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Correspondence to the Editor. Legionnaires’ disease is an often severe form of pneumonia that is typically acquired by susceptible persons (e.g., elderly persons and smokers) through inhalation of aerosols that contain legionella species.1-4 A cluster of cases of this disease occurred in Vila Franca de Xira, Portugal, in 2014.

The effects of adding rutin to the diet (0, 0.15 or 0.30%) of silver catfish for 21 days on blood parameters, oxidative stress biomarkers and pituitary hormones expression were investigated. Fish that received the diet containing 0.15% rutin exhibited reduced plasma cortisol levels. The levels of lipid peroxidation were lowered in the all tissues of animals receiving the diet containing rutin. Rutin increased the activity of the superoxide dismutase (SOD), catalase (CAT), nonprotein thiols (NPSH), ascorbic acid content (AA) and total reactive antioxidant potential (TRAP) in the brain; glutathione S-transferase (GST) activity and TRAP in the gills; SOD, CAT and GST activity, NPSH, AA levels and TRAP in the liver; CAT and GST activity and TRAP levels in the kidneys; and glutathione peroxidase activity, NPSH, AA levels and TRAP in the muscle. There were no changes regarding the expression of growth hormone, prolactin and somatolactin in fish fed with the diet containing rutin when compared with the control. The supplementation of rutin to the diet of fish is beneficial because it increases the antioxidant responses of tissues.

Because caspase-3 is considered a primary executioner of apoptosis and has been implicated as a mediator of luteal regression, we hypothesized that corpora lutea (CL) derived from caspase-3 null mice would exhibit a delayed onset of apoptosis during luteal regression, when compared with CL derived from wild-type (WT) mice. To test this hypothesis, ovulation was synchronized in immature (postpartum d 24 –27) WT and caspase-3-deficient female littermates by exogenous gonadotropins. Individual CL were isolated by manual dissection, 30 h after ovulation, and placed in organ culture dishes in the absence of serum and growth factors. At the time of isolation (0 h) and after 24, 48, and 72 h in culture, the CL were removed and assessed for the presence of processed (active) caspase-3 enzyme and for apoptosis by multiple criteria. There was no evidence of active caspase-3 enzyme or apoptosis in either WT or caspase-3-deficient CL before culture. However, CL derived from the WT mice exhibited a time-dependent increase in the level of active caspase-3 and apoptosis during culture. By comparison, CL derived from caspase-3-deficient mice, cultured in parallel, failed to exhibit any detectable active caspase-3 and showed attenuated rates of apoptosis. To extend these findings derived from ex vivo culture experiments, ovaries were collected from WT and caspase-3 null female littermates at 2, 4, or 6 d post ovulation, and the occurrence of apoptosis within the CL was analyzed. Whereas ovaries of WT mice had only residual luteal tissue at d 6 post ovulation, ovaries collected from caspase-3-deficient mice retained many CL, at d 6 post ovulation, that were similar in size to those observed in the early luteal phase of WT mice. Importantly, there was no dramatic increase in apoptosis in CL of caspase-3-deficient mice at any time point examined post ovulation, indicating that the involution process had indeed been delayed. In contrast, the levels of progesterone declined regardless of genotype. These data provide the first direct evidence that caspase-3 is functionally required for apoptosis to proceed normally during luteal regression. However, caspase-3 is not a direct mediator of the decrease in steroidogenesis associated with luteolysis. (Endocrinology 143: 1495–1501, 2002)

Background: Estradiol (E2) receptors mediate E2 effects on cell proliferation and apoptosis under normal and pathological conditions. However, the mechanisms involved in E2 signaling are not completely understood. The objectives in this study were to evaluate the expression of estrogen receptors (ESRs) during follicular selection in cattle, and the effect of intrafollicular injection of fulvestrant (an antagonist of ESRs) on follicular development and transcript abundance in granulosa cells. Methods: Granulosa cells were obtained from the two largest follicles around follicular deviation, after FSH treatment and after intrafollicular injection of fulvestrant. Ovarian follicular dynamics monitored by ultrasonography and quantitative real time PCR were used to validate the in vivo model and investigate the effects of FSH supplementation or ESR blockade on mRNA expression of estradiol-related genes. Results: ESR1 and ESR2 were expressed in granulosa cells of both dominant (F1) and subordinate (F2) follicles, but their transcripts levels were higher in F1 than F2 after follicular deviation. FSH treatment maintained mRNA levels of both ESR1 and ESR2 in F2 follicles at similar levels observed in F1 follicles. Intrafollicular injection of 100 μM fulvestrant inhibited follicular growth and decreased CYP19A1 mRNA levels. Transcript levels for both ESR1 and ESR2 were not affected by fulvestrant injection. Analyses of FSH-regulated genes revealed that ESRs inhibition in the dominant follicle decreased the transcript levels of the GJA1 but not those of PRKAR2B, MRO or LRP11 genes. Conclusions: Our findings indicate that: both ESR1 and ESR2 are regulated during follicular deviation and dominance in cattle and in response to FSH treatment, and ESRs are required for normal gene expression and development of the dominant follicle. Furthermore, we have validated an in vivo model to study estrogen signaling during follicular development that allows paracrine signaling between different follicular cells in a physiological endocrine environment.

Castration of male calves is necessary for trading to facilitate handling and prevent reproduction. However, some methods of castration are traumatic and lead to economic losses because of infection and myiasis. The objective of the present study was to evaluate the efficiency of intratesticular injection (ITI) of hypertonic sodium chloride (NaCl; 20%) solution in male calf castration during the first weeks of life. Forty male calves were allocated to one of the following experimental groups: negative control—surgically castrated immediately after birth; positive control —intact males; G1—ITI from 1- to 5-day old; G2—ITI from 15- to 20-day old; and G3—ITI from 25- to 30-day old. Intratesticular injection induced coagulative necrosis of Leydig cells and seminiferous tubules leading to extensive fibrosis. Testosterone secretion and testicular development were severely impaired in 12-month-old animals from G1 and G2 groups (P

Follicle development is coordinated by gonadotropins, steroids, and growth factors, which activate multiple signaling pathways. Phosphorylated-MAPK (pMAPK) level was indicated as an early marker of follicle dominance, whereas phosphorylated STAT3 (pSTAT3) was increased in granulosa cells of hypophysectomized rats. We hypothesized that MAPK3/1 and STAT3 pathways are regulated in granulosa cells during follicle deviation in cattle. Cyclic beef cows were synchronized and ovariectomized to recover the two largest follicles. Follicular diameter did not differ on Day 2 but was significantly greater in dominant follicles (DFs) than that in subordinate follicles (SFs) on Days 3 and 4 of the follicular wave. The elevated abundance of CYP19A1 mRNA expression in granulosa cells of DFs and cleaved caspase 3 in Day-4 SFs further validated our in vivo model. Before deviation, pMAPK3/1 levels were significantly higher in granulosa cells of the future DF. STAT3 mRNA and total protein (tSTAT3) were higher in granulosa cells of SFs collected on Day 4. Furthermore, levels of pSTAT3 were dramatically increased in granulosa cells of Day-4 SFs. In conclusion, pMAPK3/1 was increased in the future DF, but such differential abundance between the DF and SF was not evident after deviation. The higher abundance of pSTAT3 in granulosa cells of SFs after deviation suggests that this pathway may be involved in granulosa cell death and follicular atresia.

