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Although prostaglandins are important in the ovulation process, a precise role for prostaglandin F2α (PGF) has not been elucidated. This study aimed to evaluate the regulation of PGF receptor mRNA (PTGFR) in granulosa cells and the local effect of PGF on ovulation and luteinization. In Experiment 1, using samples collected in vivo before (Day 2), during (Day 3) and after (Day 4) follicular deviation, expression of PTGFR in bovine granulosa cells was more abundant in the dominant follicle after deviation than in subordinates (P 0.05). However, the expression of PTGFR was not regulated (P = 0.1) in preovulatory follicles at different time-points (0, 3, 6, 12 and 24 h) after ovulation induction with GnRH. In Experiment 2, to assess the role of systemic PGF treatment on luteinization and vascularization of preovulatory follicles, flunixin meglumine (FM), a nonsteroidal anti-inflammatory drug, was used to inhibit endogenous prostaglandin synthesis. Cows with preovulatory follicles were induced to ovulate with GnRH (0 h) and allocated to three groups: Control, with no further treatment; FM, treated with 2.2 mg/kg FM im 17 h after GnRH treatment; and FM + PGF, treated with FM 17 h after GnRH, followed by 25 mg dinoprost tromethamine (PGF) 23 h after GnRH treatment. FM injection was able to reduce the concentration of PGF in the follicular fluid (FF) (P 0.001). However, contrary to our hypothesis, color Doppler ultrasound evaluations revealed decreased vascular flow in FM + PGF group (P 0.05), and no effect of the treatments on intrafollicular P4 and E2 concentrations 24 h after GnRH. The prostaglandin metabolite (PGFM) concentrations in the FF were greater in cows receiving systemic PGF (P 0.001), which prompted us to further check its role on ovulation. Therefore, in Experiment 3, in a final attempt to demonstrate the local effect of PGF on ovulation, cows with preovulatory follicles received an intrafollicular injection (IFI) of PBS (Control) or 100 ng/mL purified PGF (PGF group). PGF treatment did not affect the time of ovulation after IFI (66 ± 6.4 and 63 ± 8.5 h for control and PGF, respectively; P 0.05), further suggesting that it has no direct effect in the ovulatory process. Based on our findings, we concluded that FM decreased PGF synthesis within the follicle, whereas PGF treatment decreased follicular vascularization. In addition, the in vivo model of intrafollicular injection evidenced that PGF alone is not able to locally induce ovulation.

The peroxisome proliferator-activated receptor gamma (PPARG, also called NR1C3) is a nuclear receptor of the peroxisome proliferator-activated receptor family (PPAR). PPARs are involved in the regulation of apoptosis, cell cycle, estradiol and progesterone synthesis, and metabolism. However, the role of PPARs and their regulation during follicular development and ovulation in monovular species remain poorly understood. In this study, a well-established intrafollicular injection model was used to investigate if the PPARG participates in the regulation of dominant follicle development and ovulation in cattle. Findings from this study revealed that the relative mRNA abundance of PPARG was similar between dominant and subordinate follicles around follicle deviation, decreased after the LH surge, and increased before ovulation. In addition, a quadratic correlation was found between PPARG mRNA levels in granulosa cells and progesterone concentration in the follicular fluid. Intrafollicular injection of 50 μM Troglitazone (TGZ; a PPARG agonist) inhibited follicular growth and decreased CYP19A1 mRNA abundance in granulosa cells. These findings indicate that PPARG is involved in the regulation of steroidogenesis, follicle growth and ovulation in cattle.

Oncostatin M (OSM) and its receptor (OSMR) are members of the interleukin-6 family cytokines. Although OSM and OSMR expression was detected in human ovaries, their function and regulation during follicle development, ovulation and luteolysis have not been studied in any species. The aim of the present study was to investigate the levels of OSM and OSMR mRNA in bovine ovaries and the effect of OSM treatment on cultured granulosa cells. OSM mRNA was not detected in granulosa cells obtained from follicles around the time of follicular deviation and from pre-ovulatory follicles, whereas OSMR transcript levels were greater in granulosa cells of atretic subordinate follicles (P 0.001). Abundance of OSMR mRNA increased in granulosa cells of preovulatory follicles, collected at 12 and 24 h after the ovulatory stimulus with gonadotropins (P 0.001). In the luteal tissue, OSM mRNA abundance levels were higher at 24-48 h after PGF-induced luteolysis (P 0.01) compared to 0 h, whereas OSMR mRNA was transiently increased at 2 h after PGF treatment (P 0.05). In cultured granulosa cells, 10 ng/mL OSM in the presence of FSH increased BAX/BCL2 mRNA ratio (P 0.05) compared to the control. Moreover, 100 ng/mL OSM in the presence of FSH increased OSMR (P 0.05) and decreased XIAP mRNA (P 0.05) levels, compared to the control group. These findings provide the first evidence that OSMR is regulated during follicle atresia, ovulation and luteolysis, and that OSM from other cells may mediate granulosa and luteal cell function, regulating the expression of genes involved in cell's viability.

