رامین صیقلانی

Identifying the strains carrying acquired tetracycline resistance genes is one of the important points in evaluating the safety of probiotic strains. The aim of the present study is to comprehensively evaluate tetracycline resistance in lactobacillus spp. isolated from the digestive system of Iranian backyard chickens.

In the present study, the phenotypic sensitivity patterns of 36 lactobacillus isolates belonging to four species L. reuteri, L. salivarius, L. crispatus and L. johnsonii were evaluated by measuring the Minimum Inhibitory Concentration (MIC) to tetracycline. After that, four tetracycline resistance genes tet(L), tet(M), tet(W) and tet(O) were detected in isolates with phenotypic resistance to this antibiotic based on the polymerase chain reaction method.

As a result, four isolates of L. reuteri, three isolates of L.salivarius, two isolates of L. crispatus and four isolates of L. johnsonii among the studied lactobacillus spp. showed phenotypic resistance to tetracycline. After the detection of tetracycline resistance genes in isolates with phenotypic resistance, the presence of tet(W) gene was confirmed in all investigated isolates. The simultaneous presence of tet(M), tet(L) and tet(W) genes was observed in three studied L. salivarius isolates. It was also found that the phenotypic resistance observed in three isolates of L. johnsoniiABRIG7, L. johnsoniiABRIG14 and L. johnsoniiABRIG24 is caused by the simultaneous presence of tet(O) and tet(W) genes.

This study showed that tetracycline resistance among Lactobacilli isolated from digestive system of Iranian backyard Chickens is due to the presence of single or multiple resistance genes.. We also found that tet(W) is the most widespread tetracycline resistance gene among investigated Lactobacillus isolates. Therefore, local poultry breeders should refrain from arbitrarily using tetracycline, and when the digestive system of domestic chickens is the source for probiotic microorganism selection, the evaluation of resistance to tetracycline from the phenotypic and molecular point of view should be taken into consideration in order to select safe strains.

Introduction: 

Feed additives are commonly used in poultry feed to enhance performance, promote health, and increase nutrient efficiency. The use of antibiotics as growth promoters in poultry feed has been prevalent for years. However, due to concerns regarding the accumulation of antibiotic residues in poultry products and the development of antibiotic-resistant bacterial strains, antibiotics are no longer considered desirable additives in poultry feed. As a non-therapeutic alternative to antibiotics, probiotics have been introduced as suitable candidates to promote growth. Probiotics have beneficial effects on poultry digestive enzymes, improve intestinal absorption, and neutralize toxins produced by harmful microorganisms, ultimately improving the immune system and economic performance. Among the most popular probiotic bacteria are Lactobacilli, as they are generally classified as safe bacteria.

Materials and methods:

 Lactobacillus reuteri (L. reuteri ABRIG25 (MF686485)) and salivarius (L. salivarius NABRII59 (MH595987) isolated from the digestive tract of Guilan’s native chicks and Mazandaran’s duck  respectively, were prepared up to 1.36×109 CFU using MRS medium at 37 ° C, under anaerobic conditions. 300 one-day-old male Arbor-Acres chicks were distributed in a completely randomized design, with 5 treatments, 4 replications (15 chicks per replicate). Experimental treatments were: 1- Basic diet as control group (Cont), 2- Basic diet + 100 g / ton Avilamycin as antibiotic group (Anti), 3- Basic diet + 200 g / ton commercial probiotic (Lacto-feed®) (Plac), 4- Basic diet + 1 g / Kg of L. salivarius NABRII59 (MH595987) bacterial powder (Pls1), and 5- basic diet + 1 g / kg of L. reuteri ABRIG25 (MF686485) bacterial powder (Plr1). Daily feed intake, weight gain, and feed conversion ratio of broilers were determined and recorded in starter, grower, and finisher  periods. On day 42, two chicks were slaughtered from each replicate and the weight of internal organs and carcass cuts were recorded as a percentage of carcass weight. Chicken antibody reaction were determined using SRBS suspension. On days 22 and 35 of the rearing period, two chicks were randomly selected from each cage and 0.1 cc of  SRBC solution was injected into to the wing’s vein, intravenously. Humoral immunity test was applied on days 29 and 42, using 1 cc of blood taken from the wing vein of chickens. The hemagglutination reaction was recorded based on the last two dilutions as SRBC’s antibody using the logarithm and the antibody titer against Newcastle was determined by hem agglutination inhibition (HI) test. On day 42, blood samples were taken randomly from 2 birds per replication to evaluate blood-serum parameters including glucose, cholesterol, triglyceride, HDL, LDL and VLDL. All data were analyzed using SAS software v.9.1 (2012) in GLM procedure using a completely randomized design, and comparison of statistical means was performed, using Duncan's method at the level of 0.05.

