Satyendra Pal Singh

Characterization of 5′ Upstream Region and Investigation of TTTTA Deletion in 5′ UTR of Myostatin (MSTN) Gene in Indian Goat Breeds

Myostatin (MSTN) is a negative regulator of muscle growth and development. In the present study characterization of MSTN 5′ upstream region and investigation of 5′UTR TTTTA deletion was carried out in seven different Indian goat breeds. An 1181 bp fragment of 5′ upstream region of MSTN gene was PCR amplified, cloned, and sequenced. Nucleotide sequences of MSTN 5′ upstream region exhibited a high degree (>99%) identity among Indian as well as non-Indian goat breeds. These sequences showed >96.8% and 94.8% sequence identity with those of sheep and cattle. In silico analysis of MSTN 5′ upstream region using MatInspector program revealed the presence of three TATA boxes, six E-box motifs, one CAAT box, and other important putative regulatory elements. We identified 16 nucleotide substitutions in the 5′ upstream region of Indian goat breeds. Screening for a polymorphic TTTTA deletion (Δ −10 to −6) in 5′ UTR using DraI/PCR-RFLP assay revealed the presence of only two genotypes, namely, AA (deletion homozygotes; 0.95) and AB (heterozygotes; 0.05) among the Indian breeds. The mutant allele A is almost fixed (∼0.98) among Indian goat breeds and consequently, we could not perform the association study of this deletion with growth traits.

Short-hairpin Mediated Myostatin Knockdown Resulted in Altered Expression of Myogenic Regulatory Factors with Enhanced Myoblast Proliferation in Fetal Myoblast Cells of Goats

ABSTRACT Myostatin (MSTN) is a well-known negative regulator of skeletal muscle development. Reduced expression due to natural mutations in the coding region and knockout as well as knockdown of MSTN results in an increase in the muscle mass. In the present study, we demonstrated as high as 60 and 52% downregulation (p < 0.01) of MSTN mRNA and protein in the primary fetal myoblast cells of goats using synthetic shRNAs (n = 3), without any interferon response. We, for the first time, evaluated the effect of MSTN knockdown on the expression of MRFs (namely, MyoD, Myf5), follistatin (FST), and IGFs (IGF-1 & IGF-2) in goat myoblast cells. MSTN knockdown caused an upregulation (p < 0.05) of MyoD and downregulation (p < 0.01) of MYf5 and FST expression. Moreover, we report up to ∼four fold (p < 0.001) enhanced proliferation in myoblasts after four days of culture. The anti-MSTN shRNA demonstrated in the present study could be used for the production of transgenic goats to increase the muscle mass.

Role of Candidate Genes Regulating Uterine Prostaglandins Biosynthesis for Maternal Recognition of Pregnancy in Domestic Animals

The survivability and opportunity of successful development of an embryo are influenced directly or indirectly by factors controlling uterine microenvironment. Out of all factors, hormones such as prostaglandins (PGs) released during the preimplantation period influence molecular interactions involved in maintenance of pregnancy through reciprocal interactions between the conceptus and endometrium. PGs are important regulators of female reproductive functions, namely, ovulation, uterine receptivity, implantation, and parturition. Among different classes of PGs, prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2) are main prostanoids produced by human and bovine endometrium for successful growth and development of the posthatching blastocyst. In ruminants, PGF2α produced by endometrium is the major luteolytic agent, whereas PGE2 has luteoprotective and antiluteolytic properties. Therefore, the development and maintenance of the corpus luteum (CL), as well as establishment of pregnancy, depend on the balance of luteolytic PGF2α and luteotropic PGE2. In this review, we discussed the expression and function of genes which predominantly regulate the synthesis and their secretion of PGF2α and PGES, namely, PGFS (AKR1B5/AKR1C3), PGES, PGFR, and COX-2.

Absence of NsiI Polymorphism in Growth Hormone Receptor (GHR) Gene in Indian Cattle Breeds

The objective of the present study was to investigate the polymorphism in 5′ non-coding region of growth hormone receptor (GHR) gene in Sahiwal (n = 53) and Hariana (n = 50) cattle using NsiI/PCR-RFLP assay. Amplification of DNA sample revealed 302bp product using specific primer pairs and digested by using NsiI restriction enzyme. All the screened animals were found monomorphic in nature for GHR/NsiI polymorphism. It revealed that only one type of uncut banding pattern (AA genotype); which was of 302bp. We could not identify any animal with GG and AG genotypes. Consequently, we could not perform association studies with milk production traits.

Characterization of 5’ Upstream Region and Identification of Polymorphism in Intron 1 of Prolactin (PRL) Gene using HaeIII PCR-RFLP in Indian Cattle Breeds

Prolactin (PRL) gene is an important lactogenic candidate gene, plays a crucial role in mammary gland development and in the initiation, maintenance of lactation and expression of milk protein genes. In the present study, characterization of PRL 5′ upstream region and investigation of status of intron 1 polymorphism was carried out in Indian cattle breeds. An 857 bp fragment of 5′ upstream region of PRL gene consisting of part of promoter, exon1 and partial intron 1 was amplified by PCR and subsequently sequenced in Indian breed of cattle. Nucleotide sequences of PRL 5′ upstream region exhibited a high degree (>98%) identity among Indian as well as exotic cattle breeds. HaeIII polymorphism screening in PRL intron 1 of Indian cattle breeds including Sahiwal (n = 154) and Hariana (n = 50) revealed monomorphic pattern, only, AA genotype (deletion homozygote) was found and confirmed by sequencing. The obtained sequences of PRL after aligning was revealed absence of HaeIII recognition site GGCC due to deletion of G and consequently, we could not perform the association study of this deletion with milk production traits.

Association Between Polymorphisms of Solute Crrier 27 A1 Gene with Milk Production Traits in Indian Sahiwal and Hariana Cattle

This study investigated the status of Solute carrier 27A1 (SLC27A1) polymorphism in Indian cattle breeds (n = 102) using PCR-RFLP assay and its association with milk production and reproduction traits. Two region of SLC27A1 gene consisting of exon 3 and exon 4 revealed 261 and 160 bp products, respectively. The digestion of 261 bp product using SacII restriction enzyme (RE) resulted in monomorphic pattern; only GG genotype. The BsaHI/PCR-RFLP assay revealed three types of genotypes, namely, TT (160 bp), CC (106 and 54 bp) and heterozygous CT (160, 106 and 54 bp) genotypes with frequencies 18.62, 59.80 and 21.56 %, respectively. The allelic frequency of C and T alleles were 0.71 and 0.29, respectively. Association study revealed that lactation period (LP), milk yield in 300 days (MY300), calving interval (CI), peak yield (PY) and days to reach peak yield (DRPY) had non-significant difference among all the three genotypes in first and second lactations. while, significant difference was (P=0.028) observed for total milk yield (TMY), where TMY of CC genotype was significantly higher than CT and TT genotypes in only first lactation. Results indicated that C allele for SLC27A1 gene can be used as candidate gene for marker assisted selection in Sahiwal and Hariana cattle.