Internal Molecular Mechanism In Digestive Absorption And Metabolic Pathway Associated With Lower Milk Performance In Dairy Cows Fed Cereal Straw As Forage Source

The objectives of this study were to investigate the internal molecular mechanisms in digestive absorption and metabolic pathway associated with lower milk performance in dairy cows fed cereal straw as forage source. In Part 1, a feeding trial was conducted to investigate the lactation performance when alfalfa was replaced with rice straw or corn stover in diets formulated for lactating cows. Part 2 was conducted to determine the key milk components precursors generation in gastrointestinal tracts and analysis of the function of runimal epithelium. In Part 3, amino acid metabolism and glucose metabolism were systematically analyzed; and metabolomics of mammary bloods was conducted to clarify the metabolic pathway as well.PART 1.Lactation performance in lactating dairy cows fed cereal straw as a forage source in replacement of alfalfa bayForty-five multiparous Holstein dairy cows were blocked based on days in milk (164 ± 24.8 d; mean ± standard deviation) and milk yield (29.7 ± 4.7 kg; mean ± standard deviation) and were randomly assigned to 1 of 3 treatments. Diets were isonitrogenous, with a forage-to-concentrate ratio of 45:55 [dry matter (DM) basis] and contained identical concentrate mixtures and 15% corn silage, with different forage sources (on a DM basis):23% alfalfa hay and 7% Chinese wild rye hay (AH), 30% corn stover (CS), and 30% rice straw (RS). The experiment was conducted over a 14-week period, with the first 2 week for adaptation. The DM intake of the cows was not affected by forage source. Total-tract apparent digestibilities of all the nutrients were lower in cows fed the CS and RS diet than those fed AH diet. Yield of milk, milk fat, protein, lactose, and total solids was lower in cows fed RS or CS than the AF-fed cows, with no difference between diets RS and CS. Lactose content was always lower in the cows fed RS than in AH or CS through the 12 sampling weeks. Contents of milk protein and total solids were lower in RS than in AH, with no difference between CS and AH or RS. Feed efficiency (milk yield/DMI) was lower in cows fed RS and CS in comparison with AH-fed cows.PART 2.Synthesis of milk protein precursors and changes of ruminal epithelium morphology and function in lactating dairy cows fed cereal straw as a forage source in replacement of alfalfa hayThe ruminal fermentation characteristics and metabolizable protein in small intestines were determined from the above 45 cows. After of completion of the feeding trial, a total of 18 cows (6 from each dietary treatment) were slaughtered to analysis of AA composition of milk protein precursors. The morphology and RNA-seq for runimal epithelium were also analyzed for all the slaughtered cows.2.1 Metabolizable protein and rumen fermentation characteristicsCows fed CS and RS produced microbial crude protein (MCP) less than the AH-fed cows; and runimal undegraded protein (RUP) was also lower in the straw-fed cows, resulting in lower metabolizable protein (milk protein precursors) in the cows fed CS and RS than in AH. Further analysis also found that the fermentable carbohydrate fractions were also lower in CS and RS than in AH, which may contribute to the shortage of energy supply available for ruminal synthesis of MCP in cows fed cereal straw diets.There was no significant difference in rumen pH among the 3 diets. Ammonia-N concentration was higher for RS diet than for AH and CS, with no difference between CS and AH. Concentration of total volatile fatty acids in the rumen was lower in RS and CS than in AH, with no difference between RS and CS, which was consisit with the dietary energy density and digestibility.2.2 AA composition of milk protein precursor in the gastrointestinal tractsThe rumen-undegradable protein (RUP) of all forages and the 3 diets was measured by incubation for 16 h in the rumen of 3 ruminally cannulated lactating cows. The ruminal degradability of all analyzed AA was not constant among the forages. Proline had the greatest degradability in alfalfa hay, corn stover or Chinese wild grass hay, but His was the greatest degradable for rice straw. However, the 3 diets were similar for all of the analyzed individual AA. The AA composition and ruminal degradability of RUP were different among forages, but constant AA profile and ruminal degradability were observed among the 3 diets, indicating that there may has some interactions between concentrate and forage in the ruminal AA degradability.The AA profiles of MCP were similar among the dietary treatments except for greater Arg in cows fed RS than in AH or in CS. Comparison of MCP, rumen fluid, and rumen digesta showed that essential AA (EAA) was higher in digesta than in MCP or rumen fluid, with the MCP being higher than in rumen fluid. There was linear correlation of AA profiles of diets to those in ruminal fluid. The individual AA composition was significantly different among the gastrointestinal digesta across the rumen, abomasum, duodenum and jejunum, but not among dietary treatments. It is suggested that the ruminal microbes tend to utilize EAA, and their AA profiles were relatively constant except for several AA.2.3 Change in morphology and function of ruminal epitheliumThe thickness of the rumen papillae was greater in RS-fed cows than in cows fed AH or CS, whereas the thickness of the papillary epithelium was reduced in CS-fed cows compared with those fed AH or RS. In total,37,47, and 30 differentially expressed genes were identified via pair-wise comparisons between AH vs. CS, AH vs. RS, and RS vs. CS. Functional classification at the level of biological processes and functional clustering gene ontology term analysis revealed dynamic changes within the categories of ion binding (such as DSG1), proliferation and apoptotic processes (such as BAGS), and regulation of cellular growth (such as CYR61) involving