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Effects of the Number of Subclinical Mastitis and Somatic Cell Score in Milk of Parity 1 on Somatic Cell Score of Holstein Cows for Parity 2 XIA YuXin,LIANG Yan,WANG HaiYang,GUO MengLing,ZHOU Bu,DAI Xu,YANG ZhangPing,MAO YongJiang Scientia Agricultura Sinica    2022, 55 (20): 4052-4064.   DOI: 10.3864/j.issn.0578-1752.2022.20.015 Abstract （220）   HTML （39）    PDF （980KB）（73）       Knowledge map    Save 【Objective】There was correlation between physiological status of different parities of cows. The objective of this research was to explore the effect of the number of subclinical mastitis (SCM) and somatic cell score (SCS) in milk of parity 1 on SCS of Holstein cows for parity 2. 【Method】This study collected 162 509 DHI records of Holstein cows with parity 1 and 2 from 2015 to 2020 in 13 farms in Jiangsu Province. Excel 2019 was used to pre-process and filter DHI records firstly. The mixed model of SAS (Ver 9.4) was used to explore the effects of farm size, sampling year, calving season, lactation month, the number of SCM on parity 1 and each average lactation stage of SCS on parity 1 on SCS of parity 2. At the same, the correlation coefficients between the number of SCM and the SCS for each lactation stage of parity 1 with SCS in each lactation month for parity 2 of Holstein cows were calculated.【Result】The farm size, sampling year, calving season, lactation month, the number of SCM on parity 1 and average SCS on parity 1 in different periods had extremely significant effects on the SCS of parity 2 (P<0.01). Among them, SCS with farm size over 5 000 was lower than other farm size (P<0.05). SCS in 2020 was higher than other sampling year (P<0.05). SCS of cows calving in summer and spring was higher than other calving season (P<0.05), while SCS of cows calving in winter was lower than other calving season (P<0.05). SCS in the 9th and 10th lactation month was higher than other lactation months (P<0.05), while SCS in the second lactation month was lower than other lactation months (P<0.05). The SCS in each lactation month of parity 2 showed downward firstly and then upward trend for the cows with the number of SCM less than 2 for parity 1. The SCS for parity 2 in each lactation month fluctuated greatly for the cow with the number of SCM with 3 or more than in parity 1. When the average lactation of SCS, early lactation of SCS, mid lactation of SCS, and late lactation of SCS for the cows in parity 1 were 0, 1, 2, and 3, respectively, the SCS for each lactation month in parity 2 showed downward firstly and then upward trend. When the average lactation of SCS for the cow in parity 1 was more than 3, the SCS in parity 2 in each lactation fluctuated greatly. Overall, the number of SCM and the SCS for each lactation stage of cows in parity 1 increased, and the SCS in each lactation month for parity 2 gradually also increased. There was an extremely significant positive correlation between the number of SCM on parity 1 and SCS on parity 2 in each lactation month (P<0.01). The correlation coefficient of average SCS in the lactation for parity 1 with the SCS in parity 2 lactation month was the maximum (0.238). The correlation coefficient of average SCS in the early lactation for parity 1 and the SCS in parity 2 in each lactation month was the minimum (0.104). Among them, the correlation coefficient of the number of SCM for parity 1 with the SCS for parity 2 in the 3rd lactation month was the maximum. The correlation coefficient of the number of SCM for parity 1 with the SCS for parity 2 in the 10th lactation month was the minimum. The correlation coefficient of average SCS in the lactation for parity 1 with the SCS for parity 2 in the 5th lactation month was the maximum. The correlation coefficient of average SCS in the lactation for parity 1 with the SCS for parity 2 in the 5th lactation month was the minimum. The correlation coefficient of early lactation of SCS for parity 1 with the SCS for parity 2 in the 5th lactation month was the maximum, however, the SCS for parity 2 in the 1st and 10th lactation month was the minimum. The correlation coefficient of mid-lactation of SCS for parity 1 with the SCS for parity 2 in the 5th lactation month was the maximum, while the SCS for parity 2 in the 1st lactation month was the minimum. The correlation coefficient of late lactation of SCS for parity 1 with the SCS for parity 2 in the 5th lactation month was the maximum, while the SCS for parity 2 in the 1st lactation month was the minimum. 【Conclusion】The number of SCM and each average lactation stage of SCS for parity 1 had extremely significant effects on SCS in lactation for the cows of parity 2. There was an extremely significant positive correlation between the numbers of SCM for parity 1 with the SCS for parity 2 in each lactation month. The results provided references for improving quality of raw milk of Holstein cows in parity 2 in the future. Table and Figures | Reference | Related Articles | Metrics

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Effects of Amino Acid By-Products on Fermentation Quality and Digestibility of White Sorghum Silage NUERHATI·Silafuer ,WUSIMAN·Yimiti Scientia Agricultura Sinica    2022, 55 (20): 4065-4074.   DOI: 10.3864/j.issn.0578-1752.2022.20.016 Abstract （220）   HTML （36）    PDF （2148KB）（72）       Knowledge map    Save 【Objective】 The aim of this study was to investigate the effects of appropriate amino acid by-products (ABP) on fermentation quality and digestibility of white sorghum, so as to provide ideas for reducing environmental pollution as well as developing and utilizing new feed additives.【Method】In the study, the control group without any additives and the two experimental groups with ABP and ABP+ forage bacteria were used to carry out the experiment of white sorghum silage fermentation. The effects of ABP on the fermentation quality and digestibility of silage were obtained by measuring the feed composition and in vitro digestibility, and scanning electron microscopy (SEM) was used to observe the mechanism of ABP on improving feed fermentation quality and digestibility.