Growth Hormone Gene Polymorphism and Its Association with Partial Cumulative Milk Yields of Holstein Friesian Dairy Cattle

Growth hormone gene (GH gene) plays an important role in regulating body growth and in developing mammary gland, similar with its interaction to specific receptors. The GH gene has been considered as one of candidate gene associated with selection on lactation trait and milk production. This study was aimed to determine genetic polymorphism of the GH-AluI gene and to associate its genotype variants on various 15-d partial cumulative milk yields in Holstein Friesian (HF) dairy cows. A number of 370 blood samples were collected from six HF populations, respectively from small dairy farmer under the supervision of the North Bandung Milk Cooperation (NBMC) in Cilumber (98) and Pasir Kemis village (96), Dairy Cattle Breeding and Improvement Station (Cikole DCBIS) Cikole (88), Lembang Artificial Insemination Center (Lembang AIC) (17), Singosari Artificial Insemination Center (Singosari AIC (32), and Cipelang Livestock Embryo Center (Cipelang LEC) (40). A polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) method was used to identify variant genotypes of the GH gene using AluI restriction enzyme. Genotyping results produced only two genotypes, i.e. LL and LV genotypes, without VV genotype. Frequency of the former was dominant, whilst that was low for the latter (89% vs. 11%); leading to the frequency of L allele was very high (94%) compared to that of V allele (6%). No significant association between variant genotypes (LL and LV) and various 15-d partial cumulative milk yields.


INTRODUCTION
Indonesian dairy cattle population is currently around 597,129 heads (DGLAH, 2011). Almost all of those dairy cattle, as producers of fresh milk in the country, are Holstein Friesian (HF) of Bos taurus dairy cattle. Raising dairy cattle is mostly concentrated in Java Island. Therefore, the highest volumes of producing fresh milk are from East Java, Central Java and West Java, for respectively 268,042; 100,350; and 536,458 tons. However, the capacity of milk production of dairy cattle should be increased, as the milking ability of HF dairy cows has just met around 35% of the national milk demand. Attempts to make genetic improvement will increase milk yield of dairy cattle permanently, because genetic superiority of milk production will be passed from parent to offspring. One effort that can be done to improve genetic ability of HF cows in producing high milk yield is through a selection method.
Selection is commonly done by selecting superior bulls and cows to be used as sources of genetic material for the next generation. Selection in dairy cows is mainly based on the level of milk production. Milk production is a quantitative trait controlled by many genes and its expression is the accumulation of the factors of genetic, environment, and their interaction. Curently selection can be assisted by using molecular techniques. Selection based on genetic markers for a particular trait makes selection occuring early. Application of the genetic markers into livestock breeding programs can accelerate genetic improvement in livestock.
The GH gene has an important role in growth and development of postnatal longitudinal, growth of mammae and reproduction tissues, as well as metabolisms of protein, lipid, carbohydrate (Akers, 2006). Effects of the GH gene on the growth are observed in several tissues, including bone, muscle, and adiposa. In ruminants, the GH gene contributes to the development of udder glands (Akers, 2006). Growth hormone is an anabolic hormone that is synthesized and secreted by the anterior lobe cells in pituitary somatotrop. In bovine, the GH gene is located on chromosome 19 with a length of about 280 bp, composed by 5 exons and 4 introns. The GH protein consists of 191 amino acids with a molecular weight of 2 kDa (Ayuk & Sheppard, 2006).
The GH gene has been used as a genetic marker for the growth traits in some species such as cattle (Zhou et al., 2005;Jakaria et al., 2007 andKatoh et al., 2008), sheep (Marques et al., 2006), and goats (Boutinaud et al., 2003). Growth hormone (GH), growth hormone receptor (GHR) and other hormones such as Insulin-Like Growth Factor 1 (IGF1) are widely used as candidate genes of production traits in livestock and subsequently used as a genetic marker for selection. This is because these hormones are regulators of growth and development of the body (Zakizadeh et al., 2006). Studies on genetic polymorphism of the GH gene and its relationship to milk production in dairy cattle have been observed in Hungary Holstein Friesian (Balogh et al., 2009), Iranian Holstein (Mohammadabadi et al., 2010, and Poland Holstein Friesian (Olenski et al., 2010). Based on the results of those several studies it was known that the GH gene together with the GHR gene play an important role in regulating the growth of mammary gland and milk production, metabolism, lactation, and body composition (Kovács et al., 2006). This research had specific purposes to identify genetic polymorphism of the GH-AluI gene and to association variant genotypes of this gene to various 15-d milk yields in Holstein Friesian (HF) cattle.

