Bioinformatics Analysis of Upstream Region and Protein Structure of Fungal Phytase Gene.

Phytase increases the bioavailability of phytate phosphorus in seed-based animal feeds and reduces the phosphorus pollution of animal waste. Since most animal feeds for pellets are heated up to 65-80 °C, the production of a thermostable structure for phytase can be useful. In this study, we sought to perform bioinformatics analysis of the upstream region and protein structure of fungal phytase to improve its expression and thermostability properties. We used bioinformatics methods such as similarity search, multiple alignment, statistical analysis of physicochemical properties of amino acids, pattern recognition, and protein modeling to find out the effective factors in heat resistance of phytase. Change in Gibbs free energy (ΔG) of the best pattern promoter resulting from the interaction between RNA polymerase and the promoter sequences of modified genes of phytase was equal to -9 kcalmol-1, which is lower compared to other interactions. The evaluation of the three-dimensional structure of new phytases showed that amino acid substitutions aimed at improving thermostability did not change the form and structure of the protein. The results of Prochek, Whatcheck, and ERRAT for structural analysis and verification were 84, 72, and 70, respectively, that were satisfactory.

Growth performance, feed digestibility, body composition, and feeding behavior of high- and low-residual feed intake fat-tailed lambs under moderate feed restriction.

Two experiments were conducted to evaluate the effect of moderate feed restriction on productivity of lambs classified on the basis of phenotypic expression of residual feed intake (RFI). In Exp. 1, 58 fat-tailed Kurdi ram lambs (32.1 ± 4.2 kg BW) were individually fed, ad libitum, a pelleted diet (35% alfalfa hay and 65% concentrate). Feed intake and ADG were determined for a 6-wk period and 3 feed efficiency measures including RFI, G:F, and partial efficiency of maintenance (PEM) were calculated. The lambs were sorted based on RFI and the 16 highest RFI (RFI ≥ mean + 0.5 SD) and 16 lowest RFI (RFI ≤ mean - 0.5 SD) lambs were subjected to body composition (BC) and DM digestibility (DMD) analysis. Feeding behavior traits (FB) were also evaluated for 24 h using a regular 5-min interval observation method. The high- and low-RFI lambs (14 lambs/RFI group) so classified in Exp. 1 were used in Exp. 2. Half of the lambs in each RFI group were randomly selected to be fed ad libitum or 85% of ad libitum (restricted feeding), which resulted in 4 experimental groups: 1) ad libitum high-RFI, 2) feed restricted high-RFI, 3) ad libitum low-RFI, and 4) feed restricted low-RFI. The lambs were fed the same diet as Exp. 1, and growth efficiency during a 6-wk test period as well as BC, DMD, and FB were also determined in Exp. 2. In Exp. 1, the low-RFI lambs consumed 14% ( < 0.01) less feed than high-RFI lambs. Differences were also observed between high- and low-RFI groups for G:F ( = 0.01), RFI ( < 0.01), and PEM ( < 0.01) in Exp. 1, but no differences were detected between high- and low-RFI lambs for ADG ( = 0.79), DMD ( = 0.42), BC ( > 0.72), and FB ( > 0.24). In Exp.2, the restriction feeding regime negatively affected ADG ( < 0.01) and G:F ( = 0.02) in low-RFI lambs, whereas G:F ( = 0.02) and PEM ( < 0.01) were improved in high-RFI lambs under the feed restriction condition. No effects of feed restriction on DMD ( = 0.87) and BC ( > 0.05) were observed. The lambs fed at the restricted level of intake presented a greater time ( < 0.01) and rate ( = 0.01) of eating than those fed ad libitum. Although bunk visits and feeding events were decreased ( < 0.01) with feed restriction, no interaction ( > 0.05) was detected between RFI phenotype and feeding regime for FB. In summary, feeding high-RFI lambs at 85% of ad libitum level improved G:F with no effect on ADG, whereas growth performance was reduced by feeding low-RFI lambs at 85% of ad libitum. However, these changes in feed efficiency were not related to DMD, BC, or FB.

Distribution of BoLA-DRB3 allelic frequencies and identification of a new allele in the iranian cattle breed sistani (Bos indicus).

The distribution of the frequencies of BoLA-DRB3 gene alleles in the Iranian cattle breed Sistani was studied by the PCR-RFLP ("hemi-nested") assay using restriction endonucleases RsaI, HaeIII and BstYI. In the examined cattle breed (65 animals) 32 alleles have been identified one of which being described for the first time (6.15% frequency). The nucleotide sequence of the polymorphic region of exon 2 of this allele has been determined and submitted in the GeneBank database under accession number DQ486519. The submitted sequence has maximum homology (92%) with the previously described sequence DRB3-mRNA from Bos indicus (AccN X79346) and differs from it by 24 nucleotide substitutions which result in 16 amino acid substitutions. The peptide (on the basis of the reconstructed amino acid sequence) has 89% identity to the sequence encoded by the BIDRBF 188 locus (Bos indicus). The results obtained permit the sequence described by us to be considered as a new allele of the BoLA-DRB3 gene (DRB3.2**X). The total frequency of the main six alleles (DRB3.2*X, *10, *11, *20, *34 and *X) occurring with a frequency of over 5% is about 60% in Iranian Sistani cattle. Fifteen alleles have <1% frequency. The highest frequency was observed for DRB3.2*8 allele (21.54%) like in other previously described breeds of Bos indicus (up to 23.07%). The Iranian breed Sistani has a high level of similarity by the spectrum of BoLA-DRB3 alleles and their frequencies to other Bos indicus breeds and significantly differs by these criteria from the Bos taurus breeds. The Iranian Sistani herd under study includes alleles associated with to resistance to leukemia (DRB3.2*ll and *23) and to different forms of mastitis (DRB3.2*2, *7, *11, *23 and *24) although their frequencies are low (from 0.77 to 5.37%). On the whole, a high level of diversity of BoLA-DRB3 gene alleles and the availability of alleles associated with resistance to different diseases makes this breed of interest for breeding practice.

