Genome wide scan for quantitative trait loci affecting tick resistance in cattle (Bos taurus × Bos indicus)

Abstract Background In tropical countries, losses caused by bovine tick Rhipicephalus (Boophilus) microplus infestation have a tremendous economic impact on cattle production systems. Genetic variation between Bos taurus and Bos indicus to tick resistance and molecular biology tools might allow for the identification of molecular markers linked to resistance traits that could be used as an auxiliary tool in selection programs. The objective of this work was to identify QTL associated with tick resistance/susceptibility in a bovine F2 population derived from the Gyr (Bos indicus) × Holstein (Bos taurus) cross. Results Through a whole genome scan with microsatellite markers, we were able to map six genomic regions associated with bovine tick resistance. For most QTL, we have found that depending on the tick evaluation season (dry and rainy) different sets of genes could be involved in the resistance mechanism. We identified dry season specific QTL on BTA 2 and 10, rainy season specific QTL on BTA 5, 11 and 27. We also found a highly significant genome wide QTL for both dry and rainy seasons in the central region of BTA 23. Conclusions The experimental F2 population derived from Gyr × Holstein cross successfully allowed the identification of six highly significant QTL associated with tick resistance in cattle. QTL located on BTA 23 might be related with the bovine histocompatibility complex. Further investigation of these QTL will help to isolate candidate genes involved with tick resistance in cattle.

Patterns of cell proliferation and apoptosis by topographic region in normal Bos taurus vs. Bos indicus crossbreeds bovine placentae during pregnancy

Background: Placental and fetal growth requires high rates of cellular turnover and differentiation, which contributes to conceptus development. The trophoblast has unique properties and a wide range of metabolic, endocrine and angiogenic functions, but the proliferative profile of the bovine placenta characterized by flow cytometry analysis and its role in fetal development are currently uncharacterized. Complete understanding of placental apoptotic and proliferative rates may be relevant to development, especially if related to the pathogenesis of pregnancy losses and placental abnormalities. Methods: In this study, the proliferation activity and apoptosis in different regions of normal bovine placenta (central and boundary regions of placentomes, placentomal fusion, microplacentomes, and interplacentomal regions), from distinct gestation periods (Days 70 to 290 of pregnancy), were analyzed by flow cytometry. Results: Our results indicated that microplacentomes presented a lower number of apoptotic cells throughout pregnancy, with a higher proliferative activity by the end of gestation, suggesting that such structures do not contribute significantly to normal of placental functions and conceptus development during pregnancy. The placentome edges revealed a higher number of apoptotic cells from Day 170 on, which suggests that placentome detachment may well initiate in this region. Conclusion: Variations involving proliferation and apoptotic rates may influence placental maturation and detachment, compromising placental functions and leading to fetal stress, abnormalities in development and abortion, as frequently seen in bovine pregnancies from in vitro fertilization and cloning procedures. Our findings describing the pattern of cell proliferation and apoptosis in normal bovine pregnancies may be useful for unraveling some of the developmental deviations seen in nature and after in vitro embryo manipulations.

Genetic diversity of the Brazilian Creole cattle Pe-duro assessed by microsatellites and mitochondrial DNA

The objective of this study was to describe the genetic diversity and structure of the largest Pe-duro population by assessing variation at ten autosomal microsatellite (STR) loci and mitochondrial DNA (mtDNA) sequences. The mean expected heterozygosity was 0.755, the mean observed heterozygosity was 0.600 and significant inbreeding coefficient (Fis) and deviations from the Hardy-Weinberg equilibrium in most of analyzed loci demonstrate the impact of inbreeding and homozygosis on this population. A more in-depth genetic analysis could be achieved by expanding the STR list. The analysis of mtDNA provided evidence of ancestral African taurine haplotypes in Pe-duro and excluded maternal Zebuine introgression. In this report, the main Pe-duro population is genetically portrayed by sampling approximately 40% of it. As this herd represents the core of the Pe-duro conservation program, these findings are of outstanding value for the management and preservation of this Brazilian 'native' cattle breed.

