Responses of Dairy Cows During Early Lactation to Ruminal or Abomasal Administration of L-Carnitine1

Responses of Dairy Cows During Early lactation to Ruminal or Abomasal Administration of L-Carnltine1 D. W. LaCOUNT,2 J. K. DRACKLEY,3 and D. J. WEIGEL Department of Animal Sciences University of Illinois Urbana 61801 ABSTRACT

tended to increase when carnitine was administered, primarily because of greater retained N when carnitine was administered ruminally. Excretion of carnitine in milk and urine increased when carnitine was administered at either site. Carnitine supplementation increased concentrations of carnitine in plasma and liver and improved lipid digestibility. (Key words: carnitine, dairy cows, dietary fat)

Six multiparous Holstein cows were used in a replicated Latin square to investigate the effects of carnitine administration into the rumen or abomasum. Treatments were 1) control, 2) twice daily ruminal administration of carnitine, and 3) continuous abomasal infusion of carnitine. Cows not receiving abomasal carnitine were infused continuously with an equal volume of water. Carnitine dosage was constant for both treatments (226 mglkg of DMI; ca. 6 gld). The diet fed to all cows contained 3% added fat. Carnitine concentrations in plasma and liver increased when carnitine was administered into either the rumen or abomasum, indicating that both sites of administration were equally effective at increasing carnitine concentrations in tissue. Milk yield, milk composition, and DMI were unaffected by carnitine supplementation, except for increased SNF content. Apparent digestibilities of lipid, energy, and total fatty acids increased with carnitine administration at either site. The concentration of VFA in ruminal fluid tended to increase with ruminal carnitine, and the percentage of propionate increased when carnitine was administered at either site. Retained N

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INTRODUCTION

Received August 4, 1994. Accepted April 11, 1995. lSupported by Hatch funds appropriated to the llIinois Agricultural Experiment Station (Project 35-0352) and by a gift from Lonza Inc. (Fair Lawn, NJ). 2Supported by a Jonathan Baldwin Turner Graduate Fellowship from the College of Agriculture, University of Illinois. 3Address correspondence and reprint requests to James K. Drackley, Department of Animal Sciences, University of lllinois. 260 Animal Sciences Lab, 1207 West Gregory Drive, Urbana 61801. 1995 J Dairy Sci 78:1824-1836

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Abbreviation key: EE ether extract, FA fatty acid, ME metabolizable energy, SAM = S-adenosyl methionine.

High yielding dairy cows typically have difficulty consuming enough DM to meet their energy requirements; therefore, fat often is included in their diets. Yield responses to supplemental dietary fat have been inconsistent (7). Utilization of dietary fat by cows might be limited by an inadequate supply of precursors and metabolites needed for processes of fatty acid (FA) metabolism. Oxidation of long-chain FA by ruminants is low relative to that of nonruminants (28), which may result in suboptimal utilization of dietary fat by ruminants. One possibility is that oxidation of long-chain FA might be limited by insufficient amounts of carnitine available for the transport of longchain FA into the mitochondrial matrix. Addition of L-carnitine to in vitro incubations of liver slices from dairy cows increased oxidation of palmitate and decreased palmitate esterification (8); however, whether increasing carnitine availability to tissues would increase oxidation of long-chain FA in vivo is unknown. During early lactation dairy cows secrete as much as 500 nmo1 of carnitine/ml of milk (12). This loss may impose a substantial drain on

1824

1825

CARNITINE FOR DAIRY COWS

the methyl pool of high yielding dairy cows because de novo synthesis of camitine requires the transfer of three methyl groups from Sadenosyl Met (SAM) to protein-bound Lys residues found primarily in skeletal muscle proteins (2). Theoretically, supplementation of 6 g1d of camitine to lactating dairy cows could spare enough Met for the synthesis of 185 g1d of milk protein if exogenous camitine decreased endogenous synthesis of carnitine. The decreased milk protein content that is often associated with supplemental dietary fat for dairy cows has been alleviated by provision of protected Met and Lys in the diet (6). If Met is the first-limiting amino acid for milk protein synthesis, supplemental carnitine may spare enough Met to alleviate the milk protein decrease typically observed when fat is fed. Little information is available about the effects of administration of camitine to lactating dairy cows. Subcutaneous injection of carnitine (1.0 mg of carnitine/l00 kg of BW) into Holstein cows did not affect milk fat percentage. plasma glucose concentration, or yields of milk fat and SCM; however, the percentage of SNF in milk was lower for cows injected with camitine than for untreated cows (38). Also, intravenous infusion of carnitine (20 g1d) did not affect OMI, nutrient digestibilities, or milk yield and composition (14); however, the concentration of camitine in milk was greater when cows were infused intravenously with camitine than with saline. Supplementation of camitine in the diets of other species has improved weight gain (25, 26) and decreased carcass lipid content (26, 42). Our primary objective was to compare the effects of camitine administration into either the rumen or abomasum on concentrations of camitine in plasma, milk, muscle, and liver to determine if site of carnitine administration affected carnitine status of cows. Milk yield and composition were measured to assess the effect of camitine supplementation on milk protein synthesis because of the potentially decreased demand for Met for de novo camitine synthesis. Finally. the effects of dietary carnitine on N and energy balances were monitored because camitine is an essential compound for mitochondrial oxidation of longchain FA, which could alter utilization of energy and N.

