Eprinomectin in dairy zebu Gobra cattle (Bos indicus): plasma kinetics and excretion in milk

The pharmacokinetics and mammary excretion of eprinomectin were determined in zebu Gobra following topical administration of 0.5 mg kg⁻¹. The kinetics of plasma and milk was analysed using a one-compartment model. The maximum plasma concentration of 8.83±2.15 ng ml⁻¹ occurred 1.30 days post-administration. The area under the plasma concentration–time curve was 30.63±5.56 ng day⁻¹ ml⁻¹ and the mean residence time was 3.38±0.60 days. Eprinomectin was detected in milk at the first sampling time and thereafter for at least 8 days. The systemic availability of eprinomectin was significantly lower than that for other breeds of cattle. Comparison of the milk and plasma data demonstrated the parallel disposition of the drug in the milk and plasma with a milk/plasma ratio of 0.094. The very low extent of mammary excretion supports the permitted use of eprinomectin in lactating zebu Gobra.     

Oocyte structure in dominant and subordinate follicles in zebu cattle (Bos indicus)

The objective of the study was to investigate the light and ultrastructural morphology of dominant and subordinate oocytes from zebu (Bos indicus) cattle. Healthy cycling animals, which had a well-developed corpus luteum as judged by rectal palpation, were administered cloprostenol to induce luteolysis and therefore ovulation. The animals were slaughtered at days 3–11 post-ovulation, but those slaughtered at days 8–11 received a second injection of cloprostenol at day 7. Cumulus-oocyte complexes were aspirated from the largest (dominant) and the second largest (subordinate) follicles, and processed for transmission electron microscopy. Up to day 7, the dominant oocyte presented a peripherally located spherical oocyte nucleus with a compact dense fibrillar nucleolus. After day 7, the nuclear envelope became undulated and the nucleolus vacuolated. The nuclei contained an average of four nucleoli. In addition to vesicles, mitochondria, smooth endoplasmic reticulum, Golgi complexes and cortical granules, the ooplasm contained annulate lamellae and microtubules. Moreover, mitochondrial granules and pleomorphic forms of mitochondria were commonly observed. Some subordinate oocytes exhibited advanced stages of meiotic maturation. It is concluded that (1) the dominant oocyte undergoes certain prematurational changes, including nucleolus vacuolation, in the period from luteolysis up to the presumptive occurrence of the LH peak and (2) subordinate oocytes may undergo meiotic maturation.     

Distribution of bovine lymphocyte antigen (BoLA-DRB3) alleles in Brazilian dairy Gir cattle (Bos indicus).

Brazilian dairy Gir (Bos indicus) cattle are a tropical, well-adapted breed, for which no information on the major histocompatibility complex (MHC) is presently available. The second exon of the bovine lymphocyte antigen (BoLA-DRB3) was amplified by polymerase chain reaction (PCR) of DNA samples from 28 Brazilian dairy Gir cows. Two experimental genotyping approaches were used: direct sequencing of PCR gene products (PCR-DS) and sequencing of cloned PCR fragments (S-CLO). Results demonstrate the viability of both typing approaches. PCR-DS allowed typing of 39% of the animals while the remainder were genotyped by S-CLO. Seventeen BoLA-DRB3 alleles were assigned, including some that were only recently described for zebu cattle. Allelic frequencies ranged from 0.02 to 0.18. The most frequent alleles were *3601 (frequency = 0.18), *2201 (0.14) and *2101 (0.11).     

Distribution of bovine lymphocyte antigen (BoLA‐DRB3) alleles in Brazilian dairy Gir cattle (Bos indicus)

Brazilian dairy Gir (Bos indicus) cattle are a tropical, well‐adapted breed, for which no information on the major histocompatibility complex (MHC) is presently available. The second exon of the bovine lymphocyte antigen (BoLA‐DRB3) was amplified by polymerase chain reaction (PCR) of DNA samples from 28 Brazilian dairy Gir cows. Two experimental genotyping approaches were used: direct sequencing of PCR gene products (PCR‐DS) and sequencing of cloned PCR fragments (S‐CLO). Results demonstrate the viability of both typing approaches. PCR‐DS allowed typing of 39% of the animals while the remainder were genotyped by S‐CLO. Seventeen BoLA‐DRB3 alleles were assigned, including some that were only recently described for zebu cattle. Allelic frequencies ranged from 0.02 to 0.18. The most frequent alleles were *3601 (frequency = 0.18), *2201 (0.14) and *2101 (0.11).     

