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The growing and widespread use of glyphosate-based herbicides (GBHs) has raised an intense public debate about the impact of environmental contamination on animal and human health, including male fertility. The aim of this study was to deepen the impact of glyphosate (Gly) and GBHs on mammalian sperm investigating the effect of in vitro exposure of stallion spermatozoa to Gly and to its commercial formulation Roundup® (R). Spermatozoa were incubated at 37 °C with different Gly or R concentrations (from 0.5 to 720 μg/mL Gly or R at the same Gly-equivalent concentrations). After 1 h of incubation motility, viability, acrosome integrity, mitochondrial activity and ROS production were assessed. Gly, at all the concentrations tested, did not induce any detrimental impact on the sperm quality parameters evaluated. Conversely, R starting from 360 μg/mL (Gly-equivalent dose) significantly (P < 0.05) decreased total and progressive motility, viability, acrosome integrity, mitochondrial activity and the percentage of live spermatozoa with intact mitochondria not producing ROS. Our results indicate that the commercial formulation R is more toxic than its active molecule Gly and that the negative impact on stallion sperm motility might be likely due to a detrimental effect mainly at membrane and mitochondrial level and, at least in part, to redox unbalance. Moreover, based on the data obtained, it can be hypothesized a species-specificity in sperm sensitivity to Gly and GBHs as horse spermatozoa were negatively influenced at higher concentrations of R compared to those reported in literature to be toxic for human and swine male germ cells.

Sperm cells rely on different substrates to fulfil thei energy demand for different functions and diverse moments of their life. Species specific mechanism involve both energy substrate transport and their utilization: hexose transporters, a protein family of facilitative passive transporters of glucose and other hexose, have been identified in spermatozoa of different species and, within the species, their localization has been identified and, in some cases, linked to specific glycilitic enzyme presence. The catabolism of hexose sources for energy purposes has been studied in various species, and recent advances has been made in the knowledge of metabolic strategies of sperm cells. In particular, the importance of aerobic metabolism has been defined and described in horse, boar and even mouse spermatozoa; bull sperm cells demonstrate to have a good adaptability and capacity to switch between glycolysis and oxidative phosphorylation; finally, dog sperm cells have been demonstrated to have a great plasticity in energy metabolism management, being also able to activate the anabolic pathway of glycogen syntesis. In conclusion, the study of energy management and mitochondrial function in spermatozoa of different specie furnishes important base knowledge to define new media for preservation as well as newbases for reproductive biotechnologies.

This study evaluated the bioenergetic map of mitochondria metabolism in cryopreserved bovine sperm. The detected oligomycin-sensitive basal respiration supported ATP production; frozen-thawed spermatozoa were found to have a coupling efficiency higher than 0.80. Cell respiration, however, was not stimulated by the protonophoric action of FCCP, as its titration with 1, 2, 4 and 6 μM did not stimulate the uncoupling activity on oxidative phosphorylation as highlighted by unresponsive oxygen consumption. The unusual effect on the stimulation of maximal respiration was not related to fibronectin- or PDL-coated plates used for cellular metabolism analysis. Conversely, irradiation of frozen-thawed bovine sperm with the red light improved mitochondrial parameters. In effect, the maximal respiration of red-light-stimulated sperm in PDL-coated plates was higher than the non-irradiated. In spite of this, red-light irradiation had no impact on membrane integrity and mitochondrial activity evaluated by epifluorescence microscopy Open Access Funding provided by Universita degli Studi di Bologna within the CRUI-CARE Agreement

The aim of this study was to verify the reliability of an open access CASA software (BGM) to evaluate the sperm motility of cattle and buffalo, comparing motility and kinematic parameters to those of a commercial one (HTM). Thirty frozen-thawed samples for each species were analyzed with both HTM and BGM, after 1 h of incubation at 37 °C. Sperm viability and mitochondrial membrane potential (MMP) were evaluated through flow cytometric analysis. Agreement of all motility variables between the two systems was assessed. Correlation analysis was performed to identify relationships between motion parameters and sperm viability and MMP. Bland Altman analysis showed good agreement between methods for all motility parameters except for curvilinear velocity (VCL) in cattle, and for average path (VAP), VCL and (amplitude of lateral head displacement) ALH in buffalo, that showed a proportional bias (P > 0.05). In both systems, positive correlation between both viability and high MMP and total and progressive motility of cattle spermatozoa were found; viability and the sperm with high MMP were positive correlated only with VAP, straight-line (VSL), VCL and ALH evaluated with HTM system. Different results were found for buffalo sperm motility parameters, since viability had positive correlations and mitochondrial activity negative ones. Results suggested that motility assessment performed by these two systems are comparable. The discrepancy of VCL, VAP, and ALH could be due to the difference in the algorithms between software. The open-access CASA plug-in is a reliable alternative to the expensive commercial CASA system for sperm motility assessment in cattle and buffalo.

