Effects of a Duodenal Glucose Infusion on the Relationship Between Plasma Concentrations of Glucose and Insulin in Dairy Cows

Effects of a Duodenal Glucose Infusion on the Relationship Between Plasma Concentrations of Glucose and Insulin in Dairy Cows S. LEMOSQUET,* N. RIDEAU,† H. RULQUIN,* P. FAVERDIN,* J. SIMON,† and R. VERITE* *Station de Recherches sur la Vache Laitie`re, Institut National de la Recherche Agronomique, 35 590 St-Gilles, France †Station de Recherches Avicoles, Institut National de la Recherche Agronomique, 37 380 Nouzilly, France

ABSTRACT The effects of duodenal infusion of glucose on the relationship between plasma concentrations of glucose and insulin and on milk composition were investigated in a crossover design. Eight dairy cows were continually infused with water (control) or glucose (1.5 kg/d). Cows received diets consisting of dehydrated whole-plant maize in restricted amounts to equalize the energy supply between treatments. Basal (before meal) plasma concentrations of glucose and insulin were increased, but concentrations of nonesterified fatty acids (NEFA) were decreased, by glucose treatment. During the first 2 h after feed distribution, plasma insulin increased, and plasma glucose and NEFA decreased, in both control and treated cows. Afterward, plasma glucose increased in treated cows but further decreased in control cows. The difference reached 8 mg/100 ml without any change in plasma insulin. During the meal, concentrations of growth hormones in plasma were inhibited to a similar extent in both groups. In response to intravenous glucose or insulin challenges, changes in plasma glucose, NEFA, and insulin stimulated by glucose were also very similar in both groups. In conclusion, duodenal infusion of glucose increased basal plasma concentrations of glucose and insulin, increased postprandial plasma glucose, and decreased NEFA without inducing insulin resistance. Glucose treatment did not change milk yield but decreased milk fat yield, mainly through a decrease in the yield of C18 fatty acids that were derived from circulating fatty acids. In the absence of insulin resistance, the decrease in the yield of C18 fatty acids might be attributed to an inhibition of adipose lipolysis or an increase in adipose lipogenesis. ( Key words: lactating dairy cow, milk fat, glucose, insulin)

Received September 6, 1996. Accepted July 7, 1997. 1997 J Dairy Sci 80:2854–2865

Abbreviation key: a-AN = a-amino nitrogen, AUC = area under the response curve, PDI = protein truly digested in the small intestine. INTRODUCTION In lactating dairy cows, circulating insulin concentrations, pancreas reactivity, and tissue insulin response may greatly influence the partitioning of nutrients between the mammary gland and other tissues (47), even though insulin has no direct effect on the uptake of nutrients by the mammary gland. Therefore, any change in insulin release in response to dietary treatments may affect the availability of milk precursors and possibly milk composition. To date, few studies [see review (21)] have focused on the effect of nutritional manipulation on insulin secretion. An increase in the starch content of the rations of dairy cows decreases milk fat yield (10). Several mechanisms were proposed to explain milk fat depression based on the different modifications of digestive end products of various diets (10, 19, 23). Among those explanations was the glucogenic theory (29), which assumes that an increase in propionic acid or glucose availability obtained through the digestion of starch would increase insulin secretion. As a consequence, an increase in insulin would decrease lipolysis or increase lipogenesis and decrease the availability of milk fat precursors. To verify the glucogenic theory, glucose has been directly infused postruminally (9, 12, 13, 18, 24, 25, 33, 34, 38, 40, 42, 48, 49). Most of the time, those treatments significantly decreased milk fat content (12, 13, 18, 33, 34, 48). However, the negative effect of postruminal infusions of glucose (24, 25) on milk fat yield is less clear. In addition, a few studies have already measured plasma insulin and glucose concentrations (13, 18). None of those studies was developed to characterize the reactivity of the pancreas to glucose and the response of tissue to insulin, and, at present, no clear conclusion has been established.

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When insulin was measured in response to postruminal infusions of glucose (13, 18), the total supply of energy was different in the experimental treatments (including glucose infusion and diet) than in the control treatments. Such experimentation does not allow the differentiation of the effects of general energy intake from the specific effect of the glucose nutrient infusion on insulin secretion. Plasma insulin concentration increases as energy intake increases (6). In the present study, the effect of a duodenal infusion of glucose on the relationship between plasma glucose and insulin was further investigated using eight lactating dairy cows in two trials for which the same procedure was repeated. The amount of glucose infused (1.5 kg) represented about half of the daily glucose requirement. The energy equivalent supplied by glucose was removed from the solid diet so that the total energy intake was similar in treated and control cows. Concentrations of plasma insulin, glucose, and NEFA were monitored under standardized conditions, including basal and postprandial status. In addition, possible changes in pancreas reactivity to glucose and the sensitivity of tissues to insulin were explored through i.v. challenge with glucose and insulin. MATERIALS AND METHODS Treatments, Design, and Management Treatments consisted of continuous duodenal infusions of either glucose (1.5 kg/d) or water with identical supplies of NEL and protein truly digested in the small intestine ( PDI) . The treatments were compared in a single crossover design; two 4-wk periods were used. The experiment was conducted in 1994

(trial 1 ) and 1995 (trial 2 ) on a total of eight lactating Holstein cows (four per trial) that were fitted with a ruminal cannula and a T-shaped duodenal cannula. At the onset of the experiment, six cows ranged from 22 to 30 wk postpartum, weighed 633 ± 63 kg, and yielded 26 ± 2.5 kg/d of milk; the two other cows were 10 wk postpartum, weighed 732 ± 16 kg, and yielded 43.2 ± 2.5 kg/d of milk. Cows received restricted amounts of feed to meet requirements for NEL and PDI (26). The control diet consisted of a mixture containing (DM basis) 60% dehydrated whole-plant maize, 20% dehydrated alfalfa, 15% concentrate, and 5% treated oil meal (Table 1). To equalize energy and protein supplied by the experimental treatment with those supplied by the control treatment, during glucose infusion, 3.7 kg of DM of the mixture of dehydrated maize, alfalfa, and concentrate were removed from the daily control diet and were replaced with 0.9 kg of oil meal and 1.5 kg of glucose, assuming that 1 kg of glucose provided 2.75 Mcal of NEL ( 3 ) . Diets were administered twice daily (60% at 1000 h and 40% at 1800 h ) and were available for only 4 h. Water solution (20 kg/d) was continuously infused with or without glucose [1.65 kg of D (+)-monohydrate glucose 8346.905; Merck Clevenot, Nogent sur Marne, France] using a peristaltic Masterflex pump (Bioblock Scientific, Illkirch, France). Infusion buckets were loaded daily at 1430 h. Cows were milked at 0630 and 1730 h. Before the experiment, cows were adapted to the control diet for 6 wk, and the amount of feed offered was adjusted to exact requirements at 2 wk before the start of experiment. Changes in diets and infusions between treatments were made during the 1st wk of each period. Two days before the first blood sampling of each period ( d