【Result】The study has shown that the addition of 2.0% ABP to white sorghum straw could reduce the pH of the feed to 3.65, which was significantly different from the control group (5.13) (P<0.05). The sensory score belonged to the quality silage interval. The lactic acid content of each experimental group (ABP:11.95 g·kg-1; MIX:15.14 g·kg-1) was significantly higher than that of the control group (3.54 g·kg-1) (P<0.01), the content of acetic acid and butyric acid (AA: ABP:2.87 g·kg-1, MIX:2.75 g·kg-1; BA: ABP:0.72 g·kg-1, MIX:0.78 g·kg-1) was significantly lower than that of the control group (acetic acid:3.85 g·kg-1; butyric acid: 1.39 g·kg-1) (P<0.05), and the lactic acid content of the experimental group of ABP+ forage bacteria was 327.85% higher than that of the control group; the content of dry matter (DM) in each group did not change significantly (P>0.05). Neutral detergent fiber (NDF) (ABP:58.67%; MIX:57.67%), acid detergent lignin (ADL)(ABP:4.77%; MIX:4.27%) and ash (Ash) (ABP : 1.56%; mixed: 2.04%) lower than the control group (NDF:63.66%; ADL:5.15%; Ash:2.76%), but the difference was not significant (P>0.05), the acid detergent fiber (ADF) (ABP:35.77%; MIX:28.63%) was significantly lower than that of the control group (40.58%) (P<0.01), and the crude protein content (ABP: 9.65%, MIX:9.67%) was significantly higher than the control group (6.88%) (P<0.01); the in vitro digestibility of each experimental group was DM (ABP: 74.66%; MIX: 80.03%), NDF (ABP: 72.74%; MIX: 83.08%) and ADF (ABP: 68.29%; MIX: 79.56%), which were significantly higher than the control group (DM: 60.67%, NDF: 48.06%; ADF: 44.81%) (P<0.05); the results showed that ABP significantly improved and increased the fermentation quality and digestibility of silage. From the SEM results, it was found that the cross-section and surface structure of the control group were small, and the number of adhering microorganisms was small too, while the wax layer of the surface structure of the treatment group was destroyed and adhered to a large number of forage bacteria, cross-section cells or a large amount of forage bacteria adhered inside the tissue. Therefore, it was preliminarily informed that ABP improved and increased the fermentation quality and digestibility of silage. In addition, the carbon and nitrogen sources provided by the feed bacteria, the wax layer on the surface of the feed was destroyed to promote the adhesion of the feed bacteria and degrade the cell wall cellulose.【Conclusion】2.0% ABP added to white sorghum silage could significantly improve the fermentation quality and digestibility, and had great economic and social significance for the reuse of ABP, the reduction of environmental pollution, and the development and utilization of new feed additives. Table and Figures | Reference | Related Articles | Metrics

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Evolution of Human H9N2 Avian Influenza Virus in China from 1998 to 2021 WANG YanWen,WANG MengJing,ZHANG Hong,GAO XinXin,GUO Jing,LI XuYong Scientia Agricultura Sinica    2022, 55 (20): 4075-4090.   DOI: 10.3864/j.issn.0578-1752.2022.20.017 Abstract （365）   HTML （48）    PDF （4859KB）（136）       Knowledge map    Save 【Objective】 The epidemiological characteristics of H9N2 avian influenza virus were clarified by analyzing the onset time, province, age, gender and other information of human cases infected with H9N2 avian influenza from 1998 to 2021 in China. By analyzing the genetic characteristics of human H9N2 subtype avian influenza virus, the genetic evolution law of human H9N2 subtype avian influenza virus was elucidated. This study could provide data support for the early warning and control of H9N2 subtype avian influenza virus cross-species transmission. 【Method】 According to the case report, literature data and gene bank, the case information and strain sequence data of human infection with H9N2 subtype avian influenza virus in China from 1998 to 2021 were obtained. The infection cases were analyzed from the time distribution, space distribution, gender and age distribution, and the epidemiological characteristics of human-derived H9N2 subtype avian influenza virus infection were found. The MegAlign software in DNASTAR were used to analyze Nucleotide sequence of the homology of various gene fragments of human H9N2 isolates, a phylogenetic tree was constructed by MEGA7.0 software, and the key sites of viral proteins were analyzed. The genetic evolution trend of viral proteins and the variation of key amino acid sites were clarified. The 2019-2021 H9N2 subtype avian influenza virus nucleotide sequence in China was downloaded from the GISAID website. After mafft alignment, the key amino acid site mutation differences between human and avian H9N2 viruses were examined in MEGA7.0, the potential risks brought by current human and avian H9N2 viruses were revealed. 【Result】 From1998 to 2021, a total of 71 cases of human infection with the H9N2 subtype avian influenza virus occurred in China. From the spatial distribution analysis, the cases were distributed in 16 provinces and cities, of which 91.55% of the cases were from 12 provinces and cities in the south. In terms of time, the number of reported infections has been on the rise after 2013, and the cumulative number of infections from 2013 to 2021 accounted for 61.97% of the total number of infections. From the analysis of gender and age distribution, the ratio of male to female was 1:1.68. Human infection with H9N2 virus was mainly seen in infants and young children, accounting for 74.14% of the total number of cases. Genome comparison analysis of human H9N2 viruses found that these viruses belonged to the Eurasian branch, but the nucleotide sequence of homology of these viruses gene fragments was quite different. The nucleotide homology of HA, NA, PB2, PB1, PA, NP, M, and NS were 75.3%-100%, 80.1%-100%, 78.7%-100%, 82.5%-100%, 72.6%-100%, 74.1 -100%, 65.5%-100%, and 82.0%-100%, respectively. According to the genealogy of human isolates, 22 human isolates with complete gene fragments could be divided into 8 genotypes, genotypes in 2003, 2008, and 2013 were significantly different from genotypes in 1999. A total of 42 human H9N2 virus strains uploaded HA sequences from 1998 to 2021, and 38 of them had the mutation of Q226L in the HA protein; a total of 30 human virus strains uploaded PB2 sequence from 1998 to 2021, and 9 strains of which had the mutation of E627V in the PB2 protein, 1 of which had the mutation of E627K in the PB2 protein; the 701 site of the PB2 protein of one virus had the mutation of D701N, a total of 31 virus strains uploaded NS and M sequence from 1998 to 2021, the 42nd positions of the NS1 protein were all S, and the amino acids at positions 30th and 215th of the M1 protein were D and A, respectively. 【Conclusion】 Since 2013, the number of human infections with H9N2 subtype avian influenza reported in China has been on the rise, with significant differences in geographic, age and gender distribution. Since 1998, the genetic homology between human H9N2 isolates was quite different, and the viral gene rearrangement between different branches was frequent, forming a complex genotype. It was suggested that the H9N2 subtype avian influenza virus was constantly evolving. Mutations in key amino acid sites of human H9N2 virus and mutation rate of human viruses was higher than avian H9N2 viruses from 2019 to 2021, suggesting that the potential of H9N2 subtype avian influenza virus to infect humans was gradually increasing. This result enriched the knowledge of human H9N2 virus and provided an important reference for the prevention and control of H9N2 subtype avian influenza virus. Table and Figures | Reference | Related Articles | Metrics