The objectives of the present study were to estimate the number of colonies forming units (CFU) from penile mucosa and semen, the effect of two antiseptic solutions used to flush the preputial cavity to reduce the bacterial counts from those sites, and compare them. Six clinically healthy bulls between 15 and 16 mo old declared satisfactory potential breeders were used. A prospective, randomized, and controlled cross-over design was performed, in which each bull was first sampled from the penile mucosa and semen without treatment (control group) and 24 h later, after antiseptic preputial flushing (treated group). In the treated group, the preputial area was cleaned, the preputial hair was cut, urination was stimulated, prepuce area was scrubbed twice, and the preputial cavity was flushed with either 1% of povidone-iodine solution (POI; 500 mL) or 0.05% chlorhexidine digluconate (CHG; 500 mL), maintained for 10 min. Then, the preputial cavity was emptied and flushed with 500 mL of sterile saline solution. Next, the accessory sexual glands were massaged per rectum. Finally, protrusion, erection, and ejaculation were obtained by electroejaculation, and samples from penile mucosa and semen were collected for microbiological culture. The number of CFU was determined for each sample by enumerate total aerobic bacteria using Standard Plate Surface Count cultured for 48 h. In the first replicate, half of the bulls were treated with CHG, and the other half were treated with POI. After 58.8 ± 5.3 days (x ± SD) of wash-out period, the treatments were reverted, and the same protocols were applied again. In the control group, the median number of CFUs from the penile mucosa was 750,000 (range from 60,000 to 1,800,000) and the median number of CFUs in semen was 8,000,000 (700,000-45,000,000). The CFU in semen was higher than the penile mucosa (P = 0.005). Both antiseptic solutions reduced the median number of CFUs on the penile mucosa to 915 (P = 0.002) and in semen to 1,680 (P = 0.002). The antiseptic effect on the penile mucosa was higher for CHG solution (490) than for POI solution (6,650; P = 0.05). The antiseptic effect on semen of CHG was also greater (200) than for the POI solution (31,000; P = 0.05). It can be concluded that the median number of CFU was higher in semen compared with penile mucosa, and flushing the preputial cavity either with 0.05% CHG or 1% POI maintained for 10 min reduced the number of CFUs from penile mucosa and semen. The level of antiseptic activity was higher for CHG than for POI.

In this study boar sperm mitochondrial activity was studied and deepened in order to delineate the main metabolic strategies used by boar sperm to obtain energy and to link them to sperm function. Boar spermatozoa were collected, diluted at 30 × 106 spz/mL and incubated for 1 h with: Rotenone (ROT), complex I inhibitor, Dimethyl-malonate (DMM), complex II inhibitor, antimycin A (ANTI), complex III inhibitor, oligomycin (OLIGO), ATP synthase inhibitor, Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), uncoupling agent, 2-deoxy-glucose (2DG), glucose agonist, and Dimethyl sulphoxide (DMSO) as control vehicle. Viability and mitochondrial membrane potential (Sybr14/PI/JC1 staining) and sperm motility (using CASA system) were assayed after incubation. ROT, ANTI, OLIGO and CCCP significantly reduced total and progressive motility as well as cell velocities; ANTI and CCCP depressed mitochondrial membrane potential but did not affect cell viability. Cluster analysis of kinematic parameters showed some interesting features of sperm subpopulations: ANTI and CCCP caused a shift in sperm subpopulation towards “slow non progressive” cells, OLIGO and ROT caused a shift towards “average” and “slow non progressive” cells, while DMM and 2DG increased the “fast progressive” cells subpopulation. Sperm mitochondrial respiration and substrate oxidation, assayed polographically and spectrofluorimetrically, respectively pointed out a high ATP turnover and a low spare respiratory capacity, mainly linked to the NADH-O2 oxidase activity. Therefore, boar spermatozoa heavily rely on mitochondrial oxidative phosphorylation, and especially on Complex I activity, to produce ATP and fuel motility.

Equine spermatozoa highly rely on oxidative phosphorylation for their energy management. The present work aimed to characterize the role of mitochondria on horse sperm motility and ROS production by incubating spermatozoa with specific inhibitors of the different mitochondrial complexes. Equine spermatozoa were incubated 1 h and 3 h at 37 °C with: complex I inhibitor rotenone (5 μM, ROT), complex II inhibitor dimethyl-malonate (10 mM, DMM), complex III inhibitor antimycin A (1.8 μM, ANTI), the uncoupling agent carbonyl cyanide m-chlorophenyl hydrazine (5 μM, CCCP), ATP synthase inhibitor oligomycin (5 μM, OLIGO), and 2 μL vehicle DMSO (control, CTL). Samples were analyzed for sperm motility and for mitochondrial membrane potential (MMP), mitochondrial integrity, mitochondrial O2•– production, and cytoplasmic H2O2. A multivariate analysis was performed on the data. CCCP caused a pronounced MMP reduction at both time points while ROT and ANTI showed the same effect at 3 h. All treatments at 3 h incubation significantly reduced the percentage of sperm with early changes in membrane permeability with active mitochondria. The H2O2 production of live cells was low at 1 h incubation in all treatments; after 3 h a slight decrease in the percentage of low-H2O2 producing cells was recorded. All treatments, except DMM, induced a significant decline in sperm motility and kinematics and modified the pattern of sperm subpopulations. The effect of DMM was evident only after 3 h, increasing the percentage of slow sperm subpopulation. In conclusion, the disruption of mitochondrial integrity induces an increase of mitochondrial ROS production that could be detrimental for cell function and survivior.

Context While conventional semen analysis is a simple, time-saving, and economical means to evaluate sperm quality, it leaves biochemical and metabolic characteristics of spermatozoa aside. To address this issue, the use of fluorescent probes assessing functional sperm parameters, such as JC-1, DiOC6(3) and MitoTracker, has increased over the last decades. Apparently contradictory observations have nevertheless fostered an ongoing debate on their sensitivity and ability to evaluate the mitochondrial membrane potential (MMP) of sperm cells, thus warranting a re-examination of these probes. Aims The present study aims to elucidate the suitability and sensitivity of each probe to evaluate the MMP of bovine spermatozoa by flow cytometry. Methods Cryopreserved spermatozoa from ten bulls were thawed, stained with JC-1/SYTOXRed, DiOC6(3)/propidium iodide (PI) or MitoTracker Deep Red (MTDR)/PI, and evaluated with flow cytometry and fluorescence microscopy. Key results DiOC6(3), JC-1 and MTDR can be simultaneously co-stained with a viability marker. The results of the present study support the ability of DiOC6(3)/PI and JC-1/SYTOXRed, but not that of MTDR/PI, to monitor the MMP of spermatozoa. Conclusions JC-1/SYTOXRed assessed by flow cytometry was found to be the most sensitive and robust fluorescent probe to assess MMP. Moreover, DiOC6(3)/PI could be a suitable alternative when the flow cytometer is not equipped with a red laser and/or an adequate optical filter. Implications Both DiOC6(3) and JC-1, but not MTDR, could be used as probes to assess the mitochondrial membrane potential of bovine spermatozoa.

