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In several mammalian species, acute endometritis driven by the recruitment of polymorphonuclear cells (PMN) occurs in response to semen. These PMNs release DNA to form neutrophil extracellular traps (NETs) in cattle, horse and human, leading to sperm entrapment. While there is no evidence of this phenomenon occurring in donkeys, artificial insemination (AI) with frozen-thawed semen, which results in very poor pregnancy rates, leads to a large PMN recruitment to the uterus.

Altres ajuts: Generalitat de Catalunya 2017-SGR-1229. Fundación Séneca, Región de Murcia 21453/FPI/20, This study investigated whether the activities of the antioxidant components of donkey seminal plasma (SP)-both enzymatic (superoxide dismutase (SOD), catalase-like (CAT), glutathione peroxidase-like (GPX), and paraoxonase type 1 (PON1)) and non-enzymatic (measured in terms of total thiol, copper-reducing antioxidant capacity (CUPRAC), ferric-reducing ability of plasma (FRAP), and Trolox equivalent antioxidant capacity (TEAC))-and oxidative stress index (OSI) are related to sperm cryotolerance. For this purpose, 15 ejaculates from jackasses (one per individual) were collected and split into two aliquots. The first one was used for measuring the activities levels of enzymatic and non-enzymatic antioxidants and OSI in SP, whereas the other aliquot was cryopreserved. Before cryopreservation, sperm quality parameters (concentration, motility, and viability) were evaluated. After thawing, sperm motility, plasma membrane integrity, lipid disorder, mitochondrial membrane potential, reactive oxygen species (ROS), and calcium intracellular levels were also determined. Based on the percentages of total motility (TM) and of sperm with an intact plasma membrane (SYBR14 + /PI −) after thawing, samples were classified as good-freezability (GFE) or poor-freezability (PFE) ejaculates through cluster analyses. The SP activity levels of enzymatic (SOD and PON1) and non-enzymatic antioxidants (CUPRAC, FRAP, and TEAC) were higher (p < 0.05) in GFE than in PFE, whereas SP-OSI was higher (p < 0.05) in PFE than in GFE. In addition, the activity levels of SOD, PON1, GPX, CUPRAC, FRAP, and TEAC were positively (p < 0.05) related to post-thaw sperm motility and plasma membrane integrity and negatively to intracellular ROS levels. The SP-OSI was negatively correlated (p < 0.05) to post-thaw sperm quality parameters and positively to intracellular ROS levels. It can thus be concluded that donkey SP antioxidants are related to sperm cryotolerance and that measurements of antioxidants PON1, SOD

Funding: GENCAT (2017-SGR-1229)., In donkeys, the use of frozen-thawed sperm for artificial insemination (AI) leads to low fertility rates. Furthermore, donkey sperm produce a large amount of reactive oxygen species (ROS), and post-AI inflammation induces the formation of neutrophil extracellular traps (NETosis), which further generates many more ROS. These high ROS levels may induce lipid peroxidation in the sperm plasma membrane, thus affecting its integrity. Enzymatic and non-enzymatic antioxidants, mainly found in the seminal plasma (SP), are responsible for maintaining the redox balance. However, this fluid is removed prior to cryopreservation, thereby exposing sperm cells to further oxidative stress. The exogenous addition of antioxidants to the freezing medium can reduce the detrimental effects caused by ROS generation. Therefore, the aim of this study was to evaluate how the addition of different reduced glutathione (GSH) concentrations (control, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM) to fresh sperm affect their cryotolerance. Total and progressive motility, kinematic parameters and motile sperm subpopulations were significantly (p < 0.05) different from the control in treatments containing 8 mM and 10 mM GSH, but not at lower concentrations. Plasma and acrosome membrane integrity, mitochondrial membrane potential (MMP) and intracellular superoxide levels (O ) were not affected (p > 0.05) by any GSH concentration. Interestingly, however, the addition of 8 mM or 10 mM GSH reduced (p < 0.05) the percentages of viable sperm with high overall ROS levels compared to the control. In conclusion, frozen-thawed donkey sperm are able to tolerate high GSH concentrations, which differs from what has been observed in other species. This antioxidant capacity suggests that ROS could be important during post-AI and that the impact of using exogenous antioxidants like GSH to improve the sperm resilience to freeze-thawing is limited in this species.

