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94:2374–2382
doi:10.3168/jds.2010-3860
© American Dairy Science Association®, 2011.
opment of insulin resistance, greater concentration of DM concentration was determined by drying samples
lactate in the plasma, and lower circulating BHBA. at 135°C for 2 h (AOAC, 1990). Ash concentration was
Nevertheless, a direct association between the rise determined after 5 h at 500°C in a furnace. Methods of
of free endotoxin, released into rumen fluid during the Van Soest et al. (1991) were used in analyses of NDF
feeding of diets high in starch, and the perturbations and ADF using heat-stable amylase and sodium sulfite
of plasma variables related to carbohydrate and lipid in the case of NDF. Ingredients and nutrient compo-
metabolism in dairy cows has not yet been document- sition of the TMR are in Table 1. All experimental
ed. We hypothesized that alterations in the ruminal procedures were approved by the University of Alberta
environment, including a rise in the concentration of Animal Care and Use Committee for Livestock and ani-
endotoxin, in response to feeding graded amounts of mals were cared for in accordance with the guidelines of
barley grain, might play a role in oscillations of differ- the Canadian Council on Animal Care (1993).
ent plasma metabolites. Therefore, the main objectives
were to investigate the effects of feeding graded amounts Sample Collection
of barley grain on rumen pH patterns, concentration of
endotoxin in the rumen fluid, and the profile of selected Blood samples were obtained from the tail vein on d
plasma metabolites in dairy cows. Furthermore, asso- 1, 3, 5, 7, and 10 of the measurement period at 0800
ciations among grain-induced rise in the concentration h, shortly before the morning feeding. Sodium hepa-
of rumen endotoxin with different plasma metabolites rinized 10-mL Vacutainer tubes (Becton Dickinson,
allied to carbohydrate and lipid metabolism in lactat- Franklin Lake, NJ) were used to collect blood samples.
ing dairy cows were evaluated. Immediately after collection, blood samples were stored
on ice and plasma was separated within 20 min from
MATERIALS AND METHODS withdrawal by centrifuging at 3,000 × g at 4°C for 20
min (Rotanta 460 R, Hettich Zentrifugan, Tuttlingen,
Animals and Diets Germany). Plasma samples were stored at −20°C until
analysis.
This investigation was part of a larger study designed Samples from rumen fluid (100 mL) were obtained on
to investigate the immune and metabolic events associ- d 1, 3, 5, 7, and 10 of the measurement period shortly
ated with the feeding of high-starch diets in lactating before the morning feeding (i.e., 0800 h). All rumen
dairy cows at Dairy Research and Technology Centre, fluid samples were collected through the cannula us-
University of Alberta (Edmonton, AB, Canada). Re- ing a tube fitted with a strainer and a syringe into
sults related to acute phase response, plasma miner- a 140-mL plastic container. Subsequently, rumen fluid
als, milk production, and rumen metabolomics were samples were centrifuged at 6,000 × g for 15 min, and
published previously (Emmanuel et al., 2008; Ametaj the supernatant was stored at −20°C until analysis for
et al., 2010). In brief, 8 rumen-fistulated (Ø 100 mm, concentration of endotoxin. Other samples of rumen
Bar Diamond, Parma, ID) primiparous Holstein cows fluid were obtained at 0, 2, 4, 6, 8, 10, and 12 h after
were used in a replicated 4 × 4 Latin square design. the morning feeding on d 21 to determine the post-
The experimental period was 21 d in duration with the prandial changes of rumen pH. The pH of rumen fluid
first 11 d used for diet adaptation. The herd veterinary was measured immediately after collection by a mobile
technician supervised cows daily; all cows remained pH meter (Accumet AP61, Fischer Scientific, Ottawa,
clinically healthy throughout. At the start, the cows Ontario, Canada).