The present study investigated the effect of native probiotic isolates (Plr1, Pls1) compared to commercial probiotics (Plac) and antibiotics (Anti) on the growth performance of chickens. The results showed no significant differences in daily feed intake, weight gain, and feed conversion ratio between experimental treatments. However, two native probiotic isolates (Plr1, Pls1), the commercial probiotic (Plac), and the antibiotic (Anti) resulted in a significant reduction (P <0.05) in proventriculus weight, while there were no significant differences in the relative weight of other organs. Both the commercial probiotic (Plac) and the native Lactobacillus isolates (Plr1 and Pls1) significantly improved total immunoglobulin and immunoglobulin G (IgG) levels after both the first and second injections (P <0.05). Lactobacillus salivarius (Pls1) isolate also significantly improved immunoglobulin M (IgM) levels after the second injection. However, there were no significant differences between treatments and the control group in terms of antibody titer against Newcastle disease vaccine, blood glucose, cholesterol, triglyceride, VLDL, and LDL levels. Probiotics can affect gut microbiota by competing for nutrients and attachment sites on the intestinal epithelial cells. Additionally, they may improve blood parameters and stimulate immune system cells to produce cytokines, which play an important role in inducing and regulating immune responses in poultry. Probiotics support lactic acid-producing bacteria and stabilize the gut microflora, which has beneficial effects on feed conversion ratio by stimulating the production of digestive enzymes. Furthermore, probiotics may reduce cholesterol synthesis by fermenting indigestible carbohydrates, leading to the production of short-chain fatty acids and ultimately lowering cholesterol levels in the host bird. Discrepancies in results reported by different researchers may partly be due to differences in chick’s age and breed, level of stress, diet composition, consumption period and duration, type of commercial probiotics, dose or amount of probiotic intake, management skills, and environmental conditions in different experiments.

The general conclusion is that the probiotic isolates used in this study are competitive with the commercial type of probiotics as well as the antibiotic used and are promising as probiotic candidate with beneficial effects on broiler’s performance, blood biochemical parameters and immune system

There is much information about the regulation of gene expression in response to various stresses at the transcriptional level. Nevertheless, there is limited information about this process at the post-transcriptional level. The diversity and complexity of miRNA regulation indicates their importance in biological processes. Many miRNA regulatory modules can form a complex miRNA-mRNA regulatory network. Therefore, research on miRNA-mRNA regulatory networks can provide valuable information for understanding complex biological processes. These data are very important to further study the stress tolerance mechanisms in plants, especially in rapeseed. In this research, the selection of miRNAs related to drought and salinity stress was made by reviewing the articles on abiotic stresses. Then the target genes were identified using the sequences of mature miRNAs and psRNATarget online software. A gene list of 225 identified target genes was prepared using the UniProt database. Their functional pathway was identified utilizing the DAVID bioinformatics database and KEGG database according to default parameters. Investigations showed that these target genes were involved in several biological pathways including ribosome, spliceosome, proteasome, purine metabolism, selenocompound metabolism, and sulfur metabolism. In addition, the STRING database was used to check co-expression genes. Our result indicated the existence of 37 co-expression genes among the identified target genes.

