Benefits of heat-killed Lactobacillus acidophilus on growth performance, nutrient digestibility, antioxidant status, immunity, and cecal microbiota of rabbits.

Introduction: Heat-killed probiotics, as a type of inactivated beneficial microorganisms, possess an extended shelf life and broader adaptability compared to their live counterparts. This study aimed to investigate the impact of heat-killed Lactobacillus acidophilus (L. acidophilus, LA) - a deactivated probiotic on the growth performance, digestibility, antioxidant status, immunity and cecal microbiota of rabbits. Methods: Two hundred weaned Hyla rabbits were randomly allocated into five equal groups (CON, L200, L400, L600, and L800). Over a 28-day period, the rabbits were fed basal diets supplemented with 0, 200, 400, 600, and 800 mg/kg of heat-killed LA, respectively. Results: Results revealed a significant reduction in the feed-to-gain ratio (F/G) in the L600 and L800 groups (p < 0.05). Additionally, the L800 group exhibited significantly higher apparent digestibility of crude fiber (CF) and crude protein (CP) (p < 0.05). Regarding digestive enzyme activities, enhanced trypsin and fibrinase activities were observed in the L600 and L800 groups (p < 0.05). Concerning the regulation of the body's antioxidant status, the L800 group demonstrated elevated levels of superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) in both serum and ileal tissue (p < 0.05). In terms of immune capacity modulation, serum tumor necrosis factor-α (TNF-α) levels were significantly lower in the L600 and L800 groups (p < 0.05), while immunoglobulin A (IgA) and immunoglobulin M (IgM) levels were higher (p < 0.05). Additionally, the L800 group exhibited a substantial increase in secretory immunoglobulin A (SIgA) levels in the intestinal mucosa (p < 0.05). In comparison to the CON group, the L800 group exhibited a significant increase in the relative abundance of Phascolarctobacterium and Alistipes in the cecum (p < 0.05). Phascolarctobacterium demonstrated a positive correlation with SIgA (p < 0.05), IgM (p < 0.01), and Glutathione peroxidase (GSH-Px) (p < 0.05), while displaying a negative correlation with TNF-α levels (p < 0.05). Concurrently, Alistipes exhibited positive correlations with IgA (p < 0.05), IgM (p < 0.05), SIgA (p < 0.01), GSH-Px (p < 0.05), SOD (p < 0.05), and T-AOC (p < 0.01), and a negative correlation with TNF-α (p < 0.05). Discussion: In conclusion, the dietary incorporation of 600 mg/kg and 800 mg/kg of heat-killed LA positively influenced the growth performance, nutrient digestibility, antioxidant status, immune capacity and cecal microbiota of rabbits. This highlights the potential benefits of utilizing heat-killed probiotics in animal nutrition.

Curcumin alleviates cecal oxidative injury in diquat-induced broilers by regulating the Nrf2/ARE pathway and microflora.

This study evaluated the alleviative effect of curcumin (CUR) on the diquat (DQ)-induced cecal injury in broilers. A total of 320 one-day-old Cobb broilers were selected and randomly divided into 4 treatments, namely control, DQ, CUR 100, and CUR150 groups. The control and DQ groups were fed a basal diet, while the CUR 100 and CUR150 groups were fed the basal diet supplemented with 100 and 150 mg/kg CUR, respectively. Each group had 8 replicates, with 10 broilers per replicate. On day 21 of the experiment, 1 broiler was selected from each replicate and intraperitoneally injected 20 mg/kg body weight of DQ for DQ, CUR 100, and CUR 150 groups. Broilers in control group received equivalent volume of saline. Broilers were euthanized 48h postinjection for tissue sampling. The results showed that DQ injection could cause oxidative stress and inflammatory reactions in the cecum, affecting the fatty acid production and flora structure, thus leading to cecum damage. Compared with the DQ group, the activity of superoxide dismutase, the level of interleukin 10, acetic acid, and total volatile fatty, and the abundance of nuclear factor E2-related factor 2, copper and zinc superoxide dismutase and catalase mRNA in the cecal mucosa of broilers in the CUR group increased significantly (P < 0.05). However, the levels of malondialdehyd, reactive oxygen species, tumor necrosis factor-alpha, and the expression of cysteine-aspartic acid protease-3 and tumor necrosis factor-alpha decreased significantly (P < 0.05) in the CUR group. In addition, CUR treatment alleviated the damage to the cecum and restored the flora structure, and Lactobacillus and Lactobacillaceae promoted the alleviative effect of CUR on DQ. In summary, CUR could alleviate the cecal injury caused by DQ-induced oxidative damage and inflammatory reactions by regulating the Nrf2-ARE signaling pathway and intestinal flora, thus protecting the cecum.

