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Effects of dietary energy and protein density on plasma concentrations of leptin and metabolic hormones in dairy heifers
J. Dairy Sci. 92:1430–1441 doi:10.3168/jds.2008-1385 © American Dairy Science Association, 2009.
Effects of dietary energy and protein density on plasma concentrations of leptin and metabolic hormones in dairy heifers P. K. Chelikani,*1 D. J. Ambrose,†‡ D. H. Keisler,§ and J. J. Kennelly‡ *Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada, T2N 4N1 †Agriculture Research Division, Alberta Agriculture and Rural Development, Edmonton, Alberta, Canada, T6H 5T6 ‡Department of Agricultural, Food and Nutritional Science, Faculty of Agricultural, Life and Environmental Sciences, University of Alberta, Edmonton, Canada, T6G 2P5 §Division of Animal Sciences, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia 65211
ABSTRACT
The hormonal and metabolic signals that communicate the level of body energy reserves to the reproductive-mammary axis remain undefined in dairy cattle; consequently, our hypothesis was that leptin may fulfill this role. Our objectives were to determine the effects of diets differing in energy and protein density on dry matter intake (DMI), growth traits [body weight (BW), body condition score (BCS), back-fat (BF) thickness], and temporal changes in plasma concentrations of leptin, insulin, growth hormone (GH), insulin-like growth factor-1 (IGF-1), glucose, and nonesterified fatty acids (NEFA) in dairy heifers during the pre- and postpubertal periods. In period 1, heifers were randomly allotted (n = 10/diet) at 103 kg of BW to diets for a predicted average daily gain of 1.10 (high, H), 0.80 (medium, M), or 0.50 kg/d (low, L). Five heifers in each of the H and L groups were further studied during period 2, either at 12 mo of age (HA, LA) or at 330 kg of BW (HW, LW). The data provide evidence that 1) DMI (18%), BW (17%), and BF (5%) together explained 40% of the variation in plasma leptin concentrations (r2 = 0.396); 2) unlike the acute postprandial increase in plasma insulin as a result of increased nutrient density (H 1.42 ± 0.09, M 1.02 ± 0.09, L 0.68 ± 0.11 ng/mL), plasma leptin concentrations did not respond acutely with a distinct postprandial profile; 3) although plasma leptin concentrations increased with age, leptin at puberty did not differ among treatment groups (H 5.63 ± 2.48, M 4.28 ± 0.55, L 4.12 ± 0.72 ng/mL) and there was no evidence of an abrupt transition in prepubertal plasma leptin concentrations; 4) plasma leptin concentrations may not be a critical trigger for puberty in rapidly growing heifers, but are apparently essential for puberty in heifers with normal or restricted growth rates; and 5) plasma concentrations of insulin (H 0.59 Received May 22, 2008. Accepted November 11, 2008. 1 Corresponding author: [email protected]
± 0.07, M 0.43 ± 0.09, L 0.30 ± 0.09 ng/mL), IGF-1 (H 151.08 ± 16.47, L 82.51 ± 17.47 ng/mL), and glucose (H 81.35 ± 3.39, M 73.59 ± 2.34, L 68.25 ± 3.39 mg/ dL) reflected nutrient density, whereas GH (H 1.82 ± 0.23, L 5.87 ± 0.45 ng/mL) and NEFA (H 209.54 ± 50.83, L 234.93 ± 48.97 μM) were inversely related to the plane of nutrition. Collectively, these data suggest that plasma concentrations of leptin may play a role in long-term regulation of energy reserves and puberty in growing Holstein heifers. Key words: dairy heifer, nutrition, leptin, puberty INTRODUCTION
The ruminant growth phase is characterized by hormonal and metabolic adaptations that coordinate somatic tissue accretion with reproductive maturation and mammary development. The pre- and peripubertal periods appear as critical periods of growth in dairy heifers, because accelerated growth rates during these periods are invariably associated with increased adiposity, earlier puberty, and impaired development of mammary secretory tissue (Davis Rincker et al., 2008). Although mammary development is linked to the maturity of the reproductive tract in prepubertal heifers (Lammers and Heinrichs, 2000), the hormonal and metabolic signals that communicate the level of body energy reserves to the reproductive-mammary axis remain undefined. There is substantial evidence on the effects of nutritional manipulation on the hormonal and metabolite milieu in peripubertal heifers. In dairy heifers, a high plane of nutrition during the pre- and postpubertal periods was often associated with increased circulating concentrations of insulin, glucose, and IGF-1, and decreased concentrations of growth hormone (GH) and NEFA (Petitclerc et al., 1983; Lacasse et al., 1994; Capuco et al., 1995). Among the hormonal signals, a prepubertal increase in circulating concentrations of GH and IGF-1 is believed to play a role in determining the onset of puberty in beef heifers (Yelich et al., 1995,
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1996). In addition to these hormones, leptin, a hormone secreted primarily from adipose tissue, is sensitive to dietary manipulation and appears to play an important role in transmitting the status of energy reserves to the central nervous system to regulate feed intake and reproductive function in ruminants (Zieba et al., 2005). A linear prepubertal increase in plasma leptin concentrations was found in beef heifers (Garcia et al., 2002, 2003), whereas in dairy heifers a similar increase was found in one study (Diaz-Torga et al., 2001), but not in another (Block et al., 2003). Given that restricted growth rates delay the onset of puberty in heifers (Yelich et al., 1995), and that nutrient restriction decreases plasma leptin concentrations in cattle (Block et al., 2003; Brown et al., 2005), it was hypothesized that a relative deficiency of leptin may play a role in the delayed puberty of slowly growing heifers. Yet, we are not aware of studies that examined the effects of chronic dietary restriction and realimentation on circulating leptin concentrations and its relationship to feed intake and reproductive function in dairy heifers. Our objectives were to determine the effects of diets differing in energy and protein density on DMI, growth measures [BW, back-fat (BF) thickness, and BCS] and temporal changes in plasma concentrations of leptin, insulin, GH, IGF-1, glucose, and NEFA in dairy heifers during the pre- and postpubertal periods. We assessed the effects of realimentation of feed-restricted heifers on DMI, BW, and hormonal and metabolic adaptations. MATERIALS AND METHODS Animals and Treatments
This study was conducted at the Dairy Research Unit of the University of Alberta, Edmonton, Alberta, Canada; all experimental procedures were approved by the University of Alberta Animal Policy and Welfare Committee. The experimental design and treatments were described previously (Chelikani et al., 2003). Briefly, this was a 2-phase study involving 30 Holstein heifers (Figure 1). In period 1, following an initial 10-d adaptation period, the heifer calves (103 ± 2 kg of BW and 104 ± 2 d of age) were assigned at random to 1 of 3 dietary treatments (n = 10/treatment). The experimental diets (Table 1) were formulated using the Cornell Net Carbohydrate and Protein System (Van Amburgh et al., 1998) for a predicted ADG of 0.50 kg/d (low, L), 0.80 kg/d (medium, M), or 1.10 kg/d (high, H). Heifers were housed in tie-stalls, exercised thrice weekly between 0900 and 1000 h, and individually fed a TMR daily at approximately 1000 h with free access to water. Daily feed consumption was recorded for each heifer. In period 1, based on the weekly BW, the heifers were feed
restricted at varying levels to reach the predicted ADG. It was predicted that the M heifers would achieve a postpubertal BW of 330 kg at approximately 12 mo of age, at which stage they were allowed ad libitum access to a single period-2 diet formulated to maintain their ADG of 0.8 kg/d. To compare treatment differences at a similar age and BW, 5 heifers each within the H and L groups were assigned to period 2 at either 12 mo of age (HA and LA) or entered period 2 when they attained 330 kg of BW with ad libitum access to a single period-2 diet (HW and LW). Measurements and Sampling Measurements of DMI, BW, BCS, and BF
The amount of feed consumed by each heifer was recorded daily. Samples of TMR, ingredients, and orts were collected once weekly, dried at 55°C for 72 h, ground through a 1-mm screen (Thomas-Wiley Laboratory Mill Model 4, Philadelphia, PA), and composited monthly for proximate analysis (Chelikani et al., 2003). The animals were weighed on 2 d consecutively each week before feeding and the mean weekly BW calculated. Once a heifer reached 200 kg of BW, BCS and BF thickness (between 12th and 13th rib) were recorded every 2 wk. The BCS was assessed on a scale of 1 to 5 (Edmonson et al., 1989) by 2 individuals, and BF thickness was measured transcutaneously using an ultrasound scanner (Aloka 500V, Aloka Co. Ltd., Tokyo, Japan) equipped with a 7.5-MHz linear array transducer (Brethour, 1992). The temporal changes in structural growth measures were reported previously (Chelikani et al., 2003). Blood Sampling
Blood samples were collected from all heifers, before feeding, by jugular vein puncture at 2-wk intervals. Samples were placed on ice in 10-mL Vacutainer tubes (Becton Dickinson, Sparks, MD) without or with appropriate anticoagulants (EDTA for leptin, IGF-1, and progesterone; sodium heparin for insulin and GH; sodium fluoride for glucose; and serum for NEFA). Plasma was separated by centrifugation at 1,500 × g for 20 min and stored at –20°C for leptin assays; samples were analyzed for insulin, GH, IGF-1, glucose, and NEFA every 4 wk. Further, beginning at 200 kg of BW, for determining the onset of puberty, blood samples were collected twice weekly (Monday and Friday) via jugular venipuncture from all heifers into 10-mL Vacutainer tubes containing EDTA for progesterone assay. A heifer was considered pubertal, and first ovulation had occurred, when plasma progesterone concentrations exceeded 1 ng/mL for the first time; this was further confirmed by the formation Journal of Dairy Science Vol. 92 No. 4, 2009
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Figure 1. Overview of experimental design. In period 1 (solid lines), heifer calves (103 ± 2 kg BW and 104 ± 2 d of age) were assigned at random to 1 of 3 dietary treatments (n = 10/treatment) formulated for a predicted ADG of 1.10 kg/d (high, H), 0.80 kg/d (medium, M), or 0.50 kg/d (low, L) growth rates. The M heifers were expected to achieve a postpubertal BW of 330 kg at approximately 12 mo of age, at which stage they were allowed ad libitum access to a single period-2 diet (dashed lines) formulated to maintain their ADG of 0.8 kg/d. To compare treatment differences at a similar age and BW, 5 heifers each within the H and L groups were assigned to period 2 at either 12 mo of age (HA, LA) or entered period 2 when they attained 330 kg of BW (HW, LW). Frequent blood sampling for determining plasma LH and GH pulse characteristics, and for assessing the postprandial changes in plasma leptin and insulin concentrations, were done in period 1 at 8 and 10 mo of age from a subset (n = 5) of H, M, and L heifers. Adapted with permission from Chelikani et al. (2003).
