A New Tandem Repeat in Bovine Fibrinogen Aα Gene

In this study, we describe intraspecies variation in the αC connector region of the bovine fibrinogen Aα gene. Sequencing and genotyping of six bovine breeds revealed 7 to 10 tandem repeats in the αC connector region. In addition, we observed length differences between B. indicus and B. taurus, with the B. indicus having longer fibrinogen αC connectors (10-repeat alleles) than B. taurus (7- and 9-repeats). The difference in tandem repeats may be related to the function of blood coagulation system.     

Heteroplasmy in bovine fetuses produced by intra- and inter-subspecific somatic cell nuclear transfer: neutral segregation of nuclear donor mitochondrial DNA in various tissues and evidence for recipient cow mitochondria in fetal blood

Varying degrees of mitochondrial DNA (mtDNA) heteroplasmy have been observed in nuclear transfer embryos, fetuses, and offspring, but the mechanisms leading to this condition are unknown. We have generated a clone of 12 bovine somatic cell nuclear transfer fetuses, using nuclear donor cells, recipient oocytes, and recipient heifers with defined mtDNA genotypes, to study nuclear-mitochondrial interactions and the origins of mtDNA heteroplasmy. Embryos were reconstructed from granulosa cells with Bos taurus mtDNA type A and recipient oocytes collected from three different maternal lineages with B. taurus mtDNA type B, B. taurus mtDNA type C, or B. indicus mtDNA. Sequence differences in the control region (CR) of B. taurus mtDNAs ranged from 6 to 11 nucleotides and differences between B. taurus and B. indicus CRs from 45 to 50 nucleotides. Fetuses were recovered from recipient heifers with B. taurus mtDNA type B on Day 80 after nuclear transfer (eight B. taurus A/B, two B. taurus A/C, and two B. taurus A/B. indicus). Agarose gel analysis of the CR by polymerase chain reaction-based restriction fragment length polymorphism failed to detect nuclear donor mtDNA in 11 investigated tissues of 10 viable fetuses and in DNA samples of two fetuses in resorption (one B. taurus A/B and one B. taurus A/C). A more sensitive analysis of 1801 plasmid clones with CR inserts derived from tissues of a B. taurus A/B. indicus fetus detected no or very low levels of heteroplasmy (0.5-0.7%). However, the analyses detected considerable amounts ( approximately 2.5% and 5%) of recipient heifer mtDNA in blood samples from two fetuses. Our data do not suggest a replicative advantage of somatic nuclear donor cell mtDNA in bovine transmitochondrial clones produced with oocytes from domestic forms of the same or a different aurochs (B. primigenius) subspecies. Detection of mtDNA from the recipient animal in the circulation of two fetuses points to leakage of the placental barrier, mimicking heteroplasmy.     

Interaction of fibrin(ogen) with fibronectin: further characterization and localization of the fibronectin-binding site

The interaction of fibronectin with fibrin and its incorporation into fibrin clots are thought to be important for the formation of a provisional matrix that promotes cell adhesion and migration during wound healing. However, it is still unclear whether fibronectin interacts with both fibrin and fibrinogen or fibrin only and whether fibronectin binds exclusively to the fibrin(ogen) alphaC domains. To address these questions, we studied the interaction of fibronectin with fibrinogen, fibrin, and their proteolytic and recombinant fragments. In both ELISA and surface plasmon resonance (SPR) experiments, immobilized fibrinogen did not bind fibronectin at all, but after conversion to fibrin, it bound fibronectin with high affinity. To test which regions of fibrin are involved in this binding, we studied the interaction of fibronectin with the fibrin-derived D-D:E(1) complex and a recombinant alphaC fragment (residues Aalpha221-610) corresponding to the alphaC domain that together encompass the whole fibrin(ogen) molecule. In ELISA, when fibronectin was added to the immobilized D-D:E(1) complex or the immobilized alphaC fragment, only the latter exhibited binding. Likewise, when fibronectin was immobilized and the complex or the alphaC fragment was added, only the latter was observed to bind. The selective interaction between fibronectin and the alphaC fragment was confirmed by SPR. The fibronectin-binding site was further localized to the NH(2) terminal connector region of the alphaC domain since in ELISA, the immobilized recombinant Aalpha221-391 sub-fragment bound fibronectin well while the immobilized recombinant Aalpha392-610 sub-fragment exhibited no binding. This finding was confirmed by ligand blotting analysis. Thus, the results provide direct evidence for the existence of a cryptic high-affinity fibronectin-binding site in the Aalpha221-391 region of the fibrinogen alphaC domain that is not accessible in fibrinogen but becomes exposed in fibrin.     

