Evaluation of candidate gene for fecundity in Gaddabolli sheep of Andhra Pradesh

Introduction

Over generations of natural selection and domestication, India's diverse sheep breeds have evolved within specific agro-climatic zones each uniquely adapted to survive and thrive in its specific local environment. While India's sheep germplasm is vast, with 60 genetic groups as reported by the FAO (Scherf, 2000) [1] , only 46 have been recognized to date (ICAR-NBAGR, 2025) [2] , underscoring the need for the identification and characterization of lesser-known sheep. Gaddabolli sheep is one such lesser-known genetic group (Kondapu et al., 2025) [3] , that are distinct in their phenotype and are distinguishable from Nellore breed with its home tract in the dry land areas of Prakasam, Bapatla, Guntur and Palnadu districts of Andhra Pradesh. The local farmers prefer this sheep to Nellore due to high twinning rate which is about 20 to 25 per cent in the flocks that is uncommon in most of the Indian sheep breeds. The FecB (Booroola) gene, a point mutation in the BMPR1B (Bone Morphogenetic Protein Receptor Type 1B) gene, represents one of the most significant genetic discoveries in small ruminant science. Originally identified in the Booroola Merino but tracing its roots to the Indian Garole, this autosomal dominant mutation fundamentally alters the TGF-$\beta$ signaling pathway in the ovaries. The high prolificacy in sheep is preferred as it benefits the farmer in terms of lamb production. GDF9 gene (FecG locus) is a critical regulator of fertility in sheep that operates through a unique overdominance mechanism for high prolificacy. Hence, the study on Gaddabolli sheep to explore the genetic cause of prolificacy was undertaken by screening for the presence of mutation in Booroola gene (FecB/BMPR1B) and Growth differentiating factor gene (FecG H /GDF9).

Materials and Methods

Experimental animals and sample collection

The present study does not warrant Institutional Animal Ethics Committee approval as no experimentation is conducted on the animals. Four milliliter of blood sample was collected from each of the 210 Gaddabolli sheep (Table 1) reared by farmers in their home tract with their consent in Guntur, Bapatla, Palnadu and Prakasam districts of Andhra Pradesh.The collected blood samples were transported to the laboratory in an ice packed container and stored at -20 C.

DNA extraction

Genomic DNA was isolated from whole blood by high salt method (Milleret al., 1988) [4] , with modifications to suit the lab conditions. The genomic DNA was verified using 1% agarose gel electrophoresis and was quantified by using Nano spectrophotometer (Venchal Scientific, USA).

Selection of Fecundity genes for screening

Owing to their significance for prolificacy in Sheep FecB and FecG H genes were considered for the study. All the ewes and rams having twin lambs or a history of twinnings were screened for mutations in FecB using the primersF:5'-CCAGAGGACAATAGCAAAGCAAA-3', R:5'-CAAGATGTTTTCATGCCTCATCAACAGGTC-3' (Abouheif et al., 2011) [5] , and for FecG H using the primers F:5'-GAAGACTGGTATGGGGAAATG -3', R:5'-CCAATCTGCTCCTACACACCT -3' (Hanrahan et al., 2004) [6] . The cycling conditions for amplification of FecB included initial denaturation for 3 min at 94 °C, followed by 35 cycles comprising of 45 s at 94 °C (denaturation), 45 s at 52 °C (annealing), and 45 s at 72 °C (extension) followed by final extension for 7 min at 72 °C. The corresponding cycling conditions for amplification of FecG were 94 o C / 3 min, 94 o C / 30 s, 58 o C / 30 s, 72 o C / 30 s and 72 o C / 7 min. PCR products were subjected to 2% agarose gel. Subsequently, the PCR amplicons were subjected to RFLP with AvaII on FecB and HhaI for FecG respectively to observe the polymorphic patterns and thus genotype the animals for the respective locus. Seven milliliters of the digested samples was separated on 3.5 percent agarose gel for genotyping. Gels were visualized with Omega Fluor™ Plus Documentation Systems (BioExpress, USA). The agarose gels were scored based on the pattern observed as wild type, mutant and heterozygotes.

