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SummaryThe Hippo pathway is involved in the proliferation of intrafollicular cells and in early embryonic development, mainly because effectors of this pathway are key transcription regulators of genes such as CTGF and CYR61, which are involved in cell proliferation. Recent studies by our group found that fibroblast growth factor 18 (FGF18) is present in the fallopian tube during early embryonic development, leading to the hypothesis that FGF18 may have a role during embryonic development. Therefore, the aim of the following study was to determine whether FGF18 modulates the expression of Hippo pathway target genes, CTGF and CYR61, during oocyte maturation and early embryonic development. Three experiments were carried out, with in vitro maturation (IVM) of cumulus–oocyte complexes (COCs) and embryo culture. In experiment one, FGF18 (100 ng/ml) induced an increase (P < 0.05) in CTGF gene expression at 12 h post-exposure. In experiment two, FGF18 (100 ng/ml) induced a reduction (P < 0.05) in CTGF expression at 3 h post-exposure. In the third experiment, day 7 embryos exposed to FGF18 during oocyte IVM expressed greater CTGF mRNA abundance, whereas FGF18 exposure during embryo in vitro embryo culture did not alter CTGF expression in comparison with untreated controls. The preliminary data presented here show that FGF18 modulates CTGF expression in critical periods of oocyte nuclear maturation, cumulus expansion and early embryonic development in cattle.

Neospora caninum is the main etiologic agent of neosporosis in domestic animals and its pathogenesis comprises two characteristic phases: acute and chronic. Rodents are used as experimental models to mimic acute and chronic bovine neosporosis. In this study, we inoculated a total of 27 female gerbils, with different doses of N. caninum tachyzoites aiming to induce chronic disease. DNA was extracted from different organs of each animal after spontaneous death or euthanasia. Encephalic tissues were submitted to a highly sensitive real time PCR aiming to detect chronically infected animals. All the other samples were submitted to standard PCR. A total of 11 gerbils died due to acute neosporosis, as confirmed by N. caninum DNA detection in organs. 5x103 tachyzoites/mL of N. caninum was the dosage of antigen that can induce chronic infection in gerbils. In the encephalon sections of some animals that showed clinical signs of persistent infection, we found 70% positive for the anterior encephalon section, suggesting this area as preferential for cyst formation. Therefore, we determined the doses of tachyzoites that cause acute or chronic infection and detection of positive tissues, preferably, systemic organs during acute and encephalon in chronic phases.

SummaryThis study aimed to assess the effects of the inhibition of nitric oxide synthase (NOS) on events that modulate bovine in vitro oocyte maturation. Cumulus–oocyte complexes (COCs) were cultured with hemisections (HSs) of the follicular walls in a maturation medium supplemented with different concentrations (0.1–10.0 mM) of Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME). Controls consisted of COCs cultured in the presence (+HSs) or absence of HSs (–HSs) with no additional l-NAME supplementation. The following parameters were assessed: oocyte nuclear maturation stage; cumulus cell (CC) membrane integrity; nitrate/nitrite, progesterone, and estradiol concentrations in the culture medium at 22 h of cultivation; and the concentrations of cGMP and cAMP in COCs during the first hour of maturation. The addition of 1.0 mM l-NAME increased the percentage of oocytes that reached metaphase II (MII) and the percentage of intact CCs (P < 0.05). All l-NAME concentrations reduced the nitrate/nitrite concentrations (P < 0.05), but none affected steroid concentrations compared with control +HSs (P > 0.05). The addition of 1.0 mM l-NAME reduced cGMP concentrations at 3 h and increased cAMP concentrations in the first hour of culture (P < 0.05). Our findings suggest that the NOS/NO/cGMP pathway participates in meiosis progression (MI to MII) of the bovine oocytes matured in vitro in the presence of hemisections of the follicular walls. Lastly, the mechanisms that lead to the progression of meiosis after NOS inhibition do not involve changes in steroid production.

Dermoid and epidermoid cysts are rare in the head and neck, specially in the oral cavity, accounting for 1.6 %. It represents an even smaller percentage of all the oral cysts, less than 0.01%. Usually asymptomatic, however, occasionally, obstructive symptoms like dysphagia or speech difficulties are found. We present a case of a 32 years old female patient with a sublingual epidermoid cyst. The patient was referred to our ENT department with complaints of an oral mass, speech difficulties and solid food dysphagia for 1 month. Physical examination revealed a well circumscribed symmetrical mass on the floor of the mouth with no inflammatory signs, tender and fluctuant on palpation. Computed tomography (CT) was requested. CT scan showed a sublingual cystic mass inferiorly limited by the mylohyoid muscle. Aspiration was performed to improve symptoms until surgery. The liquid that resulted from aspiration showed a granulated white cheesy material. Enucleation of the cyst was performed intraorally in the operating room. Histopathologic investigation confirmed the diagnosis of epidermoid cyst. The patient fully recovered and there was no recurrence. Although rare, cases like this exist. There are several differential diagnoses and a high level of suspicion is necessary for a correct workup, diagnosis and follow-up. Surgical excision is usually necessary and curative.

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.

There is growing evidence that histone lysine demethylases (KDMs) play critical roles in the regulation of embryo development. This study investigated if KDM7A, a lysine demethylase known to act on mono-(me1) and di-(me2) methylation of H3K9 and H3K27, participates in the regulation of early embryo development. Knockdown of KDM7A mRNA reduced blastocyst formation by 69.2% in in vitro fertilized (IVF), 48.4% in parthenogenetically activated (PA), and 48.1% in somatic cell nuclear transfer (SCNT) embryos compared to controls. Global immunofluorescence (IF) signal in KDM7A knockdown compared to control embryos was increased for H3K27me1 on D7, for H3K27me2 on D3 and D5, for H3K9me1 on D5 and D7, and for H3K9me2 on D5 embryos, but decreased for H3K9me1, me2 and me3 on D3. Moreover, KDM7A knockdown altered mRNA expression, including the downregulation of KDM3C on D3, NANOG on D5 and D7, and OCT4 on D7 embryos, and the upregulation of CDX2, KDM4B and KDM6B on D5 embryos. On D3 and D5 embryos, total cell number and mRNA expression of embryo genome activation (EGA) markers (EIF1AX and PPP1R15B) were not affected by KDM7A knockdown. However, the ratio of inner cell mass (ICM)/total number of cells in D7 blastocysts was reduced by 45.5% in KDM7A knockdown compared to control embryos. These findings support a critical role for KDM7A in the regulation of early development and cell lineage specification in porcine embryos, which is likely mediated through the modulation of H3K9me1/me2 and H3K27me1/me2 levels, and changes in the expression of other KDMs and pluripotency genes.