Importance of sperm genotype (indicus versus taurus) for fertility and embryonic development at elevated temperatures

Heat stress has negative effects on bovine reproduction, particularly for European breeds (Bos taurus taurus) that are less thermotolerant than zebu cattle (Bos taurus indicus). Here, the evidence that spermatozoa and oocyte both contribute to early embryonic resistance to heat shock is demonstrated. In addition, the use of reproductive biotechnologies to improve bovine thermotolerance, are outlined by comparing data from taurus, indicus and crossbred genotypes.     

Complete mitochondrial genomes of Bos taurus and Bos indicus provide new insights into intra-species variation, taxonomy and domestication

The taurine and zebuine cattle breeds comprise the majority of the world cattle population but their taxonomic status is still controversial. The two forms of cattle are currently classified as Bos taurus and Bos indicus species and are differentiated primarily by the presence or absence of a hump. However, these two species hybridize readily, producing fully fertile offspring. We have determined and analyzed complete B. taurus and B. indicus mitochondrial genome sequences to investigate the extent of sequence divergences and to study their taxonomic status by molecular dating. The sequences encompassed 16,338 and 16,339 nucleotides, respectively, and differed at 237 positions. Estimated divergence times indicated that the two cattle lineages separated 1.7-2.0 million years ago. Combined phylogenetic analyses of 18 new and 130 previously reported extant B. taurus and B. indicus control region sequences with data from 32 archaeological specimens of the extinct wild aurochs (Bos primigenius) identified four major maternal lineages. B. primigenius haplotypes were present in all but the B. indicus lineage, and one B. taurus sequence clustered with B. primigenius P haplotypes that were not previously linked with domestic cattle. The B. indicus cluster and a recently reported new B. primigenius haplotype that represents a new lineage were approximately equidistant from the B. taurus cluster. These data suggest domestications from several differentiated populations of B. primigenius and a subspecies status for taurine (B. primigenius taurus) and zebuine (B. primigenius indicus) cattle.     

Structural organization of the fibrin(ogen) alpha C-domain

We hypothesized that the alpha C-domain of human fibrinogen (residues hA alpha 221-610) and of other species consists of a compact COOH-terminal region (hA alpha 392-610) and a flexible NH(2)-terminal connector region (hA alpha 221-391) which may contain some regular structure [Weisel and Medved (2001) Ann. N.Y. Acad. Sci. 936, 312-327]. To test this hypothesis, we expressed in E. coli recombinant fragments corresponding to the full-length human alpha C-domain and its NH(2)- and COOH-terminal regions as well as their bovine counterparts, bA alpha 224-568, bA alpha 224-373, and bA alpha 374-568(538), respectively, and tested their folding status by fluorescence spectroscopy, circular dichroism (CD), and differential scanning calorimetry (DSC). All three methods revealed heat-induced unfolding transitions in the full-length bA alpha 224-568 and its two COOH-terminal fragments, indicating that the COOH-terminal portion of the bovine alpha C-domain is folded into a compact cooperative structure. Similar results were obtained by CD and DSC with the full-length and the COOH-terminal h392-610 human fragments. The NH(2)-terminal fragments of both species, b224-373 and h221-392, did not exhibit any sign of a compact structure. However, their heat capacity functions, CD spectra, and temperature dependence of ellipticity at 222 nm were typical for peptides in the extended helical poly(L-proline) type II conformation (PPII), suggesting that they contain this type of regular structure. This is consistent with the presence of proline-rich tandem repeats in the sequence of both bovine and human connector regions. These results indicate that both bovine and human fibrinogen alpha C-domains consist of a compact globular cooperative unit attached to the bulk of the molecule by an extended NH(2)-terminal connector region with a PPII conformation.     