Polymorphism within the intron region of the bovine leptin gene in Iranian Sarabi cattle (Iranian Bos taurus).

The main goal of breeders is breeding superior animals. Most traits of the economic importance in farm animals are determined by polygenic loci with environmental factors. Progress in animal breeding may be improved by combining traditional performance data with molecular genetic information on quantitative loci in selection index. Candidate genes are chosen for study on the basis of known relationships between biochemical and physiological processes. Leptin is 16 KD protein that is synthesized by adipose tissue and it is involved in the regulation of feed intake, energy balance, fertility and immune functions. In cattle, the leptin gene is located on chromosome 4. It consists of 3 exons and 2 introns of which only two exons are translated into protein. Sixty six animals were genotyped for this project. A PCR was carried out between 2 exon (intron 2). A strategy employing polymerase chain reaction was used to amplify a 422 bp from blood, semen, hair root and milk DNA. Digestion of polymerase chain reaction products with Sau3AI revealed two alleles: Allele A was 390, 32 fragments and allele B was 303, 88, 32 (only 303 fragment visible on the gel). Three patterns were observed and frequencies were 0.31, 0.43, and 0.14 for AA, AB and BB respectively. This polymorphism could be further evaluated for marker assisted selection and developed PCR methodology would expedite screening for large numbers of animals required for such studies.

Analysis and frequency of bovine lymphocyte antigen (BoLA-DRB3) alleles in Iranian Holstein cattle.

The bovine lymphocyte antigen (BoLA-DRB3) gene encodes cell surface glycoproteins that initiate immune response by presenting processed antigenic peptides to CD4 T helper cells. DRB3 is the most polymorphic bovine MHC class II gene which encodes the peptide-binding groove. DRB3 gene has been extensively evaluated as a candidate marker for association with various bovine diseases and immunological traits. This study describes genetic variability in the BoLA-DRB3 in Iranian Holstein cattle. This is the first study of the DNA polymorphism of the BoLA-DRB3 gene in Iranian Holstein cattle. Hemi-nested PCR-RFLP method is used for identification the frequency of BoLA-DRB3 alleles. The BoLA-DRB3 locus is highly polymorphic in the studied herd (26 alleles). Almost 67% of the alleles were accounted for four alleles (BoLA-DRB3.2*8, *24, *11 and *16) in Iranian Holstein cattle. The DRB3.2*8 allele frequency (26.6%) was higher than the others. The frequencies of the DRB3.2*54, *37, *36, *28, *25, *14, *13, *10, *1 alleles were lower than 1%. Significant distinctions have been found between Iranian Holstein cattle and other cattle breeds studied. In Iranian Holstein cattle the alleles (BoLA-DRB3.2*22, *2 and *16) associated with a lower risk of cystic ovarian disease in Holstein cattle are found. The alleles associated with the resistance to mastitis and to bovine leukemia virus infection BoLA-DRB3.2*11 and *23 are detected with the frequencies 10.4% and 4.4%, respectively. Thus in the Iranian Holstein cows studied are found alleles which are associated with resistance to various diseases. The method of DNA-typing of animals can be used in agricultural practice for BoLA-DRB3 allele genotyping of cattle in order to reduce spreading of alleles providing susceptibility to mastitis or leukemia in cattle herds.

Identification of bovine leucocyte adhesion deficiency (BLAD) carriers in Holstein and Brown Swiss AI bulls in Iran.

BLAD is a hereditary disease in Holstein dairy cattle. The defective allele of CD18 gene which is responsible for this disease has a recessive inheritance. The recessive homozygous form (BL/BL) is lethal and since carrier animals have viability, BLAD frequency increases by use of carrier bulls in Artificial Insemination (AI). BLAD carriers can be detected easily by means of polymerase chain reaction followed by restriction analysis of the amplicons. In this study DNA samples from Holstein (n = 30) and Brown Swiss (n = 10) bulls from Abbas Abad AI center (Khorasan state of Iran) were analysed. A 101 bp fragment from the polymorphic region of CD18 gene located on chromosome 1 was amplified by PCR. Restriction enzymes TaqI and HaeIII were used to identify genotypes. Digestion products were screened by electrophoresis on 8% non-denaturing polyacrylamide gel and visualized by ethidium bromide staining. Frequencies of BL/TL (carrier) genotypes in Holstein and Brown Swiss bulls were 3.33% and 0%, respectively. Our pedigree studies of the carrier bull in this experiment revealed that the mutation inherited to him from Hawkeye bull (CANM 369995, BL). Although the elimination of BLAD-carrier bulls from the Holstein world would be the most efficient method to control this genetic disorder, many BLAD-carrier bulls are still listed commercially for AI and BLAD is still occurring in Iran. Monitoring the prevalence of BLAD-carriers in random selected herds may be helpful in judging the effectiveness of the BLAD-control program.