Biochemical profile and oocyte quality of primiparous Bos indicus cows submitted to a timed artificial insemination protocol.

The objective of this study was to verify the causes of the lower response of primiparous Bos indicus cows to the ovulation synchronization protocol. Two experiments were performed to evaluate the biochemical profile, oocyte and follicular cell quality (Experiment 1) and pregnancy (Experiment 2). In Experiment 1, suckled primiparous (n = 24) and multiparous cows (n = 24) were submitted to ovum pick up (OPU). On Day 0 (D0), all cows received 2 mg of estradiol benzoate (EB) and an intravaginal progesterone insert (P4). Five days (D5) after the first hormonal administration (EB + P4), all follicles larger than 3 mm were counted on each ovary, and ovum pick-up (OPU) was performed. On day 12 (D12), the intravaginal progesterone insert was removed, and measurement and aspiration of the largest follicle were performed. Blood samples were collected on D5 and D12 to evaluate the concentrations of glucose, cholesterol, NEFA, IGF-1 and insulin. In Experiment 2, suckled primiparous (n = 50) and multiparous (n = 50) cows were subjected to an ovulation synchronization protocol based on E2/P4 (D0: 2 mg EB plus P4 intravaginal insert; D8: 500 µg of cloprostenol, 1 mg cypionate estradiol and 300UI of eCG; D10: artificial insemination). In addition, blood samples were collected on D10 for evaluation of the same hormones and metabolites described in Experiment 1. In all studies, calves remained with the cows during the experimental period. In experiment 1, the number of oocytes grade 1 (P = 0.83), grade 2 (P = 0.23) and grade 3 (P = 0.51), total number of retrieved oocytes (P = 0.14), oocyte quality index (P = 0.93) and total viable oocytes (P = 0.38) did not differ between primiparous and multiparous cows. The number of follicles at the time of follicular aspiration (20.7 ± 1.5 vs. 18.0 ± 1.9; P = 0.05) and the diameter of the largest follicle on D12 (13.5 ± 0.6 vs. 11.4 ± 0.6; P = 0.04) were greater in multiparous cows, and the number of degenerated oocytes was greater in primiparous cows (1.9 ± 0.7 vs. 1.2 ± 0.3; P = 0.05). On D5, the concentrations of IGF-1 (P = 0.47), insulin (P = 0.08), total cholesterol (P = 0.47), NEFA (P = 0.77) and glucose (P = 0.55) in the blood and IGF-1 (P = 0.97) and insulin (P = 0.11) in the follicular fluid did not differ between parity groups. On D12, there was no difference in the concentrations of IGF-1 (P = 0.26), total cholesterol (P = 0.32), NEFAs (P = 0.31) and glucose (P = 0.93) in the blood between primiparous and multiparous cows, however, the serum insulin concentration (P = 0.04) was higher in primiparous cows. There was no correlation between serum and follicular fluid insulin concentrations (r = 0.17; P = 0.31), however, there was a low correlation between serum and follicular fluid IGF-1 concentrations (r = 0.47; P = 0.002). Quantification of transcripts did not differ between parity groups. In experiment 2, concentrations of NEFA (P = 0.12) and insulin (P = 0.16) in the blood and P/AI (P = 0.93) did not differ between parity [60 % (30/50) primiparous vs. 60 % (30/50) multiparous]. In contrast, blood concentrations of IGF-1 (P = 0.0001), total cholesterol (P = 0.005) and glucose (P = 0.01) were greater in primiparous cows. It was concluded that the oocyte quality and expression of the genes evaluated in the granulosa cells were not different between primiparous and multiparous cows. Unexpectedly, the pregnancy rate did not differ between parity. Nevertheless, the blood concentrations of IGF-1, total cholesterol and glucose were greater in primiparous cows.

Aspectos que influenciam a contagem bacteriana total e a contagem de células somáticas do leite de propriedades rurais do interior do estado de São Paulo, Brasil / Aspects that influence milk total bacterial count and somatic cell count from farms in interior of São Paulo State, Brazil / Aspectos que influyen el contaje bacteriano total y el contaje de células somáticas de la leche de propiedades rurales del interior del estado de São Paulo, Brasil

Este estudo objetivou avaliar aspectos que exercem influência sobre a contagem bacteriana total (CBT) e a contagem de células somáticas (CCS) do leite e avaliar a adequação das amostras do produto à Instrução Normativa nº 76, de 26 de novembro de 2018, do Ministério da Agricultura e Pecuária do Brasil (IN 76). A pesquisa foi realizada em propriedades rurais da microrregião de Birigui, São Paulo, Brasil, e se baseou em coleta de dados, por entrevista estruturada, e coletas de leite nas propriedades. Foi realizada amostragem por conveniência, sendo escolhidos 15 produtores de cada estrato (de acordo com a produção diária de leite: pequeno - até 100 L; médio - 101 a 300 L; grande - mais de 300 L), totalizando 45 produtores de leite. Participaram da pesquisa somente propriedades com base da alimentação volumosa em sistema de pastejo ou em semiconfinamento. Para avaliar a influência de diferentes fatores sobre a contagem bacteriana total (CBT) e a contagem de células somáticas (CCS), separadamente, realizou-se regressão multivariada. Ainda, foi determinado o coeficiente de correlação entre variáveis desta pesquisa. As variáveis resposta foram transformadas em logaritmo para normalização dos dados. Foi feita comparação entre os resultados da pesquisa e os parâmetros da IN 76 para verificar o cumprimento das normas pelos produtores. Foram encontrados valores médios de 6.986.977,818.961.790,7 UFC/mL para CBT e de 608.911,1 ± 414.802,9 CS/mL para CCS. A utilização de tanque de resfriamento individual mostrou-se favorável à baixa CBT, em comparação com não uso de tanque ou uso de tanque comunitário, conforme o esperado, pois quanto mais tempo leva para resfriar o leite, maior é a proliferação de bactérias no produto. Foi verificado que o leite de produtores que realizam o California Mastitis Test (CMT) com maior frequência apresentou CCS mais elevada, o que não era esperado. As análises físico-químicas médias foram densidade 1,031 g/mL a 15°C, índice crioscópico -0,538 °H, teor de proteína 3,58%, teor de gordura 4,06%, extrato seco total 12,75%, extrato seco desengordurado 8,69%, acidez titulável 16,2 °D e estabilidade ao álcool (Alizarol 72%) 100%. Em 13,3% (6/45) das amostras foi detectada presença de antibióticos. Considerando a IN 76, o leite de somente 8,9% (4/45) dos produtores apresentou-se adequado simultaneamente para todos os parâmetros analisados (CBT, CCS, densidade, índice crioscópico, proteína, gordura, extrato seco total, extrato seco desengordurado, acidez titulável, estabilidade ao alizarol e pesquisa de antibióticos). A utilização de tanque de resfriamento individual e a realização de CMT favoreceram menor CBT e maior CCS, respectivamente.

