Repeated inoculation with rumen fluid accelerates the rumen bacterial transition with no benefit on production performance in postpartum Holstein dairy cows

Background The dairy cow’s postpartum period is characterized by dramatic physiological changes,therefore imposing severe challenges on the animal for maintaining health and milk output.The dynamics of the ruminal microbiota are also tremendous and may play a crucial role in lactation launch.We aim to investigate the potential benefits of early microbial intervention by fresh rumen microbiota transplantation(RMT)and sterile RMT in postpartum dairy cows.Twelve fistulated peak-lactation dairy cows were selected to be the donors for rumen fluid collection.Thirty postpartum cows were divided into 3 groups as the transplantation receptors respectively receiving 10 L fresh rumen fluid(FR),10 L sterile rumen fluid(SR),or 10 L saline(CON)during 3 d after calving.Results Production performance,plasma indices,plasma lipidome,ruminal microbiome,and liver transcriptome were recorded.After fresh and sterile RMT,we found that the molar proportion of propionic acid was increased on d 7 in the FR and SR groups and the bacterial composition was also significantly changed when compared with the CON group.A similarity analysis showed that the similarities between the CON group and FR or SR group on d 7 were 48.40%or 47.85%,whereas the similarities between microbiota on d 7 and 21 in the FR and SR groups were 68.34%or 66.85%.Dry matter intake and feed efficiency were not affected by treatments.Plasmaβ-hydroxybutyrate concentration in the FR group was decreased and significantly different lipids mainly included phosphatidylcholine and lysophosphatidylcholine containing polyunsaturated fatty acids.Hepatic transcriptomics analysis indicated acutephase response pathways were upregulated in the SR group.Conclusions Our study suggests that RMT can shorten the transition process of the ruminal microbiota of postpartum dairy cows with no benefit on dry matter intake or feed efficiency.Inoculation with rumen fluid may not be a useful approach to promote the recovery of postpartum dairy cows.

Multi‑omics reveals that the host‑microbiome metabolism crosstalk of differential rumen bacterial enterotypes can regulate the milk protein synthesis of dairy cows

Background Dairy cows’lactation performance is the outcome of the crosstalk between ruminal microbial metabo-lism and host metabolism.However,it is still unclear to what extent the rumen microbiome and its metabolites,as well as the host metabolism,contribute to regulating the milk protein yield(MPY).Methods The rumen fluid,serum and milk of 12 Holstein cows with the same diet(45%coarseness ratio),parity(2–3 fetuses)and lactation days(120–150 d)were used for the microbiome and metabolome analysis.Rumen metabolism(rumen metabolome)and host metabolism(blood and milk metabolome)were connected using a weighted gene co-expression network(WGCNA)and the structural equation model(SEM)analyses.Results Two different ruminal enterotypes,with abundant Prevotella and Ruminococcus,were identified as type1 and type2.Of these,a higher MPY was found in cows with ruminal type2.Interestingly,[Ruminococcus]gauvreauii group and norank_f_Ruminococcaceae(the differential bacteria)were the hub genera of the network.In addition,differential ruminal,serum and milk metabolome between enterotypes were identified,where the cows with type2 had higher L-tyrosine of rumen,ornithine and L-tryptophan of serum,and tetrahydroneopterin,palmitoyl-L-carnitine,S-lactoylglutathione of milk,which could provide more energy and substrate for MPY.Further,based on the identi-fied modules of ruminal microbiome,as well as ruminal serum and milk metabolome using WGCNA,the SEM analysis indicated that the key ruminal microbial module1,which contains the hub genera of the network([Ruminococcus]gauvreauii group and norank_f_Ruminococcaceae)and high abundance of bacteria(Prevotella and Ruminococcus),could regulate the MPY by module7 of rumen,module2 of blood,and module7 of milk,which contained L-tyrosine and L-tryptophan.Therefore,in order to more clearly reveal the process of rumen bacterial regulation of MPY,we established the path of SEM based on the L-tyrosine,L-tryptophan and related components.The SEM based on the metabolites suggested that[Ruminococcus]gauvreauii group could inhibit the energy supply of serum tryptophan to MPY by milk S-lactoylglutathione,which could enhance pyruvate metabolism.Norank_f_Ruminococcaceae could increase the ruminal L-tyrosine,which could provide the substrate for MPY.Conclusion Our results indicated that the represented enterotype genera of Prevotella and Ruminococcus,and the hub genera of[Ruminococcus]gauvreauii group and norank_f_Ruminococcaceae could regulate milk protein synthesis by affecting the ruminal L-tyrosine and L-tryptophan.Moreover,the combined analysis of enterotype,WGCNA and SEM could be used to connect rumen microbial metabolism with host metabolism,which provides a fundamental understanding of the crosstalk between host and microorganisms in regulating the synthesis of milk composition.