TABLE 1. Ingredient and chemical composition of diets. Ingredient

Dehydrated whole-plant maize3 Dehydrated lucerne Concentrate4 Oil meal5 Soybean meal

Trial

1 and 2 1 2 1 and 2 1 2

CP

8.1 23.5 16.8 27.9 47.6 48.9

OM

Cellulose

ADF

95.8 85.2 86.5 89.9 92.5 93.6

( % of DM) 19.3 21.7 20.8 22.6 28.5 33.2 6.1 7.1 8.9 11.3 6.3 7.3

NDF

44.8 36.0 46.0 14.8 24.3 15.4

EE1

Energy

PDI2

2.6 2.8 1.5 1.6 2.4 2.5

(Mcal/kg of DM) 1.58 1.48 1.12 1.84 1.95 2.02

(g/kg of DM) 85 115 87 112 351 352

1Ether

extract. truly digested in the small intestine. 3Pelleted dehydrated whole-stalk and ear-chopped corn. 4Contained 45% peas, 27.5% barley, 15% soybean meal, 4% beet molasses, 2.0% urea, and 6.5% mineral premix (Centrale Coope ´ rative de Productions Animales, Janze´, France). 5Oil meal treated with formaldehyde, contained 80% soybean meal and 20% rapeseed oil meal. 2Protein

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10), two catheters were inserted in both jugular veins of each cow. The catheters were composed of a 0.5-m silastic catheter (1.02 mm i.d. and 2.03 mm o.d.; Dow Corning Corp., Midland, MI) and a removal connector (1.02 mm i.d. and 2.5 mm o.d.; Vygon Bionector, Ecouen, France). Between sampling sessions, the catheters were filled with 0.15 M physiological sterile saline containing 200 IU of heparin, 0.01 ml of procamycine (Rhoˆne Merieux, Lyon, France), and 0.01 ml of benzyl alcohol/ml of saline. Sampling and Measurements To allow for adaptation, all reported measurements were made after d 12 only. However, additional blood samples were taken before d 12 in trial 2, but those data are not reported. Twice daily the amount of feed given and orts were weighed, and milk yield was recorded and assayed for fat and protein contents. Morning milk samples were taken on d 13 and 28 to analyze the fatty acid composition. During each period, blood was monitored at four occasions: during kinetics after the morning feed distribution on d 13 and 28 and during a glucose and insulin challenge (between d 15 and 26). Before each of these measurements, two blood samples (collected 10 to 15 min apart) were taken to measure basal concentrations; in trial 2, additional basal samples were taken on 3 separate d. For each cow, two consecutive sampling sessions were conducted at a 48-h interval, and the glucose challenge always preceded the insulin challenge. To describe postprandial changes of hormones and metabolites, blood was collected at 15, 45, 60, 90, 120, 180, 240, 300, and 360 min after the morning feed distribution using syringes containing EDTA for hormone analyses and heparin for other analyses (1.2 to 2 mg/ml of Sarstedt 04.1069 EDTA-K and 12 to 30 IU/ ml of Sarstedt 02.1065 heparin lithium; Sarstedt, Nu¨mbrecht, Germany). Challenges were performed in the morning before feed distribution. The amount of glucose injected i.v. was chosen to increase plasma glucose concentration twofold, which is much higher than the usual diurnal variation observed in ruminants. Glucose (0.1 g/kg of BW) was administered into the first jugular vein through a catheter as a 30% (wt/vol) solution (Bruneau Braun, Boulogne-Billancourt, France) over 1.5 min. Insulin injected i.v. was chosen to create a substantial, but moderate, hypoglycemia (–20 to –30% of the basal concentrations). Bovine insulin (0.6 mg/kg of BW, 10 UI/ml; Organon Technika-Cappel, Fresnes, France) was administered using the same method over 1 min and was immediately followed by 6 ml of sterile saline Journal of Dairy Science Vol. 80, No. 11, 1997

(9%). During challenges, blood was drawn using a peristaltic Ismatec pump (Bioblock Scientific) and was collected into 16-ml tubes containing 30 IU of heparin (0.3 ml) on a fraction collector. A catheter of 5-m tubing joined the second jugular vein catheter to the fraction collector. During glucose challenge, 15 samples were taken at 3, 6, 9, 12, 15, 18, 21, 24, 30, 35, 40, and 45 min after i.v. injection using the fraction collector and at 55, 65, and 75 min after injection using syringes. During insulin challenge, 12 samples were taken in the same manner at 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, 65, and 75 min after i.v. injection. Blood was kept on ice and centrifuged at 2500 × g for 12 min at 4°C. Plasma was aliquoted and stored at –20°C. Samples were assayed for glucose, NEFA, and insulin. Insulin was not assayed during insulin challenge. During glucose challenge, NEFA were only analyzed at 6-min intervals. Additionally, plasma aamino nitrogen ( a-AN) was measured on basal samples. In trial 2, additional analyses were performed on basal plasma samples for acetate, BHBA, total glycerides (triglycerides plus free glycerol), and growth hormone and on plasma kinetic samples after feed distribution for growth hormone; three samples of ruminal fluid were also taken 0.5 h before the morning meal and 3 and 6 h after the meal to analyze VFA ( d 13 and 28). Chemical Analysis Feed DM content was determined weekly. Milk was analyzed for fat and protein contents by infrared analysis (Milkoscan; Foss Electric, Hillerød, Denmark), and cream was analyzed for fatty acid composition (23). Ruminal VFA samples were preserved with a solution composed of HgCl2 (1%, wt/wt) and H3PO4 (5%, vol/vol) in distilled water, which was added at 1:9 (vol/vol) with the samples. The VFA were determined by gas chromatography (23). Plasma was deproteinized with HCLO4 (50%, vol/vol) and filtrated for analysis of a-AN, acetate, BHBA, and glucose. Concentrations of acetate, BHBA, total glycerides, glucose, and NEFA were measured using an Isamat autoanalyzer (Instruments Socie´te´ Anonyme Biologie, Cachan, France) as described by Hurtaud et al. ( 2 3 ) and Bareille and Faverdin ( 5 ) . The a-AN was measured using a continuous flux analyzer (Technicon Industrial Systems, New York, NY) by a colorimetric reaction with trinitrobenzene sulfonate (35). Insulin was measured by the Rosselin radioimmunoassay method adapted to bovine insulin ( 1 6 ) using 125I-labeled porcine insulin (CIS BIO International, Gif-sur-Yvette, France) as the tracer. A guinea