This study aimed to evaluate aspects that exerts influence on the total bacterial count (TBC) and somatic cell count (SCC) of milk and evaluate the conformity of the samples to the Normative Instruction no. 76, from November 26, 2018, of the Brazilian Agriculture and Livestock Ministry (NI 76). The research was done in farms around Birigui micro-region, São Paulo State, Brazil, and was based on data collection obtained by structured interviews and milk collection in the farms. Convenience sampling was carried out from 15 producers of each stratum (according to daily milk production: small - up to 100 L; medium - 101 to 300 L; large - more than 300 L), totalizing 45 milk producers. Only farms with grazing or semi-feedlot system for roughage feeding base were included. To evaluate the influence of different factors on Total Bacterial Count (TBC) and Somatic Cell Count (SCC), separately, a multivariate regression was done. Moreover, the correlation coefficient among variables was determined. The response variables were transformed into logarithms for the normalization of the data. A comparison between results and NI 76 parameters was done to verify the standard compliance by producers. Mean TBC was 6.986.977,8 ± 18.961.790,7 CFU/mL and mean SCC was 608.911,1414.802,9 cells/mL. The use of individual cooler tank was favorable to lower TBC, compared with the non-use of the tank or with the use of the communitarian tank, as expected, since the longer it takes to cool the milk, the higher the proliferation of bacteria in the product. It was verified that milk from farmers that more frequently perform California Mastitis Test (CMT) showed higher SCC, which was not expected. The average physicochemical analyzes were density 1.031 g/mL at 15°C, cryoscopic index -0.538 °H, protein content 3.58%, fat content 4.06%, total dry extract 12.75%, dry extract defatted 8.69%, titratable acidity 16.2 °D and alcohol stability (Alizarol 72%) 100%. The presence of antibiotics was detected in 13.3% (6/45) of the samples. Considering NI 76, milk from only 8.9% (4/45) farmers were proper simultaneously for all the parameters analyzed (TBC, SCC, density, cryoscopic index, protein, fat, total dry extract, defatted dry extract, titratable acidity, alizarol stability and antibiotics). The use of individual cooling tank and the CMT performance support lower TBC and higher SCC respectively.

Este estudio tuvo como objetivo evaluar los aspectos que influyen en el contaje bacteriano total (CBT) y el contaje de células somáticas (CCS) de la leche y evaluar la adecuación de las muestras del producto a la Instrucción Normativa nº 76, de 26 de noviembre de 2018, del Ministerio de Agricultura y Ganadería en Brasil (IN 76). La investigación se llevó a cabo en propiedades rurales en la microrregión de Birigui, São Paulo, Brasil, y se basó en la recolección de datos, a través de entrevistas estructuradas, y colectas de leche en las propiedades. Se realizó un muestreo por conveniencia, escogiendo 15 productores de cada estrato (de acuerdo con la producción diaria de leche: pequeño - hasta 100 L; medio - 101 hasta 300 L; grande ­ más de 300 L), totalizando 45 productores de leche. Participaron de la investigación únicamente propiedades basadas en alimentación voluminosa en sistema de pastoreo o, a lo sumo, en semiconfinamiento. Para evaluar la influencia de diferentes factores sobre el contaje bacteriano total (CBT) y el contaje de células somáticas (CCS), por separado, se realizó una regresión multivariada. Asimismo, se determinó el coeficiente de correlación entre las variables de esta investigación. Las variables de respuesta se transformaron en logaritmos para la normalización de datos. Se realizó una comparación entre los resultados de la investigación y los parámetros de la IN 76 para verificar el cumplimiento de las normas por parte de los productores. Se encontraron valores medios de 6.986.977,818.961.790,7 UFC/mL para CBT y de 608.911,1 +414.802,9 CS/mL para CCS. El uso de un tanque de enfriamiento individual se ha mostrado favorable a la baja CBT, en comparación con no usar un tanque o usar un tanque comunitario, conforme lo esperado, ya que cuanto más se tarda en enfriar la leche, mayor es la proliferación de bacterias en el producto. Se verificó que la leche de los productores que realizan el California Mastitis Test (CMT) con mayor frecuencia presentó CCS más alto, lo cual no se esperaba. Los análisis fisicoquímicos promedio fueron densidad 1,031 g/mL a 15°C, índice crioscópico -0,538 °H, contenido de proteína 3,58%, contenido de grasa 4,06%, extracto seco total 12,75%, extracto seco desgrasado 8,69%, acidez titulable 16,2°D y estabilidad del alcohol (Alizarol 72%) 100%. En el 13,3% (6/45) de las muestras se detectó la presencia de antibióticos. Considerando la IN 76, la leche de solo el 8,9% (4/45) de los productores se presentó adecuado simultáneamente para todos los parámetros analizados (CBT, CCS, densidad, índice crioscópico, proteína, grasa, extracto seco total, extracto seco desgrasado, acidez titulable, estabilidad al alizarol e investigación de antibióticos). El uso de un tanque de enfriamiento individual y la realización de CMT favorecieron un CBT más bajo y un CCS más alto respectivamente.

