Postprandial blood hormone and metabolite concentrations influenced by feeding frequency and feeding level in veal calves

Available online at www.sciencedirect.com

Domestic Animal Endocrinology 34 (2008) 74–88

Postprandial blood hormone and metabolite concentrations influenced by feeding frequency and feeding level in veal calves夽 T. Vicari a,1 , J.J.G.C. van den Borne b , W.J.J. Gerrits b , Y. Zbinden a , J.W. Blum a,∗ a

Veterinary Physiology, Vetsuisse Faculty, University of Bern, Bremgartenstrasse 109a, CH-3012 Bern, Switzerland b Animal Nutrition Group, Wageningen University, NL-6700 AH Wageningen, The Netherlands Received 28 August 2006; received in revised form 18 November 2006; accepted 27 November 2006

Abstract This study hypothesized that increased feeding frequency (FF) decreases problems with glucose homeostasis seen at high feeding levels (FL) in heavy veal calves. Effects of FF and FL on hormone and metabolite concentrations were studied in 15 heavy veal calves fed once (FF1; at 12:00), twice (FF2; at 12:00 and 24:00) or four times daily (FF4; at 06:00, 12:00, 18:00 and 24:00). In period 1, all calves were fed at a low FL (FLlow ; 1.5 × metabolizable energy requirements for maintenance, MEm ). In period 2, FF2 and FF4 calves were fed at high FL (FLhigh ; 2.5 × MEm ), whereas FF1 calves were still fed at FLlow . Blood was sampled every 30 min from 12:00 to 18:00 and postprandial integrated plasma hormone and metabolite concentrations (AUC12–18 h ) were calculated. Glucose AUC12–18 h increased with increasing FL, but decreased with increasing FF, urea AUC12–18 h increased with increasing FL, whereas non-esterified fatty acid AUC12–18 h were unaffected by FL and FF. Insulin AUC12–18 h decreased with increasing FF and decreasing FL. Glucagon AUC12–18 h increased with increasing FL and FF. Growth hormone AUC12–18 h decreased, whereas insulin-like growth factor-1 and leptin AUC12–18 h increased with increasing FL. Mean thyroxine and 3,5,3 -triiodothyronine concentrations were modified by FF and FL. There were no FF × FL interactions, except for plasma glucose. In conclusion, postprandial hormone and metabolite responses were differentially affected by FF and (or) FL. Glucose and insulin concentrations were maximally increased at high FL and low FF. Hyperglycemia, glucosuria and excessive insulinemia were prevented by increasing FF and decreasing FL. © 2006 Elsevier Inc. All rights reserved. Keywords: Hormones; Metabolites; Nutrition; Veal calf

1. Introduction Feeding frequency (FF) and feeding level (FL) are well known to influence animal performance and health.

夽

The data have been presented at the bi-annual meeting of the “Fachgruppe f¨ur Physiologie und Biochemie, Deutsche Gesellschaft f¨ur Veterin¨armedizin”, Giessen, Germany, 20–21 February 2006. ∗ Corresponding author. Tel.: +41 26 4077298; fax: +41 26 4077297. E-mail addresses: [email protected], [email protected] (J.W. Blum). 1 Part of a thesis for Dr. med. vet. of T.V., accepted in October 2006 to the Vetsuisse Faculty, University of Bern, Switzerland. 0739-7240/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.domaniend.2006.11.002

Calves suckling on their dam ingest colostrum or milk several times per day and basically ad libitum. Once or twice daily feeding of milk or milk replacer, as is mostly the case with bucket feeding, may not be physiologically adequate [1–4]. This is particularly the case in veal calves that are fed up to slaughter mainly with highly digestible liquid feed and at a very high feeding intensity. Furthermore, if skimmed milk protein in veal calf diets is replaced by non-milk proteins or whey, milk clotting in the abomasum is reduced or absent, and protein and fat are more rapidly transported from the abomasum to the absorptive sites in the small intestine [5,6]. It can be expected that glucose, fatty acids and amino acids

T. Vicari et al. / Domestic Animal Endocrinology 34 (2008) 74–88

(AA) under these conditions are absorbed and appear in blood more rapidly and (or) in greater amounts than if derived from casein. This may result in metabolic stress. Thus, twice daily fed veal calves towards the end of fattening often develop postprandial hyperglycemia, glucosuria, galactosuria, hyperinsulinemia, and insulin resistance [2,3,7–11], in contrast to postweaning calves [10]. Increasing the FF at the same FL improved blood glucose homeostasis, reduced postprandial insulin levels and lowered plasma urea concentrations, suggesting that nitrogen (N) utilization was enhanced [11]. Moreover, an increased FF enhanced the efficiency of energy and protein utilization in heavy veal calves [12]. Although various aspects of FF and FL, especially on growth performance, have been studied in veal calves [12–18], several things remain open. Effects of FF were in many studies, as in [11], confounded with the type of feeding system. In addition, a clotting protein source (whole milk and skimmed milk powder) was used in studies on FF and FL, as in [11,15,16], which does not correspond with the contemporary composition of milk replacer diets, in which skimmed milk protein is largely been replaced with rapidly hydrolyzed proteins. Moreover, FL × FF interactions have not been studied, except in [12]. Studies on FF and FL have often been performed in neonatal and young calves [18,19], while metabolic problems especially occur in heavy calves (>100 kg body weight). Recently it was shown that an increased FF enhanced the efficiency of energy and protein utilization, whereas a high FL enhanced the fat and protein retention in veal calves [12]. Studies on the effect of the pattern and level of nutrient intake on metabolic and endocrine profiles are lacking in heavy veal calves. Based on these premises we have studied effects of FF at two FL, as well as FF × FL interactions, on growth performance, protein and energy balances and on postprandial changes of selected metabolites and hormones in heavy veal calves. As effects of FL and FF on performance and protein and energy balances have been presented elsewhere [12], and effects were not due to improved digestibility of nutrients, it was hypothesized that they were possibly mediated by endocrine changes. Therefore, the present study focuses on the effects of FF and FL, and on FF × FL interactions, on plasma concentrations of metabolites (glucose, nonesterified fatty acids (NEFA) and urea) and of hormones (insulin, glucagon, growth hormone (GH), insulin-like growth factor-1 (IGF-1), thyroxine (T4 ), and 3,5,3 triiodothyronine (T3 )). We hypothesized that spreading the nutrient load over multiple meals would improve glucose metabolism (i.e. lower plasma glucose and insulin

75

concentrations and a lower insulin/glucose ratio) in heavy veal calves, which are known to develop insulin resistance. 2. Material and methods 2.1. Animals, husbandry, experimental design, and experimental procedures The experiment was conducted with 18 male Holstein-Friesian calves at the experimental farm “De Haar” of the Wageningen University, the Netherlands. Experimental protocols were approved by the Ethical Committee of the Wageningen University. The experimental design was described in detail previously [12] and is summarized in Table 1. Briefly, 18 Holstein Friesian calves of 136 ± 3 kg body weight (BW) were assigned to different FF (1, 2 or 4 meals daily; FF1, FF2 and FF4, respectively). Calves at FF1 were fed at 12:00, calves at FF2 at 12:00 and 24:00, and calves at FF4 at 06:00, 12:00, 18:00, and 24:00. Calves were fed at each of two FL (FLlow and FLhigh ; 1.5 and 2.5 × metabolizable energy requirements for maintenance (MEm ), respectively), except for the calves fed once daily (FF1), which were only fed at FLlow because a high FL in just one meal (at 2.5 × MEm ) is not feasible. The calculations for MEm were based on the weekly measured BW and were adjusted daily for growth. The MEm were assumed to be 460 kJ/(kg0.75 day) [20]. Calves at FF1 served as a control to evaluate the effect of age (i.e. of experimental period). After birth calves were reared for 10 weeks, then a pre-period of 28 days was allowed for the calves to adjust to the assigned FF and FL. During the first 2 weeks of the pre-period calves were housed in large individual pens and during the second 2 weeks of the pre-period in metabolic cages. This was followed by a first experimental period (P1) of 14 days, consisting of a balance period Table 1 Experimental design to study effects of feeding frequency and feeding levels on metabolic and endocrine traits in heavy veal calves (n = 6 for each group) Group

FF1 FF2 FF4

Feeding frequency (FF; meals per day)

1 2 4

Feeding level (g dry matter/ (kg body weight0.75 day)) Period 1 (P1)

Period 2 (P2)

36.6 36.6 36.6

36.6 61.1 61.1

FF, feeding frequency: FF1, fed at 12:00; FF2, fed at 12:00 and 24:00; FF4, fed at 06:00, 12:00, 18:00, and 24:00.