Reproductive cycles in Bos indicus cattle

Several studies using transrectal ovarian ultrasonic scanning in Bos taurus (B. taurus) cattle and more recently in Bos indicus (B. Indicus) females evaluated the reproductive cycles of heifers and cows under different conditions. In general, B. indicus cattle have more follicles and more follicular waves during the estrous cycle and ovulate from smaller follicles than B. taurus. Consequently B. indicus females have smaller corpora lutea and it is assumed circulating concentrations of estradiol and progesterone are also less. However, these findings may vary depending on the nutritional status and regimen in which the animals are managed. Moreover, there are significant differences between B. taurus and B. indicus regarding follicle size at the time of deviation of the dominant follicle. These differences in ovarian function between B. indicus and B. taurus, e.g. greater antral follicle population are, probably, the main reasons for the great success of in vitro embryo production programs in Zebu cattle, especially in Brazil.     

Nuclear-cytoplasmic interactions affect in utero developmental capacity, phenotype, and cellular metabolism of bovine nuclear transfer fetuses

We generated a clone of bovine somatic cell nuclear transfer embryos using oocyte pools from defined maternal sources to study nuclear-cytoplasmic interactions. Nucleocytoplasmic hybrids were reconstructed with Bos taurus (Brown Swiss) granulosa cells and oocytes that contained B. taurus A (Simmental), B. taurus B (Simmental), or Bos indicus (Dwarf Zebu) cytoplasm. Another set of embryos was reconstructed with randomly selected Brown Swiss (B. taurus R) oocytes. Embryo transfer resulted in nine (12.5%), nine (13.8%), three (50%), and 11 (16.7%) Day 80 fetuses, of which eight (11.1%), three (4.6%), three (50%), and 10 (15.2%) were viable, respectively. The proportion of viable fetuses was affected by cytoplasm (likelihood ratio test, P < 0.02) and was higher for embryos with B. indicus cytoplasm than for the B. taurus A (P < 0.05) and B (P < 0.01) groups. Furthermore, the proportion of surviving Day 80 fetuses was reduced for B. taurus B as compared with B. taurus A and B. taurus R cytoplasm (P < 0.05 and P < 0.02). Body weight of nucleocytoplasmic hybrid fetuses was not significantly different from Brown Swiss control fetuses produced by artificial insemination (AI), but fetuses reconstructed with random cytoplasts of the same breed as the nuclear donor exhibited overgrowth (P < 0.01) and a higher coefficient of variation in weight. Furthermore, body weight, crown rump length, thorax circumference (P < 0.05), and femur length (P < 0.01) of fetuses with B. taurus A cytoplasm differed from fetuses with B. taurus R cytoplasms. Fetal skin, heart, and liver cells with B. indicus cytoplasm showed a greater increase in number per time period (P < 0.001) and oxygen consumption rate per cell (skin and liver, P < 0.001; heart, P < 0.08) in comparison with their counterparts with B. taurus A cytoplasm. These data point to complex oocyte cytoplasm-dependent epigenetic modifications and/or nuclear DNA-mitochondrial DNA interactions with relevance to nuclear transfer and other reproductive technologies such as ooplasmic transfer in human assisted reproduction.     

Nucleotide Sequence and Variation of IGF2 Gene Exon 6 in Bos Taurus and Bos Indicus Cattle

The major assumption of this study is that polymorphism of a gene could be used to investigate its allele-specific expression as well as its methylation and imprinting status. Therefore, the aim of this study was to analyze the polymorphism of the coding region of the bovine IGF2 gene and to determine the sequence of its gene exon 6 in Bos taurus and Bos indicus cattle. A single nucleotide “C” deletion/insertion polymorphism was found in both cattle subspecies and a G/T transversion (RFLP-MboII) in the Bos indicus IGF2 gene. A 407-bp fragment of bovine IGF2 exon 6 was sequenced and the sequences (including variable nucleotides) were deposited in the GenBank database. A comparative analysis was performed for this fragment from different species; 99.5% identity was found between Bos taurus and Bos indicus cattle.     

Nucleotide sequence and variation of IGF2 gene exon 6 in Bos taurus and Bos indicus cattle

The major assumption of this study is that polymorphism of a gene could be used to investigate its allele-specific expression as well as its methylation and imprinting status. Therefore, the aim of this study was to analyze the polymorphism of the coding region of the bovine IGF2 gene and to determine the sequence of its gene exon 6 in Bos taurus and Bos indicus cattle. A single nucleotide "C" deletion/insertion polymorphism was found in both cattle subspecies and a G/T transversion (RFLP-MboII) in the Bos indicus IGF2 gene. A 407-bp fragment of bovine IGF2 exon 6 was sequenced and the sequences (including variable nucleotides) were deposited in the GenBank database. A comparative analysis was performed for this fragment from different species; 99.5% identity was found between Bos taurus and Bos indicus cattle.     