Ovarian follicular development, hormonal and metabolic profile in prepubertal ewe lambs with moderate dietary restriction and lipid supplementation.

To evaluate the effects of moderate dietary restriction and lipid supplementation on ovarian follicular development, hormonal and metabolic profile, thirty-five prepuberal ewe lambs were blocked by body weight and randomly assigned to treatments: ALUS (control) - unsupplemented-diet ad libitum (3.5% ether extract, n = 9); R-US - intake restricted to 85% of the ALUS diet (n = 9); AL-LS - lipid-supplemented-diet ad libitum (9.8% ether extract, n = 8); R-LS - intake restricted to 85% of the ALLS diet (n = 9), from 95 ± 8 days of age until estrus or 7 months of age. Lipid supplementation did not reduce dry matter intake. Daily weight gain was greater in lambs fed ad libitum. Plasma glucose was greater in the RLS treatment group, while serum insulin was less with lipid supplementation. There was a treatment by age interaction on total cholesterol, HDL cholesterol and triglyceride serum concentrations. Estrus was detected in 43% of the animals and the overall ovulation rate was 60%. The number of follicles, diameter of the largest follicle, body weight, age and serum progesterone at puberty did not differ among treatment groups. The mean diameter of the largest follicle was greater in lambs having than in those not having ovulations and increased with age in both groups. There was an interaction between the effects of occurrence of ovulation and age on the number of follicles between 3 and 5 mm and > 5 mm. Lipid supplementation and dietary restriction altered the metabolic profile in ewe lambs with no concomitant changes in values for reproductive variables.

Anti-Müllerian hormone (AMH) as a predictor of antral follicle population in heifers.

Antral follicular population (AFP) and anti-Müllerian hormone (AMH) concentration are strongly associated. Thus, analyzing AMH concentration is a reliable method to predict animals with high AFP, an important feature to select donor cows for embryos and oocytes. However, not much is known about this parameter in heifers, particularly in crossbred animals. The aim of this study was to assess AFP in heifers and cows and its relation to serum AMH levels. Bos taurus indicus cows (36-60-months-old; n = 30) and heifers (12-14-months-old; n = 17) of the same breed were evaluated. A single procedure of follicular counting was performed by ultrasound for each animal. Random sampling was performed to analyze serum AMH concentration. Cows showed higher AFP than heifers; nonetheless, plasma AMH concentration did not differ between the categories. A high correlation of AFP with plasma AMH concentration was observed in Girolando heifers. Thus, it is suggested that AMH concentration could be a reliable and less invasive method for selecting heifers with high AFP.

Corpus luteum dynamics after ovulation induction with or without previous exposure to progesterone in prepubertal Nellore heifers.

The objectives of this study were 1) to monitor corpus luteum (CL) dynamics after two different protocols of ovulation induction in prepubertal Nellore heifers, and 2) to determine differences in luteal function. Fifty-seven heifers (weight 289.61 ± 32.28 kg, BCS 5.66 ± 0.65, age 17.47 ± 0.81 months) were divided into two groups: GP4+GnRH received a progesterone (P4) device of 3rd use for 10 days, followed by the administration of 0.02 mg buserelin acetate (GnRH) 48 h after removal of the device, and GGnRH received only GnRH. The CLs formed were monitored by ultrasonography every 2 days until their functional regression (decrease in the color Doppler signal and serum P4 concentration < 1 ng/mL), determining their diameter and area, numerical pixel value (NPV), pixel heterogeneity, and vascularization percentage. The peak systolic velocity, end diastolic velocity, resistivity index and pulsatility index (PI) of the ovarian artery and serum P4 concentration were also measured. A lifespan of the CL of more than 16 days was classified as normal-function and of less than 16 days as premature regression. The variables were compared between treatments, CL categories (normal-functional, prematurely regressed or non-functional), days of evaluation, and their interactions using the MIXED procedure of the SAS program (p ≤ 0.05). Three animals of each group (6/57 = 11%) did not respond to treatment, corresponding to an ovulation rate of 89%. There was a higher percentage of normal-function CLs in GP4+GnRH (81%) and a higher percentage of non-functional CLs in GGnRH (52%; P4 concentration < 1 ng/mL in all assessments). Normal-function CLs exhibited a greater area, vascularization percentage and P4 concentration than prematurely regressed and non-functional CLs. Lower diameter, area, NPV and P4 concentration were observed for non-functional CLs, but there was no difference in vascularization percentage compared to prematurely regressed CLs. Progesterone concentration was efficient in diagnosing CL function and was positively correlated with CL area (r = 0.62; p < 0.001) and vascularization percentage (r = 0.38; p < 0.001). Diameter and PI were important for the early diagnosis of non-functional and prematurely regressed CLs, respectively. In conclusion, luteal function differed for the first CL that develops after ovulation induction in prepubertal heifers. Ultrasonographic parameters (diameter, area, NPV, vascularization percentage, and PI) can be used to predict CL function.

