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The worldwide chicken gene pool encompasses a remarkable, but shrinking, number of divergently selected breeds of diverse origin. This study was a large-scale genome-wide analysis of the landscape of the complex molecular architecture, genetic variability, and detailed structure among 49 populations. These populations represent a significant sample of the world's chicken breeds from Europe (Russia, Czech Republic, France, Spain, UK, etc.), Asia (China), North America (USA), and Oceania (Australia). Based on the results of breed genotyping using the Illumina 60K single nucleotide polymorphism (SNP) chip, a bioinformatic analysis was carried out. This included the calculation of heterozygosity/homozygosity statistics, inbreeding coefficients, and effective population size. It also included assessment of linkage disequilibrium and construction of phylogenetic trees. Using multidimensional scaling, principal component analysis, and ADMIXTURE-assisted global ancestry analysis, we explored the genetic structure of populations and subpopulations in each breed. An overall 49-population phylogeny analysis was also performed, and a refined evolutionary model of chicken breed formation was proposed, which included egg, meat, dual-purpose types, and ambiguous breeds. Such a large-scale survey of genetic resources in poultry farming using modern genomic methods is of great interest both from the viewpoint of a general understanding of the genetics of the domestic chicken and for the further development of genomic technologies and approaches in poultry breeding. In general, whole genome SNP genotyping of promising chicken breeds from the worldwide gene pool will promote the further development of modern genomic science as applied to poultry.

Whole Genome Screening Procures a Holistic Hold of the Russian Chicken Gene Pool Heritage and Demographic History.

A study for genomic variation that may reflect putative selective signaling and be associated with economically important traits is instrumental for obtaining information about demographic and selection history in domestic animal species and populations. A rich variety of the Russian chicken gene pool breeds warrants a further detailed study. Specifically, their genomic features can derive implications from their genome architecture and selective footprints for their subsequent breeding and practical efficient exploitation. In the present work, whole genome genotyping of 19 chicken breeds (20 populations with up to 71 samples each) was performed using the Chicken 50 K BeadChip DNA chip. The studied breed sample included six native Russian breeds of chickens developed in the 17th-19th centuries, as well as eight Russian chicken breeds, including the Russian White (RW), created in the 20th century on the basis of improving local chickens using breeds of foreign selection. Five specialized foreign breeds of chickens, including the White Leghorn (WL), were used along with other breeds representing the Russian gene pool. The characteristics of the genetic diversity and phylogenetic relationships of the native breeds of chickens were represented in comparison with foreign breeds. It was established that the studied native breeds demonstrate their own genetic structure that distinguishes them from foreign breeds, and from each other. For example, we previously made an assumption on what could cause the differences between two RW populations, RW1 and RW2. From the data obtained here, it was verified that WL was additionally crossed to RW2, unlike RW1. Thus, inherently, RW1 is a purer population of this improved Russian breed. A significant contribution of the gene pool of native breeds to the global genetic diversity of chickens was shown. In general, based on the results of a multilateral survey of this sample of breeds, it can be concluded that phylogenetic relationships based on their genetic structure and variability robustly reflect the known, previously postulated and newly discovered patterns of evolution of native chickens. The results herein presented will aid selection and breeding work using this gene pool.

Effects of Trehalose Supplementation on Lipid Composition of Rooster Spermatozoa Membranes in a Freeze/Thaw Protocol.

The plasma membrane of spermatozoa plays an important role in the formation and maintenance of many functions of spermatozoa, including during cryopreservation. As a result of chromatographic analysis, the content of lipids and fatty acids in the membranes of spermatozoa of roosters of two breeds was determined under the influence of cryoprotective media containing trehalose LCM-control (0 mM), Treh20 (9.5 mM), and Treh30 (13.4 mM). The use of the cryoprotective diluent Treh20 made it possible to maintain a dynamic balance between the synthesis and degradation of phospholipids and sterols in the plasma membranes of frozen/thawed spermatozoa, close to that of native spermatozoa. This contributed to an increase in the preservation of frozen/thawed spermatozoa membranes from 48.3% to 52.2% in the egg breed and from 30.0% to 35.1% in the meat- and-egg breed. It was also noted that their kinetic apparatus (mobility indicators) remained at the level of 45.6% (egg breed) and 52.4% (meat-and-egg breed). An increase in the concentration of trehalose to 13.4 mM in a cryoprotective diluent for rooster sperm resulted in a decrease in the morphofunctional parameters of frozen/thawed spermatozoa.