Previous research demonstrated that seminal plasma, the fluid that together with sperm composes the semen, modulates the interaction between sperm and polymorphonuclear neutrophils (PMN) within the donkey uterus. This study sought to evaluate whether a specific set of proteins or the entire seminal plasma is involved in that modulation. Seminal plasma was fractioned into six portions: <3, 3-10, 10-30, 30-50, 50-100, and >100 kDa. Sperm incubated with two specific fractions, containing 30-50 kDa and 50-100 kDa proteins, exhibited the highest motility and viability after co-incubation with PMN for 4 h, and the highest sperm-PMN interaction at the beginning of the experiment. These results suggest that proteins of 30-50 and 50-100 kDa, rather than the entire seminal plasma, appear to be involved in the inflammatory reaction within the donkey endometrium. While artificial insemination (AI) with frozen-thawed sperm results in low fertility rates in donkeys, the addition of seminal plasma, removed during cryopreservation, partially counteracts that reduction. Related to this, an apparent inflammatory reaction in jennies is induced following AI with frozen-thawed sperm, as a high amount of polymorphonuclear neutrophils (PMN) are observed within the donkey uterus six hours after AI. While PMN appear to select the sperm that ultimately reach the oviduct, two mechanisms, phagocytosis and NETosis, have been purported to be involved in that clearance. Remarkably, sperm interacts with PMN, but the presence of seminal plasma reduces that binding. As seminal plasma is a complex fluid made up of different molecules, including proteins, this study aimed to evaluate how different seminal plasma fractions, separated by molecular weight (<3, 3-10, 10-30, 30-50, 50-100, and >100 kDa), affect sperm-PMN binding. Sperm motility, viability, and sperm-PMN binding were evaluated after 0 h, 1 h, 2 h, 3 h, and 4 h of co-incubation at 38 °C. Two seminal plasma fractions, including 30-50 kDa or 5

Collagen fibers and inflammatory cells are the basis for jenny endometrium Kenney and Doig's classification developed for the mare. The infiltration of a large number of eosinophils in the jenny endometrium is intriguing. Eosinophil and fibroblast produced IL33, which has been related to fibrosis development and chronicity. This work on the endometrium consisted of (i) quantification of collagen type I (COL1A2), type III (COL3A1), and IL33 transcripts; (ii) histological localization and quantification of COL1 and COL3 proteins; and (iii) eosinophil and neutrophil count and correlation with collagen area and IL33 transcripts. Localization of COL protein in the jenny endometrium was also compared to the mare endometrium. As fibrosis increased, eosinophil and neutrophil count decreased (P < 0.05). A 5-fold increase in IL33 transcripts was noted from categories IIA to III. There was a tendency toward a positive correlation between eosinophil count and IL33 transcripts in category IIA endometrium (P = 0.055). Neither transcripts of COL1A2 nor COL3A1 nor the areas of COL1 or COL3 differed with endometrial categories. Unlike for the mare, and regardless of the jenny endometrium classification, COL3 was always found to different extents in the stratum compactum, while COL1 was mainly present in deep stroma. As fibrosis progressed in the mare, an extensive increase in COL1 fibers was notorious under the surface epithelium. Correlations between neutrophil count and COL1 and COL3 areas were observed in the jenny endometrium, although no correlation was found for eosinophil count. Neutrophil count positive correlation with the COL1 area and negative correlation with the COL3 area in endometria with mild lesions suggest that neutrophils in the jenny endometrium may be involved in fibrogenesis. In addition, when eosinophilia subsides, the endometrium reacts with fibrosis establishment, which could be stimulated by the pro-fibrotic cytokine IL33, whose release might then be ascrib

