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Aim: The objective of this study was to develop a simplified, efficient, and accurate protocol for sexing of cattle meat based on the amelogenin (AMELX/AMELY) gene using PCR technique which is superior to earlier work in terms of band patterns. Materials and Methods: Based on the amelogenin gene located on the conservation region of X and Y chromosomes, a pair of primers was designed and the system of PCR was established to amplify a 241-bp fragment from the X chromosome in female cattle, and a 241-bp fragment from X chromosome and 178-bp fragment from the Y chromosome in male cattle, respectively. The accuracy and specificity of the primers was assessed using DNA template extracted from cattle meat samples of known sex. The protocol was subjected to a blind test showed 100% concordance, proving its accuracy and reliability. Results: PCR products of cattle meat samples after electrophoresis showed two bands (241, 178-bp) for tissue from male while female tissue resulted in only one (241-bp) band. Conclusions: Our findings show that the PCR assay based on the amelogenin gene is reliable for sex determination in cattle meat.


Introduction
-MS/MS) [3] and gas chromatography-mass spectrometry (GC-MS) [4]. Most of the methods Determination of sex origin of cattle meat has proved to be highly specific and sensitive, but were not been always of public interest in country like India performed on a regular basis for meat sexing due to the where slaughter of cow (female cattle) is banned technical limitations or the expensive equipments because of religious beliefs and laws thereby. The required. economic aspect allied with such discriminatory Over the last few decades, DNA-based techniques, slaughter policy also gets support as male beef is especially polymerase chain reaction (PCR)-based designated to be of higher quality than cow meat and methods for meat sexing have received particular therefore yield higher prices [1] particularly in attention, and have proved to be reliable, sensitive, and European countries. To implement the regulations fast [5,6]. DNA regions that differ between male and pertaining to such issues and to assure consumers of female individuals are essential features in PCR sex accurate labeling meat analysts need to have reliable, determination. In general, these methodologies were authentic and simple method for determining the sex developed mainly based on amelogenin gene of cattle meat.
the bovine-specific repetitive sequence BRY-1 [17] In general terms, hormone-based methods include and the single copy sequence BOV97 M [18] are also immunochemical determination using ELISA [2], and used in the analysis for the sexing of cattle. More chromatographic techniques with mass-spectrometric recently, based on real time PCR, both SYBR Green detection, such as high performance liquid chromato- [19] and TaqMan [20] technology for bovine sex graphy-mass spectrometry/mass spectrometry (HPLC Gel electrophoresis: The PCR product obtained was Subsequently, the quality of genomic DNA was analyzed by agarose gel electrophoresis in 2% agarose assessed by agarose gel electrophoresis using 0.8% gels (AMRESCO Inc., USA) stained with ethidium agarose gel (AMRESCO, USA) stained with ethidium bromide. A 100 bp DNA ladder (O'Gene Ruler, Fermentas) bromide. The purity and concentration of DNA was ® was electrophoresed simultaneously in order to assess estimated spectrophotometrically using Nanodrop the size of amplification product. The gels were ND-1000 spectrophotometer (Thermo Scientific) at visualized in automatic gel documentation system 260 and 280 nm. The DNA sample showing the OD (MiniBis, DNR Bio-Imaging Systems) and the size of 260:280 nm value of 1.70-1.90 was considered as the amplicon was determined using software available good quality.
with the gel documentation system. Design of PCR primers: As target for PCR primers (AMELX/AMELY) using PCR technique which is AMELX and AMELY, this gene is used as a target for superior to earlier work in terms of band patterns. sequence containing the Y-linked genes (SRY) and an In conclusion our findings show that the PCR autosomal sequence that acts as a control for the presence assay based on the amelogenin gene is reliable for sex of DNA [21]. In the present study, we employed determination in cattle meat. Due to the relatively primers derived from a sequence for X and Y specific short size (<250-bp) of the products compared to amelogenin, and verified the accuracy of the assay by earlier work, this method can be successfully applied evaluating genomic DNA from 8 males and 8 females.
to sex determination of cattle meat samples from The overall amplification products obtained showed 100% accuracy. This assay provides a rapid and highly degraded DNA and also in a shorter period of sensitive method for sexing, because of the presence time. The PCR product comparatively shorter in size, of the X chromosome band. This result was superior to size difference between band from AMELX and those reported by other authors [7,8]. Ennis and AMELY was more prominent on resolution, ensuring Gallagher [7] reported the primer pairs which yielded no ambiguity. Also, the advantage of this assay is that 280 and 217 bp, whereas the primer pairs, suggested neither additional control amplicons with a second by Chen et al [8] resulted in amplified product of 417 locus-specific autosomal primer pair nor restriction and 340bp in cattle. However we have attempted to endonuclease steps are necessary for sex determiexplore the amelogenin gene for designing the nation and control of the PCR reaction. The method primers, which could amplify the relative bands of proved to be reliable, cheap and is potentially suitable smaller size and our self-designed primers effectively for routine analyses. amplified the PCR products below 250 bp.

Author's contribution
Amelogenin gene encodes for a protein found in developing tooth enamel which belongs to the family P. Gokulakrishnan carried out the experiment and drafted the manuscript. R.R. Kumar, B.D. Sharma, of extra cellular matrix proteins [22]. In most mammals S.K. Mendiratta guided during the research and helped the amelogenin gene is located on both X and Y in drafting of manuscript. D. Sharma provided the chromosomes (AMELX and AMELY) [23], but a 63 laboratory facilities and guided the study. O.P. Malav bp deletion in exon 5 of the AMELY gene in comparison helped in collection of sample. All authors read and to the AMELX homologue yields two different size approved the final manuscript. bands in male and two similar size bands (which appear as a single band on resolution) in female [22]. Acknowledgements Due to an insertion in the region amplified in the X The authors gratefully acknowledge Indian specific gene (AMELX) or a deletion in the Y specific Veterinary Research Institute (IVRI) and Central gene (AMELY), the amplified fragments are of Avian Research Institute (CARI), Izatnagar, India for different sizes. Due to length polymorphism between www.veterinaryworld.org Veterinary World, Vol.5 No.9 September 2012 528