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pig anti-bovine insulin serum (Miles Laboratories Ltd., Slough, England) was used as the antibody, and monocomponent bovine insulin (Novo, Copenhagen, Denmark) was the standard. Free insulin and insulin bound to antibodies were separated using the charcoal method. The interassay coefficient of variation was 8% in trial 1 and 6.5% in trial 2. Samples of growth hormone were analyzed according to the radioimmunoassay technique using 125I-labeled-ovine growth hormone (1.5 IU/ng), a rabbit anti-ovine growth hormone serum, and an ovine anti-rabbit serum to precipitate the complex. The intraassay coefficient of variation was 2.3%. Data Analysis Nutrient supply values and lactational performances were averaged from d 13 and 28 of each period. Variations of each plasma parameter during analysis of kinetics after feed distribution and challenges were integrated as the area under the response curve ( AUC) . The AUC was obtained by the trapezoidal rule, after correcting for basal values, and was calculated over 360 min following distributions of the feed or over 75 min following i.v. injections. During glucose challenge, the peak value of insulin was calculated by averaging the values determined from 6 to 18 min. The glucose fractional rates ( K) were calculated using linear regressions of glucose concentrations after Log (base e ) transformation. Glucose K was estimated from sample results taken 6 to 30 min after i.v. injection of glucose and those taken 0 to 20 min after i.v. injection of insulin. Data were subjected to ANOVA using the general linear models procedure of SAS (41). Trials, cows within trials, periods within trials, treatments, and residual effects were the sources of variation. Data were Log (base e ) transformed before the ANOVA for hormone concentrations, NEFA concentrations, and AUC. Differences between means were declared at P < 0.05, and tendencies were declared at P < 0.1. None of the kinetic parameters after feed distribution were significantly different between d 13 and 28; therefore, final analysis was made on the values averaged on d 13 and 28. RESULTS Nutrient Supply and Milk Yield and Composition Glucose treatment provided the same amount of NEL and PDI that the control treatment did and slightly increased the net energy balance (Table 2).

TABLE 2. Effect of glucose treatment on nutrient supply and milk yield and composition. Treatment Control Glucose1 DMI, kg/d NEL Intake, Mcal/d Balance,2 Mcal/d PDI3 Intake, g/d Balance, g/d Milk Yield, kg/d 4% FCM, kg/d Fat g/d % True protein g/d %

19.9

17.1

30.4 1.9

30.3 3.4

1981 306

2002 278

SE

0.3 1.2 47 31

P

0.58 0.06 0.42 0.13

26.3 25.9

25.5 23.7

1.5 1.3

0.33 0.02

1025 3.95

901 3.59

53 0.13

<0.01 <0.01

801 3.10

815 3.25

18 0.09

0.19 0.03

1Glucose (1.5 kg/d) was continually infused into the duodenum, and glucose treatment (diet and infusion) supplied the same energy level as did the control treatment. 2Difference between intake and requirement for milk yield. 3Protein truly digested in the small intestine.

The difference in dietary intake that was imposed to equalize total energy intake did not significantly change the concentration (91 mmol/L) or the proportion of VFA in the rumen (62.5% acetate, 19.2% propionate, and 15.4% butyrate), which was only measured in trial 2. Glucose treatment hardly affected milk yield but increased protein content (0.15 percentage units) and decreased milk fat content (0.36 percentage units). Milk protein yield was slightly, but not significantly, increased, and milk fat yield was significantly decreased (–124 g ) for cows infused with glucose. Glucose treatment also modified milk fatty acid composition (Table 3): the concentration (grams per 100 g ) of the shortest chain fatty acids ( C 4 to C8) and C18 fatty acid fractions (–4 percentage units) decreased. Conversely, the concentration of the other fatty acids (mainly the C16 fatty acid fraction) increased. Glucose treatment decreased daily secretion of C18 fatty acids in milk fat by 61 g (Table 3), which represented about half of the decrease in milk fat yield (–12 kg). Plasma Insulin Concentrations and Metabolites Glucose infusion significantly increased basal plasma concentrations of glucose and insulin and significantly decreased basal a-AN (Table 4). Basal NEFA were also decreased by glucose infusion. However, the high heterogeneity of basal NEFA did Journal of Dairy Science Vol. 80, No. 11, 1997

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not permit them to reach significance; this effect was only due to one cow at 10 wk of lactation that exhibited very high NEFA during the control treatment. Basal concentrations of BHBA and total glycerides (measured only in trial 2 ) also decreased after glucose treatment. In addition, acetate and growth hormone were slightly, but not significantly, decreased (Table 4). After feed distribution, plasma insulin increased to the same extent in both treated and control cows soon after feeding, despite a slightly lower feed supply for cows treated with glucose (Figure 1). The AUC ( 0 to 360 min) for insulin was similar (Table 4). Plasma glucose concentrations started to decrease very early after feed distribution but remained significantly higher in cows that were infused with glucose (Figure 1). Two hours after feed distribution, plasma glucose concentrations started to increase in treated cows but continued to decrease in control cows. As a result, the difference in concentrations of plasma glucose between the two treatments increased and reached 8 mg/100 ml from 180 to 360 min after the beginning of

TABLE 3. Effect of glucose treatment on composition of milk fatty acids. Treatment Milk fatty acid

Control Glucose1

SE

P

(%) C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 iso-C15:0 C15:0 C15:1 C16:0 C16:1 iso-C17:0 C17:0 C17:1 C18:0 C18:1 C18:2 C18:1:C18 C4 to C122 C142 C15 and C172 C162 C182

3.12 2.49 1.70 4.11 5.01 13.80 1.21 0.30 1.27 0.50 37.43 1.56 0.34 0.58 0.65 8.39 14.79 2.83 1.78 146 132 31 343 230