of a detectable corpus luteum in the heifers that were subjected to transrectal ultrasonography (Chelikani et al., 2003). These samples were used for determining leptin concentrations; for each heifer the mean weekly plasma leptin concentrations during the pre- and peripubertal period were calculated from the twice-weekly samples for ascertaining the leptin levels relative to the onset of puberty. To determine pulsatile characteristics of GH and LH, blood samples from 5 heifers from each of the H, M, and L treatment groups in period 1 were taken via jugular catheters at 15-min intervals for 8 h at 8 mo and again at 10 mo of age. The distribution of the heifers in period 1 for serial bleeding that entered period 2 was as follows: HA = 3, HW = 2, LA = 2, LW = 3, and M = 5. Two baseline samples were taken at −30 and −15 min relative to feeding, followed by serial 15-min sampling for 8 h. The baseline (15 min before feeding) and hourly plasma samples were analyzed for leptin and insulin concentrations. Data on LH pulsatility were described previously (Chelikani et al., 2003). The Kushler-Brown PulseFit program (Kushler and Brown, 1991), a stepwise selection algorithm for identifying hormone pulses, was used to characterize GH Journal of Dairy Science Vol. 92 No. 4, 2009
pulsatility and obtain values for pulse frequency, amplitude, mean, and basal concentrations. The criteria used for LH pulse characterization (Chelikani et al., 2003) were applied for determining GH pulse measures. Hormone and Metabolite Assays
Plasma leptin concentrations were determined by a highly sensitive ovine leptin RIA validated for bovine plasma (Delavaud et al., 2000). All the samples were analyzed in 8 assays. Assay sensitivity at 95% of total binding was 0.15 ng/mL, intraassay coefficient of variation (CV) was 3.3%, and interassay CV was 5.6%. Plasma concentrations of insulin and GH were determined by homologous double-antibody RIA, and IGF-1 by heterologous double-antibody RIA, as described previously (Chelikani et al., 2004). For insulin, all the samples were analyzed in 3 assays, the intra- and interassay CV were 8.7 and 11%, respectively, assay sensitivity at 94% of total binding was 0.08 ng/mL, and recovery was 93%. For IGF-1, all the samples were analyzed in 2 assays, the intra- and interassay assay CV were 6.45 and 7.49%, radioinert recovery was 94%, and
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Table 1. Ingredient and chemical composition (mean ± SEM) of diets1 Period-1 diet Item Ingredient (% of DM) Barley silage Alfalfa silage Grass hay Alfalfa hay Rolled barley Canola meal Corn gluten meal Tallow Fortified mineral salt2 Vitamin ADE premix3 Chemical composition DM (%) OM (% of DM) CP (% of DM) Crude fat (% of DM) NDF (% of DM) ADF (% of DM) Lignin (% of DM) Ash (% of DM) ME (Mcal/kg of DM) NEG (Mcal/kg of DM)
High
Medium
Low
Period-2 diet
31.8 — — 23.9 19.8 7.8 13.5 2.9 0.1 0.1
36.6 — — 32.6 17.2 6.4 5.5 1.5 0.1 0.1
48.3 — — 40.9 10.6 — — — 0.1 0.1
28.2 28.2 14.1 — 29.4 — — — 0.1 0.1
59.3 ± 1.0 90.7 ± 0.2 20.9 ± 0.7 4.6 ± 0.2 44.1 ± 1.2 25.7 ± 0.9 6.2 ± 0.3 9.3 ± 0.1 2.62 0.97
57.4 ± 1.2 90.8 ± 0.2 18.1 ± 0.5 3.4 ± 0.1 47.8 ± 1.3 28.3 ± 1.2 6.9 ± 0.4 9.2 ± 0.1 2.42 0.93
52.9 ± 1.4 90.4 ± 0.3 13.5 ± 0.2 1.6 ± 0.1 54.3 ± 1.2 33.6 ± 1.1 8.5 ± 0.4 9.6 ± 0.1 2.28 0.85
52.9 ± 1.8 90.1 ± 0.2 13.9 ± 0.4 2.1 ± 0.1 48.9 ± 1.0 29.0 ± 1.0 6.7 ± 0.7 9.9 ± 0.1 2.29 0.86
1 Adapted with permission from Chelikani et al. (2003). In period 1, heifers were randomly allotted (n = 10/diet) at 103 kg of BW to diets for a predicted ADG of 1.10 (high), 0.80 (medium), or 0.50 kg/d (low). The period-2 diet was formulated to maintain ADG of 0.8 kg/d. 2 Contained 37% Na, 58% Cl, 80 mg of CO/kg, 400 mg of Cu/kg, 200 mg of I/kg, 6,000 mg of Mn/kg, 400 mg of Se/kg, 800 mg of Zn/kg; Champion Feed Services Ltd., Edmonton, Alberta, Canada. 3 Contained 3,846 kIU vitamin A/kg, 2,292 kIU vitamin D/kg, and 6,539 IU vitamin E/kg; Champion Feed Services Ltd.
assay sensitivity at 88% of total binding was 20.94 ng/ mL. For GH, all the samples were analyzed in 4 assays, the intra- and interassay CV were 2.7 and 6.8%, and assay sensitivity at 95% of total binding was 0.06 ng/ mL. Plasma NEFA and glucose concentrations were determined by enzymatic assays (Chelikani et al., 2004). The intra- and interassay CV for NEFA were 5 and 4%, and for glucose were 2.5 and 2.6%, respectively. Statistical Analyses
Data were analyzed using the MIXED procedure of SAS (SAS Institute, Cary, NC). Repeated measures on DMI, BW, BF, BCS, plasma hormones (leptin, insulin, IGF-1, GH) and metabolites (glucose and NEFA) were analyzed using mixed model analyses as described previously (Chelikani et al., 2003). Briefly, the model included treatment, time, and their interaction as fixed effects, and heifer within treatment as the random effect. Plasma concentrations of insulin, GH, and NEFA were log-transformed before analyses. Stepwise multiple linear regression analysis was used to determine the relative contribution of DMI, BW, BF, BCS, plasma hormones and metabolites to the variation in plasma leptin concentrations.
RESULTS DMI, BW, and BF Thickness
At the beginning of the study (15 wk of age), heifers in all treatment groups had similar (P > 0.10) DMI and BW (DMI: H 3.12 ± 0.47, M 2.97 ± 0.33, and L 2.49 ± 0.50 kg/d; BW: H 105 ± 18, M 101 ± 13, and L 105 ± 19 kg). The HA, LA, and M heifers entered period 2 at 54 wk of age, the HW heifers began period 2 at 48 wk of age, and the LW heifers entered period 2 at 76 wk of age. Temporal changes in DMI during both experimental periods, and the entire study, are in Figure 2. In period 1, as expected, the DMI of the H heifers increased with age, and was greater than that of the L and M heifers. The DMI of the H heifers was 52 and 28% greater than that of the L and M heifers by 25 and 29 wk of age, respectively; the M heifers consumed 25% more than L heifers by 31 wk of age (treatment by age, P < 0.001, Figure 2A). During period 2, the DMI was similar between the HA and HW heifers (HA 10.37 ± 0.49, HW 10.25 ± 0.56 kg/d; P > 0.10); however, the intakes of both sub-groups were nearly 20% greater than that of M heifers (HA 10.0 ± 0.48, HW 10.14 ± 0.47, M 8.43 ± 0.33 kg/d) for the first 4 wk of period 2. Although the DMI of the HA heifers was greater than
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that of the LA heifers (HA 10.52 ± 0.48, LA 7.68 ± 0.47 kg/d) for the first 14 wk of period 2 (treatment by age, P < 0.01), this difference was temporally attenuated with the DMI of LA heifers catching up with the HA heifers after 15 wk of realimentation in period 2 (P > 0.10). Similarly, the difference in DMI between the HW and LW heifers (HW 10.20 ± 0.48, LW 6.51 ± 0.53 kg/d) was gradually attenuated by 10 wk of realimentation in period 2. Compared with M heifers, the DMI of the LA and LW heifers remained lower (P < 0.01) for 18 (M 10.16 ± 0.36, LA 7.88 ± 0.47 kg/d) and 10 wk (M 9.82 ± 0.34, LW 6.51 ± 0.53 kg/d), respectively, after realimentation in period 2. The DMI of the LA heifers was greater than that of the LW heifers (LA 6.92 ± 0.47, LW 4.97 ± 0.53 kg/d) for the first 8 wk of
realimentation; thereafter, the DMI of the LW heifers accelerated over that of the LA heifers from wk 12 to 21 (LA 8.63 ± 0.47, LW 11.08 ± 0.58 kg/d) of period 2 (treatment by age, P < 0.001). In period 1, the BW of the H heifers increased with age and was greater than that of the L and M heifers by 24 (H 168 ± 18, L 134 ± 19 kg) and 29 wk of age (H 208 ± 18, M 180 ± 13 kg), respectively; the M heifers were heavier than L heifers by 29 wk of age (M 180 ± 13, L 174 ± 18 kg; treatment by age, P < 0.001; Figure 2B). In period 2, the BW of the HW and LW heifers did not differ from that of the M heifers (HW 396 ± 23, M 395 ± 16, LW 373 ± 22 kg; P > 0.10); however, the HA heifers were heavier than the HW and LA heifers (HA 448 ± 18, HW 396 ± 23, LA 317 ± 18 kg), and the LW
Figure 2. Effect of dietary energy and protein density on temporal changes in A) DMI, B) BW, C) back-fat (BF) thickness, and D) BCS of Holstein heifers. During period 1, all heifers were fed to gain at either high (H, 1.10 kg/d), medium (M, 0.80 kg/d), or low (L, 0.50 kg/d) growth rates. During period 2, the M heifers were fed ad libitum to maintain their growth rate; half the H and L heifers were switched to the period-2 diet either at 12 mo of age (HA, LA) or at 330 kg of BW (HW, LW). The SEM for DMI, BW, BF thickness, and BCS were 0.48 kg, 18.59 kg, 0.35 mm, and 0.13, respectively. Journal of Dairy Science Vol. 92 No. 4, 2009
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animals were heavier than the LA heifers (LW 373 ± 22, LA 317 ± 18 kg; treatment by age, P < 0.001). Similar to BW changes, the BF thickness of the H heifers increased with age and was greater than that of the L and M heifers (H 2.63 ± 0.29, M 2.14 ± 0.22, L 1.85 ± 0.37 mm), by 30 wk of age, and the M heifers had more BF than the L heifers by 32 wk of age (M 2.75 ± 0.23, L 1.87 ± 0.33 mm; treatment by age, P < 0.001; Figure 2C). In period 2, the BF of the HA heifers increased rapidly and was greater than that of the LA and M heifers but did not differ from that of HW heifers (HA 5.72 ± 0.33, HW 4.05 ± 0.39, M 4.33 ± 0.23, LA 3.27 ± 0.26 mm). During period 2, the HW heifers had similar BF to M heifers, but had more BF compared with LW heifers (3.11 ± 0.39 mm); the BF of the LW and LA heifers did not differ (treatment by age, P < 0.001). The BCS followed a similar a pattern as BF (treatment by age, P < 0.001; Figure 2D); however, the BCS changes appeared to be less robust and sensitive than the BF changes. Plasma Leptin Concentrations
Temporal changes in plasma leptin concentrations during both experimental periods and the entire study are shown in Figure 3. The postprandial profile of plasma leptin concentrations at 8 and 10 mo of age are in Figure 4, and the changes in plasma leptin concentrations relative to the onset of puberty are in Figure 5. Plasma leptin concentrations closely followed the changes in DMI, BW, and BF thickness (Figures 2 and 3). In period 1, plasma leptin concentrations of the H heifers increased with age and were greater than those of the L and M heifers by 34 (H 3.86 ± 0.07, L 2.81 ± 0.07 ng/mL) and 44 wk of age (H 5.42 ± 0.06, M 4.54 ± 0.04 ng/mL), respectively; the M heifers had greater circulating leptin levels than L heifers from 37 wk of age (M 4.08 ± 0.04, L 3.11 ± 0.06 ng/mL; treatment by age, P < 0.001; Figure 3). The postprandial changes in plasma leptin concentrations did not differ among the H, L, and M heifers at 8 mo (32 wk; H 3.90 ± 0.24, M 3.48 ± 0.15, L 3.20 ± 0.33 ng/mL) of age (Figure 4A; P > 0.10); however, at 10 mo (40 wk) of age postprandial plasma leptin concentrations were greater in H compared with L and M heifers (H 4.94 ± 0.29, M 3.96 ± 0.26, L 3.68 ± 0.43 ng/mL; Figure 4B), without an apparent postprandial surge. After switching to the period-2 diet, in general, plasma leptin concentrations rapidly increased in the H and M heifers, but the response was delayed in the L heifers. In period 2, the plasma leptin concentrations of LA and LW heifers increased gradually reaching the levels of M heifers following 12 (LA 5.63 ± 0.06, M 7.71 ± 0.05 ng/ mL) and 15 wk (LW 5.72 ± 0.07, M 6.94 ± 0.07 ng/
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Figure 3. Effect of dietary energy and protein density on temporal changes in plasma concentrations of leptin in Holstein heifers. During period 1, all heifers were fed to gain at either high (H, 1.10 kg/d), medium (M, 0.80 kg/d), or low (L, 0.50 kg/d) growth rates. During period 2, the M heifers were fed ad libitum to maintain their growth rate; half the H and L heifers were switched to the period-2 diet either at 12 mo of age (HA, LA) or at 330 kg of BW (HW, LW). The SEM for leptin was 1.03 ng/mL.