76

T. Vicari et al. / Domestic Animal Endocrinology 34 (2008) 74–88

(separate collection of feces and urine while calves were held in metabolic cages within respiration chambers) followed by blood sampling. After a second pre-period of 28 days, a second experimental period (P2) of 14 days followed with the same calves (repeated use of calves) and the same experimental procedures as in P1. The ambient temperature in the chambers was kept constant at 18 ◦ C, relative humidity was 65%, and air velocity was <0.2 m/s. Between 05:30 and 18:30 and between 00:00 and 00:30 calves were exposed to 50 lx of light, the rest of the time to a darkness of 6 lx. There were exclusions from the study of one calf because it became sick and of one calf at FF1 and of one calf at FF2 because they refused >10% of the feed supply in P2. One calf, at FF4, was replaced by a substitute calf between the two experimental periods. The experimental milk replacer contained whey protein as the only protein source (Table 2). It did not contain antibiotics. Prior to feeding, 140 g of the milk replacer was dissolved in 1 L of warm water (55 ◦ C). The milk replacer was supplied at a temperature of 40–43 ◦ C. Roughage was never offered. Calves were fed by bucket, which was emptied automatically in a receptacle for feed refusals at 15 min after feeding. Urine was collected

quantitatively during the 10-day balance period and was analyzed for glucose concentration. For blood sampling, calves were prepared with a central venous catheter (16G/1.70 mm × 1.60 mm; Secalon T, Becton Dickinson, Alphen aan de Rijn, The Netherlands) in the left jugular vein. The catheter was attached to the skin using Vicryl suturing (Ethicon, Sumerville, NJ) and extended with a 200 cm three-layer extension tube (Vygon, Valkenswaard, The Netherlands) and a 25 cm Lectroflex extension (Vygon). Tape (Fermoflex and Elastoplast, Instruvet, Cuijk, The Netherlands) was used to cover the catheter and to maintain the extensions in their position. Blood samples (8 mL) were obtained every 30 min from 10:30 to 16:00 and every hour from 16:00 to 18:00 to study postprandial responses of metabolites and hormones to the 12:00 meal. After collection, blood was immediately transferred to tubes (Vacutainer, Becton Dickinson, Alphen aan de Rijn, The Netherlands) containing lithium heparin (17 IU/mL) that were held on crushed ice until centrifugation within 1 h at 1500 × g for 10 min. Plasma was stored at −20 ◦ C until analyzed. Concentrations of insulin, (pancreatic) glucagon, GH, glucose and NEFA were measured in all samples. Con-

Table 2 Ingredients and analyzed nutrient composition of the experimental diet and nutrient intake at the two feeding levels Ingredientsa

g/kg

g/kg BW0.75 Energy intake

Fat filled whey powder Delactosed whey powder Whey powder Whey protein concentrate (35%) dl-Methionine (99%) Monopotassium phosphate Calcium carbonate Magnesium oxide (59%) Vitamin and mineral premixb Analyzed nutrients

380.0 70.0 211.2 322.0 1.56 2.60 6.96 0.84 4.80 g or kJ/kg dry matter

1.5 × ME/day

2.5 × ME/day

13.9 2.6 7.7 11.8 0.057 0.10 0.25 0.03 0.18

23.2 4.3 12.9 19.7 0.10 0.16 0.43 0.05 0.29

g/kg BW0.75 Energy intake

Dry matter (DM) Nitrogen (N)-free extract Crude protein (N × 6.25) Crude fat Crude ash Gross energy a

978 530 195 200 75 21,090

1.5 × ME/day

2.5 × ME/day

35.8 19.4 7.1 7.3 2.7 773

59.7 32.4 11.9 12.2 4.6 1288

As fed. Vitamin A, 25,000 IU/kg; Vitamin D3 , 2000 U/kg; Vitamin E, 80 mg/kg; Vitamin C, 130 mg/kg; zinc, 80 mg/kg; copper, 8 mg/kg; manganese, 12 mg/kg; selenium, 0.1 mg/kg; iron, 48 mg/kg. b

T. Vicari et al. / Domestic Animal Endocrinology 34 (2008) 74–88

centrations of urea, IGF-1 and leptin were measured at 10:30 and 11:00 and then hourly until 18:00. Concentrations of T3 and T4 were measured in pools that were prepared from plasma samples obtained during the preand postprandial period. 2.2. Laboratory analyses Hormones in blood plasma were measured by radioimmunoassay (RIA) as described and (or) cited [11,21–24]. Determinations from one animal of each experimental group were performed within one assay. Commercial or self-made quality control samples with low and high concentrations were included. Concentrations of hormones (except for insulin; see below) were mostly in the middle range, i.e. in the most sensitive and precise regions of standard curve concentrations. Intraand interassay coefficients of variation for all RIA were below 10 and 15%, respectively. Insulin concentrations were measured with a homologous double-antibody system using bovine insulin (25.7 IU/mg; Sigma, St. Louis, MO) for standards and for iodination and guinea-pig anti-bovine insulin (#5506, lot GP23; Bioyeda, K. Weizmann Institute, Rehovot, Israel). The antibody did not cross-react with human C-peptide. Precipitating anti-guinea-pig ␥-globulin (Calbiochem, La Jolla, CA) was used as second antibody to separate antibody-bound from free hormone. The sensitivity was <2 ␮U/mL plasma and this allowed to measure basal (preprandial) plasma levels (that in cattle, as in many other species, range normally from 5 to 20 ␮U/mL). When (based on pre-tests) the postprandial insulin concentrations were expected to be above the highest standard of the calibration curve (1000 ␮U/mL), plasma samples were diluted (1:10, v/v) with assay buffer and ovalbumin (35 mg/mL). Dilutions of the plasma that contained high amounts of insulin paralleled the standard curve. This also indicated immunological similarity of standard and endogenous bovine insulin. (Pancreatic) glucagon concentrations were measured using a kit (# GL-32 K; Linco Research Inc., St. Charles, MO). Recombinant glucagon was used as standard and for iodination and the guinea-pig antibody 100% cross-reacted with human glucagon and 0.1% with oxyntomodulin (the primary gut glucagon), but did not cross-react with human (pro-)insulin, human C-peptide, somatostatin and human pancreatic polypeptide. A goat anti-guinea-pig antibody was used to separate antibodybound and free hormone. Dilution of bovine plasma that contained high amounts of glucagon paralleled the standard curve, indicating immunological similarity of