2.72 2.29 1.63 4.21 5.71 14.00 1.63 0.26 1.51 0.42 40.42 2.08 0.29 0.55 0.59 5.90 13.26 2.58 2.27 (g) 129 121 28 327 169

0.16 0.08 0.05 0.22 0.23 0.42 0.09 0.04 0.03 0.05 0.76 0.19 0.03 0.02 0.03 0.28 0.60 0.15 0.13 8 5 2 12 23

<0.01 <0.01 0.04 0.40 <0.01 0.38 <0.01 0.13 <0.01 0.02 <0.01 <0.01 0.02 0.02 0.01 <0.01 <0.01 0.02 <0.01 <0.01 <0.01 <0.01 0.05 <0.01

1Glucose (1.5 kg/d) was continually infused into the duodenum, and glucose treatment (diet and infusion) supplied the same energy level as did the control treatment. 2Sum of fatty acids, including isomers indicated.

Journal of Dairy Science Vol. 80, No. 11, 1997

Figure 1. Comparison of glucose ( ⁄) or control ( ◊) treatment responses during morning meals. Growth hormone was only measured during trial 2. †P ≤ 0.1, *P ≤ 0.05, and **P ≤ 0.01.

GLUCOSE INFUSION AND INSULIN SECRETION

2859

the meal. During the same time, both treated cows and control cows exhibited identical insulin concentrations (1.48 ng/ml; P > 0.5). Plasma NEFA concentrations decreased after feed distribution, reducing the initial difference observed between treated cows and control cows. Plasma NEFA concentrations were significantly lower in treated cows most of the time. However, the negative AUC for NEFA was not significantly different between both groups. Plasma growth hormone, which was only measured in trial 2, decreased in response to the meal up to 4 h. The response was similar in both groups (Table 4; Figure 1). Responses to Glucose and Insulin Challenges In response to the i.v. injection of glucose (Figure 2), plasma glucose concentrations reached about 160 mg/100 ml at 3 min after the beginning of the injection and then decreased similarly up to 45 min after the injection. Insulin concentrations increased, peaked at 12 min, and then decreased to return to the basal concentration at 45 min in both groups. Around the peak (from 6 to 18 min), insulin concentrations were significantly higher in treated cows (Table 5). However, when the entire time course (from 0 to 75 min) was considered, neither AUC for insulin and glucose nor the glucose K value (calculated from 6 to 30 min) was modified by glucose treatment. Finally, plasma concentrations of NEFA were consistently and significantly lower in treated cows most of the time, and NEFA exhibited parallel changes in both groups throughout glucose disposal and during recovery. The injection of insulin i.v. (0.6 mg/kg of BW) created a moderate, but significant, decrease in plasma concentrations of glucose and NEFA (Figure 3). The minimum decrease in glucose concentrations occurred between 25 and 30 min after the injection and was similar in both groups (50.7 and 53.8 mg/100 ml, respectively, in control and treated cows; P > 0.5). The glucose K value (from 0 to 20 min) as well as the AUC for glucose and NEFA (from 0 to 75 min) remained unchanged by glucose treatment. Recovery of basal concentrations of NEFA and glucose occurred at the same rate in both control and treated cows. Throughout the experiment, plasma concentrations of glucose and, to a larger extent, NEFA were higher and lower, respectively, in treated cows than in control cows. DISCUSSION Insulin and Glucose Relationships Glucose treatment increased basal plasma concentrations of glucose and insulin by 5 and 25%, respec-

Figure 2. Comparison of glucose ( ⁄) or control ( ◊) treatment responses after glucose challenge (0.1 g/kg of BW; i.v. injection over 1.5 min). †P ≤ 0.1, *P ≤ 0.05, and **P ≤ 0.01. Journal of Dairy Science Vol. 80, No. 11, 1997

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tively (relative increase compared with that of the control treatment), and decreased NEFA concentrations by 44%. Following feed distribution, concentrations of insulin increased and were similar from 60 to 360 min in both treatments. In response to this early increase in plasma insulin, plasma concentrations of glucose decreased in both treatments after the beginning of the meal as was previously observed in ruminants: lactating goats (11), lactating cows (6, 16), and sheep (32). At the same time, NEFA decreased to a large extent in the control group and only to a minor extent in the treated group, which suggests that, in the treated group, lipolysis was largely inhibited before access to feed. From 120 to 360 min, plasma glucose continued to decrease in the control cows and was rapidly restored to basal concentrations in the treated cows. As a result, there was an increase in the difference in plasma glucose between treatments. Plasma concentrations of glucose reached

at least 8 mg/100 ml, which in dairy cows represents a large change. During the same time (from 120 to 360 min), plasma concentrations of insulin were maintained at a high but similar concentration in both groups. The relationships observed between glucose and insulin concentrations in response to glucose treatment from 120 to 360 min after the beginning of the meal might indicate peripheral insulin resistance coupled with a decrease in pancreas sensitivity to glucose. Both glucose and insulin challenges led to the opposite conclusion; pancreas reactivity to glucose, glucose disposal, and the hypoglycemic effect of insulin were not modified by glucose treatment. Pancreas reactivity was, in fact, slightly enhanced early

TABLE 4. Effects of glucose treatment on basal concentrations of nutrients, insulin, and growth hormone and plasma kinetics of nutrients, insulin, and growth hormone after the morning feed distribution. Treatment Control Glucose1 Basal concentration Glucose, mg/100 ml Insulin, ng/ml a-AN,3 mg/100 ml NEFA, mmol/L BHBA,4 mg/100 ml Total glycerides,4,5 mmol/L Acetate,4 mg/100 ml Growth hormone,4 ng/ml Plasma kinetics after feed distribution DMI,6 kg AUC7 Insulin, ng/ml × min Glucose, mg/ml × min NEFA, mmol/ml × min Growth hormone, ng/ml × min

65.9 0.82 26.0 73.4 4.8 66 3.6 4.83

69.2 1.02 24.2 41.3 3.8 52 3.2 3.95

11.9

10.3

171 –23.5 –14.8

153 –12.1 –5.1

–524

–386

SE2

P2

1.39 0.10 1.13 0.44 0.3 0.5 0.3 0.15

<0.01 <0.01 0.02 0.07 0.06 <0.01 0.22 0.14

0.53 0.82 0.87

0.54 0.03 0.23

0.99

0.81

1Glucose

(1.5 kg/d) was continually infused into the duodenum, and glucose treatment (diet and infusion) supplied the same energy level as did the control treatment. 2Standard error and the treatment effect are the results of the ANOVA after a Log (base e ) transformation of data for basal concentrations of insulin and growth hormone and for area under the response curve of all the parameters. 3a-Amino nitrogen. 4Only measured during trial 2. 5Sum of glycerol plus triglycerides. 6DMI during the corresponding morning meals. 7Area under the response curve calculated from 0 to 360 min after feed distribution after correcting for basal concentrations. Journal of Dairy Science Vol. 80, No. 11, 1997

Figure 3. Comparison of glucose ( ⁄) or control ( ◊) treatment responses after insulin challenge (0.6 mg/kg of BW; i.v. injection over 1 min). †P ≤ 0.1, *P ≤ 0.05, and **P ≤ 0.01.