mL) of realimentation to period-2 diet; however, the leptin levels of LA and LW heifers remained lower than that of HA and HW heifers for the entire duration of period 2 (HA 7.99 ± 0.06, HW 7.97 ± 0.07, LA 5.75 ± 0.07, LW 4.33 ± 0.07 ng/mL; treatment by age, P < 0.001; Figure 3). Stepwise multiple regression analysis revealed that of all the variables considered, DMI (18%), BW (17%), and BF (5%) together explained 40% of the variation in plasma leptin concentrations (r2 = 0.396; P < 0.001). When leptin concentrations were aligned to the week of puberty, there was a trend for an effect of week (P = 0.09), but treatment and treatment by week interactions were not significant (P > 0.10, Figure 5A). Therefore, the data from all treatments were collapsed and regressed on age at onset of puberty. The pooled data revealed a significant (P < 0.0001), but relatively low (r2 = 0.13) linear increase in leptin relative to the timing of puberty (Figure 5B). A plot (Figure 5C) of the raw distribution of plasma leptin concentrations during the week before and after the first luteal cycle of each individual heifer revealed that the plasma leptin concentrations of the L and M heifers were less variable, whereas the H heifers exhibited a high degree of variability (mean ± SE: H 5.70 ± 2.48, M 4.28 ± 0.61, and L 4.07 ± 0.51 ng/mL); however, across all heifers the plasma leptin concentrations were >2.9 ng/mL during the peripubertal period. Journal of Dairy Science Vol. 92 No. 4, 2009
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Plasma Concentrations of Insulin, IGF-1, GH, Glucose, and NEFA
Temporal changes in plasma concentrations of metabolic hormones and metabolites at 4-wk intervals are in Figure 6. During period 1, plasma insulin concentrations of the H heifers were greater than that of L and M heifers by 31 (H 0.46 ± 0.10, L 0.30 ± 0.17 ng/ mL) and 53 wk (H 0.59 ± 0.07, M 0.43 ± 0.09 ng/ mL) of age, respectively, and insulin levels of L heifers were reduced compared with M heifers by 37 wk of age (M 0.40 ± 0.07, L 0.31 ± 0.09 ng/mL) (treatment by age, P < 0.001). The postprandial changes in plasma insulin concentrations did not differ among the H, L, and M heifers at 8 mo (32 wk) of age (H 0.65 ± 0.09,
M 0.61 ± 0.11, L 0.53 ± 0.11 ng/mL; Figure 4A; P > 0.10). At 10 mo (40 wk) of age, postprandial plasma insulin concentrations of H heifers were greater than L heifers from 2 to 5 h following feeding, and were also greater than that of M from 3 to 4 h following feeding; the postprandial insulin concentrations of the M and L heifers did not differ (treatment by time, P < 0.001; Figure 4B). During period 2, plasma insulin concentrations of LA and LW heifers increased rapidly and were similar to those of HA and HW heifers (HA 0.49 ± 0.15, HW 0.37 ± 0.17, LA 0.48 ± 0.15, LW 0.53 ± 0.17 ng/mL), respectively, and M heifers (0.44 ± 0.11 ng/ mL), within a week of realimentation to the period-2 diet (P > 0.10).
Figure 4. Effect of dietary energy and protein density on postprandial plasma concentrations of leptin and insulin at 8 (panels A and C) and 10 (panels B and D) mo of age. Blood samples were collected at baseline (time 0 = 15 min before feeding) and hourly for 8 h for determining plasma leptin and insulin concentrations. During this period, all heifers were fed to gain at high (1.10 kg/d), medium (0.80 kg/d), or low (0.50 kg/d) growth rates. The SEM for plasma insulin and leptin were 0.10 and 0.29 ng/mL, respectively. Journal of Dairy Science Vol. 92 No. 4, 2009
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During period 1, plasma IGF-1 concentrations of the H heifers increased rapidly and were greater than that of L heifers by 17 wk of age (H 151.08 ± 16.47, L 82.51 ± 17.47 ng/mL); the IGF-1 concentrations of the M heifers were similar to those of H heifers after 17 wk of age (H 188.19 ± 18.63, M 151.45 ± 16.12 ng/mL), but were greater than those of L (84.72 ± 17.47 ng/mL) heifers by 19 wk of age (Figure 6B; treatment by age, P < 0.001). In period 2, plasma IGF-1 concentrations of LA and LW heifers increased rapidly and were similar to those of HA and HW heifers (HA 205.22 ± 16.47, HW 198.97 ± 16.47, LA 179.86 ± 18.44, LW 188.96 ± 18.11 ng/mL), respectively, as well as the M heifers (M 197.95 ± 12.17 ng/mL), within 2 wk of realimentation to the period-2 diet (P > 0.10). Despite a significant treatment by age (P < 0.001) interaction, the temporal profile of plasma GH concentrations exhibited considerable variability; plasma GH levels were higher in L compared with H heifers (H 4.42 ± 1.95, L 8.59 ± 1.97 ng/mL), and were reduced with age (data not shown). The pulsatile characteristics of plasma GH at 8 and 10 mo of age are in Figure 7. The mean GH concentrations of the H heifers were lower than those of L heifers at both 8 (H 1.82 ± 0.23, L 5.87 ± 0.45 ng/mL) and 10 mo (H 1.70 ± 0.44, L 4.12 ± 0.51 ng/mL) of age. The basal concentrations and the number and amplitude of GH pulses did not differ among the L and H heifers (P > 0.10). There were significant effects of age (P < 0.0001) and treatment by age (P < 0.001) interactions for plasma concentrations of glucose and NEFA. In period 1, plasma glucose concentrations of H heifers were higher than those of L heifers after 17 wk of age (H 81.35 ± 3.39, L 68.25 ± 3.39 mg/dL), and differed from that of M heifers from 17 to about 35 wk of age (H 80.72 ± 3.30, M 73.59 ± 2.34 mg/dL). In period 2, the plasma glucose concentrations of LA heifers increased rapidly to the levels of HA heifers within 1 wk (HA 71.46 ± 2.90, LA 73.32 ± 2.90 mg/dL), whereas the glucose levels of LW heifers gradually approached the levels of HW heifers at about 6 wk (HW 79.86 ± 3.62, LW 72.63 ± 3.25 mg/dL) of realimentation to the period-2 diet (Figure 6C). Plasma NEFA concentrations exhibited considerable variability, and were, in general, higher in the L groups relative to the H groups (H 209.54 ± 50.83, L 234.93 ± 48.97 μM), but were reduced with time and after changing to the period-2 diet in all groups (Figure 6D). DISCUSSION
There is evidence that rapid prepubertal growth rates hasten the onset of puberty and have detrimental effects on mammary development in dairy heifers (Davis Rincker et al., 2008); however, the hormonal and
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Figure 5. A) Plasma leptin concentrations during the peripubertal period in heifers fed to gain at high (1.10 kg/d), medium (0.80 kg/d) or low (0.50 kg/d) growth rates. As plasma leptin concentrations did not differ among the treatments around puberty, the data from all treatments were collapsed and regressed on week relative to puberty (panel B). The distribution of plasma leptin concentrations for 2 wk before the first luteal cycle (onset of puberty) for each treatment group is shown in the box and whisker plot (panel C). Upper border, middle line, and lower border of each box indicate 75th percentile, median, and 25th percentile, respectively; whiskers (error bars) above and below the box indicate 90th and 10th percentiles. Journal of Dairy Science Vol. 92 No. 4, 2009
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metabolic signals that mediate these effects remain undefined. There is some evidence that leptin, a hormone produced primarily from adipose tissue, may play a role in determining the timing of puberty and mammary development (Garcia et al., 2002, 2003; Block et al., 2003). Nevertheless, there is limited information on the effects of long-term nutritional manipulation on changes in circulating leptin concentrations during the pre- and peripubertal period in heifers. Plasma leptin concentrations did not differ among treatments when Holstein heifers fed to grow at either 1.2 kg/d (high) or 0.4 kg/d (medium) from 7 to 14 wk of age (Brown et al., 2005), or when fed to grow at 0.9 kg/d with diets containing calcium salts of either palm fatty acids or conjugated linoleic acid from 10 to 30 wk of age (Block et al., 2003). In contrast to these studies, with
our dietary regimen we were able to produce significant differences among the 3 treatment groups in growth rates, and the temporal profile of plasma leptin concentrations showed a marked, although delayed, separation among the treatments from approximately 34 wk of age. In support of other studies in ruminants (Delavaud et al., 2007), plasma leptin levels did not show a distinct postprandial increase in the prepubertal heifers despite overall differences among treatments. Thus, to our knowledge, this is the first study to provide a detailed characterization of plasma leptin concentrations in growing dairy heifers over a range of average daily gains (0.5 to 1.2 kg/d) during the pre- and peripubertal periods. The hormonal and metabolic signals that gauge the level of body reserves and convey this information to
Figure 6. Effect of dietary energy and protein density on temporal changes in plasma concentrations of A) insulin, B) IGF-1, C) glucose, and D) NEFA in Holstein heifers. During period 1, all heifers were fed to gain at high (H, 1.10 kg/d), medium (M, 0.80 kg/d), or low (L, 0.50 kg/d) growth rates. During period 2, M heifers were fed ad libitum to maintain their growth rate; half the H and L heifers were switched to the period-2 diet either at 12 mo of age (HA, LA) or at 330 kg of BW (HW, LW). The SEM for insulin, IGF-1, glucose, and NEFA were 0.17 ng/ mL, 17.82 ng/mL, 3.22 mg/dL, and 45.95 μM, respectively. Journal of Dairy Science Vol. 92 No. 4, 2009
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Figure 7. Effect of feeding high (H, 1.10 kg/d), medium (M, 0.80 kg/d), or low (L, 0.50 kg/d) energy and protein dense diets on plasma growth hormone (GH) characteristics in dairy heifers. Blood was sampled at 15-min intervals for 8 h at 8 and 10 mo of age in a subset of heifers (n = 5/treatment). Mean (panel A) and basal (panel B) concentrations of GH, GH pulse frequency (panel C), and amplitude (panel D) are shown (least squares means ± SEM). a,bFor a given age, means with different letters are different (P < 0.05).