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Analysis of Indirect Genetic Effects on Body Weight of 42 Day-Old Rugao Yellow Chickens GUO Jun,WANG KeHua,HAN Wei,DOU TaoCun,WANG XingGuo,HU YuPing,MA Meng,QU Liang Scientia Agricultura Sinica    2022, 55 (19): 3854-3861.   DOI: 10.3864/j.issn.0578-1752.2022.19.014 Abstract （222）   HTML （37）    PDF （617KB）（85）       Knowledge map    Save 【Background】 In addition to regulating its own phenotype, the genotype of an individual animal also affects the performance of other animals within a social group through resource allocation or behavioral interaction, and this phenomenon is called an indirect genetic effect. In the animal breeding, if the genetic model harbored the indirect genetic effects, it will not only improve the social relationship between individuals, but also obtain more genetic gains. 【Objective】 In this study, Rugao Yellow Chickens raised in group cages were used as the test animals, and the indirect genetic model was used to evaluate body weight data, aiming to provide a flexible model to select the Rugao Yellow Chickens. 【Method】The body weight data was collected from the breeding group of Rugao Yellow Chickens. The fowls were weighed at the age of 42 days-old, and 11 983 raw data were collected. The data cleaning procedure included: i. removing outlier beyond three standard deviations either side of the mean; ii. eliminate the fowl without marker; iii. get rid of unknown sexed fowls; iv. the fowls with less than 4 records within a social group were also excluded. The pedigree data consisted of 12 208 fowls, including 11 735 chickens with body weight records and 473 chickens without records, 10 560 chickens without progeny and 1 648 with progeny, and the progeny of them included 208 male breeders and 1 440 female breeders. With SPSS software packages, ANOVA was used to test the influence of environmental factors on body weight and determine the factors included in the fixed effects. The classic animal model and indirect genetic model were used to analyze the variance components and genetic parameters of Rugao Yellow Chickens, and to test whether there was a dilution effect on the indirect genetic variance. The genetic model included the general fixed effects, fixed regression terms, additive genetic effects, indirect genetic effects, common environmental effects and residuals. In this study, the fixed regression term included cage sizes, and random terms included additive genetic effects, indirect genetic effects and common environmental effects. The initial value of the dilution parameter was set to 0, and it was step increased to 1.0 in increments of 0.1. After evaluating with AIC and BIC standard, the dilution parameter should be set to 0. Accounting for heterogeneous errors did not alter the estimates of genetic parameters and variance components. Therefore, the homogeneous error was assumed. Using WOMBAT software, estimates of variance components and genetic parameters converged for both classic and indirect genetic models (with or without dilution effect). 【Result】The fixed effects included the combination of laying batch-row-sex level. The significant indirect genetic variance for body weight of 42-day-old Rugao Yellow Chickens was found, for the additive heritability was 0.54±0.02, and the total heritable variation was 0.66±0.06. The corporation relationship between individuals presented in the same cage of Rugao Yellow Chickens. The direction of additive genetic and indirect genetic selection was the same, and the genetic correlation coefficient was 0.41. There was no dilution effect in the indirect genetic variance of Rugao Yellow Chickens. The indirect genetic variances were distinguished between sexes, and so on heritability and genetic correlation coefficient. 【Conclusion】Indirect genetic model could be used for the genetic evaluation and selection of body weight during the rearing period. Compared with those classic animal models, the indirect genetic models could achieve an additional genetic gain. Table and Figures | Reference | Related Articles | Metrics

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Study on Seasonal Grazing Management Optimal Model in Alpine Desert Steppe HU ZhiQiang,SONG XiaoYu,QIN Lin,LIU Hui Scientia Agricultura Sinica    2022, 55 (19): 3862-3874.   DOI: 10.3864/j.issn.0578-1752.2022.19.015 Abstract （199）   HTML （38）    PDF （1202KB）（101）       Knowledge map    Save 【Objective】In order to protect the ecological environment of China’s alpine desert steppe and guide the comprehensive and sustainable development of grassland pastoral areas, this study took the alpine desert steppe as the research object, and the optimization simulation calculation and dynamic balance analysis of grassland and livestock were carried out. 【Method】In this paper, based on the characteristics of seasonal rotational grazing and livestock production rhythms, the spatial and temporal segregation of grassland resources and the dynamic changes in livestock numbers were considered, and the two pasture grazing time periods of warm season and cold season were refined. On the basis of the existing reasonable livestock carrying capacity calculation method, the maximum livestock carrying capacity was the goal, the dynamic grass-livestock balance was the constraint, and the turnout time and slaughter rate were the optimization parameters. A grazing-management optimization model was established, and the Ulan County Pastoral area of Qinghai Province was selected as the typical representative of alpine desert seasonal pastoral area. Based on the genetic algorithm optimization, the optimal grazing conditions under the three precipitation scenarios of wet, normal and dry in the pastoral area of Ulan County were determined, a simulation to optimize the forage-livestock balance and dynamic forage-livestock balance analysis was carried out, and then the results with those before optimization were compared. 【Result】 The simulation results of the grazing-management optimization model were better for different precipitation scenarios in the grazing area. Based on the lowest amount of unutilized forage, the optimal grazing conditions under the three precipitation scenarios of wet, normal and dry in the pastoral area of Ulan County were determined to be the transition of livestocks on November 1 with a slaughter rate of 43.4%, the transition of livestocks on November 3 with a slaughter rate of 38.2% and the transition of livestocks on November 3 with a slaughter rate of 36.7%, respectively. The optimal proper carrying capacity was 1.099 million sheep unit, 0.961 million sheep unit and 0.836 million sheep unit in high, normal, and low flow years, respectively, which were 11.75%, 10.44% and 10.43% higher than the proper carrying capacity before optimization, respectively. In addition to the livestock slaughter rate and the transition time, the precipitation was also an important factor affecting the proper carrying capacity in pastoral areas. Before and after optimization, the proper carrying capacity of Ulan County’s pastoral area in wet years was 0.983 and 1.099 million sheep unit, respectively, which was 30% higher than the proper