• Animal Bioscience
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• v.36 no.12
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• pp.1831-1841
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• 2023

Objective: This study was realized to evaluate the effects of supplementation with blends of water and fat-soluble vitamins on animal performance and carcass traits of young Nellore bulls. Methods: Forty-three Nellore bulls, with an initial weight of 261±27.3 kg and a mean age of 8±1.0 months, were used. Five animals were slaughtered at the beginning of the experiment (reference group), to determine the initial empty body weight of the bulls that remained in the experiment. The remaining 38 bulls were fed ad libitum and distributed in a completely randomized design in a 2×2 factorial scheme, with or without supplementation of water-soluble (B-blend+ or B-blend-) and fat-soluble (ADE+ or ADE-) vitamin blends. Diets were isonitrogenous (120 g of crude protein/kg dry matter [DM] of total mixed ration) and consisted of a roughage:concentrate rate of 30:70 based on total DM of diet. The experiment lasted 170 days, with 30 days of adaptation and 140 days for data collection. At the beginning and end of the experimental period, the bulls were weighed to determine the average daily gain. To estimate the apparent digestibility of nutrients and microbial efficiency, spot collections of feces and urine were performed for five consecutive days. Results: DM, ashes, organic matter, crude protein, ethereal extract, neutral detergent fiber corrected for residual ash and residual nitrogenous, and N intake and apparent digestibility were not influenced by vitamin supplementation, but total digestible nutrients intake and non-fibrous carbohydrates digestibility were influenced by B complex vitamin supplementation. Nitrogen balance, microbial efficiency, and performance data were not influenced (p>0.05) by vitamin supplementation. Conclusion: Vitamin supplementation (a blend of water-soluble and fat-soluble vitamins or their combinations) does not influence the animal performance and carcass traits of young Nellore bulls.

• Animal Bioscience
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• v.36 no.12
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• pp.1775-1784
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• 2023

Objective: The aim of this study was to reveal the role and regulatory mechanism of miR-188-5p in the proliferation and differentiation of goat muscle satellite cells. Methods: Goat skeletal muscle satellite cells isolated in the pre-laboratory were used as the test material. First, the expression of miR-188-5p in goat muscle tissues at different developmental stages was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). In addition, miR-188-5p was transfected into goat skeletal muscle satellite cells by constructing mimics and inhibitors of miR-188-5p, respectively. The changes of differentiation marker gene expression were detected by qPCR method. Results: It was highly expressed in adult goat latissimus dorsi and leg muscles, goat fetal skeletal muscle, and at the differentiation stage of muscle satellite cells. Overexpression and interference of miR-188-5p showed that miR-188-5p inhibited the proliferation and promoted the differentiation of goat muscle satellite cells. Target gene prediction and dual luciferase assays showed that miR-188-5p could target the 3'untranslated region of the calcium/calmodulin dependent protein kinase II beta (CAMK2B) gene and inhibit luciferase activity. Further functional studies revealed that CAMK2B promoted the proliferation and inhibited the differentiation of goat muscle satellite cells, whereas si-CAMK2B restored the function of miR-188-5p inhibitor. Conclusion: These results suggest that miR-188-5p inhibits the proliferation and promotes the differentiation of goat muscle satellite cells by targeting CAMK2B. This study will provide a theoretical reference for future studies on the molecular mechanisms of skeletal muscle development in goats.

• Animal Bioscience
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• v.36 no.12
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• pp.1853-1859
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• 2023

Objective: This study aimed to explore the effects of different types of xanthophyll extracted from marigold on the growth performance, skin color, and carcass pigmentation. Methods: A total of 192 healthy 60-day-old yellow-feathered broilers weighing an average of 1,279±81 g were randomly allocated to 4 groups, each with 6 replicates and 8 broilers. The 4 treatments were as follows: i) CON group, fed with basal diet; ii) LTN group, supplemented with lutein; iii) MDP group, supplemented with monohydroxyl pigment including dehydrated lutein, β-cryptoxanthin, and α-cryptoxanthin; iv) LTN+MDP group, supplemented with lutein and monohydroxyl pigment in proportion to 1:1. The supplementary content of LTN, MDP, and LTN+MDP was 2 g/kg. Skin color was measured after 7, 14, 21, and 28 days of feeding the dietary treatments. The breast, thigh, and abdominal fat of slaughtered chickens were stored in cold storage at 4℃ for 24 hours and then the meat color of lightness (L^*), redness (a^*), and yellowness (b^*) values was determined. Results: The results showed that all treatments enhanced the yellow scores of subwing skin on day 14, 21, and 28 (p<0.05), and the mixture of lutein and monohydroxyl pigment promoted the yellow scores of shanks on day 14, 21, and 28 (p<0.05). The mixture of lutein and monohydroxyl pigment increased the yellow scores of beaks and all treatments enhanced the yellow of shanks on day 28 (p<0.05). In addition, all treatments improved the yellow (b^*) values of breast and thigh muscle, moreover, the monohydroxyl pigment and the mixture of lutein and monohydroxyl pigment enhanced the values of redness (a^*) and yellow (b^*) of abdominal fat (p<0.05). Conclusion: In summary, different types of xanthophyll extracted from marigold significantly increased the yellow scores of skin color and the yellow (b^*) values of carcass pigmentation. Especially, the mixture of lutein and monohydroxyl pigment was more efficient on skin color.

