Post-thawed sperm quality parameters vary across different species after cryopreservation. To date, the molecular mechanism of sperm cryoinjury, freeze-tolerance and other influential factors are largely unknown. In this study, significantly dysregulated microRNAs (miRNAs) and mRNAs in boar and giant panda sperm with different cryo-resistance capacity were evaluated. From the result of miRNA profile of fresh and frozen-thawed giant panda sperm, a total of 899 mature, novel miRNAs were identified, and 284 miRNAs were found to be significantly dysregulated (195 up-regulated and 89 down-regulated). Combined analysis of miRNA profiling of giant panda sperm and our previously published data on boar sperm, 46, 21 and 4 differentially expressed (DE) mRNAs in boar sperm were believed to be related to apoptosis, glycolysis and oxidative phosphorylation, respectively. Meanwhile, 87, 17 and 7 DE mRNAs in giant panda were associated with apoptosis, glycolysis and oxidative phosphorylation, respectively. Gene ontology (GO) analysis of the targets of DE miRNAs showed that they were mainly distributed on membrane related pathway in giant panda sperm, while cell components and cell processes were tied to the targets of DE miRNAs in boar sperm. Finally, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DE mRNAs indicated that most of these DE mRNAs were distributed in membrane signal transduction-related pathways in giant panda sperm, while those in boar sperm were mainly distributed in the cytokine-cytokine receptor interaction pathway and inflammatory related pathways. In conclusion, although the different freezing extenders and programs were used, the DE miRNAs and mRNAs involved in apoptosis, energy metabolism, olfactory transduction pathway, inflammatory response and cytokine-cytokine interactions, could be the possible molecular mechanism of sperm cryoinjury and freeze tolerance.

MiR-26a is associated with sperm metabolism and can affect sperm motility and apoptosis. However, how miR-26a affects sperm motility remains largely unknown. Our previous study indicated that the PDHX gene is predicted to be a potential target of miR-26a, which is responsible for pyruvate oxidative decarboxylation which is considered as a key step for connecting glycolysis with oxidative phosphorylation. In this study, we first reported a potential relationship between miR-26a and PDHX and their expressions in fresh, frozen-thawed, and epididymal boar sperm. Then, sperm viability and survival were determined after transfection of miR-26a. mRNA and protein expression level of PDHX in the liquid-preserved boar sperm after transfection were also determined by RT-qPCR and Western Blot (WB). Our results showed that expression level of PDHX was significantly increased during sperm transit from epididymal caput to corpus and cauda. Similarly, expression of PDHX was significantly higher (P < 0.05) in fresh sperm as compared to epididymal cauda and frozen-thawed sperm. However, the expression of miR-26a in epididymal corpus sperm was significantly higher (P < 0.05) than that of caput and cauda sperm. Furthermore, after transfection of boar sperm with miR-26a mimic and inhibitor under liquid storage, the lowest and highest sperm viability was observed in miR-26a mimic and inhibitor treatment (P < 0.05), respectively. The protein levels of PDHX, after 24 and 48 h of transfection of miR-26a mimics and inhibitor, were notably decreased and increased (P < 0.05), respectively, as compared to negative control (NC) group. In conclusion, the novel and enticing findings of our study provide a reasonable evidence that miR-26a via PDHX, a link between glycolysis and oxidative phosphorylation, could regulate the glycometabolic pathway which eventually affect boar sperm viability and survival.

The health of pets is becoming a growing concern for the pet industry and its owners. Immunity is one of the foundational supports for health, thus developing a functional bioactive substance that can boost pets’ immunity is essential. Many studies have shown that casein phosphopeptide (CPP) and selenium (Se) can individually regulate immunity in many species, but there has been no reported research on the immunomodulatory function of casein phosphopeptide–selenium complex (CPP-Se). The objective of this study was to investigate the function of CPP-Se on immunomodulation in dogs. Twenty Beagle dogs were equally divided into two groups and fed either a control snack or a test snack supplemented with 0.03% CPP-Se for 30 days. Anticoagulated blood, serum and peripheral blood lymphocytes (PBL) were collected from dogs at 0 d, 10 d, 20 d and 30 d to detect the change in the number of immune cells and the expression of cytokine-related mRNAs and proteins. PBL isolated from blood were exposed to CPP-Se in vitro to measure the proliferative responses and cytokine-related mRNAs expression. During the time the test snack was fed, the number of lymphocytes increased significantly, whereas neutrophils and monocytes remained unaltered. The expression of interleukin-4 (IL-4), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), CD4 molecule (CD4) and CD8α molecule (CD8α) was up-regulated, while interleukin-1β (IL-1β) was down-regulated, and interleukin-10 (IL-10) declined initially and subsequently increased. ELISA detection revealed a significant increment in serum IL-4, IL-6, Immunoglobulin M (IgM) and IFN-γ, except for IgG. Furthermore, CPP-Se treatment increased the proliferation and the expression of cytokine-related mRNAs in PBL cultured in vitro. This is the first study to demonstrate that CPP-Se can improve immunity in the dog.