Background Disruption of the balance between the production of ROS and their removal through enzymatic and non-enzymatic (antioxidant) processes has been proposed as a new mechanism in the pathology of polycystic ovary syndrome (PCOS). Evidence from animal models of PCOS (prenatally androgenized sheep) has suggested that treatment with insulin sensitizers, but not antiandrogens, can reduce increases in ROS. Materials and methods In the present study, we investigated the effects of neonatal treatment with a gonadotropin-releasing hormone (GnRH) agonist (leuprolide acetate) on prenatally androgenized sheep with testosterone propionate to determine its impact on oxidative stress molecules (ferric reducing antioxidant power [FRAP], advanced oxidation protein product [AOPP], nitric oxide [NOx], albumin) at 8, 12, and 18 months of age. Results Androgenized ewes (but not leuprolide-treated ewes) showed reduced total cholesterol levels associated with a decrease in the ratio of visceral to subcutaneous adiposity (adjusted to abdominal area) as determined by computed tomography. In androgenized ewes at 12 months of age, an increase in subcutaneous fat and relative decrease in the visceral fat compartment did not affect the expression of REDOX markers. At 18 months of age, however, the levels of NOx metabolites decreased in androgenized animals, but remained close to normal in ewes subjected to neonatal treatment with leuprolide acetate. Other oxidative stress parameters (FRAP, AOPP, albumin) did not vary among groups. Conclusion Our results demonstrate that the GnRH agonist leuprolide (as a single dose after birth) had weak effects on markers of the oxidative stress balance.

Failure to ovulate is a major cause of infertility. The critical pathway that induces ovulation involves the EGF and MAPK phosphorylation, but studies in rodents have suggested that the Hippo activator, YAP, is also involved. It is unknown whether YAP-dependent transcriptional activity is important for the LH- or EGF-induced ovulatory cascade in monovulatory species such as the cow. Using a well-defined preovulatory GC culture system, we employed pharmacological inhibitors to demonstrate that YAP signaling is critical for expression of EGFR and downstream target genes EREG, EGR1 and TNFAIP6. Most importantly, by using an ultrasound guided follicle injection system, we also showed that the classic Hippo signaling inhibitor Verteporfin inhibits GnRH-induced ovulation in vivo in cattle. In conclusion, YAP transcriptional activity is critical for EGF-like cascade induced by LH to promote ovulation in a monovulatory species.

There is growing evidence that greater than homeostatic blood concentrations of nonesterified fatty acids (NEFAs) and β-hydroxybutyrate (BHBA) have negative consequences on dairy cow’s fertility, but effects on cell homeostasis in the reproductive system is not completely understood. In this study, lipids accumulation, reactive oxygen species (ROS) concentrations, abundance of gene transcripts, and immunofluorescence signal of H3K4me3 and H3K9me3 were evaluated in endometrial epithelial cells of cattle cultured with NEFAs (Oleic (OA), Stearic (SA) and Palmitic (PA) acids), BHBA, NEFAs + BHBA or each of the three NEFAs alone. The cellular lipids were in greater concentrations as a result of NEFAs + BHBA, NEFAs, SA or OA supplementation, but not by BHBA or PA. The ROS concentrations were greater when there were treatments with NEFAs + BHBA, NEFAs or BHBA. The relative mRNA abundance for genes involved in the regulation of apoptosis (XIAP), glucose transport (GLUT3), and DNA methylation (DNMT1) were greater when there were NEFAs + BHBA, but not NEFAs, BHBA, OA, SA or PA treatments. The immunofluorescence signal for H3K9me3 was greater when there were NEFAs + BHBA, NEFAs or PA, but not by BHBA, OA or SA treatments. These findings indicate that NEFAs and BHBA have an additive effect on endometrial cells of cattle by altering epigenetic markers and the expression of genes controlling important cellular pathways. Furthermore, there was cellular lipid accumulation and increased H3K9me3 in cultured bovine endometrial cells that was mainly induced by OA and PA treatments, respectively.

Background The incidence of invasive meningococcal disease due to serogroup C (MenC) decreased in Portugal since the introduction of the conjugate vaccine (MCC) in the free market in 2001 and in the National Immunisation Plan in 2006. Considering the potential waning of the antibody response reported in the literature, the different vaccination schemes that were used in our country over the past decade, and that Neisseria meningitidis serogroup C continues to circulate, the Portuguese population may currently be at increased risk of infection. In the absence of national data, we evaluated the seroprotection level of the Portuguese population against MenC, in order to identify the protected fraction of the population and ponder on the necessity of a booster dose of the MCC vaccine. Methods We measured serum bactericidal antibody levels against MenC in a representative sample of the population (n = 1500) aged 2–64 years who participated in the 2015/2016 National Serological Survey. Results A total of 31.1% (466/1500, 95%CI: 29–33%) of the individuals studied were protected against MenC. The geometric mean titre was 6.5. The proportion of seroprotected was particularly low in children aged 2–4 years ( Conclusion The small fraction of population seroprotected, combined with the already known waning effect of the antibody response over time, may indicate that the Portuguese population will become progressively more exposed to the risk of infection. Taking in consideration our results, we recommend to change the current vaccination strategy and introduce a booster dose of the MCC vaccine during adolescence.

Although it is well documented that leptin signals the body nutritional status to the brain, mechanisms of leptin regulation at the ovary are not well understood. This study was conducted to determine whether there was leptin and the receptor for leptin (LEPR) in cattle ovarian follicles and to investigate potential actions of leptin on follicular growth in vivo and on regulation of granulosa cell functions in vitro. There was leptin and LEPR in granulosa and theca cells of dominant and subordinate follicles, with greater immunostaining for leptin in granulosa cells of subordinate follicles. There was a lesser relative abundance of leptin receptor gene-related protein (LEPROT) and of the adiponectin receptors 1 (ADIPOR1) and 2 (ADIPOR2) mRNA transcripts in granulosa cells of subordinate than dominant follicles (P0.05). Intrafollicular injection of either 100 or 1000 ng/mL leptin did not affect the diameter and the growth of dominant follicles (P0.05). Supplementation of in vitro culture medium with different leptin concentations did not affect (P0.05) the relative abundance of hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), signal transducer and activator of transcription 3 (STAT3) and X-linked inhibitor of apoptosis protein (XIAP) mRNA transcripts in granulosa cells. These findings indicate that leptin and LEPR are present in the follicular cells of cattle ovaries, but leptin apparently does not have essential functions in steroidogenesis and growth of dominant follicles.

