Seminal extracellular vesicles subsets modulate gene expression in cumulus cells of porcine in vitro matured oocytes.

Seminal plasma (SP), a fluid composed mainly by secretions from accessory sex glands, contains a heterogenous population of extracellular vesicles (EVs), involved in several reproductive physiological processes. Seminal plasma has been found to modulate ovary function, in terms of hormone secretion and immune regulation. This study evaluated the potential effect of SP-EV-subsets on the modulation of cumulus-oocyte-complex (COCs) physiology during in vitro maturation (IVM). Two SP-EV-subsets, small-EVs (S-EVs) and large-EVs (L-EVs), were isolated from pig SP by size-exclusion-chromatography. Next, COCs were IVM in the absence (control) or presence of each SP-EV-subset to evaluate their uptake by COCs (PKH67-EVs labelling) and their effect on oocyte and cumulus cells (CCs) (gene expression, and progesterone and estradiol-17ß levels). S-EVs and L-EVs were able to bind CCs but not oocytes. Supplementation with L-EVs induced changes (P ≤ 0.05) in the transcript levels of oocyte maturation- (HAS2) and steroidogenesis-related genes (CYP11A1 and HSD3B1) in CCs. No effect on nuclear oocyte maturation and progesterone and estradiol-17ß levels was observed when COCs were IVM with any of the two SP-EV-subsets. In conclusion, while SP-EV-subsets can be integrated by CCs during IVM, they do not affect oocyte maturation and only L-EVs are able to modulate CCs function, mainly modifying the expression of steroidogenesis-related genes.

Use of specific mitochondrial complex inhibitors to investigate mitochondrial involvement on horse sperm motility and ROS production.

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.

Impact of glyphosate and its formulation Roundup® on stallion spermatozoa.

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.

Different approaches for assessing sperm function.

Different approaches can be used to assess sperm function in different conditions, i.e. sperm storage, freezing-thawing or activation by induction of capacitation and acrosome reaction. In this review we will focus on the assays routinely performed in our laboratories, giving a literature support to critically analyse different approaches. In fact, researchers usually tend to look for the "one shot" parameter that could explain itself a specific process; it is our conviction that a multiparametric approach is still more valid, as some changes in sperm function are very complex and could be explained only by operating in different ways. Sperm motility, the most evident sperm characteristic, should be assessed by computer-aided sperm analysers that permit an objective evaluation of the motility and its kinematic parameters. Commercial and open source instruments are available and could be profitably used together with specific statistical approaches. The use of microscopy, and particularly fluorescent microscopy, could be a very useful tool to assess different parameters in sperm cells both by fluorophores that give indication of a determined function, and by immunolocalization of proteins, that permits the discover of new features or to explain particular sperm functions. The same substrates could be used also in flow cytometry: the difference is that it permits to study wider sperm populations (and their sub-population distribution). Flow cytometry is undergoing a very wide use in spermatology and technical and experimental rigor is needed to obtain reliable results. Metabolic assessment of sperm features, particularly energetic supply, ATP formation and other enzyme activities, could represent a very important challenge to acquire new information and complete/integrate those derived from other techniques. Finally, functional assays such as oocyte binding and in vitro fertilization, represent a very strong tool to assess sperm function in vitro, as they could evidence the functional intactness of some pathways.

Improvement of in vitro fertilization by a tannin rich vegetal extract addition to frozen thawed boar sperm.