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GLUCOSE INFUSION AND INSULIN SECRETION TABLE 5. Effect of glucose treatment on responses to glucose or insulin challenge. Treatment

Glucose challenge AUC3 Glucose, mg/ml × min Insulin, ng/ml × min NEFA, mmol/ml × min Glucose K,4 10–3 × min–1 Insulin peak,5 ng/ml Insulin challenge AUC Glucose, mg/ml × min NEFA, mmol/ml × min Glucose K, 10–3 × min–1

Control

Glucose1

SE2

P2

15.6 79 –1.91 –20.0 3.9

14.3 98 –0.96 –18.8 5.3

0.19 0.47 0.47 2.5 0.27

0.25 0.57 0.28 0.36 0.09

–6.98 –1.44 –13.8

–7.91 –0.62 –12.5

0.22 0.72 3.6

0.30 0.84 0.49

1Glucose (1.5 kg/d) was continually infused into the duodenum, and glucose treatment (diet and infusion) supplied the same energy level as did the control treatment. 2Standard error and the treatment effect are the results of the ANOVA after a Log (base e ) transformation of data. 3Area under the response curve calculated from 0 to 75 min after the i.v. injection after correcting for basal concentrations. 4Glucose fractional rate calculated after a Log (base e ) transformation of the data from 6 to 30 min after i.v. injection of glucose and from 0 to 20 min after i.v. injection of insulin. 5Insulin peak was calculated from 6 to 18 min after i.v. injection.

during the glucose challenge in treated cows as was suggested by the insulin peak, which remains unexplained. In addition, the response to insulin for the antilipolytic effect also appeared normal; plasma concentrations of NEFA, even though initially low in treated cows, were further depressed during both challenges. Other studies have examined possible changes in insulin response using different but related protocols (either increasing the ratio of concentrate to roughage or the digestive availability of glucose). In dairy cows ( 1 5 ) and sheep (1, 27), pancreas reactivity to nutritional stimuli (1, 15) and tissue response (1, 15, 27) or sensitivity to insulin (1, 27) were unchanged, regardless of the technique used (isotopic i.v. injection, glucose challenge, hyperinsulinemic-euglycemic clamps, or hyperglycemic clamps). The origin of the large differences found in plasma glucose between the two treatments during the second part of plasma kinetics after the meal remains unexplained but may come from a decrease in glucose utilization or from an increase in glucose production rate. In the presence of the high postprandial concentration of insulin, a decrease in glucose utilization might be linked to a decrease in the number of insulin-sensitive glucose transporters (GLUT4) in muscles, which are the major insulin-dependent components for glucose utilization (22, 28). In several animal species, hyperglycemia down-regulates the

number of insulin-sensitive glucose transporters (28). However, such a down-regulation by glucose treatment is unlikely. In fact, its presence would have been evident during both glucose and insulin challenges unless the decrease in insulin-sensitive glucose transporters was moderate and only appeared after a sustained stimulation of longer duration. An increase in glucose supply from both exogenous and endogenous sources was more likely. The continuous glucose infusion provided glucose at a rate of 1 g/min in the duodenum, which was 30% higher than the blood glucose clearance rate estimated during glucose challenge (20 × 10–3/min; i.e., 0.7 g/min). Before and during the first 2 h of the morning meal, the amount of glucose infused would have accounted for the positive difference of 3.3 mg/100 ml in plasma concentrations of glucose compared with the control. Later on, further mechanisms are supposed to be responsible for the changes. Liberation of endogenous glucose from glycogenolysis would not occur at the end of plasma kinetics after the meal because of the presence of a high postprandial concentration of insulin. Conversely, the infusion of glucose i.v. has been reported to increase liver glycogen content twofold in fed lactating dairy cows (43). Gluconeogenesis is likely to be decreased in the period between meals by glucose infusion (43). However, by 120 to 360 min after the beginning of the meal, adaptive gluconeogenesis might have developed in treated cows to preJournal of Dairy Science Vol. 80, No. 11, 1997

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vent the hypoglycemic state that continued to develop in the control group. In ruminants, gluconeogenesis is continuously active but is maximal following feeding. Gluconeogenesis is considered to be regulated more by precursor availability than by hormonal status (glucagon and corticoids) ( 7 ) . In the present experiment, other lipolytic and glucogenic hormones did not appear to be involved because plasma concentrations of NEFA and growth hormone remained depressed at the end of the meal when plasma concentration of glucose was increasing in cows treated with glucose. It can be hypothesized that the rate of postprandial gluconeogenesis is regulated at a different set point in cows treated with glucose. In the absence of a clear hormonal control of postprandial gluconeogenesis ( 7 ) , the plasma concentration of glucose itself, which normally is higher with glucose treatment, might serve as a signal for gluconeogenesis and for hepatic glucose delivery after its initial decrease during the meal (43). Even though the exact mechanism is still to be identified, glucose treatment was characterized by an increase in basal plasma insulin and glucose concentrations and a decrease in basal plasma NEFA concentrations. This feature seemed to prevent lipolysis and to increase glucose availability toward the end of the postprandial period. Infusion of glucose i.v. is reported to decrease lipolysis ( 4 6 ) and to increase the activity of key lipogenic enzymes in adipose tissue (4, 39, 46). In the long term, such alterations might increase body fat deposition. Milk Composition Glucose treatments decreased milk yield slightly, but not significantly. Milk protein content was slightly increased by glucose infusion, most likely as result of opposite, but nonsignificant, changes in milk volume and milk protein yield. In contrast, milk fat content (0.36 percentage units) and milk fat yield (–124 g ) were both significantly decreased by glucose treatment. The decrease in milk fat yield observed in the present experiment was within the range of variations previously observed by Hurtaud et al. (24, 25), who infused 0.5 to 1.5 kg/d of glucose into the duodenum. In the present experiment and in the experiments of Hurtaud et al. (24, 25), the total NEL intake was equalized in glucose and control treatments. In these experiments, milk yield did not differ among treatments. Most of the time, postruminal glucose infusions administered in the form of a supplement to the diet (9, 12, 13, 18, 33, 34, 38, 40, 42, 48, 49) significantly decreased milk fat content (12, 13, Journal of Dairy Science Vol. 80, No. 11, 1997

Figure 4. Effect of postruminal infusions of glucose on milk fat yield. Numbers indicate reference numbers; ⁄ = present study.