The effect of social rank on the physiological response during repeated stressful handling in Zebu cattle (Bos indicus)

Although the order of entry to a handling chute is related to social rank, it is still not clear what the consequences are for Zebu cattle of occupying different ranks when being exposed repeatedly to a stressful handling procedure. Eighteen Brahman cows were observed for 243 h to obtain information on social interactions. From that information, indices of success in displacing other individuals of the herd were calculated to reflect social status of each cow. One week after behavioral observations, the cows were forced 19 times to enter a handling chute where they were palpated and 7 ml of blood was collected from their caudal vein. To have an adrenal activity profile along the experimental period, five blood samples (Days 2, 6, 10, 15, and 19) were chosen for cortisol determination. On each sampling day, the average time in the chute, the order of entry, and a value of entrance consistency were calculated. On average, the high-ranking cows entered the chute before the medium- and low-ranking cows. Medium-ranking cows showed higher consistency when entering the race than high- and low-ranking cows. Low-ranking cows had significantly lower cortisol levels than the other two groups since the second sample (Day 6 in the race), and had higher cortisol levels the first time handled than at subsequent sampling days. It is suggested that low-ranking cows adopt a passive strategy that allows them to have a better control over the stressful event, while high-ranking cows respond with higher cortisol levels perhaps because of the need to become aroused to deal with challenges. It was concluded that the herd (a) habituated to repeat handling in a squeeze chute and (b) low-ranking cows responded with lower cortisol concentration to handling.     

Genotyping BoLA-DRB3 alleles in Brazilian Dairy Gir cattle (Bos indicus) by temperature-gradient gel electrophoresis (TGGE) and direct sequencing.

BoLA-DRB3 is a gene of the major histocompatibility complex (MHC) in cattle. The product of the BoLA-DRB3 gene is a beta chain of an MHC class II molecule, a glycoprotein expressed on the surface of antigen-presenting cells (APCs). Responses of CD4+ T lymphocytes to peptides are dependent on the presentation of peptide ligands bound to class II molecules on APCs. Genotyping of the BoLA-DRB3 gene is relatively complex due to the extensive polymorphism of this locus. Current techniques for assignment of genotypes are polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), direct sequencing of PCR products, cloning-sequencing, polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP), and denaturant-gradient gel electrophoresis. These techniques are time-consuming, do not discriminate all possible alleles, or are not readily reproducible. The objective of this study was to genotype BoLA-DRB3 using temperature-gradient gel electrophoresis (TGGE) to separate alleles before sequencing. PCRs using 28 DNA samples from Gir Dairy cattle (a Brazilian breed of Bos indicus) were submitted to TGGE. New PCR products were generated from separated alleles, purified, and sequenced. Allele separation was possible in 21 out of 26 heterozygote samples (81%). Results indicate that two sequence reads (forward and reverse) were sufficient for accurate genotyping of BoLA-DRB3 alleles. Separation of alleles by TGGE provides high-throughput, reliable typing of BoLA-DRB3, which is critical in disease association studies in cattle.     

Sexual behavior of Zebu cattle (Bos indicus) following estrous induction by Syncro-Mate B, with or without estrogen injection