Animals and Milk Yields
Blood samples were collected from HF cattle, male and female, taken from the vena jugularis. A total number of 370 HF blood samples were taken from six populations with different management or condition. Blood samples from HF heifers and cows were collected from Cikole Dairy Cattle Breeding and Improvement Station (Cikole DCBIS) located in Lembang, West Java for 88 samples, Cipelang Livestock Embryo Center (Cipelang LEC) for 34 samples, and from North Bandung Milk Cooperation Unit (NBMCU) at small farmers in the two villages of Cilumber (Cilumber NBMCU) for 98 samples and Pasir Kemis (Pasir Kemis NBMCU) for 95 samples. Blood samples from active and non active AI services of HF bulls from Lembang Artificial Insemination Center (Lembang AIC) for 17 samples and Singosari Artificial Insemination Center (Singosari AIC) for 32 samples. The collection of blood samples from these HF bulls was intended to know their genetic potency in transmitting the GH genetic polimorphism to HF cows.
Data of milk yields were collected from 56 HF cows that were genotyped their the GH gene from Cikole DCBIS for production periods of 2008-2010. Data of milk yileds were in the range of I-4 lactation periods. Data of daily milk yields that were recorded weekly were estimated for various partially cumulative milk yields at each 15-d intervals, since the 1 st until the 12 th partial cummulative milk yields (15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, and 180 d).

Primer
Primers used to amplify mutant locus of the GH|AluI gene followed Balogh et al. (2009), with a forward primer 5'-CGGACCGTGTCTATGAGAAGCTGAAG-3' and a reverse primer 5'-GTTCTTGAGCAGCGCGTCGTCA-3'. The amplified product or amplicon had the length of 432 bp.

DNA Sample
DNA samples obtained were from blood and semen. Blood samples used as DNA sources were 353 samples originating from 5 locations, while semen samples as DNA sources were 17 samples from Lembang AIC.

DNA Extraction
DNAs was extracted from blood and semen. Extraction procedure followed the phenol-chloroform method that was modified by Andreas et al. (2010), with the following procedures: Sample preparation. Semen sample was added by alcohol 400 µl, whereas blood sample was added by alcohol 200 µl, then inserted into a 1.5 ml tube. Alcohol was then eliminated from the sample by adding distilled water until 1000 µl, and left in room temperature for 20 min. Then it was precipitated by centrifugation at a speed of 8000 rpm for 5 min.

DNA precipitation.
Samples was centrifuged at a speed of 5000 rpm for 10 min to separate water phase over phenol phase. The water phase was transferred in a new tube with the volume measured. DNA molecules were deposited by adding a 2x volume of alcohol absolute and 0.1 x volume of 5M NaCl. Then the mixture was incubated at a temperature -20 °C during the night. Subsequent DNA precipitation was centrifugated at a speed of 12000 rpm for 10 minutes. The obtained DNA precipitation was washed by 70% alcohol, then reprecipitated. The precipitated DNAs was cleaned from alcohol by adding 100 µl TE (Tris EDTA). The DNA samples were then stored at -20 °C and ready for use.

Amplification of the GH Gene
Amplification of fragments of the GH gene was done by using PCR (polymerase chain reaction) method. Reagents used for the amplification of the targetted fragment were a 2 µl sample DNA, each primer 25 pmol, 200 µM dNTPs mixture, 1 mM MgCl2, and 0.5 units of DreamTaq™ DNA Polymerase and 1x buffer (Fermentas) in total solution 25 µl. Amplification was by in vitro within GeneAmp® PCR System 9700 (Applied Biosystems™). It was done with the condition of pradenaturation at 94 o C for 5 min, 35 cycles consisting of denaturation at 94 o C for 45 s, annealing primers at 62 o C for 45 s and extention of new DNA at 72 o C for 1 min, and the final extention at 72 o C for 5 min.

