The effect of bovine interferon-alpha I1 on pregnancy rate in heifers.

Bovine interferon-alpha I1 (bIFN-alpha) may be useful for enhancing fertility in sheep and cattle because it has extensive sequence homology with ovine and bovine trophoblast protein-1 and, like those proteins, extends corpus luteum lifespan. To test the effectiveness of bIFN-alpha to enhance fertility, several experiments were performed in which inseminated heifers were given i.m. injections of bIFN-alpha approximately at the time of embryo-mediated signals that result in maintenance of the corpus luteum. In Exp. 1, heifers given 20 mg of bIFN-alpha daily from d 14 to 17 tended (P less than .07) to have lower pregnancy rates at d 110 to 112 of gestation (36/75; 48% vs 43/72; 60%). Similar results were obtained in Exp. 2 when heifers received a single injection of 40 mg of bIFN-alpha or placebo at d 13 after estrus; pregnancy rates at d 42 were 39/104 (38%) for bIFN-alpha and 47/98 (48%) for placebo. In Exp. 3, heifers were given gradually increasing doses of bIFN-alpha or placebo from d 11 to 19, because such a regimen had been shown to reduce the number of heifers experiencing hyperthermia after bIFN-alpha injection. Pregnancy rates were 42/95 (44%) for bIFN-alpha and 62/111 (56%) for placebo. Across all three experiments, pregnancy rates were lower (P less than .01) for heifers treated with bIFN-alpha (117/274; 43%) than for heifers treated with placebo (152/281; 54%). In conclusion, these results demonstrate that, under the administration systems used, bIFN-alpha does not increase pregnancy rate, but rather tends to reduce it.

Sex determination of murine and bovine embryos using cytotoxicity and immunofluorescence assays

Murine and bovine embryos were sexed by using polyclonal and monoclonal anti-H-Y antibodies in cytotoxic and immunofluorescence assays. The specificity of 3 different sources of antibodies are compared. Sexing was accomplished by complement-mediated cytotoxicity or by H-Y antigen detection using a anti-mouse Ig fluorescein conjugate. Cytogenetic ascertainment for correct sex identification yielded a score of about 85% in both species, while the immunofluorescence assay yielded a score of 83%. © 1993.

Structures of the noncovalent complexes of human and bovine prothrombin fragment 2 with human PPACK-thrombin

Both human and bovine prothrombin fragment 2 (the second kringle) have been cocrystallized separately with human PPACK (D-Phe-Pro-Arg)-thrombin, and the structures of these noncovalent complexes have been determined and refined (R = 0.155 and 0.157, respectively) at 3.3-Å resolution using X-ray crystallographic methods. The kringles interact with thrombin at a site that has previously been proposed to be the heparin binding region. The latter is a highly electropositive surface near the C-terminal helix of thrombin abundant in arginine and lysine residues. These form salt bridges with acidic side chains of kringle 2. Somewhat unexpectedly, the negative groups of the kringle correspond to an enlarged anionic center of the lysine binding site of lysine binding kringles such as plasminogens K1 and K4 and TPA K2. The anionic motif is DGDEE in prothrombin kringle 2. The corresponding cationic center of the lysine binding site region has an unfavorable Arg70Asp substitution, but Lys35 is conserved. However, the folding of fragment 2 is different from that of prothrombin kringle 1 and other kringles: the second outer loop possesses a distorted two-turn helix, and the hairpin β-turn of the second inner loop pivots at Val64 and Asp70 by 60°. Lys35 is located on a turn of the helix, which causes it to project into solvent space in the fragment 2-thrombin complex, thereby devastating any vestige of the cationic center of the lysine binding site. Since fragment 2 has not been reported to bind lysine, it most likely has a different inherent folding conformation for the second outer loop, as has also been observed to be the case with TPA K2 and the urokinase kringle. The movement of the Val64-Asp70 β-turn is most likely a conformational change accompanying complexation, which reveals a new heretofore unsuspected flexibility in kringles. The fragment 2-thrombin complex is only the second cassette module-catalytic domain structure to be determined for a multidomain blood protein and only the third domain-domain interaction to be described among such proteins, the others being factor Xa without a Gla domain and Ca2+ prothrombin fragment 1 with a Gla domain and a kringle. © 1993 American Chemical Society.

The effect of serum on in vitro maturation, in vitro fertilization and steroidogenesis of bovine oocytes co-cultured with granulosa cells.

