The study focuses on the effects of fermented feed with tea residue, soybean meal and bran as substrates on growth performance, nutrient metabolic rates and serum indexes of broiler chickens. Using single factor test design, 240 one-day-of-age Ross 308 broiler chickens with similar body mass and good health were randomly divided into five groups [control group Tl, fed the basic diet; experimental group T2, fed the basic diet＋methylene salicylic acid bacitracin (40 mg/kg); experimental groups T3, T4, T5, fed the fermented feed to replace 2%, 4% and 6% soybean meal in the basic diet, respectively] with six replicates per treatment and eight chickens per replicate. The test period was 42 d. The results showed that: 1) Compared with the control group Tl, the average daily gain from 1 to 21 days of age, 21 day-of-age and 42 day-of-age body masses of broiler chickens in the T4 group significantly increased (P＜0.05), and the feed-to-gain ratio was significantly reduced from 1 to 21 days of age (P＜0.05). 2) The apparent metabolic rates of crude protein of broiler chickens in T4 and T5 groups were significantly higher than those in T1 and T2 groups (P＜0.05). 3) Compared with the control group Tl, the serum albumin levels of the T2 and T5 groups significantly increased (P＜0.05), and the serum antioxidant indexes (total superoxide dismutase, catalase activities, and malondialdehyde content) of the T5 group were significantly different (P＜0.05). In summary, the addition of fermented feed with tea residue, soybean meal and bran as substrates in the diets can improve broiler chickens’ growth performance, slaughter performance, apparent metabolic rates of crude protein and serum index levels, reflecting growth-promoting effects similar to antibiotics.
In order to compare the morphological differences of otoliths among different Harpodon nehereus populations, five measurement indexes of sagittal otoliths including distance from geometric center to rostrum (A1), distance from geometric center to veutro (A2), distance from geometric center to dorsal (A3), distance from geometric center to excisural notch (A4) and distance from geometric center to excisural minor notch (A5) were examined by paired t-test. The results showed that the P values were greater than 0.05, indicating no significant differences between left and right otoliths. The cumulative contribution rates of the first principal component (PC1) in seven measurement indexes, including A1-A5, major axis length (L) and minor axis length (H), and the ellipticity (X1) and radial ratio (X2) were 93.114% and 99.950%, respectively. The results of one-way analysis of variance showed that the ellipticity could be used as the main feature to distinguish Zhoushan population from the others. The hierarchical clustering tree demonstrated that the specimens from the southern East China Sea and the South China Sea first gathered together into one branch, and then followed by Sanmen and Zhoushan populations. Haikou population was located at the outermost of the clustering tree and separated apart from other populations. The average discriminant accuracy rate was 80.63%, of which Zhoushan population was the highest (89.29%), while Ningde population was the lowest (71.43%). The above results suggest that the multivariate statistical analysis of morphological characteristics of otolith could be used as the basis for population identification of H. nehereus.
A headspace-gas chromatograph (HS-GC) (external standard method) was established for the determination of the volatile fatty acid (VFA) contents in rumen fluid of ruminants. Under the optimized headspace sampling conditions (balance temperature at 85 ℃ and equilibrium time of 30 min), the method was verified and used to detect the VFA. The coefficient of determination of regression equation for each compound was not less than 0.999 7. The limit of detection (LOD) was 0.036-0.329 mmol/L. The recovery rates of three different concentrations of standards were 93.540%-108.130%. The detection results showed that the relative standard deviations (RSDs) of VFA in all samples were less than 2.3%, and the method was stable within three days. The developed method is simple, which has accurate quantity and good repeatability, and suitable for the quantitative analysis of VFA in rumen fluid of ruminants.
In order to understand the annual epidemic situation of Chinese sacbrood disease and European foulbrood disease pathogens [Chinese sacbrood virus (CSBV) and Melissococcus pluton, respectively] in Chinese honeybee (Apis cerana cerana) of Chun’an County of Zhejiang Province, the workers in Chinese honeybee colonies were collected from five breeding sites in Chun’an County from January to December in 2019, and the dynamic changes of the two main disease pathogens in the Chinese honeybee colonies were monitored by polymerase chain reaction (PCR). The results showed that the colony infection rate of CSBV was the highest in March of spring (84%), and it decreased to the lowest in July of summer, then increased from autumn to winter. The infection rate of M. pluton increased from spring to summer, and then increased from autumn to the highest level of 72% in late autumn (October) and early winter (November), and decreased slightly in winter. Furthermore, there was co-infection phenomenon of these two disease pathogens in honeybee colonies in the whole year. The results of real-time quantitative PCR showed that the viral load of CSBV in colonies with M. pluton in autumn was significantly higher than that in the colonies with a single infected CSBV (P＜0.05). In conclusion, the annual epidemic dynamics of two main disease pathogens of Chinese honeybee is clarified in Chun’an County of Zhejiang Province, which can provide theoretical guidances for the prevention and control of Chinese honeybee diseases in this region.
