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,1
* Adaptation Physiology Group,
Animal Nutrition Group, and
Quantitative Veterinary Epidemiology Group, Wageningen Institute of Animal Sciences, Wageningen University, PO Box 338, 6700 AH Wageningen, the Netherlands
# Utrecht University, PO Box 80151, 3508 TD Utrecht, the Netherlands
Catholic University Leuven, Kasteelpark Arenberg 30, 3001 Heverlee, Belgium
1 Corresponding author: Ariette.vanKnegsel{at}wur.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Key Words: negative energy balance • lipogenic nutrient • glucogenic nutrient
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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In an earlier published review (van Knegsel et al., 2005) it was hypothesized that part of the metabolic effect of NEB is an imbalance in C2:C3 nutrient ratio. The C2 and C3 nutrients can split up into fragments containing 2 or 3 C atoms; C2 and C3 nutrients are directly supplied in the diet or are derived from larger compounds. This imbalance in C2:C3 nutrient ratio is induced by an excess of C2 nutrients (stimulated by body fat mobilization) that cannot be oxidized completely as a result of a deficiency in C3 nutrients. There are 3 major alternative pathways for C2 nutrients. First, an increase in incomplete oxidation of C2 nutrients may occur, which causes an increase in plasma ketone body concentration, ultimately resulting in ketosis. Second, C2 nutrients can be esterified and stored in the liver as triacylglycerides (TAG), potentially causing a fatty liver. Third, C2 nutrients can be transported to the mammary gland and transformed into milk fat (Palmquist and Mattos, 1978), resulting in increased milk fat production.
It has been suggested (Drackley, 1999; van Knegsel et al., 2005) that diets high in lipogenic nutrient content increase the severity and risk of ketosis and fatty liver by increasing the C2:C3 nutrient ratio. In dairy cows, lipogenic dietary ingredients such as dietary fat, or forages that stimulate the ruminal production of acetate and butyrate, are expected to increase the availability of C2 nutrients. In contrast, glucogenic dietary ingredients are either fermented in the rumen to produce high amounts of propionate or are digested in the small intestine and absorbed as glucose. Additionally, high-producing dairy cows experience NEB in early lactation. Negative energy status promotes mobilization of body fat (Tamminga et al., 1997) and increases the availability of C2 compounds.
The importance of an appropriate balance of lipogenic and glucogenic nutrients to optimize milk production efficiency and to decrease metabolic disorders has been addressed previously (Adler, 1970; Kronfeld, 1976; Drackley, 1999). However, studies that test this hypothesis by feeding isocaloric diets differing in lipogenic and glucogenic nutrients are scarce. The companion paper showed that dietary energy source alters energy partitioning in dairy cows in early lactation (van Knegsel et al., 2007). Although energy intake was not different between diets, cows fed a mainly lipogenic diet had higher milk fat and milk energy outputs and an increased body fat mobilization compared with cows fed a glucogenic diet. To obtain insight into the metabolic signals of this observed shift in energy partitioning, the objective of this study was to measure the effects of feeding a lipogenic or a glucogenic diet on liver TAG content, metabolites and metabolic hormones. Various studies have dealt with relationships among plasma NEFA, ketone bodies, glucose, insulin, or milk fat and milk protein and the energy balance (EB) in dairy cattle (De Vries and Veerkamp, 2000; Kokkonen et al., 2005). Such relationships were based on the estimated EB, calculated from energy intake and milk production. To our knowledge, the measured EB has not been used in testing associations with metabolite and metabolic hormone concentrations in dairy cows in early lactation. Therefore, the second objective of this study was to relate blood metabolites, metabolic hormones, and milk characteristics to the measured EB in early lactation dairy cattle.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Feeding
The complete ingredient and chemical composition of both diets are described in the companion paper (van Knegsel et al., 2007). In summary, diets were fed as TMR and formulated to be isocaloric (NE basis) and equal in intestinal digestible protein and degraded protein balance (Dutch DVE/OEB system; Tamminga et al., 1994). Postpartum, the concentrate supply increased stepwise by 0.5 kg/d until concentrate intake reached 10.0 kg/d with a concentrate to forage ratio of 40:60 (DM basis). Forage consisted of grass silage, corn silage, and chopped alfalfa hay in a ratio of 45:45:10 (DM basis). Diets were fed twice daily in equal proportions before milking.
Sampling Procedures
Liver biopsies were taken in wk –2, 2, and 4 relative to calving. Before the biopsy, the biopsy site was clipped and disinfected. A stab incision was made at the location of the greater trochanter in the 11th intercostal space on the right side of the cow. The biopsy was obtained under local anesthesia (7 mL of lidocaine-HCl 2% with adrenaline, Alfasan Nederland B.V., Woerden, the Netherlands) with a 17G x 200 mm biopsy needle. Approximately 300 mg of wet weight of liver tissue was harvested by moving the biopsy needle several times in the direction of the contralateral ulna. Tissue was kept for up to 24 h on ice in a 0.9% NaCl solution. Subsequently, connective tissue was removed, and the sample was weighed and stored at –20°C until analysis.