Este trabalho teve por objetivo avaliar a eficiência de tratamentos hormonais sobre a fertilidade de vacas de corte no pós-parto com diferentes condições corporais, durante a estação de monta de outono. Setenta e três vacas pluríparas cruzadas (Hereford x Nelore) criadas extensivamente, com condição corporal entre 2 e 4, foram pesadas e distribuídas em três grupos experimentais. O grupo GSED, constituído por 25 vacas, recebeu pessário vaginal (dia 0) contendo 250mg de acetato de medroxiprogesterona e 500mg de somatotropina bovina recombinante (bST-r). Na retirada dos pessários (dia 7), as vacas receberam 0,5mg de cipionato de estradiol e procedeu-se o desmame temporário dos bezerros por 72 horas. No grupo SED, 25 vacas receberam tratamento semelhante ao grupo GSED, porém não receberam bST-r. No grupocontrole, as 23 vacas somente foram separadas dos seus bezerros por 72h. Quando da retirada dos pessários as vacas foram colocadas com touros por 30 dias. Os animais foram pesados e avaliados quanto à condição corporal no início do experimento e na retirada dos touros (dia 37). Foi constatada perda média de peso de 0,648 kg/dia e os percentuais de estro foram de 26,1%, 33,3% e 56,5%, respectivamente, para os grupos controle, SED e GSED. O diagnóstico de gestação, realizado pela palpação retal 60 dias após a retirada dos touros, indicou percentuais de prenhez de 13,0%, 8,3% e 21,7%, respectivamente, para os grupos controle, SED e GSED (P=0,16), demonstrando que os programas hormonais adotados não foram eficientes no incremento das taxas de prenhez de vacas que perdiam peso entre 50 e 70 dias após o parto. _____________________________________________________________________________________________ ABSTRACT This study aimed to assess the efficiency of hormonal treatment in the postpartum fertility of nursing beef cows. Seventy-three cows (Hereford x Nellore) raised extensively, with body condition ranging from 2 to 4 were randomly allotted in three groups. The GSED group, with 25 cows, received an intravaginal device (ID) of 250mg of medroxiprogesteron acetate and 500mg of recombinant bovine somatotropin (bST-r; day 0). After the ID withdrawal (day 7), the cows received 0.5mg of estradiol cipionate; their calves were temporarily weaned for 72 hours. In the SED group, 25 cows received a similar treatment, however, somatotropin was not used. In the control group, 23 cows were temporarily separated from their calves for 72 hours and did not receive any hormonal treatment. The cows were put together with the bulls for 30 days just after ID withdrawal. The cows were weighted and assessed for body condition at day 0 and just after bulls withdrawal. The cows lost weight in an average of 0.648 kg/day, and 56.5%, 33.3%, and 26.1% of estrous rates were observed, respectively for the GSED, SED and control groups (GSED vs. SED, P=0.0001; GSED vs. control, P=0.0007; SED vs. control, P=0.53). The pregnancy rates were 21.7%, 8.3%, and 13.0%, respectively for the GSED, SED and control groups (P=0.16). The results showed that the hormonal program associated with temporary 72-hour calf removal did not increase conception rate of cows that loosed weight from 50 to 70 days postpartum.

Este experimento foi desenvolvido com o objetivo de avaliar a eficiência de tratamentos hormonais, associados ao desmame temporário, na indução de ovulação após o parto, em fêmeas de corte criadas extensivamente. Foram utilizadas 143 vacas (Hereford e cruzas Hereford x Nelore),pluríparas, entre 50 e 70 dias após o parto, com condição corporal (CC) 2 e 3 (1-5). O grupo SEMED (somatotropina, estradiol, medroxiprogesterona, gonadotrofina e desmame) foi constituído por 50 vacas, as quais receberam (dia 0) 500mg de somatotropina bovina recombinante (bST-r), 5mg de benzoato de estradiol e um pessário intravaginal contendo 250mg de acetato de medróxiprogesterona (MAP) e, seis dias após, (dia 6) 500UI de gonadotrofina coriônica eqüina (eCG). No momento da retirada dos pessários (dia 7), os terneiros foram separados totalmente das vacas por 96h. No grupo EMED (estradiol, medroxiprogesterona, gonadotrofina e desmame), constituído de 48 vacas, adotou-se um tratamento semelhante ao do grupo anterior, diferindo apenas na não utilização da somatotrofina. No grupo CONTROLE, 43 vacas foram unicamente separadas dos seus filhos por 96h. Logo após a retirada dos pessários vaginais e de realizado o aparte dos terneiros, as vacas foram colocadas em um piquete com touros (1:10). Após 53 dias, foi realizado o diagnóstico de gestação por palpação retal e ultra-sonografia, para detectar as vacas que conceberam no estro subseqüente aos tratamentos. Obtiveram-se índices de prenhez de 11,1% , 38,0% e 56,2% respectivamente, para os grupos CONTROLE, SEMED e EMED, cujas diferenças foram significativas (p

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

O presente experimento foi delineado com o objetivo de determinar a influência de diferentes períodos de digestão enzimática nos ovários de fetos bovinos, sobre o número total de folículos pré-antrais resgatados e o número de folículos resgatados em cada estágio de desenvolvimento. A presença da membrana basal, envolvendo o complexo formado pelas células da granulosa e o oócito, foi observada através de um estudo histológico, o que permitiu avaliar a integridade morfológica dos folículos pré-antrais isolados. Para isso, ovários de fetos bovinos, entre 150 e 270 dias de gestação, foram obtidos em frigorífico. No laboratório, os ovários foram seccionados em vários fragmentos com uma tesoura cirúrgica e dissociados com pipetas de Pasteur, com diâmetros aproximados de 1000 e 500mm. Após este processo, os fragmentos foram submetidos à digestão enzimática com colagenase em uma concentração de 200UI/ml de TCM199 modificado, por períodos de 20, 30 ou 40 minutos. Os resultados obtidos com esta técnica permitiram determinar que o número de folículos pré-antrais isolados, assim como, os estágios de desenvolvimento em que esses folículos encontravam-se no momento do isolamento, são similares nos três períodos de incubação enzimática. A estrutura morfológica desses folículos, formada pelo oócito, células da granulosa e membrana basal, manteve-se intacta após o isolamento nos três períodos de digestão enzimática. _______________________________________________________________________________ ABSTRACT The objective of this experiment was to determine the efficiency and the effect of different enzymatic digestion periods on the number and developmental stages of preantral follicles isolated from bovine fetal ovaries. The presence of basal membrane, surrounding granulosa cells-oocyte complex, was observed, after digestion, through histologycal study. For this, ovaries from bovine fetues, were collected in slaughterhouses, between 150 and 270 days of pregnancy. In the laboratory, the ovaries were cut into several fragments with surgical scissors and dissociated with Pasteur pipette, having either 1000 or 500mm diameter. After these steps, the fragments were submitted to an enzymatic digestion with collagenase in a concentration of 200IU/ml of modified TCM 199, for periods of 20, 30 or 40 minutes. The results showed that determined the total number and the quantity of the different developmental stages of isolated preantral follicles were similar in the three different periods of enzymatic digestion. The histological analyses demonstrated that the enzymatic digestion periods did not affect the morphological structure of the follicles.

O presente trabalho descreve os efeitos do estágio reprodutivo (animais impúberes, adultos não-gestantes e gestantes), posição anatômica do ovário (direito e esquerdo) presença de corpo lúteo funcional, peso do ovário e número de folículos antrais superficiais (FAS) sobre o número de folículos pré-antrais (FPA) isolados mecanicamente. Os FPA isolados foram divididos em 3 classes a saber: primordiais, primários e secundários. O número médio (± EP) de FPA isolados por ovário foi de 1.324 ± 193; 866 + 170 e 779 ± 139, respectivamente para ovários oriundos de animais impúberes, não-gestantes e gestantes, sendo observada diferença significativa somente entre animais impúberes e gestantes. A posição anatômica do ovário (exceto para os animais não-gestantes) e a presença ou ausência de corpo lúteo não exerceram nenhum efeito sobre o número e distribuição dos FPA isolados. De um modo geral, o número de folículos primários e secundários foi negativamente correlacionado com o peso ovariano. Já o número de folículos primordiais e secundários foi, respectivamente, positiva e negativamente correlacionado com o número de FAS. Analisando as categorias de animais isoladamente, observou-se que, nos animais impúberes, o número de folículos primordiais foi positivamente correlacionado com o número de FAZ, enquanto que o número de folículos primários e secundários foi negativamente correlacionado com o número de FAS. No tocante à categoria de animais gestantes, o número de folículos primários foi negativamente correlacionado com o peso ovariano. Dependendo da categoria de ovários estudada, foram observadas associações significativas entre o número de folículos primordiais, primários e secundários. _________________________________________________________________________________ ABSTRACT The aïm of this study was to describe the effects of reproductive stages (pre-pubertal, non-pregnant and pregnant adult animals), ovarian anatomic position (left or right), presence of functional corpus luteum, ovarian weight and number of superficial antral follicles on the number of preantral follicles mechanically isolated. The mean number (mean ± SEM) of isolated preantral follicles per ovary was 1,324 ± 193, 866 ±170 and 779 ± 139, respectively for prepubertal, non-pregnant and pregnant adult animals. Anatomic position (except for nonpregnant animals) and the presence or absence of corpus luteum dia nof affect the number and distribution of isolated preantral follicles. In general, the number of primary and secondary follicles was negatively correlated with the ovarian weight. The numbers of primordial and secondary follicles were, respectively, positively and negatively correlated with the number of superficial antral follicles. Analyzing animal categories separately, it was observed that in prepurbetal animals, the number of primordial follicles was positively correlated with the number of superficial antral follicles. However the numbers of primary and secondary follicles were negatively correlated with the number of superficial antral follicles. In the category of pregnant animals, the number of primary follicles was negatively correlated with the ovarian weight. According to ovary category analyzed, were observed significant correlation between the number of primordial, primary and secondary isolated follicles.