Gastrointestinal nematodes (GIN) represent a major health issue for livestock production systems worldwide. Haemonchus contortus is one of the most pathogenic GIN in small ruminants and causes serious losses to farmers, both in impaired production and in control with anthelmintics. It has long been recognized that differences in host resistance and susceptibility to parasitic infection exist in various sheep breeds and that genetics may play an important role in regulating host resistance, which has spurred efforts to control parasitic infection through selective breeding for naturally resistant sheep. A detailed understanding of the genes and mechanisms involved in expressing a resistant phenotype and the factors that regulate this response would facilitate the identification of candidate genes for selection. Recent advances in high-throughput technology, such as microarrays and RNA sequencing, have made a large number of transcriptome data accessible. As a result, researchers are now able to obtain more reliable results by integrating information from multiple sources. Accordingly, meta-analysis can be used as a useful and powerful tool to identify differential gene expression. It can also find out genes that their products are key molecules in response to the infection and use in animal breeding programs. The purpose of this study was to conduct a systematic review and meta-analysis on data collected from infected sheep with H. contortus and general analysis of changes in abomasal gene transcripts in response to infection using RNA-seq technology and bioinformatics tools. In this study, to identify infection-related genes, pathways, and molecular mechanisms underlying host resistance to this parasite, a meta-analysis was performed by combining two different datasets, including 70 samples of sheep H. contortus infection with Rankprod package of R software. After pre-processing, to remove heterogenicity across studies, batch effect correction was performed on gene expression data. The result of the principal component analysis showed that batch correction reduced the batch variation among the datasets. Meta-analysis was carried out and DEGs selected by meta-analysis were further analyzed and characterized. Enrichment analysis, as an efficient method for functional analysis of massive genetic data, was used to determine the biological process, molecular function, and cellular component of DEGs. Moreover, we searched upstream regions of DEGs for over-represented DNA motifs and functional analysis of discovered motifs. To explore the potential interaction network of the DEGs, the protein-protein interaction network among the DEGs was analyzed using the STRING database, which included direct and indirect associations of proteins. After analyzing the result derived from STRING analysis and expression change information for each DEG, the network figure was drawn for the selected DEGs (connected with one or more DEGs) by using the Cytoscape software and hub genes identified with the CytoHubba plugin of Cytoscape. Results derived from the meta-analysis showed a total of 1388 differentially expressed genes between resistant and susceptible sheep. Among them, 1137 were significantly upregulated, whereas 251 were downregulated across the datasets. In the identified DEGs, DEGs corresponding to ribosomal protein S3A, lysozyme C-1-like (LOC443320), and heterogeneous nuclear ribonucleoprotein K were the most strongly upregulated ones, while tenascin C and fibromodulin were the most strongly downregulated. Results from enrichment analysis showed these differentially expressed genes (DEGs) were involved in different biological processes such as one-carbon metabolism, translation, cell surface receptor signaling pathway, immune response, metabolic pathways, PPAR signaling pathway, etc. Moreover, searching in upstream regions of DEGs to find DNA motifs, were able to identify eight conserved sequence motifs. The functional analysis of these motifs revealed that they were involved in the positive regulation of gene expression, defense response, positive regulation of immune response, cellular calcium ion homeostasis, etc. Using the protein-protein interaction analysis also identified multiple hub genes such as albumin and CD4 which may show that improved immune response, induced by up-regulation different genes affects the creation of resistance. The mechanisms of sheep resistance to GIN infections involve complex immune responses. Our results offered overall insight into changes in the transcriptomes of resistant and susceptible sheep and molecular mechanisms of host resistance induced by H. contortus infection. We propose these DEGs as a useful resource of molecular biomarkers and potential candidate genes for breeding programs which can provide a basis for further research on this topic.