MATERIALS AND METHODS Cows and

Tr.atmen~

Six multiparous Holstein cows. fitted with ruminal cannulas. were used in a replicated 3 x 3 Latin square design with 21-d periods. Cows were blocked according to DIM (X = 37 and 65 DIM for squares 1 and 2). Cows were housed in individual stalls, milked twice daily (0530 and 1600 h), and fed a total mixed diet (Table 1) for ad libitum intake at 0630 and 1700 h daily. Treatments were 1) control (continuous abomasal infusion of 4 L of tap water and an empty gelatin capsule placed into the rumen twice daily). 2) ruminal carnitine (continuous abomasal infusion of 4 L of water and a gelatin capsule containing carnitine placed into the rumen twice daily). and 3) abomasal

TABLE I. Ingredient and nutrient composition of total mixed diet. Composition

Content (% of DM)

Ingredient Alfalfa haylage Corn silage Soyhulls. pelleted Soybean meal Corn. ground shelled Fat l Sodium bicarbonate Magnesium oxide Dicalcium phosphate Limestone Sodium chloride Mineral and vitamin mixture2 Nutrient OM, % OM CP NDF ADF Cellulose Hemicellulose

Ash Ether extract Fatty acids Gross energy. Mca1/kg

30.00 20.00 4.00 14.00 25.90 3.00 .75 .15 1.00 .85 .20 .15 66.2 91.3 19.3 30.2 165 14.7 13.6 8.7 5.7 4.6 4.52

lEnergy Booster lOOT>< (Mille Specialties Co., Dundee, IL).

2Contains 10.0% S. 7.5% K, 5.0% Mg. 3.0% Zn. 3.0% Mn, 2.0% Fe, .5% Cu, .025% I, .015% Se•.004% Co. 2200 IV of vitamin Alg. 662 IV of vitamin Dig. and 8 IV of vitamin Fig. Journal of Dairy Science Vol. 78, No.8, 1995

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laCOUNT ET AL.

carnitine (continuous abomasal infusion of carnitine in 4 L of water and an empty gelatin capsule placed into the rumen twice daily). Pure (>99%) carnitine [L-(3-carboxy-2hydroxypropyl)trimethyl-ammonium hydroxide, inner salt] was provided by Lonza Inc. (Fair Lawn, NJ). Preliminary experiments demonstrated that OMI and milk yield were not affected adversely by abomasal infusion of as much as 400 mg of carnitinelkg of OM! (data not shown). The amount of carnitine supplementation was set at 250 mglkg of OM! on the basis of previous work with growing beef cattle (S. A. Blum, 1992, personal communication). The amount of carnitine administered to each cow was calculated from the mean OM! for that cow during the 5 d before the experiment began and was held constant during both ruminal administration and abomasal infusion. Cows were in early lactation when the dosage of carnitine was determined; therefore, OMI increased as the experiment proceeded, and the actual dos~e of carnitine averaged 226 mglkg of OM! (X ± SO, 5.96 ± 1.14 gld). An infusion tube was placed into the abomasum through the ruminal cannula (37). The abomasal infusate of carnitine was prepared daily by dissolving carnitine in 4 L of tap water. Cows not receiving abomasal carnitine were infused abomasally with water (4 U d). The infusate was pumped into the abomasum over 20 to 22 hid (Rabbitt™ pumps; Rainin Instrument Co., Inc., Woburn, MA). Cows given carnitine in the rumen received one-half of their daily dose at each feeding. Milk Yield and Composition

Milk yield was measured and recorded at each milking. Milk was sampled at each milking during d 16 to 20, preserved with 2-bromo2-nitropropane-l,3-diol, and composited into a single sample in proportion to milk yield. Composite milk samples were analyzed for SNF (17) and contents of CP, true protein, and fat by midinfrared spectrophotometric (39) analysis (New York OIDA Laboratory, Ithaca, NY). Fat was measured using the A filter (39). Noncasein N was determined by Kjeldahl analysis of the filtrate after precipitation with 10% acetic acid and 1 N sodium acetate (20). Casein N was calculated as the difference between Journal of Dairy Science Vol. 78, No.8. 1995