The effect of estrogen administered with norgestomet implants in the expression of behavioral estrous of Zebu cattle was studied in a herd of 18 cycling Brahman cows, in two trials. In the first and second trials, six different cows were treated with progestagen on successive days. In addition, in the first trial, estrogen injection was applied with the treatment. With the purpose of enhancing mounting behavior in treated and/or non-treated animals, on the third day of each trial, three other cows with an active CL were injected with 25 mg of PGF2alpha. All animals were examined three times a week by rectal ultrasound, and a blood sample was also taken for progesterone analysis. Sexual activity was monitored by a closed-circuit TV from 1500 to 0600 h. Direct observation was carried out from 0900 to 1200 h. A total of 15 cows displayed 36 mounting periods (11 with ovulation). Twenty-five annovulatory estrous periods were observed, 77% supported by large follicles. More cows (66% vs. 16%) showed mounting activity when estradiol valerate was used as part of the progestagen treatment (P=.03). Ovulation rate in response to the effect of norgestomet implant was 16%, independent of treatment. It is concluded that estrogen administered with norgestomet implants increases the expression of behavioral estrous in Bos indicus cattle but does not improve ovulation rate.     

Immunological Profiles of Bos taurus and Bos indicus Cattle Infested with the Cattle Tick,Rhipicephalus (Boophilus) microplus

The cattle tick, Rhipicephalus (Boophilus) microplus, is a major threat to the improvement of cattle production in tropical and subtropical countries worldwide. Bos indicus cattle are naturally more resistant to infestation with the cattle tick than are Bos taurus breeds, although considerable variation in resistance occurs within and between breeds. It is not known which genes contribute to the resistant phenotype, nor have immune parameters involved in resistance to R. microplus been fully described for the bovine host. This study was undertaken to determine whether selected cellular and antibody parameters of the peripheral circulation differed between tick-resistant Bos indicus and tick-susceptible Bos taurus cattle following a period of tick infestations. This study demonstrated significant differences between the two breeds with respect to the percentage of cellular subsets comprising the peripheral blood mononuclear cell population, cytokine expression by peripheral blood leukocytes, and levels of tick-specific immunoglobulin G1 (IgG1) antibodies measured in the peripheral circulation. In addition to these parameters, the Affymetrix bovine genome microarray was used to analyze gene expression by peripheral blood leukocytes of these animals. The results demonstrate that the Bos indicus cattle developed a stabilized T-cell-mediated response to tick infestation evidenced by their cellular profile and leukocyte cytokine spectrum. The Bos taurus cattle demonstrated cellular and gene expression profiles consistent with a sustained innate, inflammatory response to infestation, although high tick-specific IgG1 titers suggest that these animals have also developed a T-cell response to infestation.The cattle tick Rhipicephalus (Boophilus) microplus is a major threat to the improvement of cattle production in tropical and subtropical countries worldwide. Heavy tick infestation has adverse physiological effects on the host, resulting in decreased live weight gain (21), and anemia is a common symptom of heavy infestation (35). R. microplus is also the vector of Babesia bovis, Babesia bigemina, and Anaplasma marginale, which cause tick fever in Australia. Acaricide treatment is the primary method of controlling ticks; however, populations of ticks have subsequently developed resistance to organochlorines, organophosphates, carbamates, amidines, and synthetic pyrethroids (27). Resistance to multiple classes of chemicals has also been observed (27). It is probable that with current usage new acaricides will encounter similar problems (10), and a more sustainable solution to tick control is needed.Naturally acquired host immunity has been proposed as a viable cattle tick control method because of the potential reduction in expenditure on acaricides and husbandry practices associated with chemical