Results and Discussion

Screening for FecB and FecG H gene mutation

This genetic group of sheep were reported by the farmers to be having high prolificacy at the rate of 25-30 per cent in the flocks. During the survey in thesampled27 flocks (Table 1) twinning rate was observed at 21 per cent (Figure 1). More twinnings were observed in flocks from Darsi Mandal. RFLP was employed to detect the polymorphic patterns and to determine genotypes of the animals for each of FecB and FecG H gene in these populations in the sample under study. The Gaddabolli (n = 210) ewes, rams and lambs that were from families having two lambs in a lambing were selected for blood collection during the survey. The detail of samples screened across different flocks is represented in Table 1. The study revealed the presence of only ++ and few B+ genotypes in the populations. In the present population that was screened 26 sheep (∼12.4%) were heterozygous carriers (B+) and the rest were non-carriers for the FecB mutation (Table 2) and frequency of the allele was about 0.1. In various studies on Indian sheep breeds that were non prolific viz. Deccani, Bannur (Nimbkaret al., 2003) [7] , Kenguri sheep (Asharani et al., 2018) [8] , Marwadi, Patanwadi and Dumba sheep (Mankad et al., 2020) [9] , Mecheri sheep (Sudhakar et al., 2013) [10] , Coimbatore, Mecheri and Tiruchy Black sheep (Saravanan et al., 2020) [11] , Deccani (Praveena et al, 2017) [12] , were reported to be non carriers of FecB mutation. However, all the three genotypes of FecB were detected in Bonpala sheep. In another study on forty Nellore sheep blood samples, there was only one ewe with (BB) homozygous, five ewes with (B/+) heterozygous and thirty four ewes with (+/+) homozygous genotype in FecB loci (Praveena et al., 2017) [12] .

Though, all the local Kashmir sheep were prolific, lacked the mutation indicating that the prolificacy in those animals may be due to some other Fec gene (Ganai et al., 2012) [13] .

The observation on Gaddabolli sheep in the present study was in accordance to those in Nilagiri sheep, in which 12.99% of ewes were with multiple births (Rajendran, 2005) [14] , and the frequency of the FecB allele was 0.14 (Sudhakar et al., 2013) [10] . The varied frequencies of FecB allele was reported in Kendrapada sheep (Kumar et al., 2008) [15] . In the current study, the FecB mutant allele occurred at a low frequency despite the prevalence of twin births. This suggests that other genetic factors may be at play, as previously noted by Davis et al. (2006) [16] , who reported twin-producing sheep that lacked both the FecB and Inverdale mutations. This may be due to certain genes that are yet to be identified having a role in molecular regulatory mechanisms underlying sheep prolificacy.

FecG H genotyping

A DNA fragment with the expected size of 462 bp was amplified from exon I of GDF9 gene (Figure 3a). The G→A nucleotide change in GDF9 exon I disrupts a HhaI restriction enzyme cleavage site (GCGC to GCAC). Restriction digestion of the PCR product from wild-type animals with HhaI should result in cleavage of the 462-bp product (at two internal HhaI sites) into fragments of 52, 156, and 254 bp (FecG+). But, DNA fragments containing the 'A' nucleotide was supposed to yield only two fragments of 52 and 410 bp (FecG H ). Animals heterozygous for the mutation have fragments of all four sizes (52, 156, 254, and 410 bp). Among the 210 sheep that were screened for FecG H mutation no polymorphism at GDF9/HhaI locus existed and all individuals had the wild type except one sheep that was tested positive for presence of mutation showing 52 and 410 bp fragments (Figure 3b). The sheep that was harbouring FecG H mutation was free from FecB mutation. The coexisting polymorphism of these two was reported in Kendrapada sheep (Dash et al., 2017) [17] . Mutations of FecG (five single nucleotide polymorphisms (SNPs)) are associated with increased ovulation rates in heterozygous animals and sterility in homozygous animals (Hanrahan et al. 2004; Bodin et al.

2007) [6,18] . [19] . At G1 locus of GDF9 gene in Bonpala sheep, also an Indian sheep, three genotypes, namely, wild type (GG, 0.89), heterozygous (GA, 0.10), and mutant (AA, 0.01) were detected (Roy et al., 2011) [20] . The changing concentrations of GDF9 in vivo leads to incremental changes in ovulation rate in sheep (Hanrahan et al. 2004) [6] . The details of sheep that were carrying a copy of either FecB or FecG mutations were presented in [21] , but rather reflects the low probability of capturing rare homozygotes in small sample known as sampling zero or random zero (Huang et al., 2011) [22] , due to the limits of the sample size. Among the 210 sheep that were screened for FecG H mutation no polymorphism at GDF9/HhaI locus existed and all individuals had the wild type except one sheep that was tested positive for presence of mutation.

Conclusion

The cause of frequent twinning in the flocks could be due the effect of other fecundity genes that segregate in other prolific sheep breeds, or may be a combination of gene products that stimulate/alter the ovulatory cycle. Further efforts are required to investigate the cause. However, other possible reasons may be attributed to good nutritional plan and body condition of sheep which was evident by their biometrical and growth traits that could have stimulated the ovulatory responses to some extent and in turn produce extra ova/lambs as also opined by Kandil (2002) [23] .

Acknowledgement

AcknowledgementsThe authors acknowledge Sri Venkateswara Veterinary University for providing facilities to conduct this study.

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