Padronização da quantificação do fator de crescimento semelhante à insulina I (IGF-I) em plasma bovino por ELISA / Standardization of ELISA for the measurement of Insulin-Like Growth Factor I (IGF-I) in bovine plasm

Esse estudo teve como objetivo a padronização de um ensaio imunoenzimático competitivo (cELISA) in house para a determinação das concentrações plasmáticas do fator de crescimento semelhante a insulina I (IGF-I) total para a espécie bovina, utilizando o sistema de amplificação biotina-estreptavidina peroxidase. O IGF-I foi extraído das proteínas ligadoras do fator de crescimento semelhante a insulina I (IGFBP), utilizando o tampão glicina acidificado seguido de neutralização do pH com hidróxido de sódio. As microplacas foram sensibilizadas com anti IgG de coelho, e as dosagens realizadas utilizando duas abordagens, um método com incubação prévia das amostras com o anticorpo anti-h-IGF-I e outro sem incubação prévia (adição simultânea de IGF-I biotilinado e amostra). Os melhores resultados foram obtidos utilizando o método sem incubação prévia, com a sensibilização da placa com 0,25µg/poço de anti-IgG de coelho, o anticorpo específico na diluição 1:250.000 e 0,06ng/poço de IGF-I biotinilado. O ensaio in house apresentou um limite inferior de detecção de 50ng/mL, uma correlação de 0,945 entre doses quando comparado a uma metodologia comercial. Os coeficientes de variação inter-ensaio de 12,94% (345,8ng/mL) para os controles alto e 20,71% (131,6ng/mL) para o baixo. Dessa forma, conclui-se que a metodologia imunoenzimática para quantificação de IGF-I total utilizando o sistema de amplificação biotina-estreptavidina peroxidase em um ensaio competitivo está estabelecida e apresenta-se como uma ferramenta útil para estudos que visem o monitoramento das concentrações de IGF-I.(AU)

This study aimed to standardize an in house competitive enzyme-linked immunosorbent assay (cELISA) to determine plasma concentrations of total insulin-like growth factor I (IGF-I) for the bovine specie using the amplification biotin-streptavidin peroxidase system. The IGF-I was extracted from insulin-like growth factor binding proteins (IGFBPs) using the acidified glycine buffer followed by the pH neutralization with sodium hydroxide. The microplates were coated with anti-rabbit IgG, thereafter the measurements were carried out using two approaches, one with a prior incubation of samples with the anti-h-IGF-I antibody and another without previous incubation (simultaneous addition of IGF-I and biotinylated sample). The best results were obtained using the method without the prior incubation, using the following combination of reagents: microplates were coated with 0.25µg/well of anti-rabbit IgG, the specific antibody at a dilution of 1:250,000 and 0.06ng/well of biotinylated IGF-I. The in house methodology showed sensitivity of 50ng/ml, a correlation between doses of 0.945 when compared to a commercial method. In addition, after 33 assays (quantification of 1114 samples) the proposed methodology presented a good precision, with inter-assay variation coefficients of 12.94% and 20.71% for the high and low controls, respectively. Finally, we concluded that ELISA method for the quantification of total IGF-I using the system biotin-streptavidin-peroxidase amplification in a competitive assay is established and is presented as a useful tool for studies aimed at monitoring the IGF-I concentrations.(AU)

Characterizing emergence and divergence in the first follicular wave in a tropically adapted Bos taurus breed.

Tropically adapted Bos taurus breeds are descended from the cattle which were brought from the Iberian Peninsula to Latin America by the colonizers and which adapted over time to local conditions. These breeds have become a genetic treasure that must be preserved. The objective of this study was to characterize ovarian follicular emergence and divergence in tropically adapted B. taurus cows and heifers. Cyclic heifers (n = 11) and nonlactating, multiparous cows (n = 11) were examined at 8-hour intervals using ultrasonography starting on the day following the final application of PGF2α, which was used to synchronize the estrous cycle, and ending 5 days after ovulation. Blood samples were collected immediately following the ultrasonographic examinations in order to evaluate FSH and estradiol-17ß (E2) concentrations. The interval between ovulation and follicular wave emergence (11.6 ± 3.3 hours vs. 20.3 ± 2.5 hours, P < 0.05) and ovulation and follicular wave divergence (52.4 ± 5.2 hours vs. 71.8 ± 4.1 hours, P < 0.05) was shorter in the cows than in the heifers, respectively. Plasma FSH concentrations increased (P < 0.05) and serum E2 concentrations decreased earlier in cows than in heifers before ovulation. Following follicular wave emergence, no difference in follicular development was found between the cows and the heifers. Consequently, following follicular wave emergence, the data from both the cows and the heifers were combined and categorized by dominant follicle (DF) and second largest follicle (SF). The DF and SF were identified at the same time (P > 0.05). The mean number of small (≤4 mm, 7.2 ± 5.1) and medium (4 to ≤ 8 mm, 6.8 ± 3.5) follicles was greater than that of large follicles (≥8 mm, 0.6 ± 0.5) from ovulation until 5 days after ovulation. The DF diameter (8.1 ± 1 mm) did not differ (P = 0.09) from SF diameter (7.6 ± 0.9 mm) at the time of follicular divergence (around 45 hours after follicular emergence). The DF and SF growth rates were similar (P > 0.05) until follicular divergence, at which point the SF growth rate decreased, whereas the DF growth rate remained constant. Serum E2 concentrations did not change (P > 0.05) during the divergence period, whereas FSH concentrations decreased between 48 and 32 hours before follicular wave divergence. In conclusion, cows and heifers differed only in the interval from ovulation to follicular wave emergence and divergence, which was confirmed by the different patterns of FSH and E2 concentrations.

Manifestation of estrous behavior and subsequent progesterone concentration at timed-embryo transfer in cattle are positively associated with pregnancy success of recipients.