A maturação in vitro, fecundação e as técnicas de cultivo de embriões na espécie eqüina são extremamente importantes e necessárias para se examinar as causas de repetição de cio, redução de concepção em éguas velhas e também para a preservação da espécie eqüina. Para avaliar o efeito das gonadotrofinas na regulação da maturação nuclear de oócitos eqüinos, folículos com diâmetro entre 5 a 20mm foram aspirados de 551 ovários provenientes de matadouro obtendo-se 408 oócitos aptos para cultivo. Após a aspiração, os oócitos foram avaliados no próprio líquido folicular quanto a sua integridade e distribuídos nos diferentes tratamentos. No Tratamento I (T1) - grupo controle - os oócitos (n=92) foram cultivados em TCM-199 modificado (mod.), com 25mM de HEPES, 2,2mg/ml de bicarbonato de sódio, 1 mi g/ml 17-beta estradiol, 250mi M de piruvato de sódio e 0,4% de albumina sérica bovina. No tratamento 2 (T2), os oócitos (n= 108) foram cultivados no mesmo meio TC M-199 mod. acrescido de lug/ml de hormônio luteinizante suíno (LHs). No Tratamento 3 (T3), os oócitos (n=102) também foram cultivados em TCM-199 mod. porém com 0,5mi g/ml de hormônio folículo estimulante suíno (FSHs) e no Tratamento 4 (T4) os oócitos (n= 106) foram cultivados com 1mi g/ml de LHs e 0,5mi g/ml, de FSHs. Os oócitos dos quatros tratamentos foram cultivados em estufa a 39°C com 5% de CO2 , e 95% de umidade relativa no ar, durante 24 h. Após este período, as células do Cumulus oophorus foram removidas e os oócitos fixados em solução de ácido acético glacial-metanol (1:3) em placas de Petri 10 x 35mm por 24h sendo posteriormente corados com aceto-orceína. O percentual de oócitos em telófase I / metáfase II foi de 55,6% (59/106) para o T4 (FSHs/LHs) e de 53,9% (55/102) para o T3 (FSHs), os quais não diferiram significativamente. No entanto, estes percentuais foram significativamente superiores (p

Background/Aim. The use of herbal products as a supplement to minimize the effects of chemotherapy for cancer treatment requires further attention with respect to the activity and toxicity of chemotherapy.Uncaria tomentosaextract, which contains oxindole alkaloids, is one of these herbal products. The objective of this study was to evaluate whetherUncaria tomentosaextract modulates apoptosis induced by chemotherapy exposure.Materials and Methods. Colorectal adenocarcinoma cells (HT29 cells) were grown in the presence of oxaliplatin and/orUncaria tomentosaextract.Results. The hydroalcoholic extract ofUncaria tomentosaenhanced chemotherapy-induced apoptosis, with an increase in the percentage of Annexin positive cells, an increase in caspase activities, and an increase of DNA fragments in culture of the neoplastic cells. Moreover, antioxidant activity may be related to apoptosis.Conclusion.Uncaria tomentosaextract has a role for cancer patients as a complementary therapy. Further studies evaluating these beneficial effects with other chemotherapy drugs are recommended.

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.

The effect of fetuin, an a1-glycoprotein that has protease inhibitor activity, on the biock to potyspermy was determined. Cumulus-oocyte complexes from eCG primed mice were matured in vitro in the presence of 0, 0.01, 0.1, l and 10mg/ml of fetuin in modifica TCM 199. Both in vivo and in vitro matured oocytes were fertilized in the presence of fetuin and incubated for 6 and 24h. Fetuin present in a concentration of 1 mg/ml in the ferlilization but not in the maturation medium was able to induce polyspermy in 52.4% of the eggs. There was a positive relationship between concentration of fetuin in the fertilization medium and the proportion of polyspermic eggs (p

Neste trabalho foi avaliado o efeito do estresse calórico durante a maturação de oócitos sobre a produção in vitro de embriões ovinos. Os ovários foram obtidos em abatedouro e os oócitos colhidos de folículos de 2 a 6 mm de diâmetro. Após seleção, os oócitos, em 10 replicações, foram colocados para maturação in vitro (MIV) durante 24 horas. Os oócitos submetidos ao estresse térmico de 41º C durante 3, 6, 12, 18 e 24 horas foram posteriormente transferidos para completar a MIV a 39 ºC, mesma temperatura utilizada para maturação dos oócitos do grupo controle. O desenvolvimento dos embriões foi determinado nos dias 3, 4, 5 e 8 pós-fecundação. A avaliação da qualidade dos embriões foi efetuada através da contagem total de células coradas pelo DAPI e da determinação do número de blastômeros positivos para apoptose através do teste de TUNEL. Observou-se que o estresse térmico diminuiu (P < 0,05) a capacidade de maturação dos oócitos de acordo com o tempo de exposição à temperatura de 41º C. No grupo de oócitos incubados a 39° C, 70,70% maturou, enquanto que nos grupos expostos ao estresse térmico, apenas 45,28%, 35,17%, 12,30%, 9,74% e 4,60% maturaram, respectivamente, após 3, 6, 12, 18 e 24 horas de incubação. A duração de exposição dos oócitos ao estresse calórico foi inversamente proporcional (P < 0,05) à capacidade de desenvolvimento embrionário e diretamente proporcional (P < 0,05) ao número de blastocistos positivos para apoptose. Todavia, o efeito deletério do estresse térmico sobre a clivagem e os embriões nos estádios de 8 a 16 células e de mórula foi crescente (P > 0,05) somente até 18 horas de incubação. Os resultados permitem concluir que o estresse calórico durante a maturação in vitro de oócitos reduz a quantidade e a qualidade dos embriões ovinos produzidos in vitro determinadas pela alta incidência de apoptose. The aim of this work was to evaluate the effect of heat stress during oocyte maturation on ovine in vitro embryo production. Ovaries were collected at abattoirs and oocytes retrieved from follicles ranging from 2 and 6 mm in diameter. After selection, all oocytes, in 10 replicates, were placed in in vitro maturation (IVM) during 24 hours. The oocytes were submitted to heat stress at 41º C during 3, 6, 12, 18 and 24 hours and were further transferred to 39º C in order to complete IVM, which was the temperature of maturation of control oocytes. Embryonic development was determined on days 3, 4, 5, and 8 post-fertilization. Embryo evaluation was performed as total cell count by DAPI staining and determination of positive blastomere for apoptosis by the TUNEL assay. We observed that heat stress diminishes (P < 0.05) oocyte maturation capacity in accordance with exposure time at 41º C. In the group of oocytes incubated at 39 ºC, 70.70% matured, while in the groups exposed to heat stress at 41º C, only 45.28%, 35.17%, 12.30%, 9.74% and 4.60% matured after 3, 6, 12, 18 and 24 hours of incubation, respectively. The duration of exposure to heat stress was inversely proportional (P < 0.05) to embryonic developmental capacity and directly proportional (P < 0.05) to the number of blastocysts positive to apoptosis. However, the cleavage rate and embryonic development from 8 to 16 cells and morulae stages were affected by heat stress (P > 0.05) only up to 18 hours of incubation. The results allow the conclusion that heat stress during oocyte in vitro maturation reduces the quantity and quality of ovine embryos produced in vitro determined by the high incidence of apoptosis.

