Expression of genes of immune response and adaptation and cecal microbiome composition in males and females of chickens (Gallus gallus L.) in CM5 and CM9 preparental lines of Smena 9 cross

The Cornish and Plymouthrock breeds form the basis of modern specialized meat crosses of chickens. The selection of the paternal line of the Cornish CM5 breed of the new Russian cross of meat chickens Smena 9 is carried out mainly on the basis of meat productivity, while the maternal line of the Plymouthrock CM9 breed is primarily for reproductive efficiency and viability at a lower live growth rate than that of the CM5 line masses. In the present study, we revealed for the first time that hens and roosters of the parent stock of lines CM5 and CM9 of the novel cross Smena 9 differ in the expression of some immunity and adaptation genes, as well as in the composition of the microbiome and its putative metabolic pathways. Differences are related to genotype and sex. The aim of the work was to compare the level of expression of immunity genes and genes associated with adaptive potential, respectively, in the tissues of the bursa in the liver, as well as the composition and functions of the microbiome of the caecum of the intestine in chickens and roosters of the CM5 and CM9 lines. The experiments were carried out in the vivarium of the Zagorsk EPH (Moscow Province, 2022) on the parent stock of chickens and roosters of the CM5 and CM9 lines of 39 weeks of age, kept under identical conditions and receiving the same diet. From each line and gender, tissue samples were taken from five individuals with a close live weight. Analysis of gene expression in tissue samples was performed using quantitative reverse transcription PCR (RT-qPCR). Total RNA was isolated using the Aurum™ Total RNA mini kit (Bio-Rad, USA). PCR amplification was performed using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, USA) and a detecting amplifier DTlight (DNA-Technology, Russia). In the liver tissues, we analyzed the expression of genes associated with the adaptive potential: genes CAT1 of the transporter of cationic amino acids 1, HSF1 and HSF2 - transcription factors of heat shock proteins 1 and 2, SOD - superoxide dismutase, Gpx1 - glutathione peroxidase, HO-1 - heme oxygenase-1. In the tissues of the bursa, the expression of genes associated with immunity was analyzed: genes IL8 - interleukin-8, IRF7 - regulatory factor interferon 7, PTGS2 - prostaglandin endoperoxide synthase, AvBD1 , AvBD2, AvBD9 and AvBD10 - β-defensins 1, 2, 9 and 10, Casp6 - caspase 6. A primer for the β-actin gene (ACTB) was used as a reference control. The relative level of expression was assessed by the 2-ΔΔCT method. Total DNA for analysis of the composition of the microbiome was isolated using the Genomic DNA Purification Kit (Thermo Fisher Scientific, Inc., USA). The caecal bacterial community was assessed by NGS sequencing on the MiSeq platform (Illumina, Inc., USA) with primers for the V3-V4 region of the 16S rRNA gene. The reconstruction and prediction of the functional content of the metagenome, gene families, and enzymes was carried out using the PICRUSt2 software package (v. 2.3.0). Mathematical and statistical processing of the results was carried out by the method of multivariate analysis of variance (ANOVA) in Microsoft Excel XP/2003 and R-Studio (v. 1.1.453). The results obtained showed an increase in the expression of the HSF1 and HSF2 genes in CM5 cocks compared to other groups (p ≤ 0.05), in particular, the difference with CM9 cocks was 68 and 218 %, respectively (p ≤ 0.05). The expression of the HSF1 and HSF2 genes within the CM5 line in roosters was 1.6 and 3.0 times higher, respectively, than in hens (p ≤ 0.05). Significant activation of the expression of antimicrobial peptides and pro-inflammatory genes occurred in CM9 cocks compared to CM5 cocks and hens (p ≤ 0.05). The expression of AvBD2, AvBD9, AvBD10, IL8 and PTGS2 genes in CM9 cocks increased 7.6-, 5.3-, 2.1-, 6.3- and 1.5-fold (p ≤ 0.05), respectively, compared to CM5 cocks. NGS sequencing showed that the microbiome of the caecum of the CM9 hens and roosters contained bacteria of the superphylum Elusimicrobiota (0.32±0.11 and 0.49±0.19 %, respectively). These microorganisms did not occur in CM5 roosters while in the SM5 hens, their proportion was 0.04±0.01 %. Significant (p ≤ 0.05) differences were found between the groups in 25 genera, in some genera, it depends on the genotype, in others - on the sex of the bird. For example, in cocks of the CM5 line, the abundance of microorganisms of the genera Barnesiella , Clostridia_UCG-014 and Frisingicoccus was 17.2, 2.0 and 4.9 times higher (p ≤ 0.05), respectively, than in males of the CM9 line. Members of the genus Desulfovibrio were present in the intestines of CM5 and CM9 cocks (0.25±0.08 and 0.73±5.6 %). However, we did not find these microorganisms in the intestines of hens of both lines.