Fatty acid composition of the longissimus dorsi muscle in crossbred steers fed different sources of fatty acids

The objective of this study was to evaluate the fatty acid composition of the longissimus dorsi muscle in carcasses of 3/4 Bos taurus taurus 1/4 Bos taurus indicus steers fed different sources of fatty acids. Thirty-six steers aged 14 months, with a mean live weight of 320 kg, were fed the following diets for 96 days:1) control diet, containing no supplemental fat source; 2) CaSFA, diet containing 50 g calcium salts of fatty acids per kg total dry matter; 3) CS diet, containing 210 g cottonseed per kg total dry matter. The fatty acid composition of the longissimus dorsi muscle was determined by gas chromatography. No difference in slaughter weight, carcass weight, backfat thickness, or longissimus dorsi muscle area was observed between animals receiving the diets CaSFA and CS. Animals consuming the two fat-supplemented diets presented higher concentrations of oleic (C18:1), palmitic (C16:0) and stearic (C18:0) acids, corresponding to an average 80.76% of total fatty acids, and higher concentrations of vaccenic acid (C18:1 t11) in the muscle when compared with the control group. Supplementation of the diet of feedlot crossbred steers with CaSFA or cottonseed did not promote significant alterations in the lipid composition of the longissimus dorsi muscle.

ERYTHROGRAM PROFILE DURING POSTPARTUM PERIOD IN HOLSTEIN COWS

With the intention to evaluate the erythrogram profile during postpartum period in Holstein cows, 142 blood samples were collected from cows during the first 90 days after parturition. Blood samples were collected from clinically healthy cows that were non-reactive to the antigen of bovine leukosis virus (GP 51). These animals were divided into nine experimental groups based on the time of the postpartum sample collection. The blood samples were collected with EDTA and submitted to the following tests: erythrocyte counts, packed cell volume, hemoglobin concentration and calculated blood indices (MCV, MCH, MCHC). There was influence of the postpartum period on the erythrogram. These alterations were observed during the first 24 h after parturition. The hemoconcentration observed during the parturition disappeared during the first 24h of postpartum. Variation of the values were observed: red blood, 5.08 +/- 0.60 and 6.11 +/- 0.72 cells /mu L; hemoglobin concentration, 9.85 +/- 1.6 and 11.46 +/- 1,40 g/dL; packed cell volume, 27.9 +/- 3.3 and 31.9 +/- 3.6 %; MVC, 50.10 +/- 5.90 and 55.27 +/- 6.88 fL; MHC, 18.19 +/- 1.63 and 19.50 +/- 2.76 pg; MCHC, 34.83 +/- 2.07 and 36.88 +/- 1.67 %. The values of the hematological components obtained in this study did not exceed the limits of the reference values, obtained from cows raised within the state of Sao Paulo.

MUC1 gene polymorphism in three Nelore lines selected for growth and its association with growth and carcass traits

The objective of this study was to describe the VNTR polymorphism of the mucin 1 gene (MUC1) in three Nelore lines selected for yearling weight to determine whether allele and genotype frequencies of this polymorphism were affected by selection for growth. In addition, the effects of the polymorphism on growth and carcass traits were evaluated. Birth, weaning and yearling weights, rump height, Longissimus muscle area, backfat thickness, and rump fat thickness, were analyzed. A total of 295 Nelore heifers from the Beef Cattle Research Center, Instituto de Zootecnia de Sertozinho, were used, including 41 of the control line, 102 of the selection line and 152 of the traditional. The selection and traditional lines comprise animals selected for higher yearling weight, whereas control line animals are selected for yearling weight close to the average. Five alleles were identified, with allele 1 being the most frequent in the three lines, especially in the lines selected for higher means for yearling weight. Heterozygosity was significantly higher in the control line. Association analyses showed significant effects of allele 1 on birth weight and weaning weight while the allele 3 exert significant effects on yearling weight and back fat thickness. Despite these findings, application of this marker to marker-assisted selection requires more consistent results based on the genotyping of a larger number of animals in order to increase the accuracy of the statistical analyses.

