Characterization of polymorphisms in the follicle-stimulating hormone receptor and insulin-like growth factor-1 genes and their association with fertility traits in Jawa-Brebes cows

Background and Aim: The availability of fertility markers is crucial for maintaining, protecting, and improving the genetics of Jawa-Brebes (Jabres) cows. Follicle-stimulating hormone receptor (FSHR) and insulin-like growth factor-1 (IGF-1) play critical roles in female reproductive physiology. The single-nucleotide polymorphisms (SNPs) FSHR G-278A and IGF-1 C-512T correlate with cows’ fertility traits. This study aimed to identify these SNPs and their potential associations with fertility parameters in Jabres cows. Materials and Methods: Samples were collected from 45 heads of multiparous Jabres cows aged 3–10 years with body condition scores of 2.5–5.0 on a 5-point scale in Brebes Regency, Java, Indonesia. These cows were assigned to fertile (n = 16) and infertile groups (n = 29). Polymerase chain reaction (PCR) was carried out for DNA amplification of FSHR G-278A and IGF-1 C-512T fragments. Restriction fragment length polymorphism-PCR with the restriction enzymes FaqI for the product of FSHR G-278A and SnaBI for the product of IGF-1 C-512T was used to identify SNPs. Results: The FaqI enzyme cut the 211 bp DNA fragment of FSHR G-278A in all samples into two bands of 128 bp and 83 bp (GG genotype). Meanwhile, the genotyping of amplicon products of IGF-1 C-512T generated a single 249 bp fragment (CC genotype) in both groups. Conclusion: The results showed that the FSHR G-278A/FaqI and IGF-1 C-512T/SnaBI loci were monomorphic in Jabres cows. Thus, neither FSHR G-278A/FaqI nor IGF-1 C-512T/SnaBI is a possible genetic marker for fertility in Jabres cows.


Introduction
Jawa-Brebes (Jabres) cattle, an indigenous Indonesian breed found in Brebes Regency, Java, Indonesia, are renowned for unique reproductive characteristics, including a birth rate of 15-20 calves during the lifespan and good reproductive function when fed inadequate diets [1]. This breed is classified as having a protected and preserved livestock germplasm [2] and plays vital socioeconomic roles [3]. However, there is no effective Jabres breeding program for selecting dams, leading to low pregnancy and calving rates. This contributes to shrinking the population and threatens the survival of Jabres. Indeed, the population of Jabres decreased from 13,890 heads to 12,082 heads in 2021 [4]. There is thus an urgent need for research on genetic markers of fertility to protect, preserve, and enhance the genetics and population of Jabres cows.
Follicle-stimulating hormone (FSH) is an essential glycoprotein hormone that critically controls male and female reproductive physiology [5]. It promotes follicular growth and development and facilitates the production of related proteins in parietal granulosa cells in females [6]. Follicle-stimulating hormone exerts its physiological effects through the FSH receptor (FSHR) [7]. It is mainly found in ovarian granulosa cells in female animals [8][9][10].
In female livestock, single-nucleotide polymorphisms (SNPs) in the 5'-upstream region (5'-UTR) of the FSHR gene have been significantly identified as valuable fertility indicators. For example, significant associations between SNPs in the 5'-UTR of the FSHR gene and the litter size of Chinese native ewes have been identified [11,12]. Moreover, in Chinese Holstein cows, an SNP in the 5'-UTR of the FSHR gene (FSHR G-278A) was found to correlate with the superovulation response [13]. Furthermore, the FSHR G-278A mutation was shown to influence service per conception (S/C) in Holstein dairy cows [14].
Meanwhile, insulin-like growth factor-1 (IGF-1), which is secreted by liver cells and various other cells, functions as a fundamental growth factor in numerous physiological activities, including reproduction, fetal development, and growth [15][16][17]. IGF-1 is crucial in female mammalian fertility because it regulates ovarian function, follicle development, oocyte maturation, and preimplantation embryos [18]. By interacting with gonadotropins, the IGF-1 gene stimulates the growth and steroidogenesis of ovarian cells, promoting ovarian function [19]. In addition, IGF-1 inhibits follicular atresia [20].
Based on reported studies, the IGF-1 gene is a strong putative genetic marker for the reproductive traits of female animals. Insulin-like growth factor-1 polymorphisms have been reported to be correlated with the litter size of Gulin Ma goat and Small Han Tail sheep [21,22]. In addition, SNPs in the IGF-1 gene have been shown to influence the fertility rate of Sarda ewes [23]. Mutation of T to C at position 512 in the regulatory region of the IGF-1 gene (IGF-1 C-512T) is the most studied SNP in the IGF-1 gene associated with fertility traits in cows. This SNP influences the length of the period from calving to commencement of luteal activity (CLA) postpartum, whereby CLA is essential for subsequent pregnancy after calving [24]. The IGF-1 C-512T mutation has also been reported to influence calving-first service interval in primiparous Holstein cows [25]. Moreover, several reproductive traits of Holstein cows, such as postpartum estrus and days open, are affected by the SNP IGF-1 C-512T [26].
Restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) methods with the FaqI restriction enzyme for the FSHR gene and the SnaBI restriction enzyme for the IGF-1 gene have been developed to detect fertility markers for dairy cows [13,14,[24][25][26].
However, to the best of our knowledge, no study of these has been conducted on Jabres cows. This study aimed to identify FSHR G-278A and IGF-1 C-512T SNPs and investigate their possible associations with fertility traits, including postpartum estrus, days open, and calving interval (CI), in Jabres cows.

