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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
* Department of Animal Sciences, Purdue University, West Lafayette, IN 47907; and
and
Department of Dairy and Animal Science, The Pennsylvania State University, University Park 16802
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Key Words: gene expression • monensin • transition dairy cow
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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), modifying the end products of fermentation with monensin feeding (Duffield et al., 2002), and providing specific glucogenic precursors have been used to overcome the shortfall in endogenous glucose production during the transition period (Studer et al., 1993).
Hepatic glucose output is directly proportional to propionate supply (Lomax et al., 1979; Baird et al., 1980; Wieghart et al., 1986) and propionate accounts for 50 to 60% of total glucose flux in dairy cows (Reynolds et al., 1988). Monensin increases propionate produced in the rumen (Prange et al., 1978), which may be part of its action to reduce the incidence of clinical ketosis (Duffield et al., 2002, 2003), a metabolic disorder closely linked to glucose insufficiency in dairy cows.
Pyruvate carboxylase (PC) and cytosolic phospho-enolpyruvate carboxykinase (PEPCK-C) are 2 key gluconeogenic enzymes in ruminants (Greenfield et al., 2000; Agca et al., 2002). Expression of PC mRNA is elevated at calving (Greenfield et al., 2000; Hartwell et al., 2001), but changes in PEPCK mRNA are delayed until after calving (Greenfield et al., 2000) when DMI has increased. The expression of PEPCK-C mRNA is sensitive to VFA concentrations in cultured rat liver cells (Massillon et al., 2003). Therefore, we hypothesized that altering the supply of VFA to favor more ruminal propionate production would induce PEPCK-C mRNA at calving.
The objective of this study was to determine the effect of feeding diets containing NFFS, monensin, or their combination on expression of PC and PEPCK-C mRNA in liver of transition dairy cows.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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). Cows and Dietary Treatments
Cows were ranked by previous 305-d, mature, equivalent milk yield, calving date, and BCS and were assigned randomly to treatment within each group of 4 cows. Treatments were assigned at dry-off but were applied through a nesting of treatment factors in time. At dry-off, cows (n = 20 per group) were separated into 2 prepartum, basal ration groups and fed a conventional (CONV) diet or a NFFS diet. Cows were managed in 2 groups, and diets were fed twice daily as a total mixed ration to ensure ad libitum intake.
Approximately 28 d before expected calving, cows were moved to individual tie stalls and continued to receive their previous basal diet (CONV or NFFS) treatments. Beginning at –28 d relative to calving, one-half of the cows within the CONV and NFFS groups were fed 0 (–) or 300 (+) mg of monensin/d. A total of 34 of the 40 cows chosen for the current study successfully completed the experiment and were distributed as follows: CONV–, 10 cows; CONV+, 8 cows; NFFS, 9 cows; and NFFS+, 7 cows. Cows were removed from the study for the following reasons: Four cows were removed because they had twins and calved early and were not on treatments for a long enough period before parturition. Another cow was culled early in the dry period without adequate time on treatment to permit sampling, and another cow was culled after the first week of lactation with complications associated with a retained placenta and severe infectious metritis.
Ingredients and chemical composition of the CONV and NFFS diets are shown in Table 1. The NFFS diet was formulated by replacing 30% of the forage component of the CONV diet with cottonseed hulls and soy-hulls, so that the NDF content of each diet and the proportion of grain and protein supplement in each diet were similar. Both diets were formulated to meet the requirements for dry and lactating cows (NRC, 2001). Monensin (monensin sodium, 300 mg; Elanco Animal Health, Greenfield, IN) was incorporated into a pellet containing 170.5 g of distiller’s grain as a carrier and fed as a top dress during the morning feeding. To ensure adaptation to the ionophore feeding, cows were fed 150 mg of monensin/d during the first 3 d. At calving, all cows were placed on the same diet, but lacking monensin and NFFS (Table 1). For the first 7 d after calving, 1.5 kg grass hay was offered daily along with the total mixed ration. All cows were fed to ensure ad libitum intake throughout the study.