• Animal Bioscience
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• v.37 no.3
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• pp.471-480
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• 2024

Objective: The objective of this study was to investigate the regulation relationship of Ten-eleven translocation 1 (Tet1) in DNA demethylation and the proliferation of primordial germ cells (PGCs) in chickens. Methods: siRNA targeting Tet1 was used to transiently knockdown the expression of Tet1 in chicken PGCs, and the genomic DNA methylation status was measured. The proliferation of chicken PGCs was detected by flow cytometry analysis and cell counting kit-8 assay when activation or inhibition of Wnt4/β-catenin signaling pathway. And the level of DNA methylation and hisotne methylation was also tested. Results: Results revealed that knockdown of Tet1 inhibited the proliferation of chicken PGCs and downregulated the mRNA expression of Cyclin D1 and cyclin-dependent kinase 6 (CDK6), as well as pluripotency-associated genes (Nanog, PouV, and Sox2). Flow cytometry analysis confirmed that the population of PGCs in Tet1 knockdown group displayed a significant decrease in the proportion of S and G2 phase cells, which meant that there were less PGCs entered the mitosis process than that of control. Furthermore, Tet1 knockdown delayed the entrance to G1/S phase and this inhibition was rescued by treated with BIO. Consistent with these findings, Wnt/β-catenin signaling was inactivated in Tet1 knockdown PGCs, leading to aberrant proliferation. Further analysis showed that the methylation of the whole genome increased significantly after Tet1 downregulation, while hydroxyl-methylation obviously declined. Meanwhile, the level of H3K27me3 was upregulated and H3K9me2 was downregulated in Tet1 knockdown PGCs, which was achieved by regulating Wnt/β-catenin signaling pathway. Conclusion: These results suggested that the self-renewal of chicken PGCs and the maintenance of their characteristics were regulated by Tet1 mediating DNA demethylation through the activation of Wnt4/β-catenin signaling pathway.

• Animal Bioscience
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• v.37 no.3
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• pp.437-450
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• 2024

Objective: Vanin-1 (VNN1) is a pantetheinase that catalyses the hydrolysis of pantetheine to produce pantothenic acid and cysteamine. Our previous studies have shown that the VNN1 is specifically expressed in chicken liver which negatively regulated by microRNA-122. However, the functions of the VNN1 in lipid metabolism in chicken liver haven't been elucidated. Methods: First, we detected the VNN1 mRNA expression in 4-week chickens which were fasted 24 hours. Next, knocked out VNN1 via CRISPR/Cas9 system in the chicken Leghorn Male Hepatoma cell line. Detected the lipid deposition via oil red staining and analysis the content of triglycerides (TG), low-density lipoprotein-C (LDL-C), and high-density lipoprotein-C (HDL-C) after VNN1 knockout in Leghorn Male Hepatoma cell line. Then we captured various differentially expressed genes (DEGs) between VNN1-modified LMH cells and original LMH cells by RNA-seq. Results: Firstly, fasting-induced expression of VNN1. Meanwhile, we successfully used the CRISPR/Cas9 system to achieve targeted mutations of the VNN1 in the chicken LMH cell line. Moreover, the expression level of VNN1 mRNA in LMH-KO-VNN1 cells decreased compared with that in the wild-type LMH cells (p<0.0001). Compared with control, lipid deposition was decreased after knockout VNN1 via oil red staining, meanwhile, the contents of TG and LDL-C were significantly reduced, and the content of HDL-C was increased in LMH-KO-VNN1 cells. Transcriptome sequencing showed that there were 1,335 DEGs between LMH-KO-VNN1 cells and original LMH cells. Of these DEGs, 431 were upregulated, and 904 were downregulated. Gene ontology analyses of all DEGs showed that the lipid metabolism-related pathways, such as fatty acid biosynthesis and long-chain fatty acid biosynthesis, were enriched. KEGG pathway analyses showed that "lipid metabolism pathway", "energy metabolism", and "carbohydrate metabolism" were enriched. A total of 76 DEGs were involved in these pathways, of which 29 genes were upregulated (such as cytochrome P450 family 7 subfamily A member 1, ELOVL fatty acid elongase 2, and apolipoprotein A4) and 47 genes were downregulated (such as phosphoenolpyruvate carboxykinase 1) by VNN1 knockout in the LMH cells. Conclusion: These results suggest that VNN1 plays an important role in coordinating lipid metabolism in the chicken liver.