The influence of fetal calf serum alone (FCS) or associated with proestrous (FCS+PCS), estrous (FCS+ECS) or metaestrous (FCS+MCS) cow serum added to the culture medium and of the steroids produced by co-cultured granulosa cells were evaluated in terms of the in vitro maturation (IVM) and fertilization (IVF) of bovine oocytes. Supplementation of the medium with FCS+ECS and FCS+MCS resulted in higher proportions of oocytes that reached metaphase II (96.0% and 93.3%, respectively) and in higher proportions of embryos that reached the four- and eight-cell/morula stages (51.9% and 65.6%, respectively), whereas the supplementation with FCS and FCS+PCS resulted in only 79.2% and 67.5%, respectively, of matured oocytes and 26.7% and 34.3%, respectively, of cleaved embryos. These findings show that the best IVM and IVF were obtained at lower concentrations of estradiol produced by co-cultured granulosa cells (supplementation with FCS+ECS: 10.3 ng/ml and FCS+MCS: 2.1 ng/ml), whereas the worst results in IVM and IVF occurred at higher concentrations of estradiol that were obtained with FCS (33.1 ng/ml) and FCS+PCS (19.9 ng/ml) supplementation. These data suggest an inhibitory effect of estradiol on resumption of oocyte meiosis in vitro.

Detection of leptospires in bovine semen by polymerase chain reaction

Objective: In view of the considerable importance of venereal transmission of bovine leptospirosis, the objective of the present study was to compare the polymerase chain reaction (PCR), culture/isolation and serology to detect leptospire infection in bovine semen. Design: Blood for serologic examination and semen for bacterial culture and PCR were collected from 20 bulls at artificial insemination centres in Brazil. Each animal was sampled twice for serology. Result: Forty-five percent (9/20) of the serum samples collected showed agglutinin titers to serovar hardjo in the first sample and 25% (5/20) had agglutinin titers to serovar hardjo in the second sample. Eighty percent (16/20) of semen samples were positive by PCR. Leptospires could not be isolated from any of the semen samples examined. Conclusion: Polymerase chain reaction can be a method of great potential for the detection of leptospires at artificial insemination centres.

Structure of tick anticoagulant peptide at 1.6 Å resolution complexed with bovine pancreatic trypsin inhibitor

The structure of tick anticoagulant peptide (TAP) has been determined by X-ray crystallography at t.6 Å resolution complexed with bovine pancreatic trypsin inhibitor (BPTI). The TAP-BPTI crystals are tetragonal, a = b = 46.87, c = 50.35 Å, space group P41, four complexes per unit cell. The TAP molecules are highly dipolar and form an intermolecular helical array along the c-axis with a diameter of about 45 Å. Individual TAP units interact in a head-to-tail fashion, the positive end of one molecule associating with the distal negative end of another, and vice versa. The BPTI molecules have a uniformly distributed positively charged surface that interacts extensively through 14 hydrogen bonds and two hydrogen bonded salt bridges with the helical groove around the helical TAP chains. Comparing the structure of TAP in TAP-BPTI with TAP bound to factor Xa(Xa) suggests a massive reorganization in the N-terminal tetrapeptide and the first disulfide loop of TAP (CyS5(T)- Cys 15(T)) upon binding to Xa. The Tyr1(T)OH atom of TAP moves 14.2 Å to interact with Asp189 of the S1 specificity site, Arg3(T)CZ moves 5.0 Å with the guanidinium group forming a cation-π-electron complex in the S4 subsite of Xa, while Lys7(T)NZ differs in position by 10.6 Å in TAP-BPTI and TAP-Xa, all of which indicates a different pre-Xa-bound conformation for the N- terminal of TAP in its native state. In contrast to TAP, the BPTI structure of TAP-BPTI is practically the same as all those of previously determined structures of BPTI, only arginine and lysine side-chain conformations showing significant differences.

Seroprevalence of bovine leptospirosis in Garanhuns municipal district, Pernambuco State, Brazil

The prevalence of Leptospira interrogans serovars in dairy cattle was determined by analyzing 464 serum samples from cows on 15 properties in Garanhuns municipal district, Pernambuco State, Brazil. A microscopic seroagglutination test including 12 serovars of Leptospira interrogans as antigens was used. Samples with titres 3 100 were considered positive. Two hundred and twenty-one (47.63%) of the samples were positive to one or more serovars. The prevalence of the serovars was hardjo (21.98%), bratislava (15.73%), castellonis(11.64%), tarassovi (10.56%), pyrogenes(1.72%), icterohaemorrhagiae (1.08%), pomona (0.86%), wolffi (0.86%), grippotyphosa (0.86%), djasiman (0.43%), canicola (0.21 %), and copenhageni (0.21 %).