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Dry matter intake was measured daily from 4 wk before calving through 8 wk after calving. Feed samples were collected weekly, and a monthly composite sample was made for analysis by wet chemistry methods at a commercial laboratory (Pennsylvania DHIA, University Park, PA). Cows were milked twice daily, and individual milk yields were determined electronically at each milking (Afifarm version 3.01A, S.A.E. Afikim, Germania Dairy Automation, Waunakee, WI).
Blood samples were collected by puncture of the median caudal vein or artery and collected in evacuated tubes at approximately 3 h postfeeding. Weekly blood samples were collected from 4 wk before expected calving through 1 wk before expected calving. From –7 d before expected calving through 3 wk postpartum, blood samples were taken 3 times weekly. During wk 4 and 5 postpartum, blood sampling was once per week. Blood samples for plasma glucose analysis were collected into Vacutainers (Beckton Dickinson, Rutherford, NJ) containing potassium oxalate and 4% sodium fluoride. Blood samples for plasma ß-hydroxybutyrate (BHBA) and NEFA were collected into Vacutainers containing sodium heparin. Blood samples were placed on ice immediately after collection and, within 1 h, were centrifuged at 3,300 x g for 15 min at 4°C to separate plasma. Samples were then analyzed for glucose (Stanbio Enzymatic Glucose Kit 1075, Stanbio Laboratory Inc., Boerne, TX), NEFA (Johnson and Peters, 1993; Wako NEFA C Kit, Biochemical Diagnostics Inc., Edgewood, NY), and BHBA (Standbio LiquiColor Kit 2440, Stanbio Laboratory Inc.) concentrations. Concentrations of BHBA were determined only for samples collected during the postpartum period.
Liver biopsy samples were obtained by ultrasound-guided, percutaneous needle biopsy on –28 and –14 d relative to expected calving and on d 1, 14, and 28 relative to calving. Liver samples were rinsed in saline, placed in tubes containing 10 mL of guanidinium thiocyanate solution [4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.4), 0.5% Sarcosyl, and 0.1 M ß-mercaptoethanol], snap frozen in liquid nitrogen, stored at –80°C, and shipped on dry ice to Purdue University for RNA analysis.
cDNA Probes, RNA Extraction, and Northern Analysis
Plasmids bPC1000, containing a 1,051-bp fragment of bovine PC cDNA, and bPEPCK-700, containing a 1,150-bp fragment of bovine PEPCK-C cDNA, were used in Northern blot analysis (Agca et al., 2002). Insert DNA was removed from the plasmids by restriction enzyme digestion. Insert DNA was separated by electrophoresis through 1% agarose and purified using the Wizard DNA Purification system (Promega, Madison, WI). Gamma-32P[dCTP] and a Ready-to-go DNA labeling kit (Pharmacia, Piscataway, NY) were used to generate the radiolabeled cDNA probes. The specific activity of cDNA probes was approximately 1.68 x 107 Bq/ µg of DNA.
Total RNA was extracted from liver biopsy samples, as previously described (Chomczynski and Sacchi, 1987). A 20-µg aliquot was separated through a 1% agarose gel and transferred by capillary action over a 20-h period to a Genescreen membrane (NEN Life Science Products, Boston, MA). The RNA was cross-linked to the membrane using UV light, and the membrane was baked for 2 h at 80°C to volatilize any residual formaldehyde.
Membranes were prehybridized for 15 h at 42°C, as described previously (Greenfield et al., 2000), and were hybridized with the 32P-labeled cDNA probes for 18 h at 42°C. Membranes were then washed twice with 2x SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) at room temperature for 5 min, twice with 2x SSC, 1% SDS at 65°C for 30 min, and twice in 0.1x SSC (0.015 M NaCl, 0.0015 M sodium citrate, pH 7.0) at room temperature for 30 min. The washed membrane was wrapped in plastic and exposed to Kodak X-Omat AR film (Eastman Kodak Co., Rochester, NY) to visualize the mRNA transcripts. Expression was quantified using Kodak Digital Science 1D Image Analysis software (Eastman Kodak Co.). Pooled samples of bovine liver mRNA were placed on the outside lanes of each gel to account for the variation in transfer of RNA, hybridization, and washing. Variation in sampling loading was adjusted using 18S mRNA abundance.