Samples of jugular blood were obtained weekly from wk –2 to 9 pp at 3 h after the a.m. feeding and immediately before liver biopsy if both were on the same day. Blood was collected in evacuated tubes (Vacuette, Greiner BioOne, Kremsmünster, Austria) containing either NaF for glucose determination, Li-heparin for NEFA, BHBA, cholesterol, urea, triiodothyronine (T3), and thyroxin (T4) determination, or EDTA for IGF-I and insulin determination. Plasma was obtained by centrifugation, aliquoted, and frozen at –20°C until analysis.
Analytical Procedures
Blood and liver samples were analyzed in a quality controlled veterinary laboratory (Stichting Kwaliteits-bewaking Medische Laboratoriumtechniek, Nijmegen, the Netherlands). Analyses for glucose, NEFA, BHBA, and cholesterol were performed using commercially available kits (glucose: reagent 443355 for Synchron CX7 analyzer, Beckman Instruments B.V., Mijdrecht, the Netherlands, Rukkwamsuk et al., 1999; NEFA: FA 115 kit, Randox Laboratories Ltd., Crumlin, UK, Rukkwamsuk et al., 1999; BHBA: Ranbut kit, Randox, Rukkwamsuk et al., 1998; cholesterol: cholesterol reagens for Synchron CX5, Beckman Instruments B.V.; Bouchard et al., 2002). Insulin concentration was determined using an RIA kit (Coat-a-Count Insulin, Diagnostic Products Corporation, Los Angeles, CA; Rukkwamsuk et al., 1999). Triiodothyronine, T4, and IGF-I were analyzed as described by Gerrits et al. (1998). Liver tissue was handled as described by Van den Top et al. (1995) and analyzed on a Synchron CX5 analyzer with a TAG reagent (Beckman Instruments B.V.). The accuracy of each assay was monitored with the use of a commercial reference serum sample (Bovine precision serum, Randox) and the outcome deviated <5% from the target values.
Statistical Analyses
Three cows were excluded from the experiment because of left displaced abomasums: in group 2, 1 cow fed the lipogenic diet; and in group 3, 2 cows fed the glucogenic diet. Therefore, values are based on 13 cows (glucogenic diet: n = 6; lipogenic diet: n = 7) for analysis of metabolites and metabolic hormones (model 1) or 6 climate respiration chambers (n = 3 per dietary treatment per lactation week) for analysis of relationships between ER and metabolites, metabolic hormones, milk fat, and fat to protein ratio (model 2). Although multiple measurements per animal cannot be regarded as independent units of observation, repeated-measures ANOVA (PROC MIXED of SAS, version 9.1; Littell et al., 2006) was performed. Cow (model 1) or chamber (model 2) was included as the repeated subject. First, metabolites and metabolic hormones were analyzed with diet (glucogenic or lipogenic), week (–2 to 9 pp), and their interaction included in the model as fixed effects (both as class variables; model 1a). In case of lack of significance (P > 0.05), the diet x week interaction was excluded from the model. Additionally, the same analysis with model 1 was performed for the prepartum and postpartum data separately. Second, to quantify metabolic relationships, plasma BHBA, insulin, liver TAG content, and milk fat were analyzed with NEFA and glucose included as fixed effects in the repeated measurements model 1 (model 1b) to obtain regression coefficients (ß). Diet was not significant (P > 0.05) and therefore not included as a covariable. Third, to test possible indicators for ER, energy retention as body fat (ERf) and energy retention as body protein (ERp) were analyzed with either milk fat, fat to protein ratio, plasma glucose, NEFA, BHBA, insulin, IGF-I, T3, T4, or liver TAG content included as fixed effects in a repeated measurements model (model 2) to obtain regression coefficients (ß). To avoid collinearity, week was not included in the model. Concerning the 3 mentioned analyses, a first-order autoregressive covariance structure [AR(1)] was the best fit and was used to account for within-cow (model 1a and 1b) or within-chamber variation (model 2). The only exception was liver TAG in which a compound symmetry covariance structure was best. The null model likelihood ratio test (PROC MIXED of SAS, version 9.1; Littell et al., 2006) confirmed that modeling the extra covariance was significant over the null model (P < 0.001). Model assumptions were evaluated by examining the distribution of residuals. Transformation (natural logarithm) of plasma insulin concentration was necessary to obtain normal distribution.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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), and milk protein (%) did not differ between diets. Milk fat (%) and daily milk fat yield were higher (P < 0.05) in cows fed the lipogenic diet compared with cows fed the glucogenic diet. Energy retention was numerically lower (P = 0.12) for cows fed the lipogenic diet compared with cows fed the glucogenic diet. Moreover, ERp did not differ between diets, but ERf tended to be lower (P = 0.08) for cows fed the lipogenic diet compared with cows fed the glucogenic diet.