SummaryThe Grb14 protein is a member of the Grb7 protein family. This protein family acts by binding to tyrosine kinase receptors, promoting cell proliferation and differentiation. There is evidence of the involvement of tyrosine kinase factors in the bovine oocyte maturation process. However, Grb14 has not been studied for bovine cumulus–oocyte complexes (COCs). The aim of the present study was to characterize Grb14 mRNA expression in bovine COCs during follicular development. Furthermore, we demonstrated that the expression of Grb14 mRNA is not regulated by estradiol. mRNA expression of Grb14 was assessed in 480 COCs from follicles of different sizes (1–3, 4–6, 6–8 or >8 mm) by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Grb14 mRNA expression decreased in COCs throughout follicular growth (P

Neosporosis has been considered the main cause of abortion between the first and the second trimester of pregnancy in cattle. Therefore, the objective of this study was to identify the presence of Neospora caninum DNA obtained from experimental models based on the evaluation of different areas of the fetal nervous system and organs from heifers previously inoculated with NC-1 after or before insemination. This study was performed with Hereford × Nelore (n=29) heifers and all animals were considered free of diseases at the beginning of the experiment. All animals were bred by fixed-time artificial insemination (TAI) and allocated as follows: (a) seronegative heifers subjected to TAI (TAI, n=9), (b) heifers infected with N. caninun 60 days prior to TAI (NC-1+TAI, n=9), and (c) heifers submitted to TAI and infected with N. caninum 60 days later (TAI+NC-1, n=11). The pregnancy was confirmed by transrectal ultrasonography 35 days after TAI and evaluated every 30 days until the end of gestation. Fetuses were collected surgically at 170 days of gestation, and immediately necropsied to remove tissues aseptically. Samples of the central nervous system (CNS), heart, kidney, lung, liver, skeletal muscle and caruncle were collected for DNA extraction. Days of gestation at abortion and interval from abortion to first insemination were examined by Student's t-test. At 35 days of gestation the pregnancy rates in the group NC-1+TAI (4/9, 44.4%) was lower than in the control group (8/9, 88.8%, P

Made available in DSpace on 2018-11-28T03:24:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) ACSC There is evidence that regulation of follicle selection in cattle involves locally produced growth factors. In the present study, we investigated the expression of members of the fibroblast growth factor (FGF) 7 family during follicle deviation. The largest and second largest follicles were recovered during the second day of a synchronised follicle wave and the future dominant and future subordinate follicles were identified based on diameter and cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1) mRNA levels in granulosa cells. Theca cells of the future dominant follicle contained less mRNA encoding FGF7 and FGF10 compared with those from the future subordinate follicle 2.5 days after ovulation, before a significant difference between the diameters of the future dominant and future subordinate follicles could be observed, but FGF22 mRNA levels did not change. Levels of mRNA encoding FGF receptors FGFR1B and FGFR2B in theca and granulosa cells, respectively, were lower in the future dominant follicle compared with the future subordinate follicle. Addition of FGF10 to granulosa cells in vitro significantly decreased oestradiol secretion, as well as CYP19A1, FSH receptor (FSHR) and insulin-like growth factor 1 receptor (IGF1R) mRNA abundance, whereas FGF22 had no effect. We conclude that FGF10 and FGFR2B expression is increased in the future subordinate follicle before morphological deviation, which may contribute to follicle selection. Univ Estadual Paulista, Dept Farmacol, Inst Biociencias, BR-18618970 Sao Paulo, Brazil Univ Montreal, Fac Med Vet, Ctr Rech Reprod Anim, 3200 Sicotte, St Hyacinthe, PQ J2S 7C6, Canada Univ Fed Santa Maria, Lab Biotecnol & Reprod Anim, Ave Roraima 1000, BR-97105900 Santa Maria, RS, Brazil Univ Estadual Paulista, Dept Fisiol, Inst Biociencias, BR-18618970 Sao Paulo, Brazil Univ Estadual Paulista, Dept Farmacol, Inst Biociencias, BR-18618970 Sao Paulo, Brazil Univ Estadual Paulista, Dept Fisiol, Inst Biociencias, BR-18618970 Sao Paulo, Brazil FAPESP: 2008/57365-2

Contents The aim of the present study was to examine the role of oxytocin (OT) in the progesterone (P4) and prostaglandins (PGs) pathway to induce oocyte meiotic resumption. Cumulus–oocyte complexes were co-cultured with follicular hemisections for 15 h to determine the effects of different doses of OT or atosiban (ATO; oxytocin receptor antagonist) on oocyte meiotic resumption. In another experiment, we examined the effect of the interaction between P4, OT and PGs on the regulatory cascade of the oocyte meiotic resumption. Oxytocin at 1 μm was effective in inducing meiotic resumption in oocytes co-cultured with follicular cells (84.0%), not differing from the positive control group (74.4%). Atosiban inhibited in a dose-dependent manner the positive effect of OT on the meiotic resumption (27.6% metaphase I with 10 μm of ATO, which did not differ from the 25.5% of the negative control group). Furthermore, a third experiment showed that P4 was able to induce oocyte meiotic resumption, which was inhibited by ATO. However, the OT positive effect was not blocked by mifepristone (P4 antagonist), but was inhibited by indomethacin (a non-selective PTGS2 inhibitor). Collectively, these data suggest a sequential role of P4, OT and PGs in the induction of oocyte meiotic resumption.

Hyperthyroidism may lead to a loss of sperm motility and an increase in oxidative stress (OS) in testes and may cause male reproductive disorders. Thus, the use of compounds with antioxidant properties may be a strategy for preventing these disorders. The effect of resveratrol (RSV) on sperm motility and on variables of the antioxidant status in the testes of rats with triiodothyronine-induced hyperthyroidism (100μg/kg) was investigated. Hyperthyroid rats presented lower sperm motility, higher levels of lipid hydroperoxides and thiobarbituric reactive substances, lower catalase and glutathione peroxidase activities and higher glutathione-S-transferase activity in their testes than control animals. RSV treatment (1mg/kg and 10mg/kg) was able to prevent these effects in the hyperthyroid rats and had no effect in the control animals. In conclusion, RSV might be a strategy for therapeutic intervention to preserve sperm motility and to prevent OS in testes, preserving testicular function in those with hyperthyroidism.

Fibroblast growth factors (FGFs) are involved in paracrine control of follicle development. It was previously demonstrated that FGF10 decreases estradiol (E2) secretion in granulosa cell culture and that theca cell FGF10 mRNA expression is decreased in healthy follicles from abattoir ovaries. The main objectives of this study were to evaluate FGF10 and FGFR2b mRNA expression during follicular development in vivo, to evaluate the effect of FGF10 on follicle growth using Bos taurus taurus cows as a model, and to gain more insight into the mechanisms through which FGF10 inhibits steroidogenesis. Messenger RNA encoding both FGF10 and FGFR2b (main FGF10 receptor) was significantly more expressed in subordinate follicles (SFs) than in dominant follicles (DFs). The intrafollicular injection of FGF10 into the largest growing follicle at 7–8 mm in diameter interrupted the DF growth in a dose-dependent manner (11±0.4, 8.3±1 and 5.9±0.3 mm for 0, 0.1, and 1 μg/ml FGF10, respectively, at 72 h after treatment; PLHCGR and IGFBPs, but significantly upregulated FGF10 mRNA expression. The expression of CYP19A1 mRNA in granulosa cells was downregulated by FGF10 treatment, which was accompanied by a 50-fold decrease in E2 production, and decreased cyclin D2 mRNA. These results have shown that FGF10 and its receptor FGFR2b are more expressed in SFs and provide solid in vivo evidence that FGF10 acts as an important regulator of follicular growth in cattle.

This study investigated the expression of genes controlling homologous recombination (HR), and non-homologous end-joining (NHEJ) DNA-repair pathways in bovine embryos of different developmental potential. It also evaluated whether bovine embryos can respond to DNA double-strand breaks (DSBs) induced with ultraviolet irradiation by regulating expression of genes involved in HR and NHEJ repair pathways. Embryos with high, intermediate or low developmental competence were selected based on the cleavage time after in vitro insemination and were removed from in vitro culture before (36 h), during (72 h) and after (96 h) the expected period of embryonic genome activation. All studied genes were expressed before, during and after the genome activation period regardless the developmental competence of the embryos. Higher mRNA expression of 53BP1 and RAD52 was found before genome activation in embryos with low developmental competence. Expression of 53BP1, RAD51 and KU70 was downregulated at 72 h and upregulated at 168 h post-insemination in response to DSBs induced by ultraviolet irradiation. In conclusion, important genes controlling HR and NHEJ DNA-repair pathways are expressed in bovine embryos, however genes participating in these pathways are only regulated after the period of embryo genome activation in response to ultraviolet-induced DSBs.

The growth factor receptor-bound protein 14 (Grb14) is a cellular adapter protein belonging to the Grb7 family of proteins. Studies with human and rodent cells have demonstrated that Grb14 acts as a negative regulator of tyrosine kinase receptor signalling through the MAPK and PI3K pathways. In cattle, tyrosine kinase receptors are activated during follicular development but the role of Grb14 in this process has not yet been investigated. Therefore, the aim of the present study was to characterize Grb14 mRNA expression in ovarian somatic cells during follicular growth and deviation in cattle. We found Grb14 mRNA expressed in both granulosa and theca cells derived from follicles at different stages of development (3-5 , 6-8,8 mm in diameter). The abundance of mRNA for Grb14 was higher in granulosa cells of subordinate compared with those from dominant follicles at days 3 and 4 of the follicular wave (p 0.05). Further, there was a negative correlation between the abundance of mRNA for Grb14 and P450Arom in granulosa cells (R(2) = 0.367; p 0.05) and between the abundance of mRNA for Grb14 in granulosa cells and concentration of oestradiol in follicular fluid (R(2) = 0.545; p 0.05). In theca cells, the expression of Grb14 mRNA did not differ between dominant and subordinate follicles (p 0.05). These findings suggest that Grb14 may play a regulatory role in granulosa cells during follicular deviation in cattle.

