Molecular evaluation of the corpus luteum of ewes submitted to different nutritional plans in pre and peripuberty

• Grant number: 22/02588-4
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2023
• End date: January 31, 2024
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Caliê Castilho
• Grantee: Nathalia Sant Ana de Almeida
• Host Institution: Pró-Reitoria de Pesquisa e Pós-Graduação. Universidade do Oeste Paulista (UNOESTE). Presidente Prudente , SP, Brazil

Abstract

Nutritional status is the main factor influencing an animal to reproduce. The onset of puberty and the maintenance of reproductive function are physiologically linked to nutrition and body condition. Nutritional variations influence metabolism, with consequent reflection on the concentrations of hormones and their receptors. Thus, the objective of the present project will be to investigate the influence of 3 different nutritional plans in peripubertal ewes on the gene expression of insulin-like growth factor 1 (IGF1), IGF-binding protein (IGFBP3), acute regulatory protein of steroidogenesis (STAR), 3²-hydroxysteroid dehydrogenase (HSD3B) and leptin receptor (LEPR) in corpus luteum (CLs). The field experiment has already been carried out and the CLs are stored, so below is the summary of the experiment done previously to obtain the CLs. 24 ewe lambs (7/8 Dorper) aged between 6 and 7 months were used. Sheep were randomly assigned to 1 of 3 food groups: G1 (70-80% of National Research Council [NRC] requirement), G2 (100-110% [NRC]) and G3 (140% [NRC]). Ewe lambs from G-1 (n=8) and G-2 (n=8) were kept on Panicum maximum cv. Tanzania with access to water and mineral salt ad libitum and only those in the G-2 group will receive 1.5% of the live weight of commercial feed twice a day. The lambs from G3 (n=8) were confined throughout the experimental period, receiving a total diet, in the roughage: concentrate ratio of 20:80, containing 16% CP and 72% TDN, aiming at a daily weight gain of 200d/ day according to NRC, with mineral salt ad libitum. Initially the ewes received 3.5% of the live weight of the total diet (hay + feed), and this percentage was increased until reaching an average of 4.5 to 5% of the live weight. Upon reaching a body weight of 35 kg, the ewes were synchronized by inserting a vaginal progesterone releasing device (CIDR®) for 12 days. On the day of implant removal (D12), 0.075 mg of cloprostenol and 300 IU of equine chorionic gonadotropin were administered intramuscularly and eight days later, the sheep were slaughtered and the reproductive system removed for weighing and processing of later CL samples. analysis of gene expression of hormone receptors. Fragments of LCs that were deposited in liquid nitrogen (-196oC) and stored in a -80°C freezer will be used to perform RT-qPCR. These fragments (~40 mg) will be ground in a tissue homogenizer and submitted to the Trizol® (ThermoFisher Scientific®) extraction protocol for total extraction. Reverse transcription will be performed using the Superscript® III protocol (InvitrogenTM, Termo Fisher Scientific, Brazil), following the manufacturer's protocol. The qPCR will be performed for the quantitative analysis of the relative gene expression. After normality analysis, if the data are parametric ANOVA will be used followed by Student's t test. If the data are not parametric, the Kruskal Wallis Test will be performed. All analyzes will be performed using a significance level of 5% (p<0.05).