Sample Collection and Preparation: The data of Lohi sheep were obtained from Small Ruminants Training and Research Centre, Pattoki, Pakistan and statistically analysed to evaluate the effect of environmental factors. The data concerning ADG from birthday to 90 days were obtained and analyzed to conclude the influence of dam’s age, season, sex of new born, type of birth and year of birth. After analysis, 18 animals were randomly selected from the herd, though definite correction factors were also calculated to decrease the effects of environment [10].

Procedure and Analysis: A total of 5 ml blood was collected cautiously from each animal from jugular vein using a sterile syringe and stored in 15 ml centrifuge tubes including ethylenediaminetetraacetic acid (EDTA) as anticoagulant and blood samples were stored at -20ºC. Stored blood samples were subjected to DNA extraction by using a protocol as followed by Babar et al. [11], while primers for LEP gene were designed using Primer3Plus software against NCBI gene accession no: 443534. A primer set, LEP-F “5’-TGGTAACGGAGCTCAT-3’” and LEP-R “5’-TTACCTCATCTCCCTGTC-3’”, was used for the amplification of 268bp fragment covering up the entire exon 2 and partial intron 2 of the gene. Touchdown PCR was performed with a Thermal Cycler (iCyclerBioRad, USA) with initial denaturation at 94ºC for 5 min, followed by 35 cycles of denaturation at 94ºC for 45 sec, annealing 56-46ºC for 45 sec and extension was at 72ºC for 45 sec. Final extension was given at 72ºC for 10 minutes and stored at 4ºC.

Prior to Sanger sequencing from 1^st Base Laboratories Singapore, amplicons were cleaned using TIANquick Midi Purification Kit (Tiangen Biotech Beijing Co., Ltd.). The sequences were aligned and edited using CodonCode Aligner ver. 5.0.2 and MEGA 6.0 software [12]. The following model was utilized to minimize the effect of environmental factors:

Y_i = µ + Z_i + Ɛ_i

Where, Y_i is ADG of n^th lamb; µ is overall population mean; Z_i is the effect of year, season, sex of new-born, type of birth and age of dam; at the same time Ɛ_i is a random error associated with ADG of n^th lamb. Furthermore, it was presumed that Ɛ_i was generally and autonomously distributed with mean 0 and variance σ².

Polymorphism in LEP gene was studied in Lohi animals, an important indigenous sheep breed of Pakistan. Heterozygous peaks were observed in two individuals (ID: 201312025, 201321143) and were labeled as “Y”. The comparative evaluation of individuals carrying “Y” and those lacking it based on phenotypic data did not show any significant association. An attempt was made to correlate observed mutation with ADG but due to a limited number of animals, the analysis did not show any concluding relationship. Qureshi et al. [14] reported variations in intron 2 (A2262T, C2256G, -2730C) and exon 2 (A3201G) in the LEP gene in Lohi sheep maintained at Livestock Production and Research Institute, Bahadurnagar, Okara, Pakistan. Jonas et al. [15] found variation (C11T) in exon 2 in Awassi-Merino sheep with non-significant (P>0.05) association with body weight. Similarly, Boucher et al. [16] also found SNPs in exon 2 of Dorset and Suffolk sheep breeds. Three SNPs were observed by Wang et al. [17] while studying polymorphism in LEP receptor gene in caprine and along-with association between genotypes and traits i.e. birth weights of kids and prolificacy. A relationship was observed between genotypes and weights at different ages of animals in ovines of Makooei breed [18].  Certain SNPs in intron 2 and exon 2 in goats were also reported by Singh et al. [19] and Maitra et al. [20].

Phylogenetic tree (fig. 3) shows that Lohi animals used in this study have conserved regions for exons and goats showed close relation with this. Phylogenetic tree constructed based on exons of leptin gene reconfirmed the biological classification of mammalian species. In other words, molecular classification-based leptin exons gene sequence reconfirmed the classical taxonomical classification. Phylogenetic tree showing inter-relationship of Lohi sheep animals with closely related animal species inferred from exon 2 sequences in ovine LEP gene. Tree was generated using the neighbour- joining methods. Bootstrap values expressed as percentage of 1000 replications and indicated at the nodes.

The present study may be regarded as one of the initial attempts to understand the genetic diversity of LEP gene in local sheep breeds of Pakistan. Molecular analysis of exon 2 of LEP gene in Lohi individuals showed polymorphism. At present, very less data are available to compare our results. There is need to extend this work with a higher number of animals in different breeds to have more candidate polymorphisms that may be associated with growth traits for possible use in Marker Assisted Selection (MAS) in future breeding programs.