During folliculogenesis, the luteinizing hormone (LH) surge triggers dynamic events in granulosa cells that culminate with ovulation. The aim of this study was to evaluate if the epidermal growth factor receptor (EGFR) is required for ovulation in cattle, and if it regulates the expression of the natriuretic peptide (NP) system in granulosa cells after gonadotropin-releasing hormone (GnRH)/LH stimulation. It was observed that GnRH induces amphiregulin (AREG) and epiregulin (EREG) mRNA at 3 and 6 h after in vivo treatment, but the expression of these genes was not regulated by atrial (ANP) and C-type (CNP) NPs in granulosa cells cultured in vitro. The abundance of mRNA encoding the NP receptors (NPR1, 2 and 3) was not altered by LH supplementation and/or EGFR inhibition (AG1478; AG) in granulosa cells after 6 h of in vitro culture. However, in the same conditions, mRNA encoding the natriuretic peptide precursor C (NPPC) was upregulated by LH, whereas AG (0.5 and 5 μM) inhibited the LH effect. In order to confirm those results, 5 μM AG or saline were intrafollicularly injected in preovulatory follicles and cows were simultaneously treated with GnRH intramuscularly. Granulosa cells harvested at 6 h after GnRH injection revealed higher NPR3 and lower NPPC mRNA levels in AG-treated, compared to control cows. However, intrafollicular injection of AG did not inhibit GnRH-induced ovulation. In granulosa cells cultured in vitro, ANP associated with LH increased prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA abundance. In conclusion, we inferred that LH modulated NPPC and NPR3 mRNA abundance through EGFR in bovine granulosa cells, but ovulation in cattle did not seem to depend on EGFR activation.

The objective of this study was to investigate the effects of inhibiting the epidermal growth factor receptor (EGFR) pathway on meiosis blockage and resumption, mRNA expression of genes involved in oocyte maturation and cumulus expansion, and embryo development. Bovine cumulus-oocyte complexes (COCs) were cultured for 15 h in the presence of the EGFR inhibitor (AG1478) and follicular hemisections (FHS). Most of the oocytes (89.3%) remained at the germinal vesicle (GV) stage when cultured in the presence of FHS and 5 μM AG1478. The inhibitory effect was reversible as most oocytes (83.8%) completed meiosis after additional 20 h maturation. Embryo development to the blastocyst stage was similar (P > 0.05) between FHS and 5 μM AG1478 treated (39.3%) and control (41.1%) groups. In cumulus cells, mRNA abundance of early growth response protein 1 (EGR1), tumor necrosis factor alpha-induced protein 6 (TNFAIP6) and hyaluronan synthase 2 (HAS2) genes, and phosphorylated extracellular regulated kinase (p-ERK1/2) protein were lower in COCs treated with AG1478 plus FHS compared with FHS alone (P < 0.05). In granulosa cells of FHS, AG1478 treatment reduced transcript levels of PGR and ADAMTS1 (P < 0.05). The inhibitory effect of AG1478 on meiotic progression was not reverted by treatment with angiotensin II (ANG II) or prostaglandins (PGF2α or PGE2). This study demonstrates that inhibition of EGFR in the presence of FHS is a reliable approach to promote reversible arrest of bovine oocytes at the GV stage.

Nitric oxide (NO) is identified as a signaling molecule involved in many cellular or physiological functions, including meiotic maturation of cattle oocytes. This study aimed to evaluate the effect of supplementation of culture medium with the L-arginine (L-arg, NO synthesis precursor) in nuclear maturation of oocytes, concentrations of nitrate/nitrite, progesterone (P4), and 17β-estradiol (E2) in the culture medium; and the cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) intracellular concentrations in the cumulus-oocyte complexes (COCs) during the first hours of maturation in the presence of hemisections (HSs) of the follicular wall (control −ve). The addition of 5.0-mM L-arg increased (P 0.05) at 22 hours. All concentrations of L-arg tested increased (P 0.05) the nitrate/nitrite, P4, and E2 concentrations in relation to control −ve at any of the times tested. In immature COCs, immediately after being removed from the follicles (0 hours), the intracellular concentration of cGMP in the control −ve and treatment with 5-mM L-arg progressively decreased (P

Long-term administration of valproic acid (VPA) is known to promote reproductive impairment mediated by increase in testicular oxidative stress. Vitamin E (VitE) is a lipophilic antioxidant known to be essential for mammalian spermatogenesis. However, the capacity of this vitamin to abrogate the VPA-mediated oxidative stress has not yet been assessed. In the current study, we evaluated the protective effect of VitE on functional abnormalities related to VPA-induced oxidative stress in the male reproductive system. VPA (400 mg kg −1 ) was administered by gavage and VitE (50 mg kg −1 ) intraperitoneally to male Wistar rats for 28 days. Analysis of spermatozoa from the cauda epididymides was performed. The testes and epididymides were collected for measurement of oxidative stress biomarkers. Treatment with VPA induced a decrease in sperm motility accompanied by an increase in oxidative damage to lipids and proteins, depletion of reduced glutathione and a decrease in total reactive antioxidant potential on testes and epididymides. Co-administration of VitE restored the antioxidant potential and prevented oxidative damage on testes and epididymides, restoring sperm motility. Thus, VitE protects the reproductive system from the VPA-induced damage, suggesting that it may be a useful compound to minimize the reproductive impairment in patients requiring long-term treatment with VPA.