Ethical approval and Informed consent
All experimental procedures were approved by the Research Ethics Committee of the Faculty of Veterinary Medicine, Universitas Gadjah Mada, Indonesia (00143/EC-FKH/Int./2021). In addition, this research was conducted with the verbal consent of farmers as the cows' owners.

Study period and location
The study was carried out from November 2021 to March 2022 on smallholder farmers in the sub-district of Bantar Kawung, Brebes Regency, Central Java Province, Indonesia, and Biochemistry Department, Gadjah Mada University, Yogyakarta, Indonesia.

Animals
The animals included in this study were multiparous Jabres cows aged 3-10 years with body condition scores of 2.5-5.0 on a 5-point scale. The cows were observed in the sub-district of Bantar Kawung, Brebes Regency, Central Java Province, Indonesia, where the breed originated. The reproductive characteristics recorded and used were postpartum estrus (PPE), days not pregnant (DNP), and CI. The Jabres cows were allocated to two groups: (a) Fertile, defined as PPE of <90 days, DNP of <120 days, and CI of <390 days; and (b) Infertile, defined as EPP of more than 90, DNP of more than 120 days, and CI of more than 390 days. There were 19 Jabres cows in the Fertile group and 26 in the Infertile group. The cows were reared under conditions with management by farmers. Specifically, all cows in this study were raised semi-intensively by smallholder farmers under similar conditions. All cows grazed on native pastures in the morning and were fed rice and corn straw in the afternoon. Water was provided before and after grazing.

Sample collection and DNA extraction
Blood samples (750 μL) were drawn from the jugular vein of each animal through a 5 mL sterile syringe and put into a 1.5 mL microtube containing 750 μL of absolute ethanol. Blood samples in microtubes were aerated at room temperature (25℃) for 1 d before DNA extraction. Each sample (200 μL) was used for DNA extraction using the gSYNC™ DNA Mini Extraction Kit (Geneaid Biotech Ltd., Taipei, Taiwan), in accordance with the manufacturer's protocol. Then, DNA samples were kept at −20°C until further molecular analysis.

Primers
The primers used for FSHR G-278A and IGF-1 C-512T are shown in Table-1 [14,24]. Primer synthesis was performed by the Genetika Science Company in Indonesia.

Polymerase chain reaction reactions
The PCR reaction to amplify FSHR G-278A and IGF-1 C-512T DNA for all samples comprised a total volume of 50 μL featuring 25 μL of master mix (Bioline My Taq HS Red Mix, 1 st Base, Meridian Life Science Inc., London, UK), 20 μL of distilled water, 3 μL of DNA template, and 1 μL (10 pmol) of each primer. The PCR reaction was performed using the Infinigen PCR machine (Biotech Infinigen, CA, USA) with initial denaturation at 94°C for 5 min, followed by 35 cycles at 94°C for 45 s, 55°C for 30 s, and 72°C for 45 s, with final extension at 72°C for 5 min. PCR products were electrophoresed on 1% agarose gel with a 1000 bp DNA ladder (Genaid, Taiwan). . These fragments identified for the FSHR and IGF-1 genes were putative genetic markers for fertility in cows [14,26].

Statistical analysis
Restriction fragment length polymorphism-polymerase chain reaction data were analyzed by calculating the allele and genotype frequencies as follows: Where X i is the genotype frequency, X ii is the allele frequency, n ii is the number of genotype A i A i , n ij is the number of genotype A i A j , and N is the total samples.  Figure-2. This confirmed that all of the studied samples were monomorphic and genotyped as GG genotypes (Table-2).

Restriction fragment length polymorphism-polymerase chain reaction on the IGF-1 gene
This study showed that the PCR amplification of IGF-1 C-512T produced a 249 bp DNA fragment, as displayed in Figure-3. The SnaBI restriction enzyme was used for genotyping amplicon products. However, the specific DNA-cutting sites of IGF-1 C-512T were left uncut by the SnaBI enzyme in all Jabres cows of both groups. Therefore, the genotyping of amplicon products of IGF-1 C-512T in Jabres cows generated a single 249 bp fragment, as shown in Figure-4. This indicated that all of these Jabres cows possessed the same genotype, namely, the CC genotype (Table-3).

