Estimation of Inbreeding Coefficients Using Pedigree and Microsatellite Markers and Its Effects on Economic Traits of Shirvan Kordi Sheep

INTRODUCTION Intensive selection within a single population of finite size reduces the genetic variability and increases the rate of inbreeding KEY WORDS Research Article Estimation of Inbreeding Coefficients in Shirvan Kordi Sheep In the overdominance hypothesis, inbreeding depression is attributable to higher fitness of heterozygotes for the loci in question. For the partial recessive hypothesis, negative fitness consequences are due to the fixation of recessive or partially recessive deleterious alleles Microsatellites has been widely used as reliable molecular markers to study the genetic relationship of different populations and for indirect measures of inbreeding. They are codominant, highly polymorphic, highly abundant, heritable, locus specific, and easily analyzed and therefore suitable for studies on population phylogenesis constitution This study was carried out using microsatellite markers because they are powerful tools for tracking alleles through a population and to estimate genetic variability and inbreeding Marker data collected provides information on population structure, relatedness and inbreeding MATERIALS AND METHODS Data Pedigree information from 1989 to 2009 of a flock of Kordi sheep maintained at Shirvan Sheep Breeding Station was used. Pedigree file contained information on individual identification number, sex, type of birth, dam and sire as well as birth date and included 7170 registered animals (3332 males and 3838 females), progeny of177 sires and 2182 dams. Pedigree analysis Inbreeding coefficients of the animals were computed using the CFC program Microsatellite method Blood samples (5 mL) of 100 animals were collected from the jugular vein and transferred into vacutainer tubes containing 0.5 molar EDTA as anticoagulant and frozen at -20 ˚C. Total DNA was isolated from blood samples using the Diatom DNA Kit, according to the manufacturer instructions. The quantity and quality of the isolated DNA was determined using both spectrophotometry and by 0.8% agarose gel electrophoresis. Characteristics of the microsatellite markers used in this study are listed in Approximately, 100 ng DNA (adjusted concentration) was used as template for polymerase chain reaction (PCR). The PCR reaction cycle was carried out in a the rmocycler (Biorad) by denaturation at 95 ˚C for 4 min, denaturation at 95 ˚C for 45 sec, primer annealing for 45 sec at the desired temperature (55-60 ˚C) and an extension for 1 min at 72 ˚C, repeating the cycle 35 times. The final extension step was at 72 ˚C for 4 min. The PCR amplification was conducted in a 12 μL volume. PCR products were analyzed by vertical electrophoresis in 6% non-denaturing polyacrylamide gel (170 V, 3-4 h) and bands visualized by rapid silver staining Molecular data analysis Molecular data were analysed using the POPGENE V1.32 RESULTS AND DISCUSSION Pedigree analysis The analysis of pedigree revealed that mean level of inbreeding (F) of all animals acrossall years (1989-2009) was 0.668%. Moreover, the minimum and maximum coefficientsof inbreeding for the animals in the flock was 0 and 31.25%, respectively. The low average coefficient of inbreeding could be ascribed to breeding strategies at the station for preventing mating of relatives. Totally, 23.26% of the animals (1668 out of 7170) were inbred with a mean inbreeding coefficient of 2.87%. In the pedigree, 3332 and 3838 of the animals were males and females with mean inbreeding coefficients of 0.693% and 0.646%, respectively. Out of all, 1668 animals were inbred. These included 823 males and 845 females having average inbreeding coefficient of 2.81% and 2.93%, respectively. These results indicated that fewmatings of close relatives have occurred. Descriptive statistics for inbreeding coefficients for the entire population and the inbred portion of the population are shown in 135 Estimation of Inbreeding Coefficients in Shirvan Kordi Sheep Mean of all traits decreased and increased irregularly by increasing inbreeding coefficients. This could be due to fewer records in the numerically higher classes of inbreeding. Inbreeding depression Details of the data used for the estimation of inbreeding depression are given in Regression coefficients and their standard errors on inbreeding coefficients were -0.0013 ± 0.0003 kg for BWT, 0.080 ± 0.015 kg for WWT, 0.001 ± 0.0009 kg for BW6M, -0.065 ± 0.055 kg for BW9M, -0.092 ± 0.063 kg for BW12M, 0.008 ± 0.0066kg for Wool and -0.023 ± 0.012 lambs for LS. Pre-weaning growth traits BWT: birth weight trait; WWT: weaning weight trait; BW6M: body weight at 6 months of age trait; BW9W: body weight at 9 months of age trait; BW12M: body weight at 12 months of age trait; Wool: wool produced annually and LS: number of lambs per ewe lambing. Changes for every one percent increase in inbreeding coefficient for BWT and WWT were -0.0131 and 0.0795 kg, respectively. Regression coefficient estimate for BWT Regression coefficients for BW in the 9 th and 12 th month were more than all the other studied traits. Estimates of -0.0653 and -0.0921 kg per 1% increase in inbreeding coefficient for BW in 9 th month and BW in 12 th month, were more than the values reported in other studies. Annual wool production and number of lambs per ewe lambing The effect of inbreeding on wool produced annually and the number of lambs per ewe lambing amounted to 0.0083 kg and -0.023 lambs, respectively. For the annual wool production, the linear effect of individual inbreeding, regression was positive but non-significant. On the average, an increase in 1 percent in individual inbreeding increased the wool produced annually by 0.0083 kg. A review b