Quercetagetin alleviates zearalenone-induced liver injury in rabbits through Keap1/Nrf2/ARE signaling pathway.

Introduction: This study aimed to assess the alleviative effect of quercetagetin (QG) on zearalenone (ZEN)-induced liver injury in rabbits. Methods: Ninety 41-day-old healthy Hyla rabbits were randomly assigned into three groups, including a control (fed with basic diet), ZEN addition group (fed with basic diet + 600 µg/kg ZEN), and ZEN + QG addition group (fed with basic diet + 600 µg/kg ZEN + 100 mg/kg QG), with 30 rabbits per group. The duration of the experiment was 28 days. Results: The results revealed no significant differences in the average daily gain, average daily feed intake, the gain to feed ratio and the liver, kidney and spleen organ indexes (p > 0.05) between the rabbits across the three groups. However, the sacculus rotundus index of the rabbits in the control group was significantly higher than that in the ZEN + QG group (p < 0.05). The intake of ZEN-contaminated diet also significantly increased the activities or levels of alanine transaminase, alkaline phosphatase, total bile acid (TBA), total bilirubin, malondialdehyde, and interleukin-4 (IL-4) and enhanced the abundance of kelch-like ECH-associated protein 1 (Keap1), heat shock protein 70 (HSP70) and cysteine-aspartic acid protease-3 (Caspase-3) mRNA in the blood or liver tissue in ZEN group, compared to the control group (p < 0.05). On the contrary, the activities or levels of immunoglobulin A, complement 3, total antioxidant capacity, glutathione peroxidase (GSH-Px), superoxide dismutase, interleukin-10, and the abundance of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) mRNA were significantly decreased (p < 0.05). Supplementing the diet with QG still maintained significantly higher levels of TBA and IL-4, and the abundance of GSH-Px, HSP70, IL-4, and Caspase-3 mRNA in the blood and liver of rabbits in the ZEN + QG group than in the control group (p < 0.05). At the same time, the other indicators were restored to levels in the control group (p > 0.05). Discussion: In conclusion, QG alleviated the ZEN-induced oxidative damage and liver injury caused by inflammatory reaction through the Keap1-Nrf2-antioxidant response element (ARE) signal pathway, which protected the liver. This study revealed the alleviative effect of QG on the hepatotoxicity of ZEN in rabbits for the first time, providing a new perspective for applying QG and developing a ZEN antidote.

Effects of zearalenone on liver development, antioxidant capacity and inflammatory factors of prepubertal gilts.

Zearalenone (ZEA) is a kind of mycotoxin that pose great threat to the liver of human and livestock due to its toxicity to eukaryotic cells, however, its toxicity mechanism on prepubertal gilts liver development and function is not known. The study aimed to examine the effects of ZEA on liver development, antioxidant capacity and inflammatory factors of prepubertal gilts. Forty-eight prepubertal gilts (Landrace ×Yorkshire) were randomly divided into four groups: three treatment (T1, T2 and T3) groups and a control group. Prepubertal gilts in the control group were fed with basal diet, and those in T1, T2 and T3 groups were fed with basal diets supplemented with low, medium and high doses (200 µg/kg, 800 µg/kg and 1,600 µg/kg, respectively) of ZEA during the experiment period. The results showed that diets supplemented with ZEA significantly increased the activity of alanine aminotransferase of serum in the T3 group (p < 0.05). Besides, compared to the control group, the activities of total antioxidant capacity, superoxide dismutase, the content of tumour necrosis factor-alpha of liver in the T3 group and the relative expression level of manganese-superoxide dismutase mRNA of liver in the T2 group were significantly reduced (p < 0.05). We also performed correlation analysis among caecal microorganisms and antioxidant enzyme activities and inflammatory factor concentrations of liver. In conclusion, diets supplemented with ZEA has no obvious effect on liver development, but it can cause liver damage.

Effects of ammonia on hypothalamic-pituitary-ovarian axis in female rabbits.