Plasma estradiol and progesterone (P4) concentrations during the peri-ovulatory period are positively correlated with pregnancy success in cattle. The aims of this study were to assess the effects of estrus occurrence and early diestrus P4 concentrations on pregnancy per timed-embryo transfer (P/TET). A total of 267 crossbred beef heifers [222 with corpus luteum (CL) and 45 without a CL but with a follicle >8mm at beginning of the estrous synchronization protocol) received an intra-vaginal P4 device and intramuscular administration of estradiol benzoate. Progesterone devices were removed 8 days later (Day 0), and heifers received d-cloprostenol, eCG and estradiol cypionate. Estrous behavior was monitored twice daily for 3 days after P4 device removal. Plasma P4 concentration was measured by radioimmunoassay at Day 7 and Day 9. At Day 9, heifers with a CL (n=236; i.e. submission rate of 85.5%; 236/276) undergoing TET received an in vitro-produced embryo. Heifers expressing a standing behavioral estrus had a greater P/TET than heifers that did not express a standing estrus [62.4% (106/170) compared with 47.0% (31/66)]. The probability of pregnancy was positively correlated with plasma P4 concentration at TET. When heifers were grouped by quartiles of P4 concentration at TET (Q1=0.64±0.16, Q2=1.70±0.04, Q3=2.90±0.07 and Q4=5.52±0.27ng/mL) the P/TET were 45.8% (Q1; 27/59)(c), 52.25% (Q2; 31/59)(bc), 66.1% (Q3; 39/59)(ab) and 67.8% (Q4; 40/59)(a). Additionally, heifers that became pregnant had greater P4 concentrations at TET (2.87±0.16ng/mL; n=137) than heifers that did not become pregnant (2.45±0.24ng/mL; n=99). No statistical difference was observed regarding P4 concentrations on Day 7, regardless of standing estrus or pregnancy status. In cattle, manifestation of estrous behavior and plasma P4 concentration at TET increase the probability of pregnancy in in vitro-produced embryo recipients.

Association of the leptin gene with carcass characteristics in Nellore cattle.

Advances in DNA technology have created biotechnological tools that can be used in animal selection and new strategies for increasing herd productivity and quality. The objective of the present work was to associate the genotypes of leptin gene exon 2 polymorphisms with productive traits in Nellore cattle. Blood was collected from Nellore males and PCR-RFLP reactions were performed with the restriction enzymes Cla I and Kpn 2I. The gene frequencies resulting from digestion by Cla I were 0.60 and 0.40 for allele A and T, respectively; the genotypic frequencies were AA = 0.20 and AT = 0.80. The gene frequencies from digestion by Kpn 2I were 0.81 for allele C and 0.194 for allele T; the genotypic frequencies were CC = 0.62 and CT = 0.38. The populations in both cases were not in Hardy-Weinberg equilibrium (p > 0.05), and the TT genotype was not found. Significant associations were noted between leptin gene exon 2 polymorphisms and five productive traits in Nellore cattle: carcass fat distribution, the intensity of red muscle coloration, pH, marbling, and post-slaughter fat thickness.

Seasonal differences in endocrine and ovarian patterns of Bos taurus indicus (Nelore) heifers estrous cycles / Diferenças sazonais no padrão endócrino e ovariano do ciclo estral de novilhas Bos taurus indicus (Nelore)

Estrous cycle of eight Nelore heifers were evaluated during different seasons of the year (autumn n=11; winter n=8; spring n=9 and summer n=9) with daily count and measurement of follicles ≥3mm, blood was collected every 12h for LH and progesterone (P4), and after estrous every 3h for LH peak. Five ovariectomized heifers were injected with 17β-estradiol (2μg/kg) every season and blood samples collected every 3h (for 30h) thereafter for LH quantification. The monthly percent body weight difference (Δ%) did not vary among seasons. P4 concentration was higher (p<0.01) and follicle number lower during autumn and summer compared to winter and spring. During winter there were more estrous cycles with three and during summer only cycles with two follicular waves (p<0.01). As LH secretion did not vary despite P4 concentration and as there was negative correlation between higher P4 values and daily percentile variation of photoperiod (Δ%, p<0.01; r= -0.45) it is possible to suppose that there is seasonal variation on luteal cell sensitivity to LH. In the ovariectomized Nelore heifers, the LH basal concentration (without estradiol stimulus, p=0.02) and the LH response to estradiol (p<0.01) were lower during summer, leading to the hypothesis that there is seasonal variation of hypothalamic sensitivity to estradiol. According to the present experiment there are suggestions of seasonal reproduction in Nelore heifers.

Ciclos estrais em oito novilhas Nelore foram acompanhados durante diferentes estações (outono n=11; inverno n=8; primavera n=9; verão n=9) com contagem e diâmetro de folículos ≥ 3 mm diariamente, com colheitas de sangue a cada 12 h para LH e P4, e a cada 3 h para o pico de LH. Cinco novilhas ovariectomizadas receberam 17β estradiol (2μg/kg) em cada estação, com colheitas de sangue a cada 3 h para quantificar LH. A diferença percentual (Δ%) do peso entre os meses não variou entre as estações. Durante o ciclo estral, a concentração de P4 (média e máxima) foi maior (p<0,01), e o número de folículos menor (p<0,01), no outono e verão, se comparados ao inverno e primavera. No inverno houve mais ciclos com três ondas e no verão somente ciclos com duas ondas foliculares (p<0,01). Como as características da secreção de LH não foram diferentes apesar da variação na concentração de P4, e como houve correlação negativa entre os valores máximos de P4 e a variação percentual (Δ%) diária no fotoperíodo (p<0,01; r= -0,45), supõe-se que haja variação sazonal na sensibilidade das células luteínicas ao LH. Nas novilhas ovariectomizadas, a concentração basal (sem estradiol) circanual de LH foi menor no verão (p=0,02), assim como a sua secreção em resposta ao estradiol (p<0,01), o que permite supor que também haja uma variação sazonal na sensibilidade hipotalâmica ao estradiol. De acordo com o presente experimento, há indícios de sazonalidade reprodutiva em novilhas Nelore.