control (10). Bos indicus cattle breeds are more resistant to R. microplus than are Bos taurus breeds, although considerable variation in resistance occurs between and within breeds (37, 45). Although innate immunity arising from genetic differences between B. indicus and B. taurus breeds forms the basis of whether an animal will be resistant to tick infestation, host resistance is considered to be predominantly an acquired trait because the higher level of resistance seen in B. indicus becomes apparent only following a period of initial susceptibility to primary infestation (15, 44). Host resistance to tick infestation is heritable, with a rate estimated to be between 39% and 49% for British breed animals (45) and as high as 82% in Africander and Brahman (B. indicus) crossbred animals (37). Since these initial studies, it has been shown that the resistance status of both B. taurus and B. indicus breeds can be improved by selection for increased tick resistance, as demonstrated by a breeding program that has resulted in a highly tick-resistant line of Hereford × Shorthorn (B. taurus) cattle, now known as the Belmont Adaptaur (9, 25). Identifying the mechanisms responsible for mediating naturally acquired tick resistance in cattle is an essential step in developing predictive phenotypic markers to enable rapid identification of highly resistant individuals and is potentially useful in the development of a tick vaccine. It is not known, however, which genes contribute to the resistant phenotype, nor have immune parameters involved in resistance to R. microplus been fully described for the bovine host.Studies of immune parameters of the peripheral circulation of tick-infested cattle have yielded varied and sometimes conflicting results. Cattle tick infestation has been reported to reduce the number of circulating T lymphocytes and the antibody response to ovalbumin injection in susceptible B. taurus animals compared to tick-free control animals (17). In another study, infestation with several species of African ticks resulted in higher levels of serum gamma globulin and increased numbers of circulating white blood cells (WBCs) in B. taurus animals compared with those in Brahman cattle managed under the same conditions (33). Exposure of animals to high and low levels of tick infestation has been reported to result in differential patterns of immunoglobulins specific for tick salivary proteins in resistant and susceptible cattle (7, 24). Sustained heavy infestation has been shown to alter host hemostatic mechanisms by inhibiting platelet aggregation and coagulation functions (34) and also by altering the level of acute-phase proteins in the susceptible host (4).In vitro studies of mononuclear cell populations have shown that salivary gland proteins from R. microplus can inhibit immune cell function. The proliferative response of bovine peripheral blood mononuclear cells (PBMC) to stimulation with the T-lymphocyte mitogen phytohemagglutinin (PHA) was inhibited by the addition of salivary gland protein to the culture (17), and subsequent studies showed that sufficient prostaglandin E₂ is present in tick saliva to be responsible for this inhibition (16). Turni et al. (42) found that low concentrations of R. microplus salivary gland extract (SGE) inhibited the oxidative burst capacity of monocytes and neutrophils, as well as the proliferation response of PBMC to concanavalin A (ConA) in vitro, in both B. taurus and B. indicus cattle. However, a higher concentration of SGE caused a significant difference in the degree of inhibition observed in the proliferation assay between the B. taurus and B. indicus cells: a 40.7% and an 88.5% reduction, respectively. The authors suggested that the disproportionate increase in inhibition at the higher concentration of SGE may be an indication that the mechanisms by which the two breeds resist infestation are different.Here we report the results of a study undertaken to define selected immune parameters in tick-resistant Brahman and tick-susceptible Holstein-Friesian animals following challenge infestations with R. microplus. The aim of this study was to determine whether cellular and antibody components of the peripheral circulation differed between these two breeds of highly divergent resistance following a period of tick infestations.     