total N and noncasein N, NPN was calculated as the difference between total N and true protein N, and whey protein N was calculated as the difference between true protein N and casein N. A portion of the composite milk sample was placed into a test tube and allowed to stand overnight in a cold room at 4·C. The cream layer was transferred into a clean test tube, and FA were methylated and quantified by GLC using the procedures of Sukhija and Palmquist (40), except that an external standard rather than an internal standard was used to quantify the proportions and yields of FA in milk (32). Glycerol was calculated as described by Schauff et al. (32). Oietary ingredients and orts were sampled during the last 5 d of each period. The individual samples were dried at 55'C, ground in a Wiley mill (l-mm screen; Arthur H. Thomas, Philadelphia, PA), and composited on a proportional basis according to amounts of feed offered and refused each day. The composite samples were analyzed for contents of OM, OM (550·C for 12 h), CP (1), ether extract [EE; (1)], energy (1261 Isoperibol Calorimeter; Parr Instrument Co., Moline, IL), FA (40), NDF [using a-amylase; (41)], ADF (41), hemicellulose (NDF minus ADF), and soluble residue [neutral detergent solubles minus CP and EE (24)]. As analyzed, the diet provided 19.3% CP, 16.5% ADF, 30.2% NDF, and 4.6% total FA (Table 1). Rumlnal Fermentation

On d 18 of each period, ruminal fluid was collected from each cow via the ruminal cannula by a suction pump at hourly intervals for 8 h after the a.m. feeding. Ruminal fluid pH was determined immediately by glass electrode. Ruminal fluid then was acidified to pH 2 with 50% sulfuric acid and centrifuged at 27,000 x g for 15 min; the supernatant was frozen. Hourly samples were composited on an equal volume basis and analyzed for concentrations of VFA and ammonia N. The VFA in the supernatant were determined (22) using an automated gas chromatograph (Varian model 4600; Varian, Palo Alto, CA). Concentration of ammonia N was determined according to procedures of Chaney and Marbach (5).

CARNITINE FOR DAIRY COWS

Total Tract Apparent Nutrient Digestlblllties and Utlllzatlon of Energy and N

Apparent digestibilities of nutrients in the total tract and utilization of energy and N were determined by total collection of feces and urine during d 16 through 20 of each period. Urine was collected into polyethylene containers via external urine cups (13) that were attached on d 15 of each period. To minimize loss of volatile compounds, 220 ml of concentrated HCI were added to each container. Urine was weighed, and 5% of the daily urine output was stored at 4·C until an aliquot of the composite sample was frozen at -20·e. Total fecal output was weighed daily, and 1% of the daily fecal weight was frozen at -20'C. The fecal samples were thawed and composited by cow within period, dried (55·C), and ground through a I-mm screen in a Wiley mill. Feces were analyzed for contents of DM, OM, CP, EE, energy, FA, NDF, ADF, cellulose, hemicellulose, and soluble residue as described for feed samples. Results from these analyses were used to calculate apparent digestibility coefficients. The amounts of digested soluble residue, hemicellulose, and cellulose were used to calculate methane production using the multiple regression equation derived by Moe and Tyrrell (24). Urine was analyzed for contents of energy (1261 Isoperibol Calorimeter; Parr Instrument Co.) and N (1) to determine utilization of energy and N. Apparent metabolizable energy (ME) intake was estimated by subtraction of gross energy lost in feces, urine, and methane from gross energy in feed consumed. Retained N was calculated by subtraction of N in feces, urine, and milk from N intake. Productive N was calculated as the sum of N retained in the body and N secreted in milk. Sampling and Analyses of Blood and Tissues

Blood was sampled from the coccygeal vein or artery at 0, 3, and 6 h after the a.m. feeding on d 19 of each period. Plasma was collected after the blood was centrifuged at 1500 x g for 5 min and was frozen at -20'C. Plasma was analyzed for concentrations of NEFA (9), urea N (5), glucose (kit number 315; Sigma Chemi-

1827

cal Co., 51. Louis. MO). and BHBA (4). Additional blood (20 ml) was obtained 6 h after feeding for collection of triglyceride-rich lipoproteins by ultracentrifugation (18). The concentration of triglyceride in the triglyceride-rich lipoprotein fraction was determined enzymatically (kit number 339: Sigma Chemical Co.). On d 21 of each period, a puncture biopsy of liver was obtained from each cow under local anesthesia (9). The liver was frozen immediately in liquid N2 and stored at -{)O'C until analysis for concentrations of total lipid (9) and triglyceride (15). A biopsy of the middle gluteus muscle was performed at the same time as the liver biopsy. Anesthetic (lidocaine) was injected posterior to the tuber coxae, and a 2-cm incision was made. A biopsy instrument (invertebral disc rongeur) was inserted through the incision and into the muscle. A sample of muscle (50 mg) was excised. blotted, frozen immediately in liquid N2' and stored at -{)D'C until analysis for camitine. Camitine is present in the free form and as acylcamitine esters in biological tissues and fluids. Alkaline hydrolysis produces free carnitine from acylcamitine (27). Samples of urine, milk, and liver were treated with HCI04 (300 gIL) to precipitate long-chain acylcamitine. An aliquot of the supernatant was neutralized with 3.5 M KOH and analyzed for free carnitine. Another aliquot of supernatant was alkalinized (pH 9) with 3.5 M KOH and incubated for 1 h at 40·C to hydrolyze acylcarnitine. The sample was neutralized with 3.3N HCI and analyzed for acid-soluble carnitine (free plus short-chain acylcarnitine). Short-chain acylcamitine was determined as the difference between acidsoluble camitine and free camitine. The precipitate from milk and liver samples was washed twice with water, redissolved in 3.5 M KOH, incubated for 2 h at 50'C, and neutralized with HCI04 (300 gIL) for determination of long-chain acylcamitine. Plasma, muscle, and feces were analyzed for total carnitine by alkalinization with 3.5 M KOH and incubation for 1 h at 40'C prior to treatment with HCI04 (300 gIL). The supernatant was neutralized and analyzed for camitine. The concentration of camitine in the various supernatant fractions was determined by a radiJournal of Dairy Science Vol. 78. No.8. 1995