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). Plasma BHBA, cholesterol, glucose, and IGF-I concentrations were different (P < 0.01) over time (Figure 2). Plasma NEFA concentration tended (P = 0.08) to be lower for cows fed the glucogenic diet compared with cows fed the lipogenic diet. A diet x week interaction (P < 0.01) was detected for plasma NEFA and cholesterol. Plasma NEFA concentration increased earlier (wk 0) and decreased later for cows fed the lipogenic diet compared with cows fed the glucogenic diet. Plasma insulin concentration tended (P = 0.11) to be higher for cows fed the glucogenic diet compared with cows fed the lipogenic diet. Postpartum, the plasma insulin concentration was significantly higher (P < 0.05) for cows fed the glucogenic diet. Plasma urea was lower (P < 0.01) for cows fed the glucogenic diet compared with cows fed the lipogenic diet. Liver TAG content increased from wk 2 prepartum to wk 2 pp, but the increase was similar for both diets (Figure 2). From wk 2 to 4 pp, liver TAG content decreased again. In wk 4 liver TAG content was numerically lower (P > 0.05) for cows fed the glucogenic diet.
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). Plasma glucose was negatively related to plasma BHBA, liver TAG (P < 0.01), and milk fat (P < 0.05). There was a positive relationship (P < 0.01) between plasma glucose and insulin concentration.
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). There was a tendency (P = 0.10) for a positive relationship between ER and plasma insulin. No significant relationships (P > 0.05) existed between ER and the milk fat to protein ratio, liver TAG content, plasma glucose, BHBA, IGF-I, T3, or T4; ERf and ERp showed a similar relationship as with overall ER.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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It was shown (van Knegsel et al., 2007) that feeding early lactation dairy cows a diet high in lipogenic nutrients resulted in an increase (P < 0.05) in milk fat yield. This increase was mainly caused by an increase in daily secretion of C16:0, C18:0, and C18:1 fatty acids. Dietary fat as well as mobilized body fat were higher for cows fed the lipogenic diet; both fat sources likely contributed to this increase in long-chain fatty acids. Furthermore, the contrast in total fat available from both mobilized body fat reserves and dietary fat for maintenance and milk production increased during wk 2 to 4 pp. Based on observed dietary fat intake and mobilized body fat and assuming a fat digestibility of 0.86 (NRC, 2001), we estimated that in wk 2 pp, cows fed the glucogenic diet had 1.66 kg of fat available per day for maintenance and milk production. Cows fed the lipogenic diet had 2.04 kg of fat available per day in wk 2 pp. From wk 2 to 4 pp, cows fed the glucogenic diet had a 42% decrease in fat availability to 0.96 kg/d in wk 4. However, fat availability for cows fed the lipogenic diet decreased much less in this period (13% to 1.79 kg/d in wk 4). Thus, differences in fat availability could explain the dietary difference in plasma cholesterol concentration, a tendency for a difference between diets in plasma NEFA concentration, and possibly the numerical difference between diets in liver TAG in wk 4. However, liver TAG content was not statistically different between diets. The rate of decrease in fat availability was equal for both groups after wk 4. In addition, body fat mobilization was negative in wk 9 for cows fed the glucogenic diet and the BHBA concentration was lower (P < 0.05) compared with cows fed the lipogenic diet.
The difference in mobilized body fat and EB between diets, as reported in the companion article (van Knegsel et al., 2007), may be explained by effects of dietary energy source on plasma insulin concentration. In this study, a trend was found for a higher plasma insulin concentration in cows fed the glucogenic diet compared with cows fed the lipogenic diet. The difference between diets in plasma insulin concentration was significant when only the postpartum data were taken into account. Dietary effects on plasma insulin concentration were more pronounced postpartum, which can be explained by an increase in contribution of the experimental concentrates to the TMR postpartum. Insulin is known as a key regulator of nutrient repartitioning from body tissue to milk in the transition period from pregnancy to lactation (Laarveld et al., 1981). Low plasma insulin concentrations reduce glucose uptake by muscle and adipose tissue and facilitate increased uptake of glucose by the mammary gland, which is not insulin-responsive (Bauman and Elliot, 1983). A hyper-insulinemic-euglemic clamp in dairy cows in early lactation was shown to decrease milk yield and positively affect the EB (Butler et al., 2003). In the current study, the negative relationship (P < 0.05) between plasma NEFA and insulin and a tendency for a negative relationship between ERf and insulin confirm the importance of insulin status in relationship to the EB in dairy cows in early lactation. In addition, insulin has been suggested to restore hepatic growth hormone responsiveness in dairy cows in NEB (Butler et al., 2003). Although no diet effect or increase with week pp on plasma IGF-I concentration was found, it can be speculated that the diet effect on insulin concentration or the increase in insulin concentration with week pp for cows fed the glucogenic diet was not enough to restore growth hormone responsiveness during the first 9 wk of lactation.
