Molecular and serological characterization of Leptospira interrogans serovar Canicola isolated from dogs, swine, and bovine in Brazil

The identification of Leptospira clinical isolates through genotyping and serotyping, besides the recognition of its reservoirs, are important tools for understanding the epidemiology of leptospirosis, and they are also keys for identifying new species and serovars. Fourteen clinical isolates from animals were characterized by means of single enzyme amplified length polymorphism, variable number of tandem repeat analysis, pulsed field gel electrophoresis, and serotyping. All isolates were identified as Leptospira interrogans, serovar Canicola. Infections by this serovar occur in urban regions, where dogs represent the main maintenance hosts, whereas bovine and swine may act as reservoirs of serovar Canicola in rural areas. Both urban and rural aspects of leptospirosis, and the role of domestic animals as maintenance hosts, cannot be neglected in developing and developed countries.

Norepinephrine stimulates progesterone production in highly estrogenic bovine granulosa cells cultured under serum-free, chemically defined conditions

Background: Since noradrenergic innervation was described in the ovarian follicle, the actions of the intraovarian catecholaminergic system have been the focus of a variety of studies. We aimed to determine the gonadotropin-independent effects of the catecholamine norepinephrine (NE) in the steroid hormone profile of a serum-free granulosa cell (GC) culture system in the context of follicular development and dominance. Methods: Primary bovine GCs were cultivated in a serum-free, chemically defined culture system supplemented with 0.1% polyvinyl alcohol. The culture features were assessed by hormone measurements and ultrastructural characteristics of GCs. Results: GCs produced increasing amounts of estradiol and pregnenolone for 144h and maintained ultrastructural features of healthy steroidogenic cells. Progesterone production was also detected, although it significantly increased only after 96h of culture. There was a highly significant positive correlation between estradiol and pregnenolone production in high E2-producing cultures. The effects of NE were further evaluated in a dose response study. The highest tested concentration of NE (10 (-7) M) resulted in a significant increase in progesterone production, but not in estradiol or pregnenolone production. The specificity of NE effects on progesterone productio n was further investigated by incubating GCs with propranolol (10 (-8) M), a non-selective beta-adrenergic antagonist. Conclusions: The present culture system represents a robust model to study the impact of intrafollicular factors, such as catecholamines, in ovarian steroidogenesis and follicular development. The results of noradrenergic effects in the steroidogenesis of GC have implications on physiological follicular fate and on certain pathological ovarian conditions such as cyst formation and anovulation.

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.

Impact of experimental Nano-HAP pastes on bovine enamel and dentin submitted to a pH cycling model

This in vitro study evaluated the preventive potential of experimental pastes containing 10% and 20% hydroxyapatite nanoparticles (Nano-HAP), with or without fluoride, on dental demineralization. Bovine enamel (n=15) and root dentin (n=15) specimens were divided into 9 groups according to their surface hardness: control (without treatment), 20 Nanop paste (20% HAP), 20 Nanop paste plus (20% HAP + 0.2% NaF), 10 Nanop paste (10% HAP), 10 Nanop paste plus (10% HAP + 0.2% NaF), placebo paste (without fluoride and HAP), fluoride paste (0.2% NaF), MI paste (CPP-ACP, casein phosphopeptide-amorphous calcium phosphate), and MI paste plus (CPP-ACP + 0.2% NaF). Both MI pastes were included as commercial control products containing calcium phosphate. The specimens were treated with the pastes twice a day (1 min), before and after demineralization. The specimens were subjected to a pH-cycling model (demineralization–6-8 h/ remineralization-16-18 h a day) for 7 days. The dental subsurface demineralization was analyzed using cross-sectional hardness (kgf/mm 2 , depth 10-220 µm). Data were tested using repeated-measures two-way ANOVA and Bonferroni's test (p<0.05). The only treatment able to reduce the loss of enamel and dentin subsurface hardness was fluoride paste (0.2% NaF), which differed significantly from the control at 30- and 50-µm depth (p<0.0001). The other treatments were not different from each other or compared with the control. The experimental Nanop pastes, regardless of the addition of fluoride, were unable to reduce dental demineralization in vitro.