Effects of omega-3 supplementation on components of the endocannabinoid system and metabolic and inflammatory responses in adipose and liver of peripartum dairy cows

Background:Dietary supplementation of omega-3 fatty acids can reduce the activation of the endocannabinoid system(ECS)by decreasing the availability of arachidonic acid,thus lowering endocannabinoids(e CBs)levels.The ECS is a modulator of energy metabolism,stress response and inflammation in mammals,yet there is little information on the roles of the ECS in transition dairy cows.During the periparturient period,the adipose tissue and liver are the main metabolic organs that participate in the adaptations of dairy cows to onset of lactation;however,exceeded adipose tissue lipolysis and accumulation of lipids in the liver have adverse effects on cows'physiology.Here we aimed to examine whether omega-3 supplementation during the transition period will modulate ECS activation and affect metabolic and inflammatory indices in postpartum dairy cows,by supplementing twenty-eight transition Holstein dairy cows with either saturated fat(CTL)or encapsulated flaxseed oil(FLX).Components of the ECS,metabolic and inflammatory markers were measured in blood,liver,and subcutaneous adipose tissue.Results:FLX supplementation reduced feed intake by 8.1%(P<0.01)and reduced plasma levels of arachidonic acid(by 44.2%;P=0.02)and anandamide(by 49.7%;P=0.03)postpartum compared to CTL.The m RNA transcription levels of the cannabinoid receptor 1(CNR1/CB1)tended to be lower(2.5 folds)in white blood cells of FLX than in CTL(P=0.10),and protein abundance of ECS enzyme monoacylglycerol lipase was higher in peripheral blood mononuclear cells of FLX than in CTL(P=0.04).In adipose tissue,palmitoylethanolamide levels were lower in FLX than in CTL(by 61.5%;P=0.02),relative m RNA transcription of lipogenic genes were higher,and the protein abundance of cannabinoid receptor 2(P=0.08)and monoacylglycerol lipase(P=0.10)tended to be higher in FLX compared to CTL.Hepatic 2-arachidonoylglycerol tended to be higher(by 73.1%;P=0.07),and interlukin-6 m RNA transcription level was 1.5 folds lower in liver of FLX than in CTL(P=0.03).Conclusions:Nutritional supplementation of omega-3 fatty acids seems to partly modulate ECS activation,which could be related to lower feed intake.The altered ECS components in blood,adipose tissue and liver are associated with moderate modulations in lipid metabolism in the adipose and inflammation in liver of peripartum dairy cows.

Predicting nitrogen use efficiency,nitrogen loss and dry matter intake of individual dairy cows in late lactation by including mid‑infrared spectra of milk samples

Background Nitrate leaching to groundwater and surface water and ammonia volatilization from dairy farms have negative impacts on the environment.Meanwhile,the increasing demand for dairy products will result in more pollution if N losses are not controlled.Therefore,a more efficient,and environmentally friendly production system is needed,in which nitrogen use efficiency(NUE)of dairy cows plays a key role.To genetically improve NUE,extensively recorded and cost-effective proxies are essential,which can be obtained by including mid-infrared(MIR)spectra of milk in prediction models for NUE.This study aimed to develop and validate the best prediction model of NUE,nitrogen loss(NL)and dry matter intake(DMI)for individual dairy cows in China.Results A total of 86 lactating Chinese Holstein cows were used in this study.After data editing,704 records were obtained for calibration and validation.Six prediction models with three different machine learning algorithms and three kinds of pre-processed MIR spectra were developed for each trait.Results showed that the coefficient of determination(R2)of the best model in within-herd validation was 0.66 for NUE,0.58 for NL and 0.63 for DMI.For external validation,reasonable prediction results were only observed for NUE,with R2 ranging from 0.58 to 0.63,while the R2 of the other two traits was below 0.50.The infrared waves from 973.54 to 988.46 cm−1 and daily milk yield were the most important variables for prediction.Conclusion The results showed that individual NUE can be predicted with a moderate accuracy in both within-herd and external validations.The model of NUE could be used for the datasets that are similar to the calibration dataset.The prediction models for NL and 3-day moving average of DMI(DMI_a)generated lower accuracies in within-herd validation.Results also indicated that information of MIR spectra variables increased the predictive ability of models.Additionally,pre-processed MIR spectra do not result in higher accuracy than original MIR spectra in the external validation.These models will be applied to large-scale data to further investigate the genetic architecture of N efficiency and further reduce the adverse impacts on the environment after more data is collected.