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LaCOUNT ET AL.

oenzymatic method (27). An aliquot of the neutralized supernatant was incubated with [114C]acetyl-coenzyme A (lCN Biomedicals, Inc., Costa Mesa, CA) and acetylcamitine transferase (EC 2.3.1.7; Sigma Chemical Co.) for 30 min at room temperature (25°C). The incubation mixture was applied to a 5-cm column of Dowex 2X8-200 (Sigma Chemical Co.) to remove unreacted [l_14C]acetylcoenzyme A. Scintillation fluid (Scintiverse IT; Fisher Scientific, Fair Lawn, NJ) was added to the effluent from the column, and [114C]acetylcamitine was quantified by liquid scintillation. Camitine concentrations were determined by comparison with a standard curve of known camitine concentrations after corrections for sample blanks (without acetylcamitine transferase) that were specific to each sample type. Statistical Analysis

Data were analyzed as a replicated Latin square design using the general linear models procedure of SAS (31). Repeated measurements such as DMI and milk yield were reduced to period means for each cow before statistical analyses. The repeated measurements of ruminal pH and blood metabolites were analyzed as a split plot in time (16) within the Latin squares. Means were separated by use of orthogonal contrasts: 1) control versus both camitine treatments and 2) ruminal

versus abomasal camitine administration. Significance was P :5 .05; contrasts with P :5 .10 are discussed as trends. RESULTS AND DISCUSSION Concentrations of Carnltlne In Plasma, Liver, and Muscle

Compared with untreated cows, administration of camitine into either the rumen or abomasum of lactating dairy cows increased total camitine concentrations in plasma to the same extent (Table 2). The significant increase in plasma camitine concentrations with either treatment indicates that camitine was absorbed from the gastrointestinal tract, independent of the site of administration. The concentrations of camitine in plasma measured in this experiment are higher than those reported previously [about 10 nmoVml in whole blood; (12, 35»). The differences may be due to the relatively low milk yield for the cows used in earlier studies or to the use of a diet containing supplemental fat in our study. The concentration of total camitine in muscle was not increased significantly by camitine administration (Table 2). The measured concentrations of camitine in muscle fall within reported ranges for cattle (34). Muscle carnitine also was not greatly increased by exogenous camitine in rodents (30), evidently because the muscle camitine transporter is saturated at nor-

TABLE 2. Concentrations of carnitine in plasma, muscle, and liver and concentrations of lipid in liver for cows administered carnitine into the rumen or abomasum. Contrast Treatment Item

Control

Rumen

63.5 4.75

76.7 5.21

Abomasum

SEM

Control vs. carnitine

Rumen vs. abomasum p

Plasma carnitine, nmoVrnl Muscle camitine,l I'moVg Liver carnitine 1 Free, nmoVg Short-chain acyl, nmoVg Long-chain acyl. nmoVg Liver total lipid.! % Liver triglyceride,. %