Dairy cows frequently undergo a state of negative energy balance (NEB) after parturition and some have impaired ovarian functions that result in delayed resumption of estrous cyclicity and development of follicles without ovulation occurring. During the postpartum period, cows undergo body-fat store losses, hormonal changes, fat mobilization and increases in nonesterified fatty acid (NEFAs) concentrations in blood and follicular fluid. The effect of NEFAs on follicular development and function of follicular cells, however, is not fully understood. The aim of this study, therefore, was to study the effect of an intrafollicular injection of a mixture of oleic, stearic and palmitic NEFAs on dominant follicle development and function of granulosa cells in cows that were not in a NEB state. Follicular size was less at 24 and 48 h after administration of NEFAs compared to that of control follicles injected with vehicle only. At 24 h after intrafollicular injection, the relative mRNA transcript abundance for proteins involved in steroidogenesis (CYP19A1, 3BHSD, STAR, FSHR), metabolism (GLUT1, GLUT3, INSR, IRS1, IRS2, SLC27A1, PPARG), and cell proliferation and apoptosis (CCND2; XIAP) in granulosa cells, as well as estradiol concentrations in follicular fluid were similar in control and NEFA-treated follicles. In conclusion, the results of this study indicate increased intrafollicular concentrations of NEFAs in cows that are not in a NEB state has a detrimental effect on follicle development. We propose intrafollicular injection is a useful approach to further investigate the local effects of NEFAs on the function of follicular cells.

The main clinical feature associated with hyperandrogenism in polycystic ovary syndrome (PCOS) in humans is hirsutism, where hair increases its length, pigmentation, and particularly its diameter. Currently, it is not known whether PCOS animal models also exhibit changes in the hair. Therefore, the aim of this study was to explore the wool characteristics in sheep prenatally androgenized (PA) with testosterone propionate. After 4 and 13 months of life, wool was collected from the top of the shoulder of both females and males (both androgenized and controls). The offspring sheep were followed for up to 19 months of life to evaluate testosterone and androstenedione serum levels by ultra-high-performance liquid chromatography-tandem mass spectrometry, determine insulin and glucose response to intravenous glucose tolerance test, and address estrus cyclicity during the second breeding season. PA male animals showed a reduction in wool fiber diameter at 4 months of age compared with controls (P = 0.02) but not at 13 months, whereas PA females showed increased hair diameter at 13 months (P = 0.002), with no difference at 4 months. No substantial changes in other hair parameters (length, color, and medullation) were identified. In addition, increased levels of serum testosterone were observed in PA female sheep compared with controls at 12 months (P = 0.03). Our results indicate for the first time, to our knowledge, that changes in wool fiber diameter observed in PA ewes replicate, at the translational level, the increase in hair diameter in hirsute women with PCOS.

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.

In aquaculture, nutrition and supplemented diets have been shown to affect broodstock reproductive performance. In this study, we investigated the effects of dietary supplementation with Cymbopogon flexuosus essential oil (CFEO) microcapsules on reproductive-related parameters in silver catfish (Rhamdia quelen) male broodfish. Adult male broodstocks were separated into three groups according to the concentrations of supplemented CFEO (0.0 = control; 1.0 or 3.0 mL per kg of diet). After 20 days under experimental conditions, the animals were euthanized and the gonads were harvested for gonadosomatic index, sperm analysis, oxidative stress, and histopathology; testosterone levels were measured in the plasma; gene expression of prl, smtl, pomca, and pomcb was assessed in the pituitary gland by real-time PCR. The results showed no alterations on reproductive parameters in R. quelen males treated with Cymbopogon flexuosus essential oil compared to the control-diet animals. In conclusion, CFEO microcapsules supplied for 20 days in the concentrations of 1.00 or 3.00 mL per kilogram of diet did not affect the reproduction criteria evaluated in this study in male silver catfish.

Lady Serrano Mujica,1 Alessandra Bridi,1,2 Ricardo Della Méa,1 Vitor Braga Rissi,1 Naiara Guarda,3 Rafael Noal Moresco,3 Melissa Orlandin Premaor,4 Alfredo Quites Antoniazzi,1 Paulo Bayard Dias Gonçalves,1 Fabio Vasconcellos Comim1,4 1Laboratory of Biotechnology and Animal Reproduction, BioRep, Federal University of Santa Maria, Santa Maria, Rio Grande do Sul, 2Department of Veterinary Medicine, University of São Paulo, São Paulo, 3Laboratory of Clinical Biochemistry, Department of Clinical and Toxicological Analysis, 4Department of Clinical Medicine, Federal University of Santa Maria, Santa Maria, Rio Grande do Sul, Brazil Background: Several studies have described an enhanced inflammatory status and oxidative stress balance disruption in women with polycystic ovary syndrome (PCOS). However, there is scarce information about redox markers in the blood of androgenized animal models. Here, we evaluated the serum/plasma oxidative stress marker and metabolic parameter characteristics of prenatal (PreN) and postnatal (PostN) androgenized rat models of PCOS. Materials and methods: For PreN androgenization (n=8), 2.5mg of testosterone propionate was subcutaneously administered to dams at embryonic days 16, 17, and 18, whereas PostN androgenization (n=7) was accomplished by subcutaneously injecting 1.25mg of testosterone propionate to animals at PostN day 5. A unique control group (n=8) was constituted for comparison. Results: Our results indicate that PostN group rats exhibited particular modifications in the oxidative stress marker, an increased plasma ferric-reducing ability of plasma, and an increased antioxidant capacity reflected by higher albumin serum levels. PostN animals also presented increased total cholesterol and triglyceride–glucose levels, suggesting severe metabolic disarrangement. Conclusion: Study findings indicate that changes in oxidative stress could be promoted by testosterone propionate exposure after birth, which is likely associated with anovulation and/or lipid disarrangement. Keywords: animal models of PCOS, oxidative stress, prenatal, postnatal