Bovine gene polymorphisms related to fat deposition and meat tenderness

Leptin, thyroglobulin and diacylglycerol O-acyltransferase play important roles in fat metabolism. Fat deposition has an influence on meat quality and consumers' choice. The aim of this study was to determine allele and genotype frequencies of polymorphisms of the bovine genes, which encode leptin (LEP), thyroglobulin (TG) and diacylglycerol O-acyltransferase (DGAT1). A further objective was to establish the effects of these polymorphisms on meat characteristics. We genotyped 147 animals belonging to the Nelore (Bos indicus), Canchim (5/8 Bos taurus + 3/8 Bos indicus), Rubia Gallega X Nelore (1/2 Bos taurus + 1/2 Bos indicus), Brangus Three-way cross (9/16 Bos taurus + 7/16 Bos indicus) and Braunvieh Three-way cross (3/4 Bos taurus + 1/4 Bos indicus) breeds. Backfat thickness, total lipids, marbling score, ribeye area and shear force were fitted, using the General Linear Model (GLM) procedure of the SAS software. The least square means of genotypes and genetic groups were compared using Tukey's test. Allele frequencies vary among the genetic groups, depending on Bos indicus versus Bos taurus influence. The LEP polymorphism segregates in pure Bos indicus Nelore animals, which is a new finding. The T allele of TG is fixed in Nelore, and DGAT1 segregates in all groups, but the frequency of allele A is lower in Nelore animals. The results showed no association between the genotypes and traits studied, but a genetic group effect on these traits was found. So, the genetic background remains relevant for fat deposition and meat tenderness, but the gene markers developed for Bos taurus may be insufficient for Bos indicus.     

Mapping of quantitative trait loci controlling tick [Riphicephalus (Boophilus) microplus] resistance on bovine chromosomes 5, 7 and 14

Differences in domestication and selection processes have contributed to considerable phenotypic and genotypic differences between Bos taurus and Bos indicus cattle breeds. Of particular interest in tropical and subtropical production environments are those genetic differences between subspecies that underlie the phenotypic extremes in tolerance and susceptibility to parasite infection. In general, B. taurus cattle are more susceptible to ectoparasites than B. indicus cattle in tropical environments, and much of this difference is under genetic control. To identify genomic regions involved in tick resistance, we developed a B. taurus x B. indicus F(2) experimental population to map quantitative trait loci (QTL) for resistance to the Riphicephalus (Boophilus) microplus tick. About 300 individuals were measured for parasite load in two seasons (rainy and dry) and genotyped for 23 microsatellite markers covering chromosomes 5, 7 and 14. We mapped a suggestive chromosome-wide QTL for tick load in the rainy season (P < 0.05) on chromosome 5. For the dry season, suggestive (P < 0.10) chromosome-wide QTL were mapped on chromosomes 7 and 14. The additive effect of the QTL on chromosome 14 corresponds to 3.18% of the total observed phenotypic variance. Our QTL-mapping study has identified different genomic regions controlling tick resistance; these QTL were dependent upon the season in which the ticks were counted, suggesting that the QTL in question may depend on environmental factors.     

Association between Sscp Haplotypes at the Bovine Growth Hormone Gene and Milk Protein Percentage

The bovine Growth Hormone gene (bGH) is an attractive candidate gene for milk production in cattle. Single-strand conformation polymorphisms at bGH were identified and used to define haplotype configurations at this gene in the Israeli Holstein dairy cattle population (Bos taurus) and in the parent animals of the International Bovine Reference Family Panel (a collection of B. taurus and B. indicus crosses). B. taurus and B. indicus haplotypes at the bGH gene differed qualitatively, confirming the previously proposed long evolutionary separation of these cattle subraces. Only a small number of bGH haplotypes were present in the Israel Holstein population. One of the haplotypes, apparently of B. indicus origin, was found to have a highly significant positive effect on milk protein percentage. This illustrates the utility of the haplotype approach for uncovering candidate gene involvement in quantitative genetic variation in agricultural populations. The strong effect of an indicine haplotype in a taurine background raises the possibility that indicine alleles at other candidate genes may comprise a genetic resource for improvement of taurine populations. It is proposed that haplotype analysis may be a useful adjunct to measures of genetic distance for evaluating rare breeds with respect to gene conservation.     

