Identification of Polymorphism of Fsh Beta-subunit Gene as Sperm Quality Marker in Bali Cattle Using Pcr-rflp

The aim of study was to identify the association of FSH beta-subunit gene polymorphisms withsperm quality traits. A total of 470 samples of normal mature bull from several breeds were used forpopulation study and 127 bulls from National and Regional AI centre of Indonesia for association study.To amplify, a PCR-RFLP method was used and digested with Pst1 restriction enzyme. The allelefrequency of the A and B in Bali cattle were (0.000) and (1.000), respectively. The absence of otherallele A suggested that the Bali cattle was monomorphic, while Brahman, FH, Simmental and Limousinewere polymorphic. The highest observed heterozygosity were found in Limousine (0.318) and thehighest expected heterozygosity were in Simmental (0.420). The higher incident of percentage of spermabnormalities were found in Simmental, Limousin, Brahman compared to Bali and FH. Among all typesof sperm abnormalities, the abaxial and microcephalus were found in highest number.


INTRODUCTION
Follicle stimulating hormone (FSH) is a glycoprotein hormone expressed in the pituitary, and it is responsible for regulating reproduction in mammals (McLahlan and Meachem, 2003;Otha et al. 2007). In males, FSH incombination with testosterone is the most important tropic hormone regulating Sertoli cell function, requires for the initiation and maintenance of the quality and quantity sperm in spermatogenesis processes. Follicle stimulating hormone (FSH) acts on the germinal cells in the seminiferous tubules of the testis and is responsible for spermatogenesis up to the secondary spermatocyte stage, and androgens from the testis support the final stages of spermatogenesis.
The genetic factors influence on production and sperm quality have been reported by several studies (Kim et al., 1988;Fries, 1989). Dai et al. (2009) and Dai et al. (2011) examined the gene polymorphism FSH beta-sub unit of production and sperm quality in Simmental, Charolais and Limousin. Some researchers also associate the diversity of FSH beta-sub unit genes with litter size in sow (Yaofeng et al., 1998;Li et al., 2000;Li et al., 2008;Liu et al., 2009), sperm quality in boar (Wimmer et al., 2005;Lin et al., 2006), litter size in ewes (Xiaopeng et al., 2010;Zhang et al., 2011) and equine sperm quality (Samper, 2010). FSH beta-sub unit gene specific unit is located on chromosome 15 that comprises two introns and three exons with a length of 6601 bp (Kim et al., 1988;Fries, 1989;Hediger et al., 1991). Variants of this gene was three alleles, namely A, B, and C. Dai et al. (2009) found 9 SNPs mutations, i.e. 4 mutations in promoter section ('5 URR), 3 in intron 2, and 2 in exon 3. Polymorphism of FSH beta-sub unit gene in exon 3 on the study significantly influenced the fresh and frozen semen quality. The AA and AB genotypes showed better quality of fresh as well as in frozen semen when compared to the BC. Based on the previous study in Bos taurus and Bos indicus, this research aimed to investigate the polymorphisms of FSH beta-sub unit gene as marker of sperm quality in Bali cattle (Bos javanicus).

Sources of Animal and Data Collection
A total of 470 heads of normal mature bulls (225 Bali, 69 Brahman, 92 FH, 44 Limousin and 40 Simmental) for population studied. While, for association studied were used normal mature bulls from National and Regional AI centre of Indonesia (24 Bali,6 Brahman,13 FH,44 Limousin and 40 Simmental).

Isolation of Genomic DNA
Blood samples were collected from each bull in 10-ml non anticoagulan polypropylene tubes. Blood samples then were mixed with 96% ethanol.
Genomic DNA was isolated by the phenol-chloroform extraction method (Sambrook et al., 1998: Andreas et al., 2010 and then were dissolved in TE buffer (10mM Tris-HCl, pH 8.0; 1mM EDTA). Genomic DNA was stored at −20 o C until amplification with polymerase chain reaction (PCR).