Selective footprints and genes relevant to cold adaptation and other phenotypic traits are unscrambled in the genomes of divergently selected chicken breeds.

BACKGROUND: The genomes of worldwide poultry breeds divergently selected for performance and other phenotypic traits may also be affected by, and formed due to, past and current admixture events. Adaptation to diverse environments, including acclimation to harsh climatic conditions, has also left selection footprints in breed genomes. RESULTS: Using the Chicken 50K_CobbCons SNP chip, we genotyped four divergently selected breeds: two aboriginal, cold tolerant Ushanka and Orloff Mille Fleur, one egg-type Russian White subjected to artificial selection for cold tolerance, and one meat-type White Cornish. Signals of selective sweeps were determined in the studied breeds using three methods: (1) assessment of runs of homozygosity islands, (2) FST based population differential analysis, and (3) haplotype differentiation analysis. Genomic regions of true selection signatures were identified by two or more methods or in two or more breeds. In these regions, we detected 540 prioritized candidate genes supplemented them with those that occurred in one breed using one statistic and were suggested in other studies. Amongst them, SOX5, ME3, ZNF536, WWP1, RIPK2, OSGIN2, DECR1, TPO, PPARGC1A, BDNF, MSTN, and beta-keratin genes can be especially mentioned as candidates for cold adaptation. Epigenetic factors may be involved in regulating some of these important genes (e.g., TPO and BDNF). CONCLUSION: Based on a genome-wide scan, our findings can help dissect the genetic architecture underlying various phenotypic traits in chicken breeds. These include genes representing the sine qua non for adaptation to harsh environments. Cold tolerance in acclimated chicken breeds may be developed following one of few specific gene expression mechanisms or more than one overlapping response known in cold-exposed individuals, and this warrants further investigation.

Influence of Technological Stages of Preparation of Rooster Semen for Short-Term and Long-Term Storage on Its Quality Characteristics.

There is a problem of declining quality of rooster semen in the "native semen-equilibrium-short-term and long-term storage (cryopreservation)" cycle. The aim of this study was to determine the effects of various methods of preparing rooster semen on its qualitative characteristics, taking into account the method of removing possible contaminants (centrifugation or filtration), and to evaluate the change in the composition of the cytosol of the spermatozoon of the native semen, during equilibration of the diluted semen and during short-term storage. In this study, semen from roosters (n = 22) of the Russian White breed was used. Experiment 1: semen was divided into 3 aliquots: I-was diluted with synthetic cryoprotective medium (1:1 with LCM control, II-was filtered (membrane pore Ø 0.2 µm), and III-was centrifugated (at 3000 rpm for 10 min). Native and frozen/thawed semen was evaluated. Experiment 2: the composition of carbohydrates and polyols of the spermatozoa of native semen was evaluated during equilibration and after storage (3 h). The results of Experiment 1 showed an advantage in the quality of filtered semen compared to centrifuged in terms of progressive motility (41.0% vs. 27.0%) and chromatin integrity (56.6% vs. 33.6%). Results from frozen/thawed samples of filtered semen compared to centrifuged in terms of progressive motility were 25.5% vs. 5.5%, respectively, and in terms of chromatin integrity-83.5% vs. 64.4%, respectively. The results of Experiment 2 showed the main component in the composition of the native spermatozoa cytosol in assessing the content of carbohydrates and polyols was inositol-75.6%. The content of inositol decreased during storage by 6.5 times (from 0.030 mg/mL to 0.007 mg/mL), proposing the role of inositol as the main antioxidant in the cytosol of spermatozoa, which makes it biologically justified to introduce inositol into the composition of synthetic diluents, including cryoprotective ones.

Identification of Key Candidate Genes in Runs of Homozygosity of the Genome of Two Chicken Breeds, Associated with Cold Adaptation.