the brain to trigger puberty in dairy cattle are not clearly understood. Foster and Nagatani (1999) postulated that for an endogenous signal to act as a trigger for puberty, the circulating concentrations of the signal should preferentially increase before the onset of puberty. Whether leptin fulfils such a criterion in ruminants remains controversial. A prepubertal increase in plasma leptin levels was observed in grazing Holstein heifers (Diaz-Torga et al., 2001); however, a commercial and relatively nonspecific RIA system was used to quantify circulating leptin levels in that study. In contrast, using an RIA specific for bovine leptin Block et al. (2003) reported that a linear increase in plasma leptin concentrations during the prepubertal period was found only in Holstein heifers in which puberty was delayed, but not in heifers that were younger at puberty, sug-
gesting that plasma leptin is not a critical determinant of puberty in dairy heifers. Importantly, none of the aforementioned studies examined the effects of different growth rates on plasma leptin profiles during the preand postpubertal periods in heifers. The overall combined data did not reveal a distinct prepubertal increase in plasma leptin concentrations in the heifers. This is in partial agreement with Block et al. (2003); unlike the latter study, we did not partition heifers within each treatment into early or late pubertal animals because of insufficient sample size. In the current study, a closer examination of the distribution of plasma leptin concentrations in each treatment for 2 wk before the timing of first ovulation revealed considerable variability in leptin levels of H heifers, but not M and L heifers (Figure 5C). These data suggest that in rapidly Journal of Dairy Science Vol. 92 No. 4, 2009
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growing heifers, plasma leptin concentrations may not be an important determinant of puberty or that other hormonal and metabolic signals predominate over leptin to trigger puberty in these animals. If leptin levels are not important for pubertal transition in heifers with normal or limited growth rates, then we should have observed a similar degree of variability in leptin profiles of M and L as in H heifers. The lack of such variability in leptin concentrations in the L and M heifers (Figure 5C), together with a tight temporal coupling of plasma leptin concentrations and BW (r2 = 0.396) in these groups that attained puberty at different chronological ages, suggests that plasma leptin concentrations might be more important than other hormonal and metabolic signals for pubertal transition in heifers in which growth rates are normal or restricted. Nonetheless, systemic injections of leptin that produced 2- to 5-fold elevations in circulating levels failed to advance the onset of puberty in growth-restricted beef heifers, suggesting that leptin alone is insufficient to trigger puberty in heifers (Zieba et al., 2003, 2004). The effects of long-term feed restriction and realimentation on the temporal changes in plasma leptin levels in cattle have received little attention. In the current study, ad libitum feeding on the period-2 diet resulted in rapid increases in plasma leptin concentrations of the H and M group heifers (Figure 3). Still, in the L group heifers, realimentation to the period-2 diet resulted in a delayed increase in plasma leptin concentrations with the delay being approximately proportional to the duration of feed restriction; that is, the LA and LW heifers that were realimented to the period-2 diet at about 54 and 76 wk of age responded with an increase in leptin levels by 12 and 15 wk of ad libitum feeding, respectively. A similar delay in plasma leptin response to realimentation was observed in adult sheep that were previously chronically underfed or had low adipose reserves (Delavaud et al., 2007). The growth and hormonal profiles in period 2 (Figures 2 and 3) reveal 2 interesting findings: 1) the plasma leptin concentrations and DMI of the LA heifers appeared to plateau at about 12 to 15 wk of realimentation coincident with slowing of BW gain, and 2) early in period 2, the delayed leptin response of the LW heifers coincided with an accelerated DMI suggesting that the initial hypoleptinemia of the LW heifers may have contributed to their exaggerated hyperphagia. In contrast, the hyperleptinemia of the H groups was not associated with a reduction in DMI or reduced weight gain of the H group heifers. Although these data do not prove a cause-effect relationship, the tight coupling of leptin with DMI and BW in growing heifers suggests that relative hypoleptinemia may be a more important signal for promoting increased DMI and weight gain, whereas hyperleptinemia may Journal of Dairy Science Vol. 92 No. 4, 2009
not be critical for producing anorexia and weight loss. As DMI, BW, and BF are significant predictors of plasma leptin concentrations in the stepwise regression analyses, it is likely that leptin concentrations reflected the alterations in DMI, BW, BF, and other measures. Thus, our data reinforce the concept that leptin plays an important role in the long-term regulation of DMI and BW in cattle. In the current study, we did not observe a marked postprandial increase in plasma leptin concentrations in heifers across treatments, supporting the results of Delavaud et al. (2007) but not those of Marie et al. (2001). In contrast to leptin, circulating insulin concentrations increased within 2 to 3 h postfeeding in H compared with L or M heifers at 40 wk of age, which supports other reports on postprandial increases in plasma insulin concentrations in cattle (Chelikani et al., 2004; Wylie et al., 2008). The increase in plasma concentrations of insulin, IGF-1, and glucose and the reciprocal decrease in plasma GH and NEFA levels in heifers on high compared with medium or low growth rates reflects dietary nutrient density and is confirmatory of other studies in dairy heifers (Petitclerc et al., 1983; Lacasse et al., 1994; Yelich et al., 1996). In the current study, the L group heifers had higher mean GH concentrations than the H group, but we were unable to detect significant alterations in GH pulse characteristics in the H group heifers from 8 to 10 mo of age. Although this may suggest that GH pulsatility is not a critical determinant for onset of puberty in dairy heifers, it is more likely that insufficient statistical power may have contributed to the lack of differences. The rapid increase in plasma concentrations of glucose, insulin, and IGF-1, and decrease in NEFA, on realimentation to the period-2 diet is consistent with previous reports in growing cattle (Yelich et al., 1995, 1996). In summary, the current study profiles with a high degree of temporal resolution the long-term changes in plasma concentrations of leptin, metabolic hormones, and other metabolites in Holstein heifers fed at varying levels with diets differing in energy and protein density during the pre- and postpubertal periods. The data demonstrate that 1) the temporal profile of plasma leptin concentrations closely paralleled the changes in DMI, BW, BF, and BCS in growing heifers; 2) unlike the postprandial increase in plasma insulin concentrations that reflects nutrient density in prepubertal heifers, plasma leptin concentrations did not display a distinct postprandial profile; 3) although plasma leptin concentrations increased with age, the leptin concentrations at puberty did not differ among treatment groups and there was no evidence of a prepubertal spurt in plasma leptin concentrations; 4) plasma leptin concentrations may not be a critical trigger for puberty in rapidly
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growing heifers, but a certain threshold of leptin levels appears important for puberty especially in heifers with normal or restricted growth rates; and 5) plasma concentrations of insulin, IGF-1, and glucose reflected nutrient density, whereas GH and NEFA levels were inversely related to the plane of nutrition. Together, the data provide evidence that dietary energy and protein density, as well as total intake, are important regulators of plasma leptin concentrations, which in turn may play a role in the long-term regulation of energy reserves and puberty in growing Holstein heifers. ACKNOWLEDGMENTS
Financial assistance for this research was provided by Alberta Agricultural Research Institute and Alberta Milk Producers. We appreciate the help of the farm staff at the Dairy Research and Technology Centre, especially Pavol Zalkovic, Harold Lehman, and Wanda Semple for the feeding and care of the experimental animals. The assistance of fellow researchers and graduate students in sample collection, Shirley Shostak (Department of Agricultural, Food and Nutritional Sciences, University of Alberta) in hormone assays, and Pat Marceau (Department of Agricultural, Food and Nutritional Sciences, University of Alberta) in feed and metabolite analyses is much appreciated. The authors thank the National Institute of Diabetes and Digestive and Kidney Diseases-National Hormone and Pituitary Program (NIDDK-NHPP) and A. F. Parlow for assay reagents for GH and IGF-1. REFERENCES Block, S. S., J. M. Smith, R. A. Ehrhardt, M. C. Diaz, R. P. Rhoads, M. E. Van Amburgh, and Y. R. Boisclair. 2003. Nutritional and developmental regulation of plasma leptin in dairy cattle. J. Dairy Sci. 86:3206–3214. Brethour, J. R. 1992. The repeatability and accuracy of ultrasound in measuring backfat of cattle. J. Anim. Sci. 70:1039–1044. Brown, E. G., M. J. Vandehaar, K. M. Daniels, J. S. Liesman, L. T. Chapin, D. H. Keisler, and M. S. Nielsen. 2005. Effect of increasing energy and protein intake on body growth and carcass composition of heifer calves. J. Dairy Sci. 88:585–594. Capuco, A. V., J. J. Smith, D. R. Waldo, and C. E. Rexroad Jr. 1995. Influence of prepubertal dietary regimen on mammary growth of Holstein heifers. J. Dairy Sci. 78:2709–2725. Chelikani, P. K., J. D. Ambrose, D. H. Keisler, and J. J. Kennelly. 2004. Effect of short-term fasting on plasma concentrations of leptin and other hormones and metabolites in dairy cattle. Domest. Anim. Endocrinol. 26:33–48. Chelikani, P. K., J. D. Ambrose, and J. J. Kennelly. 2003. Effect of dietary energy and protein density on body composition, attainment of puberty, and ovarian follicular dynamics in dairy heifers. Theriogenology 60:707–725. Davis Rincker, L. E., M. S. Weber Nielsen, L. T. Chapin, J. S. Liesman, K. M. Daniels, R. M. Akers, and M. J. Vandehaar. 2008. Effects of feeding prepubertal heifers a high-energy diet for three, six, or twelve weeks on mammary growth and composition. J. Dairy Sci. 91:1926–1935. Delavaud, C., F. Bocquier, Y. Chilliard, D. H. Keisler, A. Gertler, and G. Kann. 2000. Plasma leptin determination in ruminants: Effect of nutritional status and body fatness on plasma leptin
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concentration assessed by a specific RIA in sheep. J. Endocrinol. 165:519–526. Delavaud, C., F. Bocquier, R. Baumont, E. Chaillou, T. Ban-Tokuda, and Y. Chilliard. 2007. Body fat content and feeding level interact strongly in the short- and medium-term regulation of plasma leptin during underfeeding and re-feeding in adult sheep. Br. J. Nutr. 98:106–115. Diaz-Torga, G. S., M. E. Mejia, A. Gonzalez-Iglesias, N. Formia, D. Becu-Villalobos, and I. M. Lacau-Mengido. 2001. Metabolic cues for puberty onset in free grazing Holstein heifers naturally infected with nematodes. Theriogenology 56:111–122. Edmonson, A. J., I. J. Lean, L. D. Weaver, L. Farver, and G. Webster. 1989. A body condition scoring chart for Holstein dairy cows. J. Dairy Sci. 72:68–78. Foster, D. L., and S. Nagatani. 1999. Physiological perspectives on leptin as a regulator of reproduction: role in timing puberty. Biol. Reprod. 60:205–215. Garcia, M. R., M. Amstalden, C. D. Morrison, D. H. Keisler, and G. L. Williams. 2003. Age at puberty, total fat and conjugated linoleic acid content of carcass, and circulating metabolic hormones in beef heifers fed a diet high in linoleic acid beginning at four months of age. J. Anim. Sci. 81:261–268. Garcia, M. R., M. Amstalden, S. W. Williams, R. L. Stanko, C. D. Morrison, D. H. Keisler, S. E. Nizielski, and G. L. Williams. 2002. Serum leptin and its adipose gene expression during pubertal development, the estrous cycle, and different seasons in cattle. J. Anim. Sci. 80:2158–2167. Kushler, R. H., and M. B. Brown. 1991. A model for the identification of hormone pulses. Stat. Med. 10:329–340. Lacasse, P., E. Block, and D. Petitclerc. 1994. Effect of plane of nutrition before and during gestation on the concentration of hormones in dairy heifers. J. Dairy Sci. 77:439–445. Lammers, B. P., and A. J. Heinrichs. 2000. The response of altering the ratio of dietary protein to energy on growth, feed efficiency, and mammary development in rapidly growing prepubertal heifers. J. Dairy Sci. 83:977–983. Marie, M., P. A. Findlay, L. Thomas, and C. L. Adam. 2001. Daily patterns of plasma leptin in sheep: Effects of photoperiod and food intake. J. Endocrinol. 