A B S T R A C T - Background: We describe the investigation and control of an outbreak of Legionnaires’ disease in Portugal in October, November and December 2014. Methods: Confirmed cases were individuals with pneumonia, laboratory evidence of Legionella pneumophila serogroup 1 and exposure, by residence, occupational or leisure to the affected municipalities. 49 possible sources were reduced to four potential sources, all industries with wet cooling system, following risk assessment. We geo-referenced cases’ residences and the location of cooling towers defining four study areas 10 km buffer centered on each cooling tower system. We compared the number of cases with expected numbers, calculated from the outbreak’s attack rates applied to 2011 census population. Using Stones’ Test, we tested observed to expected ratios for decline in risk, with distance up to 10 km four directions. Isolates of Legionella pneumophila were compared using molecular methods. Results: We identified 403 cases, 377 of which were confirmed, 14 patients died. Patients became ill between 14 October and 2 December. A NE wind and thermal inversion were recorded during the estimated period of exposure. Disease risk was highest in people living south west from all of the industries identified and decreased with distance (p < 0.001). 71 clinical isolates demonstrated an identical SBT profile to an isolate from a cooling tower. Whole genome sequencing identified an unusual L. pneumophila subsp. fraseri serogroup 1 as the outbreak causative strain, and confirmed isolates’ relatedness. Conclusions: Industrial wet cooling systems, bacteria with enhanced survival characteristics and a combination of climatic conditions contributed to the second largest outbreak of Legionnaires’ disease recorded internationally. R E S U M O Contexto: Descrevemos a investigação epidemiológica e medidas de controlo de um surto de doença dos Legionários, ocorrido em Portugal em outubro, novembro e dezembro de 2014. Métodos: A definição de caso englobou doentes com critérios clínicos de pneumonia aguda, com provas imagiológicas compatíveis e confirmação laboratorial para a identificação de Legionella pneumophila (L. pneumophila) serogrupo 1, para além do critério epidemiológico de exposição, quer por motivos de residência, ocupacional ou lazer nas freguesias suspeitas. Quarenta e nove possíveis fontes de infeção foram reduzidas a 4 potenciais fontes, após avaliação de risco, todas as indústrias com sistema de torres de arrefecimento. A georreferenciação por residência dos casos e localização de torres permitiu definir 4 áreas de investigação num perímetro de 10 km centrado em cada uma daquelas 4 torres. Comparouse o número de casos observados com o número de casos esperados, calculados a partir de taxas de ataque do surto aplicadas à população. Usando o teste de Stones, testou-se a razão entre casos observados e casos esperados e declínio do risco em relação à distância de até 10 km em 4 direções. As amostras de L. pneumophila foram comparadas utilizando métodos moleculares. Resultados: Foram identificados 403 casos, dos quais 377 foram confirmados, tendo ocorrido 14 óbitos. Os doentes apresentaram sintomas entre 14 de outubro e 2 de dezembro. Em termos meteorológicos, foram registados ventos NE e inversão térmica durante o período estimado de exposição. O risco de doença foi maior em pessoas que vivem a sudoeste de todas as indústrias identificadas, diminuindo com o aumento da distância (p < 0,001). Amostras de 71 dos casos clínicos demonstraram um perfil SBT idêntico às amostras isoladas a partir de uma torre de arrefecimento. A sequência de genoma de L. pneumophila fraseri serogrupo 1 pouco comum como a estirpe causadora do surto confirmou a relação das amostras isoladas. Conclusões: Torres de arrefecimento industriais, agentes bacterianos com características mais desenvolvidas para elevada sobrevivência e uma rara combinação de condições climáticas, contribuíram para o segundo maior surto de doença dos Legionários registado na literatura. info:eu-repo/semantics/publishedVersion

Valproic acid (VPA) is a drug widely use for the treatment of epilepsy in both children and adults. Evidence suggests that long-term use of VPA may lead to an impairment in the male reproductive function. Oxidative stress is considered to play a major role in VPA associated toxicity. In the present work, we demonstrated that the natural antioxidant compound resveratrol (RSV) can be use to prevent VPA oxidative damage. Wistar rats treated with VPA (400 mg kg −1 ) by gavage for 28 days showed decrease in sperm motility accompanied by increase in oxidative damage to lipids and proteins. Additionally, VPA administration leaded to depletion of reduced glutathione and decrease in total antioxidant potential in testes and epididymides of Wistar rats. The co-administration of RSV (10 mg kg −1 ) efficiently prevented VPA pro-oxidant effects. In summary, RSV was shown to protect the reproductive system from the damage induced by VPA. Altogether, our data strongly suggests that RSV administration might be a valuable strategy to minimize reproductive impairment in patients requiring long-term VPA treatment.

We analysed the effects of quercetin-containing diet on blood parameters, antioxidant status and pituitary hormone expression in silver catfish. Diets containing three concentrations of quercetin (0, 0.15 and 0.30%) were provided to fish once a day. The results indicated that quercetin did not promote any significant change on the haematological and biochemical parameters measured. Fish that received the diet with quercetin presented decreased lipid peroxidation (LPO) (measured by lipid hydroperoxides and thiobarbituric acid reactive substances) in all tissues evaluated. On the other hand, the activity of antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase and glutathione S-transferase were higher in tissues of fish fed with diets containing quercetin. Additionally, the content of non-protein thiols, total reactive antioxidant potential and ascorbic acid were also higher in tissues of quercetin fed fish. Finally, there was no changes regarding cortisol levels and the expression of growth hormone, prolactin and somatolactin in fish fed with quercetin when compared with the control. Our results suggests that supplementation of silver catfish diet with quercetin is beneficial since it reduced the LPO and increased antioxidant capacity in vital tissues of fish without having any impact on haematological and biochemical parameters, and on pituitary hormone gene expression. Statement of relevance We propose quercetin as supplement in fish diets.

