association of bovine Osteopontin [OPN] gene with milk production traits in Iranian Holstein Bulls

Background: the Osteopontin [OPN] is a highly phosphorylated glycoprotein in numbers of bovine tissues and milk. OPN has been reported to be associated with milk production in cattle

Objective: the genotype and allelic frequencies for OPN and its association with milk production will be evaluated in Iranian Holstein Bulls

Materials and Methods: bulls DNA [100] was isolated. Oligo was used for primer design. Polymerase Chain Reaction was implemented to amplify a 826 bp fragment and the amplicon was digested by BsrI. Restricted Maximum likelihood [REML] method based on average information algorithm using ASRMEL programs [version 3.1] was employed to estimate the genetic parameters and variance of components. The association of OPN genotypes with milk production traits were analysed by the least square method as applied in the general linear model [GLM] procedure of SAS. Allele substitution effects were performed by regression analyses

Results: allele frequencies of T and C were 0.59 +/- 0.03 and 0.41 +/- 0.03, respectively. Genotype frequencies of TT, CT and CC were 34.69, 48.62, and 16.69, respectively. The chi-square test showed the deviation from Hardy-Weinberg equilibrium. Estimated heritability for milk yield, fat yield and its percent, protein yield and its percent were 0.28 +/- 0.0061, 0.21 +/- 0.0064, 0.22 +/- 0.0086, 0.32 +/- 0.0065 and 0.34 +/- 0.0096 respectively. Allelic substitution effects and differences between genotypes were not significant for milk production traits

Conclusions: this study suggested that the C allele frequency of OPN was noticeable in Iranian proven bull Holstein population, but was not associated with milk production traits. However, before being practical for the breeding improvement of Iranian Holsteins a larger sample size is required

Evaluation of two DNA extraction methods from maternal plasma for using in non-invasive bovine fetus gender determination

Fetal DNA in maternal plasma and serum has been shown to be a useful material for prenatal fetal sex determination during early gestational ages. Non-invasive prenatal diagnosis is now possible at 8[th] week of pregnancy, by maternal blood sample testing. The purpose of this study was to evaluate two DNA extraction methods from mother plasma and its routine clinical application in bovine fetus gender determination with non-invasive method. Maternal blood samples were taken from 40 pregnant cows during the 8[th]-38[th] weeks of gestation. DNA was extracted from 350 micro l of maternal plasma with two salting-out and phenol-chloroform methods. The absorption in A[260] and purity [A[260]/A[280]] of extracted DNA were detected by ultraviolet spectrophotometer. Three micro l of the extracted DNA with phenol-chloroform method was used as a template. The PCR reaction was carried out to amplify the fragments of X and Y chromosomes of amelogenin, TSPY and BC1.2 genes. The difference between the mean absorption of DNA extracted by phenol-chloroform method and salting-out method was not significant in A260 [p>0.05, p=0.3549], but the difference between mean purity [A260/A280] of DNA extracted by phenol-chloroform method and salting-out method was significant [p<0.001]. X chromosome fragment was detected in all 40 samples and Y chromosome fragments were detected in 25 plasma samples which were delivered a male calf. The sensitivity and specificity of test was 100% with no false negative and false positive results. The results showed that phenol-chloroform method is a simple and sensitive method for isolation of fetal DNA in maternal plasma

low-cost efficient multiplex PCR for prenatal sex determination in bovine fetus using free fetal DNA in maternal plasma

In order to establish a reliable non-invasive method for sex determination in a bovine fetus in a routine setting, the possibility of identifying specific sequence in the fetal X and Y-chromosomes has been evaluated in maternal plasma using conventional multiplex polymerase chain reaction [PCR] analysis. The aim of this study was to provide a rapid and reliable method for sexing bovine fetuses. In this experimental study, peripheral blood samples were taken from 38 pregnant heifers with 8 to 38 weeks of gestation. DNA template was extracted by phenol-chloroform method from 350 micro l maternal plasma. Two primer pairs for bovine amelogenin gene [bAML] and BC1.2 were used to amplify fragments from X and Y chromosomes. A multiplex PCR reaction has been optimized for amplification of 467 bp and 341 bp fragments from X and Y bAML gene and a 190 bp fragment from BC1.2 related to Y chromosome. The 467 bp fragment was observed in all 38 samples. Both 341 and 190 bp fragments were detected only in 24 plasma samples from male calves. The sensitivity and specificity of test were 100% with no false negative or false positive results. The results showed that phenol-chloroform method is a simple and suitable method for isolation of fetal DNA in maternal plasma. The multiplex PCR method is an available non-invasive approach which is cost efficient and reliable for sexing bovine fetuses