77

standard and endogenous bovine glucagon. The sensitivity was <20 pg/mL. Growth hormone concentrations were determined using recombinant bovine GH (rbGH; kindly donated by Calbiochem, La Jolla, CA) for standards and for iodination. The antiserum against rbGH was raised in a rabbit (kindly donated by D. Schams, Institute of Physiology, Technical University of Munich, Freising, Germany). Precipitating goat-anti-rabbit-␥-globulin was used as second antibody to separate (together with 6% polyethyleneglycol) antibody-bound and free hormone. The rbGH paralleled the standard curve obtained from hypothalamic extracts (USDA-bGH-B-1 AFP-5200). Mean recoveries of rbGH for 1 and 10 ng added (10 ␮L) to 1 mL bovine serum were 117 and 107%, respectively. The sensitivity was <1 ng/mL. Insulin-like growth factor-1 concentrations were measured after extraction of the samples (50 ␮L) with formic acid (12.5 ␮L, 2.4 M) and absolute ethanol (250 ␮L). Based on column chromatography this completely removed IGF-1 from binding proteins in calves. Recombinant human IGF-1 (kindly donated by Novartis AG, Basel, Switzerland) was used as standard and for iodination. A monoclonal antibody against human IGF-1 (kindly donated by D. Kerr, Department of Animal Science, Vermont University, Burlington, VT), raised in mice hybridoma cells, was used as first antibody together with normal mouse serum. The antibody cross-reacted with des-3N-IGF-1 (40%), slightly cross-reacted with recombinant bovine IGF-2 (8%), but barely cross-reacted (<0.1%) with rat multiplication stimulating activity, bovine GH, bovine prolactin, and insulin. Sheep-anti-mouse serum (100 ␮L) was used (together with 1000 ␮L 6% polyethyleneglycol) to separate antibody-bound and free hormone. Recovery of added recombinant human IGF-1 to neonatal calf plasma was 95–107%. If plasma from a calf with high endogenous IGF-1 concentrations was diluted, IGF-1 concentrations paralleled the standard curve, indicating immunological similarity of standard and endogenous IGF-1. The sensitivity was <6.5 ng/mL. Leptin concentrations were measured using ovine leptin (Diagnostic Systems Laboratories, Webster, TX) for standards and for iodination. An anti-ovine (Ab 8172) leptin antiserum (kindly donated by Y. Chilliard, Herbivore Research Unit, National Institute for Agricultural Research, St-Gen`es-Champanelle, France) 100% crossreacted with bovine leptin. Precipitating goat anti-rabbit IgG was used to separate antibody-bound and free hormone. Concentrations of total T4 and of total T3 were measured based on solid-phase RIA kits (#TKT35 for

78

T. Vicari et al. / Domestic Animal Endocrinology 34 (2008) 74–88

T3 and #TKT45 for T4 ; Diagnostic Products Corporation, Los Angeles, CA), in which the antibody was bound to assay tubes and in which binding to carrier proteins was inhibited. In T3 assays, the antibody barely cross-reacted with reverse T3 (0.014%), l-T4 (0.5%), tetraiodothyroacetic acid (0.44%), diiodo-ltyrosine (0.007%), and monoiodotyrosine (0.001%). If plasma with high endogenous T3 concentrations was diluted, T3 concentrations paralleled the standard curve. The sensitivity was <0.1 pmol/mL. In T4 assays, the antibody cross-reacted with tetraiodothyroacetic acid (100%), but barely cross-reacted with l-T3 (2%) and triiodothyroacetic acid (2%), and did not cross-react with diiodo-l-tyrosine and monoiodotyrosine. If plasma with high endogenous T4 concentrations was diluted, T4 concentrations paralleled the standard curve. The sensitivity was <3.3 pmol/mL. Concentrations of plasma glucose and urea were measured using kits from Bio-M´erieux, Marcy l’Etoile, France (#61270 and #61974, respectively) and of plasma NEFA with a kit from Wako Chemicals, Neuss, Germany (#994-75406) using a Cobas Mira Plus automatic analyzer (Roche, Basel, Switzerland). Urinary glucose concentrations were measured enzymatically using the glucose oxidase method (GOD-PAP, Roche Diagnostics, Basel, Switzerland). Analyses of the experimental diet have been described previously [12]. 2.3. Statistical analyses Values are expressed as means ± S.E.M. Statistical analyses were conducted using SAS (Version 8.02, SAS Institute, Cary, NC). Data were subjected to descriptive statistical analysis. Normal distribution of the data was evaluated with the Shapiro–Wilk test. Non-normally distributed data were logarithmically transformed in order to meet assumptions of normal distribution. Transformed data were only used for statistics, but values shown in tables and figures are without transformation. The individual data on BW, average daily gain, dry matter (DM) intake, gross energy intake, energy retention, N intake, and N retention were calculated and analyzed as described previously [12]. Differences between means of preprandial blood plasma values from postprandial values (12:00–18:00) were evaluated by paired t-test (version 7.0; Systat Software Inc., Point Richmond, CA). Areas under the concentration curves (AUC12–18 h ; 6 h periods from 12:00 to 18:00) were calculated by the trapezium method (using Graph Pad Prism, version 1.03; Graph-Pad Software, San Diego, CA) and served as a measure of integrated postprandial concentrations of blood plasma metabolites and hor-

mones. Episodic secretion of GH (mean concentrations, basal concentrations, peak heights, and peak frequencies) was analyzed according to Merriam and Wachter [25]. Feed intake, energy retention, growth performance, urinary glucose excretion, peak values and AUC12–18 h of plasma metabolites and hormones, and episodic secretion of GH were analyzed for the effects of FF and FL, interactions between FF and FL, and period by analysis of variance (ANOVA) in two ways. Because except for BW effects of period were neither significant nor relevant, effects of FF and FL were tested in a mixed model with repeated measurements within calves, using PROC GLM, including fixed effects of FF and FL and random effect of each calf. The model used was Yijk = μ + FFi + FLk + (FF × FL)ik + εijk , where Yijk is the dependent variable, μ the average intercept, FFi the effects of different feeding frequencies i (i = 1, 2, 4), FLk the effects of different feeding levels k (k = 1 and 2), (FF × FL)ik the FF × FL interactions, and εijk is the error term, which represents the random effect of calf within feeding frequency (j = 1, . . ., 6). In this procedure P2 is not considered for FF1. The difference between the two experimental periods (P1, P2) was only analyzed in the group fed once daily (FF1) because in FF2 and FF4 effects of period and FL could not be differentiated. The model used was Yij = μ + Pj + εij , where Yij is the dependent variable, μ the average intercept, Pj the effect of experimental period (=1 and 2), and εjj is the error term, which represents the random effect of calves within feeding period (j = 1, 2, 3, 4). Because Littell et al. [26] have shown that for analyzing mixed models using PROC MIXED might be more appropriate than PROC GLM, we additionally analyzed the models also in PROC MIXED. However, both procedures yielded identical results. Treatment effects were studied by pairwise comparison using the Bonferroni method. Results are presented only from the first approach, i.e. using models without period effect. Overall differences were considered significant if P < 0.05. 3. Results 3.1. Feed intake, energy retention, growth performance and urinary glucose excretion An enhanced FF did not affect average daily gain, DM intake, gross energy intake and N intake, but positively affected (P < 0.05) energy retention and N retention, although the effect could not be assigned to individual groups (Table 3). In contrast, increasing the FL affected average daily gain, N retention, gross energy intake, and energy retention (P < 0.001). The average BW of

T. Vicari et al. / Domestic Animal Endocrinology 34 (2008) 74–88

79

Table 3 Influences of feeding frequency (FF) and feeding level (FL) on average body weight, dry matter intake, nitrogen intake and nitrogen retention, gross energy intake, and energy retention during a 9-day balance period in heavy veal calves fed once, twice or four times daily (FF1, FF2, and FF4, respectively) Trait

Number of calves Average body weight (kg) Average daily gain (kg) Dry matter intake (g/(kg BW0.75 × day)) Nitrogen intake (g/(kg BW0.75 × day)) Nitrogen retention (g/(kg BW0.75 × day)) Gross energy intake (kJ/(kg BW0.75 × day)) Energy retention (kJ/(kg BW0.75 × day)) Urinary glucose losses (mmol/day)