In situ effect of chewing gum containing CPP–ACP on the mineral precipitation of eroded bovine enamel

Objectives: Stimulation of salivary flow is considered a preventive strategy for dental erosion. Alternatively, products containing calcium phosphate, such as a complex of casein phosphopeptide–amorphous calcium phosphate (CPP–ACP), have also been tested against dental erosion. Therefore, this in situ study analyzed the effect of chewing gum containing CPP–ACP on the mineral precipitation of initial bovine enamel erosion lesions. Methods: Twelve healthy adult subjects wore palatal appliances with two eroded bovine enamel samples. The erosion lesions were produced by immersion in 0.1% citric acid (pH 2.5) for 7 min. During three experimental crossover in situ phases (1 day each), the subjects chewed a type of gum, 3 times for 30 min, in each phase: with CPP–ACP (trident total), without CPP–ACP (trident), and no chewing gum (control). The Knoop surface microhardness was measured at baseline, after erosion in vitro and the mineral precipitation in situ. The differences in the degree of mineral precipitation were analyzed using repeated measures (RM-) ANOVA and post hoc Tukey’s test ( p < 0.05). Results: Significant differences were found among the remineralizing treatments ( p < 0.0001). Chewing gum (19% of microhardness recovery) improved the mineral precipitation compared to control (10%) and the addition of CPP–ACP into the gum promoted the best mineral precipitation effect (30%). Conclusions: Under this protocol, CPP–ACP chewing gum improved the mineral precipitation of eroded enamel. Clinical significance: Since the prevalence of dental erosion is steadily increasing, CPP–ACP chewing gum might be an important strategy to reduce th eprogression of initial erosion lesions.

Invasion of Ureaplasma diversum in bovine spermatozoids

Background Ureaplasma diversum has been associated with infertility in cows. In bulls, this mollicute colonizes the prepuce and distal portion of the urethra and may infect sperm cells. The aim of this study is to analyze in vitro interaction of U. diversum isolates and ATCC strains with bovine spermatozoids. The interactions were observed by confocal microscopy and the gentamycin internalization assay. Findings U. diversum were able to adhere to and invade spermatozoids after 30 min of infection. The gentamicin resistance assay confirmed the intracellularity and survival of U. diversum in bovine spermatozoids. Conclusions The intracellular nature of bovine ureaplasma identifies a new difficulty to control the reproductive of these animals.

Evaluation of inductively coupled plasma mass spectrometry for determining Ca, Cu, Fe, Mg, Mn, Se and Zn in bovine semen samples using a simple sample dilution method

A simple and fast method for the determination of Ca, Cu, Fe, Mg, Mn, Se and Zn in bovine semen by quadrupole inductively coupled plasma spectrometry (q-ICP-MS) is described. Prior to analysis, samples (200 µL) were diluted 1:50 in a solution containing 0.01% v/v Triton® X-100 and 0.5% v/v nitric acid and directly analyzed by ICP-MS. The limits of detection of the method are 0.3, 0.03, 0.2, 0.04, 0.04, 0.03 and 0.03 µg L-1 for 44Ca, 63Cu, 57Fe, 24Mg, 64Zn, 82Se and 55Mn, respectively. For purposes of comparison and method validation, four ordinary bovine semen samples were directly analyzed by ICP-MS and by flame atomic absorption spectrometry (FAAS) or graphite furnace atomic absorption spectrometry (GF AAS), with no statistical difference between the techniques at the 95% level when applying the t-test. Then, the proposed method was applied in the determinations of Ca, Cu, Fe, Mg, Mn, Se and Zn in collected samples of bovine semen from different breeds, which are used in reproduction programs and artificial insemination.

Exploring anorganic bovine bone granules as osteoblast carriers for bone bioengineering: a study in rat critical-size calvarial defects

It is known that current trends on bone bioengineering seek ideal scaffolds and explore innovative methods to restore tissue function. In this way, the objective of this study was to evaluate the behavior of anorganic bovine bone as osteoblast carrier in critical-size calvarial defects. MC3T3-E1 osteoblast cells (1x10(5) cells/well) were cultured on granules of anorganic bovine bone in 24-well plates and after 24 h these granules were implanted into rat critical-size calvarial defects (group Biomaterial + Cells). In addition, other groups were established with different fillings of the defect: Blood Clot (negative control); Autogenous Bone (positive control); Biomaterial (only granules) and Cells (only MC3T3-E1 cells). After 30 days, the animals were euthanized and the calvaria were technically processed in order to allow histological and morphometric analysis. It was possible to detect blood vessels, connective tissue and newly formed bone in all groups. Particularly in the Biomaterial + Cells group, it was possible to observe a profile of biological events between the positive control group (autogenous bone) and the group in which only anorganic bovine granules were implanted. Altogether, the results of the present study showed that granules of anorganic bovine bone can be used as carrier to osteoblasts and that adding growth factors at the moment of implantation should maximize these results.

