Validação de radioimunoensaio para quantificação de leptina plasmática bovina

Due necessity of better understanding leptin and reproduction relations, a specific radioimmunoassay (RIA) to bovine leptin was avalidated. 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), however, temperatures above 37°C impaired the bovine leptin recuperation. The use of assay buffer with or without bovine plasma did not show any 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.

Use of LH or eCG in the last day of superestimulatory treatment

In the last decades several hormonal treatments to induce multiple ovulation and embryo transfer (MOET) have been developed. Tight control of the time of ovulation allowed the use of fixed-time artificial insemination (FTAI) in embryos donors, facilitating animal management. Although, protocols that allow FTAI have evolved and yield as much embryo as conventional protocols that requires estrus detection, substantial increase in viable embryo production has not been observed in superestimulated bovine cattle. The present mini-review put emphasis on superstimulatory protocols in which the last two doses of pFSH are replaced by eCG or LH. Recent results indicate that an extra LH stimulus (using eCG or LH), on the last day of P-36 superestimulatory treatment, seems to improve transferable embryo yield in both Bos taurus and Bos indicus cattle.

Application of molecular markers on genetic improvement and reproduction in the post-genomic Era: Emerging biotechnologies and perspectives in livestock

Background: New challenges are rising in the animal protein market, and one of the main world challenges is to produce more in shorter time, with better quality and in a sustainable way. Brazil is the largest beef exporter in volume hence the factors affecting the beef meat chain are of major concern in countrýs economy. An emerging class of biotechnological approaches, the molecular markers, is bringing new perspectives to face these challenges, particularly after the publication of the first complete livestock genome (bovine), which has triggered a massive initiative to put in practice the benefits of the so called the Post-Genomic Era. Review: This article aimed at showing the directions and insights in the application of molecular markers on livestock genetic improvement and reproduction as well at organizing the progress so far, pointing some perspectives of these emerging technologies in Brazilian ruminant production context. An overview on the nature of the main molecular markers explored in ruminant production is provided, which describes the molecular bases and detection approaches available for microsatellites (STR) and single nucleotide polymorphisms (SNP). A topic is dedicated to review the history of association studies between markers and important trait variation in livestock, showing the timeline starting on quantitative trait loci (QTL) identification using STR markers and ending in high resolution SNP panels to proceed whole genome scans for phenotype/genotype association. Also the article organizes this information to reveal how QTL prospection using STR could open ground to the feasibility of marker-assisted selection and why this approach is quickly being replaced by studies involving the application of genome-wide association using SNP research in a new concept called genomic selection. Conclusion: The world's scientific community is dedicating effort and resources to apply SNP information in livestock selection through the development of high density panels for genomic association studies, connecting molecular genetic data with phenotypes of economic interest. Once generated, this information can be used to take decisions in genetic improvement programs by selecting animals with the assistance of molecular markers.

Somatotropina bovina recombinante (rbST) e grupos raciais sobre a precocidade reprodutiva de cabras

The objective of this work was to evaluate the effect of rbST and breed group on the reproductive precocity of goat females. Eighteen goat females of Alpine breed and breed groups 1/2 Boer + 1/2 Alpine (1/2 BA) and 3/4 Boer + 1/4 Alpine (3/4 BA) were used, distributed in two homogeneous groups of 9 animals, received treatments: Group 1-0,3 mg of recombinant bovine somatotropin by kg of body weight each 14 days and Group 2 - control and aplication was subcutaneous. Animals from control group received saline solution in same dose and interval. The administration of hormone begins after weaning of animals (approximate 45 days of age). The train of the development of females was made by weighed weekly until reaching the previously definitive weight of 30 kg of live weight (LW). For evaluation of the reproductive performance it was made observations of the signals related to the oestrus. The characteristics evaluated was: Weight gain at born until weaning, weaning at 120 days, 150 and 180 days of age, weight to the oestrus, age to the rutting and the 30 age to kg of LW. It was not observed influence of growth hormone and the breed group for weight to rutting, age to rutting and age to reach 30 kg LW. The use of rbST in growth females did not modify the parameters of sexual precocity.