In order to explore the regulation of nucleobindin-2 (NUCB2)/Nesfatin-1 in the growth and development of Chinese sucker (Myxocyprinus asiaticu), the full-length cDNA sequence of NUCB2 was cloned by rapid amplification of cDNA ends (RACE) technology, and the expression and distribution of Nesfatin-1 in the diencephalon and hepatopancreas were observed by quantitative real-time polymerase chain reaction (qPCR) and immunofluorescence technique, respectively. The results showed that the full-length cDNA of NUCB2 was 2 090 bp, including a 99 bp 5′ untranslated region (UTR), a 1 449 bp open reading frame (ORF) and a 542 bp 3′ untranslated region. The NUCB2 gene encoded 482 amino acids, consisted of a signal peptide region of 23 amino acids and a NUCB2 peptide region of 459 amino acids. Two propeptide cleavage sites were in NUCB2 peptide region, lysine (Lys) 106-arginine (Arg) 107 and Lys179-Arg180, then three functional peptides of Nesfatin-1, Nesfatin-2, and Nesfatin-3 were divided according to the two cleavage sites. Phylogenetic tree analysis showed that the sequence of M30, the domain region of Nesfatin-1, was highly conserved in fish. In the same age, the expression level of Nesfatin-1 mRNA in the hepatopancreas was highly significantly higher than that in the diencephalon (P＜0.01); however, in the same tissue, such as diencephalon and hepatopancreas, no significant differences were found between three and six-year-old M. asiaticus (P＞0.05). The strong positive immunofluorescence signal was detected in the pancreatic cells instead of hepatocytes in the hepatopancreas. In the diencephalon, positive immunofluorescence signals were only detected in the lateral tuberal nucleus, anterior periventricular nucleus and preoptic nucleus magnocelluar part. In conclusion, the expression of Nesfatin-1 in the diencephalon is significantly lower than that in the hepatopancreas during two typical growth stages of Chinese sucker, indicating that the central and peripheral regulation of Nesfatin-1 on the growth and development of Chinese sucker has obvious difference.
Guinea pig is a laboratory animal model to study the illness and stimulation of infection. The previous studies showed that the sensitivity of Zmu-1∶DHP outbred guinea pig to foot-and-mouth disease virus was 100%, which was significantly different from DHP (parental strain). This study took three strains of guinea pigs (Zmu-1∶DHP outbred line, Zmu-2∶DHP outbred line and DHP strain) as the experimental groups. The RNA extracted from spleen of different strains of guinea pigs was transcribed into cDNA and amplified by polymerase chain reaction. The amplified products were sequenced by high-throughput sequencing to analyze the structure, haplotype and polymorphism of MHC classⅠgene in guinea pigs, so as to detect whether there were differences in the expression of MHC classⅠgene among different strains. The results showed that eight MHC classⅠsequences in guinea pig were found and some of these haplotypes had 23 amino acids deletion in exon 3. There were significant differences in the frequencies of haplotype expression reads among some strains (P＜0.05), which suggested that the difference of MHCclassⅠhaplotype expression might be related to the difference of immune ability among different strains. The research provides a theoretical basis for the application of guinea pig strains in disease model and vaccine development.
Haemonchosis is caused by Haemonchus contortus parasitic in the ruminant abomasum. In order to study the function of zinc metalloproteinase NAS-31 gene (Hc-nas-31) in the free-living period (especially L3) of this kind of worm, real-time quantitative polymerase chain reaction (qRT-PCR) was conducted to detect the transcription level of the target gene in different developmental stages of H. contortus. Rapid amplification of cDNA ends (RACE), genome walking and fusion PCR methods were used to amplify Hc-nas-31 gene and the gene structure was analyzed after that. Then, prokaryotic expression plasmid was constructed and transformed into BL21 to express the recombinant protein. A New Zealand white rabbit was immunized with the purified recombinant Hc-NAS-31 (rHc-NAS-31) to prepare polyclonal antibodies, which were used to analyze the expression pattern of Hc-NAS-31 in H. contortus by indirect immunofluorescence assay. The results showed that Hc-nas-31 was transcribed in all stages of the worms, while the transcription level of L3 was the highest. Polyclonal antibody was made successfully and its specific binding with Hc-NAS-31 natural protein was confirmed by Western blotting. Indirect immunofluorescence analysis showed that Hc-NAS-31 protein was mainly expressed in the epithelial syncytia of L3, while in adults, it was distributed in the intestines, gonads, muscle tissues and early eggs. In summary, we confirmed the expression pattern of NAS-31 in H. contortus. This experiment lays the foundation for further research on the biological functions of Hc-nas-31.