The role of superoxide dismutase manganese dependent enzyme (SOD2) in colorectal cancer is presently insufficiently understood. Some studies suggest that high SOD2 levels found in cancer tissues are associated with cancer progression. However, thus far, the role of colorectal cancer superoxide-hydrogen peroxide imbalance has not yet been studied. Thus, in order to address this gap in extant literature, we performed an in vitro analysis using HT-29 colorectal cell line exposed to paraquat, which generates high superoxide levels, and porphyrin, a SOD2 mimic molecule. The effect of these drugs on colorectal cancer cell response to oxaliplatin was evaluated. At 0.1 μM concentration, both drugs exhibited cytotoxic and antiproliferative effect on colorectal cancer cells. However, this effect was more pronounced in cells exposed to paraquat. Paraquat also augmented the oxaliplatin cytotoxic and antiproliferative effects by increasing the number of apoptosis events, thus causing the cell cycle arrest in the S and M/G2 phases. The treatments were also able to differentially modulate genes related to apoptosis, cell proliferation and antioxidant enzyme system. However, the effects were highly variable and the results obtained were inconclusive. Nonetheless, our findings support the hypothesis that imbalance caused by increased hydrogen peroxide levels could be beneficial to cancer cell biology. Therefore, the use of therapeutic strategies to decrease hydrogen peroxide levels mainly during oxaliplatin chemotherapy could be clinically important to the outcomes of colorectal cancer treatment.

This study investigated (1) the effects of cilostamide and follicular hemisections on in vitro oocyte meiotic resumption during a pre-maturation period of 12 h, (2) the levels of cAMP in bovine cumulus oocyte complexes (COCs) after pre-maturation, and (3) the expression of mRNA for CONNEXIN 43(CX43), HAS2, MATER, NPM2 and ZAR1 in denuded oocytes and cumulus cells from COCs that were subjected to both pre-maturation and maturation in vitro . To evaluate the effects of follicular hemisections and cilostamide on meiosis resumption, COCs were subjected to pre-IVM for 12 h in TCM-199 alone or in presence of 10 µM cilostamide, follicular hemisections and both cilostamide and follicular hemisections. After 6 h, COCs were used to measure the levels of cAMP, while after 12 h of pre-IVM, COCs were fixed to assess meiotic progression or stored to evaluate mRNA expression. At the end of this pre-IVM period, COCs were matured in vitro and meiotic progression was evaluated. The results showed that the presence of both follicular hemisections and cilostamide in pre-maturation medium increased significantly the percentages of oocytes at germinal vesicle stage (94%) when compared with COCs cultured in control medium alone or in presence of either follicular hemisections or cilostamide. After this pre-maturation period, 75% of oocytes reached metaphase II after 16 h of maturation, emphasizing that cilostamide did not have a toxic effect on oocytes. Moreover, oocytes cultured in medium containing cilostamide and follicular hemisections had significantly higher levels of cAMP when compared to other treatments. The presence of both cilostamide and follicular hemisections also increased the levels of mRNA expression for NPM2 and maintained those of MATER , ZAR1 , HAS2 and CX43 similar to control. In conclusion, cilostamide and follicular hemisections interact and promotes the maintenance of oocytes at germinal vesicle stage by increasing the levels cAMP in cultured COCs.

Busulfan is a chemotherapy drug that has side effects on spermatogonial stem cells (SSC). The effects of bulsufan treatment on male germ cells and fertility vary significantly between individuals. In this study, we have used molecular, cellular and histopathology approaches to investigate the effects of a single intraperitoneal dose of busulfan (40 mg kg −1 ) in two mice strains, Balb/C and Swiss, at two different periods after treatment, 30 and 90 days. Testicular degeneration was observed in both Balb/C and Swiss mice after busulfan injection. Interestingly, testicular functions and fertility recovered spontaneously post busulfan treatment in Swiss mice, but not in Balb/C mice. Abnormal fertility induced by busulfan in Balb/C mice was associated with altered seminiferous tubules, sperm morphology and transcript levels of Nanos2, Nanos3 , Gdnf and Plzf genes. These findings revealed that SSC of Balb/C mice are more sensitive to the toxic effects of busulfan then those of Swiss mice.

The aim of the present study was to evaluate the expression of mRNA encoding natriuretic peptides (NPs) and their receptors in the cumulus-oocyte complex in cattle, a monovular mammalian species, and also to investigate the role of NPs in oocyte meiotic resumption in vitro. mRNA was observed for the NP precursor type-A (NPPA), type-C (NPPC), NP receptor-1 (NPR-1), receptor-2 (NPR-2) and receptor-3 (NPR-3) in bovine cumulus cells, and NPR-2 mRNA was observed in oocytes. These results are different from those obtained in mouse and pig models. The effects of NPPA, NP precursor type-B (NPPB) and NPPC on the resumption of arrested meiosis maintained by forskolin were studied at three different doses (10, 100 and 1000nM) with a 12h culture system. The germinal vesicle breakdown rates were greater (P≤0.05) in oocytes that were cultured in the presence of one or a combination of NPs (from 44% to 73%) than the negative control (from 24% to 27%). Additionally, it was demonstrated that the concentration of cyclic guanosine 3',5'-monophosphate (cGMP) is increased by NPPA and NPPC in oocytes and cumulus cells after 3h of in vitro maturation. However, in both groups, the concentration of cyclic adenosine 3',5'-monophosphate (cAMP) in the oocyte did not increase between 3 and 6h of culture, even when forskolin was used. In summary, we observed the presence of mRNA for NPs and their receptors in the bovine cumulus-oocyte complex and demonstrated that, in vitro, NPPA, NPPB and NPPC stimulate oocyte meiotic resumption in a monovular species.