• Animal Bioscience
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• v.37 no.4
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• pp.689-696
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• 2024

Objective: Our study examined the impact of propriety blends of Bacillus strain probiotics on the performance, egg quality, and faecal microflora of laying hens. Methods: A total of 183 Institut de selection Animale (ISA) brown laying hens aged 23 weeks with an average body weight of 1,894±72 g were randomly allocated into 3 groups as control (corn-soybean meal based diet, CON), 0.5 g/kg Enterosure probiotics (ET1, 3×10⁸ colony-forming unit [CFU]/kg feed), and 5 g/kg Enterosure probiotics (ET2, 3×10⁹ CFU/kg feed) administered in mashed form. At the completion of each phase hen day egg production (HDEP), average egg weight (AEW), feed intake, and faecal microbiota were evaluated. Results: HDEP and AEW were higher (p<0.05) in the ET2-supplemented diet in phase 3 (week 9 to 12) compared with CON. Egg mass (EM) was higher (p<0.05) in phase 2 at ET2, and also higher (p<0.05) in phase 3 at the ET1 and ET2-supplemented diets compared with CON. Feed conversion ratio was lower (p<0.05) in phase 3 at the ET1 and ET2-supplemented diets, with ET2 being the lowest compared with ET1 and CON. Yolk colour was higher (p<0.05) in the ET-supplemented diets at phase 3 compared with CON. Bifidobacterium spp. was higher (p<0.05) in the ET2- supplemented diet compared with CON in phase 2, while in In phase 3, Lactobacillus spp. and Bifidobacterium spp. were higher (p<0.05) in the ET-supplemented diets compared with CON. Coliforms were lower (p<0.05) in the ET-supplemented diets compared with CON in phase 3. Conclusion: The propriety blends of Bacillus strain probiotics supplements at 0.5 g/kg and 5 g/kg could improve the production and quality of eggs with more significance at 5 g/kg for HDEP, AEW and EM, which was achieved via the increase of beneficial microbiomes such as Lactobacillus spp., Bifidobacterium spp., and the decrease of pathogenic microbiomes like Escherichia coli and Coliforms which was speculated to improve gut barrier function and the reproductive hormone.

• Animal Bioscience
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• v.37 no.4
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• pp.709-717
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• 2024

Objective: Abnormally increased somatic cell counts (SCCs) in milk is usually a sign of bovine subclinical mastitis. Mutual interaction between the host and its associated microbiota plays an important role in developing such diseases. The main objective of this study was to explore the difference between cows with elevated SCCs and healthy cattle from the perspective of host-microbe interplay. Methods: A total of 31 milk samples and 23 bovine peripheral blood samples were collected from Holstein dairy cattle to conduct an integrated analysis of transcriptomic and metagenomics. Results: The results showed that Ralstonia and Sphingomonas were enriched in cows with subclinical mastitis. The relative abundance of the two bacteria was positively correlated with the expression level of bovine transcobalamin 1 and uridine phosphorylase 1 encoding gene. Moreover, functional analysis revealed a distinct alternation in some important microbial biological processes. Conclusion: These results reveal the relative abundance of Ralstonia and Sphingomonas other than common mastitis-causing pathogens varied from healthy cows to those with subclinical mastitis and might be associated with elevated SCCs. Potential association was observed between bovine milk microbiota composition and the transcriptional pattern of some genes, thus providing new insights to understand homeostasis of bovine udder.