A forma de abertura, o comprimento, o diâmetro e o número de dobras de 272 cérvices, provenientes de ovelhas abatidas, foram examinados, sendo 134 da raça Ideal e 138 da Corriedale. Adicionalmente, a forma de abertura de 136 ovelhas Corriedale, em serviço de inseminação artificial, foi examinada. A forma de flap foi a mais freqüente em ambas as raças, com uma média de 52,2%, confirmado também nos animais inseminados. O comprimento cervical médio foi de 5,9cm para a raça Ideal e 5,7cm para a raça Corriedale (p

It is generally understood that angiotensin II (AngII) promotes follicle atresia in rats, although recent data suggested that this may not be true in cattle. In this study, we aimed to determine in vivo whether AngII alters follicle development in cattle, using intrafollicular injection of AngII or antagonist into the growing dominant follicle or the second largest subordinate follicle. Injection of saralasin, an AngII antagonist, into the growing dominant follicle inhibited follicular growth, and this inhibitory effect was overcome by systemic FSH supplementation. Injection of AngII into the dominant follicle did not affect follicular growth, whereas injection of AngII into the second largest follicle prevented the expected atresia of this subordinate follicle, and the treated follicle grew at the same rate as the dominant follicle for the next 24 h. Inhibition of AngII action in the dominant follicle decreased estradiol concentrations in follicular fluid and the abundance of mRNA encoding aromatase, 3β-hydroxysteroid dehydrogenase, LH receptor, and cyclinD2 in granulosa cells, with minimal effects on theca cells. The effect of AngII on aromatase mRNA levels was confirmed using an in vitro granulosa cell culture system. In conclusion, these data suggest that AngII signaling promotes follicle growth in cattle and does so by regulating genes involved in estradiol secretion and granulosa cell proliferation and differentiation.

O objetivo deste trabalho foi avaliar a utilização de dispositivos intravaginais (DIV) para o controle da reprodução em suínos. Porcas aos 112 dias de gestação receberam injeção de PGF2α (controle, n = 15) ou PGF2α com inserção de DIV contendo acetato de medroxiprogesterona (grupo DIV, n = 14) por 48 horas. As fêmeas iniciaram o parto 27,7±1,6 e 82,3±3,8 horas após aplicação de PGF2α nos grupos controle e tratado, respectivamente. Quanto ao controle do estro, dez porcas receberam DIV por 12 dias, iniciando imediatamente após o desmame, e o estro foi confirmado aos 17,25±0,17 dias após o desmame, em comparação a 4±0,25 dias no grupo controle. Dispositivos intravaginais com progestágeno podem ser utilizados no controle da reprodução em suínos. The objective of this work was to evaluate the use of intravaginal devices (IVD) for the control of reproductive events in swine. Sows at 112 days of pregnancy received an injection of PGF2α (control, n = 15) or PGF2α plus an IVD containing medroxyprogesterone acetate (IVD group, n = 14) for 48 hours. Sows initiated labor 27.7±1.6 and 82.3±3.8 h after PGF2α aplicaction, in control and treated groups, respectively. Regarding control of estrus cycle, ten sows received IVD for 12 days starting immediately after weaning, and estrus was confirmed 17.25±0.17 days after weaning, in comparison to 4.00±0.25 days for the control group. Intravaginal devices with progestagen can be applied for the control of reproduction in swine.

SummaryThe objective of this study was to test the hypothesis that equine growth hormone (eGH), in combination with insulin growth factor-I (IGF-I), influences positively in vitro nuclear and cytoplasmic maturation of equine oocytes. Cumulus–oocyte complexes were recovered from follicles that were < 25 mm in diameter, characterized by morphology and were allocated randomly as follow: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 400 ng/ml anti-IGF-I antibody. Oocytes were matured for 30 h at 38.5°C in air with 5% CO2 and then stained with 10 μg/ml propidium iodide (PI) to evaluate nuclear status and 10 μg/ml Lens culinaris agglutinin-fluorescein complex (FITC-LCA) to assess cortical granule migration by confocal microscopy. The proportion of immature oocytes that developed to the metaphase II (MII) stage in the eGH + IGF-I group (15 of 45) was greater than in the groups that were treated only with IGF-I (7 of 36, p = 0.03). Oocytes that reached MII in the control group (20 of 56; 35.7%) showed a tendency to be different when compared with eGH + IGF-I group (15 of 45; 33.3%, p = 0.08). The treated group that contained anti-IGF-I (15 of 33; 45.4%) decreased the number of oocytes reaching any stage of development when compared with eGH (47 of 72; 65.3%) and eGH + IGF-I (33 of 45; 73.3%) groups (p = 0.05) when data from MI and MII were combined. We concluded that the addition of eGH to in vitro maturation (IVM) medium influenced the in vitro nuclear and cytoplasmic maturation of equine oocytes. The use of GH and IGF-I in vitro may represent a potential alternative for IVM of equine oocytes.

The kallikrein-kinin system (KKS) has been described as an important mediator of physiologic processes. Kallikreins use kininogen (KNG) as substrate to generate bradykinin, the main active peptide of the KKS that acts through two types of receptors, the B(1)R and the B(2)R. The goal of this study was to characterize some components of the KKS in different compartments of the ovary during the bovine ovulation process. The KNG, B(1)R and B(2)R mRNA expression patterns were assessed in theca and granulosa cells, as well as the bradykinin concentration and kallikrein-like activity in follicular fluid of bovine periovulatory follicles. To obtain a periovulatory follicle (≥12 mm), twenty-seven cows were submitted to estrus synchronization protocol and ovariectomized by colpotomy at 0, 3, 6, 12 or 24h after a GnRH-analog injection (gonadorelin; 100 μg, IM). Follicular fluid was aspirated for enzymatic assays while granulosa and theca cells were harvested for mRNA analysis. The mRNA expressions in follicular cells were evaluated by real-time RT-PCR and data representation related to the cyclophilin housekeeping gene. The bradykinin concentration and kallikrein-like activity were measured in follicular fluid by enzymatic immunoassay and selective substrate cleavage, respectively. The B(2)R expression in theca cells and B(1)R expression in theca and granulosa cells showed different profiles during the periovulatory period (P0.05). The bradykinin concentration and kallikrein-like activity in the follicular fluid were different (P0.05) due to the time during the ovulation process. KNG mRNA expression was similar for both follicular cell types (P0.05). Taken together, these results provide an important characterization of the presence and possible KKS regulation during the bovine ovulation.

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

Nitric oxide (NO) is a potential regulator of ovarian follicle growth, and ovarian granulosa cells reportedly generate NO in response to gonadotrophins, suggesting that the regulated form of nitric oxide synthase (iNOS) is present. The objectives of the present study were to gain insight into the expression and role of iNOS in the follicle. Messenger RNA encoding iNOS was detected in granulosa cells, and abundance was higher in growing dominant follicles compared to subordinate follicles (P

The use of oil to avoid water evaporation from cell culture has several disadvantages, amongst which there is the migration of compounds from media to oil and from oil to media. The aim of this study was to evaluate the osmolality of a culture system using four-well plates with water in the central hole as an alternative to in vitro bovine embryo production (IVP). In addition, the osmolality changes of the oocyte washing medium were assessed in 35mm dishes with or without 2 mL of silicon oil overlay. Osmolality of oocyte washing medium changed a great deal over time after 60 minutes on a 39°C heated plate (291 mOsm kg-1), which was not detected when the medium was overlaid with silicon oil (280 mOsm kg-1; P0.05). Blastocyst rates were higher when embryos were cultured in presence of water or oil (29.7 and 29.9% for water and 33% in oil conventional microdrop system), except in the group that oocytes were washed in hyperosmotic washing medium (15.1%; P