Participação da via alfa-2 adrenérgica no controle da secreção do hormônio do crescimento no período pré-púbere em novilhas da raça Nelore / Participation of the pathway alfa-2: adrenergic in the control of growth hormone secretion in the pre-puberty period of the Nellore heifers

O objetivo deste trabalho foi investigar a variação na secreção de GH em resposta ao tratamento com clonidina, agonista alfa-2 adrenérgico, no período pré-púbere de novilhas da raça Nelore, e desta forma obter informações neuroendócrinas envolvidas no processo de maturação sexual destes animais. A administração de clonidina (10 µg/kg, I.V., amostras 15 min por 4h) foi feita nas novilhas aos oito (n =4), 12(n = 5) e 15 meses de idade (n = 4). A concentração de GH foi quantificada por radioimunoensaio (sensibilidade = 0,25 ng/mL, CV = 16%). Aos oito meses, a administração do estimulador alfa-2 adrenérgico aumentou a concentração de GH, área total de picos, área total de secreção de GH e amplitude do maior pico e reduziu o tempo para aparecimento de pico (P < 0,05). A administração de clonidina aumentou a concentração de GH aos 15 meses, e aos 12 meses, em intervalos restritos (P < 0,05). O uso da clonidina estimulou a secreção de GH em novilhas Nelore pré-púberes. Este efeito foi mais evidente nas novilhas aos oito meses, comparado aos 12 e 15 meses de idade.

This study investigated GH secretion after clonidine (alpha-2 adrenergic agonist) treatmentin pre-pubertal Nelore heifers. Clonidine (10mg/kg, IV, 15 min samples for 4h) was administrated in the same Nelore heifers at eight (n = 4), 12 (n = 5) and 15 (n = 4) months of age. The GH concentration was measured by radioimmunoassay (sensivity = 0.25 ng/mL, CV = 16%). At eight months, clonidine increased GH average concentration, total area of peaks, the total area of GH secretion and increased peak amplitude and reduced time to onset of peak (P < 0.05). At 15 months, the administration of clonidine increased the GH average concentration and at 12 months the increased occurred only in restricted intervals (P <0.05). Clonidine injection stimulated GH secretion in prepubertal heifers and this effect was more evident in Nelore heifers at eight months compared to 12 and 15 months of age.

Validação de radioimunoensaio para quantificação de leptina plasmática bovina / Imunoradioassay validation to plasmatic bovine leptin quantification

Devido à necessidade de compreender melhor as interações entre leptina e reprodução, um ria específico para a leptina bovina foi validado. Primeiro, um protocolo para produção de anticorpos foi desenvolvido por meio da inoculação de leptina recombinante equina em um coelho, que resultou em 28,05% de ligação máxima (MB) 105 dias após o inicio do protocolo. Os testes de validação verificaram paralelismo entre a curva padrão e as diluições dos controles alto e baixo (p<0,01). O anticorpo contra leptina equina mostrou especificidade para a leptina bovina (p<0,01). A taxa de recuperação da leptina bovina pelo anticorpo contra leptina recombinante equina foi de 98,4 a 101,6% (p < 0, 01). Quando as amostras foram armazenadas em temperatura ambiente ou refrigeradas à 4°c, foi verificado estabilidade de ligação (p > 0,2), no entanto temperaturas acima de 37°C interferiram negativamente na recuperação da leptina bovina. O uso do tampão de ensaio com ou sem a adição de plasma não apresentou diferenças (p > 0,3). Esses resultados demonstraram que o anticorpo produzido em coelho contra leptina equina foi capaz de detectar a leptina plasmática bovina, e que o ria para a quantificação da leptina bovina apresentou características adequadas para o desenvolvimento de um ensaio válido.

Due necessity of better understanding leptin and reproduction relations, a specific radioimmunoassay (RIA) to bovine leptin was validated. First, an antibody production protocol was developed using recombinant equine leptin inoculated in a rabbit, that results in 28,05% of maximum binding (MB) 105 days after the protocol beginning. The tests of validations verified parallelism between standard curve and dilutions of high and low controls (P < 0,01). Antibody against equine leptin showed specificity to bovine leptin (P < 0,01). The recuperation tax of bovine leptin by antibody against recombinant equine leptin was from 98,4 to 101,6% (P < 0, 01). When the samples were stored in ambient temperature or refrigerated to 4°C, ligation stability was verified (P > 0,2), howether, temperatures above 37°C impaired the bovine leptin recuperation. The use of assay buffer with or without bovine plasma did not show difference (P > 0,3). These results showed that the antibody produced in rabbit against equine leptin were able to detect plasmatic bovine leptin, and that the RIA to bovine leptin quantification had adequate characteristics to the development of a valid assay.

LH surge in Nelore cows (Bos indicus), after induced estrus or after ovarian superestimulation.