18, 33, 34, 48, 49) but did not change ( 1 8 ) or nonsignificantly decreased (13, 33, 34, 48, 49) milk fat yield. For example, in response to 2.15 kg of glucose infused abomasally, milk fat yield was unchanged, and milk fat content decreased, because of an increase in milk yield (18). The results of Frobish and Davis ( 1 8 ) and Clark et al. ( 9 ) did not support the negative effect of glucose on milk fat yield, which suggests that results were dependent both on the energy supply and the balance between milk yield and milk fat secretion. However, the global analysis of 14 trials [(9, 12, 13, 18, 24, 25, 33, 34, 38, 40, 42, 48, 49) and the present experiment] showed a clear tendency for glucose infusions to decrease milk fat yield (Figure 4). In this comparison, the results of Frobish and Davis ( 1 8 ) appeared to be quite different from the results of others. The negative effect of postruminal glucose infusions on milk fat yield has already been suggested (8, 45). When high starch diets were administered, milk fat content and yield were generally decreased to a greater extent (8, 10) than that when postruminal infusions of glucose were administered. However, other mechanisms and factors other than glucose alone are involved in milk fat depression when such diets are administered (10, 18, 19, 36). Among these factors, inhibition of de novo fatty acid synthesis in the mammary gland is often suggested. When milk fat depression is observed, the concentrations of all

GLUCOSE INFUSION AND INSULIN SECRETION

short-chain fatty acids often decrease, but C18:1 fatty acids increase (19, 36). This increase in C18:1 fatty acids is attributed to an increase in trans-C18:1 that had a specific inhibitory effect on de novo fatty acid synthesis. In the present experiment, concentration of the shortest chains of fatty acids ( C 4 to C8) and of all C18 ( C 18:0 and C18:1) fractions were decreased by duodenal infusion of glucose, but the concentrations of C10 to C16 fatty acids were increased. The present glucose treatment did not have any direct inhibitory effect on de novo fatty acid synthesis in the mammary gland, suggesting that duodenal glucose infusion decreased milk fat yield through a mechanism other than high starchy diets that induced milk fat depression (36). Dhiman et al. ( 1 2 ) observed very similar changes in milk fatty acid composition when abomasal infusion of glucose and protein supplementation were compared with protein supplementation alone. Glucose, Insulin, and Milk Fat Yield In the present experiment, the decrease in shortand long-chain fatty acids might be partially attributed to the reduction of feed intake. However, this decrease is unlikely to explain the total decrease (–124 g), because of a decrease in the C18 fatty acid fraction (–61 g ) that was derived from circulating concentrations of fatty acids. Using the C18 fatty acid composition of the feed and the predicted digestibility (0.82) ( 1 4 ) as the exogenous contribution of linoleic acid to milk fat (0.44) (37), the alimentary decrease in C18 fatty acids could only explain one-fourth (–14 g ) of the decrease in the yield of C18 fatty acids. In addition, infusion of glucose i.v. administered as a supplement to the diet decreased milk fat yield mainly through a decrease in the yield of C18 fatty acids (44). The decrease in the yield of C18 fatty acids might then result from a decrease in adipose lipolysis. Similarly, the decrease in the yield of the shortest chain fatty acids ( C 4 to C8) might partially result from an increase in lipogenesis ( 2 ) . As discussed previously, plasma NEFA were always consistently low, and, in trial 2, basal BHBA and total glycerides were significantly decreased. Infusions of glucose i.v., which also decreased milk fat, similarly lowered blood plasma triglycerides, NEFA, and BHBA (17, 29) and increased lipogenesis (46). In addition, postruminal infusion of glucose has been reported to inhibit lipolysis (46). Those observations and the present results sustained the hypothesis that an increase in glucose and insulin concentrations decreased the availability

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of fatty acid precursors and thereby decreased milk fat yield (29). The fact that glucose treatment enhanced basal plasma concentrations of glucose and insulin without inducing insulin resistance might be the critical mechanism that inhibits lipolysis. However, an increase in plasma concentrations of insulin (about fivefold) and the maintenance of basal plasma concentrations of glucose ( ±10%) during a 4-d hyperinsulinemic-euglycemic clamp only transiently, and not significantly, decreased milk fat content and milk fat yield but did increase milk yield (30, 31). McGuire et al. ( 3 0 ) suggested that the increase of lipid synthesis in adipose tissue during milk fat depression was a consequence of reduced mammary synthesis of milk fat and increased energy intake from a high concentrate content in the diet. In the present experiment, net energy intake was similar in both treatments. Energy balance was slightly more positive, resulting from the decrease in milk fat yield, which might have caused the increase in plasma insulin and induced the inhibition of adipose lipolysis. However, the analysis of milk fatty acid composition suggested no direct inhibitory effect of glucose treatment on milk fat synthesis. In the present experiment, the increase in basal plasma concentration of insulin as basal plasma concentrations of NEFA and total glycerides decreased seemed to be a cause rather than a consequence of the decrease in milk fat yield. Moreover, the fatty acid composition of milk fat was modified during another 4-d hyperinsulinemiceuglycemic clamp ( 2 0 ) in a manner that was very similar to that observed in the present experiment, which also suggested an inhibition of lipolysis or an increase in lipogenesis caused by the high concentrations of insulin during the clamp. Interestingly, other changes were observed at the end of the 4-d clamp. Despite a decrease in feed intake, milk protein yield and plasma IGF-I concentrations increased, and plasma concentrations of IGF-II, IGF binding protein2, and an IGF binding protein of a lower molecular mass decreased (31). In the present experiment, milk protein deposition also tended to be increased by glucose infusion (milk protein content was increased). However, most likely, both protocols (4-d clamp and duodenal glucose infusion for a month) may induce different modifications in short- and long-term metabolic control. In particular, the diurnal cycles of plasma insulin that are associated with the meals were maintained during the protocol for duodenal infusion of glucose. At the same energy level supplied by the control treatment, the glucose treatment primarily increased plasma concentrations of glucose Journal of Dairy Science Vol. 80, No. 11, 1997

