| Feeding and management of dairy cows during the perinatal period is a hot issue.Unsui table feeding and management will have adverse effects on the health and production performance of the cows and calves.Overstocking density is a common management measure in intensive pasture,but high density is considered to be a high risk factor affecting the health and performance of dairy cows.A series of changes have taken place in the body of postpartum dairy cows,one of which is the reduction of immunity.The intensive development of pastures in China is very fast,but there is no study on the effect of feeding density on the perinatal cows.It has been reported that the immune enhancers for the perinatal cows are mostly single component.In this study,a large-scale pasture was selected to study the effect of feeding density and compound immune e nhancer on cows and calves in the early stage of perinatal period by measuring the indexes of immunity,metabolism,health and production performance,and to explore its mechanism.It would provide scientific basis and theoretical support for improving the feeding and management of perinatal dairy on their management and nutrition.This study included a total of four parts.In Trial-1,48 healthy Holstein cows were selected as experimental animals,and three stocking density gradients of 80% group,100% group and 120% group were set up.The behavior,blood biochemical indexes and production performance indexes were analyzed.The results showed that:(1)With the increase of stocking density during the pre-perinatal period,the lying time and rumination time of the cows decreased significantly.The cows in the 80% group had the most lying time(P<0.05),and the lying time in the 80% group was increased by 1.2h compared to that in 100% group.Similarity,the cows in the 80% group had more rumination time(P<0.03),and the rumination time in the 80% group was increased by 0.54 h than that in 100% group.(2)Different stocking densities in the pre-perinatal period have no effect on the milk production,4% standard milk,energy-corrected milk,milk fat rate,milk protein rate,urea nitrogen and lactose,somatic cell count(SCC)indicators,average calving date(ACD),as well as colostrum qualit y and output from tested cows.However,in the comparison of the 100% and 120% groups,the milk production in the first lactation month(DIM)of the 80% group was significantly higher than that of the other two groups.(3)Dairy cows with different stocking densities in the perinatal period demonstrated a significant difference in the concentration of blood calcium(P<0.1)).Significant changes were observed on the blood cortisol,total cholesterol,triglycerides,total protein,urea nitrogen and calcium along with the time of delivery(P<0.05).The interaction between the stocking density and the number of prenatal days had mainly effects on the triglyceride and non-esterified fatty acid(NEFA)(P<0.1).(4)The pre-perinatal stocking density reduced the concentration of acetic acid and ammoniacal nitrogen in the rumen of cows following the time of delivery,while the concentration of valeric acid,isobutyric acid and isovaleric acid was increased at the same time site.In Trial-2,the healthy calves produced by the 48 Holstein cows with three different stocking densities from Trial-1 were tested for the body size,weight and blood biochemical index.The results discovered that:(1)There was no difference in the effects of different stocking densities on the concentration of cortisol,urea nitrogen,calcium and total protein in calf blood during the pre-perinatal period.But the alcohol concentration in the calf from 80% group(58.3 mmol/L)was slightly lower than that in 100% group(65.8 mmol/L)and 120% group(61.0 mmol/L).(2)The calf weight increased significantly on the 1st and 7th days after birth,although there was no difference in body length,body height,chest circumference and abdominal circumference occurred during this period(P<0.01).In Trial-3,48 healthy Holstein cows were selected as the test animals,and the compound immunomodulator(CI)was used to set up three test groups CI0,CI60 and CI90,corresponding to the daily added CI of 0g,60 g and 90 g for each cow per day,respectively.The study were tested from 60 days of antenatal period to 35 days of postnatal peroid.The dry matter intake(DMI),body weight,production parameters,blood metabolites,neutrophil phagocytic capacity and peripheral blood leukocyte-related genes were analysis at the time site of-60d?-28d?-14d?