Neospora caninum: evaluation of vertical transmission in slaughtered beef cows (Bos indicus)

Neospora caninum is a protozoan parasite that causes the most important reproductive problems in cattle worldwide. The objective of this study was to evaluate the possibility of vertical transmission of N. caninum in zebus breed beef cows (Bos indicus) submitted for slaughter at an abattoir in the northern region of the State of Paraná, southern Brazil. One hundred and fifty-nine cows were evaluated: 83 pregnant (in different stages of gestation) and 76 non-pregnant. Serum determination of N. caninum was evaluated by indirect ELISA (Idexx). Blood (with EDTA) from pregnant cows and tissue samples (brain and heart) from their fetuses were collected and used for PCR analyses. Antibodies against N. caninum were observed in 14.6% (12/83) of pregnant and in 15.8% (12/76) of non-pregnant cows. Antibodies against the parasites were detected in one fetus (1.4%). The PCR analyses revealed that 6.0% (5/83) of cows and 4.8% (4/83) of fetuses evaluated were positive to specific N. caninum primers. These positive fetuses were between 4 and 6 months of age. Thus, considering PCR and serology as an indicative of vertical transmission in fetuses, 4.8% of fetuses were infected by N. caninum during gestation.     

Genotyping BoLA‐DRB3 alleles in Brazilian Dairy Gir cattle (Bos indicus) by temperature‐gradient gel electrophoresis (TGGE) and direct sequencing

BoLA‐DRB3 is a gene of the major histocompatibility complex (MHC) in cattle. The product of the BoLA‐DRB3 gene is a beta chain of an MHC class II molecule, a glycoprotein expressed on the surface of antigen‐presenting cells (APCs). Responses of CD4⁺ T lymphocytes to peptides are dependent on the presentation of peptide ligands bound to class II molecules on APCs. Genotyping of the BoLA‐DRB3 gene is relatively complex due to the extensive polymorphism of this locus. Current techniques for assignment of genotypes are polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP), direct sequencing of PCR products, cloning‐sequencing, polymerase chain reaction using sequence‐specific oligonucleotide probes (PCR‐SSOP), and denaturant‐gradient gel electrophoresis. These techniques are time‐consuming, do not discriminate all possible alleles, or are not readily reproducible. The objective of this study was to genotype BoLA‐DRB3 using temperature‐gradient gel electrophoresis (TGGE) to separate alleles before sequencing. PCRs using 28 DNA samples from Gir Dairy cattle (a Brazilian breed of Bos indicus) were submitted to TGGE. New PCR products were generated from separated alleles, purified, and sequenced. Allele separation was possible in 21 out of 26 heterozygote samples (81%). Results indicate that two sequence reads (forward and reverse) were sufficient for accurate genotyping of BoLA‐DRB3 alleles. Separation of alleles by TGGE provides high‐throughput, reliable typing of BoLA‐DRB3, which is critical in disease association studies in cattle.     

Oocyte structure in dominant and subordinate follicles in zebu cattle (Bos indicus)

The objective of the study was to investigate the light and ultrastructural morphology of dominant and subordinate oocytes from zebu (Bos indicus) cattle. Healthy cycling animals, which had a well-developed corpus luteum as judged by rectal palpation, were administered cloprostenol to induce luteolysis and therefore ovulation. The animals were slaughtered at days 3–11 post-ovulation, but those slaughtered at days 8–11 received a second injection of cloprostenol at day 7. Cumulus-oocyte complexes were aspirated from the largest (dominant) and the second largest (subordinate) follicles, and processed for transmission electron microscopy. Up to day 7, the dominant oocyte presented a peripherally located spherical oocyte nucleus with a compact dense fibrillar nucleolus. After day 7, the nuclear envelope became undulated and the nucleolus vacuolated. The nuclei contained an average of four nucleoli. In addition to vesicles, mitochondria, smooth endoplasmic reticulum, Golgi complexes and cortical granules, the ooplasm contained annulate lamellae and microtubules. Moreover, mitochondrial granules and pleomorphic forms of mitochondria were commonly observed. Some subordinate oocytes exhibited advanced stages of meiotic maturation. It is concluded that (1) the dominant oocyte undergoes certain prematurational changes, including nucleolus vacuolation, in the period from luteolysis up to the presumptive occurrence of the LH peak and (2) subordinate oocytes may undergo meiotic maturation.     

G- and R-banded prometaphase karyotypes in cattle (Bos taurus L.)

G- and R banded cattle (Bos taurus L.) karyotypes at about 500 band level are reported and discussed in light of chromosome banding standard nomenclature discrepancies. GTG-, GBG-, RBA- and RBG-banded karyotypes were arranged according to the Reading Conference standard. The G- and R-banding patterns of small acrocentric and disputed chromosomes (25, 27, 28 and 29) are demonstrated using some bovid marker chromosomes. G- and R-banded ideogrammatic representations using only one common banding nomenclature were also performed. This study is a contribution to the construction of new and clearer standard G- and R-banded karyotypes for this important species.accepted for publication by H. C. Macgregor     

Genetic diversity and population genetic analysis of bovine MHC class II DRB3.2 locus in three Bos indicus cattle breeds of Southern India