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and insulin from basal conditions. Duodenal infusion of glucose also increased plasma concentrations of glucose during postprandial periods. These changes were small in magnitude but were likely to be important for their cumulative effect on nutrient fluxes. It is difficult to know in our experiment which modification was critical to the decreased milk fat yield: the increase in plasma glucose and insulin during basal measurement, the increase in plasma glucose during the postprandial period, or both. CONCLUSIONS When total energy supply is maintained, duodenal infusion of glucose increased basal plasma concentrations of glucose and insulin and increased postprandial plasma concentrations of glucose without inducing insulin resistance. This feature would inhibit lipolysis continuously, which in turn would account for the decrease in milk fat yield, mainly through a decrease in long-chain fatty acids ( C 18 fraction). ACKNOWLEDGMENTS The authors are grateful to J. Grizard (Laboratoire du Me´tabolisme Azote´, Institut National de la Recherche Agronomique, Theix, France) for his comments; to G. Kann (Laboratoire de Biologie Mole´culaire et Cellulaire, Institut National de la Recherche Agronomique, Jouy en Josas, France) for growth hormone analysis; to P. Lamberton, A. Cozien, and their crew for helpful assistance, cow welfare, and feeding; and to M. Ermel, M. Verite´, V. Ferre´, S. Marion, and S. Crochet for technical assistance. REFERENCES 1 Achmadi, J., H. Yanagisawa, H. Sano, and Y. Terashima. 1993. Pancreatic insulin secretory response and insulin action in heat-exposed sheep given a concentrate or roughage diet. Domest. Anim. Endocrinol. 10:279. 2 Annison, E. F., and R. Bickerstaffe. 1974. Glucose and fatty acid metabolism in cows producing milk of low fat content. J. Agric. Sci. (Camb.) 82:87. 3 Armstrong, D. G., and K. L. Blaxter. 1961. The utilization of the energy of carbohydrate by ruminants. Page 187 in Proc. 2nd Symp. Energy Metab. E. Brouwer and A.J.H. Van Es, ed. EAAP Publ. No 10. Eur. Assoc. Anim. Prod., Wageningen, The Netherlands. 4 Ballard, F. J., O. H. Fishell, and I. G. Jarett. 1972. Effects of carbohydrate availability on lipogenesis in sheep. Biochem. J. 226:193. 5 Bareille, N., and P. Faverdin. 1996. Lipid metabolism and intake behavior of dairy cows: effects of intravenous lipid and badrenergic supplementation. J. Dairy Sci. 79:1220. 6 Blum, J. W., F. Jans, W. Moses, D. Fro¨hli, M. Zemp, M. Wanner, I. C. Hart, R. Thun, and U. Keller. 1985. Twenty four-hour pattern of blood hormone and metabolite concentrations in high-yielding dairy cows: effect of feeding low or high amounts of starch or christalline fat. Zentralbl. Veterinaermed. 32:401. Journal of Dairy Science Vol. 80, No. 11, 1997

7 Brockman, R. P., and B. Laarveld. 1986. Hormonal regulation of metabolism in ruminants: a review. Livest. Prod. Sci. 14:313. 8 Clark, J. H. 1975. Lactational responses to postruminal administration of proteins and amino acids. J. Dairy Sci. 58:1178. 9 Clark, J. H., H. R. Spires, R. G. Derrig, and M. R. Bennink. 1977. Milk production, nitrogen utilization and glucose synthesis in lactating cows infused postruminally with sodium caseinate and glucose. J. Nutr. 107:631. 10 Davis, C. L., and R. E. Brown. 1970. Low milk fat syndrome. Page 545 in Physiology of Digestion and Metabolism in the Ruminant. A. T. Phillipson, ed. Oriel Press Ltd., Newcastle Upon Tyne, England. 11 De Jong, A. 1981. The effect of feed intake on nutrient and hormone levels in jugular and portal blood in goats. J. Agric. Sci. (Camb.) 96:643. 12 Dhiman, T. R., C. Cadorniga, and L. D. Satter. 1993. Protein and energy supplementation of high alfalfa silage diets during early lactation. J. Dairy Sci. 76:1945. 13 Dhiman, T. R., C. Cadorniga, and L. D. Satter. 1993. Protein as the first-limiting nutrient for lactating dairy cows fed high proportions of good quality alfalfa silage. J. Dairy Sci. 76:1960. 14 Doreau, M., Y. Chilliard, D. Bauchart, and P. Morand-Fehr. 1987. Besoins en lipides des ruminants. Bull. Tech. Centre Rech. Zootech. Vet. Theix, Inst. Natl. Rech. Agron. 70:91. 15 Evans, E., J. G. Buchanan-Smith, G. K. McLeod, and J. B. Stone. 1975. Glucose metabolism in cows fed low- and highroughage diets. J. Dairy Sci. 58:672. ` res 16 Faverdin, P. 1985. Regulation de l’ingestion des vaches laitie en de´but de lactation. Ph.D. Diss., Inst. Natl. Agron., ParisGrignon, France. 17 Fisher, L. J., and J. M. Elliot. 1966. Effect of intravenous infusion of propionate or glucose on bovine milk composition. J. Dairy Sci. 49:826. 18 Frobish, R. A., and C. L. Davis. 1977. Effects of abomasal infusions of glucose and propionate on milk yield and composition. J. Dairy Sci. 60:204. 19 Gaynor, P. J., D. R. Waldo, A. V. Capuco, R. A. Erdman, L. W. Douglass, and B. Teter. 1995. Milk fat depression, the glucogenic theory, and trans-C18:1 fatty acids. J. Dairy Sci. 78:2008. 20 Griinari, J. M., D. E. Bauman, and M. A. McGuire. 1996. Recent developments in altering milk composition and quality. Page 12 in Symp. Milk Synthesis, Secretion and Removal in Ruminants. J. W. Blum, ed. Univ. Berne, School Vet. Med., Berne, Switzerland. 21 Harmon, D. L. 1992. Impact of nutrition on pancreatic exocrine and endocrine in ruminants: a review. J. Anim. Sci. 70:1220. 22 Hocquette, J. F., F. Bornes, B. Graulet, D. Dardevet, M. Vermorel, Y. Geay, and P. Ferre. 1994. Nutritional regulation of insulin regulable glucose transporter in bovine muscle. Reprod. Nutr. Dev. 34:628. 23 Hurtaud, C., H. Rulquin, and R. Ve´rite´. 1993. Effect of infused volatile fatty acids and caseinate on milk composition and coagulation in dairy cows. J. Dairy Sci. 76:3011. 24 Hurtaud, C., R. Ve´rite´, and H. Rulquin. 1992. Effects on milk yield and composition of infusions of different levels and natures of energy into the digestive tract of dairy cows. Ann. Zootech. (Paris) 41:105.(Abstr.) 25 Hurtaud, C., R. Ve´rite´, and H. Rulquin. 1994. Effects on milk yield and composition of infusions of graded levels of glucose into the duodenum of dairy cows. Ann. Zootech. (Paris) 43: 309.(Abstr.) 26 Institut National de la Recherche Agronomique. 1989. Ruminant Nutrition: Recommended Allowances and Feed Tables. R. Jarrige, ed. John Libbey Eurotext, London, England. 27 Janes, A. N., T.E.C. Weekes, and D. G. Armstrong. 1985. Insulin action and glucose metabolism in sheep on dried-grass or ground, maize-based diets. Br. J. Nutr. 54:459. 28 Klip, A., T. Tsakiridis, A. Marette, and P. A. Ortiz. 1994. Regulation of expression of glucose transporters by glucose: a review of studies in vivo and in cell cultures. FASEB J. 8:43. 29 McClymont, G. L., and S. Vallance. 1962. Depression of blood glycerides and milk-fat synthesis by glucose infusion. Proc. Nutr. Soc. 21:xli.

