CRISPR-Cas基因编辑技术在羊生产应用中研究进展

基因编辑是一种能够进行基因组修饰的基因工程技术。近年来,随着分子生物学技术的飞速发展,成簇规则间隔短回文重复序列相关蛋白系统(clustered regularly interspaced short palindromic repeats associated protein,CRISPR-Cas)作为新发展起来的一种强大的基因编辑工具,因具有高效性、精确性和灵活性而被广泛应用。CRISPR-Cas系统通过在特定基因组位点引入插入、缺失或单碱基替换等方式实现位点的特异性修饰,在畜牧生产的诸多领域做出了重大贡献。在羊生产应用方面,通过建立改善生产经济性状和抗病性的绵山羊动物模型,可以对关键基因的功能进行研究,从而加速性状的改良。本文主要综述了CRISPR-Cas系统的机制与功能及其在羊繁殖性状、肉用性状、产毛性状、泌乳性状和抗病性状生产应用与创建羊动物模型方面的研究进展。

Rumen microbiome structure and metabolites activity in dairy cows with clinical and subclinical mastitis

Background:Due to the high prevalence and complex etiology,bovine mastitis(BM)is one of the most important diseases to compromise dairy cow health and milk quality.The shift in milk compositions has been widely investigated during mastitis,but recent studies suggested that gastrointestinal microorganism also has a crucial effect on the inflammation of other peripheral tissues and organs,including the mammary gland.However,research focused on the variation of rumen inner-environment during mastitis is still limited.Therefore,the ruminal microbial profiles,metabolites,and milk compositions in cows with different udder health conditions were compared in the present study.Furthermore,the correlations between udder health status and ruminal conditions were investigated.Based on the somatic cell counts(SCC),California mastitis test(CMT)parameters and clinical symptoms of mastitis,60 lactating Holstein dairy cows with similar body conditions(excepted for the udder health condition)were randomly divided into 3 groups(n=20 per group)including the healthy(H)group,the subclinical mastitis(SM)group and the clinical mastitis(CM)group.Lactation performance and rumen fermentation parameters were recorded.And rumen microbiota and metabolites were also analyzed via 16S rRNA amplicon sequencing and untargeted metabolomics,respectively.Results:As the degree of mastitis increased,rumen lactic acid(LA)(P<0.01),acetate,propionate,butyrate,valerate(P<0.001),and total volatile fatty acids(TVFAs)(P<0.01)concentrations were significantly decreased.In the rumen of CM cows,the significantly increased bacteria related to intestinal and oral inflammation,such as Lachnospiraceae(FDR-adjusted P=0.039),Moraxella(FDR-adjusted P=0.011)and Neisseriaceae(FDR-adjusted P=0.036),etc.,were accompanied by a significant increase in 12-oxo-20-dihydroxy-leukotriene B4(FDR-adjusted P=5.97×10^(−9))and 10beta-hydroxy-6beta-isobutyrylfuranoeremophilane(FDR-adjusted P=3.88×10^(−10)).Meanwhile,in the rumen of SM cows,the Ruminiclostridium_9(FDR-adjusted P=0.042)and Enterorhabdus(FDR-adjusted P=0.043)were increased along with increasing methenamine(FDR-adjusted P=6.95×10^(−6)),5-hydroxymethyl-2-furancarboxaldehyde(5-HMF)(FDR-adjusted P=2.02×10^(−6))and 6-methoxymellein(FDR-adjusted P=2.57×10^(−5)).The short-chain fatty acids(SCFAs)-producing bacteria and probiotics in rumen,including Prevoterotoella_1(FDRadjusted P=0.045)and Bifidobacterium(FDR-adjusted P=0.035),etc.,were significantly reduced,with decreasing 2-phenylbutyric acid(2-PBA)(FDR-adjusted P=4.37×10^(−6)).Conclusion:The results indicated that there was a significant shift in the ruminal microflora and metabolites associated with inflammation and immune responses during CM.Moreover,in the rumen of cows affected by SM,the relative abundance of several opportunistic pathogens and the level of metabolites which could produce antibacterial compounds or had a competitive inhibitory effect were all increased.