In the recent years, many countries have banned or restricted the usage of antibiotics as growth-promoting, due to their undesirable effects on human and animal health. Using probiotics as an alternative to growth-promoting antibiotics play an important role in improving performance, health and increasing the quality of poultry products. Lactobacilli are the most popular probiotic bacteria that have beneficial effects on host’s performance and health. In this study, the effect of two native strains of Lactobacillus ruteri isolated from the gastrointestinal tract of native chickens in north-western and south-western of Iran were investigated on performance, carcass characteristics, blood parameters and immune response of broilers.

In this experiment, 300 Arbor-Acres® Plus+ male broiler chickens were divided in to 5 treatments, 4 replications (15 chickens per replication), in a completely randomized design. The experimental treatments include: 1- Basic diet as a control treatment, 2- Basic diet + Avilamycin antibiotic, 3- Basic diet + commercial probiotic (Bio-Poul®), 4- Basic diet + Lactobacillus reuteri isolate MG547731 (Plr2), and 5- Basic diet + Lactobacillus reuteri isolate MG547727 (Plr3).

Under the condition of this experiment, commercial probiotics, antibiotic and either Lactobacillus reuteri (Plr2) isolates, significantly improved daily weight gain in final period (p<0.05). Native isolates probiotics and commercial probiotics significantly (p<0.05) reduced the relative weight of gizzard and proventriculus, though no significant (p<0.05) differences were recorded for other carcass components in broiler chickens. After the first injection of SRBC, treatments include antibiotic and Lactobacillus reuteri (Plr2) isolate caused a significant increase of IgM, and after the second injection, Lactobacillus reuteri (Plr2) and commercial probiotic treatments, showed a significant (p<0.05) increase in immunoglobulin G and Total immunoglobulin. The antibody titer against Newcastle vaccine did not show a significant difference between treatments and the control group. There was no significant difference between serum glucose, cholesterol, triglyceride, VLDL and LDL between treatments and the control group. No significant difference was obtained in serum HDL level in probiotic treatments, while antibiotic treatment appeared with a significant decrease in HDL.

Addition of native Lactobacillus reuteri isolates to poultry diets can have beneficial effects similar to commercial probiotics, on performance, immune response and blood parameters of broilers.

This study was conducted to evaluate the effect of different extraction solvents, including hydro-alcoholic (H-A), ethanolic (Et-OH), and methanolic (Me-OH), on chemical composition and synergistic antibacterial activity of Frangula alnus extract against Staphylococcus aureus and Escherichia coli. The chemical composition of the F. alnus bark extract was evaluated using FTIR, UV-Vis spectroscopy, and GC/MS analysis. The synergistic antimicrobial effect of the extracts with Ciprofloxacin (CIP) and Erythromycin (ERY) was evaluated using minimum inhibitory concentrations, combined disc, and checkerboard titration method. F. alnus contained alkanes, alkenes, phenols, alcohols, esters, terpenes, fatty acid, tetrazole, halo-alkane, anthraquinone as well aromatic, and plasticizer compounds in the ethanolic, methanolic, and hydro-alcoholic extracts. Total phenolic compounds for ethanolic, hydro-alcoholic and methanolic extracts were recorded 110.92±0.01, 95.27±0.01 and 126.6±0.02 g/L Gallic acid, respectively. Et-OH and H-A extracts in both combined and synergistic forms significantly inhibited bacterial growth. Concerning the antibacterial activity of the extracts, it was assumed that the compounds present in the plant extracts would destroy the bacterial cell membranes and consequently cause leakage of intracellular contents. Therefore these antibiotics in combination with the extracts would be able to affect their target sites (CIP; DNA gyrase and ERY; 50S subunit) more effectively. According to the results, H-A, Me-OH, and Et-OH extract efficiently in the synergistic state with erythromycin and ciprofloxacin can be a promising candidate to be used against bacterial pathogens.