Polymorphisms of the ovine leptin gene and its association with growth and carcass traits in three Iranian sheep breeds

Leptin [LEP], the expression product of the obese gene produced primarily in the adipose tissue, is related to feed intake, growth and lipid metabolism. The aim of this study was to study the possible association between polymorphism of the LEP gene with growth and carcass traits among three Iranian sheep breeds of the Shal, Zandi and Zel varieties. A total of 180 purebred animals of the Shal, Zandi and Zel, breeds were chosen for this study. Three flocks, each comprising of 60 ewes of three breeds, were derived from Aboureyhan sheep populations. The Shal [n=18], Zandi [n=24] and Zel [n=17] lambs were screened for polymorphism of the LEP gene. Following genomic DNA extraction from whole blood samples, polymerase chain reaction [PCR] was carried out in order to amplify a 260 bp fragment of the target gene. Polymorphisms were detected using the single strand conformational polymorphism [SSCP] technique. Two genotypes of AA and AG with frequencies of 0.53 and 0.47 in the Shal 0.70 and 0.30 in the Zandi and 0.65, 0.35 in the Zel breed were observed, respectively. The frequencies of alleles A and G were 0.74, 0.26 in the Shal breed, 0.85, 0.15 in the Zandi breed and 0.82, 0.18 in the Zel breed, respectively. Chi-Square test [chi2] confirmed the Hardy-Weinberg equilibrium for the LEP loci. Average heterozygosity [30%] of the LEP locus for the three breeds was slightly low. Comparison of the sequence of the target gene available in the GeneBank with the results of the present study showed two single nucleotide polymorphisms [SNP] A?G and T?C transitions at 113 and 165 bp positions, respectively. In the Shal breed, the A113G SNP associated with an increase in cold carcass weight [p< 0.01], fat-tail percent [p< 0.05] and total body fat weight [p< 0.05]. In the Zel breed, the A113G SNP was associated with an increase in fat-tail percent [p< 0.05] and reduction in slaughter weight [p< 0.05], cold carcass weight [p< 0.01] and lean meat weight [p< 0.01]. Therefore, a significant association between SNP within the LEP gene and certain carcass traits in the Shal and Zel breeds is proposed. In the Zandi breed the A113G SNP was not associated with carcass traits but showed a reduction in weaning weight [P< 0.05]

Polymorphism of bovine lymphocyte antigen DRB3.2 in holstein bulls of Iran using PCR-RFLP

The Holstein bulls [n=50] were genotyped for bovine lymphocyte antigen [BoLA-DRB3.2] alleles by polymerase chain reaction and restriction fragment length polymorphism [PCRRFLP]. Genomic DNA was extracted from bull semen using phenol-chloroform method. A two-step PCR was conducted in order to amplify a 284 base-pair fragment of the target gene. Amplicons were digested by RsaI, HaeIII and BstyI restriction endonuclease enzymes. Digested fragments were electrophoresed on 8% polyacrylamide gel and visualized after silver staining. Seventeen BoLA-DRB3.2 alleles were identified with frequencies ranging from 1 to 21%. Sixteen alleles were similar to those reported previously and one was a new allele which has not been reported before. The frequencies of alleles BoLA-DRB3.2 *3, *8, *10, *11, *12, *13, *15, *16, *21, *22, *23, *24, *28, *51, *iaa, *ibb, *qbb were 2, 9, 2, 14, 1, 2, 4, 10, 1, 14, 5, 21, 6, 6, 1, 1, and 1%, respectively. The seven most frequent alleles [BoLA-DRB3.2 *8, *11, *16, *22, *24, *28, *51] accounted for 80% of alleles in the investigated population. This data indicate that the BoLADRB3.2 locus is highly polymorphic in Holstein bulls of Iran