The objective of this study was to evaluate the effect of retinol (RT) and retinoic acid (RA) on the in vitro development of pre-implantation goat embryos cultured in potassium simplex optimized medium or synthetic oviduct fluid or cocultured in oviductal cells monolayer either in potassium simplex optimized medium or synthetic oviduct fluid. A total of 2407 cumulus-oocyte complexes were aspirated from 2 to 6 mm ovarian follicles from slaughtered animals. Selected cumulus-oocyte complexes were subjected to in vitro maturation in TCM 199 for 24 h at 39 °C in an atmosphere of 5% (v/v) CO(2) in humidified air. In vitro fertilization was performed in modified defined medium. Eighteen hours after in vitro fertilization, cumulus cells were removed and presumptive zygotes were randomly distributed into experimental groups. In Experiment 1, presumptive zygotes were cultured in potassium simplex optimized medium, potassium simplex optimized medium + RT, potassium simplex optimized medium + retinoic acid, synthetic oviduct fluid, synthetic oviduct fluid + RT and synthetic oviduct fluid + RA at 39 °C in a humidified atmosphere of 5% (v/v) CO(2), 5% (v/v) O(2) and 90% (v/v) N(2). In Experiment 2, presumptive zygotes were cocultured in potassium simplex optimized medium + oviductal cells monolayer, potassium simplex optimized medium + RT + oviductal cells monolayer, potassium simplex optimized medium + RA + oviductal cells monolayer, synthetic oviduct fluid + oviductal cells monolayer, synthetic oviduct fluid + RT + oviductal cells monolayer and synthetic oviduct fluid + RA + oviductal cells monolayer in an atmosphere of 5% (v/v) CO(2) in humidified air. In both experiments, media were partially changed on day 2 after in vitro fertilization and unfertilized oocytes were excluded from the experiment. Embryos were cultured or cocultured for 8 days. In Experiment 1, there was no effect of RT or RA supplementation on the proportion of oocytes that reached the morula or blastocyst stages. By contrast, Experiment 2 demonstrated that the addition of 0.28 μg/ml RT and 0.5 μm RA to the embryo culture media stimulated (p0.05) development to the morula and blastocyst stages under the coculture conditions tested. In conclusion, retinoids play an important role in pre-implantation development of goat embryos and can be used to enhance in vitro embryo production.

The aim was to develop a timed artificial insemination (TAI) system in suckled beef cows. Cows (n=227), 60-80 days postpartum, received estradiol benzoate (5mg) and a vaginal device containing 250µg of medroxyprogesterone acetate (MPA; day 0). On day six, cloprostenol (125µg) and eCG (400IU) were administrated and calves were weaned for 88h. The devices were removed on day seven (BioRep group) or on day eight (TAI group). All cows of TAI group and cows of BioRep group that did not exhibit standing estrus received GnRH (100µg) on day 9. In experiment I, the follicular growth was monitored daily by transrectal ultrasound exams, from day 6 to day 9. The average size of the dominant follicle on day nine was 11.1±0.99mm (BioRep, n=7) and 11.5±0.65mm (TAI, n=7) and all animals ovulated. In experiment II, the BioRep group cows (n=106) were observed for estrous behavior after withdrawal of the device, twice a day for 48h, and inseminated 12h after detection. In the TAI group (n=107), the devices were withdrawn on day eight and after 24h these cows and those from the BioRep group, which were not stand in estrus, received 100µg of GnRH and TAI 16h later. The pregnancy rates were 57.6% (BioRep) and 52.3% (TAI). In conclusion, an increase on MPA exposure time did not affect the follicular dynamics and pregnancy rates and allow TAI without estrous observation. Furthermore, the treatment for eight days provides an efficient TAI system in suckled beef cows. O objetivo deste estudo foi desenvolver um protocolo de inseminação artificial com tempo fixo (IATF) em vacas de corte durante período de amamentação, avaliando o intervalo entre a retirada do progestágeno e a aplicação de GnRH sobre a dinâmica folicular e a prenhez. Para tanto, vacas (n=227) em pós-parto de 60 a 80 dias receberam benzoato de estradiol (5mg) e um pessário vaginal de acetato de medroxiprogesterona (250mg MAP; dia 0). No dia seis, os animais receberam cloprostenol sódico (125µg), gonadotrofina coriônica eqüina (400UI) e desmame temporário (88h). O MAP foi retirado no dia sete (Grupo BioRep) ou no dia oito (Grupo IATF). Todas as vacas do grupo TAI e aquelas que não manifestaram cio do grupo BioRep receberam GnRH (100µg) no dia nove. No experimento I, o monitoramento das estruturas ovarianas de 14 vacas foi realizado a cada 24h, desde o dia seis até 36h após a aplicação de GnRH em ambos os grupos. O tamanho médio do folículo dominante no dia nove foi de 11,1±0,99mm (BioRep n=7) e 11,5±0,65mm (IATF n=7) e todos os animais ovularam. No experimento II, no grupo BioRep (n=106), após a retirada do MAP, as fêmeas foram inseminadas com detecção de estro durante 48 horas. O restante dos animais do grupo BioRep e todos do grupo IATF (n=107) receberam 100µg de GnRH (dia nove) e IATF 16h depois. As taxas de prenhez foram de 57,6% (BioRep) e de 52,3% (IATF). O intervalo de 24h entre a retirada de MAP, mantido por oito dias, e a aplicação de GnRH não interfere na dinâmica folicular e na prenhez, permitindo inseminar vacas de corte amamentando sem observação de estro.

The immunohistochemical expression of p63, DeltaNp63, and p53 was studied in mixed tumors of canine mammary glands (13 benign mixed tumors and 19 carcinomas arising from benign mixed tumors) to determine the role of p63 and its isoform DeltaNp63 in the development of mixed tumors, as well as to assess its relation with p53. P63 was expressed in myoepithelial cells of all benign mixed tumors and in 18 of 19 carcinomas in mixed tumors. The p63-negative carcinoma in mixed tumors was invasive, and a loss of p63 was detected in the other malignant tumors showing a discontinuous p63-stained myoepithelial layer. DeltaNp63 was expressed in all benign mixed tumors but only in p63-positive carcinomas in mixed tumors. Despite its positive correlation with p63 expression in carcinomas in mixed tumors (r = 0.8323, P < .00001), DeltaNp63 expression showed a decrease in benign tumors. Positivity for p53 was detected in 2 of 13 and 1 of 19 benign mixed tumors and carcinomas in mixed tumors, respectively. There was no correlation between p63 or DeltaNp63 and p53 expression. Our data support the notion that the decrease of p63 expression, in particular of its isoform DeltaNp63, seems to be an important factor in the development of carcinomas in mixed tumors.

The objective of this study was to investigate the causes of the repeat breeder syndrome comparing clinical signs and postmortem findings in beef cows. The identification of factors affecting the reproductive tract can support decisions as to whether treatment of repeat breeder cows is justifiable than culling. Since all animals were submitted to clinical examination before being slaughtered, this study has a differential approach when compared with others, where genital tracts from abattoir were examined. In this study, 130 crossbred cows and heifers that have failed to conceive after three or more services were identified, submitted to a clinical examination and blood collection for karyotyping and sent to an abattoir. postmortem examinations included macroscopic evaluation of the genital tracts, bacteriology and histopathology of the uterus. Uterine alterations were predominant followed by oviduct and ovarian pathologies. Histopathological examination was more sensitive as a diagnostic tool than clinical examination. Repeat breeder cows had a predominance of uterine abnormalities (95%), such as inflammatory (42.9%) and degenerative (59.7%) conditions. Oviduct abnormalities were found in 29.8 % of animals. Furthermore, 1 out 10 karyotyped cows showed aneuploidy. Thus, this study stressed the importance of laboratory exams in the diagnosis of the causes of infertility of repeat breeder cows.

O objetivo do presente experimento foi investigar se a realização exclusiva da inseminação artificial em tempo fixo (IATF), empregando como indutor da ovulação o benzoato de estradiol (BE), proporciona taxas de prenhez semelhantes a uma associação de IA convencional e IATF com GnRH, em vacas de corte no pós-parto. Duzentos e cinqüenta vacas amamentado receberam um pessário vaginal contendo 250mg de acetato de medroxi-progesterona (MAP) e uma injeção intramuscular (IM) de 5mg de BE no dia 0. O pessário vaginal permaneceu por sete dias. No dia 6, foram aplicadas 400UI de gonadotrofina coriônica eqüina por via IM e 5mg de análogo de prostaglandina na submucosa vulvar, realizando nesse momento o desmame por 96h. Após a retirada dos pessários (dia 7), as vacas foram distribuídas em dois grupos. No grupo BioRep (n=150), as fêmeas foram observadas duas vezes por dia para detecção de estro por 48h e inseminadas 12h após sua manifestação. Os animais que não manifestaram estro nesse período receberam uma injeção IM de 100mg de GnRH, sendo submetidas à IATF, 16 a 18h após. No grupo BE (n=100), as vacas receberam uma injeção de 1mg de BE IM no dia 8 e foram inseminadas em tempo fixo no dia 9. A porcentagem de prenhez no grupo BioRep (54,7%) foi maior (P