This study was conducted to survey the antimicrobial activity of Lactococcus lactis subsp. cremoris NABRII66 lactic acid bacterial strain against common bacterial pathogens in aquaculture and human populations by in vitro experiments. L. lactis subsp. cremoris NABRII66 has been isolated from rainbow trout intestine. Two methods including double layer agar and microtiter plate assay were used to evaluate the inhibition activity of selected bacterial strain against two bacterial pathogens L. garvieae and Aeromonas salmonicida species affecting salmonids and other fish speciesand two other bacterial pathogens including Escherichia coli and Staphylococcus aureus, as two most common source of infection in human population. The results of double layer agar method showed the antimicrobial activity of L. lactis subsp. cremoris NABRII66 bacterial strain against all the tested pathogens and the highest significant growth inhibition zone was observed against A. salmonicida and S. aureus bacterial pathogens (p<0/05). The results of the microtiter plate assay method showed that cell-free supernatant of L. lactis subsp. cremoris NABRII66 bacterial strain could significantly inhibit the growth of A. salmonicida (p<0/05). This study showed that Lactococcus lactis subsp. cremoris NABRII66 bacterial strain isolated from rainbow trout intestine had antimicrobial activity against four aquaculture and human's bacterial pathogens by producing some extracellular inhibitory compounds, which should be evaluated in future studies.

The purpose of this study was to investigate the effects of the use of three lactobacillus strains (L. reuteri ABRIG17 (MF686477), L. reuteri ABRIG23 (MF686483), L. reuteri ABRIG3 (MF686463)), Isolated from duodenum and Jejunum sections of the native Guilan chickens and one isolate of L. salivarius NABRII58 (MH595986) isolated from the digestive system of the native ducks of Mazandaran, on serum lipids and immune parameters of broiler chickens. The strains were isolated from 383 gram-positive and catalase negative lactic acid bacteria in a screening procedure for the detection of bacteria with probiotic potential. In the experiment, 500 male Ross 308 male broiler chicks were used in a completely randomized design with 5 treatments, 5 replicates and 20 birds per replicate. The treatments consisted of: 1-The basal diet as control group (treatment C), 2- The basal diet + 1 g / kg of mixed powder containing MF686463 (L. Reuteri ABRIG3 (LR1), 3- The base diet + 1 g /kg of L. Reuteri ABRIG23 (MF686483) (LR2 treatment), 4- The basal diet + 1 g / kg of L. Reuteri (MF686477) ABRIG17 (LR3 treatment) and 5- The basal diet + 1 g / kg of L. Salivarius (MH595986) NABRII58 (Treatment LS). The use of bacterial strains in LR1 and LS treatments resulted in improved weight gain at the end of the experiment (P <0.05). The bacterial strains in LR1 treatment improved daily weight gain in whole experimental period (P <0.05). All three strains of Lactobacillus used, namely, LR1, LR2 and LR3 treatments, caused a significant decrease in abdominal fat pad (P <0.05). In LR3 treatment, the increase in carcass weight was observed (P <0.05). Measuring serum immunoglobulins in broiler chicks after two stages of sheep red blood cell injection showed that LR3 had the highest total immunoglobulin level after the second injection (P <0.05), and IgG levels in the LR1 treatment increased after the first injection compared to the control group (P <0.05). After the second injection, the IgG of all the four experimental groups were higher than control group (P <0.05), but IgM showed no significant difference between experimental treatments and control group. Total serum cholesterol concentration of LR2 treatment was significantly (P <0.05) lower than other treatments. There was no significant difference in serum triglyceride level between control and the LR1, LR2 and LS treatments, and only LR3 triglyceride level was higher than others (P <0.05). The level of HDL or good cholesterol in LS treatment was higher than the control group (P <0.05). Serum LDL level did not show any significant difference between the experimental and control groups. The present study revealed that probiotic bacteria can be isolated from native poultry microbial populations and isolated bacteria can improve the production and immunity of broiler chicks. It is worthy of note that the positive effect of Lactobacillus salivarius strain isolated from the native duck gastrointestinal tract on the weight gain and serum immunoglobulins in broiler chickens suggested that the gastrointestinal bacteria of a species of poultry could be used as a probiotic in other types of poultry.