Boar spermatozoa are very susceptible to cryopreservation injuries and, for this reason, pig remains one of the few species in which fresh semen is still preferred to thawed one for routine artificial insemination (AI). The present work evaluated the effect of supplementing boar sperm thawing medium with Silvafeed SP (SSP), a mixture of Chestnut and Quebracho wood extracts (60/40 w/w) rich in polyphenols (92.4% tannin content) on in vitro fertilization (IVF) and on the following sperm parameters: sperm motility (assessed by CASA), viability, acrosome integrity, mitochondrial function and lipid peroxidation (assessed by flow cytometry) and capacitation status (immunolocalization of tyrosine phosphorylated proteins). Thawed spermatozoa were incubated 1 h at 37°C in BTS without (CTR) or with (5, 10, 20 µg/mL) SSP. After incubation sperm suspension was divided in three aliquots: one was used for IVF trials, one for sperm analysis, and the last one was capacitated for 1 h at 39°C 5% CO2 in IVF medium. Sperm motility parameters, viability, acrosome integrity, mitochondrial functionality, lipid peroxidation and tyrosine phosphorylated protein immunolocalization, used as capacitation parameter, were not influenced by SSP. However, oocytes inseminated with thawed spermatozoa pretreated with all the different SSP concentrations presented a significant (P < 0.01) increase in penetration rate compared to CTR. In addition, 5 µg/mL SSP exerted a positive effect (P<0.05) on the total efficiency of fertilization. These results encourage the use of SSP in the thawing medium since post-thawing fertility is a limit for the large-scale use of boar frozen semen.

Glyphosate and its formulation Roundup impair pig oocyte maturation.

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.

Effects of Roundup and its main component, glyphosate, upon mammalian sperm function and survival.

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 GLY-equivalent 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.

Sperm function and mitochondrial activity: An insight on boar sperm metabolism.

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.

The Ramazzini Institute 13-week pilot study glyphosate-based herbicides administered at human-equivalent dose to Sprague Dawley rats: effects on development and endocrine system.

BACKGROUND: Glyphosate-based herbicides (GBHs) are broad-spectrum herbicides that act on the shikimate pathway in bacteria, fungi, and plants. The possible effects of GBHs on human health are the subject of an intense public debate for both its potential carcinogenic and non-carcinogenic effects, including potential effects on the endocrine system The present pilot study examine whether exposure to GBHs at the dose of glyphosate considered to be "safe" (the US Acceptable Daily Intake - ADI - of 1.75 mg/kg bw/day), starting from in utero life, affect the development and endocrine system across different life stages in Sprague Dawley (SD) rats. METHODS: Glyphosate alone and Roundup Bioflow, a commercial brand of GBHs, were administered in drinking water at 1.75 mg/kg bw/day to F0 dams starting from the gestational day (GD) 6 (in utero) up to postnatal day (PND) 120. After weaning, offspring were randomly distributed in two cohorts: 8 M + 8F/group animals belonging to the 6-week cohort were sacrificed after puberty at PND 73 ± 2; 10 M + 10F/group animals belonging to the 13-week cohort were sacrificed at adulthood at PND 125 ± 2. Effects of glyphosate or Roundup exposure were assessed on developmental landmarks and sexual characteristics of pups. RESULTS: In pups, anogenital distance (AGD) at PND 4 was statistically significantly increased both in Roundup-treated males and females and in glyphosate-treated males. Age at first estrous (FE) was significantly delayed in the Roundup-exposed group and serum testosterone concentration significantly increased in Roundup-treated female offspring from the 13-week cohort compared to control animals. A statistically significant increase in plasma TSH concentration was observed in glyphosate-treated males compared with control animals as well as a statistically significant decrease in DHT and increase in BDNF in Roundup-treated males. Hormonal status imbalances were more pronounced in Roundup-treated rats after prolonged exposure. CONCLUSIONS: The present pilot study demonstrate that GBHs exposure, from prenatal period to adulthood, induced endocrine effects and altered reproductive developmental parameters in male and female SD rats. In particular, it was associated with androgen-like effects, including a statistically significant increase of AGDs in both males and females, delay of FE and increased testosterone in female.

A polyphenol-rich extract from an oenological oak-derived tannin influences in vitro maturation of porcine oocytes.

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 ß-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 ß-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.

Epigallocatechin-3-gallate added after thawing to frozen dog semen: Effect on sperm parameters and ability to bind to oocytes' zona pellucida.

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.