Statistical Analysis
The data were analyzed using the MIXED procedure (SAS Inst. Inc., Cary, NC). The model accounted for the main effects of monensin (0 or 300 mg/d), basal prepartum diet (CONV or NFFS), day relative to calving (DRTC), and the 2- and 3-way interactions among monensin, basal prepartum diet, and DRTC. First-order antedependence structure gave the lowest Bayesian information criterion coefficient and was used to evaluate all of the variables measured, except for prepartum DMI and milk production. Prepartum DMI was evaluated using the compound symmetry structure and assumed equal variances. The first order, autoregressive structure was used to evaluate milk production. Means were considered different if P < 0.05 and tended to differ if 0.05 
P 0.10. When the 2- or 3- way interactions were significant, the comparisons of individual interaction means were performed using the PDIFF statement in PROC MIXED. Dry matter intake, plasma metabolites, and mRNA were also analyzed separately for pre-partum (–28 through +1 DRTC) and postpartum (+1 through +28 DRTC) intervals. The effects of monensin on PEPCK mRNA were examined as a fraction of the expression for each cow on –28 DRTC; the resulting data were analyzed using PROC MIXED and the effects indicated above. Values reported are least squares means and SE.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Although all cows experienced a reduction (P < 0.05) in DMI as parturition approached, feeding NFFS resulted in greater (P < 0.05) DMI during the prepartum period (Figure 1; Table 2). These observations are supported by an effect (P < 0.05) of DRTC, an effect (P < 0.05) of prepartum diet x DRTC during the prepartum period, and lack (P > 0.05) of effect for monensin x DRTC for the same interval. Postpartum DMI was not altered by prepartum monensin or prepartum NFFS feeding. As expected, postpartum intake increased (P < 0.05) with DRTC. There were no monensin x prepartum diet or monensin x prepartum diet x DRTC effects on prepartum or postpartum feed intake. Milk production was not affected by prepartum diet or monensin (Table 2), but there was a DRTC x prepartum diet interaction (P < 0.05) due to the more rapid increase in milk production for cows fed the CONV diet.
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Cows fed the NFFS diet had greater (P < 0.05) prepartum plasma glucose concentrations compared with cows fed the CONV diet (69.3 vs. 64.7 ± 1.0 mg/dL; Table 3). There was no effect of monensin on prepartum plasma glucose. There was a DRTC x basal diet effect (P < 0.05) on plasma glucose concentrations due to a greater increase (P < 0.05) in plasma glucose for cows fed NFFS during –3 and –1 DRTC compared with cows fed CONV diets (Figure 2). Feeding NFFS reduced (P < 0.05) prepartum NEFA concentrations compared with the CONV diet (241 vs. 166 ± 24; CONV vs. NFFS respectively). There was a DRTC x basal prepartum diet effect (P < 0.05) for plasma NEFA due to a greater increase in NEFA on –5 and –3 DRTC for cows fed the CONV diet compared with NFFS prepartum diet (Table 3, Figure 3).
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). Plasma glucose concentrations decreased (DRTC effect, P < 0.05) initially after calving and then increased slightly by d 35 of lactation. Concentrations of NEFA decreased similarly for all treatments through the first 35 d of lactation (DRTC effect, P < 0.05).
There was a tendency for an increase (P = 0.09) in postpartum plasma BHBA concentrations for cows fed the NFFS diet compared with cows fed the CONV diet (11.69 vs. 8.68 ± 1.26). There was no other main effect or any 2- or 3-way interaction effect (Figure 4) for plasma BHBA concentrations.