PCR Amplification and RLFP Analysis
The amplification of PCR was carried out using specific primer (Dai et al., 2009) for part intron 2 and complete coding of exon 3 (313 bp) ( Table 1). The PCR was performed in a final volume of 25 µl for each reaction containing 2 µl DNA templates, 0.2 µl primer, 0.2 µl dNTPs, 0.1 µl Taq polymerase, 1 µl MgCl, 2.5 µl 1 X reaction buffer and 18.1 µl dilution of sterile water. The reaction mixture was subjected to an initial 5 min of denaturation 95 o C, followed 35 cycles of denaturation at 95 o C for 30 sec, annealing 30 sec at 63 o C, extention 1 min at 72 o C and final extention 5 min at 72 o C. Amplicons were visualized by electrophoresis in 1.5 % agarose gels containing 500 ng/ml of ethidium bromide in 1× TAE buffer with a 100-bp ladder as the molecular weight marker for confirmation of the length of the PCR products. For digestion by using enzyme and determination of RFLP, 7 µl of PCR product was added to 1 µl e Pst1 enzyme and 2 µl buffer 10X with final volume 10 µl. The PCR products of FSH beta-sub unit gene digested at 37°C overnight by Pst1 enzyme. The digestion products were separated by horizontal electrophoresis (85 volts, 50 min) in 2% agarose gels in 1 × TBE and 10% ethidium bromide.

Semen Preparation
Methods of fresh semen collections and smears preparations was according to Arifiantini et al. (2010) and Bhakat et al. (2011), and were  Dai et al. (2009) performed from bull in AI centers. The samples were then delivered to the research Animal Genetic and Molecular Laboratory in Bogor Agricultural University. Sample collections were performed based on the standard protocol: a drop of semen was placed on the a glass slide, mixed with four drops of physiologic saline, homogenized by using a stick, and smeared on different glass slides. Smeared semen samples were air dried coded according to the bull ID, and packed in a glass slides box. Smeared samples were stained with carbolfuchsin-eosin according to the method described by Williams in 1920 and modified by Lagerlof in 1934 (Kavak et al., 2004). Steps in Williams staining protocol were as follow: The air-dried, thin smears of fresh semen on glass slides from AI centers were fixed in flame, washed with absolute alcohol for 4 minutes, and air dried. Then, the smears were treated with 0.5% chloramines solution for 2 minutes until the mucous disappeared and the smears looked fairly clear. The smears were washed in distilled water, rinsed in 95% alcohol, and stained with Williams for 8-10 minutes. Finally, the smears were washed in running water and allowed to dry.

Data analysis.
PCR-RFLP data were analyzed by calculating allele and genotype frequencies (Nei and Kumar, 2000). The allele and genotype frequencies were calculated by the following formulas : x i = allele frequencies n = Total sample ĥ = heterozigosity

Sperm Abnormalities
The sperm were counted on each smeared using a light microscope at 400 X magnification (Olympus CH 20). Sperm morphology was examined and all types of abnormalities were recorded and classified. The classification sperm abnormalities was based on Bart and Oko (1989), Chenoweth (2005) and Arifiantini et al. (2010) such as : abaxial, macrochepalus, microchepalus, round head and knobbed acrosome defect. Sperm abnormalities were calculated from the type of abnormality in each field of view or 500 cells. Sperm abnormalities = number of type sperm abnormalities 500 cell x 100

Amplification of FSH beta-sub unit Gene
The amplicon of FSH beta-subunit was 313 bp in lenght, in which this result was consistent with Dai et al. (2009). FSH beta-sub unit gene was located in chromosomes 15th with 3 exons and 2 introns (Kim et al., 1988;Xiao-peng et al., 2010). The nucleotide sequnces of FSH sub-beta unit gene were 6610 base pair in length (GenBank No : M83753).
In this research, it has been amplified part of intron 2 and the complete coding region of exon 3 (313 bp). The amplification fragment of FSH beta-sub unit gene carried on GeneAmp® PCR System 9700 (Applied Biosystem) is shown in Figure 1.