Milk production in Saanen goats treated with repeated low doses of intermediate-release insulin during early lactation / Produção de leite em cabras Saanen tratadas com repetidas doses baixas de insulina de liberação intermediária durante a lactação inicial

ABSTRACT: The effect of insulin administration on the productive responses of Saanen goats during early lactation was investigated. Ten of 20 adult females were subjected to subcutaneous administration of intermediate-acting insulin (0.14UI/kg body weight) at 2, 9, and 14 days postpartum. Milk yield was measured twice daily for 13 weeks and milk samples were collected to measure protein and fat contents. Plasma levels of progesterone, insulin, non-esterifies fatty acids, glucose and other metabolites were measured. Results showed a significantly increased effect of insulin treatment on the content of milk fat and protein; moreover, milk production in the first and second postpartum weeks were higher than control group. The peak of lactation in the insulin group was achieved one week earlier in comparison to the control group. In addition, the milk production rate showed lower persistency (milk yield 13 week/milk yield at peak) in the same group. During the first four weeks of postpartum, treated animals showed greater weight loss and higher non-esterified fatty acid concentration, whereas no effect was observed on the concentration of progesterone and other metabolites. The above results indicated that repeated administration of insulin in dairy goats during early lactation increase yield and qualitative components of milk, but has substantial consequences on animal productive rate and metabolic response.

RESUMO: O objetivo do estudo foi avaliar o efeito da administração de insulina sobre a resposta produtiva de cabras Saanen durante a lactação inicial. Dez de vinte fêmeas adultas foram sujeitas à administração subcutânea de repetidas e baixas doses de insulina de liberação intermediária aos 2, 9 e 14 dias pós-parto. A produção de leite foi mensurada duas vezes ao dia, por 13 semanas, e amostras de leite foram coletadas para mensurar teores de proteína e gordura. Os níveis plasmáticos de progesterona, insulina, ácidos graxos não-esterificados (AGNE), glicose e outros metabólitos foram mensurados. Os resultados mostraram um efeito significativamente maior nos animais tratados com insulina sobre o teor de gordura e proteína no leite. Além disso, a produção de leite na primeira e segunda semana pós-parto foi maior no grupo tratado do que no grupo controle. O pico de lactação no grupo insulina foi alcançado uma semana antes em comparação ao grupo controle. Além disso, a taxa de produção de leite nos animais tratados mostrou uma menor persistência de produção de leite durante o período analisado. Durante as primeiras quatro semanas pós-parto, os animais tratados com insulina mostraram maior perda de peso e maior concentração de AGNE, enquanto não se observou nenhum efeito sobre a concentração de progesterona ou outros metabólitos. Os resultados acima indicam que repetidas doses de insulina em cabras leiteiras durante a lactação inicial aumenta o rendimento de produção e concentração de componentes qualitativos do leite, mas apresenta consequências consideráveis sobre taxa de produção animal e resposta metabólica.

Combined effects of resveratrol and epigallocatechin-3-gallate on post thaw boar sperm and IVF parameters.

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.

The Ramazzini Institute 13-week study on glyphosate-based herbicides at human-equivalent dose in Sprague Dawley rats: study design and first in-life endpoints evaluation.