The aim of this study was to assess the Mn toxicity to silver catfish considering Mn accumulation and oxidative status in different tissues, as well as pituitary hormone expression after acclimation to hypoxia. Silver catfish acclimated to hypoxia for 10 days and successively exposed to Mn (9.8 mg L−1) for an additional 10 days exhibited lower Mn accumulation in plasma, liver, kidneys and brain and prevented the hematocrit decrease observed in the normoxia group. Hypoxia acclimation also modified Mn-induced oxidative damage, which was observed by lower reactive species (RS) generation in gills and kidneys, decreased lipid peroxidation (LP) levels in gills, liver and kidneys and decreased protein carbonyl (PC) levels in liver, kidneys and brain. Manganese accumulation showed positive correlations with LP levels in gills and kidneys, as well as with PC levels in gills, liver and brain. In addition, hypoxia acclimation and Mn exposure increased catalase (CAT) activity in gills and kidneys and Na+/K+-ATPase activity in gills, liver and brain. Silver catfish that were acclimated under normoxia and exposed to Mn displayed increased pituitary prolactin (PRL) and decreased somatolactin (SL) expression. Interestingly, hypoxia acclimation prevented hormonal fluctuation of PRL and SL in fish exposed to Mn. These findings indicate that while the exposure of silver catfish to Mn under normoxia was related to metal accumulation and oxidative damage in tissues together with endocrine axis disruption, as represented by PRL and SL, hypoxia acclimation reduced waterborne Mn uptake, thereby minimizing oxidative damage and changes in hormonal profile. We hypothesized that moderate hypoxia is able to generate adaptive responses, which may be related to hormesis, thereby ameliorating Mn toxicity to silver catfish.

Bone morphogenetic proteins are known to be involved in determining ovulation rate in mammals. The mechanisms through which these proteins determine follicle fate are incompletely understood. In the present study, we used cattle as a model to evaluate the regulation of BMP15 and GDF9 receptors in granulosa cells during dominant follicle (DF) selection. Before follicular deviation (day 2 of the follicular wave), BMPR2 mRNA abundance tended to be higher in the second largest follicles (F2; P0.1) compared to the future dominant follicle (F1). At the expected time of follicular deviation (day 3), BMPR2 and BMPR1B mRNA levels were higher in subordinate follicles (SFs; P0.05) compared to dominant follicles (DFs). After deviation (on day 4), BMPR1B mRNA and protein were significantly more abundant in atretic SFs (as assessed by cleaved caspase 3) than in DFs. The fact that BMPR1B is more expressed in atretic follicles was further confirmed by using intrafollicular treatment with two agents known to induce atresia, namely an estradiol receptor antagonist (fulvestrant) and FGF10. In conclusion, the fact that BMPR-1B and -2 are more expressed in the second largest follicles before and at the expected time of follicular deviation is indicative of their inhibitory role in follicle differentiation and steroidogenesis. BMPR1B also seems to have a pivotal role during follicle regression since it is upregulated in advanced atretic follicles.

The objective was to determine the effects of eCG given on the day of, or 2 days before removal of an intravaginal progestin device, on ovarian follicle diameter, luteal volume, serum progesterone (P4) concentrations, and pregnancy per insemination in a fixed-time AI (FTAI) protocol. Lactating, anestrous, multiparous Bos taurus cross beef cows, 40 to 60 days postpartum, were given estradiol benzoate (2 mg im) and a progestin intravaginal device containing 250 mg of medroxyprogesterone acetate on Day 0 and cloprostenol (0.265 mg) on Day 6. Intravaginal devices were removed on Day 8 and GnRH (100 μg im) was given on Day 9, with timed AI 16 hours later. In experiment 1, cows were randomly assigned to receive 400 IU im eCG on Day 6 (eCG6; N = 8) or Day 8 (eCG8; N = 8), or to not receive eCG (control; N = 8). Dominant follicle diameter on Day 9 in the eCG6 group (10.0 ± 0.5 mm) was larger (P < 0.05) than in the eCG8 (8.6 ± 0.2 mm) or control (8.5 ± 0.4 mm) groups. Corpora lutea (CL) in all cows in the control group underwent premature luteolysis within 10 days after ovulation. Luteal volumes and P4 concentrations 10 and 15 days after ovulation were higher (P < 0.05) in the eCG6 group than in the eCG8 group. In experiment 2, the eCG6 (N = 121) and eCG8 (N = 125) protocols were compared in lactating anestrous cows that underwent FTAI. Pregnancy rate was higher (P < 0.05) in the cows that received eCG on Day 6 (27.3%; 33/121) than on Day 8 (16.0%; 20/125). Furthermore, CL volumes and P4 concentrations were higher (P < 0.05) in the eCG6 group (5784.0 ± 857.3 mm3 and 8.1 ± 1.3 ng/mL, respectively) than in the eCG8 group (3220.9 ± 505.1 mm3 and 4.5 ± 0.7 ng/mL, respectively). We concluded that eCG given 2 days before progestin removal in this FTAI protocol for anestrous beef cows increased diameter of the dominant follicle, luteal volume, serum P4 concentrations, and pregnancy rates.