The effect of ensiled paulownia leaves in a high-forage diet on ruminal fermentation,methane production,fatty acid composition,and milk production performance of dairy cows

Background:The use of industrial by-products rich in bioactive compounds as animal feeds can reduce greenhouse gas production.Paulownia leaves silage(PLS)was supplemented to dairy cows'diet and evaluated in vitro(Exp.1;Rusitec)and in vivo(Exp.2,cannulated lactating dairy cows and Exp.3,non-cannulated lactating dairy cows).The study investigated the PLS effect on ruminal fermentation,microbial populations,methane production and concentration,dry matter intake(DMI),and fatty acid(FA)proportions in ruminal fluid and milk.Results:Several variables of the ruminal fluid were changed in response to the inclusion of PLS.In Exp.1,the p H increased linearly and quadratically,whereas ammonia and total volatile fatty acid(VFA)concentrations increased linearly and cubically.A linear,quadratic,and cubical decrease in methane concentration was observed with increasing dose of the PLS.Exp.2 revealed an increase in ruminal p H and ammonia concentrations,but no changes in total VFA concentration.Inclusion of PLS increased ruminal propionate(at 3 h and 6 h after feeding),isovalerate,and valerate concentrations.Addition of PLS also affected several populations of the analyzed microorganisms.The abundances of protozoa and bacteria were increased,whereas the abundance of archaea were decreased by PLS.Methane production decreased by 11%and 14%in PLS-fed cows compared to the control in Exp.2 and 3,respectively.Exp.3 revealed a reduction in the milk protein and lactose yield in the PLS-fed cows,but no effect on DMI and energy corrected milk yield.Also,the PLS diet affected the ruminal biohydrogenation process with an increased proportions of C18:3 cis-9 cis-12 cis-15,conjugated linoleic acid,C18:1 trans-11 FA,polyunsaturated fatty acids(PUFA),and reduced n6/n3 ratio and saturated fatty acids(SFA)proportion in milk.The relative transcript abundances of the 5 of 6 analyzed genes regulating FA metabolism increased.Conclusions:The dietary PLS replacing the alfalfa silage at 60 g/kg diet can reduce the methane emission and improve milk quality with greater proportions of PUFA,including conjugated linoleic acid,and C18:1 trans-11 along with reduction of SFA.

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.

Accuracy comparison of dry matter intake prediction models evaluated by a feeding trial of lactating dairy cows fed two total mixed rations with different forage source

Dry matter intake （DMI） prediction models of NRC （2001）, Fox et aL （2004） and Fuentes-Pila et aL （2003） were targeted in the present study, and the objective was to evaluate their prediction accuracy with feeding trial data of 32 lactating Holstein cows fed two total mixed rations with different forage source. Thirty-two cows were randomly assigned to one of two total mixed ration groups： a ration containing a mixed forage （MF） of 3.7% Chinese wildrye, 28.4% alfalfa hay and 26.5% corn silage diet and another ration containing 33.8% corn stover （CS） as unique forage source. The actual DMI was greater in MF group than in CS group （P=0.064）. The NRC model to predict DMI resulted in the lowest root mean square prediction error for both MF and CS groups （1.09 kg d-1 vs. 1.28 kg d-1） and the highest accuracy and precision based on concordance correlation coefficient for both MF and CS diet （0.89 vs. 0.87）. Except the NRC model, the other two models presented mean and linear biases in both MF and CS diets when prediction residuals were plotted against predicted DMI values （P〈0.001）. The DMI variation in MF was caused by week of lactation （55.6%）, milk yield （13.9%）, milk fat percentage （7.1%） and dietary neutral detergent fiber （13.3%）, while the variation in CS was caused by week of lactation （50.9%）, live body weight （28.2%）, milk yield （8.4%）, milk fat percentage （5.2%） and dietary neutral detergent fibre （3.8%）. In a brief, the NRC model to predict DMI is comparatively acceptable for lactating dairy cows fed two total mixed rations with different forage source.