• Animal Bioscience
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• v.37 no.3
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• pp.522-535
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• 2024

Objective: Transition period is considered from 3 weeks prepartum to 3 weeks postpartum, characterized with dramatic events (endocrine, metabolic, and physiological) leading to occurrence of production diseases (negative energy balance/ketosis, milk fever etc). The objectives of our study were to analyze the periodic concentration of serum beta-hydroxy butyric acid (BHBA), glucose and oxidative markers along with identification, and validation of the putative markers of negative energy balance in buffaloes using in-silico and quantitative real time-polymerase chain reaction (qRT-PCR) assay. Methods: Out of 20 potential markers of ketosis identified by in-silico analysis, two were selected and analyzed by qRT-PCR technique (upregulated; acetyl serotonin o-methyl transferase like and down regulated; guanylate cyclase activator 1B). Additional two sets of genes (carnitine palmotyl transferase A; upregulated and Insulin growth factor; downregulated) that have a role of hepatic fatty acid oxidation to maintain energy demands via gluconeogenesis were also validated. Extracted cDNA (complementary deoxyribonucleic acid) from the blood of the buffaloes were used for validation of selected genes via qRTPCR. Concentrations of BHBA, glucose and oxidative stress markers were identified with their respective optimized protocols. Results: The analysis of qRT-PCR gave similar trends as shown by in-silico analysis throughout the transition period. Significant changes (p<0.05) in the levels of BHBA, glucose and oxidative stress markers throughout this period were observed. This study provides validation from in-silico and qRT-PCR assays for potential markers to be used for earliest diagnosis of negative energy balance in buffaloes. Conclusion: Apart from conventional diagnostic methods, this study improves the understanding of putative biomarkers at the molecular level which helps to unfold their role in normal immune function, fat synthesis/metabolism and oxidative stress pathways. Therefore, provides an opportunity to discover more accurate and sensitive diagnostic aids.

• Animal Bioscience
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• v.37 no.4
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• pp.609-621
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• 2024

Objective: Hair follicle stem cells (HFSCs) differentiation is a critical physiological progress in skin hair follicle (HF) formation. Goat HFSCs differentiation is one of the essential processes of superior-quality brush hair (SQBH) synthesis. However, knowledge regarding the functions and roles of miR-133a-3p and miR-145-5p in differentiated goat HFSCs is limited. Methods: To examine the significance of chi-miR-133a-3p and chi-miR-145-5p in differentiated HFSCs, overexpression and knockdown experiments of miR-133a-3p and miR-145-5p (Mimics and Inhibitors) separately or combined were performed. NANOG, SOX9, and stem cell differentiated markers (β-catenin, C-myc, Keratin 6 [KRT6]) expression levels were detected and analyzed by using real-time quantitative polymerase chain reaction, western blotting, and immunofluorescence assays in differentiated goat HFSCs. Results: miR-133a-3p and miR-145-5p inhibit NANOG (a gene recognized in keeping and maintaining the totipotency of embryonic stem cells) expression and promote SOX9 (an important stem cell transcription factor) expression in differentiated stem cells. Functional studies showed that miR-133a-3p and miR-145-5p individually or together overexpression can facilitate goat HFSCs differentiation, whereas suppressing miR-133a-3p and miR-145-5p or both inhibiting can inhibit goat HFSCs differentiation. Conclusion: These findings could more completely explain the modulatory function of miR-133a-3p and miR-145-5p in goat HFSCs growth, which also provide more understandings for further investigating goat hair follicle development.

• Animal Bioscience
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• v.37 no.3
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• pp.509-521
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• 2024

Objective: It was shown that microRNAs (miRNAs) play an important role in milk protein synthesis. However, the post-transcriptional regulation of casein expression by exogenous miRNA (xeno-miRNAs) in ruminants remains unclear. This study explores the regulatory roles of alfalfa xeno-miR162 on casein synthesis in bovine mammary epithelial cells (bMECs). Methods: The effects of alfalfa xenomiR-162 and G protein subunit gamma 11 (GNG11) on proliferation and milk protein metabolism of bMECs were detected by 5-Ethynyl-2'-Deoxyuridine (EdU) staining, flow cytometry, cell counting kit-8 (CCK-8), enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. Dual-luciferase reporter assay was used to verify the targeting relationship between GNG11 and xenomiR-162. Results: Results showed that over-expression of xenomiR-162 inhibited cell proliferation but promoted apoptosis, which also up-regulated the expression of several casein coding genes, including CSN1S1, CSN1S2, and CSN3, while decreasing the expression of CSN2. Furthermore, the targeting relationship between GNG11 and xenomiR-162 was determined, and it was confirmed that GNG11 silencing also inhibited cell proliferation but promoted apoptosis and reduced the expression of casein coding genes and genes related to the mammalian target of rapamycin (mTOR) pathway. Conclusion: Alfalfa xenomiR-162 appears to regulate bMECs proliferation and milk protein synthesis via GNG11 in the mTOR pathway, suggesting that this xeno-miRNA could be harnessed to modulate CSN3 expression in dairy cows, and increase κ-casein contents in milk.