Made available in DSpace on 2013-09-27T14:54:28Z (GMT). No. of bitstreams: 1 WOS000304231600006.pdf: 4200149 bytes, checksum: da68c3a014ed77979dfeca212097e21a (MD5) Previous issue date: 2012-06-01 Made available in DSpace on 2013-09-30T18:37:40Z (GMT). No. of bitstreams: 1 WOS000304231600006.pdf: 4200149 bytes, checksum: da68c3a014ed77979dfeca212097e21a (MD5) Previous issue date: 2012-06-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:50:02Z No. of bitstreams: 1 WOS000304231600006.pdf: 4200149 bytes, checksum: da68c3a014ed77979dfeca212097e21a (MD5) Made available in DSpace on 2014-05-20T13:50:02Z (GMT). No. of bitstreams: 1 WOS000304231600006.pdf: 4200149 bytes, checksum: da68c3a014ed77979dfeca212097e21a (MD5) Previous issue date: 2012-06-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Oocyte meiotic resumption is triggered by the ovulatory gonadotropin surge; in cattle, angiotensin II (AngII) and prostaglandins (PG) are key mediators of this gonadotropin-induced event. Here, we tested the hypothesis that progesterone (P-4) is also involved in oocyte meiotic resumption induced by the gonadotropin surge. In Experiment I, P-4 induced nuclear maturation in a dose-dependent manner using a coculture of follicular hemisections and cumulus-oocyte complexes. In the second experiment, using an in vivo model, an injection of mifepristone (MIFE; P-4 receptor antagonist) at the antrum of preovulatory follicles prevented GnRH-induced oocyte meiotic resumption in vivo. In Experiment III (coculture system similar to that of Experiment I), MIFE prevented stimulatory effects of AngII on resumption of meiosis, but saralasin (AngII receptor antagonist) did not inhibit P-4 actions. In Experiments IV and V, fibroblast growth Factor 10 (FGF10; known to suppress steroidogenesis in granulosa cells), blocked AngII-but not P-4-induced oocyte meiotic resumption. Therefore, we inferred that AngII is upstream to P-4 in a cascade to induce meiotic resumption. Previously, we had reported that AngII acted throughout the PGs pathway to modulate nuclear progression. In Experiment V, indomethacin inhibited resumption of meiosis induced by P-4, providing further support to the AngII-P-4, sequential effect on meiotic resumption. In conclusion, we inferred that AngII, P-4, and PGs are sequential steps in the same pathway that culminates with bovine oocyte maturation. (C) 2012 Elsevier B.V. All rights reserved. Universidade Federal de Santa Maria (UFSM), Lab Biotechnol & Anim Reprod BioRep, BR-97119900 Santa Maria, RS, Brazil State Univ São Paulo UNESP, Dept Physiol, Botucatu, SP, Brazil State Univ São Paulo UNESP, Dept Physiol, Botucatu, SP, Brazil

Objectives Aspartame is one of the most popular sweeteners in the world. Several studies have linked its use to a dysregulation of glutathione homeostasis in the brain, making it more susceptible to oxidative stress. The aim of the present study was to evaluate the effects of N-acetylcysteine (NAC) on thioredoxin and glutathione-related antioxidant systems in brain regions of aspartame-treated mice. Methods Adult Swiss mice were treated with aspartame (80 mg/kg, v.o.) for 90 days, and from day 60 to day 90, immediately after aspartame treatment, the mice received NAC (163 mg/kg, i.p.). After the last doses, the animals were anesthetized and euthanized for brain removal. The analyzes were performed in cortex, cerebellum and hippocampus. Aspartame administration caused a severe depletion of non-protein thiols levels, as well as glutathione peroxidase activity. Both changes were restored by NAC treatment. Aspartame also triggered a decrease in the mRNA levels of catalytic subunit of glutamate cysteine ligase (Gclc) and cystathionine gamma-lyase (Cth). Conclusion NAC treatment restored Gclc mRNA levels. NAC treatment restored most of changes in glutathione-related system in brain regions of mice after chronic intake of aspartame.

The objective was to evaluate the effect of estradiol benzoate (EB), in association with three progestin protocols, on ovarian follicular regression of suckled beef cows treated at three stages of follicular development (pre-deviation, deviation, or post-deviation). Thirty-six suckled beef cows (60–90 d postpartum, given 125 μg cloprostenol on two occassions, 12 h apart). Forty-eight hours after the first cloprostenol treatment, all follicles >5 mm were ablated and transrectal ultrasound scanning (8 MHz) was performed every 24 h until Day 7 (Day 0 = treatment). When the largest follicle reached a designated diameter of 5–7, 8–10 or >10 mm, cows were randomly allocated to receive 2 mg of EB im in association with an intravaginal device containing 250 mg of medroxyprogesterone acetate (MPA) with or without 100 mg of progesterone (P 4 ) given im, or an intravaginal device containing P 4 (3 × 3 factorial design). Treatments induced follicular regression in all cows, independent of follicular stage or treatment. There was no interaction between progestin treatment and follicular stage, nor was there any difference in the time of follicular regression or new wave emergence among follicular stages. Treatment with MPA plus P 4 delayed follicular regression. In conclusion, EB in association with various progestins induced regression of growing follicles and emergence of a new follicular wave in postpartum beef cows, regardless of the stage of follicular development.