BACKGROUND: As one of the most harmful gases in the livestock house, ammonia is recognized as an environmental stressor by Environmental Protection Agency (United States). The study aimed to explore the effect of ammonia on hypothalamic-pituitary-ovarian (HPO) axis of rabbits. A total of ninety two-month-old female IRA rabbits were randomly divided into three groups, and were kept in animal environment control rooms for four weeks at college of animal science and technology, Hebei Agricultural University (Baoding, China). The rabbits in the control group were kept under ammonia concentration of < 3 ppm. The two treatment groups were kept under ammonia concentration of 30 ppm and 50 ppm. Hypothalamus, pituitary, and ovary were collected for hematoxylin and eosin (HE) staining, immunohistochemistry, terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Serum was collected for enzyme-linked immunosorbent assay (ELISA). RESULTS: Histopathological examination revealed that exposed to excess ammonia damaged the morphology and structure of hypothalamus, pituitary, and ovary. TUNEL assay revealed that apoptosis rate increased in hypothalamus, pituitary, and ovary. The protein expression levels of Bcl-2associated X protein (Bax) and Caspase-9 increased, while B-cell lymphoma-2 (Bcl-2) decreased, resulting in apoptosis. Moreover, the concentration of gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and progesterone (PROG) reduced in plasma. The mRNA expression of FSH and LH in pituitary and follicle-stimulating hormone receptor (FSHR), E2, PROG in ovary as well as decreased, indicated hormone secretion disorder. CONCLUSIONS: The results indicated that ammonia exposure damaged hypothalamus, pituitary, and ovary, caused hormone secretion disorder and apoptosis.

Differential expression of COL4A3 and collagen in upward and downward progressing types of nasopharyngeal carcinoma.

Upward (local growth and invasion of the base of skull), downward (distant metastasis) and mixed progressing types of nasopharyngeal carcinoma (NPC) have been identified and are distinctly different with respect to clinical symptoms, therapeutic strategies and prognosis. The present study aimed to identify the genetic difference and collagen expression levels in the upward and downward progressing types of NPC. Whole exon sequencing (WES) was used to detect genes differentially mutated between the upward and downward progressing types of NPC. Collagen deposition in the upward and downward progressing types of NPC was determined using Masson trichromatic staining, while the protein expression level of COL4A3 was detected using immunohistochemistry. Survival analysis was also performed using the Kaplan-Meier Plotter database to examine the role of COL4A3 expression level in the prognosis of head and neck squamous cell carcinoma. Knockdown of COL4A3 was performed using short interfering (si)RNA-COL4A3 in a 5-8F NPC cell line. Reverse transcription-quantitative PCR and western blot analyses were utilized to analyze the mRNA and protein expression levels of COL4A3, respectively. The roles of COL4A3 in the migration and invasion of the 5-8F cell line were examined using wound-healing Transwell and Matrigel assays, respectively. A total of 21 genes were differentially mutated between the upward and downward progressing types of NPC. The COL4A3 was investigated further, as it was found to be associated with extracellular matrix deposition and cancer metastasis. The COL4A3 gene was markedly downregulated in the downward progressing type compared with that in the upward progressing type (2.161±1.306 vs. 5.077±3.619; P<0.05). In addition, the deposition of collagen in the downward progressing type was also significantly decreased compared with that in the upward progressing type (5.63±6.83 vs. 10.94±9.60; P<0.05). Kaplan-Meier analysis indicated that high expression level of COL4A3 was positively associated with a favorable prognosis of head and neck squamous cell carcinoma (HR, 0.69; 95% CI, 0.49- 0.97; P=0.031). To confirm the role of COL4A3, the expression level of COL4A3 was knocked down using siRNA in the 5-8F cell line and the results showed that the invasion and migration was significantly increased when the expression of COL4A3 was inhibited (P<0.0001). In conclusion, the gene mutation patterns were significantly different between the upward and downward progressing types of NPC. In addition, the expression level of the COL4A3 gene was decreased in the downward progressing type, which might promote NPC metastasis through the downregulation of extracellular collagen expression.

Effects of zearalenone on genital organ development, serum immunoglobulin, antioxidant capacity, sex hormones and liver function of prepubertal gilts.

The study aimed to examine the effects of zearalenone on genital organ development, serum immunoglobulin, antioxidant capacity, sex hormones and liver function of prepubertal gilts. Forty-eight prepubertal gilts (Landrace × Yorkshire) were randomly divided into three treatment (T1, T2 and T3) groups and a control group (12 replicates per group, 1 gilt per replicate). Prepubertal gilts in the control group were fed with basal diet, and those in T1, T2 and T3 groups were fed with basal diets supplemented with 200 µg/kg, 800 µg/kg and 1600 µg/kg zearalenone during the experiment period, which lasted for 14 d. Feed intake was counted and vulvar area was measured. The blood samples were collected from the anterior vena cava of 6 prepubertal gilts in each group, and immunoglobulins, antioxidant indexes, inflammatory cytokines, genital hormones, and biochemical indexes were analyzed by enzyme-linked immunosorbent assay. The results showed that the average daily feed intake of prepubertal gilts in each group had no significant change (p > 0.05). On 14 d, compared with the control group, the vulva area of prepubertal gilts in each treatment group was significantly increased (p < 0.05). Compared with the control group, the serum immunoglobulin G content in the T3 group was significantly reduced (p < 0.05). The activities of total antioxidant capacity and the superoxide dismutase of serum in the T3 group were significantly reduced (p < 0.05). Compared with the control group, the serum interleukin-4 content in each test group were extremely significantly increased (p < 0.01). The serum contents of luteinizing hormone in the T2 and T3 groups and estradiol in the T3 group were significantly reduced (p < 0.05) than that of control group. Compared with the control group, the activity of aspartate aminotransferase in T3 group was significantly increased (p < 0.05). In conclusion, zearalenone has no significantly effect on the feed intake of prepubertal gilts, but it can reduce its serum immunoglobulin contents and antioxidant properties, disrupt the secretion of sex hormones, increase the vulva area, produce reproductive toxicity and cause liver damage. Therefore, in pig production, the use of antimould reagent together with products of immunity-boosting, antioxidant, anti-inflammatory and hepatoprotective may enhance protection.

