The effects of dietary L-carnitine and rumen-protected choline on growth performance, carcass characteristics and blood and rumen metabolites of Holstein Young bulls

Abstract

An experiment was conducted to determine the effects of L-carnitine (LC) and rumen-protected choline (RPC) on growth performance, carcass characteristics and blood and rumen metabolites of finishing bulls. Twenty-eight Holstein young bulls (average initial weigh 310±24 kg) were used in a completely randomized design with four treatments and seven replicates with 2×2 factorial arrangement for 90 days. Basal diets were formulated for 14% crude protein and 1.14 Mcal/kg of net energy for growth and contained 24% of dietary dry matter (DM) as corn silage, 6.8% alfalfa hay, 46.50% barley grain and 9.5% canola meal. Treatments included: (1) without LC and RPC or control; (2) 4g/d LC head⁻¹; (3) 5 g/d RPC head^-1; (4) 4 g/d LC head⁻¹ and 5 g/d RPC head⁻¹. Calves were fed ad libitum and had free access to fresh water. There were no differences between diets for DM intake, average daily gain and gain:feed. Plasma concentrations of glucose, insulin, nonesterified fatty acid, β-hydroxybutyrate, cholestrol and aspartate aminotransferase were not affected by treatments, but plasma triglyceride concentration decreased in calves fed RPC (P < 0.05). Longissimus muscle area, pelvic, kidney, heart and rumen and intestine fats were not affected by treatments. Subcutaneous fat and backfat thickness decreased in calves fed RPC (P < 0.05). LC and RPC had no significant effects on chemical composition of meat and liver (protein, fat, ash and dry mater). There was no significant difference between diets for rumen liquor volatile fatty acid and pH. This study showed that LC and RPC supplements cannot affect growth performance in Holstein young bulls, but lipid metabolism may be affected by LC and RPC.

Keywords:

• L-carnitine
• rumen-protected choline
• performance
• blood metabolites
• Holstein Young bulls

1. Introduction

Carnitine is a vitamin-like compound that facilitates the transport of medium- and long-chain fatty acids across the mitochondrial membrane for β-oxidation. Hepatic β-oxidation of long-chain fatty acids is stimulated by exogenous carnitine in several species (Drackley et al. 1991; Owen et al. 2001; Li et al. 2012). Further, as a result of enhanced long-chain fatty acids β-oxidation, carnitine supplementation has been shown to increase hepatic glucose production by stimulating the flux of metabolites through pyruvate carboxylase (Owen et al. 2001). Carnitine also appears to be involved in nitrogen (N) metabolism (Chapa et al. 1998; Chapa et al. 2001; White et al. 2001). Supplemental L-carnitine (LC) has improved growth in grazing beef calves fed liquid supplements containing urea and was associated with reduced ruminal ammonia N levels (White et al. 2001). In finishing experiments, carnitine had no effect on average daily gain (ADG) but reduced fat deposition in heifers (Hill et al. 1995a) and increased fat deposition and longissimus area in heifers with no effect on steers (Hill et al. 1995b). In dairy cattle, Carlson et al. (2007) reported that, by decreasing liver lipid accumulation and stimulating hepatic glucose output, LC supplementation might improve glucose status and diminish the risk for developing metabolic disorders during early lactation.

Choline is sometimes classified as a B vitamin, even though it does not fulfil the standard vitamin definition and is a small water-soluble molecule that has been found in all mammalian cells (NRC 2001). Choline is a nutrient involved in with the transport of fat from the liver, synthesized in part from methionine and required for the synthesis of phosphatidylcholine, a phospholipid found in the membranes of very low density lipoprotein (NRC 2001; Sales et al. 2010). In finishing steers, fed 0.25% rumen-protected choline (RPC) increased ADG, but responses diminished with increasing RPC level and feed:gain was improved with 0.25% RPC compared with 0% RPC, and responses diminished with increasing RPC level (Bryant et al. 1999). Cooke et al. (2007) suggest that RPC can prevent and possibly alleviate fatty liver induced by feed restriction. They expressed one of the potential effects of choline on liver triacylglycerol (TAG) accumulation could be related to fatty acid oxidation by means of increasing carnitine levels. Addition of carnitine to culture media of liver slices was found to increase palmitate oxidation (Drackley et al. 1991).