FF1

FF2

FF4

FLlow

FLlow

FLhigh

FLlow

4 121.5 ± 5.5 0.5 ± 0.06 36 ± 0

5 143.1 ± 3.2 y 0.6 ± 0.15 y 34 ± 2 y

5 174.5 ± 5.3 x 1.8 ± 0.04 x 58 ± 0 x

6 133.3 ± 3.6 y 0.6 ± 0.04 y 35 ± 0 y

1.11 ± 0.01

1.05 ± 0.05 y

1.80 ± 0.01 x

0.48 ± 0.03

0.51 ± 0.04 y

0.83 ± 0.04 x

756 ± 2

744 ± 23 y

121 ± 8

119 ± 18 y

419 ± 14 x

58.4 ± 12.1

58.3 ± 36.7 y

343.4 ± 71.2 x

1,229 ± 2 x

P-Value FF

FL

FF × FL

6 173 ± 6.8 x 1.7 ± 0.14 x 56 ± 1 x

ns ns ns

***

ns ns ns

1.09 ± 0.01 y

1.75 ± 0.02 x

ns

***

ns

0.55 ± 0.01 y

0.93 ± 0.03 x

*

***

ns

ns

***

ns

*

***

ns

ns

***

ns

FLhigh

756 ± 6 y

1,228 ± 16 x

159 ± 8 y

453 ± 16 x

8.7 ± 8.7 y

175.2 ± 61.4 x

*** ***

Values are means ± S.E.M. Data of FF1 of experimental periods 1 and 2 were combined. FF, feeding frequency: FF1, fed once daily at 12:00; FF2, fed twice daily at 12:00 and 24:00; FF4, fed four times daily at 06:00, 12:00, 18:00, and 24:00. FL, feeding level: FLlow , low feeding level (1.5 × MEm /day); FLhigh , high feeding level (2.5 × MEm /day). ANOVA: FF, effect of feeding frequency; FL, effect of feeding level; FF × FL, interaction between FF and FL; * P < 0.05; *** P < 0.001; ns, not significant (P > 0.05). Values with different letters are significantly different (P < 0.05) among groups with different feeding levels.

Table 4 Effects of feeding frequency and feeding level on integrated postprandial concentrations from 12:00 to 18:00 (AUC12–18 h ) of blood plasma metabolites and hormones in veal calves Item

Number of calves Metabolites Glucose (mmol/(L × 6 h)) NEFA (mmol/(L × 6 h)) Urea (mmol/(L × 6 h)) Hormones Insulin (␮g/(L × 6 h)) IGF-1 (␮g/(L × 6 h)) Glucagon (ng/(L × 6 h)) GH (␮g/(L × 6 h)) Leptin (␮g/(L × 6 h)) T3 (nmol/L) T4 (nmol/L)

FF1

FF2

FLlow

FLlow

FLhigh

FLlow

FLhigh

4

5

5

6

6

45.5 ± 4.1 0.7 ± 0.0 9.5 ± 0.5 148.6 1330 407.5 56.6 18.8 1.7 57.7

± ± ± ± ± ± ±

52.7 123 71.2 a 11.3 1.3 0.1 3.6

FF4

43.5 ± 1.0 y 0.7 ± 0.0 9.8 ± 0.5 y 93.9 1584 243.6 43.9 17.6 2.3 74.4

± ± ± ± ± ± ±

35.7 y 153 y 26.6 ab 6.3 2.7 0.2 5.7

61.5 ± 3.5 ax 0.8 ± 0.1 14.9 ± 0.6 x 349.5 2497 460.6 30.8 25.5 2.6 82.1

± ± ± ± ± ± ±

53.6 ax 134 x 113.7 6.2 2.8 0.1 5.6

P-Value

40.5 ± 0.6 0.8 ± 0.1 10.9 ± 0.4 y 19.9 1862 177.9 64.9 16.2 2.1 75.3

± ± ± ± ± ± ±

6.1 146 24.0 b 6.0 1.0 y 0.1 y 4.0 y

FF

47.5 ± 1.9 b 0.9 ± 0.1 14.3 ± 0.5 x 146.0 2486 265.2 41.5 29.4 2.8 98.0

± ± ± ± ± ± ±

11.7 b 172 20.4 7.2 2.8 x 0.1 x 5.4 x

FL

FF × FL

**

***

*

ns ns

ns

ns ns

***

***

ns

***

***

**

ns ns

*

*

***

*

**

***

***

ns ns ns ns ns ns ns

Values of glucose, non-esterified fatty acids (NEFA), insulin, glucagon and growth hormone (GH) are means ± S.E.M. of the area under the concentration curves of samples obtained every 30 min from 12:00 to 18:00. Urea, insulin-like growth factor-I (IGF-1) and leptin are means ± S.E.M. of the area under the concentration curves (AUC) of samples obtained every hour from 12:00 to 18:00 (AUC12–18 h ). Values of 3,5,3 -triiodothyronine (T3 ) and thyroxine (T4 ) are means ± S.E.M. of pooled plasma samples, collected during the pre- and postprandial periods. Data of FF1 of experimental periods 1 and 2 were combined. FF, feeding frequency: FF1, fed once daily at 12:00; FF2, fed twice daily at 12:00 and 24:00; FF4, fed four times daily at 06:00, 12:00, 18:00, and 24:00. FL, feeding level: FLlow , low feeding level (1.5 × MEm /day); FLhigh , high feeding level (2.5 × MEm /day). ANOVA: FF, effect of feeding frequency; FL, effect of feeding level; FF × FL, interaction between FF and FL; * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant (P > 0.05). Values with different letters (a and b) are significantly different (P < 0.05) among groups with different feeding frequencies. Values with different letters (x and y) are significantly different (P < 0.05) among groups with different feeding levels.

80

T. Vicari et al. / Domestic Animal Endocrinology 34 (2008) 74–88

group FF1 was greater (P < 0.05) in period 2 than in period 1 (142.9 and 117.5 kg in P1 and P2, respectively). With 48 mg iron/kg milk replacer the average daily iron intakes at FLlow and FLhigh were 67 and 133 mg, respectively. Urinary glucose excretion rates in FF2 and FF4 were greater (P < 0.01) at FLhigh than at FLlow and were greater (P < 0.05) in P2 than in P1 (168 ± 32 and 58 ± 12 mmol/day, respectively) in FF1. 3.2. Blood plasma metabolite concentrations Mean preprandial glucose concentrations (Fig. 1) were similar in all groups. After feeding, concentrations increased (P < 0.05) within 30 min and reached peak concentrations between 1 and 1.5 h in all groups. At FLlow , peak concentrations were similar in FF1, FF2 and FF4. Peak concentrations were higher (P < 0.05) at FLhigh than at FLlow in FF2. At FLlow , postprandial concentrations returned earlier to preprandial levels in FF4 than in FF2, and at FLhigh concentrations in FF2 remained higher (P < 0.05) than preprandial levels up to 6 h, whereas concentrations returned to preprandial levels within 4 h after feeding in FF4. Concentrations at FLhigh from 3 to 5 h after feeding were higher (P < 0.001) in FF2 than in FF4, and from 2 to 5 h after feeding concentrations were higher (P < 0.05) at FLhigh than at FLlow in FF2. The AUC12–18 h at FLhigh were greater (P < 0.01) in FF2 than in FF4 and in FF2 were also greater (P < 0.001) at FLhigh than at FLlow (Table 4). Glucose was the only trait for which significant FF × FL interactions were found. Mean preprandial NEFA concentrations (Fig. 2) were highest in FF1 in P2, but differences among FF1, FF2 and FF4 were not significant. After feeding, concentrations within 30 min decreased in all groups (P < 0.05) and remained low up to 6 h after feeding in FF1 and FF2, whereas in FF4 concentrations slowly returned towards preprandial levels. At FLlow , concentrations were higher (P < 0.01) in FF4 than in FF2 at 17:00. The AUC12–18 h (Table 4) was not affected by FF and FL. Mean preprandial urea concentrations (Fig. 3) were higher (P < 0.05) in FF4 than in FF1 at FLlow , and were higher (P < 0.001) at FLhigh than at FLlow . At FLlow , concentrations steadily increased (P < 0.05) after feeding in FF1, and increased in FF2 towards preprandial levels by 18:00, whereas at FLhigh concentrations remained more stable. The AUC12–18 h (Table 4) increased with increasing FL (P < 0.001), but was not affected by FF. Concentrations at all time-points from 0 to 6 h after feeding were higher (P < 0.001) and AUC12–18 h were greater (P < 0.001) at FLhigh than at FLlow .