170:277–286. Petitclerc, D., L. T. Chapin, R. S. Emery, and H. A. Tucker. 1983. Body growth, growth hormone, prolactin and puberty response to photoperiod and plane of nutrition in Holstein heifers. J. Anim. Sci. 57:892–898. Van Amburgh, M. E., D. G. Fox, D. M. Galton, D. E. Bauman, and L. E. Chase. 1998. Evaluation of National Research Council and Cornell Net Carbohydrate and Protein Systems for predicting requirements of Holstein heifers. J. Dairy Sci. 81:509–526. Wylie, A. R., S. Woods, A. F. Carson, and M. McCoy. 2008. Periprandial changes in metabolite and metabolic hormone concentrations in high-genetic-merit dairy heifers and their relationship to energy balance in early lactation. J. Dairy Sci. 91:577–586. Yelich, J. V., R. P. Wettemann, H. G. Dolezal, K. S. Lusby, D. K. Bishop, and L. J. Spicer. 1995. Effects of growth rate on carcass composition and lipid partitioning at puberty and growth hormone, insulin-like growth factor I, insulin, and metabolites before puberty in beef heifers. J. Anim. Sci. 73:2390–2405. Yelich, J. V., R. P. Wettemann, T. T. Marston, and L. J. Spicer. 1996. Luteinizing hormone, growth hormone, insulin-like growth factor-I, insulin and metabolites before puberty in heifers fed to gain at two rates. Domest. Anim. Endocrinol. 13:325–338. Zieba, D. A., M. Amstalden, M. N. Maciel, D. H. Keisler, N. Raver, A. Gertler, and G. L. Williams. 2003. Divergent effects of leptin on luteinizing hormone and insulin secretion are dose dependent. Exp. Biol. Med. (Maywood) 228:325–330. Zieba, D. A., M. Amstalden, S. Morton, M. N. Maciel, D. H. Keisler, and G. L. Williams. 2004. Regulatory roles of leptin at the hypothalamic-hypophyseal axis before and after sexual maturation in cattle. Biol. Reprod. 71:804–812. Zieba, D. A., M. Amstalden, and G. L. Williams. 2005. Regulatory roles of leptin in reproduction and metabolism: A comparative review. Domest. Anim. Endocrinol. 29:166–185. Journal of Dairy Science Vol. 92 No. 4, 2009
Effects of dietary energy and protein density on plasma concentrations of leptin and metabolic hormones in dairy heifers P. K. Chelikani,*1 D. J. Ambrose,†‡ D. H. Keisler,§ and J. J. Kennelly‡ *Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada, T2N 4N1 †Agriculture Research Division, Alberta Agriculture and Rural Development, Edmonton, Alberta, Canada, T6H 5T6 ‡Department of Agricultural, Food and Nutritional Science, Faculty of Agricultural, Life and Environmental Sciences, University of Alberta, Edmonton, Canada, T6G 2P5 §Division of Animal Sciences, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia 65211
ABSTRACT
The hormonal and metabolic signals that communicate the level of body energy reserves to the reproductive-mammary axis remain undefined in dairy cattle; consequently, our hypothesis was that leptin may fulfill this role. Our objectives were to determine the effects of diets differing in energy and protein density on dry matter intake (DMI), growth traits [body weight (BW), body condition score (BCS), back-fat (BF) thickness], and temporal changes in plasma concentrations of leptin, insulin, growth hormone (GH), insulin-like growth factor-1 (IGF-1), glucose, and nonesterified fatty acids (NEFA) in dairy heifers during the pre- and postpubertal periods. In period 1, heifers were randomly allotted (n = 10/diet) at 103 kg of BW to diets for a predicted average daily gain of 1.10 (high, H), 0.80 (medium, M), or 0.50 kg/d (low, L). Five heifers in each of the H and L groups were further studied during period 2, either at 12 mo of age (HA, LA) or at 330 kg of BW (HW, LW). The data provide evidence that 1) DMI (18%), BW (17%), and BF (5%) together explained 40% of the variation in plasma leptin concentrations (r2 = 0.396); 2) unlike the acute postprandial increase in plasma insulin as a result of increased nutrient density (H 1.42 ± 0.09, M 1.02 ± 0.09, L 0.68 ± 0.11 ng/mL), plasma leptin concentrations did not respond acutely with a distinct postprandial profile; 3) although plasma leptin concentrations increased with age, leptin at puberty did not differ among treatment groups (H 5.63 ± 2.48, M 4.28 ± 0.55, L 4.12 ± 0.72 ng/mL) and there was no evidence of an abrupt transition in prepubertal plasma leptin concentrations; 4) plasma leptin concentrations may not be a critical trigger for puberty in rapidly growing heifers, but are apparently essential for puberty in heifers with normal or restricted growth rates; and 5) plasma concentrations of insulin (H 0.59 Received May 22, 2008. Accepted November 11, 2008. 1 Corresponding author: [email protected]
± 0.07, M 0.43 ± 0.09, L 0.30 ± 0.09 ng/mL), IGF-1 (H 151.08 ± 16.47, L 82.51 ± 17.47 ng/mL), and glucose (H 81.35 ± 3.39, M 73.59 ± 2.34, L 68.25 ± 3.39 mg/ dL) reflected nutrient density, whereas GH (H 1.82 ± 0.23, L 5.87 ± 0.45 ng/mL) and NEFA (H 209.54 ± 50.83, L 234.93 ± 48.97 μM) were inversely related to the plane of nutrition. Collectively, these data suggest that plasma concentrations of leptin may play a role in long-term regulation of energy reserves and puberty in growing Holstein heifers. Key words: dairy heifer, nutrition, leptin, puberty INTRODUCTION
The ruminant growth phase is characterized by hormonal and metabolic adaptations that coordinate somatic tissue accretion with reproductive maturation and mammary development. The pre- and peripubertal periods appear as critical periods of growth in dairy heifers, because accelerated growth rates during these periods are invariably associated with increased adiposity, earlier puberty, and impaired development of mammary secretory tissue (Davis Rincker et al., 2008). Although mammary development is linked to the maturity of the reproductive tract in prepubertal heifers (Lammers and Heinrichs, 2000), the hormonal and metabolic signals that communicate the level of body energy reserves to the reproductive-mammary axis remain undefined. There is substantial evidence on the effects of nutritional manipulation on the hormonal and metabolite milieu in peripubertal heifers. In dairy heifers, a high plane of nutrition during the pre- and postpubertal periods was often associated with increased circulating concentrations of insulin, glucose, and IGF-1, and decreased concentrations of growth hormone (GH) and NEFA (Petitclerc et al., 1983; Lacasse et al., 1994; Capuco et al., 1995). Among the hormonal signals, a prepubertal increase in circulating concentrations of GH and IGF-1 is believed to play a role in determining the onset of puberty in beef heifers (Yelich et al., 1995,
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1996). In addition to these hormones, leptin, a hormone secreted primarily from adipose tissue, is sensitive to dietary manipulation and appears to play an important role in transmitting the status of energy reserves to the central nervous system to regulate feed intake and reproductive function in ruminants (Zieba et al., 2005). A linear prepubertal increase in plasma leptin concentrations was found in beef heifers (Garcia et al., 2002, 2003), whereas in dairy heifers a similar increase was found in one study (Diaz-Torga et al., 2001), but not in another (Block et al., 2003). Given that restricted growth rates delay the onset of puberty in heifers (Yelich et al., 1995), and that nutrient restriction decreases plasma leptin concentrations in cattle (Block et al., 2003; Brown et al., 2005), it was hypothesized that a relative deficiency of leptin may play a role in the delayed puberty of slowly growing heifers. Yet, we are not aware of studies that examined the effects of chronic dietary restriction and realimentation on circulating leptin concentrations and its relationship to feed intake and reproductive function in dairy heifers. Our objectives were to determine the effects of diets differing in energy and protein density on DMI, growth measures [BW, back-fat (BF) thickness, and BCS] and temporal changes in plasma concentrations of leptin, insulin, GH, IGF-1, glucose, and NEFA in dairy heifers during the pre- and postpubertal periods. We assessed the effects of realimentation of feed-restricted heifers on DMI, BW, and hormonal and metabolic adaptations. MATERIALS AND METHODS Animals and Treatments
This study was conducted at the Dairy Research Unit of the University of Alberta, Edmonton, Alberta, Canada; all experimental procedures were approved by the University of Alberta Animal Policy and Welfare Committee. The experimental design and treatments were described previously (Chelikani et al., 2003). Briefly, this was a 2-phase study involving 30 Holstein heifers (Figure 1). In period 1, following an initial 10-d adaptation period, the heifer calves (103 ± 2 kg of BW and 104 ± 2 d of age) were assigned at random to 1 of 3 dietary treatments (n = 10/treatment). The experimental diets (Table 1) were formulated using the Cornell Net Carbohydrate and Protein System (Van Amburgh et al., 1998) for a predicted ADG of 0.50 kg/d (low, L), 0.80 kg/d (medium, M), or 1.10 kg/d (high, H). Heifers were housed in tie-stalls, exercised thrice weekly between 0900 and 1000 h, and individually fed a TMR daily at approximately 1000 h with free access to water. Daily feed consumption was recorded for each heifer. In period 1, based on the weekly BW, the heifers were feed
restricted at varying levels to reach the predicted ADG. It was predicted that the M heifers would achieve a postpubertal BW of 330 kg at approximately 12 mo of age, at which stage they were allowed ad libitum access to a single period-2 diet formulated to maintain their ADG of 0.8 kg/d. To compare treatment differences at a similar age and BW, 5 heifers each within the H and L groups were assigned to period 2 at either 12 mo of age (HA and LA) or entered period 2 when they attained 330 kg of BW with ad libitum access to a single period-2 diet (HW and LW). Measurements and Sampling Measurements of DMI, BW, BCS, and BF
The amount of feed consumed by each heifer was recorded daily. Samples of TMR, ingredients, and orts were collected once weekly, dried at 55°C for 72 h, ground through a 1-mm screen (Thomas-Wiley Laboratory Mill Model 4, Philadelphia, PA), and composited monthly for proximate analysis (Chelikani et al., 2003). The animals were weighed on 2 d consecutively each week before feeding and the mean weekly BW calculated. Once a heifer reached 200 kg of BW, BCS and BF thickness (between 12th and 13th rib) were recorded every 2 wk. The BCS was assessed on a scale of 1 to 5 (Edmonson et al., 1989) by 2 individuals, and BF thickness was measured transcutaneously using an ultrasound scanner (Aloka 500V, Aloka Co. Ltd., Tokyo, Japan) equipped with a 7.5-MHz linear array transducer (Brethour, 1992). The temporal changes in structural growth measures were reported previously (Chelikani et al., 2003). Blood Sampling
Blood samples were collected from all heifers, before feeding, by jugular vein puncture at 2-wk intervals. Samples were placed on ice in 10-mL Vacutainer tubes (Becton Dickinson, Sparks, MD) without or with appropriate anticoagulants (EDTA for leptin, IGF-1, and progesterone; sodium heparin for insulin and GH; sodium fluoride for glucose; and serum for NEFA). Plasma was separated by centrifugation at 1,500 × g for 20 min and stored at –20°C for leptin assays; samples were analyzed for insulin, GH, IGF-1, glucose, and NEFA every 4 wk. Further, beginning at 200 kg of BW, for determining the onset of puberty, blood samples were collected twice weekly (Monday and Friday) via jugular venipuncture from all heifers into 10-mL Vacutainer tubes containing EDTA for progesterone assay. A heifer was considered pubertal, and first ovulation had occurred, when plasma progesterone concentrations exceeded 1 ng/mL for the first time; this was further confirmed by the formation Journal of Dairy Science Vol. 92 No. 4, 2009
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Figure 1. Overview of experimental design. In period 1 (solid lines), heifer calves (103 ± 2 kg BW and 104 ± 2 d of age) were assigned at random to 1 of 3 dietary treatments (n = 10/treatment) formulated for a predicted ADG of 1.10 kg/d (high, H), 0.80 kg/d (medium, M), or 0.50 kg/d (low, L) growth rates. The M heifers were expected to achieve a postpubertal BW of 330 kg at approximately 12 mo of age, at which stage they were allowed ad libitum access to a single period-2 diet (dashed lines) formulated to maintain their ADG of 0.8 kg/d. To compare treatment differences at a similar age and BW, 5 heifers each within the H and L groups were assigned to period 2 at either 12 mo of age (HA, LA) or entered period 2 when they attained 330 kg of BW (HW, LW). Frequent blood sampling for determining plasma LH and GH pulse characteristics, and for assessing the postprandial changes in plasma leptin and insulin concentrations, were done in period 1 at 8 and 10 mo of age from a subset (n = 5) of H, M, and L heifers. Adapted with permission from Chelikani et al. (2003).