125.5 28.0 105.3 4.38 .49

lWet weight basis. Journal of Dairy Science Vol. 78, No.8, 1995

144.1 36.5 105.5 5.73 1.31

79.7 4.92 144.3 43.0 109.5 5.03 .42

2.7 .24 2.8 12.4 7.1 .62 .60

.005 .31

.48 .41

.002 .45 .81

.96 .67 .71

.23 .63

.45 .33

CARNITINE FOR DAIRY COWS

mal plasma carmtme concentrations (30); therefore, any increased uptake by muscle occurs via passive diffusion. Liver samples were analyzed for concentrations of free, short-chain, and long-chain acylcarnitine (Table 2). Free carnitine in liver was higher when cows were administered carnitine at either site than when cows received the control treatment. The concentrations of shortand long-chain acylcarnitine in liver were unaffected by carnitine supplementation. The liver of rodents rapidly accumulates carnitine in response to exogenous carnitine administration (30). Snoswell et al. (33) reported that lactating dairy cows had significantly lower concentrations of free and short-chain acylcarnitine in liver than did nonlactating cows; however, the carnitine concentrations measured in that study were two- to threefold lower than those measured in our experiment. Erfle et al. (12) reported liver total carnitine concentrations of 160 nmol/g of wet tissue for lactating and nonlactating cows. which is in closer agreement with our data. Long-chain acylcarnitine concentrations in liver seem high relative to those reported for sheep and goats [.1 to 25 nmol/g of wet tissue; (34)]; however, reports of long-chain acylcarnitine in liver of cattle are unavailable. The increased concentrations of carnitine in plasma and liver that occurred with carnitine supplementation establish that carnitine administration into either the rumen or abomasum was equally effective at increasing the carnitine content of tissues and plasma of lactating dairy cows. L-Carnitine (1 mM) added to in vitro incubations of bovine liver slices increased oxidation and decreased esterification of palmitate (8). Whether the increase of hepatic carnitine concentration caused by carnitine supplementation could alter hepatic FA metabolism in vivo is not known. Concentrations of total lipid and triglyceride in liver were not affected by carnitine supplementation (Table 2); however. the effects of carnitine supplementation on liver FA metabolism should be determined during the transition from the nonlactating to lactating states, when mobilization of FA from adipose tissue and hepatic lipid accumulation are greatest. OMI, Milk Yield, and Composition

The OMI was unaffected by carnitine supplementation (Table 3). Intravenous infusion of

1829

carnitine (20 g/d) also had no effect on OMI (14). Yields of milk and 3.5% FCM as well as percentages and yields of fat, SNF, and N fractions in milk were unaffected by carnitine (Table 3), except for SNF content, which increased with carnitine administration at either site. Yields of 3.5% FCM and fat were lower when cows were infused with carnitine in the abomasum than when carnitine was administered ruminally. Site of carnitine administration did not affect proportions or yields of other milk components. Subcutaneous injection of carnitine (1.0 mg/l00 kg of BW) to cows fed a low energy diet had no effect on milk yield or fat percentage; however, SNF content of milk increased (21). Subcutaneous injection of carnitine [1 mg/l00 kg of BW; (38)] or intravenous infusion of carnitine [20 g/d; (14)] had no effect on milk or component yields; however, SNF percentage was lower when carnitine was administered (38). Contents of individual FA and glycerol in milk fat were not affected by carnitine supplementation except for decreased concentration of C6:0 and increased concentration of C16:1 when carnitine was administered at either site (data not shown). These changes in milk fat composition were very small and probably of little biological significance. Apparent Total Tract Nutrient Oigestlblllties

Measurements of N and energy utilization required the determination of digestibilities of nutrient fractions. Oigestibilities of gross energy. EE, total C16 FA, and total FA increased, and those of OM (P < .08) and total C18 FA (P < .10) tended to increase, when carnitine was supplemented at either site (Table 4). Digestibilities of CP, AOF. NDF, ash, C12:o, and C14:0 were unaffected by carnitine administration. Total tract digestibilities of all nutrient fractions were similar for both sites of carnitine administration. The increased total tract digestibilities of energy and FA when carnitine was administered at either site were not anticipated. Carnitine is involved in absorption of palmitic acid from isolated intestinal segments of rats (23) and may playa role in FA absorption as a component of bile. Gudjonsson et al. (19) showed that the quantity of carnitine secreted in bile. primarily as long-chain acylcarnitine, Journal of Dairy Science Vol. 78, No.8, 1995

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was similar to urinary carnitine excretion in rats. The role of biliary carnitine in micelle formation and FA absorption from the intestine is unknown. Alternately, carnitine could improve FA absorption from the small intestine by alleviating a drain on the methyl pool, which might increase phophatidylcholine synthesis. Phosphatidylcholine is necessary for micelle formation, lipid digestion, and lipid transport in the small intestine and also requires SAM for its synthesis from phosphotidylethanolamine. Hypothetically, provision of dietary carnitine could decrease the need for camitine synthesis and thus spare SAM for phosphatidylcholine synthesis. The potential limitations of Met and methyl group

metabolism in ruminants have been discussed elsewhere (36). Rumlnal Fermentation Characteristics

Because we anticipated that camitine would be degraded by ruminal microbes, we monitored effects of carnitine on ruminal fermentation characteristics. The pH of ruminal fluid and concentrations of ammonia N were unaffected by carnitine supplementation (Table 5). The concentration of total VFA in ruminal fluid tended (P < .09) to be higher when carnitine was administered into the rumen than when carnitine was administered into the

TABLE 3. Effects of administration of camitine into the rumen or abomasum on OMI, milk yield, and milk composition. Contrast Treatment Item