Fig. 1. Plasma glucose concentrations during the preprandial period (10:30–12:00) and during the 6 h postprandial period (from 12:00 to 18:00) in heavy veal calves. Veal calves received the daily amount of feed in either one (A: FF1; n = 4; at 12:00), two (B: FF2; n = 5; at 12:00 and 24:00) or four meals (C: FF4; n = 6; at 06:00, 12:00, 18:00 and 24:00). Data are shown after feeding at 12:00 (↓). In the first experimental period (䊉; P1), calves were fed at a low feeding level (FLlow ) at 1.5 × metabolizable energy requirements for maintenance (MEm ). In the second experimental period (; P2), calves of FF1 were continued to be fed at 1.5 × MEm , whereas calves of FF2 and FF4 were fed at a high feeding level (FLhigh ) at 2.5 × MEm . Data are means ± S.E.M. Plasma samples were analyzed from 10:30 to 16:00 every 30 min and from 16:00 to 18:00 every hour. Values with different letters (a and b) indicate significant differences (P < 0.05) between calves at different feeding frequencies (FF1, FF2 and FF4). Values with different letters (x and y) indicate significant differences (P < 0.05) between calves at the same feeding frequency but at different feeding levels (FF2: 1.5 and 2.5 MEm /day in P1 and P2, respectively; FF4: 1.5 and 2.5 MEm /day in P1 and P2, respectively), or the same feeding level (FF1: 1.5 MEm /day in both P1 and P2) to indicate an effect of period. Means with open symbols (, ) are significantly different (P < 0.05) from the mean of preprandial values; means with closed symbols (䊉, ) are not significantly different from preprandial values (P > 0.05).

T. Vicari et al. / Domestic Animal Endocrinology 34 (2008) 74–88

Fig. 2. Plasma non-esterified fatty acid (NEFA) concentrations during the preprandial period (10:30–12:00) and the 6 h postprandial period (from 12:00 to 18:00) in veal calves. For further details see the legend of Fig. 1.

3.3. Blood plasma hormone concentrations Mean preprandial insulin concentrations (Fig. 4) were not affected by FF, but increased with increasing FL (P < 0.001) and were higher (P < 0.05) at FLhigh than at FLlow . After feeding, concentrations increased (P < 0.05) within 30 min. Times needed to reach peak concentrations decreased with increasing FF (4, 3 and 1.5 h in FF1, FF2 and FF4 at FLlow , respectively; 4 and 2.5 h at FLhigh , respectively). In FF2, postprandial peak concentrations were higher (P < 0.05) at FLhigh than at FLlow , and at FLlow postprandial peak concentrations were reached earlier (P < 0.001) in FF4 than in FF1. Concentrations returned to preprandial levels at 6 h after feeding, except in FF2 at FLhigh . The rise of insulin concentrations was

81

Fig. 3. Plasma urea concentrations during the preprandial period (10:30–12:00) and the 6 h postprandial period (from 12:00 to 18:00) in veal calves. For further details see the legend of Fig. 1.

more transient in FF2 and FF4 than in FF1 at FLlow . At FLlow , concentrations at 5 and 6 h after feeding were higher (P < 0.01) in FF1 than in FF2 and from 2.5 to 6 h after feeding they were higher (P < 0.001) in FF1 and FF2 than in FF4. Concentrations were higher (P < 0.05) at FLhigh than at FLlow at 2 h and from 3 to 6 h after feeding, while in FF4 concentrations were higher (P < 0.05) at FLhigh than at FLlow in FF2 (at 2 and 3–6 h after feeding) and in FF4 (from 0 to 6 h after feeding). At FLlow , AUC12–18 h (Table 4) decreased with increasing FF. At FLhigh , AUC12–18 h in FF2 were greater (P < 0.01) than in FF4, and in FF2 were greater (P < 0.001) at FLhigh than at FLlow . Mean preprandial IGF-1 concentrations (Fig. 5) were not affected by FF, but were enhanced by increasing FL (P < 0.001) and were higher (P < 0.05) at FLhigh than at FLlow in FF4. Postprandial concentrations stayed sta-

82

T. Vicari et al. / Domestic Animal Endocrinology 34 (2008) 74–88

Fig. 4. Plasma insulin concentrations during the preprandial period (10:30–12:00) and the 6 h postprandial period (from 12:00 to 18:00) in veal calves. For further details see the legend of Fig. 1.

Fig. 5. Plasma insulin-like growth factor-1 (IGF-1) concentrations during the preprandial period (10:30–12:00) and the 6 h postprandial period (from 12:00 to 18:00) in veal calves. For further details see the legend of Fig. 1.

ble and were not affected by FF, but were enhanced by increasing FL (P < 0.001). In FF2, postprandial concentrations from 12:00 to 18:00 and AUC12–18 h (Table 4) were greater (P < 0.01) at FLhigh than at FLlow . In FF4, concentrations at 13:00 and 14:00 were significantly higher (P < 0.05) and AUC12–18 h tended to be greater (P < 0.1) at FLhigh than at FLlow . Mean preprandial glucagon concentrations (Fig. 6) were similar among groups and were not significantly affected by FF and FL. After feeding, concentrations increased in FF1 (P < 0.05 in P1) and in FF2 (P < 0.05), whereas concentrations remained relatively stable in FF4. At FLlow , concentrations were higher (P < 0.05) in FF1 than in FF2 at 6 h after feeding and were higher (P < 0.05) in FF1 than in FF4 from 3 to 6 h after feeding. At FLhigh , concentrations were higher (P < 0.05) in

FF2 than in FF4 at 3 and 4 h after feeding. Concentrations were higher (P < 0.05) at FLhigh than at FLlow at 0.5 and 1 h after feeding in FF2. The AUC12–18 h (Table 4) decreased with increasing FF (P < 0.001), whereas it was enhanced by increasing FL (P < 0.01). Preprandial GH concentrations were not affected by FF and FL. After feeding, GH concentrations decreased inconsistently (data not shown). The AUC12–18 h (Table 4) and mean concentrations (Table 5) were not affected by FF, but decreased with increasing FL (P < 0.05). There were no effects of FF or FL on basal concentrations, peak amplitudes and peak frequencies (Table 5). Mean preprandial leptin concentrations (Fig. 7) were not affected by FF, but increased with increasing FL (P < 0.001). After feeding, concentrations remained sta-

T. Vicari et al. / Domestic Animal Endocrinology 34 (2008) 74–88

Fig. 6. Plasma glucagon concentrations during the preprandial period (10:30–12:00) and the 6 h postprandial period (from 12:00 to 18:00) in veal calves. For further details see the legend of Fig. 1.

83

Fig. 7. Plasma leptin concentrations during the preprandial period (10:30–12:00) and the 6 h postprandial period (from 12:00 to 18:00) in veal calves. For further details see the legend of Fig. 1.

Table 5 Effects of feeding frequency and feeding level on secretory profiles of growth hormone (GH) in veal calves fed once, twice or four times daily by bucket Item

Number of calves Mean (␮g/L) Basal level (␮g/L) Peak amplitude (␮g/L) Peak frequency (×h−1 )

FF1

FF2

FLlow

FLlow

4 10.7 ± 8.2 ± 17.0 ± 0.15 ±

2.2 1.2 6.8 0.06

5 10.2 ± 4.6 ± 19.2 ± 0.21 ±

FF4 FLhigh 0.9 1.0 5.2 0.06

5 8.2 ± 4.6 ± 12.7 ± 0.24 ±

ANOVA (P)

FLlow 2.2 2.0 2.3 0.04

6 12.7 ± 9.7 ± 14.6 ± 0.14 ±

FLhigh 1.3 2.4 5.3 0.05

6 7.5 ± 3.3 ± 20.2 ± 0.20 ±

1.3 0.6 5.8 0.02

FF

FL

FF × FL

ns ns ns ns

*

ns ns ns

ns ns ns ns

Values are means ± S.E.M. of 15 samples obtained every 30 min during the pre- and postprandial periods. Data of FF1 of experimental periods 1 and 2 were combined. Mean concentrations, basal values, peak amplitudes and peak frequencies were calculated according to Merriam and Wachter [25]. Basal values are calculated after exclusion of peak values. FF, feeding frequency: FF1, fed once daily at 12:00; FF2, fed twice daily at 12:00 and 24:00; FF4, fed four times daily at 06:00, 12:00, 18:00, and 24:00. FL, feeding level: FLlow , low feeding level (1.5 × MEm /day); FLhigh , high feeding level (2.5 × MEm /day). ANOVA: FF, effect of feeding frequency; FL, effect of feeding level; FF × FL, interaction between FF and FL; * P < 0.05; ns, not significant (P > 0.05).