-7d during prenatal period(“-d” means days during postnatal period)and 1d,7d,14 d,and 28 d during postnatal period(“d” means days during postnatal period).The results demonstrated that:(1)Different additive volume of CI had no influence on the DMI of cows.DMI was changed along with the deliver time,it was decreased before delivery and increased after delivery,but there was no significant difference occurred among the three groups.(2)The differ level of CI presented significant difference on the body weight of perinatal dairy cows.The percentage of weight change(PWC)from CI0 at 1w before delivery was significant difference with that in CI60(P<0.05),and the percentage of weight change(PWC)from CI0 at 14 d after delivery was significantly higher than that in CI60(P<0.01)and CI90(P<0.05).(3)In the 35 d of postnatal period,the different additive volume of CI had distinguished influences on the production performance.The milk production,4% standard milk production,and all milk components all existed time effect(P<0.01),and the milk somatic cell count(SCC)from three groups observed significantly different between each other(P<0.01).SCC in CI0 after delivery was significant higher than the SCC from CI60 and CI90(P<0.05).The different additive volume of CI did not impact the production and quality of colostrum,but CI90 had an increasing trend of Ig G expression,in the comparison of CI0(P<0.1).(4)Different level of CI presented impressive influence on the blood biochemical indexes.For instance,the content of cortisol,non-esterified fatty acids(NEFA)and ?-hydroxybutyric acid(BHB)was impacted by CI during both prenatal and postnatal period,the concentration of these indexes were tested highly,especially around delivery time;the concentration of haptoglobin was dropped(P<0.1)following the increased addition of CI at delivery time,and the level of haptoglobin in CI0 was significant higher than that in CI90(P<0.05);the total protein in serum was not affected by the adding volume of CI,but an uptrend of total protein in serum was observed following feeding CI at postnatal period(P<0.1),and the total protein level in CI90 was significant higher than that in CI0(P<0.05)and CI60(P<0.01);the content of seralbumin was dropped along with the adding of CI at prenatal period,and a significant decrease was found at 14 d of prenatal period(P<0.05),the level of seralbumin in CI0 significant higher than CI60 and CI90(P<0.05);the seroglobulin of CI60 and CI90 was increased obviously with feeding CI(P<0.05),the seroglobulin in CI0 was lower than that in CI60 during prenatal period(P<0.05)and was lower than that in CI90 during postnatal(P<0.05);the content of calcium in serum showed a decreased trend at prenatal period(P<0.05)and an increased trend(P<0.05)at postnatal period(P<0.1),the calcium level of serum in CI0 was higher than that from CI60 and CI90 at 4w/-28d(P<0.05),but was lower than that from CI60(P<0.05)and CI90(P<0.1)at 1w/7d.The calcium level of serum in CI60 and CI90 dropped significantly at 4w of prenatal period(P<0.05),while increased at 1w/7d(P<0.05).(5)The phagocytic ability of neutrophils(NEUT)to Escherichia coli(E.coli)and Staphylococcus aureus was effected by different perinatal time points and experimental treatment(P<0.05).Among 1d and 7d of lactation period,the phagocytic ability of NEUT in three groups to E.coli were significant difference between each other(P<0.05).At 1d following childbirth,CI60 showed the highest phagocytic ability of NEUT to E.coli(P<0.01),the phagocytic ability of CI90 and CI60 were higher than CI0 at 7d,and CI60 were higher than CI0 at 35d(P<0.05).In the case of the phagocytic ability of NEUT to Staphylococcus aureus,CI60 were highest at 1d following childbirth(P<0.01),while CI60 and CI90 were similar to each other and both of them were higher than CI0 at 35d(P<0.05).(6)The feeding dosage of CI had a significant effect on the expression of Chemokine(C-X-C motif)ligand 8(CXCL8)and L-selectin(SELL)(P<0.05).The expression of CXCL8 in CI0 was lower than that in CI60 at-14 d and 35d(P<0.05).The expression of SELL were impacted obviously by time(P<0.01),the SELL level of three groups were increased significantly at-14 and 35d(P<0.05),moreover,there was an up-trend of SELL level among these groups from-60 d to 28d(P<0.1),thereinto,the level of SELL in CI60 was higher than that in CI0(P<0.01);the level of SELL in CI60 was higher than that in CI0 at-14d(P<0.05),and the SELL level were tested highest in CI90 at 35d(P<0.05).In Trial-4,three cows from each stocking density group of 80% group and 120% group in Trail-1 and three cows from each group of CI0 and CI60 in Trial-3 were selected for collecting the peripheral blood to extract the immune cells,these cells were prepared for