It is well known that the chicken gene pools have high adaptive abilities, including adaptation to cold environments. This research aimed to study the genomic distribution of runs of homozygosity (ROH) in a population of Russian White (RW) chickens as a result of selection for adaptation to cold environments in the early postnatal period, to perform a structural annotation of the discovered breed-specific regions of the genome (compared to chickens of the Amroks breed) and to suggest key candidate genes associated with the adaptation of RW chickens to cold environments. Genotyping of individual samples was performed using Illumina Chicken 60K SNP BeadChip® chips. The search for homozygous regions by individual chromosomes was carried out using the PLINK 1.9 program and the detectRuns R package. Twelve key genes on breed-specific ROH islands were identified. They may be considered as potential candidate genes associated with the high adaptive ability of chickens in cold environments in the early postnatal period. Genes associated with lipid metabolism (SOCS3, NDUFA4, TXNRD2, IGFBP 1, IGFBP 3), maintaining body temperature in cold environments (ADIPOQ, GCGR, TRPM2), non-shivering thermogenesis (RYR2, CAMK2G, STK25) and muscle development (METTL21C) are perspectives for further research. This study contributes to our understanding of the mechanisms of adaptation to cold environments in chickens and provides a molecular basis for selection work.

A successful protocol for achieving anhydrobiosis of Gallus Gallus Domesticus spermatozoa while maintaining their fertility IN VIVO.

Lyophilization of avian semen is a new method for gene pool preservation. The goal of this study was to develop a protocol for the lyophilization of rooster semen with preserved fertility. Red Rhode Island rooster ejaculates (n = 20) were assessed by volume, motility, and concentration of spermatozoa. They were pooled and diluted 1:1 with a medium LCM-T20 containing trehalose (9.5 mM), exposed at 5 °C for 40 min and centrifuged, and then the supernatant was removed. Media LCM-T with trehalose (1.75 M) was added and exposed for 10 min. The semen was frozen in a thin layer in glass vials. Samples were lyophilized for 2 h at -150 … -50 °C. The water content of the samples after lyophilization was 6.1 ± 0.5% (CV 20%). The sample was rehydrated with a medium LCM-GA5 containing hyaluronic acid (40mg/100 mL media). The total motility of the spermatozoa was 1.0 ± 0.3%. From artificial insemination of virgin hens (n = 12) with rehydrated semen, one fertilized egg was obtained from eight laid eggs. All samples of perivitelline membranes of the obtained eggs had points of interaction with the spermatozoa (7-37 pcs/cm2), which confirmed the presence of viable rehydrated spermatozoa in the genital tract of the hen. To create a dry biobank for poultry, the first protocol for lyophilization of rooster semen was developed to ensure sperm fertility in vivo.

Role of Mono- and Disaccharide Combination in Cryoprotective Medium for Rooster Semen to Ensure Cryoresistance of Spermatozoa.

The combination of saccharides in the composition of a cryopreservation medium may represent a promising method for the preservation of the reproductive cells of male birds. In the current study, cryoprotective media with a combined composition of mono- and di-saccharides were developed. The degree of penetration of reducing saccharide molecules (maltose-Mal20 medium) and non-reducing disaccharide molecules (trehalose-Treh20 medium) from the cryoprotective medium into the cytosol of rooster spermatozoa was studied. LCM control media without disaccharides were used as the control. The number of maltose molecules penetrating from the outside into the cytosol of the spermatozoon was 1.06 × 104, and the number of trehalose molecules was 3.98 × 104. Using a combination of maltose and fructose, the progressive motility of frozen/thawed semen and the fertility rates of eggs were significantly higher ((p < 0.05) 40.2% and 68.5%, respectively) than when using a combination of trehalose and fructose in a cryoprotective diluent (33.4% and 62.4%, respectively). A higher rate of chromatin integrity at the level of 92.4% was obtained when using Treh20 versus 74.5% Mal20 (p < 0.05). Maltose positively affected the preservation of frozen/thawed sperm in the genital tract of hens. On the seventh day from the last insemination when using Mal20, the fertilization of eggs was 42.6% and only 27.3% when using Treh20. Despite the same molecular weight, maltose and trehalose have different physicochemical and biological properties that determine their function and effectiveness as components of cryoprotective media.

Unveiling Comparative Genomic Trajectories of Selection and Key Candidate Genes in Egg-Type Russian White and Meat-Type White Cornish Chickens.