In situations where an excessive generation of reactive oxygen species overwhelms antioxidant capacity, a harmful effect on sperm function is exerted. Antioxidants are molecules capable of minimizing this detrimental effect, which is important in pig sperm due to the high content of polyunsaturated fatty acids in their plasma membrane. The present systematic review aims at evaluating whether supplementing semen extenders (for liquid storage at 17 °C) or freezing and/or thawing media (for cryopreservation) with antioxidants influences sperm quality and functionality parameters, and in vitro/in vivo fertility outcomes. We defined inclusion and exclusion criteria in a PICOS table according to PRISMA guidelines, and conducted a literature search through MEDLINE-PubMed in November 2020. After systematic selection, 75 studies were included: 47 focused on cryopreservation and 28 on liquid storage at 17 °C. More than 70% of the studies included in this review showed that adding semen extenders for liquid storage and/or freezing/thawing media for cryopreservation with antioxidants enhances sperm quality and functionality parameters. In addition, this supplementation improves in vivo/in vitro fertility outcomes, supporting the hypothesis that the beneficial effect observed upon sperm quality has a positive impact on reproduction outcomes.

Horse practitioners have difficulties with undesirable behaviors exhibited by their horses ( Equus caballus ), such as cribbing, weaving, or head shaking. It has been suggested that most of behavioral disorders orig- inate from the animals’ attempt to cope with a suboptimal environment. To evaluate animal welfare, it is important to consider both behavioral and physiological parameters. Studies on animal welfare underline the importance of the monitoring of hormonal concentrations, particularly hormones that indicate stress, such as cortisol. The aims of this research were to compare cortisol and dehydroepiandrosterone (DHEA) concentrations of 14 healthy horses (HH) and 40 horses displaying behavioral pathologies (BPHs), to cal- culate the cortisol-DHEA ratio as marker of chronic stress and to find a relationship between aspects of horses management and the presence of BPHs. The methodology of radio-immuno-assay was used, to de- termine cortisol and DHEA levels in blood and hair. A questionnaire was completed by horse owners or barn managers and used to investigate the different characteristics of boarding barns, where the horses were housed. BPH horses registered a significantly ( P < 0.05) higher plasma cortisol/DHEA ratio value than HH horses. It is possible that BPH horses suffer chronic stress, which might cause a dysfunction of hypothalamic-pituitary-adrenal. A positive correlation was found ( P < 0.05) between the presence of BPH, time spent in stall and the frequency of work. Moreover, a low fiber, high-energy diet appeared to contribute to the development of BPHs. This study underlines the importance of a multidisciplinary approach to the evaluation of horse welfare. The improvement of welfare is closely correlated to a de- creased risk of the development of stereotypic or undesirable behaviors, and a better understanding of equine ethological needs is fundamental to advances in this area.

Mammalian semen is a physiological fluid composed of a cellular fraction (spermatozoa), and a liquid fraction (seminal plasma). Once delivered to the female genital tract, spermatozoa should be able to capacitate; a process which involves a plethora of biochemical and physiological changes required to fertilize the oocyte. Sperm production (spermatogenesis) occurs in the testes, whereby pluripotent spermatogonia differentiate to form the most morphologically specialized cells in the body. Further maturation of spermatozoa occurs in the epididymis, where they are stored prior to ejaculation. During this whole process, spermatozoa are exposed to different environments and cellular processes which may expose them to substantial levels of oxidative stress. To avoid damage associated with the unchecked production of reactive oxygen species (ROS), both spermatozoa, and the parts of the male genital tract in which they reside, are furnished with a suite of antioxidant molecules which are able to provide protection to these cells, thereby increasing their chance of being able to fertilize the oocyte and deliver an intact paternal genome to the future offspring. However, there are a host of reasons why these antioxidant systems may fail, including nutritional deficiencies, genetics, and disease states, and in these situations, a reduction or abolition of fertilizing capacity may result. This review paper focuses on the endogenous antioxidant defences available to spermatozoa during spermatogenesis and sperm maturation, the site of their production and their physiological role. Furthermore, we revised the causes and effects of antioxidant deficiencies (congenital or acquired during the animal's adulthood) on reproductive function in different animal species.