Effects of far-off and close-up transition cow feeding on uterine health,postpartum anestrous interval,and reproductive outcomes in pasture-based dairy cows

Background:In seasonal,pasture-based,dairy production systems,cows must recover from calving and become pregnant within two to 3 months.To achieve this,the uterus must involute and ovulation must occur and continue at regular intervals.As these processes are affected by the cows’nutritional or metabolic status post-calving,the objective of this study was to evaluate the effect of cow feeding strategies on uterine health,the length of postpartum anestrous interval,and reproductive outcomes.The treatments consisted of two feeding strategies during late-lactation and early dry period(far-off period;starting 4-month pre-calving)and three close-up dry period feeding regimes(1-month pre-calving)in a 2×3 factorial arrangement.We randomly assigned 150 cows to one of two far-off treatments.During late lactation,the herds(n=75 cows per herd)were either control-fed(Controlfed)or over-fed(Overfed)to achieve a low or high body condition score(4-month pre-calving;BCS;~4.25 and^4.75;10-point scale)at cessation of lactation.Within each of these treatments,three feeding levels were applied during the close-up period(1-month pre-calving):~65%(Feed65),~90%(Feed90),or^120%(Feed120)of metabolizable energy(ME)intakes relative to pre-calving requirements.Results:Uterine health improved(i.e.polymorphonucleated(PMN)cells declined)with increased feeding during the close-up period for cows in the Overfed group.The reverse was evident for the Controlfed group with the greatest PMN at the highest intakes during the close-up period.The postpartum anoestrous interval(PPAI)was shorter in cows from the Overfed group when moderately fed(Feed90)during the close-up period;in comparison,the PPAI was shorter in the Controlfed group,when those cows were overfed in the close-up period(Feed120).The cows in the Overfed treatment had greater conception and pregnancy rates if cows had moderate dry matter intakes(Feed90)during the close-up period;these reproductive variables were less under excessive feed intakes(Feed120);yet,close-up dry matter intake had little effect on conception and pregnancy rates for the Controlfed group.Conclusions:The far-off feeding strategies increased early reproductive outcomes at 3 weeks of mating.Additionally,the interaction between far-off and close-up feeding strategies resulted in high six-week pregnancy rate with a slight restriction during the close-up period but only in the far-off Overfed group.Thus,our hypothesis is supported under these conditions.

Effects of Bupleurum extract on blood metabolism, antioxidant status and immune function in heat-stressed dairy cows

This experiment was conducted to evaluate the effects of Bupleurum extract （BE） on blood metabolites, antioxidant status, and immune function in dairy cows under heat stress. Forty lactating Holstein cows were randomly assigned to 1 of 4 treatments. The treatments consisted of 0, 0.25, 0.5, and 1.0 g of BE kg-1 dry matter. Supplementation with BE decreased （P〈0.05） blood urea nitrogen （BUN） contents and increased blood total protein （TP） and albumin （ALB） levels compared with control cows, but it had no effects （P〉0.05） on blood glucose （GLU）, nonesterified fatty acid （NEFA）, total triglycedde （TG）, low-density lipoprotein cholesterol （LDL-C）, and high density lipoprotein cholesterol （HDL-C）. Compared with control cows, cows fed BE had higher （P〈0.05） superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px） activity. However, supplementation with BE had no effect （P〉0.05） on total antioxidant capacity （T-AOC） or malondialdehyde （MDA） levels. The immunoglobulin （Ig） A and G contents increased （P〈0.05） in cows fed 0.25 or 0.5 g of BE kg-1. Interleukin （IL）-2 and IL-4 levels were higher （P〈0.05） in cows fed 0.5 and 1.0 g of BE kg-1, and IL-6 was significantly elevated （P〈0.05） in cows fed 0.5 g of BE kg-1. There were no treatment effects （P〉0.05） on the CD4＋ and CD8＋ T lymphocyte ratios, CD4＋/CD8＋ratio, or tumor necrosis factor-α （TNF-α） level among the groups. These findings suggest that BE supplementation may improve protein metabolism, in addition to enhancing antioxidant activity and immune function in heat-stressed dairy cows.