Genotyping by RFLP Method
Determination of genotypes of each individual cattle was done by using restriction fragment length polymorphism (RFLP) using AluI enzyme as a restriction enzyme. Visualization was conducted on 2% agarose gel with 0.5 x TBE buffer (tris borate EDTA) at 100 V for 40 min. Gel was stained with an ethidium bromide, and visualized in UV transuliminator, alpha innotech alpha imager.

Data Analysis
Genotype frequency represents the ratio of a genotype to total population. Allele frequency is a ratio of an allele to the overall allele at a locus in the population. Mathematic models for genotype and allele frequencies (Nei & Kumar, 2000) were as follows: x ii = (n ii /N) x 100% x i = (2n ii + n ij ) / 2N x ii = ii th genotype frequency x i = i th allele frequency n ii = number of individual with ii genotype n ij = number of individual with ij genotype N = total number of individuals Degree of heterozigosity both observed (h o ) and expected (h e ) were calculated with formula as follows: h o = observation heterozigosity h e = expected heterozigosity x i = i th allele frequency n = total number of individuals For study of the associations was analyzed by the General Linear Model (GLM) with one factor. Parameter observed was various partially 15-d cumulative milk yields from 56 heads of the genotyped HF cows from the Cikole DCBIS. Mathematic model (Matjik & Sumertajaya, 2006) was represented as follows: γ ij = µ+ α i + ε ij γ ij = a certain partial cumulative milk yield μ = average α i = additive effect from i th genotype ε ij = observed error

Amplification of the Growth Hormone Gene
The amplified fragments were visualized on a 1.5% agarose gel (Figure 1). The amplified product (amplicon) of the GH gene had a fragment length of 432 bp, including 55 bp of 4 th exon, 4 th intron and 99 bp of 5 th exon (Balogh et al., 2009). One of key factor in determining the success of amplification is annealing temperature. Annelaling temperature is a temperature allowing the primers attaching on DNA templates during a PCR process. The annealing temperature 60 °C for 1 min in this study was accordance with those of some previous studies (Balog et al., 2009;Mohammadabadi et al., 2010, andAndreas et al., 2010).

Identification of the GH Gene Polymorphism
The AluI enzyme as a restriction enzyme cut the recoqnized site of AC|GT bases. There are three AluI restriction sites that produce fragment lengths of 20, 51,    (Balogh et al., 2009). Genotyping the GH|AluI gene resulted two genotypes, i.e. LL and LV (Figure 3). VV genotype was not found for all samples observed. Visualization of the genotypes of the GH|AluI gene was presented in Figure  3. These results were in line with the study by Pereira et al. (2005) by obtaining two genotypes (LL and LV) in Brazilian Canchim cattle. Another study by Curi et al. (2006) did not find the VV genotype in Brazilian Zebu cattle and its crosses. However, the results of current study were not similar to that study by Dybus et al. (2002) that identified LL, LV, and VV genotypes of the GH|AluI gene in Polish Black and White cattle. The differences could be caused by breed of cattle, breeding system, and samples genotyped.

Genetic Diversity of GH|AluI Gene within Holstein Friesian
Frequencies of genotypes and alleles of the GH|AluI gene from all of HF cattle observed were presented in Table 1. Genotyping results on the GH|AluI gene showed that frequencies of the L allele were higher than those of the V allele. Frequencies of the L allele of HF cattle observed from all locations ranged from 0.92 to 0.98. Higher frequencies of the L allele in the observed HF cattle resulted higher frequencies of the LL genotype contrasted to those for the LV genotype. No existence of the VV genotype in all of HF cows observed both in breeding station and small farmers could be influenced by HF bulls used for AI services. All of active AI-HF bulls came from the two national AICs (Lembang AIC and Singosari AIC). Genotyping of all HF bulls from both AICs proved that no bull having the VV genotype, whereas the V allele frequencies of those HF bulls were low.
The results from this studi corresponded with the study by Sorensen et al. (2002) by obtaining the higher frequenci of the LL genotype (0.85%) than that of the LV genotype (0.15%) of the GH|AluI gene in Danish Holstein cattle. The results of this study, however, contrasted with the study by Grochowska et al. (2001) in Polish Friesian cattle that reported LL, LV, and VV genotypes, with the highest frequency for the LL genotype (51%) and the lowest for the VV genotype (13%). Another study by Sabour et al. (1997) in Ayrshire, Holstein and Jersey dairy cattle also identified LL, LV and VV genotypes, with the frequencies were 0.29, 0.09, and 0.24 respectively.