Variation Regulation and Models of Raw Milk Composition of Chinese Holstein Cattle in Tianjin

Based on raw milk DHI data of Chinese Holstein cattle in northern China,milk composition (milk protein percentage and milk fat percentage) of lactating cow is grouped into parity 1 to 4. After preprocessing original data,6114 data records of milk protein percentage and 5871 data records of milk fat percentage were obtained. This study discusses effects of natural months,lactation parity and their interaction on changes of milk protein percentage and milk fat percentage,and the model is established using GLM procedure of SAS software. At last,results are as follows: (i) Duncan multiple comparison of natural months,regardless of parity (only parity 1 to 4) ,indicates that milk composition takes on significant difference between different months (P < 0. 05) . And milk protein percentage reaches highest in September (3. 187%), drops to the lowest in July (3. 016%); the milk fat percentage reaches highest in February (4. 137%),and drops to the lowest in July (3.845%) . (ii) Duncan multiple comparison of different parity,regardless months (January to December) ,shows that milk composition of different parity also takes on significant difference (P < 0. 05) although the difference between parities are not significant; milk protein percentage reaches highest in the 2nd parity (3. 114%)and drops to the lowest in the 4th parity (3. 066%); milk fat percentage reaches highest in the 2nd and 3rd parity (3. 983% and 3. 973%),and drops to the lowest in the 4th parity (3. 923%). (iii) Using Wood model,the relational expression between milk protein percentage (MPP,%)and milk fat percentage (MFP,%)of different parity and natural month,i. e. MPP = 3. 094x - 0. 046 4 × e 0. 011 7x and MFP = 4. 211 6x - 0. 034 4 × e 0. 027 6x (x stands for month) . According to the above results,it is concluded that natural months,lactation parity and their interaction significantly influence milk protein percentage and milk fat percentage (P < 0. 001) ,and milk protein percentage and milk fat percentage take on Wood model change characteristics with natural months respectively. This study is intended to explore change regulation of milk composition,and to provide decision reference for properly regulating feeding management and nutrition supply of cattle,and thereby guaranteeing the quality of raw milk in certain month reach sales standard.

Recent Advance in Division of Carbohydrate and Protein Fractions of Ruminant Feed and Their Metabolism in Digestive Tract

Accurate assessment of feed's Carbohydrate( CHO) and protein nutritional values and rumen metabolism are significant for dairy production. Cornell Net Carbohydrate and Protein System( CNCPS) as an important method to evaluate feedstuff nutritional values,hasn't been widely used in China. In order to illustrate updates of CNCPS systems deeply,the following sections were reviewed:( i) CHO and protein fractions were updated,CA was subdivided into CA1,CA2,CA3 and CA4 in CNCPS v6. 1,protein was reclassified into PA1,PA2,PB1,PB2 and PC after CNCPS v6. 1. Content of CHO and protein fractions vary in different feedstuff and affected by feed processing;( ii) Degradation rates( Kd) values for the new CA expanded scheme were updated to 0,7,5,40- 60 % h^(-1)respectively,Kd for PA and PB1 decreased to 200 % h^(-1)and 10- 40 % h^(-1);( iii) Equations for passage rate( Kp) initially includes Kpf( Kp of forages) and Kpc( Kp of concentrates),and adjusted by effective NDF( e NDF),while in CNCPS v5. 0,Kpl( Kp of liquids) equation was added and e NDF was replaced by physically effective NDF( pe NDF). In CNCPS v6. 1,Fp BW and Cp BW were integrated into Kp equations and pe NDF was abandoned.( iv)The relationship and difference among Weende system of proximate analysis,Van Soest fiber analysis~[35],NRC( 2001)~[28]and CNCPS were analyzed. The first two systems laid the foundation for NRC( 2001) and CNCPS system. The latter two systems are different in CHO and protein division,also NRC( 2001) developed separate Kp equations for wet and dry forages but no equation for Kpl. CNCPS developed a Kp equation that work for wet and dry forages,and Kpl equation was established. In conclusion,the division and development of CHO and protein fractions,the update of Kd and Kp equation were reviewed systematically.

Vision-based measuring method for individual cow feed intake using depth images and a Siamese network

Feed intake is an important indicator to reflect the production performance and disease risk of dairy cows,which can also evaluate the utilization rate of pasture feed.To achieve an automatic and non-contact measurement of feed intake,this paper proposes a method for measuring the feed intake of cows based on computer vision technology with a Siamese network and depth images.An automated data acquisition system was first designed to collect depth images of feed piles and constructed a dataset with 24150 samples.A deep learning model based on the Siamese network was then constructed to implement non-contact measurement of feed intake for dairy cows by training with collected data.The experimental results show that the mean absolute error(MAE)and the root mean square error(RMSE)of this method are 0.100 kg and 0.128 kg in the range of 0-8.2 kg respectively,which outperformed existing works.This work provides a new idea and technology for the intelligent measuring of dairy cow feed intake.