Characterization and mitigation option of greenhouse gas emissions from lactating Holstein dairy cows in East China

Background:This study investigated greenhouse gas(GHG)emission characteristics of lactating Holstein dairy cows in East China and provided a basis for formulating GHG emission reduction measures.GreenFeed system was used to measure the amount of methane(CH_(4))and carbon dioxide(CO_(2))emitted by the cows through respiration.Data from a commercial cow farm were used to observe the effects of parity,body weight,milk yield,and milk component yield on CH_(4) and CO_(2) emissions.Results:Mean herd responses throughout the study were as follows:111 cows completed all experimental processes,while 42 cows were rejected because they were sick or had not visited the GreenFeed system 20 times.On average,lactating days of cows was 138±19.04 d,metabolic weight was 136.5±9.5 kg,parity was 2.8±1.0,dry matter intake(DMI)was 23.1±2.6 kg/d,and milk yield was 38.1±6.9 kg/d.The GreenFeed system revealed that CH_(4) production(expressed in CO_(2) equivalent,CO_(2)-eq)was found to be 8304 g/d,CH_(4)_(CO_(2)−eq)/DMI was 359 g/kg,CH_(4)_(CO_(2)−eq)/energy-corrected milk(ECM)was 229.5 g/kg,total CO_(2) production(CH_(4) production plus CO_(2) production)was 19,201 g/d,total CO_(2)/DMI was 831 g/kg,and total CO_(2)/ECM was 531 g/kg.The parity and metabolic weight of cows had no significant effect on total CO_(2) emissions(P>0.05).Cows with high milk yield,milk fat yield,milk protein yield,and total milk solids yield produced more total CO_(2)(P<0.05),but their total CO_(2) production per kg of ECM was low(P<0.05).The total CO_(2)/ECM of the medium and high milk yield groups was 17%and 27%lower than that of the low milk yield group,respectively.Conclusions:The parity and body condition had no effect on total CO_(2) emissions,while the total CO_(2)/ECM was negatively correlated with milk yield,milk fat yield,milk protein yield,and total milk solids yield in lactating Holstein dairy cows.Measurement of total CO_(2) emissions of dairy cows in the Chinese production system will help establish regional or national GHG inventories and develop mitigation approaches to dairy production regimes.

Effect of dietary phosphorus content on milk production and phosphorus excretion in dairy cows

Background：Phosphorus（P） supplementation is costly and can result in excess P excretion.This study investigated the effects of reducing dietary P on milk production and P excretion in dairy cows over a full lactation.Method：Forty-five multiparous Holstein dairy cows were divided into 15 blocks according to expected calving date and previous milk yield,and assigned randomly to one of the three dietary treatments：0.37,0.47,and 0.57%P（DM basis）;these P levels represent the NRC recommendations,Chinese recommendations,and the amount of dietary P commonly fed by Chinese dairy farmers,respectively.Average daily feed intake was calculated from monthly data on feed offered and refused.Milk yields of individual cows were recorded weekly,and milk samples were taken for analysis of protein,fat,solids-not-fat,lactose,and somatic cell count.Blood samples were collected on days-6,-3,0,3,6 relative to calving,and then monthly throughout lactation,and analyzed for P and Ca concentrations.Spot samples of feces and urine were collected for 3 consecutive d during weeks 12,24,and 36,and P concentrations were analyzed.Reproduction and health data were recorded.Results：Dietary P did not affect dry matter intake or milk yield（P〉 0.10）.Milk fat content was slightly higher in cows fed 0.37%P than in cows fed 0.47%P（P = 0.05）.Serum concentrations of P and Ca did not reflect dietary P content（P〉 0.10）.Fecal and urinary P both declined linearly（P〈 0.05） as dietary P decreased from 0.57 to0.37%.Fecal P content was 25%less when dietary P was 0.37%compared to 0.57%.Health events and reproductive performance were not associated with dietary P content（P〉 0.05）.Conclusions：Lowering dietary P from 0.57 to 0.37%did not negatively affect milk production,but did significantly reduce P excretion into environment.

Effects of Dietary Supplement of Fermented Tomato Pomace on Dry Matter Intake and Nutrient Apparent Digestibility of Dairy Cows

This study was carried out to evaluate the effects of replacing corn silage with ensiled tomato pomace on dry matter intake and apparent digestibility of diets.Eight close up dry Holstein cows were used in 2 × 2 Latin square design.The animals were fed control(0% tomato pomace silage) and experimental(10% corn silage replaced by tomato pomace silage) diets.The result showed that dry matter intake in experimental group was 9.77 ± 0.44 kg/d,which was significantly higher than in control group(p < 0.05).Dry matter digestibility in experimental and control group was 73.9% and 70.2%,respectively,but there was no difference between two groups(p > 0.05).The substitution of tomato pomace silage in diet was associated with an increase of dry matter intake,dietary supplement of ensiled tomato pomace without any adverse effect on their health performance.

