We investigated means to improve the production of the indigenous Naked Neck chicken in Afghanistan. Specifically, we analyzed single nucleotide polymorphisms (SNPs) in the prolactin (PRL) (24 bp indel), growth hormone (GH) (T185G), and pituitary specific transcript factor 1 (PIT-1) (intron 5) genes. Blood samples were collected from 52 birds and genomic DNA was extracted. Polymorphisms in the mentioned loci were analyzed by PCR, allele-specific PCR, and PCR-restriction fragment length polymorphism (RFLP) using TaqI and MspI endonucleases. Cloning followed by DNA sequencing was performed to ascertain the accuracy of the PCR-RFLP analysis for PIT-1. Two alleles were found for the PRL 24 bp indel, GH (T185G), and PIT-1/TaqI, with the following respective allelic frequencies: PRL-In 0.64 and PRL-Del 0.36, GH-T 0.91 and GH-G 0.09, and PIT-1-A 0.64 and PIT-1-B 0.36. Regarding the PIT-1/MspI polymorphism, three novel MspI recognition sites, as well as two reported MspI recognition sites, were detected in intron 5. Moreover, during sequence screening, two novel SNPs were found that generated restriction sites for MseI. Therefore, our results suggest that the PRL indel, GH T185G, and PIT-1/TaqI polymorphisms may be used as selection markers for Afghanistan Naked Neck chickens. Intron 5 of PIT-1 in the Afghani Naked Neck chicken was highly polymorphic compared to the reported Gallus gallus PIT-1 gene (GenBank accession no. NC_006088.4).

The reproductive system in female birds arises as bilateral asymmetrical anlagen, excluding the birds of prey. Earlier, histological and messenger RNA (mRNA) expression profile studies of several genes related to gonadal sex differentiation in chicken embryos tried to elucidate the query of this asymmetry in a scattered manner. To understand the matter precisely, we have focused on mRNA expression of a cohort of genes (FSHR, CYP19A1, caspase 3, caspase 8) in second half of the embryonic days (E10-E18). The established role of leptin in development of the embryo and its expression in the embryonic ovary also drove us to check leptin receptor (LEPR) expression in the ovary. Increased expression of FSHR and CYP19A1 in the left ovary compared with that in the right ovary was identified (P < 0.05), promoting preferential left ovarian development and functionality. Significant high expression (P < 0.05) of the apoptotic genes in the right ovary were also involved here. Leptin probably has no direct influence on ovarian asymmetry as no significant variation in gonadal mRNA expression of LEPR was observed within the same experimental days. We propose that asymmetric expression of this cohort of genes (FSHR, CYP19A1, caspase 3, caspase 8) leads to the development of dimorphic gonads during embryogenesis.

Leptin and insulin-like growth factor I (IGF-I) regulate follicle development and reproduction in vertebrates. This study investigated the role played by leptin and IGF-I in primordial follicle activation in the ovary of 7-day-old chicks. Different doses of leptin were intraperitoneally administrated to female layer chicks and further analyses were performed. While leptin administration did not affect hepatic leptin receptor (LEPR), growth hormone receptor (GHR), or IGF-I, the lower dose of leptin significantly increased the mRNA expression of IGF-I, IGF-IR, and IGF-binding protein (IGFBP)-2 and attenuated anti-Müllerian hormone (AMH) gene expression in the ovary. Furthermore, the ovaries of the same age chicks were challenged with leptin and/or IGF-I in vitro. Leptin at a lower dose increased the mRNA expression of IGF-I, LEPR, and leptin; 100 ng/mL leptin and 10 ng/mL IGF-I alone or combined with leptin reduced IGFBP-2 mRNA expression. AMH gene expression was also reduced by all doses except 10 ng/mL leptin. Histological studies showed that a lower dose of leptin injection induced the primordial follicle growth in the ovary in vivo, and the number of primordial follicles was higher in all leptin treatments over control in vitro. Moreover, the luciferase assay revealed that leptin enhanced IGF-I promoter activity in LEPR-expressing CHO-K1 cells. Collectively, the present results indicate that leptin directly affects the IGF-I/IGFBPs system and promotes primordial follicular growth in the ovary of early posthatch chicks. In addition, the follicular development by leptin-induced IGF-I is, at least in part, caused by the suppression of AMH in the ovary.