Control

Rumen

26.3 41.6 42.2

26.3 42.2 43.1

Abomasum

SEM

Rumen vs. abomasum

Control vs. carnitine

p OMI, kgld Milk, kgld 3.5% FCM, kgld

26.0 41.0 40.8

.59 .68 .41

.89 .99 .64

.79 .26 .01

Fat % kgld

3.59 1.49

3.64 1.53

3.47 1.42

.06

.62 .41

.II

.01

SNF % kgld

7.94 3.31

8.16 3.45

8.09 3.32

.05 .07

.02 .41

.34 .25

Total N % gld

.485 201.8

.482 203.8

.483 198.7

.003 3.6

.57 .89

.84 .35

True protein N % gld % of Total N

.451 188.0 93.16

.450 190.0 93.21

.450 185.0 93.2

.003 3.3 .08

.63 .92 .67

.89 .33 .95

Casein N % gld % of Total N % of True protein N

.353 147.2 72.98 78.34

.353 149.1 73.34 78.71

.349 143.4 72.23 77.51

.002 2.1 .42 .46

.33 .73 .72 .69

.15 .10 .11 .12

.098 40.8 20.18 21.66

.096 40.9 19.86 21.30

.101 41.7 20.96 22.49

.002 1.4 .43 .46

.80 .79 .67 .69

.21 .69 .12 .12

.033 13.9 6.84

.033 13.9 6.80

.033 13.6 6.80

.001 .30 .08

.37 .66 .67

.66 .65 .95

.002

Whey protein N %

gld % of Total N % of True protein N

NPN % gld % of Total N

Journal of Dairy Science Vol. 78. No.8, 1995

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CARNITINE FOR DAIRY COWS

TABLE 4. Apparent total tract digestibilities of nutrient fractions for cows administered carnitine into the rumen or abomasum. Contrast Treatment Item

Control

Rumen

Abomasum

SEM

Control vs. carnitine p

(%)

OM OM CP Ether extract ADF NDF Ash Energy C 12:0 C14:0 Total C 16 FAI Total CiS FA Total FA 1Fatty

63.4 65.2 63.4 74.4 34.0 44.0 46.5 61.9 89.1 49.0 57.2 58.1 58.9

65.6 67.1 66.6 78.7 38.4 46.9 51.4 64.1 89.1 51.8 59.7 60.3 61.3

Rumen vs. abomasum

64.5 66.3 64.5 79.3 34.6 44.1 46.4 63.3 89.9 49.3 59.4 61.6 61.3

.8 .6 1.1 1.0 1.8 1.2 2.9 .5 .5 1.1

.7 1.2 .8

.34 .41 .26 .70 .19 .15 .26 .33 .35 .14 .77 .49 .98

.13 .08 .18 .008 .29 .36 .52 .03 .53 .28 .04 .10 .05

acids.

abomasum. The molar percentage of acetate tended (p < .08) to decrease, and the molar percentage of propionate increased, with carnitine administration (Table 5); these changes tended (P < .08) to cause a decreased acetate to propionate ratio. The molar percentage of isovalerate was decreased, and those of butyrate and valerate were unaffected, by carnitine administration. Site of carnitine administration had no effect on molar percentages of

individual VFA or the acetate to propionate ratio. Compounds In Plasma and Balances of N and Energy

Concentrations of NEFA (181.5, 167.3, and 163.9 ",eqlL for control, ruminal, and abomasal treatments, respectively), glucose (81.0, 82.4, and 82.5 mg/dl), triglyceride-rich lipoprotein

TABLE 5. Ruminal fennentation characteristics for cows that were administered carnitine into the rumen or abomasum. Contrast Treatment Item

Control

Rumen

Abomasum

pHI Ammonia N, mgldl Total VFA, mM VFA, moVl00 mol Acetate (A) Propionate (P) Butyrate Isovalerate Valerate

5.75 10.2 120.2

5.68 9.2 125.5

5.71 9.2 120.2

58.5 28.7 10.0

57.2 30.4 9.7 1.0 1.6

57.5 29.8 10.0 1.0 1.7

SEM

Control vs. carnitine

Rumen vs. abomasum p

A:P

1.1

1.7 2.06

1.90

1.96

.05

.7 1.9 .5 .4 .3 .04 .05 .05

.42 .26 .29

.70 .94 .09

.08 .05

.30

.61 .35 .48 .74 .57

.08

.43

.84 .02

IThe interaction of treatment and time after feeding was not significant; means of 12 hourly samples. Journal of Dairy Science Vol. 78, No.8, 1995

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laCOUNT ET AL.

triglycerides, BHBA, and urea N in plasma were unaffected by camitine administration at either site. The concentration of cholesterol tended (P = .07) to be lower when cows received camitine in the abomasum than in the rumen (224, 225, and 210 mgldl). Others (11, 21, 38) also have reported no effect of carnitine administration on blood glucose concentrations in lactating dairy cows. Erfle et al. (11) reported that the concentration of NEFA in plasma decreased when 23.8 gld of carnitine was infused intravenously to feed restricted cows. Utilization of N by cows was not affected by camitine administration (Table 6). Retained N (grams per day) and retained and productive N as percentages of intake N tended to increase when carnitine was administered; however, improvements in N utilization occurred primarily when cows received carnitine in the rumen. Retained N and productive N, expressed as grams per day, and productive N, as a percentage of intake N, tended to be higher when cows were administered carnitine rumi-