Serum Ion Concentrations and Antioxidation of Dairy Cows with Retained Fetal Placenta

[ Objective] TO further explore the mechanism of retained fetal membrane （RFM） of dairy cows in Qinghai Province. [ Methed] As many as 15 dairy cows with retained fetal placenta and 15 without retained fetal placenta were selected. The serum concentrations of eight metal ions, namely, potassium, sodium, calcium, magnesium, copper, iron, zinc and manganese at parturition and 12 h post partum were determined by atomic absorption spectrometry. The anti-oxidative indexes at parturition were determined by UV-1601 doub｜e-beam visib｜e spectrophotometer. ~=- sultl In the dairy cows with retained fetal placenta, the concentrations of calcium, magnesium, copper, zinc and manganese were decreased signif- icantly （ P 〈 0.05） ; that of iron was increased significantly （ P 〈 0.01 ） ; those of potassium and sodium did not change greatly; the activities of su- peroxide dismutase and glutathione peroxidase were decreased significantly （ P 〈 0.01 ） ; and the MDA content was increased significantly （ P 〈 0.05）. However, the concentrations of the eight metal ions and anti-oxidative indexes did not change in the dairy cows without retained fetal placen- ta, [ CondusionJ The RFM of dairy cows has some relationships with the serum concentrations of metal ions and antioxidation.

A Complex Interrelationship between Rectal Temperature and Dairy Cows’ Performance under Heat Stress Conditions

Upper limit of thermal stability and subsequent rise of thermoregulatory functions are affected by body temperature. This study was designed to determine the effects of rectal temperature (RT) on dairy cows’ performance (heart rates (HR), respiratory rates (RR), milk yield (MY), dry matter intake (DMI), digestibility, plasma concentration of vitamin C under hot climate. This study was carried out in 2009, in north-west of Tunisia using 30 Holstein cows in mid lactation. The experiment was performed in spring (15th of February-15th of March: P1) and summer (1st-30th of August: P2). On each test day, temperature-humidity index (THI), RT, HR, RR, MY, DMI, digestibility and plasma VC concentration were determined. All this parameters were affected (P < 0.001) when the THI increased from 65.62 (P1) to 83.27 (P2). Regression analyses were carried out between THI index and some parameters (HR, RR, MY, DMI, digestibility, plasma concentration of vitamin C) and between RT and same parameters (HR, RR, MY, DMI, digestibility, plasma concentration of vitamin C). Characteristics of regression analyses in the two modes were different as also were R2 and r (correlation coefficient) of the regressions. R2 in regressions on RT (R2 (RT, DMI) = 0.92 (P < 0.01);R2 (RT, MY) = 0.91 (P < 0.001)) was markedly higher relative to R2 in regressions on THI (R2 (THI, DMI) = 0.76 (P < 0.001);R2 (THI, MY) = 0.63 (P < 0.001)). The two regressions modes suggest that increasing R2 in regressions on RT confirms that rectal temperature constitutes a larger component of total variance of responses in dairy cows to hot environmental temperature.

Effects of Supplementary Feed with Different Apple Pomace-to-Alfalfa Meal Ratios on Milk Performance of Dairy Cows

[Objective] To study the effects of apple pomace on milk performance of dairy cows. ~Method] A total of 48 Holstein dairy cows at the same parity and with close lactation stage and similar daily milk yield were randomly assigned to six groups. The supplementary feed was composed of apple pomace, alfalfa meal, com and premix, and it was processed into pellets. In the groups I - V, the percentage of apple pomace was 17.5%, 35.0%, 52.5%, 70.0% and 0%, respectively. Each cow was fed with supplementary feed （3 kg/d）. [ Result] The milk yield in the group II and group V was increased significantly （ P 〈 0.05）, and the fat content in milk of the group III and V was significantly higher than that of the con- trol group （P 〈 0.05）. [ Conclusion] The supplementary apple pomace can improve the milk performance of dairy cows effectively.