extracting the total RNA and protein to sequence and analyze the related transcriptome and proteome,respectively.The results demonstrated that,(1)In the comparison of different stocking destiny groups,12,237 genes were tested,and 10,334 shared genes were found in 80% group and 120% group,which account for a percentage of 84.4% of the total tested genes.Among these shared genes,7,351 genes expressed no difference,accounting for 71.1% of the shared genes,1,732 genes were up-regulated(?1.5,16.8%)and the 1,251 genes were down-regulated(?0.67,12.1%).Meanwhile,there were 9,290 proteins were detected in the selected stocking density groups,and a total of 8,006 shared proteins were found in 80% group and 120% group which account for 86.2% of the total tested proteins.Among them,6,112 proteins expressed no difference which account for 76.3% of the shared proteins,and there were 1,108 up-regulated proteins(?1.5,13.8%)wand 846 proteins were down-regulated(?0.67,9.9%).There were 456 up-regulated genes(19.1% of the total up-regulated genes)and 412 down-regulated genes(24.5% of the total down-regulated genes)tested in the transcriptome and the proteome.(2)In the case of the comparison among different CI group,a total of 11,175 genes were detected.There were 9,164 shared genes in CI0 group and CI60 group,accounting for 82.0% of the total number of tested genes.Among these shared genes,7,542 genes expressed no difference which account for 82.3% of the shared genes,669 genes were up-regulated which account for 7.3% of the shared genes,and the 953 genes were down-regulated,accounting for 10.4% of the shared genes.In CI0 and CI60,7,546 shared prote ins were discovered,accounting for 82.6% of the total number of proteins.Among these shared proteins,6,308 proteins expressed no difference,accounting for 83.6% of the shared proteins,and 567 proteins of these proteins were up-regulated(7.51%)and 671 proteins were down-regulated(8.89%).In addition,289 up-regulated genes(30.5% of total up-regulated genes)and 367 down-regulated genes(29.2% of total down-regulated genes)were discovered in both transcriptome and proteome.(3)Comparing these differential genes of each group by using GO annotation and KEGG pathway analysis,results verified that these genes contained the similar annotation function and pathway,which mainly involved into the inflammation-related pathways(such as Jak/Stat pathway,NF-?B pathway,MAPK pathway),metabolism-related pathways(such as glucose metabolism,energy metabolism,lipid metabolism),and immune-related pathways(such as Toll-like receptor pathway,B cell receptor pathway,T cell receptor pathway).In summary,dairy cows with the 80% stocking density had longer lying down and ruminating time,which was more beneficial to the health and welfare of the dairy cows,while the perinatal stocking density had no significant effect on the growth performance and immunity of the lactating calves.Providing the CI at the beginning of the perinatal period can alleviate negative energy balance and immunosuppression,regulate the inflammation related to childbirth,and contribute to post-natal recovery and health improvement.The recommended feeding dosage of CI is 60 g/head/day.Meanwhile,periparturient cows feeding with CI could effectively improve the phagocytosis of neutrophil on Escherichia coli(E.coli)and Staphylococcus aureus,as well as enhance the expression of CXCL8 and SELL in peripheral blood leukocytes,and then improve the immunity of periparturient cows and reduce the incidence of mastitis.Moreover,the high-stocking destiny among perinatal period impacted the performance and health of cow on the mainly basis of inhibiting the genes and pathways that were related to the metabolism,inflammation and immunity in cow cells,however,the adding CI could active the genes and pathways were connected with metabolism and immunity to balance the substance metabolism,enha nce the body immunity,and promote the production performance and health in cows.More research work are still going on the regulating mechanism and its coping strategies of high-stocking destiny impact the production performance and health of periparturient cows,as well as CI improve and regulate the mechanism of specific pathways related to the metabolism and immunity of cows.In the future,it is expected to reveal the regulation mechanism from the molecular cell level to the whole organism level through multi-group sequencing(genome,transcriptome,proteome and metabolome)and its integrated analysis,and to realize the healthy breeding without antibiotic resistance. |
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