This work analyzes the effects of red LED light on mammalian sperm mitochondrial function, using the pig as an animal model. Liquid-stored pig semen was stimulated with red-light for 1, 5 and 10 min in the presence or absence of oligomycin A, a specific inhibitor of mitochondrial ATP synthase, or carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), a specific disruptor of mitochondrial electron chain. Whereas exposure for 1 and 5 min significantly (p < 0.05) decreased total motility and intracellular ATP levels, irradiation for 10 min induced the opposite effect. Oligomycin A abolished the light-effects on intracellular ATP levels, O2 consumption and mitochondrial membrane potential, whereas compared to non-irradiated samples, FCCP significantly (p < 0.05) increased O2 consumption when sperm were irradiated for 1 min. Both oligomycin A and FCCP significantly (p < 0.05) decreased total motility. Red-light increased cytochrome c oxidase activity with a maximal effect after 5 min of irradiation, which was abolished by both oligomycin A and FCCP. In conclusion, red-light modulates sperm mitochondrial function via electron chain activity in an exposition, time-dependent manner.

Altres ajuts: Generalitat de Catalunya, Donkey Artificial Insemination (AI) with frozen/thawed semen results in poor fertility outcomes. Jennies show a significant post-AI endometrial reaction, with a large amount of defense cells-polymorphonuclear neutrophils (PMN)-migrating to the uterine lumen. Seminal plasma (SP) has a detrimental effect on sperm conservation and its removal is a necessary step in the semen freezing protocol. However, several SP proteins seem to control sperm-PMN binding. Single layer centrifugation (SLC) with colloids, which has been used to select spermatozoa and improve reproductive performance in different species, is known to remove SP proteins attached to the sperm membrane. In this study, two experiments were performed. The first one compared the quality of SLC-selected and non-selected fresh donkey spermatozoa. In the second experiment, PMN obtained from the peripheral blood were co-incubated with selected and unselected spermatozoa, and the interaction between PMN and spermatozoa was analyzed. In conclusion, SLC of fresh donkey semen increases the proportion of functionally intact spermatozoa and appears to remove the SP proteins that inhibit sperm-PMN binding, thus increasing sperm phagocytosis by PMN. This study sought to determine whether single layer centrifugation (SLC) of fresh donkey semen with Equicoll has any impact on sperm quality parameters and on the modulation of endometrial reaction following semen deposition using an in vitro model. Seventeen ejaculates from five jackasses were obtained using an artificial vagina and diluted in a skim-milk extender. Samples were either selected through SLC (Equicoll) or non-treated (control). Two experiments were performed. The first one consisted of incubating selected or non-selected spermatozoa at 38 °C for 180 min. Integrity and lipid disorder of sperm plasma membrane, mitochondrial membrane potential, and intracellular levels of calcium and reactive oxygen species were evaluated at 0, 60, 120, and 180 min. In the second e

The objective of the present study was to determine the physiological role of voltage-gated hydrogen channels 1 (HVCN1 channels) during in vitro capacitation of pig spermatozoa. Sperm samples from 20 boars were incubated in capacitating medium for 300 minutes (min) in the presence of 2-guanidino benzimidazole (2-GBI), a specific HVCN1-channel blocker, added either at 0 min or after 240 min of incubation. Control samples were incubated in capacitating medium without the inhibitor. In all samples, acrosomal exocytosis was triggered with progesterone after 240 min of incubation. Sperm viability, sperm motility and kinematics, acrosomal exocytosis, membrane lipid disorder, intracellular calcium levels and mitochondrial membrane potential were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. While HVCN1-blockage resulted in altered sperm viability, sperm motility and kinematics and reduced mitochondrial membrane potential as compared to control samples, at any blocker concentration and incubation time, it had a non-significant effect on intracellular Ca 2+ levels determined through Fluo3-staining. The effects on acrosomal exocytosis were only significant in blocked samples at 0 min, and were associated with increased membrane lipid disorder and Ca 2+ levels of the sperm head determined through Rhod5-staining. In conclusion, HVCN1 channels play a crucial role in the modulation of sperm motility and kinematics, and in Ca 2+ entrance to the sperm head.