High-resolution melting curve analysis: a novel method for identification of Mycoplasma species isolated from clinical cases of bovine and porcine respiratory disease

Mycoplasma species cause wide ranges of infectious diseases in human and animals. The aim of the present study was to evaluate a real-time polymerase chain reaction (RT-PCR) followed by a high-resolution melting curve assay (HRM) for rapid differentiation of Mycoplasma species isolated from clinical cases of bovine and porcine respiratory disease. Lung samples from suspected cases to respiratory infections from cows and pigs were cultured on specific media, and the extracted DNA were tested by conventional polymerase chain reaction (PCR) assays for Mycoplasma. A set of universal primers specific for the 16S ribosomal RNA gene was designed and used for RT-PCR and HRM. The HRM analysis was able to differentiate between five different species of Mycoplasmas, namely, M. hyopneumoniae, M. bovis, M. hyorhinis, M. hyosynoviae and other uncultured Mycoplasma. All results were confirmed based on 16S rRNA gene sequencing. This rapid and reliable assay was as a simple alternative to PCR and sequencing, differentiating bovine and porcine mycoplasmas in species level.Ania Ahani Azari, Reza Amanollahi, Razi Jafari Jozani, Darren J. Trott, Farhid Hemmatzade

Application of an indirect MilA ELISA for the detection of Mycoplasma bovis antibodies in bovine milk

The objective of this study was to detect Mycoplasma bovis specific antibodies using a recently reported MilA ELISA with the aim to detect M. bovis antibodies in milk. An indirect ELISA, based on a recombinant fragment of the Mycoplasma immunogenic lipase A (MilA) protein, was conducted on 291 milk samples for the detection of M. bovis antibodies. Samples were also tested with conventional Mycoplasma culture and M. bovis PCR. Samples were collected from individual cows from 2 commercial dairy herds in South Australia. Of 291 samples tested, 68 (23.4%) were detected positive for M. bovis antibodies, 150 (51.5%) were positive for M. bovis in PCR and 166 (57.0%) in bacterial culture. These results indicate that MilA indirect ELISA can be utilized for the detection of M. bovis antibodies in milk

The Effect of Cinnamon Extract on Spermatogenesis and

Genotypes of Iranian Zel sheep for Calpastatin (CAST) locus were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) methods and for Calpain (CAPN) locus by PCR-SSCP. Blood samples were collected from 200 purebred Zel sheep of Zel Breeding Station located in Golestan province in northeast of Iran. Extraction of genomic DNA was based on modified salting out method. The digestion of PCR products of CAST gene by MspI and NcoI restriction enzymes revealed two alleles M and N, with frequencies 85.5 and 14.5%, respectively. Frequencies were 75, 21 and 4% for MM, MN and NN genotypes, respectively. Alternatively, using PCR-SSCP method, four genotypes including AA, AB, BB and AC with frequencies of 71, 21, 4 and 4%, respectively, were observed in this population. Analyzing CAPN gene by the PCR-SSCP method, revealed two different conformational patterns (AA and AB) with frequencies of 69 and 31% for AA and AB, respectively. Average heterozygosity for both loci was low (0.28 and 0.25% for CAST using PCR-SSCP and PCR-RFLP, and 0.26% for CAPN). Yearling weights (YW) were analyzed by a statistical model comprising PCR-SSCP and as a result CAPN genotypes had significant effect (P<0.01) on YW. A Chi-square test confirmed Hardy-Weinberg (H-W) equilibrium for the CAST locus using PCR-SSCP method but not for PRC-RFLP method and CAPN locus. Totally, the investigated herd had little genetic diversity and different factors disturb H-W equilibrium and PCR-RFLP and PCR-SSCP might be used successfully in these studies