We hypothesized that carnitine and choline supplementation might increase lean deposit in cattle by increasing energy availability in the tissue level as a result of increasing fatty acid oxidation. Also choline serves as methyl donor in the synthesis of carnitine, which is essential for fatty acid oxidation and supplementing RPC may increase carnitine accretion and fatty acid oxidation, leading to a decrease nonesterified fatty acid (NEFA) concentrations. The objectives of present study were to determine the effects of LC, RPC and blend of LC and RPC on performance, carcass characteristics and blood and rumen metabolites.

2. Material and methods

2.1. Treatments and animal management

The experimental protocols for this research were approved by the University of Tehran Institutional Animal Care and Use Committee, Tehran, Iran. Twenty-eight Holstein young bulls (average initial weight 310±24 kg) were assigned randomly to one of the four treatment groups with seven replicates in a completely randomized design with 2×2 factorial arrangement for 90 days to investigate the effects of LC and dietary RPC on growth performance, carcass characteristics and blood and rumen metabolites. Holstein young bulls were fed 70% concentrate diet based on barley (NRC 1996) and had free access to balanced total mixed ration (TMR; Table 1) and fresh water. All bulls were individually fed and 10 d before the start of the experiment were adapted to the experimental diet (70% concentrate). Treatments included: (1) control, (2) fed 4 g/d LC head⁻¹ (20 g/d^.head⁻¹ Carniking 20%, Lohmann animal health [LAH], Cuxhaven, Germany), (3) fed 5 g/d RPC head⁻¹ (20 g/d^.head⁻¹ Reashure® Choline 25%, BalchemCorp., Slate Hill, NY, USA), (4) fed 4 g/d LC head⁻¹ (20 g/d^.head⁻¹ Carniking 20%, LAH, Cuxhaven, Germany) and 5 g/d RPC head⁻¹ (20 g/d^.head⁻¹ Reashure® Choline 25%, Balchem Corp., Slate Hill, NY, USA). Carniking contains at least 20% LC based on manufacture comments (LAH, Cuxhaven, Germany) and was developed for the use in premix, mineral feed and compound feed for animals. In study of Carlson et al. (2007), the researchers reported that approximately 80% of Carniking could be degraded in the rumen. The RPC product (Balchem Corp., Slate Hill, NY, USA) is a rumen-protected source of choline chloride. RPC is produced by encapsulating choline chloride with a coating matrix able to resist rumen breakdown and release choline in the intestine. Based on the comments of manufacture, 30% of Reashure Choline is degraded after 24 h of incubation in the rumen. LC and RPC were top dressed on TMR. The control group received the same diets but without LC and RPC. Monthly body weight (BW) changes and daily individual feed intake were recorded. BWs were taken after 16 h without access to feed and water. Bulls were fed for ad libitum intake. Total mixed diets and feed refusals were recorded once daily, allowing minimum 5% orts at each feed delivery.

Table 1. Feed ingredient and nutrient composition (DM basis) of the experimental diet.

Ingredient	Percentage of dietary DM	Chemical composition^a	Percentage of dietary DM
Corn silage	24.00	ME, Mcal/kg	2.66
Alfalfa hay	6.80	NEm, Mcal/kg	1.75
Barley grain	46.50	NEg, Mcal/kg	1.14
Rice bran	4.50	DM	53.00
Wheat bran	6.00	CP	13.02
Canola meal	9.50	NDF	38.00
Calcium carbonate	1.09	ADF	21.60
Salt, white	0.29	NFC	36.15
Sodium bicarbonate	0.68	Ether extract	2.66
Zeolite	0.08	Ca	0.71
Mineral and vitamin mix^b	0.56	P	0.52

^a Calculated from analysis of individual sampled two weekly and composited monthly. Values for ME, NEm, NEg, NFC, Ca and P were estimated by the NRC (1996).