carrying capacity of 0.757 and 0.836 million sheep unit in dry years, respectively. Through the analysis of pasture grass production process and livestock dynamic grass demand process, the dynamic grass-livestock balance calculation for the normal flow year in the pasture area of Ulan County was found that the available forage in the cold season pasture before optimization could not be fully utilized, the grazing process was unreasonable, and the surplus grass amount was more than 61.3 million kg, while after optimization, the problem of underutilization of available forage was effectively solved and the forage resources were efficiently utilized. For the whole grazing area of Ulan County, the adjusting grazing turnout time and livestock slaughter rate was a reliable way to improve the grazing system. The grazing-management optimization model was used to optimize the grazing process in the grazing area, which could ensure the high proper annual carrying capacity in the grazing area while being able to achieve a dynamic grass-livestock balance in the seasonal grazing area. 【Conclusion】The grazing-management optimization had the good applicability to alpine desert seasonal pasturing areas and had the certain superiority in the regulation of grazing management process and the optimization of forage-livestock balance in rotating pastoral areas. The grazing-management optimization model could effectively regulate the livestock carrying capacity of a grazing area, and its optimization results could provide reference for the development of a reasonable grazing system. Therefore, the model could be used to optimize the grazing process of a single herding household or ranch, to obtain the optimal grazing conditions for a single herding household or ranch, so as to develop the corresponding grazing system, which was more practical guidance and operability for the production practice of a single herding household or ranch. Table and Figures | Reference | Related Articles | Metrics

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Effects of IL-15 Overexpression on Myoblast Differentiation of Porcine Skeletal Muscle Cells MingJie XING,XianHong GU,XiaoHong WANG,Yue HAO Scientia Agricultura Sinica    2022, 55 (18): 3652-3663.   DOI: 10.3864/j.issn.0578-1752.2022.18.014 Abstract （297）   HTML （63）    PDF （2950KB）（110）       Knowledge map    Save 【Objective】The aim of this was to investigate the effects of interleukin 15 (IL-15) as a myokine on the proliferation and apoptosis of porcine skeletal muscle myoblast, so as to provide a basis for further studying on the regulation of IL-15 in animal muscle quality and the treatment of skeletal muscle diseases. 【Method】In this study, the IL-15 overexpressed lentiviral vector GV-492-IL-15 was constructed, and the porcine skeletal muscle satellite cells were aseptically isolated and cultured in vitro, then skeletal muscle cells morphology were subjected to myogenic differentiation, and the differentiated myoblast were verified by immunofluorescence staining. After myoblast differentiation, the IL-15 overexpressed recombinant lentiviral vector was transfected. A blank control group (Control), a negative control virus transfection group (IL-15-) and a GV-492-IL-15 lentivirus transfection group (IL-15+) were set for the experiment (n=3). The cells were cultured for 72 h; after growing to a certain number, the cells and the culture supernatant were harvested. Real-time quantitative PCR (qRT-PCR) and Western Blot were used to analyze the expression of target genes and proteins. ELISA kit was used to analyze the content of IL-15 in the culture medium, CCK-8 kit was used to analyze cell viability, and the flow cytometry was used to analyze the results of cell cycle and apoptosis. Moreover, Western Blot was used again to detect changes of the level of caspase-3 protein in the cells, which was closely related to apoptosis.【Result】(1) The identified plasmid was transfected with 293T cells, and as a result, a distinct green fluorescence could be observed in the cells, and a characteristic band near 20 KD could be observed by Western Blot. (2) Fusiform or fusiform porcine skeletal muscle satellite cells were obtained by microscopic observation and differentiated into tubular myoblasts after induction. The differentiated myoblasts were subjected to immunofluorescence staining with α-SMA monoclonal antibody. About 90% of the cells in the visual field were positive, and the cytoplasm stained red, indicating that the cultured cells were skeletal muscle myoblast cells. (3) After transfected with GV-492-IL-15 lentivirus, the relative mRNA and protein expression of IL-15 in myoblasts were significantly increased compared with the control group (P<0.001), however, the protein level of IL-15 in culture medium was not significantly changed (P>0.05). Compared with the control group, the early apoptosis rate of cells transfected with GV-492-IL-15 lentivirus was not significantly different (P>0.05), but the late apoptosis rate of cells was significantly decreased (P<0.05); there was a tendency for caspase-3 protein to decrease compared to the control group, but the overall difference was not significant (P>0.05). The CCK-8 assays showed that overexpression of IL-15 increased the ability of cell proliferation (P<0.05). In addition, the proportion of cells in G1 phase was significantly decreased by transfected with IL-15 overexpression lentivirus, while the proportion of cells in S phase and G2/M phase was significantly increased (P<0.05). 【Conclusion】Under the normal physiological conditions, IL-15 was localized in cells and played a role. The overexpression of IL-15 had no significant effects on the early apoptosis of porcine skeletal muscle myoblasts, but it could inhibit the late apoptosis and promote cell proliferation. This study provided a technical and theoretical basis for the positive regulation of IL-15 on pig skeletal muscle quality and the treatment of related muscle diseases. Table and Figures | Reference | Related Articles | Metrics

