Genetic effect of leptin gene variants on milk traits in Dangi cattle

Introduction

The domestication of livestock through artificial selection has significantly expanded genetic and phenotypic diversity. In India, where approximately 70% of the population resides in rural areas, the livestock sector is a cornerstone of agricultural economics, rural income, and food security. According to national census data, India's livestock population stands at 536.76 million, with cattle comprising 193.46 million (142.11 million indigenous and 51.36 million crossbred) and buffaloes representing 109.85 million. India is the world's leading milk producer, contributing 24.76% (239.30 million tonnes) to global production. To enhance native dairy productivity, the Department of Animal Husbandry and Dairying (DAHD) utilizes digital frameworks like the e-Gopala application and breeding initiatives such as the Rashtriya Gokul Mission. Molecular breeding programs leverage genetic markers, including RFLPs, microsatellites, and SNPs-to optimize marker-assisted selection (MAS) and map quantitative trait loci (QTL). India recognizes 53 distinct cattle breeds, with Maharashtra possessing seven native breeds: Deoni, Gaolao, Khillar, Red Kandhari, Dangi, Kokan Kapilla, and Kathani(ICAR-NBAGR, 2025 report). The Dangi breed is a hardy draft animal native to the heavy rainfall zones of Maharashtra's Nashik district and Gujarat's Dangas district. Protected by an oily skin secretion, they are optimized for field traction, though individual cows can yield 3 to 4 kg of milk per day under favourable management (Janmeda et al., 2013) [4] . Among candidate milk production genes (ABCG2, BoLA-DRB3, DGAT1, STAT5A, etc.), the Leptin (LEP) gene stands out as a key physiological regulator. Mapping to bovine chromosome 4q32, this three-exon gene produces a 16 kDa hormone that coordinates metabolic pathways, energy balance, immune function, reproduction, and lactogenesis. This study systematically evaluates the Leptin gene in Dangi cattle to fulfil two primary objectives: To detect genetic polymorphisms within Exon-2, Intron-2, and Exon-3 regions using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and to statistically evaluate the association between identified leptin genotypes and economic milk traits within the studied population (Ogorevc et al., 2009) [12] .

Materials and Methods Experimental Animals and Sample Collection

The field trial comprised 60 lactating Dangi cows maintained by local farmers across three distinct geographic zones within the Ahilyanagar district of Maharashtra: Kotul, Akole, and Sangamner. To ensure continuous milk availability for the six-month evaluation window, enrolment criteria restricted the cohort to cows in early lactation (3-4 months post-partum). From each animal, a 5 ml blood sample was drawn aseptically from the jugular vein into specialized EDTA-coated vacuum tubes. These samples were immediately transferred into automated cold chains and moved to the Genetic Investigation Laboratory (GIL), Department of Animal Genetics and Breeding, Mumbai Veterinary College, where they were kept at 4°C until molecular processing. Fortnightly milk sampling (10 ml) was carried out over a six-month lactation phase, with immediate compositional analysis for fat, protein, solidsnot-fat (SNF), and lactose percentages performed using a calibrated LACTOSCAN automated milk analyzer. Concurrently, baseline 5 ml jugular blood samples were drawn into sterile EDTA-coated vacutainers and kept at 4°C until molecular profiling.

Genomic Isolation and Quantification

Total genomic DNA was extracted from white blood cell fractions using a modified Phenol-Chloroform-Isoamyl Alcohol (P:C:I) methodology. The isolated DNA pellets were resuspended in sterile double-distilled water. Nucleic acid purity and concentration levels were determined using a NanoDrop ND-2000 Spectrophotometer, selecting extracts that displayed an optimal OD {260/280} ratio between 1.8 and 2.0. Structural integrity was verified via horizontal 0.8% agarose gel electrophoresis.

Amplification and RFLP Genotyping

Locus-specific oligo primers were used to amplify target fragments within a pre-programmed thermal cycler:The thermocycling protocols were tailored for each distinct domain as: Exon-2: Initial denaturation at 95°C for 5 min; 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 45 sec; followed by a final extension at 72°C for 5 min. Intron-2: Initial denaturation at 94°C for 2 min; 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min; followed by a final extension at 72°C for 15 min. Exon-3: Initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 95°C for 45 sec, annealing at 55°C for 45 sec, and extension at 72°C for 45 sec; followed by a final extension at 72°C for 10 min.Following successful amplification, the PCR products were subjected to overnight restriction digestion using specific endonucleases like Exon-2 amplicons were incubated with Hph I at 37°C, Intron-2 amplicons were incubated with Sau3AI at 37°C, Exon-3 amplicons were incubated with Hinf I at 37°C. [8] Exon-3 Forward (F)Reverse (R) 5'-GGGAAGGGCAGAAAGATAGG-3' 5'-GCCGCAACATGTCC7GTAGT-3' 538 bp Jhala et al. (2011) [5] The digested DNA fragments were separated on a 2.5% agarose gel alongside a 100 bp DNA step ladder, visualized under UV transillumination, and archived using a digital gel documentation system.

Statistical Modeling

Allelic profiles and genotypic distributions were calculated using direct allele-counting methods. The relationship between segregating intronic genotypes and individual milk parameters was analyzed using a two-sample t-test. & Pannu, 2021) [3,2] . This stands in contrast to the high genotypic diversity reported in commercial Bos taurus dairy herds (Tomka et al., 2016) [14] .

Results and Discussion

Intron-2 Locus

Digestion with Sau 3AI revealed a polymorphic pattern inside the Dangi cohort, yielding two distinct configurations: Homozygous AA Genotype: Produced fragments of 390 bp and 32 bp, dominating the cohort at a frequency of 83.33% ($n=50$), heterozygous AB Genotype: Yielded four distinct fragments at 390 bp, 303 bp, 87 bp, and 32 bp, appearing in 16.67% ($n=10$) of the cattle, homozygous BB Genotype was entirely lacking from the population.

The calculated allelic frequencies were 0.92 for the major 'A' allele and 0.08 for the minor 'B' allele. This skewed distribution mirrors patterns found in autochthonous Busha lines and native Turkish breeds, where the 'A' variant is preferentially selected in native cattle adapted to local environments (Maletic et al., 2019; Oztabak et al., 2010) [9,13] .

Exon-3 Locus

Enzymatic digestion using Hinf I cleaved the 538 bp segment into identical bands of 403 bp and 135 bp for all 60 animals. This confirms a monomorphic 'AA' genotype with an 'A' allele frequency of 1.00.This matches findings in Gir cattle and Mehsana buffaloes (Jhala et al., 2011) [5] , but differs from exotic Bos taurus lines like the Jersey, where the alternative 'B' variant occurs regularly (Choudhary et al., 2005) [1] .

Acknowledgement

Acknowledgments:The authors thank the Associate Dean, Mumbai Veterinary College, Mumbai, India for providing the necessary facilities for this study. The authors are also grateful to the advisory committee members for their technical support during the research.

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