of a detectable corpus luteum in the heifers that were subjected to transrectal ultrasonography (Chelikani et al., 2003). These samples were used for determining leptin concentrations; for each heifer the mean weekly plasma leptin concentrations during the pre- and peripubertal period were calculated from the twice-weekly samples for ascertaining the leptin levels relative to the onset of puberty. To determine pulsatile characteristics of GH and LH, blood samples from 5 heifers from each of the H, M, and L treatment groups in period 1 were taken via jugular catheters at 15-min intervals for 8 h at 8 mo and again at 10 mo of age. The distribution of the heifers in period 1 for serial bleeding that entered period 2 was as follows: HA = 3, HW = 2, LA = 2, LW = 3, and M = 5. Two baseline samples were taken at −30 and −15 min relative to feeding, followed by serial 15-min sampling for 8 h. The baseline (15 min before feeding) and hourly plasma samples were analyzed for leptin and insulin concentrations. Data on LH pulsatility were described previously (Chelikani et al., 2003). The Kushler-Brown PulseFit program (Kushler and Brown, 1991), a stepwise selection algorithm for identifying hormone pulses, was used to characterize GH Journal of Dairy Science Vol. 92 No. 4, 2009
pulsatility and obtain values for pulse frequency, amplitude, mean, and basal concentrations. The criteria used for LH pulse characterization (Chelikani et al., 2003) were applied for determining GH pulse measures. Hormone and Metabolite Assays
Plasma leptin concentrations were determined by a highly sensitive ovine leptin RIA validated for bovine plasma (Delavaud et al., 2000). All the samples were analyzed in 8 assays. Assay sensitivity at 95% of total binding was 0.15 ng/mL, intraassay coefficient of variation (CV) was 3.3%, and interassay CV was 5.6%. Plasma concentrations of insulin and GH were determined by homologous double-antibody RIA, and IGF-1 by heterologous double-antibody RIA, as described previously (Chelikani et al., 2004). For insulin, all the samples were analyzed in 3 assays, the intra- and interassay CV were 8.7 and 11%, respectively, assay sensitivity at 94% of total binding was 0.08 ng/mL, and recovery was 93%. For IGF-1, all the samples were analyzed in 2 assays, the intra- and interassay assay CV were 6.45 and 7.49%, radioinert recovery was 94%, and
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Table 1. Ingredient and chemical composition (mean ± SEM) of diets1 Period-1 diet Item Ingredient (% of DM) Barley silage Alfalfa silage Grass hay Alfalfa hay Rolled barley Canola meal Corn gluten meal Tallow Fortified mineral salt2 Vitamin ADE premix3 Chemical composition DM (%) OM (% of DM) CP (% of DM) Crude fat (% of DM) NDF (% of DM) ADF (% of DM) Lignin (% of DM) Ash (% of DM) ME (Mcal/kg of DM) NEG (Mcal/kg of DM)
High
Medium
Low
Period-2 diet
31.8 — — 23.9 19.8 7.8 13.5 2.9 0.1 0.1
36.6 — — 32.6 17.2 6.4 5.5 1.5 0.1 0.1
48.3 — — 40.9 10.6 — — — 0.1 0.1
28.2 28.2 14.1 — 29.4 — — — 0.1 0.1
59.3 ± 1.0 90.7 ± 0.2 20.9 ± 0.7 4.6 ± 0.2 44.1 ± 1.2 25.7 ± 0.9 6.2 ± 0.3 9.3 ± 0.1 2.62 0.97
57.4 ± 1.2 90.8 ± 0.2 18.1 ± 0.5 3.4 ± 0.1 47.8 ± 1.3 28.3 ± 1.2 6.9 ± 0.4 9.2 ± 0.1 2.42 0.93
52.9 ± 1.4 90.4 ± 0.3 13.5 ± 0.2 1.6 ± 0.1 54.3 ± 1.2 33.6 ± 1.1 8.5 ± 0.4 9.6 ± 0.1 2.28 0.85
52.9 ± 1.8 90.1 ± 0.2 13.9 ± 0.4 2.1 ± 0.1 48.9 ± 1.0 29.0 ± 1.0 6.7 ± 0.7 9.9 ± 0.1 2.29 0.86
1 Adapted with permission from Chelikani et al. (2003). In period 1, heifers were randomly allotted (n = 10/diet) at 103 kg of BW to diets for a predicted ADG of 1.10 (high), 0.80 (medium), or 0.50 kg/d (low). The period-2 diet was formulated to maintain ADG of 0.8 kg/d. 2 Contained 37% Na, 58% Cl, 80 mg of CO/kg, 400 mg of Cu/kg, 200 mg of I/kg, 6,000 mg of Mn/kg, 400 mg of Se/kg, 800 mg of Zn/kg; Champion Feed Services Ltd., Edmonton, Alberta, Canada. 3 Contained 3,846 kIU vitamin A/kg, 2,292 kIU vitamin D/kg, and 6,539 IU vitamin E/kg; Champion Feed Services Ltd.
assay sensitivity at 88% of total binding was 20.94 ng/ mL. For GH, all the samples were analyzed in 4 assays, the intra- and interassay CV were 2.7 and 6.8%, and assay sensitivity at 95% of total binding was 0.06 ng/ mL. Plasma NEFA and glucose concentrations were determined by enzymatic assays (Chelikani et al., 2004). The intra- and interassay CV for NEFA were 5 and 4%, and for glucose were 2.5 and 2.6%, respectively. Statistical Analyses
Data were analyzed using the MIXED procedure of SAS (SAS Institute, Cary, NC). Repeated measures on DMI, BW, BF, BCS, plasma hormones (leptin, insulin, IGF-1, GH) and metabolites (glucose and NEFA) were analyzed using mixed model analyses as described previously (Chelikani et al., 2003). Briefly, the model included treatment, time, and their interaction as fixed effects, and heifer within treatment as the random effect. Plasma concentrations of insulin, GH, and NEFA were log-transformed before analyses. Stepwise multiple linear regression analysis was used to determine the relative contribution of DMI, BW, BF, BCS, plasma hormones and metabolites to the variation in plasma leptin concentrations.
RESULTS DMI, BW, and BF Thickness
At the beginning of the study (15 wk of age), heifers in all treatment groups had similar (P > 0.10) DMI and BW (DMI: H 3.12 ± 0.47, M 2.97 ± 0.33, and L 2.49 ± 0.50 kg/d; BW: H 105 ± 18, M 101 ± 13, and L 105 ± 19 kg). The HA, LA, and M heifers entered period 2 at 54 wk of age, the HW heifers began period 2 at 48 wk of age, and the LW heifers entered period 2 at 76 wk of age. Temporal changes in DMI during both experimental periods, and the entire study, are in Figure 2. In period 1, as expected, the DMI of the H heifers increased with age, and was greater than that of the L and M heifers. The DMI of the H heifers was 52 and 28% greater than that of the L and M heifers by 25 and 29 wk of age, respectively; the M heifers consumed 25% more than L heifers by 31 wk of age (treatment by age, P < 0.001, Figure 2A). During period 2, the DMI was similar between the HA and HW heifers (HA 10.37 ± 0.49, HW 10.25 ± 0.56 kg/d; P > 0.10); however, the intakes of both sub-groups were nearly 20% greater than that of M heifers (HA 10.0 ± 0.48, HW 10.14 ± 0.47, M 8.43 ± 0.33 kg/d) for the first 4 wk of period 2. Although the DMI of the HA heifers was greater than
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that of the LA heifers (HA 10.52 ± 0.48, LA 7.68 ± 0.47 kg/d) for the first 14 wk of period 2 (treatment by age, P < 0.01), this difference was temporally attenuated with the DMI of LA heifers catching up with the HA heifers after 15 wk of realimentation in period 2 (P > 0.10). Similarly, the difference in DMI between the HW and LW heifers (HW 10.20 ± 0.48, LW 6.51 ± 0.53 kg/d) was gradually attenuated by 10 wk of realimentation in period 2. Compared with M heifers, the DMI of the LA and LW heifers remained lower (P < 0.01) for 18 (M 10.16 ± 0.36, LA 7.88 ± 0.47 kg/d) and 10 wk (M 9.82 ± 0.34, LW 6.51 ± 0.53 kg/d), respectively, after realimentation in period 2. The DMI of the LA heifers was greater than that of the LW heifers (LA 6.92 ± 0.47, LW 4.97 ± 0.53 kg/d) for the first 8 wk of
realimentation; thereafter, the DMI of the LW heifers accelerated over that of the LA heifers from wk 12 to 21 (LA 8.63 ± 0.47, LW 11.08 ± 0.58 kg/d) of period 2 (treatment by age, P < 0.001). In period 1, the BW of the H heifers increased with age and was greater than that of the L and M heifers by 24 (H 168 ± 18, L 134 ± 19 kg) and 29 wk of age (H 208 ± 18, M 180 ± 13 kg), respectively; the M heifers were heavier than L heifers by 29 wk of age (M 180 ± 13, L 174 ± 18 kg; treatment by age, P < 0.001; Figure 2B). In period 2, the BW of the HW and LW heifers did not differ from that of the M heifers (HW 396 ± 23, M 395 ± 16, LW 373 ± 22 kg; P > 0.10); however, the HA heifers were heavier than the HW and LA heifers (HA 448 ± 18, HW 396 ± 23, LA 317 ± 18 kg), and the LW
Figure 2. Effect of dietary energy and protein density on temporal changes in A) DMI, B) BW, C) back-fat (BF) thickness, and D) BCS of Holstein heifers. During period 1, all heifers were fed to gain at either high (H, 1.10 kg/d), medium (M, 0.80 kg/d), or low (L, 0.50 kg/d) growth rates. During period 2, the M heifers were fed ad libitum to maintain their growth rate; half the H and L heifers were switched to the period-2 diet either at 12 mo of age (HA, LA) or at 330 kg of BW (HW, LW). The SEM for DMI, BW, BF thickness, and BCS were 0.48 kg, 18.59 kg, 0.35 mm, and 0.13, respectively. Journal of Dairy Science Vol. 92 No. 4, 2009
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animals were heavier than the LA heifers (LW 373 ± 22, LA 317 ± 18 kg; treatment by age, P < 0.001). Similar to BW changes, the BF thickness of the H heifers increased with age and was greater than that of the L and M heifers (H 2.63 ± 0.29, M 2.14 ± 0.22, L 1.85 ± 0.37 mm), by 30 wk of age, and the M heifers had more BF than the L heifers by 32 wk of age (M 2.75 ± 0.23, L 1.87 ± 0.33 mm; treatment by age, P < 0.001; Figure 2C). In period 2, the BF of the HA heifers increased rapidly and was greater than that of the LA and M heifers but did not differ from that of HW heifers (HA 5.72 ± 0.33, HW 4.05 ± 0.39, M 4.33 ± 0.23, LA 3.27 ± 0.26 mm). During period 2, the HW heifers had similar BF to M heifers, but had more BF compared with LW heifers (3.11 ± 0.39 mm); the BF of the LW and LA heifers did not differ (treatment by age, P < 0.001). The BCS followed a similar a pattern as BF (treatment by age, P < 0.001; Figure 2D); however, the BCS changes appeared to be less robust and sensitive than the BF changes. Plasma Leptin Concentrations
Temporal changes in plasma leptin concentrations during both experimental periods and the entire study are shown in Figure 3. The postprandial profile of plasma leptin concentrations at 8 and 10 mo of age are in Figure 4, and the changes in plasma leptin concentrations relative to the onset of puberty are in Figure 5. Plasma leptin concentrations closely followed the changes in DMI, BW, and BF thickness (Figures 2 and 3). In period 1, plasma leptin concentrations of the H heifers increased with age and were greater than those of the L and M heifers by 34 (H 3.86 ± 0.07, L 2.81 ± 0.07 ng/mL) and 44 wk of age (H 5.42 ± 0.06, M 4.54 ± 0.04 ng/mL), respectively; the M heifers had greater circulating leptin levels than L heifers from 37 wk of age (M 4.08 ± 0.04, L 3.11 ± 0.06 ng/mL; treatment by age, P < 0.001; Figure 3). The postprandial changes in plasma leptin concentrations did not differ among the H, L, and M heifers at 8 mo (32 wk; H 3.90 ± 0.24, M 3.48 ± 0.15, L 3.20 ± 0.33 ng/mL) of age (Figure 4A; P > 0.10); however, at 10 mo (40 wk) of age postprandial plasma leptin concentrations were greater in H compared with L and M heifers (H 4.94 ± 0.29, M 3.96 ± 0.26, L 3.68 ± 0.43 ng/mL; Figure 4B), without an apparent postprandial surge. After switching to the period-2 diet, in general, plasma leptin concentrations rapidly increased in the H and M heifers, but the response was delayed in the L heifers. In period 2, the plasma leptin concentrations of LA and LW heifers increased gradually reaching the levels of M heifers following 12 (LA 5.63 ± 0.06, M 7.71 ± 0.05 ng/ mL) and 15 wk (LW 5.72 ± 0.07, M 6.94 ± 0.07 ng/
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Figure 3. Effect of dietary energy and protein density on temporal changes in plasma concentrations of leptin in Holstein heifers. During period 1, all heifers were fed to gain at either high (H, 1.10 kg/d), medium (M, 0.80 kg/d), or low (L, 0.50 kg/d) growth rates. During period 2, the M heifers were fed ad libitum to maintain their growth rate; half the H and L heifers were switched to the period-2 diet either at 12 mo of age (HA, LA) or at 330 kg of BW (HW, LW). The SEM for leptin was 1.03 ng/mL.