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Effects of Circadian Rhythm on Rumen Fermentation and Nutrient Digestion of Mutton Sheep ChunTao ZHANG,Tao MA,Yan TU,QiYu DIAO Scientia Agricultura Sinica    2022, 55 (18): 3664-3674.   DOI: 10.3864/j.issn.0578-1752.2022.18.015 Abstract （194）   HTML （25）    PDF （472KB）（80）       Knowledge map    Save 【Objective】The biological clock system generally exists at all levels of life, which is closely related to the digestive physiology and growth performance of animals. This experiment explored the relationship between circadian rhythm and rumen fermentation parameters, nutrient digestion and metabolism of Hu sheep, in order to explore the relationship between circadian rhythm and physiological digestion and metabolism of Hu sheep, so as to provide an important theoretical basis for improving the growth performance and nutrient utilization of fattening sheep.【Method】Forty-five healthy Hu sheep with body weight of 21.57 ± 0.77kg weight were randomly divided into 3 groups, with 15 sheep in each group. Each treatment group was used the same concentrate supplement and coarse feed. The three treatments were respectively set as follows: day treatment group(DH), that was, 70% of the total daily concentrate + 30% of the total daily coarse feed in the morning; in the evening treatment group(DL), 30% of the daily total concentrate + 70% of the daily total coarse feed were fed in the morning; the control group(CON), i.e. 50% of the daily total concentrate + 50% of the daily total crude feed in the morning and evening. After feeding for two months, the digestion and metabolism test was carried out. Total fecal collection and urine were used to determine nutrient apparent digestibility and metabolic rate. After the feeding experiment, the rumen fluid samples were collected from oral cavity 2 h before morning feeding and 2 h before evening feeding, respectively, for the determination of related indexes. 【Result】 ① The daily gain of DH was 215.00 g, the food intake was lower than that of DL and CON, and the feed conversion ratio was the best, with the value of 5.35, which was 11.19% and 16.04% better than the other two groups, respectively. ② Under different feeding modes, the digestibility of dry matter (DM), organic matter (OM), crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF) of sheep in DH group were higher than those in CON and DL groups. Compared with DL group, The DM digestibility, CP, NDF, and ADF was increased by 21.42%, 22.29%, 9.85%, and 28.69%, respectively. ③ The biological value of nitrogen in DH group was 20.31% higher than that of CON and 67.41% higher than that of DL (P>0.05). There was no significant difference in the intake of digestible nitrogen among all groups, and the total nitrogen excretion in DL group was significantly higher than that in DH group (P<0.05). The nitrogen deposition, nitrogen utilization and biological value in DH group were significantly higher than those in DL group (P<0.05) and CON group (P>0.05). ④ There were no significant differences in rumen microbial protein (MCP), acetic acid and total volatile fatty acid (TVFA) among groups with different treatments (P>0.05), however, the rumen pH, the ratio of acetic to propionic acid and butyric acid in DH group were significantly lower than those in DL group (P<0.05). With the alternation of day and night, rumen pH, NH3-N, acetic acid, propionic acid, total volatile fatty acid and the ratio of acetic to propionic acid were significantly changed (P<0.05), and the related parameters were higher in day than in night.【Conclusion】Growth performance and nutrient digestibility of sheep could be improved by changing the ration of concentrate to forage in the early and late days under the same nutrient content in the diet, and the feeding high concentrate ratio in the morning and low concentrate ratio in the afternoon. Rumen fermentation was affected by day and night, and feeding high concentrate diet in day promoted the contents of TVFA, NH3-N and MCP in rumen. In a word, it is better to increase the proportion of concentrate in the morning, which is more in line with the rhythm of digestion and absorption of animal body, and has better effect on the utilization of feed. Table and Figures | Reference | Related Articles | Metrics

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Molecular Mechanism of Regulation by H-NS on IncFⅡ Plasmid Transmission of Multi-drug Resistant Chicken Escherichia coli YaTing JIA,HuiHui HU,YaJun ZHAI,Bing ZHAO,Kun HE,YuShan PAN,GongZheng HU,Li YUAN Scientia Agricultura Sinica    2022, 55 (18): 3675-3684.   DOI: 10.3864/j.issn.0578-1752.2022.18.016 Abstract （198）   HTML （25）    PDF （854KB）（65）       Knowledge map    Save 【Objective】The objective of this study was to investigate the molecular mechanism of H-NS regulating conjugation of the IncFⅡ plasmid from a clinically isolated multi-drug resistant Escherichia coli from chicken, so as to provide a theoretical basis for controlling the rapid spread of IncFⅡ plasmid mediated multidrug resistance genes.【Method】The growth curves of Escherichia coli ATCC25922 and four recombinant strains (F25922, pBAD25922, FΔhns and FΔhns/phns) were determined to compare the influence of hns on different strains. The conjugation experiments were conducted with F25922, FΔhns and FΔhns/phns as donors and Escherichia coli J53 as recipient, then the conjugation frequency was calculated. The mRNA expression levels of IncFⅡ plasmid conjugation transfer related genes (traM, traJ and traY) in each recombinant strains (F25922, FΔhns and FΔhns/phns) were detected by RT-qPCR. The LacZ reporter strains F25922/PM(PJ/PY), FΔhns/PM(PJ/PY) and FΔhns/phns/PM(PJ/PY) were constructed to determine the β-galactosidase activity of three promoters of tra genes (traM, traJ and traY). The H-NS protein was purified by Ni-NTA resin affinity chromatography. The DNA sequences of three promoters of tra genes were amplified by PCR. The mechanism of H-NS regulating IncFⅡ plasmid transmission was identified by EMSA, and the binding sites of H-NS to different promoters were predicted and further verified by ESMA.【Result】The growth of recombinant strains F25922 and pBAD25922 were not significantly different from that of Escherichia coli ATCC25922, while the growth rate of deleted recombinant strain FΔhns and complemented strain FΔhns/phns were significantly lower than that of the control strain F25922. The results showed that the absence of hns could make the adaptability of strains worse, but did not affect the survival of the strains. The results of the conjugation test showed that the conjugation frequency of IncFⅡ plasmid in FΔhns was 1 279.33 times higher than that of the control strain F25922 (P<0.001), and the FΔhns/phns conjugation frequency of the supplementary strain was significantly lower than that of FΔhns, although it did not completely recover to the level of the control strain F25922. Similarly, the mRNA expression levels of these tra genes (traM, traJ and traY) were significantly higher in the deletion mutant FΔhns. The mRNA expression level of traJ was the highest in FΔhns, which was 1 510.14 times that of F25922, followed by traY and traM, which were 448.14 times and 81.54 times that of F25922, respectively. Compared to the deletion strain FΔhns, expression levels of the tra genes (traM, traJ and traY) in the complemented strain FΔhns/phns were significantly decreased. The β-galactosidase activities of promoters PM, PJ and PY in the reporter strains FΔhns/PM (PJ/PY) were 5.66, 10.45 and 21.91, respectively, which were significantly higher than that of the corresponding promoters of F25922/PM (PJ/PY) (P<0.001). The activities of promoters PM, PJ and PY in the complement reporter strains FΔhns/phns/PM (PJ/PY) were significantly lower than that of FΔhns/PM (PJ/PY), and there was no significant difference with the control strain F25922/PM (PJ/PY). EMSA results showed that H-NS protein could block the DNA migration of three promoters of tra genes, indicating that H-NS could directly bind to the three promoters. By predicting the binding sites and further verified by EMSA, it was confirmed that H-NS protein could bind directly to the AT enrichment region of the promoters of the three genes (traM, traJ and traY).【Conclusion】H-NS protein could bind directly to the AT enrichment region of the promoter region of IncFⅡ plasmid conjugation transfer related genes (traM, traJ and traY), and negatively regulate the conjugation transfer of IncFⅡ plasmid by inhibiting the activity of promoter. Table and Figures | Reference | Related Articles | Metrics