Occurrence of retained placenta is preceded by an inflammatory state and alterations of energy metabolism in transition dairy cows

Background： Failure to expel fetal membranes within 24 h of calving is a pathological condition defined as retained placenta（RP）. The objective of this investigation was to evaluate whether there are alterations in several selected serum variables related to innate immunity and carbohydrate and lipid metabolism that precede occurrence of RP in transition Holstein dairy cows.Methods： One hundred multiparous Holstein dairy cows were involved in the study. Blood samples were collected from the coccygeal vein during the-8 to ＋4 wks around parturition, once per week before the morning feeding.Six healthy control cows（CON） and 6 cows with RP were selected and serum samples at-8,-4, time of diagnosis of disease, and ＋4 wks relative to parturition were used for analyses. All samples were analyzed for lactate, nonesterified fatty acids（NEFA）, β-hydroxybutyrate（BHBA）, interleukin-1（IL-1）, interleukin-6（IL-6）, tumor necrosis factor（TNF）, haptoglobin（Hp）, and serum amyloid A（SAA）.Results： Cows with RP had greater concentrations of serum lactate, IL-1, IL-6, TNF, and SAA in comparison with CON cows. Intriguingly, elevated concentrations of all five variables were observed at-8 and-4 wks before the occurrence of RP compared to healthy cows. Cows with RP also had lower DMI and milk production vs CON animals; however milk composition was not affected by RP.Conclusions： Cows with RP showed an activated innate immunity 8 wks prior to diagnosis of disease. Overall results suggest that serum IL-1, IL- 6, and TNF, and lactate can be used as screening biomarkers to indicate cows that might have health issues during the transition period.

Correlation between the Insertion/Deletion Mutations of Prion Protein Gene and BSE Susceptibility and Milk Performance in Dairy Cows

Objective To investigate the 23 bp and 12 bp insertion/deletion(indel)mutations within the bovine prion protein(PRNP)gene in Chinese dairy cows,and to detect the associations of two indel mutations with BSE susceptibility and milk performance.Methods Based on bovine PRNP gene sequence,two pairs of primers for testing the 23 bp and 12 bp indel mutations were designed.The PCR amplification and agarose electrophoresis were carried out to distinguish the different genotypes within the mutations.Moreover,based on previous data from other cattle breeds and present genotypic and allelic frequencies of two indels mutations in this study,the corrections between the two indel mutations and BSE susceptibility were tested,as well as the relationships between the mutations and milk performance traits were analyzed in this study based on the statistical analyses.Results In the analyzed Chinese Holstein population,the frequencies of two"del"alleles in 23 bp and 12 bp indel muations were more frequent.The frequency of haplotype of 23del-12del was higher than those of 23del-12ins and 23ins-12del.From the estimated r2and D’values,two indel polymorphisms were linked strongly in the Holstein population(D’=57.5%,r2=0.257).Compared with the BSE-affected cattle populations from the reported data,the significant differences of genotypic and allelic frequencies were found among present Holstein and some BSE-affected populations(P<0.05 or P<0.01).Similarly,there were significant frequency distribution differences of genotypes and alleles among Chinese Holstein and several previous reported healthy dairy cattle(P<0.05 or P<0.01).Moreover,association of genotype and combined genotypes of two indel polymorphisms with milk performance and resistant mastitis traits were analyzed in Holstein population,but no significant differences were found(P>0.05).Conclusions These observations revealed that the influence of two indel mutations within the bovine PRNP gene on BSE depended on the breed and they did not affect the milk production traits,which layed the foundation for future selection of resistant animals,and for improving health conditions for dairy breeding against BSE in China.

Effects of Heat Stress on Microbial Numbers in Rumen Fluid of Dairy Cows

[Objective]This experiment was conducted to investigate the effects of heat stress on microbial numbers in rumen fluid of dairy cows during different lactating periods by real-time PCR.[Methods]A 2 ×3 crossover design was used and eighteen healthy Chinese Holstein cows were selected.The cows were allocated into three periods based on their parity,average days in milk,and milk yield：early lactation（postpartum 15 to 100 days）,mid-lactation（postpartum 112 to 175 days）and late lactation（postpartum 200 to 230 days）,and six cows in each period.A self-control trial between heat stress period and non-heat stress period was used based on temperature and humidity index（THI）measurement on site.[Results]The results showed as follows：compared with non-heat stress period,heat stress significantly increased the microbial numbers in early lactation,the Ruminococcus flavefaciens number in different lactation and Bacteroides succinogenes number in early and mid-lactation（P 〈0.01）.The total bacteria and Ruminococcus albus numbers in mid-and late lactation and Bacteroides succinogenes number in late lactation were decreased significantly by heat stress（P 〈0.01）.There were strong correlation between THI and the numbers of total bacteria（P 〈0.01）,anaerobic fungi（P 〈0.05）,Ruminococcus albus（P 〈0.01）and Ruminococcus flavefaciens（P 〈0.05）in rumen fluid of dairy cows in early lactation,but the correlation between THI and these indexes in mid-and late lactation were poor.[Conclusion]It is indicated that the rumen microbial numbers in early lactation are more sensitive to heat stress.