Estradiol-17β altera expressão proteica endometrial em fêmeas bovinas tratadas no 17 o dia do ciclo estral

In bovine females the release of prostaglandin F 2α (PGF 2α) is induced in vivo by estradiol (E 2). It is believed that E 2 stimulates the synthesis of essential proteins for the production of PGF 2α. This study aimed to evaluate the effect of E 2 in increasing the concentration of total protein and modifying the protein composition of endometrial explants from bovine females treated with E 2 at the 17 th day of estrous cycle. Crossbred heifers were treated at 17 th day of estrous cycle intravenously with 0 mg (Control Group; n = 6) or 3 mg of E 2 (E 2 Group; n = 6) and killed two hours after. Endometrial explants were isolated, subjected to extraction of total protein, quantified and were analyzed by onedimensional electrophoresis on polyacrylamide gel 10% SDS-PAGE. The concentration of total protein did not differ between groups, 6296.10 + 439.90 μg/mL for the Control Group and 8426.56 + 1156.00 μg/mL for E 2 Group (p = 0.1158). There was no significant difference (p > 0.05) in the protein profile of endometrial explants in gels stained with Coomasie Blue. In gels stained with Silver Nitrate it was verified in E 2 Group greater relative percentage of the bands referring to the molecular weight of 75 to 76 kDa (8.40% vs. 4.89% in E 2 Group and Control respectively; p < 0.05) and 108 to 110 Kda (6.85% vs. 3.84% in E 2 Group and Control respectively, p < 0.05). It was observed in E 2 Group lower relative percentage of the band referring to the molecular weight of 90 kDa (5.78% vs. 9.83% in E 2 Group and control respectively; p < 0.05). We concluded that the E 2 does not increase the protein concentration in the endometrium, however, it modifies the proteinic composition in the endometrial explants, indicating that E 2 alters the expression of specific proteins.

Forage mass production and grazing loss of sorghum hybrid in response to the density of the sowing and the spacing between planting lines

The objective of this experiment was to evaluate dry matter yield and loss of grazing due to animal trampling in response to sowing density and spacing between lines in the planting. Sorghum hybrid 1P400 was submitted to six treatments, composed of three sowing density combinations (12; 16 and 20 kg/ha of seeds) and two spacing between lines (0.40 and 0.80 m). Sorghum hybrid 1P400 was sowed in two seasons, at the end of spring (December 3rd, 2005) and the other at the end of summer (March 20th, 2006). Cultivation strategies influenced plant population in the two experimental seasons. Diameter of the stem in season 1 decreased with density increase, whereas in the second season, interaction between sowing density and spacing was significant. In the first season, 0.40-m spacing promoted greater losses due to grazing stepping, that is, 891 kg/ha of DM, whereas in the second season there was no statistical difference. There was no significant difference in forage dry matter yield in sowing densities among the two studied seasons. Dry mater production of sorghum hybrids 1P400 did not increase with the increase of the sowing density in the two sowing seasons, therefore it is recommended 12 kg/ha of seeds for the sowing. Sorghum IP400 cultivated in 0.80-m spacing resulted in lower forage loss caused by grazing bovine trampling. © 2011 Sociedade Brasileira de Zootecnia.