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Effects and Mechanisms of Exogenous GABA Against Oxidative Stress in Piglets YANG ChangPei,WANG NaiXiu,WANG Kai,HUANG ZiQing,LIN HaiLan,ZHANG Li,ZHANG Chen,FENG LuQiu,GAN Ling Scientia Agricultura Sinica    2022, 55 (17): 3437-3449.   DOI: 10.3864/j.issn.0578-1752.2022.17.014 Abstract （217）   HTML （27）    PDF （672KB）（69）       Knowledge map    Save 【Objective】 The purpose of this paper is to study the effect of exogenous γ-aminobutyric acid against oxidative stress in piglets, and the possible mediating role of hippocampal neuron γ-aminobutyric acid receptors regulating apoptosis signaling pathways in it, which will provide scientific basis for the application of γ-aminobutyric acid as animal stress regulator. 【Method】 bbbbbBased on the successful establishment of oxidative stress model in piglet and rat hippocampal neurons, the effects of exogenous GABA on piglets under oxidative stress, including oxidative / antioxidant related indexes, growth performance related indexes and GABA receptor in serum and hippocampus, along with the GABA receptor and apoptosis signal pathway related indexes in hippocampal neurons were investigated. 【Result】 The serum MDA levels of piglets in the low, medium, and high concentration GABA feeding groups (LD+OS; MD+OS; HD+OS) were extremely significantly lower than those in the oxidative stress (OS) group (P<0.01), the GSH level was extremely significantly higher than that in the OS group (P<0.01), and the T-AOC level in the HD+OS group was extremely significantly higher than that in the OS group and control group (P<0.01). And the high concentration (100 mg·kg-1 BW) of GABA more decreased the MDA level and more increased the GSH level in serum of piglets than those of the low concentration (20 mg·kg-1 BW) and middle concentration (60 mg·kg-1 BW) of GABA. Therefore, the follow-up study only investigated the anti-oxidative stress effect and mechanism of 100 mg·kg-1 BW. The daily gains of piglets at 0-7 days, 8-14 days and 0-28 days in the OS group were extremely significantly lower than those in the control group (P<0.01),the daily gains of piglets at 0-7 days, 8-14 days and 0-28 days in the HD+OS group were extremely significantly higher than those in the OS group (P<0.01), the daily gain of piglets aged 15-28 days in the OS group was significantly lower than that in the control group (P<0.05),the daily gain of piglets aged 15-28 days in the HD+OS group was extremely significantly higher than that in the OS group (P<0.01). The above results showed that the 100 mg·kg-1 BW GABA feeding extremely significantly increased the daily gain of piglets. There was no significant difference in the diarrhea rate of the control group, OS group and HD+OS group in the first, middle and late stages (P>0.05). The MDA level of hippocampus in the OS group was significantly higher than that of the control group and HD+OS group (P<0.01), the T-AOC and GSH levels were extremely significantly lower than the other two groups (P<0.01), which showed that GABA feeding would improve the antioxidant capacity of hippocampus. The levels of GABAA and GABAB receptors in the hippocampus of the HD+OS group were extremely significantly higher than those in the control group and the OS group (P<0.01), which showed that GABA feeding increased the levels of GABAA and GABAB in hippocampus. The Bcl-2 level in the hippocampus of the OS group was significantly lower than that of the control group (P<0.05), and the Bax and Caspase-3 levels were extremely significantly higher than those of the control group (P<0.01). The Bcl-2 level of the HD+OS group was extremely significantly higher than that of the OS group (P<0.01), the Bax level was extremely significantly lower than that of the OS group (P<0.01), and the Caspase-3 level was significantly lower than that of the OS group (P<0.05). Consistent with this, the Bax and Caspase-3 levels in hippocampal neurons of rats in the OS group, GABA+OS+Picrotoxin group and GABA+OS+CGP54626 group were significantly higher than those in the control group and GABA group (P<0.05), which indicated that GABA alleviated the damage of hippocampal neurons under oxidative stress, and the addition of GABA receptor inhibitors blocked the anti-stress damage effect of GABA. 【Conclusion】 GABA reduced the level of oxidative stress in the hippocampus of piglets. The anti-stress mechanism of GABA could be related to the decrease of the expression of apoptotic protein gene, while GABAA and GABAB receptors mediated this process. Table and Figures | Reference | Related Articles | Metrics

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Effects of Heat Stress on Ileal Microbiota of Broilers WANG XueJie,XING Shuang,ZHAO ShaoMeng,ZHOU Ying,LI XiuMei,LIU QingXiu,MA DanDan,ZHANG MinHong,FENG JingHai Scientia Agricultura Sinica    2022, 55 (17): 3450-3460.   DOI: 10.3864/j.issn.0578-1752.2022.17.015 Abstract （194）   HTML （26）    PDF （1273KB）（80）       Knowledge map    Save 【Objective】 To explore how heat stress influences the composition and structure of intestinal microbiota in broilers, and to provide a basis for alleviating the adverse effects of heat stress. 【Method】 144 broilers ((1 771±40) g, 35 d) were randomly divided into three groups with six replicates and raised in three controlled climate chambers from 35 to 42 days in this study. They were allocated to one of a thermoneutral control group (TC, 21℃, ad libitum), a heat stress group (HT, 31℃, ad libitum), or a pair-feeding group（PF, 21℃, pair-feeding as much as the consumption of the HT birds）. 