Sperm are redox-regulated cells, and deregulation of their redox status is considered to affect male fertility and to reduce their fertilizing ability following biotechnological procedures, such as cryopreservation. Cystine (CysS), after incorporation in sperm via SLC7A11 antiporter, has been demonstrated to increase intracellular GSH content, the most important non enzymatic antioxidant. This study was aimed at investigating the role of SLC7A11 antiporter on frozen-thawed stallion sperm ability to respond to in vitro capacitating environment after post-thaw incubation with CysS and/or Sulfasalazine (SS), a specific inhibitor of SLC7A11 antiporter. Viability, motility, immunolocalization of tyrosine phosphorylated proteins and the ability to bind to heterologous zonae pellucidae were evaluated. Thawed sperm from seven stallions (2 ejaculates/stallion) was washed and resuspended in Tyrodes media; each thawed ejaculate was divided in Control (CTR) and 3 samples supplemented with: 0.5 mM Cystine (CysS), 500 mu M Sulfasalazine (SS) and 0.5 mM CysS thorn 500 mu M SS (CysS thorn SS). After 1 h of incubation at 37 degrees C, samples were washed twice, resuspended in capacitating BWW medium and incubated at 38 degrees C under 5% CO2. After 30 and 60 min, sperm motility, viability and tyrosine phosphorylated protein immunolocalization, used as capacitation status index, were evaluated. After 30 min of capacitation, 4 x 10(5) sperm were co-incubated with denuded pig oocytes in capacitation medium for 30 min for the heterologous binding assay. None of the sperm parameters studied (motility, viability and tyrosine phosphorylation) showed any difference respective to control. The number of sperm bound per oocyte (mean +/- SEM) tended to increase in CysS group (44.0 +/- 12.3) respect CTR (40.8 +/- 10.8) while decreased in SS group (32.4 +/- 7.8) (p < 0.01). Moreover, CysS thorn SS group showed a lower binding rate (32.0 +/- 10.0) compared to CysS (p < 0.001). Our results suggest that CysS supplementation of thawed stallion sperm can influence their ability to bind to heterologous zona pellucidae as the inhibition of CysS incorporation by SLC7A11 reduced the number of sperm bound per oocyte. This effect does not seem to be ascribed to a modification of sperm motility, membrane integrity and tyrosine phosphorylation. (c) 2021 Elsevier Inc. All rights reserved.

Glyphosate, formulated as glyphosate-based herbicides (GBHs) including the best-known formulation Roundup, is the world's most widely used herbicide. During the last years, the growing and widespread use of GBHs has raised a great concern about the impact of environmental contamination on animal and human health including potential effect on reproductive systems. Using an in vitro model of pig oocyte maturation, we examined the biological impact of both glyphosate and Roundup on female gamete evaluating nuclear maturation, cytoplasmic maturation and developmental competence of oocytes, steroidogenic activity of cumulus cells as well as intracellular levels of glutathione (GSH) and ROS of oocytes. Our results indicate that although exposure to glyphosate and Roundup during in vitro maturation does not affect nuclear maturation and embryo cleavage, it does impair oocyte developmental competence in terms of blastocyst rate and cellularity. Moreover, Roundup at the same glyphosate-equivalent concentrations was shown to be more toxic than pure glyphosate, altering steroidogenesis and increasing oocyte ROS levels, thus confirming that Roundup adjuvants enhance glyphosate toxic effects and/or are biologically active in their side-effect and therefore should be considered and tested as active ingredients.

The wide use of glyphosate-based herbicides (GBHs) has become a matter of concern due to its potential harmful effects on human health, including men fertility. This study sought to investigate, using the pig as a model, the impact of pure glyphosate and its most known commercial formulation, Roundup, on sperm function and survival. With this purpose, fresh commercial semen doses were incubated with different concentrations (0-360 μg/mL) of glyphosate (GLY; exp. 1) or Roundup, at the equivalent GLY concentration (exp. 2), at 38 °C for 3 h. Glyphosate at 360 μg/mL significantly (P < 0.05) decreased sperm motility, viability, mitochondrial activity and acrosome integrity but had no detrimental effect at lower doses. On the other hand, Roundup did significantly (P < 0.05) reduce sperm motility at ≥ 5 μg/mL GLY-equivalent concentration; mitochondrial activity at ≥ 25 μg/mL GLYequivalent concentration; and sperm viability and acrosome integrity at ≥ 100 μg/mL GLY-equivalent concentration as early as 1 h of incubation. In a similar fashion, GLY and Roundup did not inflict any detrimental effect on sperm DNA integrity. Taken together, these data indicate that, while both glyphosate and Roundup exert a negative impact on male gametes, Roundup is more toxic than its main component, glyphosate Te authors would like to thank the support from Yentel Mateo-Otero, Estela Garcia, Marc Llavanera, Ariadna Delgado-Bermúdez, Beatriz Fernandez-Fuertes, Sergi Bonet and Isabel Barranco from University of Girona for their technical support. Tis research was supported by the Ministry of Science, Innovation and Universities, Spain (Grants: RYC-2014-15581 and AGL2017- 88329-R), and the Regional Government of Catalonia (2017-SGR-1229)

Although excessive ROS levels induce sperm damage, sperm capacitation is an oxidative event that requires low amounts of ROS. As the antioxidant activity of the ethanol extract (TRE) of a commercial oenological tannin (Quercus robur toasted oak wood, Tan'Activ R®) and its four fractions (FA, FB, FC, FD) has been recently reported, the present study was set up to investigate the biological effects of TRE and its fractions in an in vitro model of sperm capacitation and fertilization. Boar sperm capacitation or gamete coincubation were performed in presence of TRE or its fractions (0, 1, 10, 100 μg/ml). TRE at the concentration of 10 μg/ml (TRE10) stimulated sperm capacitation, as it increased (p

As the taste receptor for monosodium glutamate (umami) is expressed in both murine and human spermatozoa and the presence of α-gustducin and α-transducin, G proteins involved in the umami taste signaling, has been described in boar germ cells, the aim of this study was to evaluate if monosodium glutamate (MSG) would exert any effect on sperm-oocyte binding, invitro fertilization (IVF) and sperm parameters during invitro induced capacitation. For sperm-zona pellucida binding assay, boar spermatozoa were preincubated for 1h and then coincubated for 1h with denuded invitro matured oocytes in presence of different concentrations of MSG (0, 0.1, 1, 10mM). MSG 1 and 10mM significantly (P&nbsp