Bovine abortion associated with Neospora caninum: Diagnosis and epidemiological aspects of a dairy cattle herd in the northeast region of São Paulo state, Brazil

The objective of this study was to report the presence of Neospora caninum-associated abortion in bovines at a farm in the northeast region of São Paulo State. In January 2010, it was sent to the Department of Pathology, UNESP-Jaboticabal, a bovine fetus with an estimated age of seven months, which was natural of a dairy farm with 300 animals and an average daily production of 3,000 liters of milk, nearly 20 liters per cow. The animals were vaccinated against rabies, foot and mouth disease, carbuncle, brucellosis, leptospirosis, bovine herpes virus type I and bovine viral diarrhea virus. The herd consisted of purebred Holstein animals, Jersey, and mostly by crossbred animals 7-8 (gir x holstein). During necropsy, samples of the serosanguineous liquid present at the thoracic cavity and the heart of the fetus were collected for the detection of anti-Neospora caninum antibodies through Indirect Immunofluorescence Assay (IFA). Fragments of brain, cerebellum, tongue, liver, heart and kidneys were collected for the execution of histopathology (HP), immunohistochemistry (IHC) and Polymerase Chain Reaction. In order that IFA could be performed, the owner was requested blood samples without anticoagulants of the mother and other cows in the farm, with or without a history of abortion. At necropsy, it was verified a severe autolysis of the fetus. The serology of the fetus was 1:25, while the serology of the mother was 1:3,200. At HP, it was observed discrete multifocal non-suppurative encephalomyelitis characterized by gliosis and mononuclear inflammatory infiltration associated with cellular debris. DNA amplification of N. caninum was positive in fragments of brain, tongue, cerebellum, heart and kidneys. At IHC, it has been observed immunoreactivity to a cyst located in the tongue. The owner reported that his herd showed endemic episodes of abortion, while 27.69% (18/65) of the 65 animals sampled were seropositive. Although it has not been a significant difference (p>0.05), a higher seropositivity was observed in animals with a history of abortion (10/26) 38.46%, in comparison with animals without previous abortion (8/39) 20.51%. These findings show that the abortion under study was provoked by the protozoan N. caninum, while this is the first report concerning cattle in the northeast region of São Paulo State.

Chromatin modifying agents in the in vitro production of bovine embryos

The low efficiency observed in cloning by nuclear transfer is related to an aberrant gene expression following errors in epigenetic reprogramming. Recent studies have focused on further understanding of the modifications that take place in the chromatin of embryos during the preimplantation period, through the use of chromatin modifying agents. The goal of these studies is to identify the factors involved in nuclear reprogramming and to adjust in vitro manipulations in order to better mimic in vivo conditions. Therefore, proper knowledge of epigenetic reprogramming is necessary to prevent possible epigenetic errors and to improve efficiency and the use of in vitro fertilization and cloning technologies in cattle and other species. Copyright © 2011 Fabio Morato Monteiro et al.

Influência da raça do touro (Bos indicus x Bos taurus) na tolerância ao estresse térmico calórico de embriões bovinos produzidos in vitro

To better understand the differences related to HS resistance between Bos indicus and Bos taurus, we aim to verify if the HS tolerance is due mostly to the genetic contribution from the oocyte, spermatozoa or both. Oocytes from Nelore and crossbreed Holstein cows (cHST) were collected, matured and fertilized with semen from Nelore (N), Angus (An), Brahman (Bra) and Gir (Gir) bulls. Nine six hours post insemination (hpi), ≥ 16 cells embryos were separated in two groups: control and HS. In control group, embryos were cultured at 39°C, whereas in the HS group, embryos were subjected to 41°C for 12 h, and then returned to 39°C. There was no effect of HS on blastocyst and hatched blastocyst rates in all breeds analyzed. The percentage of oocytes that cleaved and reached morula stage was significantly lower (p < 0.05) in cHST x Gir as compared to the other breeds. Additionally, blastocyst rates was higher in cHST x N than in cHST x An and cHST x Gir (p < 0.05). It was concluded that the oocyte is more important than the spermatozoa for the development of thermotolerance, since the breed of the bull did not influence embryo development after HS.