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There was no main effect of prepartum diet or monensin on PC mRNA expression during the prepartum period (Table 3). Expression of PC mRNA was 1.66, 2.03, 3.97, 3.67, and 2.79 ± 0.52 (arbitrary units) for –28, –14, 1, 14, and 28 DRTC respectively. There was a DRTC effect on PC mRNA characterized by greater (P < 0.05) expression on 1 d postpartum compared with –28 d prepartum or –14 d prepartum and a decrease (P < 0.05) by 28 DRTC. There was no NFFS x DRTC or monensin x DRTC effect on PC mRNA.
There was an increase (P < 0.05) in PEPCK-C mRNA from –28 to +1 DRTC for cows fed monensin. When the prepartum and postpartum data were considered together, there was a tendency (P = 0.07) for a monensin x DRTC effect. Abundance of PEPCK-C mRNA as a fraction of –28 DRTC was increased (P < 0.05) by monensin on –14 and 1 DRTC (Figure 5), but the effects of monensin to elevate liver PEPCK-C did not persist when monensin was removed from the diet during the postpartum period.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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). The lack of monensin effect prepartum is consistent with a number of studies when transition cow diets are supplemented with monensin for 3 wk before expected calving (Ramanzin et al., 1997; Green et al., 1999; Vallimont et al., 2001). Feeding NFFS, monensin, or their combination did not alter milk production for cows used in this analysis, which is consistent with data from the larger study (Pickett et al., 2003). These data are in agreement with studies that demonstrate a lack of change in milk production in response to prepartum monensin (Green et al., 1999; Vallimont et al., 2001). Additional studies show a lack of production response to NFFS during the postpartum period (Firkins and Eastridge, 1992; Sarwar et al., 1992). Animals fed the NFFS diet in the current study had 8% greater prepartum glucose concentrations and 30% less NEFA concentrations compared with cows fed the CONV diet. Greater plasma glucose concentrations that accompany greater intakes for cows fed NFFS may indicate a change in glucose entry, glucose clearance, or both or may be a consequence of increased rumen fermentation endproducts with increased intake. Plasma BHBA was similar for all treatments at calving. Taken together, these data indicate a more favorable energy balance for cows fed NFFS during the prepartum period.
The lack of effect of prepartum monensin feeding on plasma NEFA concentrations are consistent with data from the parent study (Pickett et al., 2003) and studies in which monensin was fed only during the prepartum interval (Vallimont et al., 2001). Monensin feeding during the last week of gestation and first 3 wk of lactation reduced postpartum plasma BHBA in a dose-responsive manner (Sauer et al., 1989). Monensin administered 3 wk before calving as a controlled release capsule, to deliver approximately 335 mg of monensin per day over 90-d period, effectively decreased postpartum NEFA and BHBA levels (Duffield et al., 2003). Together, these data indicate reductions in NEFA and BHBA during monensin feeding but a lack of residual effect of prepartum monensin on postpartum plasma NEFA and BHBA.
There was an overall increase in PC mRNA 1 d postpartum compared with –28 and –14 d prepartum. Greenfield et al. (2000) reported that PC mRNA abundance increased on the day of calving compared with 28 d prepartum and returned to precalving levels by 56 d in milk. In the current study, PC mRNA expression was elevated in a similar manner at calving but was not responsive to monensin or NFFS feeding. Although PC mRNA is responsive to gross changes in DMI in dairy cows (Velez and Donkin, 2004), expression of PC mRNA does not appear to respond to the diets examined in this study.
The expression of PEPCK-C mRNA was greater at +28 compared with –28 DRTC when data for all treatment groups were combined. The increase in PEPCK to +28 DRTC has been previously characterized for transition dairy cows (Greenfield et al., 2000; Hartwell et al., 2001), and the current data fit the general pattern of change in PEPCK mRNA with DRTC. Cows fed 300 mg/d of monensin during the prepartum period had greater PEPCK-C mRNA expression at –14 DRTC and at +1 DRTC than cows fed diets lacking monensin. There was no carryover effect of prepartum NFFS or monensin on PEPCK-C expression in liver samples collected on +14 and +28 DRTC.