Exosomal miR-218 regulates the development of endometritis in dairy cows by targeting TGIF2/TGF-βpathway

Endometritis affects the reproductive capacity of dairy cows and leads to serious economic losses in dairy farming.Clarification of the pathogenesis of endometritis is necessary to improve the reproductive efficiency of dairy cows.Exosomes and their miRNAs have been proven to play an important role in inflammatory regulation.Exosomal miR-218 is a differentially expressed miRNA found in endometrial epithelial cells(EECs)under endometrial inflammation.Therefore,we investigated the expression of miR-218 in the uterine tissue of dairy cows,lipopolysaccharide(LPS)treated EECs,exosomal vesicles,and regulation of exosomal miR-218 by targeting TGIF-2 inducible factor homology frame 2(TGIF2)/transforming growth factor-beta(TGF-β).The expression of miR-218 was suppressed in inflammatory uterine tissues and LPS treated EECs.The expression of TGIF2 and TGF-βin inflammatory uterine tissues and LPS treated EECs was significantly higher than those in healthy uterine tissues and EECs(p<0.01).Interestingly,miR-218 derived from donor cells was found to regulate the expression of the target gene TGIF2 in recipient cells through the fusion of exosomes.Concurrently,the expression of its target gene TGIF2 was also suppressed by miR-218 in donor cells resulting in fewer TGIF2 being transported into recipient cells with exosomal fusion.This may be a novel mechanism of miRNAs-mediated regulation and provides a new reference for analyzing the pathogenesis of endometritis in dairy cows.

Effects of Diet Categories on Feces/Urine Features of Dairy Cows and Nitrogen Emission

[ Objective]Through study of different diet categories＇ effects on lactating cows＇ feces/urine features and nitrogen emission, and in order to reduce and control the nitrogen emission. [Method112 China Holsteins each with the weight of 514 kg and at the age of 3 to 5 years old are selected for this experiment to carry out test and research on their feces/urine features and nitrogen emission. The cows are divided into 3 groups which are fed with Diet A which takes dry corn straw as forage with forage to concentrate ratio of 40：60, Diet B and Diet C （ both take corn silage as forage） with forage to concentrate ratio of 40：60 and 60：40 respectively. [ ltesult]The results show that diet categories have certain effects on dairy cows＇ output of feces and urine. Feces output of Diet A （straw type） is more than that of Diet B （silage type with low forage to concentrate ratio） which is more than that of Diet C （silage type with high forage to concentrate ratio）. Feces output of Diet A group exceeds that of Diet B and Diet C groups by 6.10% and 10.08% respectively. Feces output of Diet B exceeds that of Diet C by 3.75%. The difference is insignificant （ P 〉 0.05）. In terms of urine output of these three diet groups, Diet C is more than that of Diet B which is more than that of Diet A. Urine output of Diet C exceeds that of Diet B and Diet A by 22.75% and 56.30% respectively. Urine output of Diet B exceeds that of Diet A by 27.33%. The difference is significant（ P 〈 0.05）. At the same time, diet categories have significant effects on dairy cow＇s nitrogen balance. Straw-typed diet＇s milk nitrogen and urinary nitrogen contents as well as nitrogen digestibility are far lower than those of silage-typed diet （ P 〈 0.01 ）. Under conditions of taking corn silage as forage, as diet＇s concentrate refines, nitrogen digestibility and urinary nitrogen both increase remarkably （ P 〈 0.01 ） while fecal nitrogen and milk nitrogen＇s increase is not obvious （P 〉 0.05）.[ Conclusion] Moreover, it shows that Straw-typed diet＇s absorbed nitrogen is mainly discharged through feces. When corn silage is taken as forage, as diet＇s concentrate refines, proportion of fecal nitrogen reduces while that of urinan/nitrogen increases, and fecal nitro clen and urinary nitrogen＇s proportions in total feces/urine nitroqen are basically the same.