Interferon tau (IFNT) is the pregnancy recognition signal in ruminants and is secreted by trophoblast cells. Paracrine action in the endometrium is well established by inhibiting luteolytic pulses of prostaglandin F2 alpha. Recently, endocrine action was documented in the corpus luteum, blood cell and liver. It was hypothesized that conditioned medium (CM) obtained from days 7, 9 and 12 parthenogenetic embryos alters luteal cell gene expression. The aim was to establish a bovine mixed luteal cell culture to evaluate cellular response associated to interferon stimulated genes, steroidogenesis and apoptosis. Conditioned medium was obtained from Days 7, 9 and 12 parthenogenetic (PA) embryos culture. Moreover, antiviral assay was performed on CM from Days 7, 9 and 12 to verify Type I interferon activity. Luteal cell culture was validated by steroidogenic and apoptotic genes (CYP11A1 , HSD3B1, BAX, BCL2, AKT and XIAP mRNA expression), and concentration of progesterone as endpoint. Luteal cell culture was treated with interferon alpha (IFNA) and CM from parthenogenetic embryos. Antiviral assay revealed Type I interferon activity on CM from embryos increasing on Days 9 and 12. ISG15 mRNA was greater in the mixed luteal cells culture treated with 1, 10 and 100ng/ml of interferon alpha (IFNA) and also on Days 7, 9 and 12 CM treatments. Concentration of progesterone was not altered in luteal cell culture regardless of treatments. Steroidogenic and apoptotic genes were similar among groups in luteal cell culture treated with different doses of IFNA or CM from PA embryos. In conclusion, parthenogenetic embryo-derived CM has antiviral activity, luteal cell culture respond to Type I interferon by expressing IGS15. These data indicate this model can be used for IFNT endocrine signaling studies.

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.

Prostaglandin F2α (PGF) induces the precipitous loss of steroidogenic capabilities and cellular death in the corpus luteum of many species, yet the molecular mechanisms underlying this event are not completely understood. Signal transducer and activator of transcription 3 (STAT3) was activated in granulosa cells during follicle atresia, whereas AKT is immediately down-regulated in the corpus luteum after PGF treatment in cattle; however, their involvement in both functional and morphological luteolysis in monovular species still need to be determined. Blood samples and corpus lutea were collected from cows before (0) and 2, 12, 24, and 48 hr after PGF treatment on Day 10 of the estrous cycle (4-5 cows per time point). Serum progesterone concentrations decreased by threefold (p < 0.05) within 2 hr, confirming functional luteolysis. The mRNA abundance of the pro-apoptotic gene BAX increased 12-48 hr post-PGF treatment (p < 0.05), while morphological luteolysis was observed 24 and 48 hr after PGF treatment, based on the loss of plasma membrane integrity, reduction of cytoplasmic volume, and pyknotic nuclei. Phosphorylated STAT3 increased, peaking at 12 hr, and remained elevated until 48 hr after PGF treatment. SOCS3 transcript abundance also increased (p < 0.05) starting at 2 hr post-PGF treatment. In contrast, AKT phosphorylation decreased by 12 hr after treatment. Thus, activation of STAT3 and inactivation of AKT signaling are involved in structural regression of the corpus luteum.

The aim of the present study was to evaluate the correlation of age and heat cycle to determine reproductive efficiency in young and aged Thoroughbred mares bred on foal heat (FH) or on second heat (SH) after foaling. Embryo mortality (EmbM) was determined every time a mare was found open after a positive pregnancy diagnosis. Parturition to breeding interval, pregnancy rate (PregR) and EmbM rate were the dependent variables and the treatments were breeding on the FH or on SH. The cutoff age to obtain above-average probability for the EmbM was 10 years old. PregR in mares bred on FH was lower compared with SH (P.01); however, it was neither affected by the age of mares (P.05) nor by the age group of mares (P.05). Regarding FH and SH, there was a difference in PregR in young mares (P.01), unlike in aged mares (P.05). EmbM rate was not different between mares bred on FH or SH (P.05) although it was affected by age of mares (P.01). EmbM was higher in oldest than young mares (P.01). Aged mares bred on FH had a significantly higher EmbM rate compared with the young group also bred on FH (P.01). In conclusion, the reproductive efficiency of Thoroughbred mares bred on FH is dependent of the age. Aged mares (≥10 years old) should be bred at their SH to reduce EmbM and improve reproductive performance.

Obesity reaches an epidemic level worldwide, and this condition is associated with chronic low-grade inflammation and secondary comorbidities, largely driven by global changes in lifestyle and diet. Various dietary approaches are proposed for the obesity treatment and its associated metabolic disorders. Good taste, antioxidant functions, and vitamins have been attributed to virgin coconut oil (VCO). However, VCO contains a large amount of saturated fatty acids, and the consumption of this fat is associated with a number of secondary diseases. We evaluate the effects of VCO supplementation on biochemical, inflammatory, and oxidative stress parameters in rats fed with high-fat diet (HFD). After feeding with HFD for 12 weeks, the animals were supplemented with VCO for 30 days. HFD+VCO group increased in diet intake, weight gain, low-density lipoprotein cholesterol level, and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. These findings were accompanied by increased in hepatic lipid profile and fat deposition in the liver. Adipocyte hypertrophy was observed in the HFD+VCO group, which was associated with elevated expression of tumor necrosis factor alpha (TNF-α) in adipose tissue. These results revealed that VCO associated with HFD induced important metabolic alterations, adipose inflammation, and hepatic lipid accumulation in rats.

Interferon tau (IFNT) is the cytokine responsible for the maternal recognition of pregnancy in ruminants and plays a role modulating embryo-maternal communication in the oviduct inducing a local response from immune cells. We aimed to investigate IFNT production, reactive oxygen species, and oxidative stress under the influence of heat stress (HS) during different stages of bovine in vitro embryo production. HS was established when the temperature was gradually raised from 38.5°C to 40.5°C in laboratory incubator, sustained for 6 hr, and decreased back to 38.5°C. To address the HS effects on IFNT production, reactive oxygen species, and oxidative stress, ovaries from a slaughterhouse were used according to treatments: control group (38.5°C); oocytes matured under HS; oocytes fertilized under HS; zygotes cultured in the first day under HS; and cells submitted to HS at oocyte maturation, fertilization, and the first day of zygote culture. The HS negatively affected cleavage and blastocyst rates, in all HS groups. On Day 7, all HS-treated embryos showed decrease IFNT gene and protein expressions, whereas reactive oxygen species were increased in comparison to the control. In conclusion, the compromised early embryo development due to higher temperatures during in vitro oocyte maturation, fertilization, and/or zygote stage have diminished IFNT expression and increased reactive oxygen species in bovine.

In this review, we discuss the utility of the cow as an in vivo model to study the regulation of ovarian functions in monovular species. It is increasingly becoming clear that besides endocrine control, locally produced factors play pivotal roles during dominant follicle selection, oocyte maturation, ovulation and luteolysis. Although in vitro culture systems have been used to study these processes, definitive understanding the interactions between endocrine and local factors requires appropriate in vivo models. Most of the experimental approaches to study ovarian functions in vivo in large animals are based on the use of ultrasonography and considerable progress in this field has been made during the last thirty years. It has been shown that cows are an excellent model to collect samples (e.g., follicular fluid, granulosa cells, oocytes) from live animals at specific stages of follicular development in order to study mechanisms of intrafollicular factors in a physiological endocrine environment. In addition to support fundamental studies, the cow model has contributed immensely to the refinement of assisted reproductive technologies, which are now widely used not only in farm animals but also in humans.