Carbon from propionate, lactate, and amino acids is metabolized through PEPCK as part of gluconeogenesis. Activity of PEPCK-C is elevated in liver of lactating cows compared with gestating and nonpregnant cows (Mesbah and Baldwin, 1983), and the increased expression of PEPCK mRNA in transition cows is matched by increased activity of the enzyme (Agca et al., 2002). Propionate is a major substrate for gluconeogenesis in the ruminant animal (Lomax and Baird, 1983; Reynolds et al., 1988) and accounts for up to 50 to 60% of total net hepatic glucose release in dairy cattle (Reynolds et al., 1988, 2003). Recent information indicates that propionate and other VFA stimulate the expression of glucose-6-phosphatase and PEPCK mRNA in rat liver cells in culture (Massillon et al., 2003). Although it is well recognized that VFA modulate gene expression in intestinal cells (Blottiere, et al., 2003) to control epithelial cell proliferation (Archer et al., 1998; Siavoshian et al., 2000) and to mute activation of genes for proinflammatory factors (Inan et al., 2000; Segain et al., 2000), the role of propionate in controlling gluconeogenesis has not been explored extensively. The latter may be particularly important in ruminants due to the continual need for gluconeogensis and extensive use of propionate for glucose synthesis. The data presented here indicate that the addition of monensin to the prepartum diet resulted in greater PEPCK-C mRNA expression. Although an increase in ruminal production of propionate has been observed with ionophore feeding, the corresponding rates of ruminal VFA production were not measured in the current study. Therefore additional data is necessary to determine if the effect of monensin feeding on hepatic PEPCK-C expression is directly mediated by propionate supply.
Greater expression of PEPCK-C mRNA in lactating cows compared with nonlactating cows (Greenfield et al., 2000; Hartwell et al., 2001) coincides with a marked increase in net portal-drained visceral flux of propionate in the immediate postpartum interval (Reynolds et al., 2003). In light of the induction of PEPCK-C by propionate in rat hepatocytes, it is reasonable to conclude that a portion of the postpartum increase in PEPCK-C mRNA previously observed in lactating cows (Greenfield et al., 2000; Hartwell et al., 2001) is due to increased supply of propionate. Furthermore, because monensin feeding increases ruminal propionate production (Prange et al., 1978), the increase in PEPCK-C with monensin in prepartum dairy cows may be due to increased propionate flux from the rumen. Likewise, total VFA production and therefore propionate production is directly related to feed intake; however, the profile of VFA resulting from increased intake tends to be unchanged (Martin et al., 2001). Replacing forage with soyhulls in diets fed to dairy cattle usually increases the molar proportion of propionate in ruminal fluid, but changes in molar percentages of acetate and butyrate are infrequent or inconsistent (Ipharraguerre and Clark, 2003). In contrast, feeding monensin increases rumen propionate production at the expense of rumen acetate production and tends to increase butyrate production (Armentano and Young, 1983). Therefore, the induction of PEPCK-C expression with monensin feeding during the prepartum period in transition cows may be a reflection of the profile, rather than quantity, of VFA absorbed. Measures of ruminal VFA flux coupled with simultaneous measures of liver PEPCK-C in liver with monensin feeding are necessary to more adequately determine the relationship between ruminal propionate supply and PEPCK-C expression.
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IMPLICATIONS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Footnotes |
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2 Current address: 313 Kildee Hall, Iowa State University, Ames 50010.
3 Corresponding author: sdonkin{at}purdue.edu
Received for publication June 7, 2006. Accepted for publication September 11, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
CONV–, 
CONV+, 
NFFS–, and 
NFFS+. There was an effect of DRTC (P < 0.05), prepartum diet (P <0.05), and prepartum diet x DRTC (P< 0.05) for prepartum intake (–28 DRTC through calving), and a DRTC effect (P < 0.05) from calving through 56 d in milk.