In the recent years, there is evidence of training a red type of zebrafish which differs from wild-type in body color. There is not any document how it reaches to the ornamental fish farms of Iran but at first, it was a doubt it belongs to a morphotype or genetic modification (GM). First of all, a set primer was designed to validate zebrafish species. Mitochondrial 16srDNA was selected and amplified. The validation of 16S rDNA gene sequences data detected three haplotypes deposited in the GenBank. By seeking scientific articles about transgenic zebrafish, the first attention went to Glowfish (color transgenic zebrafish). We decided to build a foundation of PCR based detection for finding a gene correspondence red color of zebrafish. This sequence is specific for producing a red color in the sea anemone and it was not in genome database of wild zebrafish. A set primer was designed according to the red color of sea anemone (dsRed) to make a fragment of 680 bp in red zebrafish and cream zebrafish specimens (they had red parents). Fragment sequencing (ds/Red) revealed that it belongs to the sea anemone and none of the samples nucleotides differ from each order and positive sequence from a plasmid containing dsRed. Also, protein samples from cream and red zebrafish were extracted and evaluated with fluorescence microscope by TxRed control color. A dark and a red spread was detected for protein smear of the cream and redfish respectively. A dark spread was detected for cream fish protein smear but a red smear was marked for redfish protein. This research simply demonstrates the existence of transgenic zebrafish in Iranian ornamental fish industry.

The nuclear polyhedrosis virus (NPV), producing grasserie disease in silkworm, is accounted as one of the most important reason for production loss in sericulture industry. Cross breeding is one of the most important and effective breeding methods which could improve silkworm performance. In the current study, the resistance of larvae to NPV in 104 and 154 Chinese based lines and their hybrids in F1 and F2 generation were investigated. Fourth instar larvae were inoculated by NPV and their mortality were checked. A great value of heterosis and heterobeltiosis was observed in F1 hybrids, due to the susceptibility of line 104 and the tolerance of line 154 to NPV. The value of heterosis and heterobeltosis in reciprocal crosses varied from 82.91 to 86.19 and 27.26 to 29.54, respectively. Reciprocal crosses, however, did not declared significant difference in the susceptibility of larvae to NPV. Heterosis was declined in F2 and maternal heterosis was completely expressed on this generation (13.80 to 17.89 percent). Reciprocal cross did not have considerable effect on the maternal heterosis which indicates that the use of tolerant line as maternal line or paternal line do not make considerable difference.

Honeybee is a pollinator that has prominent role in agriculture products and nourishment for human. So, the most number of studies like genetic improvement were done on honeybee. Recent advances in knowledge of molecular genetics of bee emphasize the potential for using genetic markers to select favored traits. This research was done to determine polymorphism of loci of first foraging age (AFF) trait in Iranian honeybee (Apis mellifera meda). Two colonies with high production- long longevity and low production- short longevity were selected from 11th generation of “Iranian Breeding Honeybee Project”. In the high production colony, foraging initiation had started sooner (age of 9 33 day). Also‚ the length of foraging initiation period in comparison with low production colony (age of 12-29 day) was longer. Genetic polymorphism of the loci was assessed by using 4 primer pairs. PCR products were detected using polyacrylamide gel. 26 polymorphic alleles were observed using AFLP markers. Totally‚ the most amount of Shanon Index is related to E3M2-1 locus in low production population (0. 693) and also E M3-4 position in high production population (0. 693). Therefore‚ these two loci have shown most polymorphism. The least amount of Shanon Index is related to E6M3-5 locus in low production population (0. 156). The highest effective allele was belong to E3M3-4 position in high production colony and the lowest was related to E6M3-5 position in low production population. Heterozygosity was diverse between (0. 053) in E6M3-5 to (0. 05) in E3M2-1. Mean of (He) in low production colony was (0. 321) and in high production colony was (0. 324). Mean of Shanon Index were (0. 490) and (0. 491) respectively. Genetic variance between 2 populations was only ٪10. There was difference in production in the 2 populations but, genetic diversity is not very impressive. Genetic similarity in them may is because of using the small number of samples in this research.