84

T. Vicari et al. / Domestic Animal Endocrinology 34 (2008) 74–88

Fig. 8. Insulin to glucose ratios during the 6 h postprandial period (from 12:00 to 18:00) in veal calves. Values with different letters (a and b) indicate significant differences (P < 0.05) between calves at different feeding frequencies (FF1, FF2 and FF4). Values with different letters (x and y) indicate significant differences (P < 0.05) between calves at the same feeding frequency but at different feeding levels or the same feeding level to indicate an effect of period. For further details see the legend of Fig. 1.

ble in FF1 and FF4, but transiently decreased (P < 0.05) in FF2, and more at FLhigh than at FLlow (P < 0.05). The AUC12–18 h (Table 4) was not affected by FF, but was enhanced by increasing FL (P < 0.001). Concentrations were higher (P < 0.05) at FLhigh than at FLlow in FF2 (at 6 h after feeding) and in FF4 (before and from 0 to 6 h; AUC12–18 h ). Mean plasma concentrations of T3 and T4 (Table 4) were enhanced by increasing FF (P < 0.05) and FL (P < 0.01) and were higher (P < 0.01 and P < 0.05 for T3 and T4 , respectively) in FF4 at FLhigh than at FLlow . 3.4. Associations between plasma glucose and plasma insulin concentrations The insulin to glucose ratios (Fig. 8) were affected by FF (P < 0.001) and FL (P < 0.001). They decreased (P < 0.05) approximately six-fold from FF1 to FF4 at FLlow and about two-fold from FF2 to FF4 at FLhigh , and were greater (P < 0.05) at FLhigh than at FLlow . 4. Discussion Experimental conditions were very suitable to compare effects of different FF and FL because daily DM intakes at both FL were identical across FF. FLhigh was only studied in calves fed twice or four times daily because veal calves are not able to consume 2.5 × MEm in one single daily meal. The DM, gross energy and N intakes were experimental design variables and their dif-

ferences led to differences in average daily gain, energy and N retention, as expected. The average daily gain was measured only during a 2-week period and was therefore not representative for the entire fattening period. Nevertheless, average daily gain at FLhigh was as under practical conditions and as seen in previous experiments [2,8,11,21]. At FLlow , the average daily gain was clearly smaller than usually observed in veal calves. Other aspects of growth performance were discussed in detail in a previous paper [12]. Individual calves were used twice in this study (in P1 and P2) to exclude individual animal effects on effects of FL. A possible period (or age) effect could be evaluated because calves were fed at the same (low) FL in both experimental periods in FF1. Importantly, there were no effects of period (or age) on plasma metabolic and endocrine traits. At FLhigh calves were studied at a BW of >160 kg, i.e. they had a BW at which metabolic problems, such as hyperglycemia, glucosuria and insulin resistance could be expected [2,3,8,9,11]. Thus, the experimental conditions were favourable to study effects of FF and FL especially on aberrant glucose homeostasis. Iron deficiency (frequently observed in veal calves and enhanced by intensive blood sampling) is associated with enhanced insulin-dependent glucose utilization in veal calves [27]. To avoid such a deficiency, the iron content was set at 48 mg/kg diet, which is sufficient to prevent calves from iron deficiency [28]. Postprandial metabolite and hormone concentrations were measured from 12:00 to 18:00. However, because the experimental treatments differently affected diurnal patterns of metabolite and hormone concentrations over 24 h, extrapolations from the studied period to average daily values need to be made with caution. Whereas preprandial plasma glucose concentrations were not affected by FF and FL, postprandial plasma glucose concentrations decreased with increasing FF, but increased with increasing FL. This indicates that glucose homeostasis in veal calves fed especially at low FF and high FL was stressed for several hours postprandially, but not preprandially, in agreement with [2,3]. In the present study postprandial plasma glucose concentrations, even at FLlow (mean: 7.4 ± 0.1 mmol/L), were well above levels that are considered as normal for calves (4.4–6.9 mmol/L; [29]), were higher than in suckling calves [30], and higher than in postweaning calves [10]. Peak levels increased with increasing FL because of a greater intake of lactose at FLhigh than at FLlow [3]. Surprisingly, FF and FL did not affect the time needed to reach peak postprandial plasma glucose concentrations. However, concentrations returned

T. Vicari et al. / Domestic Animal Endocrinology 34 (2008) 74–88

to preprandial levels more rapidly at both FL if FF had been increased. In agreement, postprandial plasma glucose concentrations increased less if a clotting milk replacer was fed more frequently in veal calves [11]. In humans, too, plasma glucose was suppressed with increasing FF [31]. Importantly, mean plasma glucose levels after feeding exceeded the renal glucose threshold of 8.3 mmol/L [2] in FF1 in P2 and in FF2 and FF4 at FLhigh . The 24 h urinary glucose losses were greater at FLhigh than at FLlow , were greatest and lowest at highest and lowest AUC12–18 h glucose concentrations, respectively, and in FF1 increased with age (losses in P2 > P1). This confirms previous studies [2,3,8,11] and indicates that veal calves have problems with glucose homeostasis and excrete glucose in urine when a low FF is combined with a high FL and that urinary glucose losses increase with age. Carbohydrates are a main energy substrate in veal calves, because postprandial respiratory quotients approach 1.0 [16]. The efficiency of glucose utilization is thus of great importance and is decreased if glucose is excreted in urine. In the present study the amounts of glucose excreted in urine may have contributed to the lower energy retention with decreasing FF [12]. Plasma concentrations of NEFA are well known to respond quickly to changes in energy intake. The higher pre- than postprandial concentrations mirrored a reduced energy balance between 10:30 and 12:00, i.e. shortly before feeding at 12:00. The rapid postprandial decrease of NEFA concentrations was as expected [2,3,9,11,22]. Because no significant effects of FF and FL on pre- and postprandial concentrations were found, inhibition of lipolysis and (or) enhanced tissue uptake and utilization of NEFA were obviously very similar among experimental treatments. With respect to FF this was in agreement with studies in veal calves that were fed twice daily by bucket or could ingest milk replacer ad libitum from an automatic feeder [11]. In studies with humans, however, an increased FF reduced plasma NEFA concentrations [31]. The postprandial rise of NEFA concentrations up to the next meal (at 18:00) in FF4 at FLlow suggests enhanced NEFA mobilization, most likely because energy intake at 12:00 was insufficient for the entire 6 h postprandial period, in contrast to FF1 and FF2. Conversely, data of Leibholz [32] suggested that veal calves fed only once daily pass through a period of energy deprivation and Williams et al. [16] showed evidence of enhanced fat oxidation with decreasing FF, which is expected to be associated with increased NEFA concentrations. Plasma urea concentrations, under the condition of normal kidney and liver functions, are well known to mirror AA oxidation. Explanations for the higher