^b 1 kg of mineral and vitamin mix contained 180 g Ca, 70 g P, 30 g Mg, 50 g Na, 4 g Fe, 0.3 g Cu, 3 g Zn, 5 g Mn, 0.1 g I, 0.1 g Co, 0.02 g Se, 0.4 g Antioxidant, 400,000 IU vitamin A, 100,000 international unit (IU) vitamin D, 200 IU vitamin E.

2.2. Diet composition

In order to determine diet composition, individual samples from diet was taken every other weeks and composited monthly. Samples of diet dried at 55^°C, were placed in air oven for 72 h to determine dry matter (DM) concentration (AOAC 2000) and ground through a 1-mm screen before analysis. Feed samples analyzed for ash (AOAC 2000), neutral detergent fiber (NDF) without ash and without using sodium sulfite and α-amylase (Van Soest et al. 1991), crude protein (CP) (AOAC 2000; KjeltecAuto 1030), and ether extract (EE) (AOAC 2000; Soxtec 1043) in nutrition laboratory of Tehran university. Nonfiber carbohydrate content (percentage of DM) of diets was calculated by 100 – (NDF% + CP%+ EE% + ash%).

2.3. Ruminal fluid sampling and analyses

For measurements of ruminal parameters, ruminal liquor collected by stomach tube at 0 h (just before the morning feeding), 3 h and 6 h after morning meal in the last day of experiment. Rumen liquor pH was immediately determined using a portable pH meter (Metrohm, 827, Swiss). A 50 mL subsample of strained ruminal fluid was mixed with 1-mL chilled 50% sulfuric acid (H₂SO₄) and stored at −20°C for later determination of volatile fatty acid (VFA) and N-NH3 concentration. At the end of the trial, frozen ruminal fluid subsamples were thawed at room temperature and then centrifuged. Ruminal VFA were separated and quantified by gas chromatography (Varian 3700; Varian Specialties Ltd., Brockville, Ontario, Canada) with a 15-m (0.53 mm i.d.) fused silica column (DB®free fatty acid phase (DBFFAP) column; J and W Scientific, CA, Folsom) with a 15-m (0.53 mm i.d.) fused silica column (DBFFAP column; J and W Scientific, CA, Folsom). Ammonia content of ruminal samples was determined using the method described by Weatherburn (1967) modified to use a microtiter plate reader.

2.4. Blood and carcass sampling and analyses

At the last day of the experiment, blood samples were collected from the jugular vein, approximately at 3 h after morning meal into tubes containing 12 mg of ethylene diamine tetra acetic acid (EDTA), and plasma was separated by centrifuged (at 3000×g for 20 min at 4°C) and stored at −20°C until analysis of plasma. Plasma samples were analyzed for glucose, insulin, NEFA, β-hydroxybutyrate (BHBA), cholesterol and aspartate aminotransferase (AST). Blood glucose was analyzed according to colorimetric methodology of glucose oxidase. For this determination, commercial kit (Parsazmoon, Iran, Tehran) was used by an automatic biochemical analyzer (Biotecnica, Targa 3000, Rome, Italy). BHBA and NEFA were determined by a D-3-hydroxybutyrate kit and an NEFA Kit (Randox Laboratories Ltd, rumlin, UK). Analyses AST and cholesterol were performed using commercial kits on an autoanalyzer TARGA 3000, Italy (Parsazmoon, Tehran, Iran). Insulin concentration was determined using an radioimmunoassay (RIA) kit (Coat-a-Count Insulin, Diagnostic Products Corporation, Los Angeles, CA).

Calves were weighted monthly and in the last day of experiment, calves slaughtered after 16 h fasting. Hot carcass weight (HCW) was determined on the day of slaughter. Heart (Pericardial) and pelvic fats were removed and weighed separately. The fat surrounding the rumen and intestine was weighed and considered as rumen + intestine fat. Also, the fat surrounding the kidney was weighed and considered as kidney fat. Subcutaneous fat separated from carcass and weighed. Backfat thickness was measured with calipers between 12th and 13th ribs. Longissimus muscle area was traced on a paper, and its area was determined using a planimeter. Meat samples took from Longissimus muscle between 12th and 13th ribs. Liver samples took from same part of liver in all samples. Representative samples (100 g) were ground by mill with a 4-mm orifice. Subsamples were analyzed for moisture (AOAC 2000; air-oven method), CP (AOAC 2000; KjeltecAuto 1030), EE (AOAC 2000; Soxtec1043) and ash contents (AOAC 2000).