Considering that there is limited information about the preovulatory LH surge in Zebu cattle (Bos indicus), the purpose of the present work was to assess the LH surge in Nelore cows during the estrous cycle and after ovarian superestimulation of ovarian follicular development with FSH. This information is particularly important to improve superovulatory protocols associated with fixed-time artificial insemination. Nelore cows (n=12) had their estrus synchronized with an intravaginal device containing progesterone (CIDR-B) associated with estradiol benzoate administration (EB, 2.5 mg, i.m., Day 0). Eight days later all animals were treated with PGF2alpha (Day 8) in the morning (8:00 h) and at night, when CIDR devices were removed (20:00 h). Starting 38h after the first PGF2alpha injection, blood sampling and ovarian ultrasonography took place every 4h, during 37 consecutive hours. Frequent handling may have resulted in a stress-induced suppression of LH secretion resulting in only 3 of 12 cows having ovulations at 46.7+/-4.9 and 72.3+/-3.8 h, respectively, after removal of CIDR-B. Thirty days later, the same animals received the described hormonal treatment associated with FSH (Folltropin), total dose=200 mg) administered twice a day, during 4 consecutive days, starting on Day 5. Thirty-six hours after the first injection of PGF2alpha, to minimize stress, only seven blood samples were collected at 4h interval each, and ultrasonography was performed every 12 h until ovulation. In 11 of 12 cows (92%) the LH surge and ovulation were observed 34.6+/-1.6 and 59.5+/-1.9 h, respectively, after removal of progesterone source. The maximum values for LH in those animals were 19.0+/-2.6 ng/ml (mean+/-S.E.M.). It is concluded that, in Nelore cows submitted to a ovarian superstimulation protocol, the LH surge occurs approximately 35 h after removal of intravaginal device containing progesterone, and approximately 12h before the LH surge observed after an induced estrus without ovarian superstimulation.

Perfil de FSH e LH na divergência folicular em novilhas Nelore (Bos indicus) / FSH and LH profiles during follicle deviation in Nelore heifers (Bos indicus)

O presente estudo teve como objetivo traçar o perfil de gonadotrofinas em 12 novilhas Nelore, a fim de testar a hipótese de que há declínio nas concentrações de FSH e elevação transitória nos níveis de LH durante a seleção folicular. A partir da ovulação (D0) foram colhidas amostras de sangue da veia jugular a cada 12 horas até o D5, para a dosagem de FSH e LH plasmáticos. Empregou-se o método de radioimunoensaio de duplo anticorpo. A sensibilidade do ensaio de LH foi de 0,02ng/ml e a de FSH de 0,005ng/ml. Os coeficientes de variação intra-ensaio foram 13,6% e 18,8%, respectivamente. Os dados (média±EPM) foram normalizados para o momento da divergência folicular e, posteriormente, analisados por ANOVA e por regressão inear, cúbica e quadrática. Também foi empregado o Teste t para comparação entre o ponto mais alto e o mais baixo da curva. Não se verificou efeito de tempo sobre as concentrações plasmáticas de FSH e de LH quando se utilizou análise de variância e de regressão. Entretanto, através do Teste t pontual, o FSH atingiu as menores concentrações plasmáticas 36 (0,40±0,05ng/ml) e 60 horas (0,42±0,04ng/ml) após a divergência, comparativamente às 36 horas anteriores ao desvio, quando as concentrações foram máximas (0,63±0,08ng/ml). Conclui-se, portanto, que há declínio nas concentrações plasmáticas de FSH, contudo, não foi comprovada elevação transitória nas concentrações de LH próximo ao momento do desvio folicular em fêmeas Nelore.

Present study aimed to evaluate gonadotropins profiles in 12 Nelore heifers, in order to test the hypothesis that FSH concentrations decrease and LH presents a transient increase during follicle selection. Blood samples from jugular vein were harvested twice daily starting at the time of ovulation (D0) until D5. Plasma samples were assayed for FSH and LH by double antibody radioimmunoassay method. LH and FSH assay sensitivity was 0,02ng/ml and 0,005ng/ml, respectively. The intraassay coefficient of variation was 13,6% and 18,8%, respectively. Data (mean±SEM) were normalized to follicle deviation and analyzed by ANOVA and by linear, cubic, and quadratic regressions. Comparisons between higher and lower FSH values were also performed by T-test. There was no effect of time in plasmatic FSH and LH circulating levels when variance analysis or regression analysis were performed. However, by T-test, FSH concentrations reached the lowest plasmatic levels 36 (0,40±0,05ng/ml) and 60 hours (0,42±0,04ng/ml) after follicular deviation, comparatively to 36 hours before deviation, when the concentrations were maximal (0,63±0,08ng/ml). In conclusion, there is a FSH decrease, although a transient LH elevation has not been confirmed encompassing follicle deviation in Nelore females.

Mecanismos neuroendócrinos envolvidos na puberdade de novilhas / Neuroendocrine mechanisms in puberty heifers / Mecanismos neuroendocrinos involucrados en la pubertad de novillas

Sob o ponto de vista fisiológico, a puberdade em novilhas Bos taurus e Bos indicus caracteriza-se por um aumento na concentração e freqüência pulsátil de LH e um decréscimo na sensibilidade do hipotálamo aos esteróides gonadais, com participação ou não de neurotransmissores com capacidade de estimular ou inibir a secreção de LH, o que resultará na primeira ovulação. Os eventos fisiológicos relacionados à primeira ovulação são similares, tanto nas novilhas Bos taurus, quanto nas novilhas Bos indicus, ocorrendo mais tardiamente nesta última. O sistema extensivo de criação do Zebu, adotado na maioria dos países sul-americanos, contribui para que a puberdade aconteça mais tardiamente, refletindo na idade ao primeiro parto. A idade à primeira ovulação é uma característica de alta herdabilidade, sendo a seleção genética, através da precocidade sexual, uma ferramenta para reduzir a idade ao primeiro parto e melhorar a eficiência reprodutiva do sistema de produção. Aliado à seleção genética, o uso de técnicas de manejo melhorando a qualidade nutricional e a oferta de alimentos, o cruzamento entre raças, a exposição das novilhas a touros e o uso de biotecnologias que permitam manipular a primeira ovulação, podem contribuir para o aumento da produtividade. Esta revisão tem como objetivo abordar aspectos fisiológicos relacionados à puberdade de novilhas Bos taurus e Bos indicus.