Implementation of DNA markers to produce genomically - Enhanced EPDs in nellore cattle

Background: The sequencing and publication of the cattle genome and the identification of single nucleotide polymorphism (SNP) molecular markers have provided new tools for animal genetic evaluation and genomic-enhanced selection. These new tools aim to increase the accuracy and scope of selection while decreasing generation interval. The objective of this study was to evaluate the enhancement of accuracy caused by the use of genomic information (Clarifide® - Pfizer) on genetic evaluation of Brazilian Nellore cattle. Review: The application of genome-wide association studies (GWAS) is recognized as one of the most practical approaches to modern genetic improvement. Genomic selection is perhaps most suited to the improvement of traits with low heritability in zebu cattle. The primary interest in livestock genomics has been to estimate the effects of all the markers on the chip, conduct cross-validation to determine accuracy, and apply the resulting information in GWAS either alone [9] or in combination with bull test and pedigree-based genetic evaluation data. The cost of SNP50K genotyping however limits the commercial application of GWAS based on all the SNPs on the chip. However, reasonable predictability and accuracy can be achieved in GWAS by using an assay that contains an optimally selected predictive subset of markers, as opposed to all the SNPs on the chip. The best way to integrate genomic information into genetic improvement programs is to have it included in traditional genetic evaluations. This approach combines traditional expected progeny differences based on phenotype and pedigree with the genomic breeding values based on the markers. Including the different sources of information into a multiple trait genetic evaluation model, for within breed dairy cattle selection, is working with excellent results. However, given the wide genetic diversity of zebu breeds, the high-density panel used for genomic selection in dairy cattle (Ilumina Bovine SNP50 array) appears insufficient for across-breed genomic predictions and selection in beef cattle. Today there is only one breed-specific targeted SNP panel and genomic predictions developed using animals across the entire population of the Nellore breed (www.pfizersaudeanimal.com), which enables genomically - enhanced selection. Genomic profiles are a way to enhance our current selection tools to achieve more accurate predictions for younger animals. Material and Methods: We analyzed the age at first calving (AFC), accumulated productivity (ACP), stayability (STAY) and heifer pregnancy at 30 months (HP30) in Nellore cattle fitting two different animal models; 1) a traditional single trait model, and 2) a two-trait model where the genomic breeding value or molecular value prediction (MVP) was included as a correlated trait. All mixed model analyses were performed using the statistical software ASREML 3.0. Results: Genetic correlation estimates between AFC, ACP, STAY, HP30 and respective MVPs ranged from 0.29 to 0.46. Results also showed an increase of 56%, 36%, 62% and 19% in estimated accuracy of AFC, ACP, STAY and HP30 when MVP information was included in the animal model. Conclusion: Depending upon the trait, integration of MVP information into genetic evaluation resulted in increased accuracy of 19% to 62% as compared to accuracy from traditional genetic evaluation. GE-EPD will be an effective tool to enable faster genetic improvement through more dependable selection of young animals.

produção de mudas de maracujazeiro amarelo submetidas à salinidade em solo com biofertilizante bovino

The experiment was conducted in a greenhouse from May 1 to July 31, 2008, in Areia county, Paraiba State, PB, Brazil, in order to evaluate the effects of irrigation water salinity on initial growth of the passionfruit seedlings in non-saline substrate with and without bovine biofertilizer. The treatments were distributed in a completely randomized design, with three replications and twelve plants per plots, in a factorial arrangement 5 × 2 × 2, corresponding the former to the levels of salinity in the irrigation water: 0.5; 1.0; 2.0; 3.0 and 4.0 dS m-1, in soils with and without bovine biofertilizer applied at two moments (25 and 65 days after seedling emergence). The growth of the seedlings and the soil electrical conductivity were evaluated at the end of the experiment. The biofertilizer was diluted in a low saline water at a 1:1 ratio and was applied once two days before sowing, corresponding to 10% of the substrates volume. The increase in water salinity inhibited the growth in height of plants, leaf area and root length, but always to a lesser extent in the treatments with bovine biofertilizer. The increase in electrical conductivity of the irrigation water elevated the soil salinity, independently of the addition of biofertilizer.