2.5. Statistical analyses

In present experiment, a completely randomized design used with four treatments (diets) and seven replicates (bulls) with 2×2 factorial arrangement. The data were analyzed using the MIXED procedure of SAS (SAS 2003) treating cow as a random effect. The general linear mixed model included the main effects of LC, RPC and their interaction. The initial BW of bulls was included in the model as a covariate effect for analyzing dry matter intake (DMI), ADG and feed:gain. Initial analyses indicated that HCW was not a significant covariate (P > 0.05) for either carcass characteristics and meat and liver chemical composition. Significance level was set at P<0.05 and individual comparison of treatments was made by Tukey's standardized multiple comparison tests.

3. Results and discussion

3.1. Growth performance

There were no differences in DMI, ADG and gain:feed caused by treatments. There were no interaction of the effects of LC and RPC combined on DMI, ADG and gain:feed. Also initial and final BW was not affected by LC and RPC supplementation (Table 2). Same as present study, growth efficiency was not affected by carnitine supplementation (Hill et al. 1995a, 1995b; Greenwood et al. 2001). In growing Holstein calves, LC had no effect on feed consumption of calves fed the broiler litter diets. Although not reflected in the ADG difference, LC elicited an 8% increase in the efficiency of feed conversion (Bunting et al. 2002). In one of the two experiments, LC improved the growth rate of grazing cattle fed liquid supplement containing urea as the only source of CP, but, when fish meal was added to the liquid supplement, LC depressed gains (White et al. 2001). Bryant et al. (1999) reported that diets containing RPC-increased ADG and improved feed efficiency in finishing beef steer fed high-concentrate diet, but responses depended on RPC level (0%, 0.25%, 0.5% and 1.0% of dietary DM). The optimum response to RPC was noted with a diet that contained 0.25% (DM basis) RPC. Same as present study in others experiments (Bryant et al. 1999; Bindel et al. 2000; Ye et al. 2010), DMI was not affected by RPC. In the present study, LC and RPC did not improve growth performance in finishing Holstein calves.

Table 2. Effects of dietary LC and RPC on DMI and performance of Holstein young bulls.

	Treatments^a					P-value
Variable	Control	LC	RPC	LC + RPC	SEM^b	LC	RPC	LC×RPC
Initial BW, kg	311.7	310.9	310.7	310.4	23.8	0.98	0.99	0.99
Final BW, kg	416.7	418.0	403.3	415.4	21.8	0.76	0.72	0.81
Daily DMI, kg	9.10	9.01	8.62	9.13	0.21	0.34	0.42	0.18
ADG, kg	1.16	1.19	1.11	1.19	0.05	0.33	0.59	0.57
Gain: Feed	0.129	0.134	0.132	0.131	0.004	0.74	0.93	0.61

^a Control = no LC and RPC; LC = 4 g/d LC; RPC = 5 g/d RPC; LC + RPC = combination of 4 g/d LC and 5 g/d RPC.

^b Standard error of treatment means.