Effect of beverages on bovine dental enamel subjected to erosive challenge with hydrochloric acid

This study evaluated by an in vitro model the effect of beverages on dental enamel previously subjected to erosive challenge with hydrochloric acid. The factor under study was the type of beverage, in five levels: Sprite® Zero Low-calorie Soda Lime (positive control), Parmalat® ultra high temperature (UHT) milk, Ades® Original soymilk, Leão® Ice Tea Zero ready-to-drink low-calorie peach-flavored black teaand Prata® natural mineral water (negative control). Seventy-five bovine enamel specimens were distributed among the five types of beverages (n=15), according to a randomized complete block design. For the formation of erosive wear lesions, the specimens were immersed in 10 mL aqueous solution of hydrochloric acid 0.01 M for 2 min. Subsequently, the specimens were immersed in 20 mL of the beverages for 1 min, twice daily for 2 days at room temperature. In between, the specimens were kept in 20 mL of artificial saliva at 37ºC. The response variable was the quantitative enamel microhardness. ANOVA and Tukey's test showed highly significant differences (p<0.00001) in the enamel exposed to hydrochloric acid and beverages. The soft drink caused a significantly higher decrease in microhardness compared with the other beverages. The black tea caused a significantly higher reduction in microhardness than the mineral water, UHT milk and soymilk, but lower than the soft drink. Among the analyzed beverages, the soft drink and the black tea caused the most deleterious effects on dental enamel microhardness.

Phylogenetic relation among bovine rabies cases from Minas Gerais and Sao Paulo States, Brazil: a possible common origin

Acknowledgements: CAPES, FAPESP process number 2011/06236-0

Storage of bovine reproductive tissues and RNA extracts on ice for 24h or repeated freeze–thaw cycles do not affect RNA integrity

The aims of this study were to test (i) the effect of time of tissue and RNA extracts storage on ice and (ii) the effect of repeated freeze–thaw cycles on RNA integrity and gene expression of bovine reproductive tissues. Fragments of endometrium (ENDO), corpus luteum (CL) and ampulla (AMP) were subdivided and incubated for 0, 1, 3, 6, 12 or 24 h on ice. RNA extracts were incubated on ice for 0, 3, 12 or 24 h, or exposed to 1, 2, 4 or 6 freeze–thaw cycles. RNA integrity number (RIN) was estimated. Expression of progesterone receptor (PGR) and cyclophilin genes from RNA extracts stored on ice for 0 or 24 h, and 1 or 6 freeze–thaw cycles was measured by qPCR. Tissue and RNA extract incubation on ice, and repeated freeze–thaw cycles did not affect RIN values of RNA from ENDO, CL or AMP. Storage on ice or exposure to freeze–thaw cycles did not affect Cq values for PGR or cyclophilin genes. In conclusion, neither generalized RNA degradation nor specific RNA degradation was affected by storage of tissue or RNA extracts on ice for up to 24 h, or by up to 6 freeze–thaw cycles of RNA extracts obtained from bovine ENDO, CL and AMP.

Heparin and penicillamine–hypotaurine–epinephrine (PHE) solution during bovine in vitro fertilization procedures impair the quality of spermatozoa but improve normal oocyte fecundation and early embryonic development

The presence of heparin and a mixture of penicillamine, hypotaurine, and epinephrine (PHE) solution in the in vitro fertilization (IVF) media seem to be a prerequisite when bovine spermatozoa are capacitated in vitro, in order to stimulate sperm motility and acrosome reaction. The present study was designed to determine the effect of the addition of heparin and PHE during IVF on the quality and penetrability of spermatozoa into bovine oocytes and on subsequent embryo development. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes and mitochondrial function, was diminished (P<0.05) in the presence of heparin and PHE. Oocyte penetration and normal pronuclear formation rates, as well as the percentage of zygotes presenting more than two pronuclei, was higher (P<0.05) in the presence of heparin and PHE. No differences were observed in cleavage rates between treatment and control (P>0.05). However, the developmental rate to the blastocyst stage was increased in the presence of heparin and PHE (P>0.05). The quality of embryos that reached the blastocyst stage was evaluated by counting the inner cell mass (ICM) and trophectoderm (TE) cell numbers and total number of cells; the percentage of ICM and TE cells was unaffected (P>0.05) in the presence of heparin and PHE (P<0.05). In conclusion, this study demonstrated that while the supplementation of IVF media with heparin and PHE solution impairs spermatozoa quality, it plays an important role in sperm capacitation, improving pronuclear formation, and early embryonic development

Effects of flavomycin on ruminal fermentation, In situ degradability and In vivo digestibility in bovine fed sugarcane diets