85

preprandial concentrations with increasing FF at FLlow may include an enhanced AA oxidation for energy purposes. Increased pre- and postprandial urea concentrations at FLhigh than at FLlow were most likely the consequence of a higher protein intake. Postprandial plasma urea concentrations and N retention were both higher at FLhigh than at FLlow . In contrast to FF2 and FF4, concentrations postprandially increased slowly in FF1, most likely also as a consequence of higher amounts of protein or N fed per meal compared with FF2 and FF4. However, different FF had no significant effects on AUC12–18 h . This does not correspond with the increased N retention with increasing FF, and suggests that the differences in diurnal pattern of urea concentrations as affected by FF complicate the extrapolation to 24 h N balances, in agreement with other studies [5]. The enhanced preprandial insulin concentrations if FL increased were in contrast to the unaffected plasma glucose concentrations, indicating that the higher preprandial insulin concentrations at FLhigh than at FLlow were not directly dependent on the plasma glucose status. Preprandial concentrations at FLhigh (means: 2.2 ␮g/L) were above previously published values in veal calves (0.3–1.5 ␮g/L; [2,3,8]). The extremely high postprandial insulin concentrations at FLlow (means: 37 and 58 ␮g/L in periods 1 and 2 in FF1, respectively; maxima: 106 ␮g/L) and especially at FLhigh in FF2 (means: 91 ␮g/L; maxima: 134 ␮g/L) were in agreement with other studies in veal calves [2,3,11], but much higher than in suckling calves [30] and in postweaning calves (range: 0.8–1.5 ␮g/L at the age of 3–4 months; [10]). Postprandial insulin concentrations increased with a delay relative to those of glucose, as expected [2,3], and peak insulin concentrations were reached earlier when FF increased, whereas glucose peak levels were reached consistently between 1 and 1.5 h after feeding. The AUC12–18 h was especially great at FLhigh at FF2, a feeding situation seen under practical conditions. Many factors enhance the secretion of insulin and the exaggerated responses after feeding cannot be explained by high plasma glucose levels only [2,3,9]. Apart from hyperglycemia, AA that circulate in blood in increased amounts after intestinal absorption most likely also increased insulin secretion. After ingesting proteins from non-clotting milk replacers, as in our study, a rapid absorption of AA occurs [33,34]. A high fat intake, which allows a high average daily gain in veal calves, may be associated with the development of insulin resistance [35,36], but protein and lactose intakes were changed as well, i.e. nutritional effects were confounded. The very high postprandial plasma insulin levels relative to glucose levels (i.e. high insulin/glucose ratios) indicate insulin resistance, which

86

T. Vicari et al. / Domestic Animal Endocrinology 34 (2008) 74–88

develops postprandially and is transient in veal calves [2,3,7,8,11]. As expected, postprandial insulin/glucose ratios were increased by FLhigh compared with FLlow . On the other hand, increasing the FF reduced postprandial insulin concentrations and insulin/glucose ratios, in agreement with previous studies in veal calves [11] and in humans [31]. It seems important to note that insulin did not result in a simultaneous postprandial reduction of plasma NEFA concentration as a consequence of enhanced NEFA tissue uptake, which is known to be mainly the result of insulin effects, in agreement with [2,3]. Insulin is well known to reduce AA oxidation and thus plasma urea concentrations and to enhance tissue AA uptake and thus N retention. That plasma urea levels did not increase despite of decreased plasma insulin levels, if FF increased at FLlow , may express an enhanced insulin sensitivity with increasing FF. However, the highest urea concentrations were found in FF2 at FLhigh and this was associated with very high insulin concentrations and the highest insulin/glucose ratios, suggesting that (under conditions of relatively low FF and high feeding intensity) AA oxidation may be enhanced due to low insulin sensitivity. Reduced post-absorptive AA utilization due to decreased insulin sensitivity and (or) responsiveness was considered as one of several factors that may contribute to the low efficiency with which an increased intake of indispensable AA is retained in heavy veal calves [37]. Concentrations of IGF-1 barely changed postprandially, in agreement with [2,3,11,22] and due to the fact that free IGF-1 is transported together with binding proteins (especially IGF binding protein 3; [38]) that dampen fluctuations of total plasma IGF-1. The higher IGF-1 concentrations at FLhigh than at FLlow were likely the consequence of an enhanced energy, protein, but not protein-free (fat and lactose) intake [39–41]. Whereas preprandial (pancreatic) glucagon concentrations were not affected by FF and FL, postprandial concentrations were influenced by both FF and FL. Glucagon is well known to have antagonistic effects on insulin [33] and one may therefore expect that plasma glucagon concentrations would change inversely to insulin. This was, however, not the case. Glucagon concentrations increased postprandially in FF1 and FF2, but not in FF4, i.e. changes were not associated with plasma glucose concentrations. Increased plasma AA concentrations, especially after protein-rich meals, are known to markedly stimulate both the secretion of glucagon and insulin [33,42,43]. In the present study, calves were fed highly digestible whey proteins, which are known to rapidly transfer to digestive and absorptive sites, followed by a marked rise of plasma AA and stimu-

lation of the secretion of glucagon. Therefore, it would be expected that glucagon concentrations are more elevated at low than at high FF and are higher at FLhigh than at FLlow because under these conditions greater amounts of AA are ingested per meal. In agreement, the AUC12–18 h of glucagon decreased with increasing FF at FLlow . This corresponds with [11] who found lower plasma glucagon concentrations in veal calves that could suckle several times daily on an automatic feeder compared with calves that were provided the same amounts of feed by bucket only twice daily. However, against expectation, glucagon levels were only in FF2 higher at FLhigh than at FLlow and differences were also only seen at few time-points during the postprandial period. Mean preprandial concentrations, AUC12–18 h and indexes of pulsatile secretion of GH were inconsistently affected by FF and FL. Concentrations of GH decrease with increasing intakes of energy in steers [44], and GH concentrations decrease shortly after ingestion of high amounts of milk or milk replacer in veal calves [11], as observed in the present study in FF2 at FLhigh . Plasma leptin concentrations are well known to be enhanced by high energy intakes [45,46]. In accordance, leptin concentrations were more increased at FLhigh than at FLlow (significantly in FF4). Leptin in young calves seems to be differently regulated compared with older cattle and immediate postprandial responses in milk-fed calves are small or absent [23], as in the present study. Concentrations of T4 and T3 were affected by FF and FL. The higher T4 and T3 concentrations with increasing FL were expected [40,44]. Whether this effect was due to the higher energy or protein intake is unclear. T4 was found to respond to protein, but not to lactose and fat intake [40]. For T3 , the situation was less clear because there were strong positive effects of lactose and fat, and weak positive effects of protein intake. In conclusion, the present study shows that metabolic and endocrine factors are differently affected by FF and FL. There were no FF × FL interactions, except for plasma glucose, indicating separate effects of FF and FL. Overloading of glucose metabolism (hyperglycemia, glucosuria) and excessive insulinemia in heavy veal calves could be prevented by increasing the FF and decreasing the FL. References [1] Abe M, Iriki T, Konduh K, Shibui H. Effects of nipple or bucket feeding of milk-substitute on rumen by-pass and on rate of passage in calves. Br J Nutr 1979;41:175–81. [2] Hostettler-Allen RL, Tappy L, Blum JW. Insulin resistance, hyperglycemia, and glucosuria in intensively milk-fed calves. J Anim Sci 1994;72:160–73.

