Detection of Mendelian and Genotype Frequency of Growth Hormone Gene in Ongole Crossbred Cattle Mated by the Artificial Insemination Technique

The objectives of this study were to detect the Mendelian mode inheritance of growth hormone(GH) and to establish genotype frequency of GH gene in Ongole-crossbred cattle mated by the artificialinsemination (AI) technique. Total of 76 blood samples were collected from Ongole-crossbred cows andbulls (G0), and their progenies (G1) at the Tumaratas AI service center in North Sulawesi province,Indonesia. All blood samples were screened for the presence of GH locus using a PCR-RFLP methodinvolving restricted enzyme Msp1 on 1.2 % of agarose gel. Data were analyzed using statistical programfunction in Excel XP. The results showed that GH locus using alleles of Msp1+ and Msp1- enzymerestriction in Ongole-crossbred cows and bulls was inherited to their Ongole-crossbred progeniesfollowing the Mendelian mode inheritance. This Mendelian inheritance generated by AI technique wasnot under genetic equilibrium for the Msp1 genotype frequencies in groups of G0 and G1. The breedingprogram using genotypes of bulls and cows (G0) for generating the genotype of GH Msp1 enzymerestriction by AI technique should be maintained to increase these various allele dispersion rates forbreeding under genetic equilibrium of the Ongole-crossbred cattle population.


INTRODUCTION
Growth hormone (GH) is an anabolic hormone synthesized and secreted by the somatotroph cells of the anterior lobe of the pituitary in a circadian and pulsatile manner, the Genotype Frequency of Growth Hormone Gene in Ongole Crossbred Cattle (U. Paputungan et al.) pattern of which plays an important role in postnatal longitudinal growth and development, tissue growth, lactation, reproduction, as well as protein, lipid and carbohydrate metabolism (Ayuk and Sheppard, 2006). GH gene with its functional and positional potential has been widely used for marker in several livestock species including the Indonesia local cattle (Jakaria et al., 2009;Sutarno, 2010).
Ongole-crossbred cattle give a significant contribution to Indonesian national meat supplies to fulfill animal protein needs of people. However, the increase of cattle population is not balanced with the national needs of meat consumption due to higher increase of human population. If this condition is uncontrolled, it will lead to the loss of germplasms which is one of the national assets in the animal husbandry field. Negative selections by the breeders and a lack of observation for the crossbreeding of local cattle cause the rest of local cattle to be the inferior cattle with a low quality, will be used to serve as the parental animals in breeding program. If this conventional breeding happens continuously, it will lose the benefit due to the extinction of superior animal germplasms (Hardjosubroto, 2002).
In the wide spread use of artificial insemination (AI) techniques in cattle reproduction industry, the Ongole bulls are used widely as sperm source in crossbreeding program to improve the performance of indigenous local breeds of the cattle in North Sulawesi province. As part of the marker assisted selection (MAS) program which aimed to improve genetic traits in bulls of the Ongole-crossebred cattle, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) has been identified at GH locus with Msp1 enzyme restriction. The uncontrol breeding of selected different genotypes of parental bull and cows by the AI technique could be the factor causing genetic inequilibrium of genotype frequency in animal population as a part of non random mating system (Van Vleck et al., 1987). The study of the Mendelian mode inheritance of GH Msp1 enzyme restriction has not been widely explored in cattle.
The objectives of this study were to detect the Mendelian mode inheritance and to establish genotype frequency of growth hormone (GH) Msp1 enzyme restriction in Ongole-crossbred cattle mated by the artificial insemination (AI) technique in North Sulawesi, Indonesia.

Animals and Sample Collection
This study was carried out in North Sulawesi Province of Indonesia. The total of 74 female animals (parental cows and their progenies) were used and comprised of 37 cows (age ranging 4 to 5 years old), and their 37 female progenies of Ongole crossbred cattle (age ranging 35 to 56 days old). All cows were reared under private areas belong to farmers with unknown ancestors. Progenies were born from those cows mated by artificial insemination using germ plasms (semen) of the two Ongole bulls called "Krista" and "Tunggul" from the artificial insemination bull germ plasm center or Balai Besar Inseminasi Buatan (BBIB) in Singosari, East Java province, Indonesia.