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 8th 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 8th-38th weeks of gestation. DNA was extracted from 350 µl of maternal plasma with two salting-out and phenol-chloroform methods. The absorption in A260 and purity (A260/A280) of extracted DNA were detected by ultraviolet spectrophotometer. Three µl 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.

Bovine growth hormone(bGH) gene, is a part of the multiple gene family that contains prolactin and placental lactogens. The variations in the introns of this gene, have potential usefulness as genetic markers and could help in the genetic improvement of populations. In ordre to investigate allele frequency of MspI in introne 3 of growth hormone gene of Guilan native cattle breed, blood samples were collected from seventy heads of randomaly choosen cattles (cows). Genomic DNA was extracted from these samples, using modified salting out method. Polymerase chain-reaction (PCR) procedure was used to amplify a 345 bp segment in this region and then digested with MspI restriction enzymes. Digested samples were run on the acrylamid gel 8% to recognize their genotype. This enzyme had two restriction sites and after digestion with MspI enzyme on that fragment and 2 allels and 3 genotypes have been observed. Frequency of MspI(+) allele was estimated to 0.4643. X2 test has showed the equilibrium of frequency MspI in samples.

PPARGC1A is a member of a family of transcription coactivators that plays a central role in the regulation of cellular energy metabolism. OPN is a phosphoprotein that is synthesized in a variety of tissues and cells and secreted into body fluids. In this study DNA was isolated from blood samples collected from 398 Holstein cows of Tehran and Esfahan provinces. DNA isolation was performed using the Salting out method. RFLP-PCR method was used for genotyping of SNP T>C at position 1892 and SNP A>C at position 3359 in PPARGC1A gene, also SNPC>T at position 8514 in OPN gene. Using of Pop Gene soft ware frequencies of TT, TC and CC genotypes at position 1892 were calculated as 12, 65 and 23%, respectively. Also, Shanon and Nei indices were for this position 0.68 and 0.49, respectively. Frequencies of AA, AC and CC genotypes at position 3359 were calculated as 38, 52 and 10%, respectively. Also, Shannon and Nei indices were 0.65 and 0.46, respectively. Frequencies of CC, CT and TT genotypes of OPN gene were calculated as 19, 57 and 24%, respectively. Also, Shannon and Nei indices were 0.69 and 0.49, respectively. Hardy-Weinberg equilibrium (HWE) was evaluated using χ square test. The genotypes in all loci deviated from HWE.

Many studies have reported quantitative trait loci on chromosome 6 that affect milkproduction traits in dairy cattle. PPARGC1A gene was identified as functionalcandidate gene for a described QTL for milk fat yield because of its key role inenergy, fat, and glucose metabolism. In this study, DNA was extracted from blood samples collected from 398 Holstein cows of Tehran and Esfahan provinces. DNA extraction was performed using the Salting out method. The PCR-RFLP method was used for genotyping of SNP T/C at position 1892 and SNP A/C at position 3359 in PPARGC1A gene. Frequencies of TT, TC and CC genotypes at position 1892 were calculated as 12, 65 and 23%, respectively. Also, frequencies of AA, AC and CC genotypes at position 3359 were calculated as 38, 52 and 10%, respectively. Hardy-Weinberg equilibrium (HWE) was evaluated using chi square test. The genotypes in both loci deviated from HWE. In this study c.1892T>C genotypes indicated significant associations with FATP2X, BVM, BVFP, PRO305 and PROME traits. It was shown significant associations between c.3359A>C genotypes and FATP2X, BVFP traits. Thus, these SNPs would provide an excellent opportunity for marker assisted selection programs in dairy cattle.