The methylation patterns of the IGF2 and IGF2R genes in bovine spermatozoa are not affected by flow-cytometric sex sorting

The objectives of this study were to investigate the effect of sexing by flow cytometry on the methylation patterns of the IGF2 and IGF2R genes. Frozen-thawed, unsorted, and sex-sorted sperm samples from four Nellore bulls were used. Each ejaculate was separated into three fractions: non-sexed (NS), sexed for X-sperm (SX), and sexed for Y-sperm (SY). Sperm were isolated from the extender, cryoprotectant, and other cell types by centrifugation on a 40:70% Percoll gradient, and sperm pellets were used for genomic DNA isolation. DNA was used for analyses of the methylation patterns by bisulfite sequencing. Methylation status of the IGF2 and IGF2R genes were evaluated by sequencing 195 and 147 individual clones, respectively. No global differences in DNA methylation were found between NS, SX, and SY groups for the IGF2 (P=0.09) or IGF2R genes (P=0.38). Very specific methylation patterns were observed in the 25th and 26th CpG sites in the IGF2R gene. representing higher methylation in NS than in the SX and SY groups compared with the other CpG sites. Further, individual variation in methylation patterns was found among bulls. In conclusion, the sex-sorting procedure by flow cytometry did not affect the overall DNA methylation patterns of the IGF2 and IGF2R genes, although individual variation in their methylation patterns among bulls was observed. Mol. Reprod. Dev. 79:7784, 2012. (C) 2011 Wiley Periodicals, Inc.

Developmental and Epigenetic Anomalies in Cloned Cattle

Many of the developmental anomalies observed in cloned animals are related to foetal and placental overgrowth, a phenomenon known as the 'large offspring syndrome' (LOS) in ruminants. It has been hypothesized that the epigenetic control of imprinted genes, that is, genes that are expressed in a parental-specific manner, is at the root of LOS. Our recent research has focused on understanding epigenetic alterations to imprinted genes that are associated with assisted reproductive technologies (ART), such as early embryo in vitro culture (IVC) and somatic cell nuclear transfer (SCNT) in cattle. We have sought and identified single nucleotide polymorphisms in Bos indicus DNA useful for the analysis of parental-specific alleles and their respective transcripts in tissues from hybrid embryos derived by crossing Bos indicus and Bos taurus cattle. By analysing differentially methylated regions (DMRs) of imprinted genes SNRPN, H19 and the IGF2R in cattle, we demonstrated that there is a generalized hypomethylation of the imprinted allele and the biallelic expression of embryos produced by SCNT when compared to the methylation patterns observed in vivo (artificially inseminated). Together, these results indicate that imprinting marks are erased during the reprogramming of the somatic cell nucleus during early development, indicating that such epigenetic anomalies may play a key role in mortality and morbidity of cloned animals.

Degree of multicollinearity and variables involved in linear dependence in additive-dominant models

The objective of this work was to assess the degree of multicollinearity and to identify the variables involved in linear dependence relations in additive-dominant models. Data of birth weight (n=141,567), yearling weight (n=58,124), and scrotal circumference (n=20,371) of Montana Tropical composite cattle were used. Diagnosis of multicollinearity was based on the variance inflation factor (VIF) and on the evaluation of the condition indexes and eigenvalues from the correlation matrix among explanatory variables. The first model studied (RM) included the fixed effect of dam age class at calving and the covariates associated to the direct and maternal additive and non-additive effects. The second model (R) included all the effects of the RM model except the maternal additive effects. Multicollinearity was detected in both models for all traits considered, with VIF values of 1.03 - 70.20 for RM and 1.03 - 60.70 for R. Collinearity increased with the increase of variables in the model and the decrease in the number of observations, and it was classified as weak, with condition index values between 10.00 and 26.77. In general, the variables associated with additive and non-additive effects were involved in multicollinearity, partially due to the natural connection between these covariables as fractions of the biological types in breed composition.