Alkaline phosphatase (AP) is present in equine seminal plasma and spermatozoa, but its functional role is not fully understood yet. Being that, sperm-oocyte interaction in equine species has been reported to be enhanced at a slightly basic pH, this work aimed at verifying whether exogenous alkaline phosphatase exerts any role on stallion spermatozoa and sperm-oocyte interaction at different pHs (7.4; 8.0; 9.0). Stallion spermatozoa were capacitated in Tyrode's medium at pH 7.4, 8.0, and 9.0 for 4 hours at 38 °C, 5% CO2 with 2.5-IU AP (AP group) or without AP (capacitated spermatozoa group); viability with mitochondrial activity, motility, and acrosome integrity were measured. In addition, a homologous binding assay was carried out: stallion spermatozoa were capacitated 1 hour at 38 °C, 5% CO2 with 2.5-IU AP (AP group) or without AP (capacitated spermatozoa group). Oocytes were then added to sperm suspensions and coincubated for 1 hour. Our results indicate that AP at pH 9.0 significantly increases the percentage of living cells with active mitochondria, whereas it significantly reduces the percentage of acrosome-damaged cells at pH 8.0. No significant differences were registered in motility parameters. The homologous binding assay showed a strong effect of AP, that increased the number of sperm bound to the oocyte's zona pellucida at all pHs tested. In conclusion, AP can induce some modifications on sperm membranes thus enhancing their capacity to bind to the zona pellucida of equine oocytes.

Oxidative stress is regarded as an important cause of sperm damage during cryopreservation. However, seasonal changes in oxidative status in unfrozen and frozen-thawed stallion sperm have not been well established. We tested the hypothesis that sperm ROS concentrations and lipid peroxidation change between breeding and non-breeding seasons and influence quality of unfrozen and frozen-thawed sperm. Eighteen ejaculates from six Warmblood stallions (8-21 y) known to be fertile, were collected in winter and summer and processed for freezing. After 90 min at +4 °C, some straws from each ejaculate were not frozen (unfrozen), whereas the remainder were frozen by N2 vapors, plunged in N2 and thawed (frozen-thawed). Rapid cells (RAP; determined by CASA), plasma membraneacrosome integrity (PMAI), high mitochondrial membrane potential (Mpos), low intracellular Ca2+ concentration (Fneg), membrane lipid peroxidation (BODIPY), intracellular ROS concentrations (DCFH, MitoSOX) and chromatin fragmentation (DFI%) were evaluated by flow cytometry in both groups and at intervals during incubation at +37 °C for 24 h. Overall, ROS concentrations and lipid peroxidation were higher and faster (P < 0.0001) in winter versus summer, DFI% was lower in winter versus summer (P < 0.0001), but similar between the two groups within season. There were moderate positive correlations in both seasons between DFI% and MitoSOX, DCFH, BODIPY in both groups, whereas a negative correlation, stronger in winter, was evident between sperm quality (RAP, PMAI, Mpos, Fneg) and BODIPY, DCFH, MitoSOX. There were no differences between seasons for RAP, PMAI, Mpos and Fneg. In conclusion, ROS-related parameters were higher in winter than in summer, without a negative effect on sperm quality. We concluded that increased ROS concentrations were less deleterious to sperm than freezing-thawing. Furthermore, incubation at +37 °C and sequential analysis were useful to assess sperm resistance.

Dog sperm cryopreservation is gaining importance both in breeding dogs for commercial purposes and for pet animals. Anyway, cryopreservation of mammalian spermatozoa, including dog ones, induces some negative effect on sperm fertility, leading to a lower use of this technique and limiting its widespread use. Therefore, studies to improve the quality of canine semen after cryopreservation could have a relevant impact on both the scientific advancement and the clinical practice. The aim of the present work was to investigate the putative ameliorative effect of Epigallochatechin-3-gallate (EGCG) addition to post thawing medium on dog sperm motility, mitochondrial activity, acrosome integrity and on zona-binding ability (zona binding assay). Spermatozoa were thawed in Tris-fructose-citrate medium supplemented with EGCG (0, 25 and 50 μM) and sperm motility, mitochondrial activity and acrosome integrity were assayed at 0.5, 1.5, 3 and 6 h after post thawing incubation at 37 °C. An aliquot of semen from each treatment group after 1.5 h post thawing incubation was washed and used to perform heterologous (using porcine oocytes) or homologous zona binding assay. The results obtained showed that no significant effect is exerted by EGCG on sperm parameters analysed neither at 0.5, 1.5, 3 or 6 h after thawing excepting for the reduction of the percentage of live cells with active mitochondria at the higher dose at 6 h; furthermore, both homologous or heterologous zona binding ability, was not influenced by EGCG. In conclusion, EGCG supplementation to thawing medium does not improve dog sperm quality or zona binding capacity.