Genotype and Allele Frequencies
The PCR product that has been digested by Pst1 enzyme incubated for 14 hours at 37 o C resulted in 3 fragments. Genotype data of FSH beta-sub unit gene showed 313 bp for genotype BB, 202 bp and 99 bp for genotype AA, 313 bp, 202 bp and 99 bp for AB genotype. Visualization of RFLP result is shown in Figure 2.
Results of FSH beta-sub unit gene analysis showed that only B allele and BB genotype was found in Bali cattle. The absence of other alleles suggested that Bali was monomorphic. This result was different from Brahman (Bos indicus), FH, Simmental and Limousine (Bos taurus) showing polymorphic. There was variation of alleles A and B as well as genotype variation, those were AA, AB and BB. The highest frequency of allele A was found in FH (0.891) and the lowest one in Bali cattle (0000). The highest allele B was found in Bali (1.000) and the lowest one in Brahman cattle (0.174). These findings were similar to those previous reported in Germany FH cattle and Gelbvieh cattle (Schlee et al., 1994), frequency of allele A was 0.000 and B was 1.000. Schlee et al. (1994) found the frequencies of allele A and B in Simmental were 0.831 and 0.147, respectively; in Limmousin were 0.910 and 0.431, respectively. The allele and genotype frequency of each breed presented in Table 2.

Sequences Alignment of the FSH Beta-sub Unit Alleles
The result of PCR product sequences showed mutation. Sequences based on GenBank access number M83753, the gonotype AA at position 237 and 238 nucleotides were CA, but the BB genotype was TG (Figure 3) .

Association polymorphism FSH beta-sub unit gene with sperm abnormalities
Sperm abnormalities have long been associated with male infertility and sterility in most species studied. These abnormalities vary from morphological defects that are evident upon clinical examination, to those, which are more subtly defective. In general, sperm structure can play a substantial role in both fertilization and pregnancy outcome (Chenoweth, 2005). Eventhough the heritability of bull fertility is generally considered to be low, certain aspects of bull fertility, including sperm morphological abnormalities are under genetic control. Sperm abnormalities according to Chenoweth (2005) and  (14) Freneau et al. (2010) was divided into major (15 type) and minor abnormality (16 type), where abnormality was caused by genetic factors ie. abaxial (abormalitas on the tail), round head, microchepalus, macrochepalus, knobbed acrosome defect (abnormalities in the head). Abaxial is a type of sperm abnormality whereas the implantation fossa of the tail is off-center. Variable size is a term to describe sperm possessing head abnormalities which are bigger (macrocephalus) or smaller (microcephalus) than the normal size. Knobbed acrosome defect occurs in the acrosome region of sperm; the apex of the acrosome is flatte or inden ( Table 3).
Among all types of sperm abnormalities, the microchepalus and abaxial were found in highest number, especially in Brahman bull, in contras the round head was found in low number in Bali bull cattle. While in FH semen, knobbed acrosome defect and microchepalus were found in highest number. This research demonstrated that the Simmental and Limousin have a highest number of microchephalus and round head.  reported the sperm abnormalities from 14 AI centre in Indonesia, it was found that the Simmental, Limousine and Brahman cattle have a high number of primary sperm abnormalities which were 4.8%, 3.6%, 2.6% respectively, compare to Bali only was 1.8%, even so, its classified as low primary sperm abnormality. Classification as high primary sperm abnormality if the percentage is larger than 10.1-15 and very high if larger than >15%. Purwantara et al. (2010) found 1.00 to 8.40% primary sperm abnormality mostly pearshaped of FH bull from 20 samples collected from three different AI centers.
In this study the primary sperm abnormality such as the abaxial and microchepalus were found mostly in AA and BB genotypes and only abaxial was found the highest number in AB genotype (Figure 4). CONCLUSION The FSH beta-subunit gene in Bali cattle was monomorphic, in contrast to Brahman, FH, Limmousin and Simmental, in which were    Micro chepalus Round head Knobbed acrosome defect Sperm Abnormality (%) )))) : AA, : BB, : AB