Superovulation and embryo transfer in Bos indicus cattle

Compared to Bos taurus breeds, Bos indicus breeds of cattle present several differences in reproductive physiology. Follicular diameter at deviation and at the time of ovulatory capability are smaller in B. indicus breeds. Furthermore, B. indicus breeds have a greater sensitivity to gonadotropins, a shorter duration of estrus, and more often express estrus during the night. These differences must be considered when setting up embryo transfer programs for B. indicus cattle. In recent studies, we evaluated follicular dynamics and superovulatory responses in B. indicus donors with the objective of implementing fixed-time AI protocols in superstimulated donors. Protocols using estradiol and progesterone/progestrogen releasing devices to control follicular wave emergence were as efficacious as in B. taurus cattle, allowing the initiation of superstimulatory treatments (with lower dosages of FSH than in B. taurus donors) at a self-appointed time. Furthermore, results presented herein indicate that delaying the removal of progesterone/progestogen-releasing devices, combined with the administration of GnRH or pLH 12 h after the last FSH injection, results in synchronous ovulations, permitting the application of fixed-time AI of donors without the necessity of estrus detection and without compromising the results.     

Randomly primed PCR amplification of pooled DNA reveals polymorphism in a ruminant repetitive DNA sequence which differentiates Bos indicus and B. taurus

By amplification of pools of DNA representative of different bovine populations with single short oligonucleotide primers of random sequence, we were able rapidly to identify markers which distinguish the two major subspecies of domestic cattle, Bos taurus and B. indicus. One of the marker polymorphisms was found to be in a novel, dispersed DNA sequence which occurs in several ruminant species. The marker will assist in the detection of crossbreeding between Zebu and B. taurus types where this threatens a potentially valuable trypanosomiasis-resistant B. taurus genetic resource in West Africa. In addition, the marker will be useful for exploration of the evolutionary relationships of the major subspecies of domestic cattle. The general approach used to identify population-specific DNA polymorphisms has potentially broad application in definition of species, breeds and populations and will be of generic value in studies of genome evolution.     

Influence of sire and sire breed (Gyr versus Holstein) on establishment of pregnancy and embryonic loss in lactating Holstein cows during summer heat stress

Heat stress has negative effects on pregnancy rates of lactating dairy cattle. There are genetic differences in tolerance to heat stress; Bos taurus indicus (B. t. indicus) cattle and embryos are more thermotolerant than Bos taurus taurus (B. t. taurus). In the present study, the effects of sire and sire breed on conception and embryonic/fetal loss rates of lactating Holstein cows during the Brazilian summer were determined. In Experiment 1, cows (n=302) were AI after estrus detection or at a fixed-time with semen from one Gyr (B. t. indicus) or one Holstein sire (B. t. taurus). Pregnancy was diagnosed 80 days after AI. In Experiment 2, cows (n=811) were AI with semen from three Gyr and two Holstein sires. Pregnancy was diagnosed at 30-40 and at 60-80 days after AI. Cows diagnosed pregnant at the first examination but non-pregnant at the second were considered as having lost their embryo or fetus. Data were analyzed by logistic regression. The model considered the effect of sire within breed, sire breed, days postpartum, period of lactation, and AI type (AI after estrus versus fixed-time). There was no effect of the AI type, days postpartum or milk production on conception or embryonic loss rates. The use of Gyr bulls increased pregnancy rate when compared to Holstein bulls [9.1% (60/657) versus 5.0% (23/456), respectively, P=0.008; data from Experiments 1 and 2 combined]. Additionally, in Experiment 2, cows inseminated using semen from sire #4 (Gyr) had lower embryonic loss (10%) when compared with other B. t. indicus (35.3% and 40%) or B. t. taurus sires (18.2% and 38.5%, P=0.03). In conclusion, the use of B. t. indicus sires may result in higher conception rates in lactating Holstein cows during summer heat stress. Moreover, sire can affect embryonic loss and selection of bulls according to this criterion may result in higher parturition rates in lactating Holstein cows.     