Infecção pelo vírus da diarreia viral bovina em vacas gestantes abatidas no Estado de São Paulo-Brasil

The most important pathogens in the bovine livestock nowadays in the virus of the viral diarrhea mainly for triggered clinical manifestations related to the reproductive sphere. The infection in pregnant females, may result in abortions, embryonic resorptions, fetal mummification, birth of weak and malformation of the cattle. Moreover, their birth with persistently infected and immunotolerant virus, which the source of infection and dissemination of their disease. Nowadays, the complexity of the diagnosis and consequently its pathogenesis are tilted in the genotypic differences agent. So, this study aimed to verify the occurrence of the BVDV-1 (SINGER) and BVDV-2 (VS-253) genotypes in cows and their respective fetuses, slaughtered in an abattoir in the State of Sao Paulo. Through blood serum, using virus neutralization technique. All in all, 52,51% (115/219) of the cows which were tested reacted, but no fetus (0/219) reacted, to its virus neutralization. Through this cross-examination we observed that 42% (92/219) of cows reacted for both BVDV-1 and BVDV-2. Furthermore 4,10% (9/219) of them reacted only to the genotype BVDV-1 and 6,39% (14/219) responded only to the genotype 2 of BVDV. Therefore it was noticed that both strains are widespread in the regions studied, which justifies the use of different antigens to avoid false-negative diagnosis. Finally antibodies showed no fetus or fetal abnormalities, it is already developed and can be considered immunocompetent, independent child born to a reagent.

Effects of ascorbic acid on in vitro culture of bovine preantral follicles

The objective of this study was to evaluate the effects of adding ascorbic acid to the media for in vitro culture of cattle ovarian fragments and to determine their effects on growth activation and viability of early-stage follicles. The ovarian cortex was divided into small fragments; one fragment was immediately fixed (control) and the other fragments were cultured in minimum essential medium (MEM) supplemented or not with various doses of ascorbic acid. Ovarian tissue was processed for histology, transmission electron microscopy (TEM) and immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA). Compared with control fragments, the percentage of primordial follicles was reduced (p < 0.05) and the percentage of growing follicles had increased (p < 0.05) in cultured cortical fragments, independent of the tested medium or incubation time. Furthermore, compared with control tissue, culture of ovarian cortex for 8 days reduced the percentages of healthy, viable follicles (p < 0.05), but not when cultures were supplemented with 25, 50 or 100 μg/ml of ascorbic acid. Ultrastructural and immunohistochemical analysis of 8 day cultured ovarian cortical fragments, however, showed the integrity and viability of follicles only when fragments were cultured in presence of 50 μg/ml of ascorbic acid. In conclusion, this study demonstrated that addition of ascorbic acid to MEM at a concentration of 50 μg/ml not only stimulates the activation of 8 day in vitro cultured cattle primordial follicles and subsequent growth of activated follicles, but also safeguards the viability of these early-stage follicles. © 2012 Copyright Cambridge University Press.

Effects of vaccination against reproductive diseases on reproductive performance of beef cows submitted to fixed-timed AI in Brazilian cow-calf operations