mL) of realimentation to period-2 diet; however, the leptin levels of LA and LW heifers remained lower than that of HA and HW heifers for the entire duration of period 2 (HA 7.99 ± 0.06, HW 7.97 ± 0.07, LA 5.75 ± 0.07, LW 4.33 ± 0.07 ng/mL; treatment by age, P < 0.001; Figure 3). Stepwise multiple regression analysis revealed that of all the variables considered, DMI (18%), BW (17%), and BF (5%) together explained 40% of the variation in plasma leptin concentrations (r2 = 0.396; P < 0.001). When leptin concentrations were aligned to the week of puberty, there was a trend for an effect of week (P = 0.09), but treatment and treatment by week interactions were not significant (P > 0.10, Figure 5A). Therefore, the data from all treatments were collapsed and regressed on age at onset of puberty. The pooled data revealed a significant (P < 0.0001), but relatively low (r2 = 0.13) linear increase in leptin relative to the timing of puberty (Figure 5B). A plot (Figure 5C) of the raw distribution of plasma leptin concentrations during the week before and after the first luteal cycle of each individual heifer revealed that the plasma leptin concentrations of the L and M heifers were less variable, whereas the H heifers exhibited a high degree of variability (mean ± SE: H 5.70 ± 2.48, M 4.28 ± 0.61, and L 4.07 ± 0.51 ng/mL); however, across all heifers the plasma leptin concentrations were >2.9 ng/mL during the peripubertal period. Journal of Dairy Science Vol. 92 No. 4, 2009
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Plasma Concentrations of Insulin, IGF-1, GH, Glucose, and NEFA
Temporal changes in plasma concentrations of metabolic hormones and metabolites at 4-wk intervals are in Figure 6. During period 1, plasma insulin concentrations of the H heifers were greater than that of L and M heifers by 31 (H 0.46 ± 0.10, L 0.30 ± 0.17 ng/ mL) and 53 wk (H 0.59 ± 0.07, M 0.43 ± 0.09 ng/ mL) of age, respectively, and insulin levels of L heifers were reduced compared with M heifers by 37 wk of age (M 0.40 ± 0.07, L 0.31 ± 0.09 ng/mL) (treatment by age, P < 0.001). The postprandial changes in plasma insulin concentrations did not differ among the H, L, and M heifers at 8 mo (32 wk) of age (H 0.65 ± 0.09,
M 0.61 ± 0.11, L 0.53 ± 0.11 ng/mL; Figure 4A; P > 0.10). At 10 mo (40 wk) of age, postprandial plasma insulin concentrations of H heifers were greater than L heifers from 2 to 5 h following feeding, and were also greater than that of M from 3 to 4 h following feeding; the postprandial insulin concentrations of the M and L heifers did not differ (treatment by time, P < 0.001; Figure 4B). During period 2, plasma insulin concentrations of LA and LW heifers increased rapidly and were similar to those of HA and HW heifers (HA 0.49 ± 0.15, HW 0.37 ± 0.17, LA 0.48 ± 0.15, LW 0.53 ± 0.17 ng/mL), respectively, and M heifers (0.44 ± 0.11 ng/ mL), within a week of realimentation to the period-2 diet (P > 0.10).
Figure 4. Effect of dietary energy and protein density on postprandial plasma concentrations of leptin and insulin at 8 (panels A and C) and 10 (panels B and D) mo of age. Blood samples were collected at baseline (time 0 = 15 min before feeding) and hourly for 8 h for determining plasma leptin and insulin concentrations. During this period, all heifers were fed to gain at high (1.10 kg/d), medium (0.80 kg/d), or low (0.50 kg/d) growth rates. The SEM for plasma insulin and leptin were 0.10 and 0.29 ng/mL, respectively. Journal of Dairy Science Vol. 92 No. 4, 2009
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During period 1, plasma IGF-1 concentrations of the H heifers increased rapidly and were greater than that of L heifers by 17 wk of age (H 151.08 ± 16.47, L 82.51 ± 17.47 ng/mL); the IGF-1 concentrations of the M heifers were similar to those of H heifers after 17 wk of age (H 188.19 ± 18.63, M 151.45 ± 16.12 ng/mL), but were greater than those of L (84.72 ± 17.47 ng/mL) heifers by 19 wk of age (Figure 6B; treatment by age, P < 0.001). In period 2, plasma IGF-1 concentrations of LA and LW heifers increased rapidly and were similar to those of HA and HW heifers (HA 205.22 ± 16.47, HW 198.97 ± 16.47, LA 179.86 ± 18.44, LW 188.96 ± 18.11 ng/mL), respectively, as well as the M heifers (M 197.95 ± 12.17 ng/mL), within 2 wk of realimentation to the period-2 diet (P > 0.10). Despite a significant treatment by age (P < 0.001) interaction, the temporal profile of plasma GH concentrations exhibited considerable variability; plasma GH levels were higher in L compared with H heifers (H 4.42 ± 1.95, L 8.59 ± 1.97 ng/mL), and were reduced with age (data not shown). The pulsatile characteristics of plasma GH at 8 and 10 mo of age are in Figure 7. The mean GH concentrations of the H heifers were lower than those of L heifers at both 8 (H 1.82 ± 0.23, L 5.87 ± 0.45 ng/mL) and 10 mo (H 1.70 ± 0.44, L 4.12 ± 0.51 ng/mL) of age. The basal concentrations and the number and amplitude of GH pulses did not differ among the L and H heifers (P > 0.10). There were significant effects of age (P < 0.0001) and treatment by age (P < 0.001) interactions for plasma concentrations of glucose and NEFA. In period 1, plasma glucose concentrations of H heifers were higher than those of L heifers after 17 wk of age (H 81.35 ± 3.39, L 68.25 ± 3.39 mg/dL), and differed from that of M heifers from 17 to about 35 wk of age (H 80.72 ± 3.30, M 73.59 ± 2.34 mg/dL). In period 2, the plasma glucose concentrations of LA heifers increased rapidly to the levels of HA heifers within 1 wk (HA 71.46 ± 2.90, LA 73.32 ± 2.90 mg/dL), whereas the glucose levels of LW heifers gradually approached the levels of HW heifers at about 6 wk (HW 79.86 ± 3.62, LW 72.63 ± 3.25 mg/dL) of realimentation to the period-2 diet (Figure 6C). Plasma NEFA concentrations exhibited considerable variability, and were, in general, higher in the L groups relative to the H groups (H 209.54 ± 50.83, L 234.93 ± 48.97 μM), but were reduced with time and after changing to the period-2 diet in all groups (Figure 6D). DISCUSSION
There is evidence that rapid prepubertal growth rates hasten the onset of puberty and have detrimental effects on mammary development in dairy heifers (Davis Rincker et al., 2008); however, the hormonal and
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Figure 5. A) Plasma leptin concentrations during the peripubertal period in heifers fed to gain at high (1.10 kg/d), medium (0.80 kg/d) or low (0.50 kg/d) growth rates. As plasma leptin concentrations did not differ among the treatments around puberty, the data from all treatments were collapsed and regressed on week relative to puberty (panel B). The distribution of plasma leptin concentrations for 2 wk before the first luteal cycle (onset of puberty) for each treatment group is shown in the box and whisker plot (panel C). Upper border, middle line, and lower border of each box indicate 75th percentile, median, and 25th percentile, respectively; whiskers (error bars) above and below the box indicate 90th and 10th percentiles. Journal of Dairy Science Vol. 92 No. 4, 2009
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metabolic signals that mediate these effects remain undefined. There is some evidence that leptin, a hormone produced primarily from adipose tissue, may play a role in determining the timing of puberty and mammary development (Garcia et al., 2002, 2003; Block et al., 2003). Nevertheless, there is limited information on the effects of long-term nutritional manipulation on changes in circulating leptin concentrations during the pre- and peripubertal period in heifers. Plasma leptin concentrations did not differ among treatments when Holstein heifers fed to grow at either 1.2 kg/d (high) or 0.4 kg/d (medium) from 7 to 14 wk of age (Brown et al., 2005), or when fed to grow at 0.9 kg/d with diets containing calcium salts of either palm fatty acids or conjugated linoleic acid from 10 to 30 wk of age (Block et al., 2003). In contrast to these studies, with
our dietary regimen we were able to produce significant differences among the 3 treatment groups in growth rates, and the temporal profile of plasma leptin concentrations showed a marked, although delayed, separation among the treatments from approximately 34 wk of age. In support of other studies in ruminants (Delavaud et al., 2007), plasma leptin levels did not show a distinct postprandial increase in the prepubertal heifers despite overall differences among treatments. Thus, to our knowledge, this is the first study to provide a detailed characterization of plasma leptin concentrations in growing dairy heifers over a range of average daily gains (0.5 to 1.2 kg/d) during the pre- and peripubertal periods. The hormonal and metabolic signals that gauge the level of body reserves and convey this information to
Figure 6. Effect of dietary energy and protein density on temporal changes in plasma concentrations of A) insulin, B) IGF-1, C) glucose, and D) NEFA in Holstein heifers. During period 1, all heifers were fed to gain at high (H, 1.10 kg/d), medium (M, 0.80 kg/d), or low (L, 0.50 kg/d) growth rates. During period 2, M heifers were fed ad libitum to maintain their growth rate; half the H and L heifers were switched to the period-2 diet either at 12 mo of age (HA, LA) or at 330 kg of BW (HW, LW). The SEM for insulin, IGF-1, glucose, and NEFA were 0.17 ng/ mL, 17.82 ng/mL, 3.22 mg/dL, and 45.95 μM, respectively. Journal of Dairy Science Vol. 92 No. 4, 2009
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Figure 7. Effect of feeding high (H, 1.10 kg/d), medium (M, 0.80 kg/d), or low (L, 0.50 kg/d) energy and protein dense diets on plasma growth hormone (GH) characteristics in dairy heifers. Blood was sampled at 15-min intervals for 8 h at 8 and 10 mo of age in a subset of heifers (n = 5/treatment). Mean (panel A) and basal (panel B) concentrations of GH, GH pulse frequency (panel C), and amplitude (panel D) are shown (least squares means ± SEM). a,bFor a given age, means with different letters are different (P < 0.05).