were at 60 ± 15 (mean ± SD) d postpartum. The cows
were housed in tie stalls with free access to water and 'HWHUPLQDWLRQRI5XPHQ(QGRWR[LQ
fed once daily at 0800 h. To challenge the cows with and Plasma Metabolites
different concentrate-to-forage ratios in the diet, differ-
ent concentrate proportions (i.e., 15, 30, 45, or 60% in Concentration of cell-free endotoxin in the rumen
DM) were used in the diet. The amount of concentrate fluid supernatant, collected only before the morning
was stepped up or down during the adaptation period feeding, was determined by the Pyrochrome Limulus
to avoid potential health implications due to abrupt di- amebocyte lysate assay (Associates of Cape Cod Inc.,
etary changes. Daily ration was offered as TMR for ad East Falmouth, MA) as described previously (Em-
libitum intake to allow approximately 5% feed refusals. manuel et al., 2008). In brief, 10 mL of rumen fluid
All diets were formulated to meet or exceed the nutri- sample was centrifuged at 6,000 × g for 15 min, and
ent requirements of a 680-kg lactating cow, following the supernatant was stored at −20°C. For use in the
NRC (2001) guidelines. Diet ingredients were analyzed assay, 1.5 mL of the supernatant was centrifuged again
for concentrations of DM, ash, NDF, and ADF. The at 10,000 × g for 30 min. The supernatant was passed
Journal of Dairy Science Vol. 94 No. 5, 2011
2376 ZEBELI ET AL.
Table 1. Ingredients and nutrient composition of the 4 experimental TMR, differing in the level of concentrate inclusion
through a disposable 0.22-μm sterile, pyrogen-free filter Enzymatic quantization of BHBA by BHBA dehydro-
(Fischer Scientific, Fairlawn, NJ) and diluted 1,000-fold genase was used for quantifying plasma concentration
using pyrogen-free Limulus amebocyte lysate reagent of BHBA using a commercially available kit (Stanbio
water and pyrogen-free test tubes (Associates of Cape Laboratory, Boerne, TX). The principle of the test
Cod Inc.). Samples were tested in duplicate, and the involves conversion of BHBA in the samples to acetoac-
optical density values were read on a microplate spec- etate and NADH at pH 8.5 by BHBA dehydrogenase in
trophotometer (Spectramax 190, Molecular Devices the presence of NAD. The NADH produced reacts with
Corporation, Sunnyvale, CA) at a wavelength of 405 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium
nm. Intra-assay coefficient of variation was <10% for chloride (INT) in the presence of diaphorase to produce
all the assays. a color proportional to the concentration of BHBA in
The concentration of glucose in the plasma was the sample. Plasma BHBA was measured in duplicate
quantified by an enzymatic method using a com- by reading a microplate spectrophotometer at an opti-
mercially available kit (Diagnostic Chemicals Ltd., cal density of 505 nm. The lower detection limit of the
Charlottetown, PE). Briefly, the procedure involves assay was 0.125 μmol/L.
phosphorylation and oxidization of glucose in samples, Quantitative determination of plasma NEFA was by
resulting in the production of NADH, which produces an enzymatic colorimetric method using a commercially
a color proportional to the glucose concentration in the available kit (Wako Chemicals, Richmond, VA). The
sample. All samples were tested in duplicate and the principle of the test involves acylation of coenzyme A
plasma glucose was determined by reading on a mi- (CoA) by fatty acids in the sample in presence of acyl-
croplate spectrophotometer at an optical density of 340 CoA synthetase and production of hydrogen peroxide
nm. According to the manufacturer instructions, the in presence of acyl-CoA oxidase. Hydrogen peroxide, in
lower detection limit of the test was 0.06 mg/dL. presence of peroxidase, permits the oxidative condensa-
Journal of Dairy Science Vol. 94 No. 5, 2011
RUMEN ENDOTOXIN AND METABOLIC RESPONSE 2377
and 60% concentrate compared with those fed the diet Dietary treatment affected the overall concentration
with 15% concentrate (P < 0.05; Figure 1). Time after of cholesterol in plasma in a curvilinear fashion (P <
the morning feeding affected the ruminal pH, whereby 0.01). The analysis showed that plasma cholesterol
it reached the nadir at 8 to 10 h post-feeding (Figure concentration was lowest in cows fed 60% concentrate
1). compared with cows fed 15% or 30% grain (Table 2).