Genetic characterization of the bovine leukaemia inhibitory factor (LIF) gene: isolation and sequencing, chromosome assignment and microsatellite analysis

The bovine leukaemia inhibitory factor was isolated from a phage library and sequences for the gene, in addition to 1213bp of 5' and 432bp of 3' sequences, were obtained and compared with other mammalian leukaemia inhibitory factor genes. Comparisons indicated amino acid homologies ranging from 89·6% to 77·2% with the human and mouse homologues, respectively. Analysis of 500bp of 5' regulatory regions indicated homologies ranging from 83·6% to 74·4% with the corresponding human and sheep sequences, respectively. Additionally, bovine leukaemia inhibitory factor-specific primers were prepared, and a panel of bovine × hamster somatic cell lines were analysed by the polymerase chain reaction (PCR). Data indicated 93% concordance of leukaemia inhibitory factor with aldehyde dehydrogenase 2 located on bovine chromosome 17, and concordance of 81% with myelin basic protein situated on bovine chromosome 24. Southern analysis of selected hybrids confirmed the PCR results, thus conclusively assigning the bovine leukaemia inhibitory factor gene to chromosome 17. Sequence analysis also revealed a microsatellite in intron 2 of the bovine leukaemia inhibitory factor. Analysis of this region by PCR in 22 unrelated Bos taurus and 19 unrelated Bos indicus cattle detected nine different alleles. Polymorphic information content values were 0·53 and 0·80 in B. taurus and B. indicus , respectively. Additionally, the same leukaemia inhibitory factor primers successfully detected allelic variants at this locus in Bos javanicus , Bos guarus and Bison bison but not in Odocoileus virginianus .     

Estradiol regulation of luteinizing hormone secretion in heifers of two breed types that reach puberty at different ages

The working hypothesis was that 17 beta-estradiol (E(2)) negative feedback on the hypothalamic-pituitary axis in regulation of LH secretion decreases during peripuberty in heifers of 2 different genotypes. We investigated whether Bos indicus heifers had a period postpuberty, as compared with prepuberty, of greater E(2) inhibition of LH secretion at a time when heifers of this genotype have been reported to have a period of anestrus. Prepubertal heifers 9 mo of age of 2 genotypes (B. indicus and B. taurus) were assigned to 3 groups (6 animals/group) to either remain intact (control), be ovariectomized, or be ovariectomized and implanted with E(2). Variables evaluated from 10 to 28 mo of age were circulating concentrations of progesterone (P(4)), presence of corpora lutea, and pulsatile pattern of LH release. Results confirmed that B. taurus heifers attained puberty at younger ages (P < 0.001) and at lower live weights (P = 0.015) than did B. indicus heifers (507 +/- 37 days of age vs. 678 +/- 7 days of age; 259 +/- 14 kg vs. 312 +/- 11 kg; respectively). There was cessation of E(2) inhibition of LH pulses coincident with the onset of puberty in heifers of both breed types but at a much younger age in B. taurus heifers. There was no evidence of enhanced negative feedback of E(2) on LH secretion subsequent to puberty in B. indicus heifers nor was there cessation of estrous cycles in control heifers of either breed type after puberty.     

A polymorphic satellite sequence maps to the pericentric region of the bovine Y chromosome

Exploiting a serendipitously observed bovine male-specific signal, generated by the mouse pSP64.2.5EI minisatellite probe, we have cloned a bovine (Bos taurus) Y-specific sequence: btDYZ-1. This sequence is composed of 60 tandem repetitions of a motif consisting of two parts: a 40-bp-long unit, showing a mean divergence of 27% between repeats, separated from the next repeat by a TG-rich stretch varying in length between 12 and 63 bp. The number of copies of this repeated motif has been estimated at 6 X 10(4) per male genome. As a consequence, the corresponding satellite, DYZ-1, might represent approximately 1/20 of the bovine Y chromosome. btDYZ-1 has been mapped by in situ hybridization to the pericentric region of the Y chromosome. It is characterized by a substantial genetic polymorphism and has been shown to be conserved within the Bos and Bison genera of the Bovinae subfamily. This sequence is being used to develop a sexing procedure for bovine preimplantation embryos based on the polymerase chain reaction.