The adaptive immune system can protect the body from various pathogens by producing a variety of antibodies. The antibody repertoire is so vast that the traditional low-throughput sequencing method cannot completely sequence it. In this study, the library of porcine B cell receptor (BCR) heavy chain was amplified from porcine peripheral blood mononuclear cell (PBMC) by multiplex polymerase chain reaction (PCR). The characteristics of porcine antibody repertoire were analyzed by next-generation sequencing. The results showed that several IGHV, IGHD and IGHJ genes were preferentially used by three-week-old piglets under normal conditions, and IGHV1-4 and IGHV1S2 were the two most frequently used variable (V) genes. The total frequencies of the use of eight IGHV genes accounted for more than 80%. In addition, the length of heavy chain complementarity determining region 3 (HCDR3) was a normal distribution with the average length of 15.8 amino acids. This study successfully constructs a high-throughput sequencing method suitable for porcine antibody repertoire, which provides an effective approach for further understanding the specific composition of it in physiological and pathological states.
To investigate the antimicrobial resistance of bacteria from animals in Jinhua City and Taizhou City of Zhejiang Province in May of 2020, a total of 284 anal swab samples were randomly collected from seven livestock and poultry farms in the two cities, respectively. Escherichia coli and Enterococcus were isolated by selective culture media and were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The minimal inhibitory concentration (MIC) was determined by broth microdilution method. The results showed that the antimicrobial resistance rate of E. coli to tetracycline was the highest (87.5%), followed by sulfamethoxazole (81.2%), while the antimicrobial resistance rate to colistin was the lowest (0%) in Jinhua City. In addition, the antimicrobial resistance rate of E. coli to sulfamethoxazole was the highest (97.1%), and the resistance rates to meropenem and colistin were both 0% in Taizhou City. The antimicrobial resistance rate of Enterococcus to sulfamethoxazole was 100% in both cities, followed by tamoxifen (98.6%). No amoxicillin/clavulanate and vancomycin resistant Enterococcus was found in the two cities. In terms of the overall antimicrobial resistance rate, MIC distribution and antimicrobial resistance pattern, the level of antimicrobial resistance in Taizhou was higher than that in Jinhua. In conclusion, through monitoring and comparing the level of antimicrobial resistance in livestock and poultry breeding in two cities, it is found that the levels of antimicrobial resistance of E. coli and Enterococcus from animals are high and different. Therefore, continuous surveillance of antimicrobial resistance dynamics is essential through veterinary antibiotics reduction initiatives by agricultural authorities.
This study was designed to screen out the key genes related to intramuscular fat deposition through detecting the fat contents in longissimus dorsi muscle (LDM) and psoas major muscle (PMM), and explore the differentially expressed genes between the two types of muscle of Qianbei black pigs using high-throughput sequencing technology. Five 12-month-old healthy Qianbei black pigs were slaughtered, and LDM and PMM were collected. Some of them were used to detect total fat content, and the rest were used for total RNA extraction, library constructing and sequencing. The annotation genes were obtained by comparing the obtained unigenes with RefSeq database of pig. The differentially expressed mRNAs were analyzed by DESeq2 1.26.0 software, and moreover gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis of differentially expressed mRNAs were analyzed by GOseq 1.15.5 software and KOBAS 3.0 software. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the candidate genes related to fat deposition. The results showed that the fat content in the PMM was significantly higher than that in the LDM (P＜0.01). A total of 162 differentially expressed genes were obtained by high-throughput sequencing, of which 66 were up-regulated and 96 were down-regulated. Six differentially expressed genes associated with fat deposition were screened out by GO, KEGG analysis combined with the function searching. qRT-PCR results showed that the expression of LPL, PPARA, COX2, PRKAG2 and SCD existed in significant differences between LDM and PMM. The above results lay a foundation for exploring the molecular mechanism of intramuscular fat deposition in Qianbei black pigs, and further provide an experimental basis for the protection, development and utilization of Qianbei black pigs.