3.2. Blood metabolites

The majority of the blood metabolites (NEFA, BHBA, glucose, insulin and AST) were not affected by LC and RPC supplementation. There were no significant interactions of the effects of LC and RPC treatment on any blood metabolite concentrations (Table 3). Plasma triglyceride concentrations decreased in calves fed RPC (P < 0.05). Combination of LC and RPC also numerically decreased triglyceride concentration. Plasma cholesterol concentration did not differ among treatments (Table 3). A similar effect of LC on plasma triglyceride concentration was observed in previous experiments (Erfle et al. 1974; Citil et al. 2009). This effect of LC could be associated with the stimulation of lipid metabolism through transfer of acyl groups across the mitochondrial membranes (Erfle et al. 1974; Owen et al. 1996). Cholesterol content in plasma decreased when about 6 g/d of carnitine was administered into abomasum but not when carnitine was administered into the rumen (LaCount et al. 1995). In the study of Bindel et al. (2000), when diet contained no added fat, triglyceride concentration increased in response to intermediate levels of RPC, but when fat was added to the diet, choline supplementation decreased concentrations of triglycerides (2% tallow diets) or did not alter them (4% tallow diets). They suggest that, without fat in the diet, RPC increased transport of lipids from the liver. The mechanism of RPC effect on decreasing plasma triglyceride concentration in present study is unknown and we did not find any explain from other researchers for this topic. The lack of an effect of LC supplementation on plasma NEFA concentration is in agreement with the observations elsewhere (LaCount et al. 1995, 1996a, 1996b; Chapa et al. 1998). Greenwood et al. (2001) observed that plasma concentration of NEFA demonstrated a treatment×time interaction; they decreased linearly in response to carnitine before feeding but increased linearly in response to carnitine at 6 h after feeding and blood BHBA concentration tended to be higher when intermediate levels of supplemental carnitine (1–2 g/d) provided. They suggest that decrease in prefeeding NEFA concentrations could reflect increased uptake and oxidation of the fatty acids in response to increased carnitine availability and the increase in plasma NEFA at 6 h after feeding. In dairy cattle, prepartum concentrations of NEFA and BHBA in plasma were not altered by dietary carnitine supplementation, but, postpartum plasma BHBA tended to be higher for medium- and high-carnitine treatments than for the low-carnitine treatment despite similar serum NEFA concentrations (Carlson et al. 2007). They suggest that a greater amount of NEFA was converted to BHBA rather than being converted to triglyceride. In agreement with our study in previous reports, NEFA and BHBA concentrations were not affected by RPC (Bryant et al. 1999; Bindel et al. 2000; Zahra et al. 2006; Davidson et al. 2008; Zom et al. 2011). Feeding RPC to periparturient cows did not alter lipid metabolism, possibly because the cows were not over-conditioned and not at a high risk for developing fatty liver (Guretzky et al. 2006) and treatment groups did not suffer from severe negative energy balance (Zom et al. 2011). In present study, calves were in positive energy balance, which may explain why the BHBA did not alter. The effect of carnitine on plasma glucose level is controversial as some reported increase (Erfle et al. 1971; Chapa et al. 2001; Greenwood et al. 2001), decrease (White et al. 2002) or unchanged (Erfle et al. 1974; LaCount et al. 1995; Bunting et al. 2002; White et al. 2002; Carlson et al. 2007). Increases in blood glucose in response to carnitine supplementation have been attributed to increased fatty acid oxidation and subsequent reduction in the oxidation of gluconeogenic precursors (Greenwood et al. 2001). In some experiments (Bindel et al. 2000; Bindel et al. 2005; Cooke et al. 2007; Chung et al. 2009) glucose and insulin did not influence by RPC.

Table 3. Effects of dietary LC and RPC on blood metabolites of Holstein young bulls.

	Treatments^1					P-value
Variable	Control	LC	RPC	LC + RPC	SEM^2	LC	RPC	LC×RPC
Glucose, mg/dl	102.4	107.1	100.9	99.8	3.49	0.60	0.22	0.42
Insulin, ng/ml	18.61	17.81	14.07	14.96	2.91	0.99	0.22	0.77
NEFA, mmol/l	0.13	0.17	0.14	0.13	0.03	0.51	0.55	0.37
BHBA, mmol/l	0.38	0.39	0.39	0.36	0.04	0.83	0.83	0.64
Triglyceride, mg/dl	24.84^a	22.43^ab	16.29^b	18.43^ab	2.06	0.94	0.01	0.28
Cholesterol, mg/dl	96.14	98.00	96.43	89.14	8.47	0.75	0.62	0.59
AST, u/l	82.57	83.85	91.00	92.28	8.58	0.88	0.33	0.99

^a,b Means within a row with different superscripts differ (P<0.05).