nally than when administered carnitine abomasally. Other measures of N utilization were unaffected by camitine supplementation at either site. The improved N retention, especially with ruminal camitine supplementation, may be an effect of altered N metabolism by the ruminal microbiota as a result of camitine supplementation; the fate of camitine in the rumen is unknown. In vitro research is necessary to determine the extent of camitine degradation by ruminal microorganisms. Energy balance was determined because of the potential improvement in utilization of long-chain FA when camitine was supplemented; however, measurements of energy metabolism generally were unaffected by carnitine administration into either the rumen or the abomasum (Table 7). Energy excretion in feces tended (P = .08) to decrease, which corresponds to the improved energy digestibility. As percentages of gross energy intake, fecal energy decreased and ME increased with carnitine supplementation. The site of camitine administration did not affect energy partitioning.

TABLE 6. Utilization of N by cows administered camitine into the rumen or abomasum. Contrast

Item

Control

Rumen

Abomasum

SEM

Control vs. carnitine

N Intake, gld

782

784

771

16

.85

.35

N Excreted, gld Feces Urine

285 228

260 228

274 227

9 10

.14 .99

.32 .93

Milk N, gld N Absorbed, gld N Retained, gld Productive N,1 gld

202 496 67 278

204 524 92 296

199 498

4 15 6 8

.96 .45 .07 .17

.35 .25 .05 .06

Treatment

Rumen vs. abomasum

p

72 271

N, % of Intake Feces Urine Milk Retained Productive

36.6 29.3 25.9 8.2 34.2

33.5 29.0 26.3 11.2 37.5

35.5 29.5 25.7 9.3 35.0

1.1 .9 .3 .7 .9

.18 .98 .76 .06 .09

.25 .75 .15 .11 .09

N % of Absorbed Urine Milk Retained Productive

46.5 40.8 12.7 53.6

43.7 39.7 16.6 56.3

45.7 40.1 14.1 54.3

1.1 .8 1.2 1.1

.23 .40 .11 .23

.23 .71 .18 .23

ISum of N retained in body and N secreted in mill.. Journal of Dairy Science Vol. 78, No.8, 1995

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CARNITlNE FOR DAIRY COWS

Carnltlne excretion

Camitine supplementation at either site increased the concentrations and yields of free and long-chain acylcamitine in milk similarly (Table 8). Short-chain acylcamitine in milk was unaffected by camitine administration. The concentration of total carnitine in milk was reported to be higher in ketotic cows than in normal cows and to decrease from approximately 500 nmol/ml at calving to approximately 300 nmollml by 8 wk postpartum (12). The concentrations of carnitine measured in milk in our experiment fall within that reported range. Earlier data from Erfle et al. (10) showed that normal cows (mixed breeds) yielding less than 22 kg/d milk had free and shortchain acylcarnitine concentrations of 62 and 57 nmollml, respectively. Snoswell and Linzell (35) reported concentrations of 107 and 11 nmol/ml for free and short-chain acylcamitine, respectively, in milk of cows yielding 10 to 20 kg/d. The concentrations of free carnitine in milk reported in those studies (10, 35) are well

below the concentrations measured in our experiment. Differences in the genetic potential, stage of lactation, diet, and methodology used to measure carnitine may explain the higher concentration of free carnitine in milk from cows used in our experiment compared with those utilized in other experiments (10, 35). Intravenous infusion of 20 g/d of camitine resulted in increased concentrations of carnitine in milk (12). The concentrations and excretion of free and short-chain acylcarnitine in urine were increased by carnitine administration (Table 8). The site of carnitine administration did not cause significant differences in urinary carnitine; however, the concentrations and excretion of free and short-chain acylcarnitine in urine were somewhat higher when cows received carnitine via the abomasum compared with ruminal administration. Increased excretion of short-chain acylcarnitine may be a primary reason that urinary energy as a percentage of gross energy intake increased with camitine

TABLE 7. Energy utilization by cows administered camitine into the rumen or abomasum. Contrast Treatment Item

Control

Rumen

Abomasum

SEM

p

(McaIld) Intake energy Fecal energy Digestible energy Urinary energy Gaseous energy 1 ME2 Maintenance energy ME above maintenance Milk energy BW. kg

119.0 45.4 73.6 4.2 5.6 63.8 15.7 48.1 29.1 577

119.2 42.7 76.5 4.4 5.8 66.3 15.6 50.7 28.8 574

Rumen vs. abomasum

Control vs. carnitine

118.0 43.3 74.7 4.7 5.6 64.4 15.5 48.9 28.3 570

2.7 .9 2.1 .2 .2 1.8 .1 1.7 .3 3

.91 .08

.46 .19 .76 .51 .21 .47 .18 .22

.78 .65 .57 .41 .56 .48 .32 .49 .33 .32

- - (% of gross energy intake) - -

Fecal energy Urinary energy Gaseous energy 1 ME Maintenance energy ME above maintenance Milk energy Crude efficiency. 3 %

38.1 3.5 4.7 53.6 13.2 40.4 24.7 64.2

35.9 3.7 4.9 55.6 13.3 42.3 24.5 58.5

36.7 4.0 4.8 54.5 13.2 41.3 24.0 58.4

.6 .2 .1 .4 .3 .6 .6 2.0

.03 .13 .33 .03 .88

.11 .64 .32

.33 .17 .31 .14 .90 .29 .60 .99

'Calculated from regression equation of Moe and Tyrrell (24). 2Metabolizable energy.