Genetic diversity of the Brazilian Creole cattle Pé-duro assessed by microsatellites and mitochondrial DNA

The objective of this study was to describe the genetic diversity and structure of the largest Pé-duro population by assessing variation at ten autosomal microsatellite (STR) loci and mitochondrial DNA (mtDNA) sequences. The mean expected heterozygosity was 0.755, the mean observed heterozygosity was 0.600 and significant inbreeding coefficient (Fis) and deviations from the Hardy-Weinberg equilibrium in most of analyzed loci demonstrate the impact of inbreeding and homozygosis on this population. A more in-depth genetic analysis could be achieved by expanding the STR list. The analysis of mtDNA provided evidence of ancestral African taurine haplotypes in Pé-duro and excluded maternal Zebuine introgression. In this report, the main Pé-duro population is genetically portrayed by sampling approximately 40% of it. As this herd represents the core of the Pé-duro conservation program, these findings are of outstanding value for the management and preservation of this Brazilian 'native' cattle breed.

Interaction of dietary vitamin D-3 and sunlight exposure on B. indicus cattle: Animal performance, carcass traits, and meat quality

Attempts to improve beef tenderness through supplementation with dietary vitamin D-3 have been challenged by null results and negative impacts on animal performance and carcass traits. Because vitamin D-3 is also synthesised by the animal via ultraviolet radiation from sunlight, the effectiveness of supplementation with dietary vitamin D-3 may be modulated by the degree of exposure of the animal to sunlight. Hence, this work aimed to verify whether dietary vitamin D-3 modifies meat quality without negatively affecting animal performance and carcass traits in B. indicus beef cattle that were either exposed to or protected from natural sunlight. Forty-two (411 +/- 38 kg) Nellore-type castrated males were fed a high-concentrate diet for 45 days after assignment to a treatment group. The treatments comprised combinations of three levels of vitamin D3 [ViTD - none (V0) or 2 x 10(6) IU of vitamin D-3 administered for either 2 (V2) or 8 (V8) consecutive days pre-slaughter] and two shading conditions (SHADE - unshaded or shaded). The post-mortem (pm) measurements were taken in the Longissimus thoracis et lumborum muscle. The animal performance and carcass traits were unaffected by ViTD or SHADE The V2 treatment increased the Myofibrillar Fragmentation Index in shaded animals compared to unshaded ones. Animals under shade had higher muscle calcium concentration. There was no effect of either ViTD or SHADE on the shear force. The L* values were higher at 24 h pm than at 0 and 1 h pm, with no differences among the animals in the ViTD or SHADE groups. Higher a* values were observed among animals in the V8 group than in the V0 group, and higher b* values were observed among animals in the V8 group than in the V2 or V0 groups, which were not different. In conclusion, ViTD and SHADE did not affect animal performance, carcass traits or shear force, whereas animals receiving a lower ViTD dosage and SHADE exhibited altered myofibrillar fragmentation. ViTD affected the colour parameters, and changes in the lightness of the beef related to the time pm were found in meat from animals under SHADE. (c) 2012 Elsevier B.V. All rights reserved.

Effects of two estradiol esters (benzoate and cypionate) on the induction of synchronized ovulations in Bos indicus cows submitted to a timed artificial insemination protocol

The effects of estradiol benzoate (EB) and estradiol cypionate (EC) on induction of ovulation after a synchronized LH surge and on fertility of Bos indicus females submitted to timed AI (TAI) were evaluated. In Experiment 1, ovariectomized Nelore heifers were used to evaluate the effect of EB (n = 5) and EC (n = 5) on the circulating LH profile. The LH surge timing (19.6 and 50.5 h; P = 0.001), magnitude (20.5 and 9.4 ng/mL; P = 0.005), duration (8.6 and 16.5 h; P = 0.001), and area under the LH curve (158.6 and 339.4 ng/mL; P = 0.01) differed between the EB and EC treatments, respectively. In Experiment 2 (follicular responses; n = 60) and 3 (pregnancy per AI; P/AI; n = 953) suckled Bos indicus beef cows submitted to an estradiol/progesterone-based synchronization protocol were assigned to receive one of two treatments to induce synchronized ovulation: 1 mg of EB im 24 h after progesterone (P4) device removal or 1 mg of EC im at P4 device removal. There was no difference (P > 0.05) between EB and EC treatments on follicular responses (maximum diameter of the ovulatory follicle, 13.1 vs. 13.9 mm; interval from progesterone device removal to ovulation, 70.2 vs. 68.5 h; and ovulation rate, 77.8 vs. 82.8%, respectively). In addition, P/AI was similar (P < 0.22) between the cows treated with EB (57.5%; 277/482) and EC (61.8%; 291/471). In conclusion, despite pharmacologic differences, both esters of estradiol administered either at P4 device removal (EC) or 24 h later (EB) were effective in inducing an LH surge which resulted in synchronized ovulations and similar P/AI in suckled Bos indicus beef cows submitted to TAI. (C) 2012 Elsevier Inc. All rights reserved.