Nutritional value, bioactivity, and application potential of Jerusalem artichoke(Helianthus tuberosus L.) as a neotype feed resource

The large-scale development of herbivorous animal husbandry in China has increased the demand for forage products.However,due to scarce land resources and poor soil quality,forage is in short supply.In particular,high-quality forage in China heavily relies on imports.The contradiction between supply and demand for forage grass products is increasingly notable.Therefore,the development of indigenous new forage resources with a strong ecological adaptability and a high nutritional value is a key to solving this problem.Jerusalem artichoke(JA,Helianthus tuberosus L.),a perennial herb of the genus Helianthus,has advantageous growth traits such as resistance to salinity,barrenness,drought,cold,and disease.The contents of crude protein,crude fiber,and calcium in the optimal harvest period of forage-type JA straw are comparable to those of alfalfa hay at the full bloom stage and the straw of ryegrass and corn at the mature stage.Inulin in JA tubers is a functional ingredient that has prebiotic effects in the gastrointestinal tract of monogastric animals and young ruminants.In addition,some bioactive substances(e.g.flavonoids,phenolic acids,sesquiterpenes,polysaccharides,and amino acids)in JA leaves and flowers have antibacterial,anti-inflammatory,and antioxidant functions as well as toxicities to cancer cells.These functional ingredients may provide effective alternatives to antibiotics used in livestock production.In this review,we summarized the potentials of JA as a feed ingredient from the aspects of nutritional value and fermenting characteristics of the straw,the functions of physiological regulation and disease prevention of inulin in the tubers,and bioactive substances in the leaves and flowers.

Dietary supplementation with inulin improves lactation performance and serum lipids by regulating the rumen microbiome and metabolome in dairy cows

This study investigated the effects of inulin on rumen fermentation parameters,ruminal microbiome and metabolites,as well as lactation performance and serum indexes in dairy cows.Sixteen Holstein dairy cows with similar body conditions were randomly divided into 2 groups(n=8 per group),with inulin addition at 0 and 200 g/d percow.The experiment lasted for 6 weeks,including a 1-week adaptation period and a 5-week treatment period.At the end of the experimental period,the milk,serum and rumen fluid were sampled and analyzed.The microbiome and metabolome in the rumen fluid were analyzed via 16 S rRNA sequencing and untargeted metabolomics,respectively.The results showed that supplementation with inulin(200 g/d per cow)increased the milk yield(P=0.001),milk protein(P=0.032),lacto se rate(P=0.004)and proportion of saturated fatty acids(SFA)in milk(P<0.001),but decreased the proportion of unsaturated fatty acids(USFA)(P=0.041).Rumen pH(P=0.040)and the concentration of NH3-N(P=0.024)were decreased;however,acetate(P<0.001),propionate(P=0.003),butyrate(P<0.001)and lactic acid(LA)(P=0.043)were increased.The total cholesterol(TC)(P=0.008)and triglycerides(TG)(P=0.01)in serum were also reduced.Additionally,inulin addition elevated the relative abundance of several beneficial symbiotic and short-chain fatty acid(SCFA)-producing bacteria,such as Muribaculaceae(false discovery rate[FDR]-adjusted P<0.01),Acetitomaculum(FDR-adjusted P=0.043),and Bu tyrivibrio(FDR-adjusted P=0.036),while elevating the levels of L-lysine(FDR-adjusted P=4.24×10^(-3)),L-proline(FDR-adjusted P=0.0158),and L-phenylalanine(FDRadj usted P=0.027).In contrast,several pathogens and ruminal bacteria abu ndant in high-fat diets,such as Escherichia-Sh igella(FD R-adj usted P=0.022),Erysipelo trichaceaeUCG-004(FD R-adjusted P<0.01)and RF39(FDR-adjusted P=0.042)were decreased along with the reduction of lysophosphatidylcholine(LysoPC)(18:1(9 Z))(FDR-adjusted P=1.03×10^(-3)),LysoPC(16:0)(FDR-adjusted P=0.0108),LysoPC(18:2(9 Z,12 Z))(FDRadjusted P=1.65×10^(-3))and 8-methylnonenoate.In conclusion,dietary inulin supplementation could increase the relative abundance of commensal microbiota and SCFA-producing bacteria,upregulate amino acidmetabolism and downregulate lipid metabolism in the rumen of dairy cows,which might further improve lactation performance and the level of serum lipids.