This study investigated differences in the distribution of acetylated histone H3 at Lysine 14 (H3K14ac) and the High-Mobility Group N2 (HMGN2) protein in the chromatin of early- (before 24 hr) and late-cleaved (after 24 hr) bovine embryos derived from small- (1–2 mm) and large-follicles (4–8 mm). The presence of HMGN2 and H3K14ac has been associated with different nuclear functions including chromatin condensation, transcription, DNA replication and repair. In vitro matured oocytes were parthenogenetically activated (PA) and cultured in synthetic oviduct fluid medium. Early- and late-cleaved embryos were fixed at 36, 50, 60, 70 and 80 hr after PA to detect the presence of H3K14ac and HMGN2. The rates of nuclear maturation (81.1% vs. 58.7%), early cleavage (46.9% vs. 38.9%), and development to blastocyst stage (34.3% vs. 18.9%) were higher (P

The aim of this study was to evaluate the efficiency of 1 M dimethylsulphoxide (DMSO), ethylene glycol (EG), propylene glycol (PROH) and glycerol (GLY) to cryopreserve primordial follicles. The first evaluation was performed soon after cryopreservation and the second evaluation after 4 days of in vitro culture, using the cryoprotectants that allowed the higher results (higher follicular survival rate) after cryopreservation. The results after follicular isolation (control) and cryopreservation using 1 M DMSO, EG, PROH and GLY showed that the mean number (+/- SEM) of live follicles per millilitre was 3204 (100%) +/- 319.27, 2798 (87%) +/- 239.14, 2492 (78%) +/- 345.8, 448 (14%) +/- 46.3 and 208 (7%) +/- 75.26, respectively. Higher follicular survival was reported when DMSO and EG were used. Control follicles and follicles cryopreserved with these two cryoprotectants were cultured and the percentage of follicular survival was 55% (control), 42% (EG) and 34% (DMSO). Similar results were found between control and follicles cryopreserved with EG. In conclusion, 1 M EG is the most effective cryoprotectant to preserve primordial follicles isolated from ovaries of sheep.

Foram investigadas três repetições curtas em tandem (STRs), BMS1074, BM1500 e IDVGA-51 e três polimorfismos de um único nucleotídeo (SNPs) LEPSau3AI (A/B), LEPSau3AI (+/-) e LEPKpn2I, ligados ao gene da leptina, verificando-se associações com o desempenho produtivo em vacas no pós-parto, em dois rebanhos de gado de corte, Aberdeen Angus (AA, n=98) e Charolês (C, n=83). Após a reação em cadeia da polimerase, os STRs foram analisados em géis de poliacrilamida e os SNPs em gel de agarose, após a clivagem com endonucleases. Na raça AA, 79% dos portadores do alelo BMS1074*151 apresentaram ganho médio de peso diário (ADG) menor, quando comparados com a média da população (103g), enquanto 62% dos nãoportadores mostraram ADG mais alto (P

The aim of the present study was to verify associations between reproductive efficiency and four microsatellite markers located in synteny with genes involved in the regulation of reproductive mechanisms. A sample of 107 females from a Brangus Ibagé population (5/8 Aberdeen Angus x 3/8 Nelore) was characterized for ETH225 (D9S1) and MM12E6 (D9S20) microsatellites, mapped on chromosome 9, and HEL5 (D21S15) and AFZ1 (D21S37) on chromosome 21. Associations between the genetic markers and reproductive efficiency were determined by one-way analysis of variance using calving interval (CI), live weight at calving (LWC), live weight at first calving (LW1C) and live weight at second calving (LW2C) as dependent variables. The genotypes were classified according to allele size into homozygous for long alleles, homozygous for short alleles and heterozygous. A longer CI was observed for individuals homozygous for long alleles at the HEL5 locus compared with the others (p = 0.022). For the AFZ1 locus, an inverse correlation between allele size and calving interval was observed (p = 0.022), suggesting that homozygosity for long alleles at this microsatellite could be advantageous. Analysis of the combined effect of favorable genotypes at HEL5 and AFZ1 indicated that animals with unfavorable genotypes (homozygous for long alleles at HEL5 and homozygous for short alleles at AFZ1) presented a significantly longer CI (p = 0.003) when compared to the other genotypes. The ETH225 and MM12E6 systems did not present any association with CI. None of the systems studied showed any significant association with LWC, LW1C or LW2C.

Oocytes were recovered by ovum pick up (OPU) from nine pairs of monozygotic twin German Simmental cows. The hypothesis was that there is less variability between identical twins versus among non-related individuals in the variation in the recovery of oocytes by OPU and in the efficiency of in vitro embryo production. Estrous cycles were synchronized with two doses of cloprostenol, 11 days apart. Beginning 3–4 days after synchronized estrus, OPU was done twice weekly (every 3 or 4 days; total of 11 sessions). The influence of repeated OPU on estrous cyclicity was established by estrus detection, plasma progesterone concentrations, and ovarian ultrasonography. There were no differences among days of collection for the number and quality of cumulus oocyte-complexes (COCs), and rates of cleavage and blastocyst formation. A total of 1661 COCs, including 657 (39.6%) good-quality COCs, were recovered. From 1457 (87.7%) cultured COCs, 827 zygotes cleaved and 314 blastocysts were produced on Day 7. The total number of COCs and the blastocyst rates varied among pairs of monozygotic twins; within pairs, only slight differences were observed. In conclusion, recovery of COCs and production of embryos had substantially less variation within pairs of monozygotic twins than among non-related cattle.

To determine the behavior of isolated primordial follicles that were exposed to different concentrations of dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PROH), and glycerol (GLY).Isolated primordial follicles were exposed to the cryoprotectant (CPA) solution and photographed to calculate their volume at different periods of exposure.Laboratorio Renzo Giuliani, University of Florence, Italy.Lambs, 30-40 days old.Isolation of primordial follicles and subsequent exposure to CPA.Follicular volume.At 2 minutes of CPA exposure, all follicles appeared to be shrunken. At approximately 5 minutes, shrinkage ceased, and follicles started to swell, absorbing the CPA and water to maintain osmotic equilibrium. When DMSO was tested, follicular dehydration in all concentrations did not exceed 17%; with PROH and EG, it reached 33% and 27%, respectively. The highest degree of dehydration (48%) was seen with GLY. In almost all tested concentrations, follicular shrinkage occurred up to 5 minutes.Volume changes in isolated primordial follicles can fluctuate according to the CPA used and its concentration.

The objective was to develop a program for inducing estrus (followed by insemination) of suckled beef cows under nutritional stress (poor body condition). A total of 123 cows, from 60 to 75 days postpartum, were classified according to their body condition score (BCS; range from 1 to 5, in increments of 0.5) and allocated into two groups. On Day 0 (without regard to stage of the estrous cycle), cows (n = 59) in the hormone induction (HI) treatment group were given an intravaginal device (IVD) containing 250 mg of medroxiprogesterone acetate (MAP) and an i.m. injection of 2.5 mg estradiol benzoate (EB). On Day 6, these cows were given 500 IU eCG i.m. and calves were weaned for 96 h. The IVD were removed on Day 7. Cows detected in estrus by 45 h after IVD removal were inseminated 12 h after standing estrus; cows not in estrus by 45 h after IVD removal received an i.m. injection of 100 microg gonadorelin (GnRH) and were inseminated 16-18 h later. In the control group (C), cows (n = 64) only had their calves weaned at Day 6 (for 96 h), with estrus detection and AI from Days 6 to 11. Overall, the BCS ranged from 2.0 to 3.0. In the treatment group, estrus and pregnancy rates in cows with BCS 2.0 (20 and 30%, respectively) was lower (P0.05) than those with BCS 3.0 (50 and 66.6%, respectively), but did not differ (P0.05) from BCS 2.5 (23.3 and 47.6%). In C group, only 2 of 66 cows were detected in estrus and bred (neither was pregnant). In conclusion, the program for induction of ovulation using MAP, EB, eCG and GnRH increased the pregnancy rate in beef cows in poor body condition, enabling AI to be done in a 63-h interval.

Cryobiology is a very important tool in reproductive biology. Research in this area focuses on the possibility of restoring fertility in women with reproductive problems or after cancer treatments. Another goal is to establish a genetic resource bank for endangered or commercially important animal species. Cryopreservation of oocytes from pre-antral follicles has been studied during the past decade. Procedures can be divided between the cryopreservation of either ovarian tissue or isolated follicles. Most studies describe a slow freezing/rapid thawing protocol to cryopreserve ovarian fragments. Histology shows that the follicles maintain their morphological integrity, and transplantation of ovarian tissue demonstrates that the follicles can restart their growth and eventually ovulate. Some research groups have obtained offspring using this procedure in mice and sheep. With regard to the cryopreservation of isolated follicles, the few studies reported in this area used the same freezing protocol, and some of them described follicular growth using in-vitro culture. The best result was obtained in mice, with animal birth after follicular cryopreservation and culture. However, additional studies are necessary for a better understanding of the events during follicular cryopreservation and to establish a standard protocol for ovarian transplantation or follicle culture.