¹ Control = no LC and RPC; LC = 4 g/d LC; RPC = 5 g/d RPC; LC + RPC = combination of 4 g/d LC and 5 g/d RPC.

² Standard error of treatment means.

3.3. Carcass characteristics

The influence of LC and RPC on carcass characteristics in finishing Holstein calves is shown in Table 4. Subcutaneous fat was lower in calves fed RPC (P < 0.05) compared with those fed other treatments. Supplementing LC and RPC or both, had no significant effect on Longissimus muscle area (LMA), pelvic, kidney, heart and rumen and intestine fats. Backfat thickness decreased with supplementation RPC and combined of LC and RPC (P < 0.05; Table 4). Few data are available to demonstrate effects of LC and RPC on carcass characteristics in finishing calves. Greenwood et al. (2001) observed that carcasses of steers supplemented with carnitine tended to be fatter than those of control steers, as indicated by tendencies for thicker backfat, higher marbling scores and higher yield grades. They noted that if carnitine increased fatty acid oxidation by increasing transport across the mitochondrial membrane, a decrease in lipid deposition should have occurred in response to carnitine supplementation. In growing-finishing pigs, LC reduced average backfat thickness, 10th-rib backfat depth and increased percentage of lean and muscle (Owen et al. 2001). Backfat decreased in offspring of sows fed LC during gestation (Musser et al. 2007). In our study, fat deposition decreased with LC, it may be depended to role of carnitine in fatty acid metabolism and muscle metabolism. Bryant et al. (1999) reported that in finishing steers, carcass yield grade increased linearly as RPC level increased, but fat thickness, LMA and percentage of kidney, pelvic and heart fat were not affected by RPC.

Table 4. Effects of dietary LC and RPC on carcass characteristics of Holstein young bulls.

	Treatments^1					P-value
Variable^2	Control	LC	RPC	LC + RPC	SEM^3	LC	RPC	LC×RPC
HCW, kg	241.0	236.3	221.3	250.0	13.4	0.39	0.82	0.25
LMA, cm^2	103.7	106.0	93.3	106.3	10.8	0.50	0.65	0.63
Pelvic fat, kg	13.66	12.76	12.00	12.60	1.21	0.90	0.47	0.55
Subcutaneous fat, kg	2.93^a	2.80^ab	2.30^b	2.41^ab	0.14	0.96	0.01	0.42
Heart fat, kg	0.34	0.49	0.41	0.33	0.07	0.63	0.54	0.16
Kidney fat, kg	4.20	2.05	3.62	3.24	0.80	0.15	0.71	0.30
Rumen + intestine fat, kg	7.31	6.46	6.66	6.41	0.95	0.58	0.72	0.76
Backfat thickness, mm	3.32^a	3.04^ab	2.76^b	2.65^b	0.21	0.15	0.01	0.52

^a,b Means within a row with different superscripts differ (P<0.05).

¹ Control = no LC and RPC; LC = 4 g/d LC; RPC = 5 g/d RPC; LC + RPC = combination of 4 g/d LC and 5 g/d RPC.

² HCW = hot carcass weight, LMA = longissimus muscle area.

³ Standard error of treatment means.

3.4. Meat and liver chemical composition

There were no significant differences in meat or liver compositions between treatments (Table 5). There were no LC×RPC interaction for meat or liver compositions. Owen et al. (2001) reported that LC had no effect on percentages of carcass CP and lipid or daily protein accretion. However, daily lipid accretion tended to decrease and then return to values similar to those for control pigs with increasing dietary LC. They suggest that providing supplemental LC affects fat metabolism. In study of Bryant et al. (1999), marbling score and tissue lipid content were not affected by RPC level, but liver lipid increased with RPC. Cook et al. (2007) reported that RPC decreased liver triglyceride. Liver lipids typically decreased because they are moved into the blood from the liver.

Table 5. Effects of dietary LC and RPC on meat and liver chemical composition of Holstein young bulls.