The aims of the present study were to determine the role of protein kinase C (PKC) on meiotic resumption and its effects on pronuclear formation and cleavage in the bovine. Oocytes were matured in the presence of 0, 1, 10 and 100 nM of phorbol 12-myristate 13-acetate (PMA), to evaluate the percentage of germinal vesicle breakdown. To study pronuclear formation and cleavage, oocytes were randomly distributed in four groups and matured in modified TCM-199 with LH and FSH (negative control); 10% of estrous cow serum (positive control); 100 nM of PMA (treatment); 100 nM of 4alpha-PDD (phorbol ester control). Oocytes were also matured in positive control medium, fertilized and transferred to KSOM with increasing concentrations of a PKC inhibitor. The protein profile and the presence of PKC at the end of maturation period were determined by SDS-PAGE followed by Silver Stain and Western blot, respectively. PMA stimulated meiotic resumption in a concentration-dependent manner. PKC stimulation during oocyte maturation caused an increase in pronuclear formation and did not cause parthenogenetic activation. Inhibitor of PKC (MyrPKC) inhibited cleavage in a dose-dependent and irreversible manner. A protein band around 74 kDa was not detected in PMA-treated oocytes and PKC was not detected by Western blot at the end of the maturation period. In conclusion, meiotic resumption was accelerated and the rate of oocytes with two pronuclei was increased when PKC was activated during oocyte maturation. Moreover, cleavage was inhibited in the presence of PMA.

The objective was to evaluate the effects of angiotensin II (Ang II), insulin-like growth factor-I (IGF-I) and insulin on the nuclear and cytoplasmic maturation of bovine oocytes in the presence of follicular cells. Cumulus-oocyte complexes (COCs) were cultured for 22h in the presence of follicular cells (control with cells) and Ang II, IGF-I or insulin (treatments), or in the absence of follicular cells (control without cells). Using these five groups, Experiment 1 was conducted with and without the addition of gonadotrophins. Only oocytes in the Ang II group resumed meiosis at rates (88.2+/-1.8% and 90.7+/-4.3% for oocytes cultured in the absence or presence of LH/FSH, respectively) similar to those observed for oocytes cultured in the absence of follicular cells (89.7+/-0.3% and 92.6+/-2.6%; P0.01). In Experiment 2, the effect of Ang II alone and in combination with IGF-I or insulin on oocyte maturation for 7h (germinal vesicle breakdown), 12h (metaphase I) and 22h (metaphase II) was evaluated in a design similar to that of the first experiment. Ang II plus IGF-I or insulin induced the resumption of meiosis, irrespective of the presence of gonadotrophins (P0.01). Experiment 3 used groups similar Experiment 2 to determine the rate of subsequent embryo development, using fetal calf serum (FCS) in the culture medium. The COCs were cultured in maturation medium for 1h (1+23h), 12h (12+12h) or 24h in the presence of follicular cells and the respective treatments and for the remaining period in the absence of follicular cells to complete 24h. In Experiment 4, BSA was used in lieu of serum in the maturation medium in a 12+12h maturation system. Oocytes matured using the 12+12h system with BSA or FCS in the presence of Ang II+IGF-I had higher rates of blastocyst formation than the other treatments (P0.05). In conclusion, Ang II reversed the inhibitory effect of follicular cells on nuclear maturation of bovine oocytes, irrespective of the presence of gonadotrophins, IGF-I and insulin. However, oocyte cytoplasmatic maturation (i.e., subsequent embryo development), was higher when Ang II and IGF-I were present in the maturation medium containing follicular cells cultured for 12+12h.

The presence of prorenin, renin, angiotensinogen, angiotensin-converting enzyme, angiotensin II (Ang II) and Ang II receptors in the ovary is suggestive of a functional ovarian renin-angiotensin system (RAS). In cattle, the expression of Ang II is greatest in large follicles, suggesting that it is important during follicular growth and maturation. The present study was designed to investigate the role of Ang II in bovine oocyte nuclear maturation. Bovine cumulus-oocyte complexes (COCs) were cultured with or without follicular cells and Ang II or saralasin (Ang II antagonist). In the absence of follicular cells, Ang II at 0, 10(-11), 10(-9) and 10(-7) M did not affect the percentage of oocytes reaching the germinal vesicle breakdown (GVBD), metaphase I (MI) and metaphase II (MII) stage after 7-h (41.3 +/- 4.3, 35.3 +/- 4.0, 31.3 +/- 9.7, 38.7 +/- 8.6), 12-h (31.6 +/- 7.0, 34.7 +/- 6.1, 31.7 +/- 5.3, 28.9 +/- 9.1; mean +/- S.E.M.) and 18-h (44.9 +/- 7.3, 58.4 +/- 8.4, 53.1 +/- 7.4, 44.9 +/- 7.3) of culture, respectively. Similarly, saralasin at 0, 10(-11), 10(-9) and 10(-7) M did not affect the percentage of oocytes reaching MII stage after 18-h of culture (37.6 +/- 7.4, 34.4 +/- 7.7, 30.0 +/- 10.8 and 31.2 +/- 5.1, respectively). The theca cells (MII = 22.9%) or medium conditioned with follicular cells (GV = 65.5%, MI = 23.6%) inhibited oocyte maturation; however, theca cells (MII = 35.5 +/- 4.9; P0.05) or medium conditioned with follicular cells (GV = 34.6%, MI = 52.7%; P0.01) were not able to inhibit nuclear maturation when Ang II (10(-11) M) was present in the culture system. Theca cells remained viable during the culture period when Ang II was present. Therefore, results supported the idea of a role of Ang II in blocking the inhibitory effect of theca cells on nuclear maturation of bovine oocytes.