A radiation hybrid map of bovine X Chromosome (BTAX)

We present a comprehensive radiation hybrid map of the bovine X chromosome (Chr) containing 20 new markers, including both microsatellites and expressed genes. This study was conducted with a 5000-rad whole genome RH cell panel consisting of 90 hybrid cell lines. Retention frequencies of individual markers range from 7.8% for XIST to 31.1% for TGLA325. Statistical analysis with RHMAPPER placed all the loci into five linkage groups under a LOD score criterion of 6.0. These groups could be oriented relative to each other because they included multiple microsatellite loci from the consensus linkage map of the X Chr. Markers included in both this RH map and the bovine cytogenetic map were in a consistent order. The comparative bovine-human map thus generated consists of five blocks of genes, the order of which is conserved, although in the opposite direction when presented as ideograms with p and q arms. Inversions of three blocks account for the difference in gene order across the entirety of the two X Chrs.

Growth hormone mRNA expression in the pituitary of Bos indicus and Bos taurus x Bos indicus crossbred young bulls treated with recombinant bovine somatotropin

The effects of breed and of recombinant bovine somatotropin (rbST) treatment on growth hormone gene expression were studied in young bulls. The experiment was completely randomized in a [2 × 2]-factorial arrangement, using two levels of rbst (0 or 250 mg/animal/14 days), and two breed groups (Nelore and Simmental x Nelore crossbred). A CDNA encoding Bos indicus growth hormone was cloned and sequenced for use as a probe in Northern and dot blot analyses. Compared to the Bos taurus structural gene, the Bos indicus CDNA was found to begin 21 bases downstream from the transcription initiation site and had only two discrepancies (C to T at position 144-His and T to C at position 354-Phe), without changes in the polypeptide sequence. However, two amino acid substitutions were found for Bubalus spp., which belong to the same tribe. The rbst treatment did not change any of the characteristics evaluated (body and pituitary gland weights, growth hormone MRNA expression level). Crossbred animals had significantly higher body weight and heavier pituitaries than Nelore cattle. Pituitary weight was proportional to body weight in both breed groups. Growth hormone MRNA expression in the pituitary was similar (P>0.075) for both breed and hormonal treatment groups, but was 31.9% higher in the pure Nelore group, suggesting that growth hormone gene transcription regulation differs among these breeds.

Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes

Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/ mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 μM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/ BSA for 18 to 20 h. For zona-free bovine oocytes, penetration rate (35%) with the combination of 8BrcAMP and ionomycin in PVA-containing medium was higher (P < 0.05) than any treatment in BSA-containing medium (5 to 6%). A similar study was conducted using equine oocytes with partially removed zonae. Sperm capacitated and used for in vitro fertilization (IVF) in PVA-containing medium had higher penetration rates (P < 0.01) than sperm in BSA-containing medium (54 vs. 11%). The effect of equine preovulatory follicular fluid on bovine oocyte penetration was assessed. Bovine oocytes were matured in tissue culture medium-199 with 0, 20, 50, or 100% equine preovulatory follicular fluid, and 1 IU/mL of equine chorionic gonadotropin. Stallion sperm were treated with 8BrcAMP + ionomycin in PVA- or BSA-containing media. The penetration rates of bovine zona-free oocytes by stallion sperm were again higher with PVA (47%) than BSA (18%; P < 0.01). Penetration rates of oocytes matured in 100% follicular fluid were higher (P < 0.05) than for oocytes matured with 0% follicular fluid. The effects of equine follicular fluid and PVA/BSA during sperm capacitation on standard bovine IVF were examined. Culture of bovine oocytes with equine follicular fluid did not affect oocyte maturation or penetration rates after IVF. Bovine sperm capacitated with heparin in PVA-containing medium yielded lower (P < 0.05) fertilization rates than those capacitated in BSA-containing medium when incubated with both zona-intact and zona-free bovine oocytes. In summary, PVA was superior to BSA for ionophore-induced capacitation of equine sperm for penetration of zona-free bovine oocytes or partially zona-removed equine oocytes, but not for standard bovine IVF with bovine sperm. Zona-free bovine oocytes may be useful for assaying in vitro capacitation and fertilization of stallion sperm. © 2003 American Society of Animal Science. All rights reserved.