Tannins have been demonstrated to have antioxidant and various health benefit properties. The aim of this study was to determine the effect of an ethanol extract (TRE) of a commercial oenological tannin (Quercus robur toasted oak wood, Tan'Activ R ® ) on female gamete using an in vitro model of pig oocyte maturation (IVM) and examining nuclear maturation, cytoplasmic maturation, intracellular GSH and ROS levels and cumulus cell steroidogenesis. To this aim, during IVM performed in medium either supplemented (IVM A) or not supplemented (IVM B) with cysteine and β-mercaptoethanol, TRE was added at different concentrations (0, 1, 5, 10, 20 μg/ml). The addition of TRE at all the concentration tested to either IVM A or IVM B, did not influence oocyte nuclear maturation. When IVM was performed in IVM A, no effect was induced on cytoplasmic maturation by TRE at the concentration of 1, 5 and 10 μg/ml, while TRE 20 μg/ml significantly reduced the penetration rate after IVF (p < 0.05) and the blastocyst rate after parthenogenetic activation (p < 0.01). Oocyte maturation in IVM B, compared to IVM A group, decreased GSH (p < 0.001) and increased ROS (p < 0.01) intracellular levels and in turn impaired oocyte cytoplasmic maturation reducing the ability to sustain male pronuclear formation after IVM (p < 0.001) and the developmental competence after parthenogenetic activation (p < 0.001). TRE supplementation to IVM B significantly reduced ROS production (5, 10, 20 μg/ml TRE) to levels similar to IVM A group, and increased GSH levels (10, 20 μg/ml TRE) compared to IVM B (p < 0.05) without reaching those of IVM A group. TRE supplementation to IVM B at the concentrations of 1, 5 and 10 μg/ml significantly improved (p < 0.001) oocyte cytoplasmic maturation enhancing the ability to sustain male pronuclear formation without reaching, however, IVM A group levels. TRE addition at all the concentration tested to both IVM A and IVM B, did not induce any effect on E2 and P4 secretion by cumulus cells suggesting that the biological effect of the ethanol extract is not exerted thought a modulation of cumulus cell steroidogenesis. In conclusion, TRE, thanks to its antioxidant activity, was partially able to reduce the negative effect of the absence of cysteine and β-mercaptoethanol in IVM B, while TRE at high concentration in IVM A was detrimental for oocyte cytoplasmic maturation underlying the importance of maintaining a balanced redox environment during oocyte maturation.

Frozen-thawed boar semen suffer a fertility decrease that negatively affects its widespread use. In recent years supplementing frozen-thawed boar sperm with different antioxidants gave interesting and promising results; the aim of the present work was to study the effect of supplementing boar sperm thawing medium for 1 h with combination of epigallocatechin-3-gallate (EGCG, 50 μM) and Resveratrol (R, 2 mM), on boar sperm motility (assessed by CASA), viability, acrosome integrity, mitochondrial function, lipid peroxidation and DNA integrity (assessed by flow cytometry), protein tyrosine phosphorylation (assessed by immunofluorescence) and on in vitro fertilization (IVF). Our results demonstrate that sperm motility is negatively affected by R (alone or associated with EGCG, p < 0.05) in comparison to control and EGCG groups both at 1 h and 4 h; this effect is evident both in average motility parameters and in single cells kinematics, studied by cluster analysis, that showed the presence of a specific cell population with simil-hyperactivated features in R group (p < 0.01). Viability, acrosome integrity, mitochondrial functionality and lipid peroxidation are not influenced by the addition of the antioxidants; finally, DNA integrity is negatively influenced by R (both alone or associated with EGCG) both at 1 h and 4 h incubation (p < 0.05). Finally, tyrosine phosphorylated protein immunolocalization, used as capacitation parameter, is not affected by the different treatments. Penetration rate is strongly enhanced by R, both alone or associated with EGCG (p < 0.05); EGCG increases penetration rate as well but to a lower extent. Our findings demonstrate that the combination of R and EGCG could positively affect frozen-thawed boar sperm fertility in vitro; the effect is evident also in R groups, thus demonstrating that this antioxidant is predominant, and no synergic effect is present. Some insights are needed to understand if, in particular R (that showed the strongest effect) could be profitably used for artificial insemination in vivo, given the detrimental effect of this molecule on both sperm motility and DNA integrity.

This study was aimed at assessing the capability of semen experimentally infected with porcine circovirus type 2 (PCV2) to produce porcine blastocysts PCR positive for PCV2. Embryos were obtained from in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes or by parthenogenesis. Sperm suspension was exposed to PCV2b and utilized for IVF. PCV2 spiked semen did not reveal any reduction in sperm viability or motility but its ability to produce infected blastocysts was irrelevant as only one out of 15 blastocysts obtained by IVF were PCV2b; however two blastocysts were PCV2a positive. Furthermore, the presence of PCV2 was demonstrated also in embryos obtained by parthenogenesis (one out of 17 was PCV2b and one PCV2a positive). Even if PCV2 firmly attaches to the surface of spermatozoa, experimentally spiked sperm were not effective in infecting oocytes during IVF and in producing PCR positive embryos. The infected blastocysts we obtained derived most probably from infected oocytes recovered at the abattoir.

In the last 30 years, encapsulation technology has been applied to different species to minimize the loss of spermatozoa after artificial insemination. In particular, the vehiculation of boar sperm cells in barium alginate membrane has proved a valid strategy to reduce the risk of polyspermy and optimize in vivo fertilizing yields. Controlled release of male gametes into the female genital tract has reduced the minimum fertilizing dose of spermatozoa. Notwithstanding these results, encapsulation has not yet reached commercial application, largely due to the additional costs of production. However, encapsulation could be useful in advanced reproductive technology, such as sex sorting, to store sorted boar semen. The controlled release of flow cytometrically sorted spermatozoa could be a promising strategy to reduce the number of cells necessary for each insemination and hence allow the widescale use of sex sorting in this species.