The objective of this study was to compare enzymatic and mechanical methods, at distinct fetal ages, on isolation of different developmental stages of preantral follicles from bovine ovaries. Fetal ovaries were obtained from pregnant cattle at 150 to 270 d of gestation, and 135,521 preantral follicles at different stages of development were studied. The dissociation of ovaries with a mechanical procedure resulted in an average of 938.16 prenatral follicles. In contrast, 3,715.56 follicles were obtained when enzymatic digestion was used (P = 0.0001). Histological evaluation confirmed follicular stages and demonstrated that both mechanical and mechanical-enzymatic procedure did not affect the cellular integrity of the follicles. Granulosa cell-oocyte complexes surrounded by a basal membrane, were considered preantral follicles in this study. The ratio of different stages of isolated preantral follicles was significantly (P = 0.0001) correlated to fetal age. The earliest fetal age at which tertiary follicles were identified was at 210 d of gestation. The results confirm previous observation that follicular development and atresia are initiated during fetal development. These data provide information on methodologies to isolate intact bovine preantral follicles for investigating the control and regulation of follicular development and the growth of preantral follicles in vitro.

Pregnancy diagnosis is a critical procedure for monitoring goat reproductive performance. In the present investigation, pregnancy-associated glycoprotein (PAG) profiles during pregnancy and first month postpartum, as well as progesterone (P4) concentrations throughout pregnancy, were determined in native goats from the north-east of Brazil. A total of 30 goats (Moxoto, n = 19 and Caninde, n = 11) were used. Peripheral blood was collected on days 0 (Day 0 = AI), 11, 16, 18, 20, 24 and 30 after AI. Samples were subsequently collected every 2 weeks until day 145, and weekly during the first month postpartum. The levels of PAG and P4 were determined by radioimmunoassay (RIA). The profiles of PAG were not different between the breeds throughout pregnancy. However, a significant effect of the week of pregnancy (p

The aim of the present study was to evaluate the effect of artificial insemination time (before or after ovulation) using either fresh or frozen-thawed boar semen on embryo viability and early pregnancy rate. Seventy-seven prepubertal crossbred (Landrace × Large White × Duroc) gilts were inseminated in 4 treatments. Artificial inseminations were performed 6 h either after (A) or before (B) ovulation using frozenthawed (A-frozen, n = 19; B-frozen, n = 19) or fresh semen (A-fresh, n = 21; B-fresh, n = 18). The gilts were induced to puberty by administration of 400 IU of eCG and 200 IU hCG (sc) followed by 500 IU of hCG (sc) 72 h later. Ovulation was predicted to occur 42 h after the second injection. All animals were slaughtered 96 h after AI. Embryos were collected and classified as viable (5- to 8-cells, morulae, compacted morulae and early blastocysts) and nonviable (fragmented, degenerated and 1- to 4-cell embryos). The total embryo viability rate was: 64.3% (A-frozen), 54.2% (A-fresh), 76.0% (B-frozen), 91.9% (B-fresh); (A-fresh vs B-fresh, P = 0.018; A-frozen vs B-frozen, P = 0.094). It was observed that AI before ovulation resulted in a higher percentage of total viable embryos than AI after ovulation (P = 0.041). The early pregnancy rate, defined as presence of at least one viable embryo, was 78.9, 80.9, 84.2 and 94.4% for A-frozen, A-fresh, B-frozen, B-fresh, respectively. There was no significant difference in the early pregnancy rate among groups. In conclusion, there was a detrimental effect upon total embryo viability rate when AI was performed after ovulation with either frozen-thawed or fresh semen. The total embryo viability rate and the early pregancy rate were not affected by AI with either frozen-thawed or fresh semen regardless of the time of AI.

Semen of four dairy bulls was inactivated by centrifugation twice through a 7% Ficoll solution. After the second centrifugation, the sperm fractions were resuspended in 14G buffer and stored for 0, 3, 6 and 9 days at room temperature. At the end of each storage period, inactivated semen was reactivated by centrifugation and the sperm plugs were resuspended to a final concentration of 400 × 106 sperm ml−1 in tris-based diluent supplemented with 5 mM caffeine or theophylline. Sperm motility and acrosome status were assessed during subsequent incubation. Sperm penetration into cervical mucus was also assessed. The fertilizing ability of the sperm was tested by using the zona-free hamster oocyte technique. The results showed that bovine semen inactivated by centrifugation through a 7% Ficoll solution could be stored beyond 9 days at room temperature. Satisfactory proportions of motile sperm and spermatozoa with intact acrosomes were recovered after re-activation. The inclusion of either caffeine or theophylline at a 5 mM level had a beneficial effect on the percentage of motile sperm (P

Two nonsurgical techniques for embryo recovery were employed and used in Days 6 and 7 of the estrous cycle. A significant difference between techniques was observed in both days of collection, with the closed system being more efficient in Day 6 and the opened one more efficient in Day 7. The bacteriological contamination of flushing medium (11% for the closed system and 21% for the opened technique) was of saprophytic bacteria and did not affect the percentage of pregnancies in either system.