Under a physiological perspective, puberty in both Bos taurus and Bos indicus heifers is characterized by an increase in the LH concentration, frequency pulses, and decreasing of the hypothalamic-pituitary axis sensibility to gonadal steroid, with, or without, the participation of the neurotransmitters capable of either stimulating or to inhibiting the LH secretion, which will result in the first ovulation. The physiological events related to the first ovulation are similar for both Bos taurus and Bos indicus, occurring later for Bos indicus. The extensive Zebu management carried out in the majority of the South American countries contributes for puberty to occur later influencing the age of the first calving. Regarding the first ovulation, age is a characteristic of high heritability, as the genetic selection, through sexual precocity, is a tool for first¬calving age reduction; and the improvement of the nutritional quality and feeding supply, crossbreeding, heifer exposition to bulls and the use o biotechnologies which ensure the manipulation of the first ovulation might contribute for the productivity increasing. This review aims at approaching physiological aspects related to the puberty of both Bos taurus and Bos indicus heifers.

Serum cortisol, lactate and creatinine concentrations in Thoroughbred fillies of different ages and states of training

Exercise can be defined as "normal stress" stimulating body functions. Some reports suggest lactate as a stimulator of cortisol levels, while creatinine varies according to the amount of muscle tissue. In the present study we investigated the relationship between creatinine, serum lactate concentration and cortisol levels in training horses. Twenty-three Thoroughbred fillies were used, divided into 3 groups according to age and training protocol: G1, 1-2 years of age (N=7) on pasture, G2, 2-3 years (N=9) starting to be mounted, and G3, 3-4 years (N=7) racing at the Jockey Club. Blood samples were collected weekly during a six-month period at about 1:00 p.m. while the animals were resting. Cortisol was quantified with a commercial kit (Coat-a Count®) and serum creatinine and lactate were evaluated with an autoanalyzer with commercial reagents. Data were evaluated using non-parametric statistical tests, with the level of significance set at P< 0.05. Cortisol concentrations were 149ª + 7, 126b + 6, and 101c + 5 nmol/l, lactate concentrations were 2.1ª + 0.1, 2.0ª + 0.1, and 1.75b + 0.1 mmol/l, and creatinine concentrations were 125ª + 2, 132ª + 2 145b + 3 mumol/l in G1, G2 and G3, respectively. Only G2 showed a low but significant positive correlation of cortisol with lactate and a negative correlation of cortisol with creatinine levels. It was possible to conclude that cortisol, lactate and creatinine varied during horse aging and physical conditioning. The decrease of cortisol concentration (G2) suggests that the better physical condition acquired during training led to the increase of creatinine concentration, possibly related to muscle mass. The lower cortisol and lactate concentrations observed in G3 animals may have been due to greater muscle mass inducing an increase in creatinine concentrations or changes in muscle fiber type during training

Post partum reproductive assessment in lowland Tapir (Tapirus terrestris): a case report

The lowland tapir is the biggest Brazilian terrestrial mammal, which belongs to the order Perissodactyla, suborder Hippomorpha, superfamily Tapiroidea and a member of the family Tapiridae. At tropical forest the tapir is involved with seed dispersal. The knowledge of this wild animal reproductive cycle is one way to help its preservation. The stress due to restrain of captive or free-ranging wild animal in order to sample collection limits endocrine study once it can be hazard for the estrous cycle. One possibility is to quantify gonadal hormones at the excreta. Progesterone milk levels were studied in a tapir housed at the Araçatuba Zoo, in Säo Paulo, Brazil. Milk samples, vaginal cytology and rectal temperature were collected during lactation. The progesterone was quantified by radioimmunoassay solid phase (Coat-a-Count, DPC®). The standard was supplied by CENA-FAO and the assay showed sensitivity of 1.25 nmol/L with intra-assay variation of 15.36 percent. During most of the lactation (November to June) the female showed no detectable levels of progesterone. After 158 days (from November to April) it was detected the first progesterone peak with 2.3 nmol/L that lasted for 5 days. The second progesterone peak of 3.54 nmol/L lasted for 23 days. The lactation ceased 74 days after the first milk progesterone surge. This animal showed a prolonged lactational anestrous period (nearly 5 months) and the return of gonadal cycle by fall suggested no positive photo-period influence. The milk progesterone quantification showed to be useful for reproductive cycle evaluation of this animal, although vaginal cytology and temperature fluctuation had no relationship with hormonal levels

Gonadal function arrest in fillies after birth, as reflected by steroid serum concentrations

Sexual maturation involves restraint of gonadal activity from birth to puberty, while somatic development continues. Fetal gonadal steroidogenesis is very important for the maintenance of pregnancy and foaling in mares. The present study was conducted to evaluate gonadal steroid serum levels in foals after birth. Five fillies born at Equília Stud Farm, Avaré, SP, Brazil, were studied. Blood samples were collected daily in the morning during the first week of life. Progesterone serum levels were measured by a commercial radioimmunoassay (RIA) kit and estradiol serum levels with a sensitized non-commercial RIA. At birth, both progesterone and estradiol serum levels were high (13.46 ñ 5.5 nmol/l and 7.95 ñ 1.5 nmol/l) and decreased to undetectable levels at the end of the first week of life. A negative correlation was found between fillie age and gonadal serum steroid concentration. Results show that fetal gonadal activity persists until birth, and decreases gradual and slowly during the first week of life, as reflected by steroid concentrations in the blood