The aim of this study was to define a protocol to store dog sperm before and after sorting to obtain an insemination dose sufficient to allow the conception by artificial insemination. Experiment 1 and 2 were performed to evaluate the more appropriate extender for preserving at room temperature dog sperm before and after sorting. Four extenders were tested: (1) Tris-fructose-citrate (TFC), (2) Tris-glucose-citrate (TGC), (3) modified Tyrode's albumin lactate pyruvate medium (mTALP), and (4) third fraction of the ejaculate (after centrifugation at 5000× g for 10 minutes; III FRAC). Experiment 3 and 4 were performed to evaluate the ability of dog semen to withstand sex sorting and freezing/thawing. Modified Tyrode's albumin lactate pyruvate medium was the best extender for canine sperm storage at room temperature (20 °C-25 °C) before (total motility: TFC, 8.3 ± 1.7; TGC, 50.0 ± 11.5; mTALP, 70.0 ± 0.1; III FRAC, 25.0 ± 1 0.4; P < 0.05) and after sorting (total motility: TFC, 7.3 ± 1.5; TGC, 10.3 ± 1.5; mTALP, 33.3 ± 6.7; III FRAC, 8.7 ± 5.8; P < 0.05), even if at 24-hour sorted sperm quality was impaired in all extenders tested herein. Sperm quality decreased after sorting (total motility: control, 92.5 ± 0.9; sorted, 52.9 ± 6.0; P < 0.05) and, especially, after freezing/thawing (total motility: frozen control, 25.7 ± 4.1; frozen sorted, 2.4 ± 1.2; P < 0.05). In conclusion, mTALP is an appropriate medium for canine sperm storage before and soon after sorting (hours), but a long storage period of sexed sperm at room temperature is not adequate. Cryopreservation greatly impaired sperm quality, and further studies are needed to optimize the freezing protocol for sexed dog sperm.

none 6 si Available online 7 October 2014 The main aim of this work was to evaluate how supplementing freezing and thawing media with reduced glutathione (GSH) and l-ascorbic acid (AA) affected the quality parameters of frozen-thawed boar spermatozoa. With this purpose, semen samples of 12 ejaculates coming from 12 boars were used. Each ejaculate was split into seven aliquots to which 5 mM of GSH and 100 μM of AA were added separately or together at two different steps of freeze-thawing. Various sperm parameters (levels of free cysteine residues in sperm nucleoproteins, sperm viability, acrosome membrane integrity, intracellular peroxide and superoxide levels [ROS], and total and progressive motility) were evaluated before freezing and at 30 and 240 minutes after thawing. Both GSH and AA significantly improved boar sperm cryotolerance when they were separately added to freezing and thawing media. However, the highest improvement was recorded when both freezing and thawing media were supplemented with 5 mM of GSH plus 100 μM of AA. This improvement was observed in sperm viability and acrosome integrity, sperm motility, and nucleoprotein structure. Although ROS levels were not much increased by freeze-thawing procedures, the addition of GSH and AA to both freezing and thawing extenders significantly decreased intracellular peroxide levels and had no impact on superoxide levels. According to our results, we can conclude that supplementation of freezing and thawing media with both GSH and AA has a combined, beneficial effect on frozen-thawed boar sperm, which is greater than that obtained with the separate addition of either GSH or AA. mixed Giaretta, Elisa; Estrada, Efrén; Bucci, Diego; Spinaci, Marcella; Rodríguez-Gil, Joan E; Yeste, Marc Giaretta, Elisa; Estrada, Efrén; Bucci, Diego; Spinaci, Marcella; Rodríguez-Gil, Joan E; Yeste, Marc

The effect of transport on Cortisol and Glucose serum concentrations were assessed in rabbits during summer and winter. Animals were divided into high (HSA, 307 cm2/rabbit), medium (MSA, 373 cm2/rabbit) and low space allowance (LSA, 475 cm2/rabbit) transport groups, and rabbits slaughtered directly in the farm were used as control group (C). During summer, cortisol and glucose concentrations were significantly higher in HSA (high space allowance), MSA and LSA than in C rabbits (P

Contents Stallion semen storage for artificial insemination is mainly based on liquid cooled storage. In many stallions this technique maintains sperm quality for an extended period of time (24–72 hr) at 7°C. While this technique is commonly used in the horse industry, there can be a decline in fertility in some stallions, due to an inability of their sperm to tolerate the cool storage process. The aim of the present work was to evaluate the effect of two natural antioxidants (epigallocatechin-3-gallate (EGCG) at 20, 60 and 120 μm and green tea polyphenols, and p at .001, .01 and .1 mg/ml) on some sperm parameters (sperm motility, viability/acrosome integrity and DNA quality) in extended semen immediately after its collection (T0) and after 2, 6, 24 and 48 hr of cool storage. Two ejaculates from three trotter stallions were analysed after 48 hr of storage at 4°C. No beneficial effect on the analysed parameters was observed: the two antioxidants were not able to improve sperm quality after 48 hr of storage. These results are in agreement with previous findings on the effect of different antioxidants reported by other researches, who have demonstrated that stallion semen keeps good antioxidant capacity after dilution for 24 hr. In conclusion, the positive effect exerted by antioxidant molecules in other species is not confirmed in the equine one.

The purpose of this study was to evaluate the long-term (12 months) efficacy and tolerability of imepitoin as first-choice treatment in 56 dogs suffering from idiopathic epilepsy and identify possible factors affecting the outcome. Primary treatment success (PTS) was defined as the achievement of a seizure-free interval three times longer than the pretreatment interictal interval (at least three months). Secondary treatment success (STS) was achieved by a decrease in seizure frequency .50 per cent compared with the pretreatment frequency. In the long-term follow-up, PTS was recorded in 14 (25 per cent) dogs and responder-dogs (PTS+STS) were 30 (54 per cent) showing significant reduction in the monthly average number of seizures (P

