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.

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.

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.

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.

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.

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.

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.

Extended ex vivo culture of fresh and cryopreserved whole sheep ovaries.

We describe an original perfusion system for the culture of whole ovine ovaries for up to 4 days. A total of 33 ovaries were divided into six groups: control (n=6), not perfused and fixed; Groups SM72 and SM72-FSH (n=6 each), perfused with a simple medium for 72h with or without FSH; Groups CM96 and CM96-FSH (n=6 each), perfused with a complex medium for 96h with or without FSH; Group CM96-FSH-cryo, (n=3) cryopreserved and perfused for 96h with Group CM96-FSH medium. Depending on the medium used, morphological parameters of cultured ovaries differed from fresh organs after 72 (SM72, SM72-FSH) or 96 (CM96, CM96-FSH) h of perfusion. Oestradiol and progesterone were secreted in all groups but FSH had an effect only on Group CM96-FSH, stimulating continued oestradiol secretion 10 times higher than in all other groups. Morphological parameters and hormone secretion of cryopreserved ovaries were not different from fresh controls. This method enables the culture of whole ovaries for up to 4 days, the time required in vivo for 0.5-mm follicles to grow to 2.2mm and then for these follicles to reach the ovulatory size of 4mm or more. It could be used as a research tool or to complement current techniques for preserving female fertility.

Effects of resveratrol on vitrified porcine oocytes.

Vitrified MII porcine oocytes are characterized by reduced developmental competence, associated with the activation of the apoptotic pathway. Resveratrol (R), a polyphenolic compound present in several vegetal sources, has been reported to exert, among all its other biological effects, an antiapoptotic one. The aim of this study was to determine the effects of R (2 µM) on the apoptotic status of porcine oocytes vitrified by Cryotop method, evaluating phosphatidylserine (PS) exteriorization and caspases activation. R was added during IVM (A); 2 h postwarming incubation (B); vitrification/warming and 2 h postwarming incubation (C); all previous phases (D). Data on PS exteriorization showed, in each treated group, a significantly higher (P < 0.05) percentage of live nonapoptotic oocytes as compared with CTR; moreover, the percentage of live apoptotic oocytes was significantly (P < 0.05) lower in all R-treated groups relative to CTR. The results on caspase activation showed a tendency to an increase of viable oocytes with inactive caspases in B, C, and D, while a significant (P < 0.05) increase in A compared to CTR was recorded. These data demonstrate that R supplementation in various phases of IVM and vitrification/warming procedure can modulate the apoptotic process, improving the resistance of porcine oocytes to cryopreservation-induced damage.

Peripartal plasma concentrations of 15-ketodihydro-PGF2α, cortisol, progesterone and 17-ß-estradiol in Martina Franca jennies.

The transition from intra- to extrauterine environment represents a very delicate phase, in which the successful coordination of maturation is strictly connected with several hormonal changes during the last weeks of gestation and at parturition. While the peripartal endocrinology in the mare has been deeply investigated, the peripartal hormonal changes in the jenny need further evaluation. The aim of this study is to evaluate the mean 15-ketodihydro-PGF(2α) (PGFM), cortisol (C), progesterone (P4), and 17ß-estradiol (E2) levels during the peripartal period in this species. Ten Martina Franca jennies, with normal gestational length and parturition, were enrolled. From each jenny, blood was collected twice a day from 10 d before to 7 d after parturition and from the plasma obtained PGFM, C, P4 and E2 were analyzed by RIA. Higher, constant PGFM concentrations were observed in the pre-foaling days compared to the decreasing levels detected the days after delivery, as previously observed in the mare. During the whole period of observation no significant differences in plasma C levels were detected. In contrast to the mare, P4 has always been detectable and the highest level found at -2.5 days was significantly different compared to samples obtained between -10 and -4.5 days and between 1.5 and 7 days after foaling. Finally, E2 showed higher concentrations before foaling, with the highest values between -3 and -1.5 days, decreasing only one day before foaling. A positive correlation was found between PGFM and P4, during the last 4 days of gestation, while a positive correlation between PGFM and E2 was observed during the prepartum. Despite some similarities with the mare exist, differences have been found in P4 and E2 profiles, underlining once more the differences in the physiology of this two species.

Daidzein does affect progesterone secretion by pig cumulus cells but it does not impair oocytes IVM.

Daidzein, an isoflavone abundant in soybeans and other legumes, displays estrogen like properties. This study was aimed at evaluating the effect of daidzein (1 and 10 microM) on nuclear and cytoplasmic maturation of pig oocytes and on steroidogenic activity of cumulus cells. Daidzein supplementation during IVM had no effect on nuclear maturation and on fertilization traits. By contrast, both concentrations significantly (P < 0.05) inhibited progesterone production by cumulus cells after 24 and 48 h of culture while they did not induce any effect on estradiol production. Furthermore, daidzein did not exert any effect on the percentage of embryos that developed to blastocyst stage, on the number of blastomeres per blastocyst, or on the level of Hsp-70 and -90 gene transcript. Overall, our data demonstrate that daidzein added during oocyte maturation does not affect pig embryo development even if it markedly inhibits progesterone production by cumulus cells. Further studies are needed to evaluate the possible effect of daidzein during embryonic development.