The present study was performed to evaluate the genetic polymorphism of BoLA‐DRB3.2 locus in Malnad Gidda, Hallikar and Ongole South Indian Bos indicus cattle breeds, employing the PCR‐RFLP technique. In Malnad Gidda population, 37 BoLA‐DRB3.2 alleles were detected, including one novel allele DRB3*2503 (GenBank: HM031389 ) that was observed in the frequency of 1.87%. In Hallikar and Ongole populations, 29 and 21 BoLA‐DRB3.2 alleles were identified, respectively. The frequencies of the most common BoLA‐DRB3.2 alleles (with allele frequency > 5%), in Malnad Gidda population, were DRB3.2*15 (10.30%), DRB3*5702 (9.35%), DRB3.2*16 (8.41%), DRB3.2*23 (7.01%) and DRB3.2*09 (5.61%). In Hallikar population, the most common alleles were DRB3.2*11 (13.00%), DRB3.2*44 (11.60%), DRB3.2*31 (10.30%), DRB3.2*28 (5.48%) and DRB3.2*51 (5.48%). The most common alleles in Ongole population were DRB3.2*15 (22.50%), DRB3.2*06 (20.00%), DRB3.2*13 (13.30%), DRB3.2*12 (9.17%) and DRB3.2*23 (7.50%). A high degree of heterozygosity observed in Malnad Gidda (H_O = 0.934, H_E = 0.955), Hallikar (H_O = 0.931, H_E = 0.943) and Ongole (H_O = 0.800, H_E = 0.878) populations, along with F_IS values close to F_IS zero (Malnad Gidda: F_IS = 0.0221, Hallikar: F_IS = 0.0127 and Ongole: F_IS = 0.0903), yielded nonsignificant P‐values with respect to Hardy–Weinberg equilibrium probabilities revealing, no perceptible inbreeding, greater genetic diversity and characteristic population structure being preserved in the three studied cattle populations. The phylogenetic tree constructed based on the frequencies of BoLA‐DRB3.2 alleles observed in 10 Bos indicus and Bos taurus cattle breeds revealed distinct clustering of specific Bos indicus cattle breeds, along with unique genetic differentiation observed among them. The results of this study demonstrated that the BoLA‐DRB3.2 is a highly polymorphic locus, with significant breed‐specific genetic diversities being present amongst the three studied cattle breeds. The population genetics and phylogenetic analysis have revealed pivotal information about the population structure and importance of the presently studied three Bos indicus cattle breeds as unique animal genetic resources, which have to be conserved for maintaining native cattle genetic diversity.     

Nitric oxide synthesis is depressed in Bos indicus cattle infected with Trypanosoma congolense and Trypanosoma vivax and does not mediate T-cell suppression.

Infection with African trypanosomes causes the diseases sleeping sickness in humans and nagana in cattle in sub-Saharan Africa. Suppression of cellular immune responses is a feature of trypanosomiasis in bovine, human, and murine hosts. Some aspects of immunosuppression in the murine model are mediated by nitric oxide (NO) produced by gamma interferon (IFN-gamma)-activated macrophages. We have investigated whether a similar mechanism is responsible for T-cell unresponsiveness in bovine trypanosomiasis. Bovine monocytes and macrophages from uninfected cattle and activated in vitro with IFN-gamma produced NO; however, this response was down-regulated in infected cattle. Similarly, the expression of inducible NO synthase messenger RNA was depressed in macrophages of infected cattle. Proliferation of mononuclear cells of trypanosome-infected cattle cultured with mitogen or trypanosome antigens was unchanged by the addition of an NO synthase inhibitor. Lymphocytes of infected cattle secreted interleukins with T-cell growth factor activity after in vitro activation with mitogens but not after activation with trypanosome antigens. Although lymph node cells secreted IFN-gamma after in vitro activation, ex vivo expression of mRNA was depressed. In contrast, the level of expression of interleukin 10 mRNA was higher during infection. We conclude that NO is not involved in the loss of T-cell proliferative function associated with trypanosomiasis in cattle and that, in contrast to the mouse model, the capacity of monocytes and macrophages to produce NO is actually down-regulated in infected cattle.     

Identification of seven new TCRBV subfamilies in cattle (Bos taurus).

In cattle, the repertoire of TCRBV genes that encode the variable region of T-cell receptor beta-chains has not been fully characterized. In this study, cattle TCRBV genes were amplified from mRNA by anchored polymerase chain reaction and sequenced. Eleven new TCRBV gene sequences were found, and classified into seven subfamilies distinct from those previously identified.