Thawing is one of the most delicate process after semen cryopreservation as spermatozoa pass from a dormant metabolic stage to a sudden awakening in cellular metabolism. The rapid oxygen utilization leads to an overproduction of reactive oxygen species that can damage sperm cells, thus causing a significant decrease of fertilizing potential of frozen-thawed spermatozoa. Resveratrol (Res) is a natural grape-derived phytoalexin and Epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis); both molecules are known to possess high levels of antioxidant activity. The objective of the present study was to assess the effect of different concentrations of Res (0.5, 1 or 2mM; Experiment 1) or EGCG (25, 50 or 100μM; Experiment 2) supplementation to thawing boar semen extender on sperm quality parameters (viability and acrosome integrity) and invitro fertilization (IVF). Semen after thawing and dilution with three volumes of Beltsville Thawing Solution (BTS), was immediately divided in control group without antioxidants addition (CTR) and either Res or EGCG groups. Sperm viability and acrosome integrity were evaluated in CTR, Res or EGCG groups after 1h of incubation at 37°C. The addition of different doses of Res or EGCG to thawing extender for 1h did not induce any effect on boar sperm viability and acrosome integrity. However, both Res and EGCG treated samples exhibited a significantly higher penetration rate compared with CTR when used for IVF. In particular the treatment with all the EGCG concentrations increased the penetration rate (P&nbsp

Objectives The aims of this study were to validate a semi-automated high-resolution electrophoretic technique to quantify urinary albumin in healthy and diseased cats, and to evaluate its diagnostic performance in cases of proteinuria and renal diseases. Methods Urine samples were collected from 88 cats (healthy; chronic kidney disease [CKD]; lower urinary tract disease [LUTD]; non-urinary tract diseases [OTHER]). Urine samples were routinely analysed and high-resolution electrophoresis (HRE) was performed. Within-assay and between-assay variability, linearity, accuracy, recovery and the lowest detectable and quantifiable bands were calculated. Receiver operating curve (ROC) analysis was also performed. Results All coefficients of variation were Conclusions and relevance HRE is an accurate and precise method that could be used to measure albuminuria in cats. UAC was useful to correctly classify proteinuria and to discriminate between healthy and diseased cats. HRE might also provide additional information on urine proteins with a profile of all proteins (albumin and globulins) to aid clinicians in the diagnosis of diseases characterised by proteinuria.

Setting an open-access computer assisted sperm analysis (CASA) may benefit the evaluation of motility in mammalian sperm, especially when economic constraints do not allow the use of a commercial system. There have been successful attempts to develop such a device in Zebra fish sperm and the system has been used in very few studies on mammalian spermatozoa. Against this background, the present study aimed at developing an open-access CASA system for mammalian sperm using the horse as a model and based upon the Image J software previously established for Zebra fish sperm. Along with determining the sperm progressive motility and other kinetic parameters (such as amplitude of lateral head displacement), the “results” window was adjusted to simplify subsequent statistical analyses. The path window was enriched with colored sperm trajectories on the basis of the subpopulation they belong to and a number that allowed the sperm track to be associated to the sperm motility data shown in the “results” window. Data obtained from the novel plugin (named as CASA_bgm) were compared with those of the commercial CASA Hamilton-Thorn IVOS Vers.12, through Bland Altman’s plots. While the percentage of total and progressive motile sperm, VCL, VAP, VSL, LIN and STR and ALH were in agreement with those obtained with the commercial system, BCF significantly differed between the two systems probably due to their settings. Interestingly, a positive and significant correlation between the percentages of total motile sperm evaluated through CASA_bgm and those showing high mitochondrial membrane potential evaluated by JC-1 staining was found. In conclusion, CASA_bgm ImageJ plugin could be useful and reliable for stallion sperm motility analysis and it is our aim to apply this system to other mammalian species.

Despite the great potential application of sex-sorted spermatozoa in swine, the technology is not practiced in the pig industry because of technical factors and species-specific issues. The susceptibility of boar spermatozoa to stresses induced by the sorting procedure, the relative slowness of the sex-sorting process together with the high sperm numbers required for routine artificial insemination in pig are some of the main factors limiting the commercial application of this technology in pigs. This review briefly describes the damage to spermatozoa during sex sorting, focusing on an additional limiting factor: increased susceptibility of sexed boar spermatozoa to injuries induced by liquid storage and cryopreservation that, in turn, impairs sperm quality leading to unsatisfactory results in vivo. Strategies to extend the lifespan of sex-sorted boar spermatozoa and to improve their fertilizing ability after liquid storage or cryopreservation need to be implemented before this technology can be used in pig farms. In this regard, encapsulation in barium alginate membranes could be a promising technique to optimize the in vivo use of sexed boar spermatozoa, by protecting, targeting, and controlling the release of sperm into the female genital tract.

Alkaline phosphatase (AP) has been studied in several situations to elucidate its role in reproductive biology of the male from different mammalian species; at present, its role in horse sperm physiology is not clear. The aim of the present work was to measure AP activity in seminal plasma and sperm extracts from freshly ejaculated as well as in frozen-thawed stallion spermatozoa and to verify whether relationship exists between AP activity and sperm quality parameters. Our data on 40 freshly ejaculated samples from 10 different stallions demonstrate that the main source of AP activity is seminal plasma, whereas sperm extracts contribution is very low. In addition, we found that AP activity at physiological pH (7.0) is significantly lower than that observed at pH 8.0, including the optimal AP pH (pH 10.0). Alkaline phosphatase did not exert any effect on sperm-oocyte interaction assessed by heterologous oocyte binding assay. Additionally, we observed a thermal stability of seminal plasma AP, concluding that it is similar to that of bone isoforms. Positive correlations were found between seminal plasma AP activity and sperm concentration, whereas a negative correlation was present between both spermatozoa extracts and seminal plasma AP activity and seminal plasma protein content. A significant decrease in sperm extract AP activity was found in frozen-thawed samples compared with freshly ejaculated ones (n = 21), concomitantly with the decrease in sperm quality parameters. The positive correlation between seminal plasma AP activity measured at pH 10 and viability of frozen-thawed spermatozoa suggests that seminal plasma AP activity could be used as an additional predictive parameter for stallion sperm freezability. In conclusion, we provide some insights into AP activity in both seminal plasma and sperm extracts and describe a decrease in AP after freezing and thawing.