【Result】 Compared with broilers in TC, the feed intake and body weight gain were significantly decreased and F/G was increased in broilers of HT (P<0.01), the feed intake and body weight gain were also significantly decreased in broilers of PF (P<0.01). The feed intake of broilers in HT was as same as it in PF, but the body weight gain in HT was lower than it in PF, and the F/G was higher (P<0.05), which indicated that the heat stress still decreased the body weight gain of broilers even at the same level of feed intake. Compared with broilers in TC, the core temperature (P=0.079) and serum corticosterone content (P=0.071) have an increasing tendency in broilers of HT, and have similar levels in PF (P>0.05). The richness of species（sobs and Chao1）and diversity indices（Shannon）of samples in the HT and PF group were higher than those in the TC group (P<0.05). There was no significant difference in terms of the diversity estimators of the ileal microbiota between the HT and the PF groups. The PCoA showed that the HT and PF groups were gathered together but were distinctly separated from the TC group. This tendency was also verified by the similarity analysis. Moreover, the feed consumption was significantly correlated with the principal coordinate 1 in the PCoA (R = 0.786, P<0.001), but the body temperature or the serum corticosterone was not correlated with the principal coordinate 1 (P>0.05). Furthermore, 18 differential genera between the HTG and the TC groups were identified by LEfSe analysis (score ≥ 3). The relative abundance of these genera in the PF and HT groups was similar but had significant differences compared with that in the TC group. In addition, among the 50 most abundant genera in the ileal microbiota, the feed consumption was significantly correlated with 30 genera, but the body temperature and serum corticosterone were significantly correlated with only two and no genera, respectively. 【Conclusion】 The core temperature and serum corticosterone content were different, the feed intake was similar between HT and PF, and the composition and community structure of ileal microbiota were similar between those two groups; the core temperature and serum corticosterone content were similar, but the feed intake was different between TC and PF, and the composition and community structure of ileal microbiota were also different between those two groups, these results indicate that the effects of constant heat stress on composition and community structure of ileal microbiota in broilers are related to reduced feed consumption. This conclusion was also proved by the results of correlation analysis. Table and Figures | Reference | Related Articles | Metrics

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Effect of Allium mongolicum Regel and Its Extracts on the Growth Performance, Carcass Characteristics, Meat Quality and Serum Biochemical Indices of Captive Small-Tailed Han Sheep LIU WangJing,TANG DeFu,AO ChangJin Scientia Agricultura Sinica    2022, 55 (17): 3461-3472.   DOI: 10.3864/j.issn.0578-1752.2022.17.016 Abstract （178）   HTML （36）    PDF （533KB）（87）       Knowledge map    Save 【Objective】 The aim of this experiment was to investigate the effects of Allium mongolicum Regel and its extracts on the production performance, carcass characteristics, meat quality and serum biochemical indexes of Small Tailed-Han Sheep, and to provide an experimental basis for the development of feed additives for captive sheep. 【Method】 Sixty 3-month-old, male, Small-Tailed Han sheep were selected and allocated into four groups in a randomized block design. There were four treatments with 3 replications each: 1) a basal diet as the control group; 2) the basal diet supplemented with 10 g•lamb-1•d-1 Allium mongolicum Regel powder as the AMR group; 3) the basal diet supplemented with 3.4 g•lamb-1•d-1 Allium mongolicum Regel water extract as the AWE group; and 4) the basal diet supplemented with 2.8 g•lamb-1•d-1 Allium mongolicum Regel ethanol extract as the AFE group. The experiment lasted for 75 days, including a 15-day preliminary feeding period for adaptation and a 60-day experimental feeding period. The production performance of small-tailed Han sheep was measured during experimental feeding period, and the serum biochemical indexes were measured by fasting jugular blood collection at the end of experimental feeding period, followed by slaughter of 2 sheep randomly selected in each replication and carcass characteristics (Hot carcass weight, Carcass yield, perirenal adipose tissue weight, dorsal subcutaneous adipose tissue thickness and firmness) were measured immediately, meat quality was determined by taking Longissimus Dorsi muscle on the left side of the carcass, and fat color was determined by taking perirenal adipose tissue, tail adipose tissue, dorsal subcutaneous adipose tissue, omental adipose tissue. 【Result】 The results showed that: compared to the control group, ① the mean daily feed intake was significantly lower (P=0.001), feed conversion ratio was significantly higher (P=0.039) and mean daily weight gain did not differ (P>0.05) for AFE group; ② the addition of Allium mongolicum Regel powder and water extracts was able to significantly reduce the shear force of lamb compared to the control group (P=0.029); ③ there was no significant difference (P>0.050) in the b* values of perirenal adipose tissue in other groups, while the b* values of perirenal adipose tissue (P=0.096) and omental adipose tissue (P=0.087) in the alcohol extract group tended to be higher compared with Allicum mongolicum Regel powder group, while the a* (P=0.044) and C* (P=0.021) values were significantly higher than those of Allicum mongolicum Regel powder group; ④ the addition of Allium mongolicum Regel and its extract significantly increased the serum total protein (P=0.006), Allium mongolicum Regel and its alcoholic extract significantly increased the serum albumin (P=0.006), and Allium mongolicum Regel alcoholic extract significantly decreased the serum urea nitrogen (P=0.024). 【Conclusion】 In conclusion, Allium mongolicum Regel and its extracts had no significant effect on carcass characteristics and fat color of captive Small Tailed-Han Sheep. The addition of Allium mongolicum Regel and its alcoholic extract to the diet significantly improved the feed conversion rate of Small-Tailed Han sheep, and Allium mongolicum Regel powder and water extract significantly reduced the shear force of meat. Meanwhile, Allium mongolicum Regel and its alcoholic extract can significantly increase the content of total protein and albumin in serum and promote the absorption of protein of meat sheep. Table and Figures | Reference | Related Articles | Metrics

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Non-Starch Polysaccharide Enzymes Cocktail of Corn-Miscellaneous Meal-Based Diet Optimization by In Vitro Method and Its Effects on Intestinal Microbiome in Finishing Pigs DENG FuLi,SHEN Dan,ZHONG RuQing,ZHANG ShunFen,LI Tao,SUN ShuDong,CHEN Liang,ZHANG HongFu Scientia Agricultura Sinica    2022, 55 (16): 3242-3255.   DOI: 10.3864/j.issn.0578-1752.2022.16.014 Abstract （514）   HTML （40）    PDF （861KB）（125）       Knowledge map    Save 【Objective】 The objective of this study was to optimize the non-starch polysaccharide (NSP) enzymes cocktail of the corn-miscellaneous meal-based diet for finishing pigs by using in vitro simulation method, and to analyze the effects of the optimal NSP enzymes cocktail (OEC) on dietary nutrient digestibility and intestinal microbial composition and structure of finishing pigs. Finally, it could provide data support and theoretical reference for efficient utilization of diets and precise feeding. 