To verify the viability of isolated primordial follicles after exposure to different concentrations of dimethyl sulfoxide (DMSO) and after cryopreservation.Randomized control trial.Laboratorio Renzo Giuliani, University of Florence, Italy.Thirty- to 40-day-old lambs.Isolated primordial follicles were stained with trypan blue to evaluate the effect of different DMSO concentrations before and after the cryopreservation. Histological structure and follicular mortality were evaluated.After the isolation procedure (control), a mean (+/-SE) of 800 +/- 203.86 live primordial follicles/mL were obtained. The number of live follicles in the toxicity test using the DMSO at 0, 0.5, 1.0, 1.5, 2.0, and 2.5 M was 782 +/- 193.96, 754 +/- 172.03, 778 +/- 191.58, 736 +/- 191.92, 476 +/- 122.9, and 316 +/- 83.52, respectively. The number of live follicles at 2.5 M was lower than that in the control procedure. After cryopreservation, the numbers decreased to 0 +/- 0, 232 +/- 44.20, 636 +/- 161.82, 628 +/- 181.28, 208 +/- 11.57, and 184 +/- 47.07, respectively at 0, 0.5, 1.0, 1.5, 2.0, and 2.5 M. The number of live follicles at 0, 0.5, 2.0, and 2.5 M were lower than that in the control procedure.After cryopreservation, only DMSO concentrations of 1.0 and 1.5 M showed a number of live follicles similar to that of the control procedure.

Specific RIA systems were developed and used to measure pregnancy-associated glycoprotein (PAG) concentrations during gestation and the postpartum period in Azawak Zebu cows. Twelve females were palpated per rectum and diagnosed as pregnant. Blood samples were taken at 5-10-day intervals from approximately Week 8 of gestation until Week 10 postpartum (pp). One Zebu cow (Z15) initially diagnosed as pregnant showed PAG concentrations lower than the assay sensitivity (0.20 ng/ml) and did not calve. Another cow (ZSand) showed abnormally high PAG concentrations during gestation and was excluded from the general PAG profile. The 10 other Zebu cows exhibited a very similar PAG profile. In these animals, concentrations increased progressively from Week 8 to 35 of gestation (from 6.0+/-4.2 to 196.0+/-34.8 ng/ml), remaining relatively constant until Week 39 (210.8+/-74.8 ng/ml), when they increased sharply to reach their highest level (1095.6+/-607.2 ng/ml) at around parturition. After delivery, PAG concentrations declined significantly (P0.05) until Week 2 postpartum (348.4+/-85.6 ng/ml) and slowly until Week 10 postpartum. Our results revealed that the PAG pattern in Zebu cattle was similar to those of taurine breeds during the first two trimesters of pregnancy, but differed in the peripartum period.

O objetivo deste experimento foi comparar a eficiência de um programa hormonal associado ao desmame temporário por 96 horas na indução do estro e ovulação com o desmame definitivo aos 60 dias em vacas de corte. Foram utilizadas 183 vacas de corte amamentando, das raças Charolês (C), Nelore (N) e suas cruzas recíprocas, as quais foram genotipadas como homozigotas (HOM) ou heterozigotas (HET) para o microssatélite (STR) BMS3004, que está localizado no mesmo cromossomo do gene da cadeia do LH. Entre 60 e 80 dias pós-parto (dia 0), as vacas foram distribuídas em dois grupos. No grupo indução hormonal (IH), as vacas (n=87) receberam (dia 0) 250 mg de acetato de medroxiprogesterona por 8 dias, 2,5 mg de benzoato de estradiol (dia 1) e 500 UI de gonadotrofina coriônica eqüina (dia 7). No dia 8, os bezerros foram desmamados por 96 horas. No mesmo dia (dia 8), as vacas (n=96) do outro grupo apenas foram submetidas ao desmame definitivo (grupo DP). Após, procedeu-se 4 dias de inseminação artificial (IA) e, passado esse período, foram entouradas. O primeiro diagnóstico de gestação (DG) foi realizado 60 dias após o período de IA e, o segundo, 60 dias após o final do entoure. As taxas de estro foram maiores nas vacas do grupo IH em relação as do grupo DP. As vacas com condição corporal 2,5 e 3,0 apresentaram menores percentuais de prenhez ao 1ºDG no grupo IH (29,6 e 46,4%) em relação ao grupo DP (56,0 e 72,2%). Os percentuais de prenhez das vacas com índice corporal 65-73 não diferiram entre os grupos IH e DP. As vacas N do grupo IH, apresentaram menor percentual de prenhez ao 1ºDG que as F1 (27,7 vs. 64,2%), mas não diferiram em relação às C (40,0%). No grupo IH, o percentual de prenhez ao 2ºDG foi menor nas vacas HOM do que nas HET. O desmame definitivo precoce mostrou-se mais eficaz no incremento dos percentuais de prenhez em vacas de corte. ___________________________________________________________________________________ ABSTRACT The aim of this experiment was to compare the efficiency of a hormonal protocol, associated to the temporary weaning for 96 hours, with the definitive weaning at 60 days in beef cows, for the induction of estrus and ovulation. One hundred and eightythree suckled beef cows were used. The breeds of the cows were Charolais (C) and Nellore (N) and their crosses. The animals were genotyped as homozygous (HOM) and heterozigous (HET) for the microsatellite BMS3004, that is localized in the same chromossome of the LH chain gene. The cows were distributed in two groups between 60 and 80 days postpartum (day 0). In the hormonal induction group (HI), the cows (n=87) received (day 0) 250 mg of medroxiprogesteron acetate for 8 days, 2.5 mg of estradiol benzoate (day 1) and 500 UI of eCG (day 7). On day 8, the calves were weaned for 96 hours. In the same day (day 8), the cows (n=96) of the other group were just submitted to early weaning (group EW). Twelve hours after weaning, artificial insemination (AI) was done during four days. After this period, they were mated. The first diagnosis of pregnancy (DP) was performed 60 days after the AI period and, the second, 60 days after the end of mating. The estrus rates were higher in cows from HI group than in those of EW group. In the HI group, the cows with body condition 2.5 and 3.0 presented lower pregnancy rates at the 1st DP (29.6 and 46.4%) than in the EW group (56.0 and 72.2%). The rates of pregnancy in cows with body index 65-73 did not differ between the HI and EW groups. The N cows of HI group presented lower pregnancy rate at 1st DP than the F1 (27.7 vs. 64.2%), but was not different than the C cows (40.0%). In the HI group, the pregnancy rate at the 2nd DP was lower in HOM cows than in the HET ones. The cows in the early definitive weaning group showed to be more efficient than in the hormonal induction group to improve the pregnancy rate.

Objective: To verify the viability of isolated primordial follicles to different propylene glycol (PROH) and glycerol (GLY) concentrations before and after cryopreservation. Design: Isolated primordial follicles were stained with trypan blue to evaluate the effect of different PROH and GLY concentrations before and after cryopreservation. Setting: Laboratorio Renzo Giuliani, University of Florence, Italy. Patient(s): Thirty- to forty-day-old lambs. Intervention(s): Isolation of primordial follicles with subsequent exposure to cryoprotectant and freezing. Main Outcome Measure(s): Histologic structure and follicular mortality. Result(s): After the isolation procedure (control), the mean number of live primordial follicles/mL was 2,688 and 4,452 in the GLY and PROH groups, respectively. When GLY was used, the number of live follicles before cryopreservation was 820, 756, 640, 524, 564, and 460 follicles/mL with concentrations of 0, 0.5, 1.0, 1.5, 2.0, and 2.5 mol/L, respectively. After cryopreservation, this number decreased to 0, 12, 36, 100, 84, and 68 follicles/mL, respectively, with the same concentrations. When PROH was used, the number of live follicles before cryopreservation was 4,216, 3,880, 3,560, 1,812, 704, and 568 follicles/mL with concentrations of 0, 0.5, 1.0, 1.5, 2.0, and 2.5 mol/L, respectively. After cryopreservation, this number decreased to 0, 116, 336, 472, 360, and 244 follicles/mL, respectively, with the same concentrations. Conclusion(s): Both cryoprotectants were shown to preserve isolated primordial follicles after cryopreservation.