DNA Extraction
The genotyping process was conducted at the Biotechnology Laboratory Department of Biological Science, Faculty of Mathematics and Natural Science, Sam Ratulangi University, Manado. Blood samples of the cows, their progenies and two Ongole bulls as source of germ plasms were collected from their Jugular vein in 10 ml EDTA (10%) tubes during July 2011 and stored in the refrigerator (4 o C) until ready for DNA isolation. Genomic DNA from their whole blood were purified by standard protocol using proteinase K digestion as described by DNA extraction kit (AxyPrep Blood Genomic DNA Miniprep kit, AXYGEN Biosciences, Union city, CA, 94587, USA).
To digest this fragment, a protocol of RFLP with restriction enzyme Msp1 was used to recognize the particular site of C↓CGG. The PCR product of GH gene was digested at 37 o C for 3 hours by Msp1 enzyme. Reaction consisted of 2 µl Buffer V2 10X, 7.5 µL H 2 O, 0.5 µL Enzim Msp1 (20 U/µL), and 10 µl PCR product. PCR protocols to amplify the fragment were done by the initial denaturation temperature step at 94 o C for 5 min for 1 cycle followed by 35 cycles of denaturation at 94 o C for 30 sec, annealing at 60 o C for 30 sec, elongation at 72 o C for 30 sec and a final extention at 72 o C for 1 minute (Dybus, 2002). Following the end of PCR and RFLP process, the products were then subsequently electrophorated using 1.2% agarose gel to identify polymorphism of allele based on the length of the band (Figure 1).

Polymorphism Detection
The Msp1 digestion of the PCR products produced the fragments of 104 bp and 223 bp for allele Msp1+ and of 327 bp for allele Msp1- (Figure 1). These alleles were the same as research reported by Zhou et al. (2005) using Msp1 enzyme restriction in Beijing Holstein and Maylinda (2011) using Msp1 enzyme restriction in Grati dairy cows. This enzyme recognized only the restriction site of four nucleotides for C↓CGG ( Figure 2). Genotype Msp1+/+ consisted of two bands (104 bp, 223 bp), genotype Msp1+/consisted of three bands (104 bp, 223 bp, 327 bp), genotype Msp1-/-consisted of one band (327 bp). The difference of these two fragments of Msp1+ and Msp1-alleles was caused by mutation of Cytosine (C) to Thymine (T) (Rifa 'i, 2010). Gene variation of GH locus for Msp1 in cattle was detected in the position of intron 3 (Rifa'i, 2010) at the sequence position of 1547 based on nucleotide sequence from GenBank, number: M57764.1 (http://www.ncbi.nlm.nih.gov) sourced in Gordon et al. (1983) accessed on March 26, 2011. Mutation occurred on DNA level due to nucleotide changes, either transition or insertion (Cambell and Reece, 2008). Based on the difference of nucleotide restriction sites of each allele, the mutation of Cytosine (C) into Thymine (T) occurred due to nucleotide transition ( Figure  2). The transition of C into T changed the restriction site of Msp1 enzyme (Rifa'i, 2010).
Theorically, the basic Mendelian mode inheritance of the crossing between all the same homozygous genotyped individuals produced all the same homozygous genotyped progenies, while the crossing between recessive homozygous genotyped individuals and dominant homozygous genotyped individuals produced all heterozygous genotyped progenies (Van Vleck, 1987). Based on the Chi square test (Table 1)

The Msp1 Genotype Frequencies of Animal Population Using AI Technique
The frequencies of cow (G0) and progeny (G1) genotypes determined in the population mated by each genotype of bull (G0) were presented in Table 2. Based on the Chi Square test (Table 2), it was found that genotype and allele frequencies of GH Msp1 were not under genetic equilibrium (P>0.05). Maylinda (2011) reported that Grati dairy cow population was in genetic equilibrium. This was supported by the fact that a population property of gene pool for GH-Msp1 under the Hardy-Weinberg equilibrium pattern was a function of both allele frequencies and biological interactions among genes (Carter et al., 2005). This inequilibrium of genotypic frequencies of GH Msp1 caused the instability of genotypic frequencies of GH gene from G0 generation to the next generation (G1) due to the breeding of selected genotypic bulls and parental cows without random mating system in animal population (Cambell and Reece, 2008;Rifa'i, 2010). The factor affecting genetic equilibrium was selection program with non random mating system, such as the artificial insemination mating system (Van Vleck et al., 1987). Jawasreh et al. (2012) reported that breeding program must be continued as the first step to increase the frequency of the favorable allele in breeding station. In North Sulawesi province, the artificial insemination service center applied the straw containing spermatozoa germplasm of Ongole bull called "Krista" and "Tunggul" from germplasm center (Balai Besar Inseminasi Buatan) Singosari, East Java province. Carter et al. (2005) reported the analysis of gene interaction and found that it might be two or more genes can interact to express a particular phenotype.

CONCLUSION
The selected growth hormone locus using alleles of Msp1+ and Msp1-enzyme restriction in Ongole-crossbred parental cows and bulls was inherited to their progenies following Mendelian mode inheritance. This Mendelian inheritance generated by AI technique was not under genetic equilibrium for Msp1 genotype frequencies in groups of parental animals (G0) and their progenies (G1). The breeding program using genotypes of bulls and cows (G0) for generating the genotype of GH Msp1 enzyme restriction by AI technique should be maintained to increase these various allele dispersion rates for breeding under genetic equilibrium of the Ongole-