FSHR gene polymorphism and its association to reproductive traits in Friesian Holstein cattle

Follicle Stimulating Hormone Receptor (FSHR) is a receptor for FSH, a hormone that stimulates reproductive cells in both male and female animals. Polymorphisms in the FSHR gene may impair cattle reproduction performance, reducing reproductive efficiency. This study aims to look into FSHR genetic variation and how it affects reproductive performance in Friesian Holstein (FH) dairy cattle. A total of 26 blood samples were collected from female FH for DNA analysis, followed by data collection on reproductive traits such as postpartum mating (PPM; days), calving interval (CI; months), and services per conception (SC). AluI was used as an enzyme restriction in PCR-RFLP to determine the FSHR genotype. The results show three genotypes: CG (highest frequency), CC, and GG, with Hardy-Weinberg analysis indicating that the population is in equilibrium. All reported reproductive traits showed no significant association with FSHR genotypes. Although not statistically significant, the GG genotype performs better in PPM and CI. Meanwhile, the CC genotype has the lowest SC among the genotypes. It is concluded that the FSHR gene may be used as a genetic marker in the reproduction performance of FH dairy cattle.


Introduction
Dairy cattle reproduction is an important aspect of dairy production efficiency because it affects the profitability of the dairy business.Good reproduction performance can reduce operating costs while increasing profitability.In the endocrine system, reproduction hormones primarily control reproduction in cattle.Follicle Stimulating Hormone is an important reproductive hormone in dairy cattle (FSH).The FSH hormone promotes De Graaf Follicle maturity development in folliculogenesis at the ovarium and spermatogenesis, which produces sperm in the testis of females and males, respectively [1].In females, the hypothalamus secretes FSH hormone, which is transported to granulosa cells within the ovary via the bloodstream.
FSH is a glycoprotein hormone composed of two subunits: alpha and beta subunit.Furthermore, the beta subunit is unique to the target cell [2].FSH has a specific receptor to a couple in the target cell, named FSH-Receptor (FSHR).FSHR is a G-protein coupled receptor (GPCR).FSHR plays a critical role in receiving, regulating, and delivering FSH to target cells [3].Moreover, the FSHR gene encodes IOP Publishing doi:10.1088/1755-1315/1341/1/012023 2 the FSHR.The FSHR gene from Bos taurus Autosome BTA 11 contained ten exons and nine introns [4].The reported alleles and genotypes of the FSHR gene were associated with reproduction traits such as the number of fertilized oocytes, estrogen levels, embryo survival, ova quantity, and superovulation response [5][6][7][8].The purpose of this research is to identify variations in the FSHR gene and their correlation with reproductive traits in FH cattle as potential candidates for reproductive genetic markers.

Animal Samples Collection and DNA Purification
Twenty-six blood samples from imported Friesian Holstein were collected from the Dairy Cattle Breeding Center in Central Java, Indonesia.According to [9], blood was successfully collected through coccygeal vein phlebotomy.A veterinarian collected and supervised the samples, which met the standards for rearing and treating farm animals outlined in the Republic of Indonesia's Law No. 18, 2009 on animal husbandry and health.This was within the veterinarian's competency as part of the animal ethical clearance procedure.
After collecting samples, the Promega Wizard® Genomic DNA Purification Kit was used to extract and purify genomic DNA.All steps were carried out in accordance with the standard protocol provided by the company, followed by a DNA quality test using agarose 1.5 % electrophoresis.

Genotyping
Genotyping and amplification were performed using the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method with the digestion enzyme AluI, followed by chain-termination PCR Sequencing to confirm the FSHR amplified sequence, as described in [9], with PCR-RFLP Primers listed in Table 1.Amplification and genotyping were performed at the Animal Production Laboratory, Department of Animal Science, Faculty of Animal Science, Universitas Sebelas Maret.The multiparous cows with a minimal 2 times of parturition (partus) were selected as samples.The data of reproduction traits were obtained between the first parturition to second parturition.The calculation of the reproduction traits is described below.Post-Partum Mating (PPM) was calculated by subtracting the first service after the parturition date from parturition date (day) as described [10].The PPM formula is described in the formula (1).PPM = First Service after Partus Date -Partus Date (1) Service per Conception (SC) was analysed by counting the number of services until conception, as described by [1].The formula (2) shows SC calculation, where the service given by insemination or natural mating is represented by S.