Heterozigosity
The degree of heterozigosity represents the mean percentage of heterozygous loci per individual or the mean percentage of heterozygous individuals in a population. Estimation of the heterozygosity degree is important to know genetic variability and to determine the level of polymorphism of alleles. High heterozygosity shows high genetic diversity within a population (Nei & Kumar, 2000).
Predicted degree of heterogozity of the GH|AluI gene was presented in Table 2. heterozygosity of the GH|AluI gene ranged between 0.050-0.156. The highest heterozygosity was found in HF bulls from Singosari AIC, whilst the lowest one was found in HF cows from Cipelang LEC. By comparing the results of observed heterozygosity analysis (H o ) and expected heterozygosity (He) at GH|AluI gene indicated no statistically difference (Table 2). Tambasco et al. (2003) stated that if the value of observed heterozygosity (H o ) is much lower compared to that value of expected heterozygosity (H e ), it might indicate a more intense selection or a higher degree of inbreeding.
Based on the heterozygosity values obtained in the GH|AluI gene from all of HF cattle observed from all locations, it could be stated that the GH|AluI gene had a low degree of genetic diversity. Selection in livestock expects high heterozygosity, as the high heterozygosity reflects genetic variation of genes in a population. A higher value of heterozygosity of genes could give a greater opportunity for selection of genes in a population.

Association between the GH Genotypes and Partial Cumulative Milk Yield
Investigation of the association between variant genotypes of the GH|AluI gene on partial cumulative milk yields of HF cows was conducted at Cikole DCBIS in Lembang, West Java. Study on the effects of the LL and LV genotypes on various partially cumulative milk yields at each 15-d interval of HF cows were presented in Table 3. The results generally seemed that the LV cows tended to have a higher milk production than those of the LL cows. These were really evident for cumulative milk yields around 135 d to 180 d of lactation. Statistical analysis however proved that those LL and LV genotypes the GH|AluI gene did not give significantly effects on all of partially cumulative milk yields observed. These results indicated that the examination on the GH gene solely did not provide sufficiently effect on milk production of HF cattle. This was because milk production is one of quantitative traits that are controlled by poly genes. Beside of that, milk production as a quantitative trait is also affected by other factors, both genetic and environment factors. Some environment factors could be possible in affecting dairy cattle milk yields, such as lactation periode (calving age), days open, days dry, calving season, and calving year (Anggraeni, 2012 Results of this study however differed from that reported by Grochowska et al. (2001) that identified the GH|AluI gene significantly influenced on 305-d milk production. The LL cows were reported producing higher milk yield by 171.7 kg compared to those LV cows. It was also reported that the first cows producing fat content of 8.8% higher than the latter cows. Yardibi et al. (2009) also reported that the variant genotypes (LL, LV, and VV) of the GH|AluI gene had positive correlation with percentages of fat content and protein content of milk, but no correlation was found between those variant genotypes with milk production of dairy cattle. Investigation on some other breeds of dairy cattle proved that the LL genotype had higher milk production than the VV genotype (Dario et al., 2008;Sadeghi et al., 2008).

CONCLUSION
The GH|AluI gen of HF cattle from six populations observed in this study had only two genotypes, i.e. LL and LV genotypes and with two types of L and V alleles. Frequencies of the LL genotype were very high (0.88-0.94), whilst those ferquencies of the LV genotype were very low (0.05 to 0.16). The values of both of observed heterozygosity (H o ) and expected heterozygosity (H e ) of the GH|AluI gene were not significantly different that could be an indication of a closer mating within population. No significant association between varian genotypes of the GH|AluI gene with partially cumulative milk yileds.