Comparison of genomic footprints in chicken breeds with different selection history is a powerful tool in elucidating genomic regions that have been targeted by recent and more ancient selection. In the present work, we aimed at examining and comparing the trajectories of artificial selection in the genomes of the native egg-type Russian White (RW) and meat-type White Cornish (WC) breeds. Combining three different statistics (top 0.1% SNP by FST value at pairwise breed comparison, hapFLK analysis, and identification of ROH island shared by more than 50% of individuals), we detected 45 genomic regions under putative selection including 11 selective sweep regions, which were detected by at least two different methods. Four of such regions were breed-specific for each of RW breed (on GGA1, GGA5, GGA8, and GGA9) and WC breed (on GGA1, GGA5, GGA8, and GGA28), while three remaining regions on GGA2 (two sweeps) and GGA3 were common for both breeds. Most of identified genomic regions overlapped with known QTLs and/or candidate genes including those for body temperatures, egg productivity, and feed intake in RW chickens and those for growth, meat and carcass traits, and feed efficiency in WC chickens. These findings were concordant with the breed origin and history of their artificial selection. We determined a set of 188 prioritized candidate genes retrieved from the 11 overlapped regions of putative selection and reviewed their functions relative to phenotypic traits of interest in the two breeds. One of the RW-specific sweep regions harbored the known domestication gene, TSHR. Gene ontology and functional annotation analysis provided additional insight into a functional coherence of genes in the sweep regions. We also showed a greater candidate gene richness on microchromosomes relative to macrochromosomes in these genomic areas. Our results on the selection history of RW and WC chickens and their key candidate genes under selection serve as a profound information for further conservation of their genomic diversity and efficient breeding.

Effects of Saccharides Supplementation in the Extender of Cryopreserved Rooster (Gallus domesticus) Semen on the Fertility of Frozen/Thawed Spermatozoa.

The aim of this study was to create balanced media for the cryopreservation of rooster semen in pellets to maintain the functional state of the sperm after thawing. Fructose was replaced by trehalose in experimental media in proportions of 10% (LCM-T10) and 20% (LCM-T20), while LCM was used as a control. After artificial insemination of the hens, the eggs were incubated (n = 400). To determine the functional safety of spermatozoa in the genital tract of hens after 5, 10, and 15 days from the last insemination, we used a method for assessing the interaction of sperm with the perivitelline membrane. Significantly higher rates of egg fertilization (82-86%) were obtained when using LCM-T10 and LCM-T20 compared to control (79%, p < 0.05). Egg fertility on the 5th day from the last insemination with the LCM-T20 diluent reached 100% versus 86% in the control; on the 10th day, the fertility rates were 55% versus 20%, respectively. The best results for fertility duration were obtained by freezing spermatozoa with LCM-T20 medium. The numbers of interaction points of spermatozoa with the perivitelline membrane were as follows: on the 5th day from the last insemination with LCM-T20-461.5 ± 11.5 holes/cm2 (LCM-control-13.7 ± 2.7 holes/cm2), p < 0.01; on the 10th day with LCM-T20-319.3 ± 12.9 holes/cm2 (LCM-control-14.9 ± 3.5 holes/cm2); and on the 15th day with LCM-T20-345.2 ± 11.1 holes/cm2 (LCM-control-0 holes/cm2). In conclusion, the use of trehalose in LCM diluent medium can increase the fertility of frozen/thawed sperm and the duration of their fertility in the genital tract of hens.

Genome-wide association studies targeting the yield of extraembryonic fluid and production traits in Russian White chickens.

BACKGROUND: The Russian White is a gene pool breed, registered in 1953 after crossing White Leghorns with local populations and, for 50 years, selected for cold tolerance and high egg production (EL). The breed has great potential in meeting demands of local food producers, commercial farmers and biotechnology sector of specific pathogen-free (SPF) eggs, the former valuing the breed for its egg weight (EW), EL, age at first egg (AFE), body weight (BW), and the latter for its yield of extraembryonic fluid (YEF) in 12.5-day embryos, ratio of extraembryonic fluid to egg weight, and embryo mass. Moreover, its cold tolerance has been presumably associated with day-old chick down colour (DOCDC) - white rather than yellow, the genetic basis of these traits being however poorly understood. RESULTS: We undertook genome-wide association studies (GWASs) for eight performance traits using single nucleotide polymorphism (SNP) genotyping of 146 birds and an Illumina 60KBeadChip. Several suggestive associations (p < 5.16*10- 5) were found for YEF, AFE, BW and EW. Moreover, on chromosome 2, an association with the white DOCDC was found where there is an linkage disequilibrium block of SNPs including genes that are responsible not for colour, but for immune resistance. CONCLUSIONS: The obtained GWAS data can be used to explore the genetics of immunity and carry out selection for increasing YEF for SPF eggs production.