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 investigated the in vitro effects of guaraná and its main metabolites (caffeine, theobromine and catechin) on cytotoxicity and cell proliferation on colorectal cancer (CRC) line HT-29 cells and on oxaliplatin sensitivity. The cells were exposed to different concentrations of guaraná extract with and without oxaliplatin. The concentrations of bioactive molecules were also estimated considering their potential proportion on guaraná hydro-alcoholic extract. Apoptosis effect was analyzed by annexin V quantification using flow cytometry, while apoptosis pathway gene modulation (p53, Bax/Bcl-2 genes ratio, caspases 8 and 3) was determined by qRT-PCR analysis. Cells exposed to guaraná at a concentration of 100 μg/mL presented a similar cytotoxic effect as HT-29 cells treated with oxaliplatin and did not affect the sensitivity of the drug. Guaraná presented cell anti-proliferative effect and increased anti-proliferative oxaliplatin sensitivity at all concentrations tested here. Guaraná was able to induce apoptosis and up-regulate the p53 and Bax/Bcl-2 genes.

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.

Cytokines are proteins secreted by cells of innate and acquired immunity, produced in response to various antigens and responsible for mediating several function of these cells. Our study evaluated the profile of cytokines interleukin 5 (IL-5) and interferon gamma (IFN-γ), induced in lymphocytes of broiler chickens in response to secondary fungal metabolites ochratoxin A (OTA) and deoxynivalenol (DON) at concentrations of 0.001, 0.01, 0.1 and 1 μg/ml. The quantification of the cytokines was analysed at 24, 48 and 72 h after incubation with mycotoxins, using real-time PCR (qRT-PCR). The results obtained showed that OTA induced mRNA synthesis of IL-5 at concentrations 0.001, 0.1 and 1 μg/ml after 24 h of lymphocyte incubation, while at 48 h only the expression of the IL-5 cytokine at a concentration of 1 μg/ml (P

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.

The aim of this study was to evaluate if the positive effects of inhibiting histone deacetylase enzymes on cell reprogramming and development of somatic cell nuclear transfer (SCNT) embryos is affected by the cell cycle stage of nuclear donor cells and host oocytes at the time of embryo reconstruction. SCNT embryos were produced with metaphase II (MII) or telophase II (TII) cytoplasts and nuclear donor cells that were either at the G1-0 or G2/M stages. Embryos reconstructed with the different cell cycle combinations were treated or not with the histone deacetylase inhibitor (HDACi) Scriptaid for 15 h and then cultured in vitro for 7 days. Embryos reconstructed with MII-G1-0 and TII-G2/M developed to the blastocyst stage with a higher frequency compared to the other groups, confirming the importance of cell cycle interactions on cell reprogramming and SCNT embryo development. Treatment with HDACi improved development of SCNT embryos produced with MII but not TII cytoplasts, independently of the cell cycle stage of nuclear donor cells. These findings provide evidence that the positive effect of HDACi treatment on development of SCNT embryos depends upon cell cycle interactions between the host cytoplast and the nuclear donor cells.

The transforming growth factors beta (TGFβ) are local factors produced by ovarian cells which, after binding to their receptors, regulate follicular deviation and ovulation. However, their regulation and function during corpus luteum (CL) regression has been poorly investigated. The present study evaluated the mRNA regulation of some TGFβ family ligands and their receptors in the bovine CL during induced luteolysis in vivo. On day 10 of the estrous cycle, cows received an injection of prostaglandin F2α (PGF) and luteal samples were obtained from separate groups of cows (n= 4-5 cows per time-point) at 0, 2, 12, 24 or 48 h after treatment. Since TGF beta family comprises more than 30 ligands, we focused in some candidates genes such as activin receptors (ACVR-1A, -1B, -2A, -2B) AMH, AMHR2, BMPs (BMP-1, -2, -3, -4, -6 and -7), BMP receptors (BMPR-1A, -1B and -2), inhibin subunits (INH-A, -BA, -BB) and betaglycan (TGFBR3). The mRNA levels of BMP4, BMP6 and INHBA were higher at 2 h after PGF administration (P

Purpose We hypothesized that vitamin D decreases rates of adenosine formation in human cutaneous melanoma cells through the inhibition of extracellular adenosine 5'-triphosphate breakdown, thereby affecting tumor cell viability. Therefore, the objective of this study was to explore the mechanisms of action of 1α, 25-dihydroxyvitamin D3 (1,25(OH)2 D3) on the activity and expression of ectonucleotidases in cutaneous melanoma cells. Methods A human melanoma cell line, SK-Mel-28, was treated with 1 to 50 nM of the active vitamin D metabolite (1,25(OH)2 D3) over 24 hours, followed by determination of NTPDase1/CD39 and ecto-5'-nucleotidase/CD73 activity and expression rates of the purinergic system-related NTPDASE1, NT5E and adenosine deaminase and vitamin D receptor. An 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay was used to evaluate cellular viability. Results 1,25(OH)2 D3 decreased adenosine monophosphate hydrolysis via ecto-5'-nucleotidase/CD73 and expression of CD73, but did not change NTPDase1/CD39 activity; it increased the CD39 expression. We also observed an increase of cell viability at 1 nM, but this viability decreased as the concentrations of vitamin D active metabolite increased to 50 nM. There were no differences in gene expression levels. Conclusion To the best of our knowledge, we showed for the first time a mechanism of control of adenosine production via modulation of the purinergic system in cutaneous melanoma cells treated with the active metabolite of vitamin D. This study provides original information regarding mechanisms, in which vitamin D plays a key role in preventing tumor progression in human melanoma cells.