Development of a monoclonal antibody against canine parvovirus NS1 protein and investigation of NS1 dynamics and localization in CPV-infected cells.

Canine parvovirus (CPV) non-structural protein-1 (NS1) plays crucial roles in CPV replication and transcription, as well as pathogenic effects to the host. However, the mechanism was not fully understood. Lack of NS1 antibody is one of the restricting factors for NS1 function investigation. To prepare NS1 monoclonal antibody (mAb), the NS1 epitope (AA461 ~ AA650) gene was amplified by PCR, and inserted into pGEX-4T-1vector to construct the prokaryotic expression vector of GST-tag-fused NS1 epitope gene. The NS1 fusion protein was expressed in E. coli, and purified with GSH-magnetic beads, and then used to immunize BALB/c mice. The mouse splenic lymphocytes were isolated and fused with myeloma cells (SP 2/0) to generate hybridoma cells. After several rounds of screening by ELISA, a hybridoma cell clone (1B8) stably expressing NS1 mAb was developed. A large amount of NS1 mAb was prepared from mouse ascites fluid. The isotype of NS1 mAb was identified as IgG1, which can specifically bind NS1 protein in either CPV-infected cells or NS1 vector-transfected cells, indicating the NS1 mAb is effective in detecting NS1 protein. Meanwhile, we used the NS1 mAb to investigate NS1 dynamic changes by qRT-PCR and location by confocal imaging in CPV-infected host cells and showed that NS1 began to appear in the cells at 12 h after CPV infection and reached the highest level at 42 h, NS1 protein was mainly located in nucleus of the cells. This study provided a necessary condition for further investigation on molecular mechanism of NS1 function and pathogenicity.

Co-Expression of Chicken IL-2 and IL-7 Enhances the Immunogenicity and Protective Efficacy of a VP2-Expressing DNA Vaccine against IBDV in Chickens.

Chicken infectious bursal disease (IBD) is still incompletely controlled worldwide. Although IBD virus (IBDV) VP2 DNA vaccine was considered a safe vaccine for IBD prevention, the immunogenicity by itself remains poor, resulting in the failure of effectively protecting chickens from infection. We and others demonstrated that chicken IL-2 (chIL-2) and chIL-7 have the capacity to enhance the immunogenicity of the VP2 DNA vaccine. However, whether chIL-2 and chIL-7 can mutually enhance the immunogenicity of VP2 DNA vaccine and thereby augment the latter's protection efficacy remains unknown. By using chIL-2/chIL-7 bicistronic gene vector to co-immunize the chickens together with the VP2 DNA vaccine, we now show that chIL-2 and chIL-7 significantly increased IBDV VP2-specific antibody titers, T cell proliferation, and IFN-γ production, resulting in the ultimate enhancement of vaccine-induced protection efficacy relative to that of chIL-2 or chIL-7 gene vectors alone. These results suggest that chIL-2 and chIL-7 can mutually enhance VP2 DNA vaccine's efficacy, thereby establishing a concrete foundation for future optimization of IBDV VP2 DNA vaccine to prevent/treat chicken IBD.

Porcine CD83 is a glycosylated dimeric protein existing naturally in membrane-bound and soluble forms.

Human and mouse CD83 have been well characteized, however, the other mammalian CD83 genes have not been cloned and characterized. In this study, the porcine CD83 (pCD83) was cloned, expressed and characterized, and showed that the pCD83 gene has 81% and 74% homologies with humans and mice, respectively, which was identified to be glycosylated when expressed in eukaryotic cells, existing naturally in two forms: membrance-bound CD83 (mCD83) and soluble CD83 (sCD83), the latter was identified to be generated mainly from mCD83 by proteolytic shedding. The pCD83 was a dimmer mediated by intermolecular disulfide bond formed by the fifth cysteine in the exrtracellular domain. Functionally, the recombinant porcine sCD83 was preliminarily tested to have the ability to inhibit DC-mediated T cell activition. This study provided necessary fundation for further investigation on pCD83 functions.