the brain to trigger puberty in dairy cattle are not clearly understood. Foster and Nagatani (1999) postulated that for an endogenous signal to act as a trigger for puberty, the circulating concentrations of the signal should preferentially increase before the onset of puberty. Whether leptin fulfils such a criterion in ruminants remains controversial. A prepubertal increase in plasma leptin levels was observed in grazing Holstein heifers (Diaz-Torga et al., 2001); however, a commercial and relatively nonspecific RIA system was used to quantify circulating leptin levels in that study. In contrast, using an RIA specific for bovine leptin Block et al. (2003) reported that a linear increase in plasma leptin concentrations during the prepubertal period was found only in Holstein heifers in which puberty was delayed, but not in heifers that were younger at puberty, sug-
gesting that plasma leptin is not a critical determinant of puberty in dairy heifers. Importantly, none of the aforementioned studies examined the effects of different growth rates on plasma leptin profiles during the preand postpubertal periods in heifers. The overall combined data did not reveal a distinct prepubertal increase in plasma leptin concentrations in the heifers. This is in partial agreement with Block et al. (2003); unlike the latter study, we did not partition heifers within each treatment into early or late pubertal animals because of insufficient sample size. In the current study, a closer examination of the distribution of plasma leptin concentrations in each treatment for 2 wk before the timing of first ovulation revealed considerable variability in leptin levels of H heifers, but not M and L heifers (Figure 5C). These data suggest that in rapidly Journal of Dairy Science Vol. 92 No. 4, 2009
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growing heifers, plasma leptin concentrations may not be an important determinant of puberty or that other hormonal and metabolic signals predominate over leptin to trigger puberty in these animals. If leptin levels are not important for pubertal transition in heifers with normal or limited growth rates, then we should have observed a similar degree of variability in leptin profiles of M and L as in H heifers. The lack of such variability in leptin concentrations in the L and M heifers (Figure 5C), together with a tight temporal coupling of plasma leptin concentrations and BW (r2 = 0.396) in these groups that attained puberty at different chronological ages, suggests that plasma leptin concentrations might be more important than other hormonal and metabolic signals for pubertal transition in heifers in which growth rates are normal or restricted. Nonetheless, systemic injections of leptin that produced 2- to 5-fold elevations in circulating levels failed to advance the onset of puberty in growth-restricted beef heifers, suggesting that leptin alone is insufficient to trigger puberty in heifers (Zieba et al., 2003, 2004). The effects of long-term feed restriction and realimentation on the temporal changes in plasma leptin levels in cattle have received little attention. In the current study, ad libitum feeding on the period-2 diet resulted in rapid increases in plasma leptin concentrations of the H and M group heifers (Figure 3). Still, in the L group heifers, realimentation to the period-2 diet resulted in a delayed increase in plasma leptin concentrations with the delay being approximately proportional to the duration of feed restriction; that is, the LA and LW heifers that were realimented to the period-2 diet at about 54 and 76 wk of age responded with an increase in leptin levels by 12 and 15 wk of ad libitum feeding, respectively. A similar delay in plasma leptin response to realimentation was observed in adult sheep that were previously chronically underfed or had low adipose reserves (Delavaud et al., 2007). The growth and hormonal profiles in period 2 (Figures 2 and 3) reveal 2 interesting findings: 1) the plasma leptin concentrations and DMI of the LA heifers appeared to plateau at about 12 to 15 wk of realimentation coincident with slowing of BW gain, and 2) early in period 2, the delayed leptin response of the LW heifers coincided with an accelerated DMI suggesting that the initial hypoleptinemia of the LW heifers may have contributed to their exaggerated hyperphagia. In contrast, the hyperleptinemia of the H groups was not associated with a reduction in DMI or reduced weight gain of the H group heifers. Although these data do not prove a cause-effect relationship, the tight coupling of leptin with DMI and BW in growing heifers suggests that relative hypoleptinemia may be a more important signal for promoting increased DMI and weight gain, whereas hyperleptinemia may Journal of Dairy Science Vol. 92 No. 4, 2009
not be critical for producing anorexia and weight loss. As DMI, BW, and BF are significant predictors of plasma leptin concentrations in the stepwise regression analyses, it is likely that leptin concentrations reflected the alterations in DMI, BW, BF, and other measures. Thus, our data reinforce the concept that leptin plays an important role in the long-term regulation of DMI and BW in cattle. In the current study, we did not observe a marked postprandial increase in plasma leptin concentrations in heifers across treatments, supporting the results of Delavaud et al. (2007) but not those of Marie et al. (2001). In contrast to leptin, circulating insulin concentrations increased within 2 to 3 h postfeeding in H compared with L or M heifers at 40 wk of age, which supports other reports on postprandial increases in plasma insulin concentrations in cattle (Chelikani et al., 2004; Wylie et al., 2008). The increase in plasma concentrations of insulin, IGF-1, and glucose and the reciprocal decrease in plasma GH and NEFA levels in heifers on high compared with medium or low growth rates reflects dietary nutrient density and is confirmatory of other studies in dairy heifers (Petitclerc et al., 1983; Lacasse et al., 1994; Yelich et al., 1996). In the current study, the L group heifers had higher mean GH concentrations than the H group, but we were unable to detect significant alterations in GH pulse characteristics in the H group heifers from 8 to 10 mo of age. Although this may suggest that GH pulsatility is not a critical determinant for onset of puberty in dairy heifers, it is more likely that insufficient statistical power may have contributed to the lack of differences. The rapid increase in plasma concentrations of glucose, insulin, and IGF-1, and decrease in NEFA, on realimentation to the period-2 diet is consistent with previous reports in growing cattle (Yelich et al., 1995, 1996). In summary, the current study profiles with a high degree of temporal resolution the long-term changes in plasma concentrations of leptin, metabolic hormones, and other metabolites in Holstein heifers fed at varying levels with diets differing in energy and protein density during the pre- and postpubertal periods. The data demonstrate that 1) the temporal profile of plasma leptin concentrations closely paralleled the changes in DMI, BW, BF, and BCS in growing heifers; 2) unlike the postprandial increase in plasma insulin concentrations that reflects nutrient density in prepubertal heifers, plasma leptin concentrations did not display a distinct postprandial profile; 3) although plasma leptin concentrations increased with age, the leptin concentrations at puberty did not differ among treatment groups and there was no evidence of a prepubertal spurt in plasma leptin concentrations; 4) plasma leptin concentrations may not be a critical trigger for puberty in rapidly
REGULATION OF PLASMA LEPTIN IN HEIFERS
growing heifers, but a certain threshold of leptin levels appears important for puberty especially in heifers with normal or restricted growth rates; and 5) plasma concentrations of insulin, IGF-1, and glucose reflected nutrient density, whereas GH and NEFA levels were inversely related to the plane of nutrition. Together, the data provide evidence that dietary energy and protein density, as well as total intake, are important regulators of plasma leptin concentrations, which in turn may play a role in the long-term regulation of energy reserves and puberty in growing Holstein heifers. ACKNOWLEDGMENTS
Financial assistance for this research was provided by Alberta Agricultural Research Institute and Alberta Milk Producers. We appreciate the help of the farm staff at the Dairy Research and Technology Centre, especially Pavol Zalkovic, Harold Lehman, and Wanda Semple for the feeding and care of the experimental animals. The assistance of fellow researchers and graduate students in sample collection, Shirley Shostak (Department of Agricultural, Food and Nutritional Sciences, University of Alberta) in hormone assays, and Pat Marceau (Department of Agricultural, Food and Nutritional Sciences, University of Alberta) in feed and metabolite analyses is much appreciated. The authors thank the National Institute of Diabetes and Digestive and Kidney Diseases-National Hormone and Pituitary Program (NIDDK-NHPP) and A. F. Parlow for assay reagents for GH and IGF-1. REFERENCES Block, S. S., J. M. Smith, R. A. Ehrhardt, M. C. Diaz, R. P. Rhoads, M. E. Van Amburgh, and Y. R. Boisclair. 2003. Nutritional and developmental regulation of plasma leptin in dairy cattle. J. Dairy Sci. 86:3206–3214. Brethour, J. R. 1992. The repeatability and accuracy of ultrasound in measuring backfat of cattle. J. Anim. Sci. 70:1039–1044. Brown, E. G., M. J. Vandehaar, K. M. Daniels, J. S. Liesman, L. T. Chapin, D. H. Keisler, and M. S. Nielsen. 2005. Effect of increasing energy and protein intake on body growth and carcass composition of heifer calves. J. Dairy Sci. 88:585–594. Capuco, A. V., J. J. Smith, D. R. Waldo, and C. E. Rexroad Jr. 1995. Influence of prepubertal dietary regimen on mammary growth of Holstein heifers. J. Dairy Sci. 78:2709–2725. Chelikani, P. K., J. D. Ambrose, D. H. Keisler, and J. J. Kennelly. 2004. Effect of short-term fasting on plasma concentrations of leptin and other hormones and metabolites in dairy cattle. Domest. Anim. Endocrinol. 26:33–48. Chelikani, P. K., J. D. Ambrose, and J. J. Kennelly. 2003. Effect of dietary energy and protein density on body composition, attainment of puberty, and ovarian follicular dynamics in dairy heifers. Theriogenology 60:707–725. Davis Rincker, L. E., M. S. Weber Nielsen, L. T. Chapin, J. S. Liesman, K. M. Daniels, R. M. Akers, and M. J. Vandehaar. 2008. Effects of feeding prepubertal heifers a high-energy diet for three, six, or twelve weeks on mammary growth and composition. J. Dairy Sci. 91:1926–1935. Delavaud, C., F. Bocquier, Y. Chilliard, D. H. Keisler, A. Gertler, and G. Kann. 2000. Plasma leptin determination in ruminants: Effect of nutritional status and body fatness on plasma leptin
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