Ovulation is triggered by gonadotropin surge-induced signaling cascades. To study the role of extracellular signal-regulated kinase 1/2 (ERK1/2) in bovine ovulation, we administered the pharmacological inhibitor, PD0325901, into the preovulatory dominant follicle by intrafollicular injection. Four of five cows treated with 50 µM PD0325901 failed to ovulate. To uncover the molecular basis of anovulation in ERK1/2-inhibited cows, we collected granulosa and theca cells from Vehicle and PD0325901 treated follicles. Next-generation sequencing of granulosa cell RNA revealed 285 differentially expressed genes between Vehicle and PD0325901-treated granulosa cells at 6 h post-GnRH. Multiple inflammation-related pathways were enriched among the differentially expressed genes. The ERK1/2 dependent LH-induced genes in granulosa cells included EGR1, ADAMTS1, STAT3 and TNFAIP6. Surprisingly, PD0325901 treatment did not affect STAR expression in granulosa cells at 6 h post-GnRH. Granulosa cells had higher STAR protein and theca cells had higher levels of STAR mRNA in ERK1/2-inhibited follicles. Further, both granulosa and theca cells of ERK1/2-inhibited follicles had higher expression of SLC16A1, a monocarboxylate transporter, transporting substances including β-hydroxybutyrate across the plasma membrane. Taken together, ERK1/2 plays a significant role in mediating LH surge-induced gene expression in granulosa and theca cells of the ovulating follicle in cattle.

Insufficient epigenetic reprogramming is incompatible with normal development of embryos produced by somatic cell nuclear transfer (SCNT), but treatment with histone deacetylases inhibitors (HDACi) enhances development of SCNT embryos. However, the mechanisms underpinning HDACi benefits in SCNT embryos remain largely uncharacterized. We hypothesized that, in addition to enhancing reprogramming, HDACi treatment may promote expression of genes not required for early development of SCNT embryos. To test this hypothesis, RNA synthesis was inhibited by treating bovine SCNT embryos with 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DBR), which were concomitantly treated or not with Scriptaid (Scrip; an HDACi). Development to the blastocyst stage was significantly increased by treatment with Scrip alone (26.6%) or associated with DRB (28.6%) compared to Control (17.9%). The total number of nuclei was significantly improved only in embryos that were treated with both Scrip + DRB. Nuclear decondensation after SCNT was significantly increased by DRB treatment either alone or associated with Scrip. The relative mRNA expression, evaluated during the embryo genome activation (EGA) transition, revealed that some KDMs (KDM1A, KDM3A, KDM4C and KDM6A) and DNMT1 where prematurely expressed in Scrip-treated embryos. However, treatment with Scrip + DRB inhibited early mRNA expression of those genes, as well as several other KDMs (KDM4A, KDM4B, KDM5A, KDM5B, KDM5C and KDM7A) compared to embryos treated with Scrip alone. These findings revealed that HDACi improved development in SCNT embryos compared to Control, but altered the expression of genes involved in epigenetic regulation and did not improve embryo quality. Inhibition of RNA synthesis during HDACi treatment enhanced nuclear chromatin decondensation, modulated gene expression and improved SCNT embryo quality.

Mutations in growth and differentiation factor 9 (GDF9) gene are associated to sterility or, paradoxically, increased ovulation rate in ewes. Despite its importance, the exact function of GDF9 in ovarian physiology is still poorly understood. This study aimed to investigate GDF9 function during dominant follicle growth and its regulation in follicular fluid. The regulation of GDF9 receptors in GnRH/LH-stimulated granulosa cells was also investigated. In a first experiment, a new follicular wave was induced and the intrafollicular GDF9 treatment into the largest growing follicle (8.5-9.5 mm) at both 100 (n = 3) and 1000ng/ml (n = 4) had no effect on follicular growth, estrus manifestation and ovulation compared to control (PBSinjected) follicles (n = 3). In a second experiment, follicles were obtained just after follicular deviation (day 4 after follicular emergence) and the abundance of GDF9 in follicular fluid did not differ between healthy dominant (n = 4) and atretic subordinate follicles (n = 4), as assessed by western blot analysis. Finally, mRNA expression of BMPR2 and TGFBR1 receptors was evaluated in granulosa cells obtained from preovulatory follicles (>12 mm diameter) obtained 0, 3, 6, 12 or 24 h after i.m. GnRH administration (n = 4-5 follicles/moment). Both receptors were significantly up regulated 12 h after GnRH treatment. Present results do not confirm the hypothesis that GDF9 inhibits dominant follicle growth and suggests a minor role in determining follicle fate. In the other hand, GDF9 receptors regulation in GnRH/LH-stimulated granulosa cells provides the first in vivo evidence of its involvement in the complex cascade of events that culminates in ovulation and luteinization in cattle.

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.

Conjugated linoleic acid (CLA) might be able to improve the cryotolerance of in vitro-produced (IVP) embryos. The effect of two CLA isomers on the cryotolerance of bovine IVP embryos, as well as that of the stage of embryonic development and the method used for cryopreservation was evaluated by three experiments. In Experiment 1, oocytes (n = 3,917) were fertilized in vitro and cultured with 0, 50, 100, or 200 ?M trans-10, cis-12 (t10, c12 CLA). In Experiment 2, fertilized oocytes (n = 2,131) were cultured with 100 ?M t10, c12 or cis-9, trans-11 (c9, t11 CLA), or a combination of both isomers. The embryos were vitrified at the blastocyst (BL) or the expanded blastocyst (EB) stage. In Experiment 3, oocytes (n = 1,720) were fertilized and cultured with or without 100 ?M t10, c12 CLA, and the blastocysts were vitrified or frozen. Blastocyst development rate as well as the rates of re-expansion and hatching after thawing was recorded. Moreover, the mean cell number and mRNA expression of acetyl-CoA carboxylase (ACC1) and stearoyl-CoA desaturase (SCD1) as well as fatty acid synthase (FASN) multienzyme complex were determined. In Experiment 1, the highest concentration of t10, c12 CLA that did not reduce blastocyst development rate was 100 ?M. In Experiment 2, the rates of re-expansion and hatching among the EBs obtained through IVP after supplementation with t10, c12 CLA (73.1% and 57.7%), with c9, t11 CLA (80.0% and 68.6%), with the combination (78.3% and 52.2%), and with the control group (85.4% and 58.3%) were similar. At the BL stage, the rates of re-expansion and hatching were lower than those at the EB stage, and CLA combination allowed a hatching rate (8.0%) lower than that observed in the control group (40.0%). In Experiment 3, the hatching rates for vitrified EBs (vitrified control; 67.4%) and vitrified CLA EBs (65.8%) were higher than those obtained for frozen EBs, exposed (13.3%) or not exposed (28.6%) to CLA. In addition, in Experiment 3, the hatching rate was higher at the EB stage in vitrified groups, while the rates of BL and EB were similar in frozen groups, thus proving that vitrification was more efficient than freezing for IVP bovine embryos. In Experiment 3, CLA isomer t10, C12 did not influence the embryonic cell number or mRNA expression of ACC1 and SCD1 enzymes, but decreased the mRNA expression of FASN. In conclusion, 100 ?M CLA did not affect subsequent embryonic development. However, neither CLA isomer improved the cryotolerance of IVP bovine embryos.