Considering the lipogenic effect of insulin, the low insulin concentration in cows fed the lipogenic diet corresponds with a tendency for higher plasma NEFA concentration. An increase in NEFA concentrations for cows fed the lipogenic diet compared with the glucogenic diet corresponds with other studies that increased the dietary fat content and also found an increase in plasma NEFA concentration (e.g., Grum et al., 1996; Salado et al., 2004). However, these studies did not feed isocaloric diets, but increased the dietary energy content with the fat addition. Other studies released a decrease in DMI with dietary fat addition (Simas et al., 1995; Beam and Butler, 1998) without compromising milk production, which resulted in more BW loss. In the current study, the higher plasma NEFA concentration in the lipogenic diet group likely originated from the mobilized body fat reserves, as indicated by the relationship between ERf and plasma NEFA concentration.
Nonesterified fatty acids, originating from body fat stores, are mobilized to compensate for NEB and to be oxidized in the Krebs cycle and respiratory chain reaction to make energy available to the body (Webster, 1993). In the Krebs cycle, oxaloacetate, a C3 nutrient, plays a crucial role. However, during periods of relative excess of NEFA (e.g., during NEB), NEFA can follow several alternative pathways. First, dairy cows in early lactation exhibit incomplete oxidation of fatty acids as indicated by high concentrations of ketone bodies (Guretzky et al., 2006). The second alternative pathway for mobilized NEFA is incorporation of NEFA into milk fat, directly or indirectly via esterification to very low density lipoproteins (VLDL), which causes an increase in long-chain fatty acids in milk (Belyea and Adams, 1990). The third alternative for NEFA is esterification to TAG in the liver, possibly causing a fatty liver (Bobe et al., 2004). Fatty liver occurs when the rate of fatty acid esterification exceeds the rate of TAG disappearance via export from the liver as a component of VLDL. It has been suggested that hepatic secretion of TAG is positively correlated with the lipogenic capacity of the liver (Pullen et al., 1990). The predominant site for lipogenesis in ruminants is adipose tissue, which might explain the low rate of hepatic secretion of TAG in ruminants (Emery et al., 1992) compared with rat (Kleppe et al., 1988), chicken, and fish (Pullen et al., 1990). Consequently, this makes ruminants more susceptible to TAG accumulation in the liver during a period of NEB, eventually causing a fatty liver.
Plasma BHBA was high (>2.0 mmol/L) for cows in both dietary treatment groups from wk 1 to 8 pp (Figure 2
). This suggests that this alternative pathway is used irrespective of dietary treatment in early lactation. In support, the nonsignificant regression coefficient of NEFA on BHBA suggests that NEFA concentrations do not determine the extent of incomplete oxidation and production of BHBA in periparturient dairy cows. In contrast, the regression coefficient of plasma glucose on BHBA was significant. This suggests that plasma glucose had a negative relationship with the plasma BHBA concentration. It has been reported that feeding cows glucogenic nutrients in early lactation decreases plasma BHBA and acetate concentrations (Pickett et al., 2003). Although we discussed the existence of a difference in fat availability from diet and mobilized body reserves between diets in the current study, the increase in lipogenic nutrients with the lipogenic diet did not result in an increase in plasma BHBA concentration as suggested earlier (Drackley, 1999; van Knegsel et al., 2005).
The second alternate pathway for NEFA seems to be used throughout the experimental period. A positive relationship was detected between plasma NEFA and milk fat yield. Body fat mobilization and NEFA were both higher in cows fed the lipogenic diet and simultaneously milk fat yield was increased by 220 g/cow per day. Glucogenic nutrients lower milk fat percentage and often lower daily milk fat yield (e.g., Pickett et al., 2003).
With regard to the third alternative pathway, the current results do not support our hypothesis (van Knegsel et al., 2005) that a diet high in glucogenic nutrients decreases hepatic TAG accumulation. This might be caused by the fact that the dietary treatment was established by feeding concentrates differing in lipogenic and glucogenic nutrient content, whereas the contribution of concentrates to the diet increased from parturition until 3 wk pp. In wk 2 pp, liver TAG content was high, but not different between diets. In wk 4 pp liver TAG was numerically lower in cows fed the glucogenic diet, but did not differ significantly from cows fed the lipogenic diet. This observation corresponds with the relative reduction in fat load in cows fed the glucogenic diet compared with cows fed the lipogenic diet as discussed above.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Received for publication July 28, 2006. Accepted for publication November 7, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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