T. Vicari et al. / Domestic Animal Endocrinology 34 (2008) 74–88 [3] Hugi DP, Bruckmaier RM, Blum JW. Insulin resistance, hyperglycemia, glucosuria, and galactosuria in intensively milk-fed calves: dependency on age and effects of high lactose intake. J Anim Sci 1997;75:469–82. [4] Jasper J, Weary DM. Effects of ad libitum milk intake of dairy calves. J Dairy Sci 2002;85:3054–8. [5] Beynen AC, Van Gils LGM. Postprandial changes in the levels of lipids, glucose, urea and nonprotein nitrogen in the serum of veal calves fed milk replacers containing either skim milk powder or soybean protein concentrate. J Anim Physiol Anim Nutr 1983;49:59–66. [6] Guilloteau P, Le Hu¨erou-Luron I, Toullec R, Chayvialle JA, Zabielski R, Blum JW. Gastrointestinal regulatory peptides and growth factors in young cattle and sheep. J Vet Med A 1997;44:1–23. [7] Hugi DP, Bracher VD, Tappy L, Blum JW. Postprandial hydrogen breath excretion, plasma lactate concentration, glucose metabolism and insulin levels in veal calves. J Anim Physiol Anim Nutr 1997;78:42–8. [8] Hugi DP, Gut SH, Blum JW. Blood metabolites and hormones – especially glucose and insulin – in veal calves: effect of age and nutrition. J Vet Med A 1997;44:407–16. [9] Hugi D, Tappy L, Sauerwein H, Bruckmaier RM, Blum JW. Insulin-dependent glucose utilization and kinetics in intensively milk-fed veal calves is modulated by supplemental lactose in an age-dependent manner. J Nutr 1998;128:1023–30. [10] Hugi DP, Blum JW. Changes of blood metabolites and hormones in breeding calves associated with weaning. J Vet Med A 1997;44:99–108. [11] Kaufhold JN, Hammon HM, Bruckmaier RM, Breier BH, Blum JW. Postprandial metabolism and endocrine status in veal calves fed at different feeding frequencies. J Dairy Sci 2000;83:2480– 90. [12] Van den Borne JJGC, Verstegen MWA, Alferink SJJ, Giebels RMM, Gerrits WJJ. Effects of feeding frequency and feeding level on nutrient utilization in heavy preruminant calves. J Dairy Sci 2006;83:3578–86. [13] Lineweaver JA, Hafez ESE. Feed intake and performance in calves fed ad libitum. J Dairy Sci 1969;52:2001–6. [14] Mitchell CD, Broadbent PJ. The effect of level and method of feeding milk substitute and housing environment on the performance of calves. Anim Prod 1973;17:245–56. [15] Ternouth JH, Ganterton P, Beattie AW. The effect of abrupt changes in the concentration and frequency of feeding milksubstitute diets on the voluntary food intake of calves. Br J Nutr 1985;55:529–36. [16] Williams PEV, Fallon RJ, Brockway JM, Innes GM, Brewer AC. The effect of frequency of feeding milk replacer to pre-ruminant calves on respiratory quotient and the efficiency of food utilization. Anim Prod 1986;43:367–75. [17] Gerrits WJJ, Tolman GH, Schrama JW, Tamminga S, Bosch MW, Verstegen MWA. Effect of protein and protein-free energy intake on protein and fat deposition rates in preruminant calves of 80 to 240 kg live weight. J Anim Sci 1996;74:2129–39. [18] Stanley CC, Williams CC, Jenny BF, Fernandez JM, Bateman II HG, Nipper WA, et al. Effects of feeding milk replacer once versus twice daily on glucose metabolism in Holstein and Jersey calves. J Dairy Sci 2002;85:2335–43. [19] Nussbaum A, Schiessler G, Hammon HM, Blum JW. Growth performance and metabolic and endocrine traits in calves pairfed by bucket or by automate starting in the neonatal period. J Anim Sci 2002;80:1545–55.

87

[20] Van Es AJH, Nijkamp HJ, Van Weerden EJ, Van Hellemond KK. Energy, carbon, and nitrogen balance experiments with veal calves. In: Blaxter KL, Kielanowski J, Thorbek G, editors. Energy metabolism of farm animals. Newcastle upon-Tyne, United Kingdom: Oriel Press; 1969. p. 197–201. [21] R´erat M, Zbinden Y, Saner R, Hammon HM, Blum JW. In vitro embryo production: growth performance, feed efficiency, and hematological, metabolic, and endocrine status in calves. J Dairy Sci 2005;88:2579–93. [22] Herrli-Gygi M, Hammon HM, Zbinden Y, Steiner A, Blum JW. Ruminal drinkers: endocrine and metabolic status and effects of suckling from a nipple instead of drinking from a bucket. J Vet Med A 2006;53:215–24. [23] Blum JW, Zbinden Y, Hammon HM, Chilliard Y. Plasma leptin status in young calves: effects of preterm birth, age, glucocorticoid status, suckling, and feeding with an automatic feeder or by bucket. Domest Anim Endocrinol 2005;28:119–33. [24] Blum JW, Elsasser TH, Greger DL, Wittenberg S, de Vries F, Distl TO. Insulin-like growth factor type-1 down-regulation associated with dwarfism in Holstein calves. Domest Anim Endocrinol; doi:10.1016/j.domaniend.2006.05.007, in press. [25] Merriam GR, Wachter KW. Algorithms for the study of episodic hormone secretion. Am J Physiol 1982;243:E310–8. [26] Littell RC, Henry PR, Ammerman CB. Statistical analysis of repeated measures data using SAS procedures. J Anim Sci 1998;76:1216–31. [27] Hostettler-Allen R, Tappy L, Blum JW. Enhanced insulindependent glucose utilization in iron-deficient veal calves. J Nutr 1993;123:1656–67. [28] Lindt F, Blum JW. Physical performance of veal calves during chronic iron deficiency anemia and after acute iron overload. J Vet Med A 1993;40:444–55. [29] Kraft W, D¨urr UM. Klinische Labordiagnostik in der Tiermedizin. New York: Schattauer Verlag Stuttgart; 1997. [30] Egli CP, Blum JW. Clinical, haematological, metabolic and endocrine traits during the first three months of life of suckling Simmental calves held in a cow-calf operation. J Vet Med A 1998;45:99–118. [31] Jenkins JA, Ocana A, Jenkins AL, Wolever TMS, Vuksan V, Katzman L, et al. Metabolic advantages of spreading the nutrient load: effects of increased meal frequency in non-insulin-dependent diabetes. Am J Clin Nutr 1992;55:461–7. [32] Leibholz J. Dietary effects on the flow of nutrients from the abomasum of the preruminant calf. Aust J Agric Res 1975;26:623– 33. [33] Fischer U. Pankreas. In: D¨ocke F, editor. Veterin¨armedizinische Endokrinologie. Jena and Stuttgart: G. Fischer Verlag; 1994. p. 609–52. [34] Frid AH, Nilsson M, Holst JJ, Bjorck IM. Effect of whey on blood glucose and insulin responses to composite breakfast and lunch meals in type 2 diabetic subjects. Am J Clin Nutr 2005;82:69– 75. [35] Doppenberg J, Palmquist DL. Effect of dietary fat level on feed intake, growth, plasma metabolites and hormones of calves fed dry or liquid diets. Livest Prod Sci 1991;29:151–66. [36] Palmquist DL, Doppenberg J, Roehrig KL, Kinsey DJ. Glucose and insulin metabolism in ruminating and veal calves fed high and low fat diets. Domest Anim Endocrinol 1992;9:233–41. [37] Van den Borne JJGC, Verdonk JMAJ, Schrama JW, Gerrits WJJ. Reviewing the low efficiency of protein utilization in heavy preruminant calves—a reductionist approach. Reprod Nutr Dev 2006;46:121–37.

88

T. Vicari et al. / Domestic Animal Endocrinology 34 (2008) 74–88

[38] Skaar TC, Baumrucker CR, Deaver DR, Blum JW. Ontogeny of circulating insulin-like growth factor (IGF) binding proteins in calves. J Anim Sci 1994;73:421–7. [39] Clemmons DR, Underwood LE. Nutritional regulation of IGF-I and IGF binding proteins. Annu Rev Nutr 1991;11:393–412. [40] Gerrits WJJ, Decuypere E, Verstegen MWA, Karabinas V. Effect of protein and protein-free energy intake on plasma concentrations of insulin-like growth factor I and thyroid hormones in preruminant calves. J Anim Sci 1998;76:1356–63. [41] Smith JM, van Amburgh ME, D´ıaz MC, Lucy MC, Bauman DE. Effect of nutrient intake on the development of the somatotropic axis and its responsiveness to GH in Holstein bull calves. J Anim Sci 2002;80:1528–37. [42] Unger RH, Ohneda A, Aguilar-Parada E, Eisentraut AM. The role of aminogenic glucagon secretion in blood glucose homeostasis. J Clin Invest 1969;48:810–22.

[43] Calbet JAL, MacLean DA. Plasma glucagon and insulin responses depend on the rate of appearance of amino acids after ingestion of different protein solutions in humans. J Nutr 2002;132:2174– 82. [44] Blum JW, Schnyder W, Kunz PL, Blom AK, Bickel H, Schuerch A. Reduced and compensatory growth: endocrine and metabolic changes during energy restriction and realimentation in steers. J Nutr 1985;115:417–25. [45] Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 1998;395:763–70. [46] Cammisotto PG, Gelinas Y, Deshaies Y, Bukowiecki LJ. Regulation of leptin secretion from white adipocytes by insulin, glycolytic substrates, and amino acids. Am J Physiol (Endocrinol Metab) 2005;289:E166–71.