The aim of this study was to evaluate the effect of the essential oil of Lippia alba (EOLA) as a feed additive on ionoregulatory and metabolic parameters and pituitary hormones expression in silver catfish, Rhamdia quelen , submitted to a stressful condition (stocking density of 10.6 kg m-3 and limited space). Fish were fed with different concentrations of EOLA (0.0 - control, 0.25 and 0.50 mL kg food-1) for 20 days. Metabolic parameters were not affected by the diet, with the exception of alanine aminotransferase, which was higher in the liver of fish fed 0.50 mL EOLA kg food-1. Plasma ions and activity of H+-ATPase did not change, but fish fed 0.25 mL EOLA kg food-1 presented higher Na+/K+-ATPase activity. Somatolactin expression in the pituitary was higher in the fish fed 0.25 mL EOLA kg food -1, but the expression of growth hormone and prolactin did not change. Therefore, dietary EOLA does not exert a protective effect in R. quelen submitted to a stressful situation because it did not alter most measured parameters. The use of 0.25 mL EOLA kg food-1 seems to be more suitable than 0.50 mL EOLA kg food-1 since the latter may be related to liver damage. RESUMO O objetivo deste estudo foi avaliar o efeito do óleo essencial de Lippiaalba (OELA) como aditivo em rações na ionoregulação, parâmetros metabólicos e expressão de hormônios hipofisários em jundiás, Rhamdiaquelen, submetidos a uma situação estressante (densidade de estocagem de 10,6 kg m-3 e espaço limitado). Os peixes foram alimentados com diferentes concentrações de OELA (0,0 - controle, 0,25 e 0,50 mL kg de ração-1) durante 20 dias. Parâmetros metabólicos não foram afetados pela dieta, com a exceção da alanina aminotransferase, que foi mais elevada no fígado dos peixes alimentados com 0,50 mL de OELA kg de ração-1. Íons plasmáticos e a atividade da H+-ATPase não apresentaram nenhuma alteração, mas os peixes alimentados com 0,25 mL OELA kg de ração-1 apresentaram maior atividade da Na+/K+-ATPase. A expressão da somatolactina na hipófise de peixes alimentados com 0,25 mL OELA kg de ração-1 aumentou, porém a expressão do hormônio de crescimento e da prolactina não mudou. Portanto, a adição do OELA na ração não tem um efeito protetor em jundiás submetidos a uma situação estressante, pois não influiu na maioria dos parâmetros medidos. O uso de 0,25 mL OELA kg de ração-1 parece ser mais adequado que 0,50 mL OELA kg de ração-1, uma vez que este nível de inclusão pode estar relacionado a danos hepáticos.

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.

Cell therapy has shown encouraging perspectives for human and veterinary medicine. Experimentally, genetic manipulation allows to mark and locate allogeneic cells. However, this makes their genotype/phenotype different from non-marked cells used clinically. Alternatively, the presence of the Y-chromosome enables male donor cells detection in female organisms. However, the concentration of engrafted cells may be minimal in tissues, due to systemic distribution. In this study, a nested-PCR multiplex test was developed, aiming to increase the sensitivity of the presence/absence diagnosis of male mice adipose-derived (ADSC-Y) and bone marrow mononuclear (BMNC-Y) cells in samples of blood and lungs from females, after endovenous transplantation. Four females received placebos; four females received ADSC-Y from two males; and four females received BMNC-Y from two males. The PCR first-step included two primer sets (multiplex): one for amplification of a Y-chromosome fragment (SRYout; 300bp); the other for amplification of an X-chromosome (DXNds3 gene) fragment. In the PCR second-step, one primer set (SRYinn) was used for amplification of a 110bp fragment, restrained in the SRYout amplification product. The PCR internal control (DXNds3 gene) was detected in all DNA samples, whereas the SRY gene external fragment (300bp) was detected exclusively in ADSC-Y and BMNC-Y pure DNA samples. The SRY gene internal fragment (110bp) was detected in 100% of the blood and lung samples from the ADSC-Y and BMNC-Y female recipients. The nested-PCR technique increased sensitivity and reliability for molecular diagnostic of presence or absence of male mice cells in body fluids and tissues of female recipients after endovenous transplantation. A terapia celular traz perspectivas encorajadoras à medicina humana e veterinária. Experimentalmente, a manipulação genética permite a marcação e a localização de células alogênicas. Porém, isso torna seu genótipo/fenótipo diferente daquelas usadas clinicamente, sem marcação. Alternativamente, a presença do cromossomo Y possibilita detectar células de doadores machos no organismo de fêmeas. Todavia, a concentração de células transplantadas pode ser mínima em certos tecidos, pela distribuição sistêmica. Neste estudo, foi desenvolvida uma nested-PCR multiplex, visando a aumentar a sensibilidade do diagnóstico de presença/ausência de células derivadas do tecido adiposo (CDTA-Y) e derivadas da fração mononuclear da medula óssea (CFMO-Y) de camundongos machos, em amostras de sangue e de pulmões de camundongos fêmeas, após transplante endovenoso. Quatro fêmeas receberam placebo; quatro fêmeas receberam CDTA-Y de dois machos; e quatro fêmeas receberam CFMO-Y de dois machos. A primeira fase da PCR teve dois pares de primers (multiplex): um para amplificação de fragmento do cromossomo Y (SRYout; 300pb); outro para amplificação de fragmento do cromossomo X (gene DXNds3). Na segunda fase da PCR, foi usado um par de primers para amplificação de fragmento de 110pb (SRYinn) interno ao produto amplificado pelo SRYout. O controle interno da reação (gene DXNds3) foi detectado em todas as amostras de DNA testadas, enquanto que o fragmento externo do gene SRY (300pb) foi detectado apenas nas amostras puras de DNA de CDTA-Y e CFMO-Y. O fragmento interno do gene SRY (110pb) foi detectado no sangue e nos pulmões de 100% das receptoras de CDTA-Y e CFMO-Y. A técnica de nested-PCR aumentou a sensibilidade e a segurança do diagnóstico molecular de presença ou ausência de células de camundongos machos em fluidos e tecidos de receptoras fêmeas após transplante endovenoso.