Problem statement: The aim of the present study was to characterize and differentiate the effects of addition of flavomycin or monensin on ruminal fermentation and degradability as well as on total digestibility in bovine. Approach: Twelve non-pregnant and non-lactating cows (736 kg of BW) were randomly assigned to three treatments: control, flavomycin (20 mg animal-1 day-1) and monensin (300 mg animal-1 day-1). The trial lasted 21 days. The last 10 days were used for external marker administration (15 g of chromic oxide animal-1 day-1). The last 5 days of the trial were used for feces collection and evaluation of corn grain, soybean meal or sugarcane ruminal degradability and the 21st day was used for ruminal fluid sampling. Results: Monensin increased 27.2%, on average, propionate molar proportion at 0, 4, 6, 8, 10 and 12 h after feeding, compared to control and flavomycin groups. When compared to control, flavomycin reduced the degradation rate of soybean meal CP in 31.0%, decreasing the effective degradability when passage rates of 5 and 8% h-1 were used. Dry matter intake, pH, total Short Chain Fatty Acids (tSCFA) or ammoniacal Nitrogen (NH3-N) concentration were not influenced by the addition of either antibiotics. Effective degradability of sugarcane NDF was not influenced by the use of either antibiotic; neither were the TDN nor the digestibility of DM, CP, EE, NFE, ADF, NDF, GE or starch of the diet. Conclusion/Recommendations: In the present study, it was possible to show the beneficial effects of monensin but not of flavomycin, on rumen fermentation

Photobiological effect of low-level laser irradiation in bovine embryo production system

The objectives of this study were to evaluate the effect of low-level laser irradiation (LLLI) on bovine oocyte and granulosa cells metabolism during in vitro maturation (IVM) and further embryo development. Cumulus-oocytes complexes (COCs) were subjected (experimental group) or not (control group) to irradiation with LLLI in a 633-nm wavelength and 1 J/cm2 fluency. The COCs were evaluated after 30 min, 8, 16, and 24 h of IVM. Cumulus cells were evaluated for cell cycle status, mitochondrial activity, and viability (flow cytometry). Oocytes were assessed for meiotic progression status (nuclear staining), cell cycle genes content [real-time polymerase chain reaction (PCR)], and signal transduction status (western blot). The COCs were also in vitro fertilized, and the cleavage and blastocyst rates were assessed. Comparisons among groups were statistically performed with 5% significance level. For cumulus cells, a significant increase in mitochondrial membrane potential and the number of cells progressing through the cycle could be observed. Significant increases on cyclin B and cyclin-dependent kinase (CDK4) levels were also observed. Concerning the oocytes, a significantly higher amount of total mitogen-activated protein kinase was found after 8 h of irradiation, followed by a decrease in all cell cycle genes transcripts, exception made for the CDK4. However, no differences were observed in meiotic progression or embryo production. In conclusion, LLLI is an efficient tool to modulate the granulosa cells and oocyte metabolism

Bovine Teeth in Age Assessment, from Medieval Cattle to Belgian Blue : Methodology, Possibilities and Limitations

The overall aim of the present thesis was to develop and characterise an age assessment method based on incremental lines in dental cementum using contemporary bovine teeth and teeth from archaeological faunal assemblages. The investigations also included two other age assessment methods: tooth wear pattern and macroscopic dental measurements. The first permanent mandibular molar and lower jaws from 70 contemporary cattle of known age and 170 archaeological molar sets from ten different Swedish archaeological sites were used. The following conclusions were drawn: • The number of incremental lines in the dental cementum varied between different parts of the tooth root as well as within one and the same individual. The results from contemporary cattle of known age showed a strong relationship between age and incremental lines in the cementum of the distal part of the mesial root (R2=65.5%) and the known ages of the animals. • With the “best” model variation in age could be explained to 65.5% (R2) by the number of incremental lines. Thus, the remaining age variation (approximately 35%) could not be explained by these lines. Other factors than must thus be responsible. However, with the exception of calves born the present material did not reveal any such significant relationship. • The results from cattle of known age indicate that the method of assessing age on the basis of cemental incremental lines is more reliable than other methods such as tooth wear or tooth measurements. However, by combining counting incremental lines and one variable assessing tooth dimension (tooth height) a slightly stronger relationship could be obtained (R2=74.5%). The results from age assessment of the medieval and post-Reformation cattle emphasize the importance of supplementing any age estimation of archaeological assemblages based on dental indicators with characteristics for the particular assessment model. Furthermore, conclusions based on age assessment with such models can not be drawn with any more detailed time scale than about 2 years leaving at best only 25% (R2) of factors influencing the dental indicator(s) utilized in the model unexplained. The accuracy of the age assessment required by the particular historical context in which the archaeological remains are found should thus decide what level of accuracy should be chosen.