Cryodamage affects the normal physiological functions and survivability of boar sperm during cryopreservation. Lysine acetylation is thought to be an important regulatory mechanism in sperm functions. However, little is known about protein acetylation and its effects on cryotolerance or cryodamage in boar sperm. In this study, the characterization and protein acetylation dynamics of boar sperm during cryopreservation were determined using liquid chromatography–mass spectrometry (LC-MS). A total of 1440 proteins were identified out of 4705 modified proteins, and 2764 quantifiable sites were elucidated. Among the differentially modified sites, 1252 were found to be upregulated compared to 172 downregulated sites in fresh and frozen sperms. Gene ontology indicated that these differentially modified proteins are involved in metabolic processes and catalytic and antioxidant activities, which are involved in pyruvate metabolism, phosphorylation and lysine degradation. In addition, the present study demonstrated that the mRNA and protein expressions of SIRT5, IDH2, MDH2 and LDHC, associated with sperm quality parameters, are downregulated after cryopreservation. In conclusion, cryopreservation induces the acetylation and deacetylation of energy metabolism-related proteins, which may contribute to the post-thawed boar sperm quality parameters.

Spexin2 (SPX2), a paralog of SPX1, is a newly identified gene in non-mammalian vertebrates. Limited studies in fish have evidenced its important role in food intake and energy balance modulation. However, little is known about its biological functions in birds. Using the chicken (c-) as a model, we cloned the full-length cDNA of SPX2 by using RACE-PCR. It is 1189 base pair (bp) in length and predicted to generate a protein of 75 amino acids that contains a 14 amino acids mature peptide. Tissue distribution analysis showed that cSPX2 transcripts were detected in a wide array of tissues, with abundant expression in the pituitary, testis, and adrenal gland. cSPX2 was also observed to be ubiquitously expressed in chicken brain regions, with the highest expression in the hypothalamus. Its expression was significantly upregulated in the hypothalamus after 24 or 36 h of food deprivation, and the feeding behavior of chicks was obviously suppressed after peripheral injection with cSPX2. Mechanistically, further studies evidenced that cSPX2 acts as a satiety factor via upregulating cocaine and amphetamine regulated transcript (CART) and downregulating agouti-related neuropeptide (AGRP) in hypothalamus. Using a pGL4-SRE-luciferase reporter system, cSPX2 was demonstrated to effectively activate a chicken galanin II type receptor (cGALR2), a cGALR2-like receptor (cGALR2L), and a galanin III type receptor (cGALR3), with the highest binding affinity for cGALR2L. Collectively, we firstly identified that cSPX2 serves as a novel appetite monitor in chicken. Our findings will help clarify the physiological functions of SPX2 in birds as well as its functional evolution in vertebrates.

Artificial insemination (AI) with liquid-stored semen is the most prevalent and efficient assisted reproduction technique in the modern pork industry. Pyruvate dehydrogenase complex component X (PDHX) was demonstrated to be associated with sperm metabolism and affected the boar sperm viability, motility, and fertility. Pyruvate Dehydrogenase Kinases (PDKs) are the key metabolic enzymes that regulate pyruvate dehydrogenase complex (PDHC) activity and also the conversion from glycolysis to oxidative phosphorylation. In the present study, two PDK inhibitors, Dichloroacetate (DCA) and Phenylbutyrate (4-PBA), were added to an extender and investigated to determine their regulatory roles in liquid-stored boar sperm at 17 °C. The results indicated that PDK1 and PDK3 were predominantly located at the head and flagella of the boar sperm. The addition of 2 mM DCA and 0.5 mM 4-PBA significantly enhanced the sperm motility, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), and ATP content. In addition, DCA and 4-PBA exerted their effects by inhibiting PDK1 and PDK3, respectively. In conclusion, DCA and 4-PBA were found to regulate the boar sperm metabolic activities via PDK1 and PDK3. These both can improve the quality parameters of liquid-stored boar sperm, which will help to improve and optimize liquid-stored boar semen after their addition in the extender.

Ducks are economically important in global poultry production and serve as valuable models for avian biology. However, the absence of a sex-specific, multi-tissue reference of duck gene expression has limited investigations into transcriptional regulation and complex traits. Here, we constructed a transcriptome atlas covering 50 tissues from six adult Nonghua Ma ducks (three males and three females). Poly(A) RNA-seq reads were mapped to the ZJU1.0 reference genome to quantify gene expression and construct co-expression networks, and expression patterns were summarized by clustering analyses. In total, 23,512 genes were detected, with tissues clustering according to organ systems. The network yielded 12,835 clusters, of which the 50 largest encompassed 61.5 % of all genes and represented major biological processes such as neuronal signaling, immune regulation, reproduction, and metabolism. A set of 306 hub genes captured key regulators of tissue functions. Furthermore, we identified a refined panel of 13 highly stable housekeeping genes (e.g., EEF2, RPL6, RPL24) that showed superior cross-tissue stability compared with commonly used references. The atlas also revealed organ-specific sex-biased expression and incomplete Z-linked dosage compensation in females, particularly evident in digestive and musculoskeletal tissues. This sex-resolved 50-tissue transcriptome atlas of the duck provides tissue-resolved expression profiles, functional modules, and validated normalization genes to support functional genomics, biomarker discovery, molecular breeding, and comparative avian studies.

This study aimed to investigate the genetic diversity and population structure of Liangshan Yanying chickens and to explore the genetic basis of dorsal plumage color changes from day-old to 12 weeks of age, thereby supporting conservation and breeding strategies. A total of 11,859,518 SNPs were obtained after quality control. The average observed heterozygosity (Ho = 0.280) was lower than the expected heterozygosity (He = 0.312). The minor allele frequency (MAF) ranged predominantly between 0.10 and 0.20 (27.3 %). The LD decay analysis showed a maximum R² of 0.27. Identity-by-state (IBS) distances suggested low kinship among individuals. A total of 1,726 ROH segments were identified, resulting in a low average inbreeding coefficient (FROH) of 0.0125, indicating moderate genetic diversity accompanied by a mild deficit of heterozygosity and suggesting the presence of mild inbreeding or past selection. GWAS revealed that candidate genes on chromosome 11 (DEF8, TCF25, TUBB3, DBNDD1, and MC1R) were associated with light/dark mottled plumage, while genes on chromosome 1 (GRM5, TYR, LOC121107450, and CTSC) were linked to the presence/absence of coloration. Overall, the Liangshan Yanying chicken population shows evidence of effective conservation. The variation in feather color is primarily associated with the MC1R, TYR, and other related regulatory genes, offering valuable genetic insights for the maintenance of color patterns and for the development of molecular-assisted breeding strategies.