Pheno and genotyping of Staphylococcus aureus, isolated from bovine milk samples from São Paulo State, Brazil

In the present study, 87 Staphylococcus aureus isolates obtained from milk samples of 87 cows with mastitis in 6 different municipal districts of 2 regions of São Paulo State, Brazil, were compared pheno and genotypically. Pulsed-field gel electrophoresis (PFGE) analysis of the strains was performed, and PCR was carried out to detect genes for a number of staphylococcal cell surface proteins, exoproteins, and 3 classes of agr genes. Nine distinct S. aureus lineages (LA-LI) were identified by PFGE. The lineages LA and LE, which accounted together for 63 strains (72.2%), were prevalent and had been collected from all of the 6 municipal districts, indicating a broad geographic distribution of these lineages; LB, LC, LD, LF, LG, LH, and LI, however, were isolated sporadically and accounted for 24 strains (27.8%). Some characteristics, like penicillin resistance and the presence of cap8 and agr class II genes, were associated with the prevalent lineages (LA and LE), and penicillin susceptibility and the presence of cna and cap5 genes were associated with sporadic lineages. According to the present results, some S. aureus lineages possess a combination of genes that confer the propensity to cause and disseminate infection, and only a limited number of clones are responsible for the cases of bovine mastitis on the various farms. © 2004 NRC Canada.

Comparative analysis of noncoding sequences of orthologous bovine and human gene pairs

Genomic sequence comparison across species has enabled the elucidation of important coding and regulatory sequences encoded within DNA. Of particular interest are the noncoding regulatory sequences, which influence gene transcriptional and posttranscriptional processes. A phylogenetic footprinting strategy was employed to identify noncoding conservation patterns of 39 human and bovine orthologous genes. Seventy-three conserved noncoding sequences were identified that shared greater than 70% identity over at least 100 bp. Thirteen of these conserved sequences were also identified in the mouse genome. Evolutionary conservation of noncoding sequences across diverse species may have functional significance, and these conserved sequences may be good candidates for regulatory elements.

The effect of adhesive systems on the pullout strength of a fiberglass-reinforced composite post system in bovine teeth

Purpose: To evaluate the pullout strength of a glass fiber-reinforced composite post (glass FRC) cemented with three different adhesive systems and one resin cement. The null hypothesis was that pullout strengths yielded by the adhesive systems are similar. Materials and Methods: Thirty bovine teeth were selected. The size of the specimens was standardized at 16 mm by sectioning off the coronal portion and part of the root. The specimens were divided into three groups, according to the adhesive system, which were applied following the manufacturers' instructions: G1, ScotchBond Multi-Purpose Plus; G2, Single Bond; G3, Tyrian SPE/One-Step Plus. The glass FRCs (Reforpost) were etched with 37% H3PO4 for 1 min and silanized (Porcelain Primer). Thereafter, they were cemented with the dual resin cement En-Force. The specimens were stored for 24 h, attached to an adapted device, and submitted to the pullout test in a universal testing machine (1 mm/min). The data were submitted to the one-way ANOVA and Tukey's test (α = 0.05). Results: G1 (30.2 ± 5.8 Kgf) displayed the highest pullout strength (p < 0.001) when compared to G2 (18.6 ± 5.8 Kgf) and G3 (14.3 ± 5.8 Kgf), which were statistically similar. Analysis of the specimens revealed that all failures occurred between the adhesive system and the root dentin (pullout of the post cement), regardless of group. Conclusion: The multiple-bottle, total-etch adhesive system provided higher pullout strength of the glass FRC when compared to the single-bottle, total-etch, and single-step self-etching adhesive systems. The null hypothesis was rejected (p < 0.001).

Bovine serum albumin displays luciferase-like activity in presence of luciferyl adenylate: Insights on the origin of protoluciferase activity and bioluminescence colours

Luciferyl adenylate, the key intermediate in beetle bioluminescence, is produced through adenylation of D-luciferin by beetle luciferases and also by mealworm luciferase-like enzymes which produce a weak red chemiluminescence. However, luciferyl adenylate is only weakly chemiluminescent in water at physiological pH and it is unclear how efficient bioluminescence evolved from its weak chemiluminescent properties. We found that bovine serum albumin (BSA) and neutral detergents enhance luciferyl adenylate chemiluminescence by three orders of magnitude, simulating the mealworm luciferase-like enzyme chemiluminescence properties. These results suggest that the beetle protoluciferase activity arose as an enhanced luciferyl adenylate chemiluminescence in the protein environment of the ancestral AMP-ligase. The predominance of luciferyl adenylate chemiluminescence in the red region under most conditions suggests that red luminescence is a more primitive condition that characterized the original stages of protobioluminescence, whereas yellow-green bioluminescence may have evolved later through the development of a more structured and hydrophobic active site. Copyright © 2006 John Wiley & Sons, Ltd.

Bio-safety technology in production of bovine herpesvirus type 5 (BoHV-5) using an alternative serum-free medium

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)