extracellular matrix proteins under different dietary particle sizes. It is indicated that forages with different particle sizes and nutrients values affected the morphology of the rumen papillae by affecting ion binding, cell growth, and cell proliferation/apoptosis.PART 3.Nutritional physiology metabolism in lactating dairy cows fed cereal straw as a forage source in replacement of alfalfa hayIn this part, the analyses were carried out for amino acid metabolism and glucose metabolism as well as metabolomics in dairy cows. In order to determine nitrogen (N) metabolism, diets, feces, urine and milk were sampled at week 6 and 10 from a total of 30 cows (10 from each group) selected from Part 1. Arterial and venous plasma were collected from the mammary gland (MG) of 30 cows (10 from each group) every 6 hours over 2 days of week 12.3.1 Nitrogen and AA metabolismReflective of crude protein digestibility, the fecal N output and its ratio to N intake were greater in cows fed CS and RS than in AH. Urinary N output and its ratio to N intake were also greater in cows fed RS than in AH and CS. The serum urea N was greater in cows fed RS than cows fed AH.The digestive AA flow predicted by the NRC model was lower in the cows fed CS or RS than in AH-fed cows. The arterial concentration of Met and Val was lower in the cows fed RS than in AH-fed cows. Mammary plasma flow was lower in the cows fed CS and RS than the AH-fed vows, with no difference between CS and TS. The mammary uptake of most essential AA, especially branched-chain AA, Met, and Arg, was smaller in cows fed CS or RS than in the AH-fed cows. The ratio of mammary Met uptake to milk Met output was lower in the RS-fed cows than that in the cows fed AH, with a value below 1 for both. It is suggested that the insufficient supply of free AA from arterial plasma to the MG and lower mammary plasma flow restrict the mammary AA uptake for milk protein synthesis when energy is limited.3.2 Glucose metabolismThe ruminal concentration of propionate (as glucose precursor) was lower in RS than in AH. The abundance of the pyruvate carboxylase mRNA in the liver (gluconeogenesis) was lower in the RS-fed cows compared to the AH or CS, whereas the mRNA abundance of mitochondrial phosphoenolpyruvate carboxykinase, insulin-like growth factors-1 receptor, and phosphofructokinase-liver,-muscle, and-platelet in the liver was lower in RS than in AH. The mammary glucose uptake was smaller in the cows fed CS or RS than in the AH-fed cows. The abundance of the glucose transporters mRNA in the MG was similar between the 3 treatments. The mRNA abundance of a-lactalbumin in the MG of the RS-fed cows tended to be higher than that of the cows fed AH or CS. Milk potassium concentration was higher in the RS-fed cows than those fed AH or CS. From the above results, it is inferred that the insufficient ruminal propionate levels in the cows fed RS were associated with decreased gluconeogenesis in the liver, resulting in the shortage of the arterial glucose supply for mammary uptake and reduced lactose synthesis.3.4 Arteial-venous metabolomicsAlobal profiling of paired arterial and venous plasma samples were collected before morning feeding for metabolomics study. Multilevel partial least squares discriminant analysis was used for paired comparisons of multivariate data. A total of 237 metabolites were identified in the arterial and venous plasma. A metabolomics analysis showed 31 significantly different metabolites (VIP> 1, P< 0.05, and Similarity> 200) in the comparison between artery and vein. Higher contents of amino acids and glucose were found in arterial plasma, possibly reflecting a higher intake of them for milk synthesis. The 3-hydroxybutyric acid, Glu, Met, Phe, glucose, methylmalonic acid, and Tyr seemed to be key metabolites in lactation.When arterial and venous blood metabolomics analysis was utilized for dietary treatments, the greater difference between cereal straws and alfalfa hay based diets was arterial plasma rather than vein, and the main difference was N metabolism (Phe, Tyr and Trp biosynthesis, Phe metabolism) and energy metabolism (TCA cycle), indicating the key role of arterial blood in the study of mammary nutrition metabolism. Phenylpropanoate were the key differential metabolites for RS and CS compared with AH, and Prostagelanndin A was only measured in RS. Compared to the AH-fed cows, the cows fed cereal straw diets had higher arterial hormones, lipids, and carbohydrates, but lower relative concentrations of organic acids and AA metabolites. Therefore, it can be speculated the two straws-based diets (especially RS), compared with AH group, were relatively in shorttage in key nutrients and energy, hence the homeostasis of cows was influenced, which may be accounted for the lower milk performance in the cows fed straw-based diets.In summary, the cows fed corn stover or rice straw to replace alfalfa hay as an alternative forage source in dairy cow diets had lower yield of milk and milk protein and lower feed efficiency. These inferior milk performances were mainly attributable to the lower production of volatile fatty acid and microbial protein in the rumen, smaller supply of dietary ruminal undegraded protein and digestive AA flow as well as their AA profiles. At metabolic level, feeding of cereal straw had slower mammary plasma flow, with RS diet being smaller gluconeogenesis in liver, thus resulting lower supply of AA and glucose and mammary uptake, and finally restricted milk synthesis. Greater difference was arterial plasma rather than vein between cereal straws and alfalfa based diets, and the main difference was nitrogen metabolism and energy metabolism. The results obtained from this study are basic information for efficient utilization of cereal straws, and provided a deep insight into the internal molecular mechanisms in digestive absorption and metabolic pathway associated with lower milk performance in dairy cows fed cereal straw as forage source.