Food deprivation stimulates the luteolytic capacity in the gilt.

The aims of this study were to study the effects of fasting on progesterone (P4) production in the pig and to verify whether fasting influences luteal expression of PGF(2alpha) receptor (FPr) and prostaglandin secretion. Superovulated prepubertal gilts were used; half of them were fasted for 72h starting on day 2 (F2) or 9 (F9) of the induced estrous cycle, respectively, while two groups (C2 and C9) served as respective controls. Plasma P4 and PGFM concentrations were determined by RIA while FPr mRNA expression in CLs collected at the end of fasting period was measured by real-time PCR. In experiment 1, plasma P4 concentrations in fasted gilts were significantly (P<0.01) higher than in controls starting from day 3 (F2; n=6) and 10 (F9; n=6). FPr mRNA expression was similar in F2 and C2 (n=6) CLs while it was significantly (P<0.05) higher in F9 than in C9 (n=6) CLs. In experiment 2, cloprostenol administered on day 12 significantly (P<0.05) increased FPr mRNA expression in CLs from both F9 (n=6) and C9 (n=6) gilts. At the time of cloprostenol injection PGFM levels were significantly higher (P<0.05) in the fasted group and cloprostenol-induced luteolysis in fasted but not in normally fed gilts. Results from this study indicate that fasting in prepubertal gilts induced to ovulate stimulates luteal P4 and PGFM production as well as FPr mRNA expression, thus increasing luteolytic susceptibility.

Hormonal and metabolic adaptation to fasting: effects on the hypothalamic-pituitary-ovarian axis and reproductive performance of rabbit does.

To assess the impact of acute caloric shortage on reproduction, rabbit does were either fed ad libitum (control, AL), or fasted for 24 (STF) or 48 h (LTF) before induction of ovulation with GnRH injection. Blood samples were collected during the last 3 h of fasting, and the following 4 h after GnRH injection, when feed was provided again, to measure plasma concentrations of LH, estradiol-17beta, leptin, insulin, T3, corticosterone, glucose, and NEFA. Before re-feeding, plasma leptin, insulin, and T3 concentrations were lower (P < or = 0.01) in both fasted groups than in controls, but then gradually increased following realimentation to match those of controls. During fasting, corticosterone levels were higher (P < or = 0.01) in LTF than in STF and AL does, but decreased to control values soon after realimentation. During fasting, plasma glucose concentrations did not differ among groups, but upon re-feeding they markedly increased (P < or= 0.01) both in STF and LTF does. NEFA levels were also more elevated (P < or = 0.01) in fasted rabbits than in controls, and rapidly decreased (P < or = 0.01) after re-feeding. Following GnRH injection, LH peak was lower (P < or = 0.01) in LTF than in AL and STF does. Estradiol-17beta showed higher pulse frequency and amplitude in AL than in STF and LTF does. Compared to controls, receptivity rate of STF and LTF artificially inseminated does declined respectively by -20.5% (P < or = 0.05) and -22.7%, and fertility rate by -23.9% (P < or = 0.05) and 21.4%, but no difference was found in ovulation rate. In summary, nutritional status of does, as modified by fasting, greatly influenced fertility, metabolic and reproductive hormones.

Fasting influences steroidogenesis, vascular endothelial growth factor (VEGF) levels and mRNAs expression for VEGF, VEGF receptor type 2 (VEGFR-2), endothelin-1 (ET-1), endothelin receptor type A (ET-A) and endothelin converting enzyme-1 (ECE-1) in newly formed pig corpora lutea.

This study was designed to verify whether fasting influences vascular endothelial growth factor (VEGF) production and VEGF, VEGF receptor-2 (VEGFR-2) as well as endothelin (ET) system members (endothelin converting enzyme-1, ECE-1; ET-1; endothelin receptor type A, ET-A) mRNA expression in pig corpora lutea; furthermore, we wanted to assess whether fasting affects steroidogenesis in luteal cells. Eight prepubertal gilts were induced to ovulate and were randomly assigned to two groups: (A) n = 4, normally fed; and (B) n = 4, fasted for 72 h starting 3 days after ovulation. At the end of fasting, ovaries were removed from all the animals and corpora lutea (CLs) were collected. VEGF and steroid levels in luteal tissue were determined by ELISA and RIA, respectively; VEGF, VEGFR-2, ET-1, ET-A and ECE-1 mRNAs expression was measured by real-time PCR. VEGF protein levels were similar in the two groups, while all steroid (progesterone, testosterone, estradiol 17beta) concentrations were significantly (P < 0.001) higher in CLs collected from fasted animals compared with those from normally fed gilts. VEGF, VEGFR-2, ET-1 and ECE-1 (but not ET-A) mRNA expression was significantly lower (P < 0.05) in fasted versus normally fed animals. The overall conclusion is that all the parameters studied are affected by feed restriction, but the mechanisms activated at luteal level are possibly not fully adequate to compensate for nutrient shortage.