SC = ∑ S (2)
Calving Interval (CI) was measured by subtracting the calving date from the date of calving before and recorded in day, as described by [10].The CI formula is expressed in the formula (3), where n is the calving number.CI = Calving Date (n + 1) -Calving Date (n) (3) Bioinformatics and Association Analysis.Unipro Ugene v.48 software performed chromatogram analysis, map to reference sequence NM 17406.1, and align amino acid with the NP 776486.1 reference sequence [11].The FSHR amino acid structure was analysed using the GPCR database (https://gpcrdb.org/)[12].The Hardy-Weinberg Equilibrium (HWE) was calculated using the Chi-Squared method, and genotype and allele frequency analysis were performed using the Genetics Package [13].The Agricolae Package with ⍺=0.05 in the R software was used to analyse the association between reproduction traits and FSHR genotype using ANOVA analysis and Duncan's Multiple Range Test (DMRT) [14,15].

Result and Discussion
Amplification by PCR successfully amplified the FSHR target fragment without contamination, yielding a 306 bp product (Figure 1).There were no bands in the negative control.The FSHR product used in this study produced results similar to those reported in previous studies in the FH population [8,9,16].Furthermore, this fragment was part of the FSHR gene's exon 10 sequence [8,[17][18][19].The FSHR genotyping results using PCR-RFLP with AluI (FSHR|AluI) revealed that the FH population was polymorphic, with three genotypes: CC, CG, and GG.The genotypes consisted of two alleles: C and G.The CC genotype has two bands (243 bp and 63 bp), the GG genotype has three bands (193 bp, 63 bp, and 50 bp), and the CG genotype has four bands (243 bp, 293 bp, 63 bp, and 50 bp).However, the 63 bp and 50 bp bands cannot be resolved clearly, but this was not a problem because the genotype can distinguish between the 143 bp and 193 bp bands, so we ignored the band under 100 bp. Figure 2 depicts the digestion-based genotype visualization.A previous study in some FH populations found similar results [8,16,17,19].Furthermore, the bands below 100 bp could not be seen clearly because the 2 percent agarose gel was unable to resolve the DNA band below 100 bp; therefore, a higher concentration (2.5-3.5 percent) of agarose gel with a longer electrophoresis time of about 35-45 minutes should be used to resolve these bands [20,21].The sequence of the FSHR fragment confirmed the RFLP results (Figure 2).The FSHR variation resulted from a substitution transversion from C to G at position 2037, or in the FSHR coding sequence NM 17406.1:c.1973C>G.Genotypes CC and GG had a single peak of base calls C and G; however, two peaks of base calls, G and C, appeared simultaneously because the CG genotype consisted of different allele combinations (C and G).The transversion substitution resulted in a missense mutation in the FSHR amino acid translation at the GPCR C-terminus region, from amino acid Threonine (T) to Serine (S) at position 368 (Figure 3).The polymorphism in FSHR exon ten was previously reported as an SNP with dbSNP ID rs209882669, located at c.1973C>G in the coding sequence and changing the amino acid at p.T658S [8,9].Furthermore, the p.T658S was a conservative mutation because threonine and serine were in the same class; they were neutral polar and small-sized amino acid groups, but threonine was an essential amino acid, whereas serine was non-essential [22,23].Furthermore, threonine and serine were important for phosphorylation; specifically, threonine was replaced by serine [3,22,24].
The CG was the highest genotype frequency (0.54), and the C was the highest allele frequency (0.65).The HWE analysis indicated that the FH population was in equilibrium (P-value 0.41) and had a Chisquare (χ2) of 0.93 (Table 2).This finding was consistent with another FH population, where the CG genotype had the highest genotype frequency (0.49), followed by the CC genotype (0.46) and the GG genotype (0.05) [16].Furthermore, previous research on the FH population revealed that the C and G alleles had the highest and lowest allele frequencies, respectively [8,18].In contrast, only the G allele and GG genotype were discovered in Bos javanicus [25].There was no significant association (P>0.05) in reproductive traits (PPM, SC, CI) among the FSHR|AluI genotype (Table 3).The CC genotype had the lowest SC (1.90 ±0.73) among the genotypes, while the GG genotype performed better in PPM (134.50 ±13.43 days) and CI (16.01 ±2.92 months).A similar result reported that the CI was not associated with genotype (P>0.05);further, a significant association in SC trait with CC genotype was the lowest genotype in an FH population raised in Türkiye [16,18].
Reproductive traits have low heritability, so the environment greatly influences reproduction performance [26].This study raised FH samples sub tropically before being imported to tropical Indonesia while pregnant.The change in climate and environment to tropical conditions most likely stressed the imported FH, affecting milk production and reproductive performance [27,28].
Finally, the FSHR|AluI in the FH population had three genotypes and two alleles.The C allele and the CG genotype had the highest frequencies, respectively.PPM, SC, and CI had no association with the FSHR|AluI genotype.Although there was no statistical association, the CC genotype had the lowest SC, while the CG genotype had the best PPM and CI performance of the genotypes.The FSHR gene is a promising marker for reproductive performance in dairy cattle, but more research into its association is needed.