Use of milk proteins as biomarkers of changes in the rumen metaproteome of Holstein cows fed low-fiber, high-starch diets.

Dietary levels of undegraded neutral detergent fiber (uNDF240) and rumen-fermentable starch (RFS) can affect the rumen microbiome and milk composition. The objective of the study is to investigate the use of milk proteins as biomarkers of rumen microbial activity through a comparative evaluation of the rumen microbial and milk protein profiles produced by Holstein cows fed diets with varying contents of physically effective uNDF240 (peuNDF240) and RFS. Eight ruminally cannulated lactating Holstein cows were included in a larger study as part of a 4 × 4 Latin square design with 4 28-d periods to assess 4 diets varying in peuNDF240 and RFS content. For this experiment, cows received one of 2 dietary treatments: (1) low-peuNDF240, high-RFS (LNHR) diet or (2) high-peuNDF240, low-RFS (HNLR) diet. Within each period, rumen fluid samples were collected from each cow on d 26 (1400 h) and d 27 (0600 h and 1000 h), and milk samples were collected from each cow on d 25 (2030 h), d 26 (0430 h, 1230 h, and 2030 h), and d 27 (0430 h and 1230 h). Microbial proteins were isolated from each rumen fluid sample. For milk samples, milk proteins were fractionated, and the whey fraction was subsequently isolated. Isolated proteins within each rumen fluid or milk sample were isobarically labeled and analyzed by liquid chromatography-tandem mass spectrometry. Product ion spectra acquired from rumen fluid samples were searched using SEQUEST against 71 composite databases. In contrast, product ion spectra acquired from milk samples were searched against the Bos taurus database. Data were analyzed using the PROC MIXED procedure in SAS 9.4 to assess the effect of diet and time of sampling. To increase stringency, the false discovery rate-adjusted P-value (PFDR) was also calculated to account for multiple comparisons. Using the mixed procedure, a total of 129 rumen microbial proteins were quantified across 24 searched microbial species. Of these, the abundance of 14 proteins across 9 microbial species was affected due to diet and diet × time interaction, including 7 proteins associated with energetics pathways. Among the 159 quantified milk proteins, the abundance of 21 proteins was affected due to the diet and diet × time interaction. The abundance of 19 of these milk proteins was affected due to diet × time interactions. Of these, 16 proteins had the disparity across diets at the 0430 h sampling time, including proteins involved in host defense, nutrient synthesis, and transportation, suggesting that biological shifts resulting from diet-induced rumen changes are not diurnally uniform across milkings. The concentration of lipoprotein lipase (LPL) was statistically higher in the milk from the cows fed with the LNHR diet, which was numerically confirmed with an ELISA. Further, as determined by ELISA, the LPL concentration was significantly higher in the milk from the cows fed with the LNHR diet at 0430 h sampling point, suggesting that LPL concentration may indicate dietary carbohydrate-induced ruminal changes. The results of this study suggest that diet-induced rumen changes can be reflected in milk in a diurnal pattern, further highlighting the need to consider sampling time points for using milk proteins as a representative biomarker of rumen microbial activity.

Influence of dietary carbohydrate profile on the dairy cow rumen meta-proteome.

Diet starch and fiber contents influence the rumen microbial profile and its fermentation products, yet no information exists about the effects of these dietary carbohydrate fractions on the metabolic activity of these microbes. The objective of this experiment was to evaluate the effects of dietary carbohydrate profile changes on the rumen meta-proteome profile. Eight cannulated Holstein cows were assigned to the study as part of a 4 × 4 Latin square design with a 2 × 2 factorial treatment arrangement including four 28-d periods. Cows received 1 of 4 dietary treatments on a dry matter (DM) basis. Diets included different concentrations of rumen fermentable starch (RFS) and physically effective undigested NDF (peuNDF240) content in the diet: (1) low peuNDF240, low RFS (LNLS); (2) high peuNDF240, low RFS (HNLS); (3) low peuNDF240, high RFS (LNHS); and (4) high peuNDF240, high RFS (HNHS). Rumen fluid samples were collected from each cow on the last 2 d of each period at 3 time points (0600, 1000, and 1400 h). The microbial protein fraction was isolated, isobarically labeled, and analyzed using liquid chromatography combined with tandem mass spectrometry techniques. Product ion spectra were searched using the SEQUEST search on Proteome Discoverer 2.4 (Thermo Scientific) against 71 curated microbe-specific databases. Data were analyzed using PROC MIXED procedure in SAS 9.4 (SAS Institute Inc.). A total of 138 proteins were characterized across 26 of the searched microbial species. In total, 46 proteins were affected by treatments across 17 of the searched microbial species. Of these 46 proteins, 28 were affected by RFS content across 13 microbial species, with 20 proteins having higher abundance with higher dietary RFS and 8 proteins having higher abundance with lower dietary RFS. The majority of these proteins have roles in energetics, carbon metabolism, and protein synthesis. Examples include pyruvate, phosphate dikinase (Ruminococcus albus SY3), 30S ribosomal protein S11 (Clostridium aminophilum), and methyl-coenzyme M reductase subunit α (Methanobrevibacter ruminantium strain 35063), which had higher abundances with higher dietary RFS. Conversely, glutamate dehydrogenase (Butyrivibrio fibrisolvens) and 50S ribosomal protein L5 (Pseudobutyrivibrio ruminis) and L15 (Ruminococcus bromii) had lower abundances with higher dietary RFS content. Among the remaining 18 proteins unaffected by RFS content alone, 5 proteins were affected by peuNDF240 content, and 13 were affected by peuNDF240 × RFS interactions. Our results suggest that the RFS content of the diet may have a greater influence on rumen microbial protein abundances than dietary peuNDF240 content or peuNDF240 × RFS interactions. This research highlights that dietary carbohydrate profile changes can influence rumen microbial protein abundances. Further research is needed to fully characterize the effects of diet on the rumen meta-proteome and manipulate the various roles of rumen microbes. This will aid in designing the strategies to maximize the efficiency of nutrient use in the rumen.