididymal sperm.