The objectives were to assess incidence of pregnancy losses, associate this outcome with immunization programs against reproductive diseases, and evaluate the effects of vaccination against bovine herpesvirus-1 (BoHV-1), bovine viral diarrhea virus (BVDV), and Leptospira spp., on reproductive efficiency of Brazilian cow-calf operations. In experiment 1, 7614 lactating Nelore cows from 18 ranches were assigned to the same estrus synchronization and fixed-time AI protocol (ESFTAI; Days -11 to 0). Pregnancy status was determined with transrectal ultrasonography on Days 30 and 120 after AI. Pregnancy loss was deemed to have occurred when cows were pregnant on Day 30 but nonpregnant on Day 120. Incidence of pregnancy loss across all ranches was 4.1%; pregnancy losses were detected (P < 0.10) in 14 ranches but not detected (P > 0.11) in four ranches. Pregnancy loss was lower (P ≤ 0.02) in ranches that vaccinated against BoHV-1, BVDV, and Leptospira spp. compared with ranches that did not vaccinate, or only vaccinated against Leptospira spp. In experiments 2 and 3, lactating Nelore cows (N = 1950 and 2793, respectively) from ranches that did not have a history of vaccinating against reproductive diseases (experiment 2), or only vaccinated against Leptospira spp. (experiment 3), were assigned to the same ESFTAI used in experiment 1. Within each ranch, cows received (VAC) or not (CON) vaccination against BoHV-1, BVDV, and Leptospira spp. at the beginning of the ESFTAI (Day -11) and 30 days after (Day 41) AI. In experiment 2, VAC cows had greater (P ≤ 0.05) pregnancy rates compared with CON on Days 30 and 120. In experiments 2 and 3, pregnancy loss was reduced (P ≤ 0.03) in primiparous VAC cows compared with CON cohorts. In experiment 4, 367 primiparous, lactating Nelore cows previously vaccinated against Leptospira spp. were assigned to the same ESFTAI used in experiment 1. Cows received VAC, or the same vaccine 30 days before (Day -41) and at the beginning (Day -11) of the ESFTAI (PREVAC). Pregnancy rates on Days 30 and 120 were greater (P ≤ 0.05) in PREVAC cows compared with VAC cows. In conclusion, pregnancy losses affected reproductive and overall efficiency of Brazilian cow-calf operations, and might be directly associated with BoHV-1, BVDV, and Leptospira spp. infections. Hence, vaccinating cows against these pathogens, particularly when both doses are administered before fixed-time AI, improved reproductive performance in Brazilian cow-calf systems. © 2013 Elsevier Inc.

Effects of heat stress on development, quality and survival of Bos indicus and Bos taurus embryos produced in vitro

Heat stress is an important cause of poor development and low survival rates in bovine embryos. Experiments were conducted to test the hypothesis that Bos indicus embryos are more resistant to heat stress than are Bos taurus embryos. In experiment 1, Nelore and Jersey embryos from oocyte pick-up-derived oocytes were submitted to heat stress (96 hours post-insemination, 41 °C, 6 hours), developmental ratios were assessed at Day 7 (Day 0 = day of fertilization), and blastocysts were frozen for RNA extraction. Experiment 2 evaluated expression of COX2, CDX2, HSF1, and PLAC8 in previously frozen blastocysts. In experiment 3, Nellore and Angus embryos from oocyte pick-up-derived oocytes were submitted to heat stress (96 hours post-insemination, 41 °C, 12 hours) and transferred to recipients on Day 7. In experiment 4, embryos developed as in experiment 3 were fixed for Terminal deoxynucleotidyl transferase dUTP nick end labeling labeling and total cell counting. In experiment 1, heat stress decreased the percentage of Jersey oocytes that became blastocysts, but had no effect on Nellore embryos (34.6%, 25.0%, 39.5%, and 33.0% for Jersey control, Jersey heat-stressed, Nellore control, and Nellore heat-stressed oocytes, respectively; P < 0.05). In experiment 2, heat stress decreased (P < 0.05) expression of CDX2 and PLAC8, with higher expression of these genes in Nellore embryos than in Jersey embryos. Heat stress also decreased (P < 0.05) expression of COX2 in Jersey embryos, but had no effect on Nellore embryos. Expression of HSF1 was decreased (P < 0.05) by heat stress in both breeds, with a greater effect in Nellore embryos. In experiment 3, heat stress tended (P = 0.1) to decrease the percentage of pregnancies among cows (Day 30 to 35) that received Angus embryos. In experiment 4, heat stress increased (P < 0.05) the percentage of apoptotic blastomeres, but had no breed-specific effects. In addition, Nellore embryos had fewer (P < 0.05) Terminal deoxynucleotidyl transferase dUTP nick end labeling- positive blastomeres than did Angus embryos. We concluded that the detrimental effects of heat stress were dependent upon embryo breed and were more evident in Bos taurus embryos than in Bos indicus embryos. © 2013 Elsevier Inc.