A novel pectic polysaccharide (HPP-1) with high immunomodulatory activity was extracted and isolated from the immature honey pomelo fruit (Citrus grandis). Characterization of its chemical structure indicated that HPP-1 had a molecular weight of 59,024 D. In addition, HPP-1 was primarily composed of rhamnose, arabinose, fucose, mannose, and galactose at a molar ratio of 1.00:11.12:2.26:0.56:6.40. Fourier-transform infrared spectroscopy, periodic acid oxidation, and Smith degradation results showed that HPP-1 had α- and β-glycosidic linkages and 1 → 2, 1 → 4, 1 → 6, and 1 → 3 glycosidic bonds. ¹³C NMR and ¹H NMR analyses revealed that the main glycogroups included 1,4-D-GalA, 1,6-β-D-Gal, 1,6-β-D-Man, 1,3-α-L-Ara, and 1,2-α-L-Rha. Immunomodulatory bioactivity analysis using a macrophage RAW264.7 model in vitro revealed that NO, TNF-α, and IL-6 secretions were all considerably increased by HPP-1. Moreover, RT-PCR results showed that HPP-1-induced iNOS, TNF-α, and IL-6 expression was significantly increased in macrophages. HPP-1-mediated activation in macrophages was due to the stimulation of the NF-κB and MAPK signaling pathways based on western blot analyses. HPP-1 extracted from immature honey pomelo fruit has potential applications as an immunomodulatory supplement.

Krill oil (KO) is a bioactive substance with antioxidant and immunomodulatory functions. This study investigated the impact of administering snacks supplemented with 0.5% KO for 8 weeks on the blood parameters, hair quality, and fecal microbiota in dogs. KO was determined to elevate the activities of serum superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and immunoglobulin G (IgG) level. Concurrently, KO markedly diminished serum tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-8 (IL-8), and malondialdehyde (MDA) levels in dogs. The KO group displayed a considerably higher ratio of ideal scores and thinner hair scales for newborn hair, as well as a significant increase in total amino acid and methionine (Met) content in comparison to the control group. Furthermore, 16S rRNA sequencing revealed the changes in the composition of fecal microbiota after KO feeding. KO significantly affected the α and β diversity of canine fecal microbiota. Moreover, KO augmented the relative abundance of Bacteroidota, Actinobacteriota, and Proteobacteria at the phylum level, and it increased the relative abundance of Allobaculum, Bifidobacterium, Prevotella_9, Collinsella, and Turicibacter at the genus level. In summary, this study provides valuable insights to further understand the role of KO in promoting canine health.

Casein phosphopeptide-selenium chelate (CPP-Se) is an organic compound produced by the chelation of casein phosphopeptide with selenium. This compound showed the ability to modulate canine immune response in our previous study; but its effect on the peripheral blood transcriptome and serum metabolome was unknown. This study aims to reveal the potential mechanism behind the immunomodulatory function of CPP-Se. We have identified 341 differentially expressed genes (DEGs) in CPP-Se groups as compared to the control group which comprised 110 up-regulated and 231 down-regulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis found that DEGs were mainly involved in immune-related signaling pathways. Moreover, the immune-related DEGs and hub genes were identified. Similarly, metabolomics identified 53 differentially expressed metabolites (DEMs) in the CPP-Se group, of which 17 were up-regulated and 36 were down-regulated. The pathways mainly enriched by DEMs were primary bile acid biosynthesis, tryptophan metabolism, and other amino acids metabolic pathways. Combined analysis of transcriptomic and metabolomic data showed that the DEGs and DEMs were commonly enriched in fatty acid biosynthesis, pyrimidine metabolism, glutathione metabolism, and glycerolipid metabolic pathways. Taken together, our findings provided a theoretical basis for further understanding of the immunomodulatory function of CPP-Se as well as a scientific reference for the future use of CPP-Se in pet foods as a dietary supplement to modulate the immunity.