portuguesEstudos anteriores avaliaram o efeito de diferentes procedimentos reprodutivos sobre marcadores de desconforto em bovinos e ovinos. Tais estudos podem estimular a adocao de tecnicas que preservem o bem-estar animal. Entretanto, os marcadores comumente utilizados apresentam alta influencia de fatores externos. Para contornar isso, a avaliacao conjunta de diferentes parâmetros sistemicos pode ser utilizada para determinar com maior precisao a presenca de estresse, dor ou inflamacao, como cortisol, proteinas de fase aguda, a bradicinina e a substância P. O objetivo desta revisao e relacionar potenciais marcadores de inflamacao e estresse, discutindo como e quando sao regulados frente aos estimulos em bovinos e ovinos. Ainda, pretende-se revisar de que forma procedimentos reprodutivos com diferentes graus de invasividade acarretam em desconforto, fornecendo informacoes para a elaboracao de estrategias que possibilitem minimiza-lo. Palavras chave: biotecnicas da reproducao; cortisol; desconforto; proteinas de fase aguda EnglishPrevious studies have evaluated the effects of different reproductive procedures on discomfort markers in sheep and cattle. Such studies may help stimulate the adoption of techniques that are more beneficial for animal welfare. However, markers that are commonly used to evaluate discomfort are highly influenced by external factors. To overcome this, several systemic markers can be evaluated to more precisely identify stress, pain, and inflammation. Such markers include cortisol, acute phase proteins, bradykinin, and substance P. We aimed to review the potential markers of stress, pain, and inflammation, and discuss how and when they are regulated after different stimuli related to reproductive procedures in cattle and sheep. Furthermore, we aimed to review how reproductive procedures with different degrees of invasiveness cause stress and provide information that may help develop strategies to limit animal discomfort. Key words: acute phase proteins; cortisol; discomfort; reproductive techniques

Prostaglandin E2 (dinoprostone) is largely used for labor induction. However, one-third of patients do not respond to treatment. One cause of this poor response may be associated with changes in regulation of prostaglandin E receptors (EP1-4). In this study, we investigated EP mRNA expression in the uterine cervix and lower uterine segment myometrium for term births. Biopsies were obtained from women with successful (responders) and failed (non-responders) dinoprostone labor induction, while women that underwent spontaneous labor were included as controls. EP1 mRNA was upregulated in the cervical tissue of women who did not respond to dinoprostone induction. In addition, in the myometrium, significantly higher levels of EP3 mRNA were observed in women treated with dinoprostone, independent of their responsiveness. Dinoprostone-responders presented 3.6-fold higher levels of EP3 mRNA expression than the spontaneous labor group. Significantly higher levels of EP3 mRNA in the myometrium of the dinoprostone-treated group indicated that dinoprostone may regulate the EP3 gene on the transcriptional level. These results highlight the relationship between EP gene expression and delivery and indicate that understanding the regulation of prostaglandin E receptors may lead to improved labor induction.

EnglishGestation length in swine has a considerable amplitude and both early and delayed parturition are common. This variation increases the occurrence of unassisted farrowing and could lead to a wide-ranging age at weaning for piglets born from one batch. Supervision of sow parturition is crucial to reduce mortality of neonate piglets. To facilitate assistance, induction of farrowing using prostaglandin F2α (PGF) has been widely used in batch farrowing systems, whereby synchronization would concentrate the time of farrowing, allowing for better organization of employees. However, a viable alternative method that can be implemented to manage farrowing is to sustain high progestagen levels in the final days of gestation and, consequently, prevent early parturition. Efficient techniques to delay farrowing such as using oral progestagen supplementation have been previously described, but are only recently being considered for commercial use. The present manuscript reviews publications regarding delaying parturition and discusses the use of intravaginal devices (IVDs) containing progestagen. There is limited data addressing the effect of progestagen treatment during gestation on productive and reproductive performance. Therefore, future studies should focus on improving synchronization protocols following progestagen supplementation and evaluating piglet viability and sow fertility, before widely using progestagen supplementation to manipulate parturition. Key words: farrowing induction; intravaginal device; progestagen; early parturiton portuguesComo a duracao da gestacao de suinos pode ter ampla variacao, e comum a ocorrencia de partos antecipados ou gestacoes prolongadas. Isso aumenta as chances de partos sem assistencia e leva a uma grande variacao de idade dos leitoes dentro do lote de producao. Portanto, a supervisao do parto e indispensavel para reduzir as perdas neonatais. Para facilitar o auxilio aos leitoes, a inducao do parto com prostaglandina F2α (PGF) e eficaz e amplamente utilizada, sendo indicada para concentrar os partos em momentos mais adequados, preferencialmente durante o horario com maior disponibilidade de colaboradores. Uma alternativa viavel e manipular o momento do parto, atraves da manutencao de niveis plasmaticos elevados de progestageno durante o final da gestacao, a fim de evitar partos antecipados. Formas eficientes de evitar o parto atraves de suplementacao oral de progestagenos foram descritas ha decadas, mas apenas recentemente tem sido cogitada a utilizacao comercial. A presente revisao aborda estudos disponiveis na literatura relacionados ao protelamento do parto, incluindo a utilizacao de dispositivos intravaginais (DIVs) impregnados com progestageno. Sao poucos os dados disponiveis relacionados ao uso de progestagenos na gestacao com indices produtivos e reprodutivos. Portanto, alguns pontos ainda devem ser melhor avaliados, especialmente com relacao a determinacao da sincronia dos partos apos o fim da suplementacao com progestagenos, a viabilidade dos neonatos e a fertilidade subsequente das femeas antes da ampla adocao desta tecnica. Palavras-chave: inducao de parto; dispositivo intravaginal; progestageno; parto antecipado.

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 of this study was to evaluate the efficiency of progestagen intravaginal devices (IVDs) in preventing parturition in sows by determining the effect of delaying parturition on the alive/total born piglets ratio. Evaluations of IVDs containing 0.5, 1.0 or 1.5g progesterone (P4) showed they were not effective in delaying parturition at any dosage tested. In a second experiment, seventy-five sows at day 112 of pregnancy were equally distributed (n=15 per group) in the following treatments: prostaglandin (PGF2α; 250µg sodium cloprostenol; control group) or PGF2α and simultaneous insertion of an IVD containing medroxyprogesterone acetate (MPA) for 48h. Control sows initiated labor 27.7±1.6h after PGF2α injection. The mean time (±SEM) between PGF2α administration and parturition was 72.1±8.8h, 72.7±3.8h, 82.7±7.1h and 81.8±3.5h for MPA 100, 200, 400 and 800mg, respectively, differing from control group (P0.05). These findings confirm that IVDs impregnated with MPA can effectively prevent parturition in sows without affecting the alive/total born piglets ratio and therefore represent an alternative to avoid weekend farrowing in swine herds.