	Treatments^a					P-value
Variable	Control	LC	RPC	LC + RPC	SEM^b	LC	RPC	LC×RPC
Meat
DM,%	28.49	27.80	27.41	26.33	0.72	0.25	0.11	0.79
CP,%	22.02	21.74	20.44	21.30	0.52	0.59	0.09	0.30
Fat (Ether extract),%	2.06	1.51	1.65	1.86	0.28	0.58	0.92	0.21
ASH,%	1.14	1.07	1.06	1.04	0.03	0.15	0.10	0.38
Liver
DM,%	30.57	29.71	29.68	29.72	0.46	0.40	0.37	0.36
CP,%	19.28	19.49	20.20	19.80	0.52	0.86	0.27	0.57
Fat (Ether extract),%	2.05	1.51	1.64	1.86	0.38	0.68	0.94	0.34
ASH,%	2.36	2.75	3.14	2.80	0.43	0.96	0.36	0.42

^a Control = no LC and RPC; LC = 4 g/d LC; RPC = 5 g/d RPC; LC + RPC = combination of 4 g/d LC and 5 g/d RPC.

^b Standard error of treatment means.

3.5. Ruminal metabolites

Ruminal VFAs concentration were reported in Table 6. There were no differences in individual VFAs concentration between treatments (Table 6). Ruminal concentration of acetate increased with add LC and decreased with add RPC. Ruminal ammonia N concentrations were reduced when diets contained LC. Rumen liquor pH was not significantly affected by RPC and LC (Table 6). In agreement with our study, LaCount et al. (1996a) observed that ruminal proportions of major VFAs were not affected by LC. Similar observations were reported in Holstein calves (Bunting et al. 2002). In study of White et al. (2002), ruminal ammonia N concentration was reduced when diets contained 50 ppm LC but increased at greater levels of LC. The molar proportions of propionate were increased by 50 ppm LC but declined at greater levels and butyrate decreased with increase in dietary LC. Supplementation of LC did not affected ruminal N and ruminal pH in lambs (Chapa et al. 2001). Same as present study, dietary choline were not affected by ruminal VFA and pH (Atkins et al. 1988).

Table 6. Effects of dietary LC and RPC on rumen metabolites of Holstein young bulls.

	Treatments^a					P-value
Variable	Control	LC	RPC	LC + RPC	SEM^b	LC	RPC	LC×RPC
Acetate,%	67.94	66.96	65.75	68.96	2.99	0.71	0.97	0.49
Propionate,%	17.04	18.49	17.95	17.87	1.73	0.70	0.93	0.66
Butyrate,%	13.04	12.54	14.28	11.21	1.40	0.21	0.97	0.37
Isovalerate,%	0.85	0.89	0.83	0.84	0.09	0.77	0.67	0.87
Valerate,%	1.12	1.12	1.18	1.11	0.11	0.76	0.79	0.76
Acetate/propionate	4.07	4.09	3.77	4.11	0.42	0.67	0.73	0.71
Ammonia N, mg/dl	6.90	6.30	6.70	5.60	0.50	0.11	0.38	0.63
pH	6.96	6.68	6.70	6.76	0.07	0.17	0.27	0.06

^a Control = no LC and RPC; LC = 4 g/d LC; RPC = 5 g/d RPC; LC + RPC = combination of 4 g/d LC and 5 g/d RPC.

^b Standard error of treatment means.

4. Conclusions

Dietary LC and RPC did not improve growth performance in Holstein young bulls. Plasma triglyceride, subcutaneous fat and backfat thickness decreased significantly in calves fed RPC. No responses to LC and RPC supplementation were observed in rumen metabolites. Taken as a whole, our data suggested that lipid metabolism of calves may be affected by RPC. RPC may have potential to improve carcass quality in young bulls. However, a combined effect of dietary LC and RPC was not evident and therefore may be not beneficial. Further researches are required to assess combined effect of LC and RPC on lipid metabolism and performance in cattle. Also future research should focus on the effects of LC and RPC supplementation to fresh cows that are mobilizing large amount of body lipid and depositing lipid in the liver.

Acknowledgements

The authors thank University of Tehran for financial support and Javaneh Khorasan Co. and Dr. Kamyab for cooperation.