The feeding of graded amounts of concentrate in- Plasma cholesterol concentration did not differ between
creased the concentration of plasma glucose (Table 2; P cows fed 45 and 60% concentrate (Table 2). The con-
< 0.01). The cows fed 15% concentrate had the lowest, centration of lactate in the plasma increased linearly
whereas the cows fed 30% concentrate in the diet had with the increasing of the amount of concentrate in the
the greatest concentration of glucose. Dietary treat- diet (P < 0.01; Table 2). Plasma lactate concentration
ment affected the overall concentration of insulin in the was lowest for cows fed 15% concentrate and increased
plasma (P < 0.01). Interestingly, cows fed 15 and 60% with the increase of the amount of grain in the diet.
concentrate showed the lowest concentration of insulin Dietary treatment affected significantly plasma con-
in the plasma, and this response was reflected by a centration of BHBA, whereby cows fed 15% concen-
quadratic effect of the treatment (P < 0.01; Table 2). trate had the greatest plasma concentration of BHBA
(Table 2). Feeding increasing proportions of concentrate
decreased the concentration of NEFA in the plasma
(Table 2). Overall, plasma NEFA concentrations were
highest in cows fed 15% concentrate, and lowest in cows
fed 60% concentrate in the diet.
DISCUSSION
vestigators suggested a decreased ketogenesis in the liver concentrations of selected metabolites related to car-
as a plausible reason for the lowered plasma BHBA con- bohydrate and lipid metabolism. Increasing rumen
centration following the administration of LPS (Naylor endotoxin concentrations correlated with the decreas-
and Kronfeld, 1986; Steiger et al., 1999; Waldron et al., ing concentrations of cholesterol, BHBA, and NEFA,
2003), this was not the case in the experimental rats as increasing concentrations of lactate, and the biphasic
reported by Lanza-Jacoby et al. (1990). The concentra- response of insulin in the plasma. Taken together, our
tion of lactate in the plasma increased with the amount data emphasize the importance of feeding diets bal-
of concentrate in the diet, with cows consuming 60% anced in forage-to-concentrate ratio to minimize the
concentrate having the greatest level of lactate in the risk of accumulation of endotoxin in the rumen and
plasma. Other investigators reported that switching ru- its resulting systemic inflammatory states to prevent
minant animals from low- to high-concentrate diets was perturbations in the metabolic profiles of dairy cows.
associated with greater plasma concentration of lactate
(Huntington et al., 1981). ACKNOWLEDGMENTS
Another finding of this study was the positive, non-
linear correlation between rumen endotoxin and plasma We acknowledge the financial support of Alberta
lactate. Because most of the variation (i.e., 93%) in Milk (Edmonton, Alberta, Canada), Alberta Livestock
the response of plasma lactate was explained by rumen Industry Development Fund (Edmonton, Alberta,
endotoxin, this suggests a major role for rumen endo- Canada), and the Natural Sciences and Engineer-
toxin and its resulting inflammatory conditions in the ing Research Council of Canada (Ottawa, Ontario,
increase of plasma lactate in cows fed large amounts Canada). The assistance of D. G. V. Emmanuel, R. P.
of cereal grain. The effect of endotoxin on plasma Pandian, S. Sivaraman, and R. Psutka (University of
lactate can be explained by the direct role of pro- Alberta, Edmonton, AB, Canada) with the sampling
inflammatory cytokines, stimulated by endotoxin, on and laboratory analyses is highly appreciated. We are
glycogenolysis and lactate metabolism (Elsasser et al., grateful to the technical staff at Dairy Technology and
2008). This assumption is supported by studies involv- Research Centre, University of Alberta for their help
ing experimental endotoxemia. For example, Steiger et and care for the cows.
al. (1999) demonstrated that heifers challenged with
endotoxin had greater concentrations of plasma lactate
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