Discussion
Follicle-stimulating hormone receptor is necessary for ovarian follicle development and ovulation [27]. Several signaling pathways that promote follicle development and estrogen synthesis are activated by FSHR [28]. Numerous studies have shown that FSHR mutations are indicators of fertility and infertility during female reproduction. In addition, the SNP FSHR rs6166 is a genetic indicator of premature ovarian insufficiency (POI) in Asian women [29]. Folliculogenesis failure caused by the mutation of the FSHR gene led to premature ovarian insufficiency in women [30]. Moreover, Lindgren et al. [31] discovered that the FSHR variant N680S is an excellent RFLP-PCR=Restriction fragment length polymorphism-polymerase chain reaction, FSHR=Follicle-stimulating hormone receptor, IGF-1=Insulin-like growth factor-1, SNP=Single-nucleotide polymorphisms Single-nucleotide polymorphisms in the FSHR gene are also significantly associated with ovarian activity in cows. For example, Yang et al. [13] reported that an SNP in the 5'-UTR region of FSHR (FSHR G-278A) is correlated with superovulation response, including the total number of ova and the number of transferable embryos, in Chinese Holstein cows. Single-nucleotide polymorphisms in the coding region (c.337C>G, c.871A>G and c.1973C>G) of the FSHR gene also influence the numbers of embryos yielded and unfertilized oocytes in Holstein cows [32]. Furthermore, Hirayama et al. [33] demonstrated that the FSHR SNP c.337C>G affected the number of embryos produced by superovulation in Japanese Black cattle. This variant was also shown to affect the ovulation rates in beef heifers [34].
Although the FSHR gene is crucial in reproduction and fertility traits, we discovered that the studied locus of FSHR G-278A in the 5'-UTR in the fertile and infertile Jabres groups was monomorphic. No variation in the FSHR G-278A/SnaBI locus was found in any Jabres cows. Follicle-stimulating hormone receptor G-278A appears to be highly conserved within the investigated Jabres cow groups. This may be due to the high inbreeding coefficient of the investigated population or the presence of a connection between the sampled animals. It has been reported that uncontrolled mating resulted in highly inbred Jabres cattle [35]. Thus, the FSHR G-278A/SnaBI locus does not affect fertility traits in Jabres cows.
Our result is similar to the finding of Abeygunawardana et al. [36], who reported that the FSHR G-278A/SnaBI locus had no association with fertility traits in crossbred multiparous dairy cows (Bos indicus × Bos taurus). Moreover, because no variation was found, it was reported that the FSHR gene was not correlated with reproductive parameters, including ovarian hypofunction in Madrasin cattle [37]. However, these findings contrast with the results of Sharifiyazdi et al. [14], who found that the FSHR G-278A/SnaBI locus affected the reproductive traits of Holstein dairy cows, such as S/C. An FSHR SNP at A-320T/TaqI of the 5'-UTR region was    Available at www.veterinaryworld.org/Vol.16/April-2023/7.pdf also reported to be associated with S/C in Antioquia Holstein cattle [38]. IGF-1 is a central regulator of many intraovarian activities during follicular development [39]. It promotes follicular development by regulating the proliferation and differentiation of granulosa cells, steroid synthesis, and gonadotropin stimulation [40]. Moreover, granulosa cells regulate oocyte development in ovarian follicles by synthesizing steroids and growth factors [41]. SNPs in IGF-1 have been reported to be closely associated with fertility in female livestock [21][22][23]42].
In our investigation, genotyping of IGF-1 C-512T with the SnaBI enzyme generated a single CC genotype with a DNA fragment of 249 bp in all Jabres cows. Consequently, only the C allele was discovered and no T allele was detected. Although our study included few samples, this work indicated no association between the SNP IGF-1 C-512T and reproductive traits in Jabres cows. Anggraeni et al. [43] also discovered no correlation between intron 1 of the IGF-1/SnaBI locus and fertility traits in Peranakan Ongole cows. The genotype of IGF-1 C-512T/SnaBI was also reported not to affect the fertility of Holstein cows reared semi-intensively or intensively [44]. However, Silveira et al. [26] found that the SNP IGF-1 C-512T/SnaBI correlated with fertility parameters of Holstein cows, including postpartum estrus and days open. Moreover, the SNP IGF-1 C-512T/SnaBI was found to influence CLA as a crucial factor for the following pregnancy after calving [24] and the calving-first service interval in Holstein cows [25].

Conclusion
Neither FSHR G-278A/FaqI nor IGF-1 C-512T/ SnaBI is a possible genetic marker for fertility in Jabres cows. Due to the crucial roles of the FSHR and IGF-1 genes, further study is needed to search for other loci within these essential genes in Jabres cows. A study with a larger sample size is also necessary to confirm the effect of specific SNPs of the FSHR and IGF-1 genes on the reproductive characteristics of Jabres cows.

Authors' Contributions
SH and AB: Designed the study and wrote, edited, and revised the manuscript. SH and RW: Conducted study in the laboratory. SH, AB, and RW: Analyzed and interpreted the data. AB, RW, and EMNS: Supervised the study and reviewed the manuscript. SH and IDP: Collected the data and blood samples. All authors have read, reviewed, and approved the final manuscripts.