GLUCOSE INFUSION AND INSULIN SECRETION 30 McGuire, M. A., J. M. Griinari, D. A. Dwyer, and D. E. Bauman. 1995. Role of insulin in the regulation of mammary synthesis of fat and protein. J. Dairy Sci. 78:816. 31 McGuire, M. A., J. M. Griinari, D. A. Dwyer, R. J. Harrel, and D. E. Bauman. 1995. Insulin regulates circulating insulin-like growth factors and some of their binding proteins in lactating cows. Am. J. Physiol. 269:E723. 32 Mineo, H., T. Oyamada, T. Yasuda, M. Akiyama, S. Kato, and J. I. Ushijima. 1990. Effect of feeding frequency on plasma glucose, insulin and glucagon concentrations in sheep. Jpn. J. Zootech. Sci. 61:411. 33 Oldham, J. D., and J. A. Bines. 1984. Milk production in cows infused abomasally with casein, glucose or aspartic and glutamic acids early in lactation. Proc. Nutr. Soc. 43:65A.(Abstr.) 34 Ørskov, E. R., D. A. Grubb, and R. N. Kay. 1977. Effect of postruminal glucose or protein supplementation on milk yield and composition in Friesian cows in early lactation and negative energy balance. Br. J. Nutr. 38:397. 35 Palmer, D. W., and J. R. Peters. 1969. Simple automatic determination of amino groups in serum/plasma using trinitrobenzene sulfonate. Clin. Chem. 15:891. 36 Palmquist, D. L., A. D. Beaulieu, and D. M. Barbano. 1993. Feed and animal factors influencing milk fat composition. J. Dairy Sci. 76:1753. 37 Palmquist, D. L., and W. Mattos. 1978. Turnover of lipoproteins and transfer to milk fat of dietary (1-carbon-14) linoleic acid in lactating cows. J. Dairy Sci. 61:561. 38 Peel, C. J., T. J. Fronk, D. E. Bauman, and R. C. Gorewit. 1982. Lactational response to exogenous growth hormone and abomasal infusion of a glucose-sodium caseinate mixture in high-yielding dairy cows. J. Nutr. 112:1770. 39 Rao, D. R., G. E. Hawkins, and R. C. Smith. 1973. Effect of glucose and insulin on lipoprotein lipase activity in adipose tissue and milk. J. Dairy Sci. 56:1415.

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40 Rogers, G. L., A. M. Bryant, and L. M. McLeay. 1979. Silage and dairy cow production. J. Agric. Res. 22:533. 41 SAS User’s Guide: Statistics, Version 6 Edition. 1987. SAS Inst., Inc., Cary, NC. 42 Spires, H. R., J. H. Clark, R. G. Derrig, and C. L. Davis. 1975. Milk production and nitrogen utilization and glucose synthesis in response to postruminal infusion of sodium caseinate in lactating cows. J. Nutr. 105:1111. 43 Stangassinger, M., and D. Giesecke. 1986. Splanchnic metabolism of glucose and related energy substrates. Page 34 in Control of Digestion and Metabolism in Ruminants. L. P. Milligan, ed. Prentice-Hall, Englewood Cliffs, NJ. 44 Storry, J. E., and J.A.F. Rook. 1965. Effects of intravenous infusions of acetate, b-hydroxybutyrate, triglyceride and other metabolites on the composition of the milk fat and blood in cows. Biochem. J. 97:879. 45 Thomas, P. C., and D. G. Chamberlain. 1984. Manipulation of milk composition to meet market needs. Page 219 in Recent Advances in Animal Nutrition. W. Haresign, ed. Butterworths, London, England. 46 Vernon, R. G. 1981. Lipid metabolism in the adipose tissue of ruminant animals. Page 280 in Lipid Metabolism in Ruminant Animals. W. W. Christie, ed. Pergamon Press, Oxford, England. 47 Vernon, R. G. 1989. Endocrine control of metabolic adaptation during lactation. Proc. Nutr. Soc. 48:23. 48 Vik-Mo, L., R. S. Emery, and J. T. Huber. 1974. Milk protein production in cows abomasally infused with casein or glucose. J. Dairy Sci. 57:869. 49 Whitelaw, F. G., J. S. Milne, E. R. Ørskov, and J. S. Smith. 1986. The nitrogen and energy metabolism of lactating cows given abomasal infusions of casein. Br. J. Nutr. 55:537.

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