BACKGROUND: Glyphosate-based herbicides (GBHs) are the most widely used pesticides worldwide, and glyphosate is the active ingredient of such herbicides, including the formulation known as Roundup. The massive and increasing use of GBHs results in not only the global burden of occupational exposures, but also increased exposure to the general population. The current pilot study represents the first phase of a long-term investigation of GBHs that we are conducting over the next 5 years. In this paper, we present the study design, the first evaluation of in vivo parameters and the determination of glyphosate and its major metabolite aminomethylphosphonic acid (AMPA) in urine. METHODS: We exposed Sprague-Dawley (SD) rats orally via drinking water to a dose of glyphosate equivalent to the United States Acceptable Daily Intake (US ADI) of 1.75 mg/kg bw/day, defined as the chronic Reference Dose (cRfD) determined by the US EPA, starting from prenatal life, i.e. gestational day (GD) 6 of their mothers. One cohort was continuously dosed until sexual maturity (6-week cohort) and another cohort was continuously dosed until adulthood (13-week cohort). Here we present data on general toxicity and urinary concentrations of glyphosate and its major metabolite AMPA. RESULTS: Survival, body weight, food and water consumption of the animals were not affected by the treatment with either glyphosate or Roundup. The concentration of both glyphosate and AMPA detected in the urine of SD rats treated with glyphosate were comparable to that observed in animals treated with Roundup, with an increase in relation to the duration of treatment. The majority of glyphosate was excreted unchanged. Urinary levels of the parent compound, glyphosate, were around 100-fold higher than the level of its metabolite, AMPA. CONCLUSIONS: Glyphosate concentrations in urine showed that most part of the administered dose was excreted as unchanged parent compound upon glyphosate and Roundup exposure, with an increasing pattern of glyphosate excreted in urine in relation to the duration of treatment. The adjuvants and the other substances present in Roundup did not seem to exert a major effect on the absorption and excretion of glyphosate. Our results demonstrate that urinary glyphosate is a more relevant marker of exposure than AMPA in the rodent model.

Biological effects of polyphenol-rich extract and fractions from an oenological oak-derived tannin on in vitro swine sperm capacitation and fertilizing ability.

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 < .001) the percentage of spermatozoa with tyrosine-phosphorylated protein positivity in the tail principal piece (B pattern) (67.0 ±â€¯10.6 vs. 48.6 ±â€¯9.0, mean ±â€¯SD for TRE10 vs. Ctr respectively). Moreover T10 significantly (p < .001) increased oocyte fertilization rate (91.9 ±â€¯4.0 vs. 69.0 ±â€¯14.8, TRE10 vs. Ctr respectively). An opposite effect of TRE at the concentration of 100 µg/ml (TRE100) on both sperm capacitation (B pattern cell percentage 33.3 ±â€¯29.2) and fertilizing ability (fertilization rate 4.9 ±â€¯8.3), associated with a higher sperm viability (66.9 ±â€¯9.3 vs. 35.4 ±â€¯10.8, TRE100 vs. Ctr respectively) (p < .001), was recorded. The potency of the TRE fractions seems to be highest in FB followed by FC, faint in FD and nearly absent in FA. Our results show that TRE and its fractions, in a different extent, exert a powerful biological effect in finely modulating capacitation and sperm fertilizing ability.

CD49f+ mammary epithelial cells decrease in milk from dairy cows stressed by overstocking during the dry period.

The work reported in this Research Communication describes the modification in epithelial cell populations during the first and the last month of milking in Holstein Friesian cows that have undergone different management during the dry period, and we report the differential expression of CD49f+ and cytokeratin18+ cell subpopulations. Twenty six cows were randomly divided into 2 balanced groups that were housed at stocking density of either 11 m2 (CTR) or 5 m2 from 21 ± 3 d before the expected calving until calving. Cells collected from milk samples taken in early lactation and late lactation were directly analysed for CD45, CD49f, cytokeratin 14, cytokeratin 18 and cell viability. We observed a differential expression with a significant reduction in CD49f+ (P < 0·01) and cytokeratin 18+ (P < 0·05) cells in early lactation. Differences were still evident in late lactation but were not significant. These observations suggest that mammary epithelial cell immunophenotypes could be associated with different animal management in the dry period and we hypothesise they may have a role as biomarkers for mammary gland function in dairy cows.

Alkaline phosphatase added to capacitating medium enhances horse sperm-zona pellucida binding.

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.

Implementing an open-access CASA software for the assessment of stallion sperm motility: Relationship with other sperm quality parameters.

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.

Porcine circovirus type 2 detection in in vitro produced porcine blastocysts after virus sperm exposure.

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.

Characterization of alkaline phosphatase activity in seminal plasma and in fresh and frozen-thawed stallion spermatozoa.

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.