Characterization of atypical Listeria innocua isolated from swine slaughterhouses and meat markets

Atypical Listeria innocua strains presenting phenotypic characteristics similar to those of Listeria monocyto genes were recently isolated from food and the environment. These isolates also tested positive for virulence genes specific to L. monocytogenes. Here we report the isolation of atypical hemolytic L. innocua strains from the environment of pork processing plants in Brazil. The strains were positive for L. monocytogenes virulence genes hly, inlA and inlB by PCR and presented genotypic similarities with human isolates of L. monocytogenes via the AFLP technique using HindIII single enzyme protocol. Phenotypic and genotypic similarities suggest that these atypical L. innocua may be pathogenic strains. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Characterization of yolk sac proteins of Bos indicus cattle embryos

The yolk sac is an embryonic membrane that is essential for the embryo's initial survival in many mammals. It also plays an important role in the production of proteins necessary for development. We studied proteins of the yolk sac in bovine embryos at up to 40 days of gestation. We examined the yolk sac of 17 bovine embryos at different gestational periods, measuring a-fetoprotein, alpha-1-antitrypsin, and transferrin. This experiment was carried out by Western blot technique, associated with electrophoresis on a 6% sodium dodecyl sulfate polyacrylamide gel. Mouse monoclonal antibody anti-human-alpha-fetoprotein, mouse antibody anti-human-transferrin and rabbit polyclonal anti-human-alpha-1-antitrypsin were used as primary antibodies, and conjugated peroxidase as a secondary antibody. We detected the three proteins in some of the yolk sac samples; however, the bands in some specimens (samples) were weak, maybe a result of poor antigen-antibody reaction, since the antibodies used in this study were not specific to bovine proteins. The fact that weak bands appeared might be due to a weak cross-reaction.

Enterococcus faecium isolated from Lombo, a Portuguese traditional meat product: characterisation of antibacterial compounds and factors affecting bacteriocin production

Strain ST211CH, identified as a strain of Enterococcus faecium, isolated from Lombo produced a bacteriocin that inhibited the growth of Enterococcus spp., Listeria spp., Klebsiella spp., Lactobacillus spp., Pseudomonas spp., Staphylococcus spp. and Streptococcus spp. The mode of action of the bacteriocin named as bacteriocin ST211Ch was bactericidal against Enterococcus faecalis ATCC19443. As determined by Tricine-SDS-PAGE, the approximate molecular mass of the bacteriocin was 8.0 kDa. Loss in antimicrobial activity was recorded after treatment with proteolytic enzymes. Maximum activity of bacteriocin ST211Ch was measured in broth cultures of E. faecium strain ST211Ch after 24 h; thereafter, the activity was reduced. Bacteriocin ST211Ch remained active after exposure to various temperatures and pHs, as well as to Triton X-100, Tween-80, Tween-20, sodium dodecyl sulfate, NaCl, urea and EDTA. Effect of media components on production of bacteriocin ST211Ch was also studied. On the basis of PCR reactions targeting different bacteriocin genes, i.e. enterocins, curvacins and sakacins, no evidences for the presence of these genes in the total DNA of E. faecium strain ST211Ch was obtained. The bacterium most probably produced a bacteriocin different from those mentioned above. Based on the antimicrobial spectrum, stability and mode of action of bacteriocin ST211CH, E. faecium strain ST211Ch might be considered as a potential candidate with beneficial properties for use in biopreservation to control food spoilage bacteria.