3Milk energylME above maintenance x 100. Journal of Dairy Science Vol. 78. No.8. 1995

1834

LaCOUNT ET AL.

administration. The increased urinary loss of short-chain acylcarnitine diminished the increase in ME that could have resulted from the increased digestible energy. The concentration and excretion of total carnitine in feces were not affected by carnitine administration at either site (Table 8). Total daily carnitine excretion (sum of milk, urinary, and fecal carnitine excretion) was determined to monitor the disposition of the exogenous carnitine administered. When cows received no supplemental carnitine (control), an average of 3.14 gld of carnitine was excreted. Crude estimates of carnitine excretion above the basal excretion were made by subtracting the amount of carnitine excreted when the cows were given the control treatment from the amount of carnitine excreted during carnitine administration. Carnitine excretion above the basal was .75 and 1.18 gld for ruminal and

abomasal carnitine administration, respectively. These excretion amounts accounted for only 11.5 and 19.7% of the carnitine administered into the rumen and abomasum, respectively. In rodents, carnitine turnover times were 4 to 5 d for muscle and 1 to 2 h for liver (3). Thus, because cows were adapted to treatments for 2 wk prior to sample collection, tissue carnitine reserves should have been saturated, and essentially all of the exogenous carnitine that was administered abomasally would be expected to be excreted. One possibility for the low recovery of carnitine is that exogenous carnitine inhibited de novo carnitine synthesis, thereby maintaining similar carnitine excretion when carnitine was supplemented. The effect of carnitine supplementation on de novo carnitine synthesis is unknown. Alternately, the apparent carnitine loss may be due to degradation

TABLE 8. Camitine excretion in milk, urine, and feces of cows administered camitine into the rumen or abomasum. Contrast Treatment Item

Control

Rumen

Abomasum

SEM

Rumen vs. abomasum

Control vs. camitine

p Milk camitine Free nrnollrnl g/d Short-chain acyl nmollrnl g/d Long-chain acyl nmollrnl g/d Urinary camitine Free nmollrnl g/d Short-chain acyl nmollrnl g/d Fecal carnitine Total nmollg g/d

345.0 2.37

357.9 2.36

15.8 .09

.004

.74 .92

69.3 .46

47.5 .29

45.9 .30

16.3 .09

.24 .18

.94 .96

14.8 .10

20.1 .14

22.1 .15

.08 .01

.0007 .0003

.14 .18

26.2 .12

66.2 .29

109.1 .51

19.5 .10

.04 .06

.17 .17

73.0 .33

121.6 .54

153.4 .70

19.8 .10

.04 .04

.30 .28

180.9 .28

178.0 .26

213.5 .32

19.2 .03

.55 .83

.24 .27

3.14

3.89 .75 11.5

4.33 1.18 19.7

.17 .17 2.8

.004

.12 .12 .09

Excreted camitine Total,l g/d Above basal. g/d % of Dose I Milk

.r:m

277.2 1.85

plus urine plus fecal.

Journal of Dairy Science Vol. 78, No.8, 1995

CARNITINE FOR DAIRY COWS

of camitine by the intestinal microflora. Microorganisms have been reported to metabolize carnitine to trimethylaminoacetone, trimethylamine, and 'Y-butyrobetaine (2), which may be excreted in urine or feces (29). More detailed balance studies of camitine metabolism in ruminants would need to include measurement of these breakdown products. CONCLUSIONS

Supplemental carnitine provided into either the rumen or abomasum was equally effective at increasing the concentrations of camitine in liver, plasma, and milk of dairy cows during early lactation. More research is necessary to determine whether this effect could alter tissue FA metabolism, particularly in cows during the peripartal period and thereby benefit health or production. Carnitine had little effect on milk yield and composition; evidence was not obtained to support the hypothesis that camitine supplementation would increase milk protein production by sparing Met. The unexpected improvements in apparent digestibilities of FA and gross energy and in N retention need to be verified with further research. The optimal amount and site of administration of carnitine also need to be determined. ACKNOWLEDGMENTS

The authors thank S. A. Blum of Lanza Inc. for funding and discussion; K. H. Kline and D. E. Grum for assistance with muscle and liver biopsies, respectively; and T. M. Cicela, J. P. Elliott, and T. R. Overton for assistance with sample collection and analysis. The authors also acknowledge the donations of soybean hulls by Archer Daniels Midland, Decatur, Illinois and Energy Booster lOOTM by Milk Specialties Co., Dundee, Illinois. REFERENCES

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