【Method】 In experiment 1, different levels of six NSP enzymes (xylanase, β-glucanase, cellulase, α-galactosidase, β-mannanase, and pectinase) were individually and respectively added to the corn-miscellaneous meal-based diet of finishing pigs. Then, in vitro ileal dry matter digestibility (IVIDMD) was determined by gastric-small intestinal simulation digestion method in vitro. When IVIDMD reached the maximum, the supplemental level of each NSP enzyme was the coding level of NSP enzyme 0. In vitro digestion experiments were carried out according to the six-element quadratic regression orthogonal rotation combination design. Meanwhile, the optimal NSP enzymes cocktail (OEC) of the corn-miscellaneous meal-based diet was selected by establishing the six-element quadratic regression equation between IVIDMD and the supplemental level of NSP enzymes. The in vitro dry matter digestibility (IVDMD), in vitro gross energy digestibility (IVGED) and in vitro digestible energy (IVDE) of diets before and after OEC addition were determined by gastric-small intestinal-large intestinal simulation digestion method in vitro to verify the effect of OEC. In experiment 2, 16 healthy castrated barrows (117.8 ± 1.66 kg) with similar body weight were randomly divided into two groups with eight pigs in each group. The pigs in the control group were fed the corn-miscellaneous meal-based diet, and the pigs in the enzyme-addition group were fed the basal diet supplemented with OEC. On the 18th day of the experiment, the fresh feces of pigs were collected by rectal wiping method, and the diversity and relative abundance of fecal microbiome were analyzed by high-throughput sequencing analysis of 16S rRNA gene, and the function was predicted. 【Result】 (1) Under the conditions of this experiment, the optimized NSP enzymes cocktail of corn-miscellaneous meal-based diet was as follows: cellulase 1 003 U·kg-1, xylanase 18 076 U·kg-1, β-glucanase 1 377 U·kg-1, β-mannanase 14 765 U·kg-1, α-galactosidase 337 U·kg-1, and pectinase 138 U·kg-1. (2) Adding NSP enzymes cocktail optimized by in vitro method in corn-miscellaneous meal-based diet significantly increased the IVDMD from 73.44% to 76.26% (P<0.01), the IVGED from 74.03% to 76.45% (P = 0.01), and the IVDE from 14.97 MJ·kg-1 to 15.58 MJ·kg-1 (P<0.01). (3) At the phylum level, a total of 12 phyla with relative abundances greater than 0.1% were selected, among which Bacteroidetes, Firmicutes, and Spirochetes were the dominant phyla, and the sum of these three phyla accounted for more than 96% in the group. (4) At the genus level, adding OEC in the diet significantly increased the relative abundance of Norank_F_F082, Norank_F_Bacteroidales_ RF16_group, Bacteroides and Roseburia (P<0.05), and Eubacterium_ruminantium_group (P = 0.083) had an increasing trend, while the relative abundance of Oscillibacter decreased significantly (P<0.05), and Clostridium_Sensu_Stricto_1 and Norank_F__Norank_O__ WCHB1-41 (P = 0.083) showed a decreasing trend (P = 0.052). 【Conclusion】 Dietary non-starch polysaccharide enzymes cocktail optimization by in vitro method increased in vitro digestibility of dry matter and energy and in vitro digestible energy of corn-miscellaneous meal-based diets in finishing pigs. It also increased the proportion of beneficial bacteria in intestinal microorganism, such as fiber decompose bacteria and butyric acid producing bacteria, and reduced the number of harmful bacteria to a certain extent, and optimized intestinal microecology. Table and Figures | Reference | Related Articles | Metrics

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Preparation and Identification of Monoclonal Antibodies to P30 Protein and Establishment of Blocking ELISA to Detecting Antibodies Against African Swine Fever Virus ZHANG FengXi,XIAO Qi,ZHU JiaPing,YIN LiHong,ZHAO XiaLing,YAN MingShuai,XU JinHua,WEN LiBin,NIU JiaQiang,HE KongWang Scientia Agricultura Sinica    2022, 55 (16): 3256-3266.   DOI: 10.3864/j.issn.0578-1752.2022.16.015 Abstract （572）   HTML （52）    PDF （1870KB）（192）       Knowledge map    Save 【Background】 African swine fever (ASF) first appeared in China in August 2018, causing great harm to the pig industry and heavy losses. At present, there is no safe and effective vaccine to prevent ASF, so the establishment of rapid and specific detection method provides an effective means for the prevention and control of ASF. 【Objective】To prepare specific monoclonal antibodies against African swine fever virus (ASFV) and establish a rapid and specific detection method for ASF. Provide technical means for the detection and prevention and control of ASF. 【Method】The expression vector pET-28a-P30 was constructed and the rP30 protein was obtained by prokaryotic expression system. The purified rP30 protein was used as antigen to immunize BALB/c mice. The specific hybridoma cell line of ASFV P30 protein was prepared by cell fusion and cell subcloning. The truncated expression of P30 protein was performed, and Western Blot and indirect enzyme-linked immunosorbent assay (iELISA) were used to identify the antigen epitope corresponding to the monoclonal antibody. The prepared monoclonal antibody was used to establish the detection method of blocking ELISA antibody for ASF. 【Result】 The results of double digestion and PCR showed that the recombinant vector pET-28a-P30 was constructed, and the sequence was not mutated by sequencing. After IPTG induction, the recombinant P30 protein was mainly expressed in the inclusion body with a molecular weight of about 33 kD. The purified P30 protein was mixed with 1﹕1 Freund's adjuvant to immunize mice. After three immunizations, the serum titer of mice reached 1﹕102 400, indicating that the expressed protein had good immunogenicity. Eight P30 protein-specific hybridoma cells were obtained by cell fusion and subcloning. The eight monoclonal antibodies obtained by Western Blot and indirect immunofluorescence assay (IFA) showed good reactivity. The superposition test showed that all the 8 monoclonal antibodies had the same antigenic sites. Different fragments of P30 protein were truncated, and the prepared 2-12B monoclonal antibodies were selected to react with different truncated P30 proteins, showing that the antigenic epitope region of the monoclonal antibodies was 187-194aa. The ASF blocking ELISA antibody detection method was successfully established by using 2-12B monoclonal antibody and optimizing the conditions. 190 clinical samples were detected and compared with commercial African swine fever ELISA antibody detection kit. The positive coincidence rate of the two methods was 90.91 %, and the total coincidence rate was 96.32 %. 【Conclusion】 In this study, ASFV P30 protein was successfully obtained. Eight specific monoclonal antibodies with good reactivity were screened by iELISA, Western Blot and IFA, and the antigen recognition epitopes were 187-194 aa. The monoclonal antibody was used to establish a high specificity and sensitivity ASFV blocking ELISA antibody detection method, which provided a means and support for the detection and prevention of ASF. Table and Figures | Reference | Related Articles | Metrics

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