Influence of dietary fat, L-carnitine and niacin on milk yield and milk composition of dairy cows in midlactation

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1 Cuban Journal of Agricultural Science, Volume 45, Number 2, th Anniversary 123 Influence of dietary fat, L-carnitine and niacin on milk yield and milk composition of dairy cows in midlactation A. R. Tasdemir 1, M. Görgülü 1, U. Serbester 2 and S. Yurtseven 3 1 University of Cukurova, Faculty of Agricultural, Department of Animal Science, Adana, Turkey 2 University of Nigde, The Vocational School of Bor, Department of Animal Breeding, Nigde, Turkey 3 University of Harran, The vocational School of Ceylanpinar, Urfa, Turkey gorgulu@cu.edu.tr Two studies were conducted to evaluate the effect of dietary fat, L-carnitine, and niacin on milk production and milk composition in midlactation dairy cows. Eight multiparous lactating dairy cows were used. The studies were carried out with 2x2 factorial arrangements in a 4x4 Latin square design. The treatments in the fat and L-carnitine study (FLC) were: 1) no fat (NF) and no L-carnitine, 2) no fat and % L-carnitine, 3) 3.6 % fat (as fed) and no L-carnitine, and 4) 3.6 % fat and % carnipass. In the L-carnitine and niacin study (LCN), rations containing dietary fat (3.6 %) were used for all treatments and treatments were: 1) no L-carnitine and no niacin, 2) no L- carnitine and % niacin, 3) % carnipass and no niacin, and 4) 0.045% carnipass and 0.045% niacin. In the FLC study, dietary fat increased milk yield (P <0.05) and tended to increase milk NPN content (P =0.10) and improved milk production efficiency (milk yield/dry matter intake, P < 0.01). In contrast, milk fat, milk protein, true protein and casein nitrogen (P < 0.05) were reduced by dietary fat. L-carnitine resulted in decrease in milk yield and casein yield (P < 0.05) in the FLC. In connection with milk yield; protein yield had a tendency to decrease (P =0.08) by L-carnitine. Only non-fat solids are affected (P < 0.01) by the interaction between the dietary fat and L-carnitine. Milk yield and milk production efficiency also tended to be lower (P = 0.12 and P = 0.07) with L-carnitine in the LCN study. Niacin had no effect on milk yield and composition, except for the proportion of casein N to total N. The proportion of casein N to total N had a tendency to be higher (P =12) with L-carnitine and niacin separately. It could be concluded that dietary fat may increase milk yield and decrease milk fat, milk protein and L-carnitine supplementation may decrease milk yield, niacin supplementation did not affect with yield, composition and other parameters of diary cows in mid lactation fed a diet containing 60 % concentrate and 40% of alfalfa hay. Key words: fat, L-carnitine, niacin, cow, milk yield, milk composition. Cows in early lactation are in negative energy balance because feed intake is low relative to high energy demand for milk yield. Therefore, fat is added to lactation diets to increase the energy density of rations and the addition fat to the diets of lactating cows becomes a common practice. Positive effects of dietary fat on milk yield in mid lactation were shown as well (DePeters et al and Schingoethe et al. 1996). Ruminants have a lower capacity to oxidize (burn) fat than nonruminants (LaCount and Drackley 1996). However, ruminants could use dietary fat efficiently when diet was supplemented with L-carnitine (Carlson et al and Carlson et al. 2007) and niacin (Pires et al and Pires and Grummer 2007). Carnitine is a vitamin-like substance that mediates transport of medium and long-chain fatty acids across the mitochondrial membrane, facilitating beta-oxidation of fatty acids (Vaz and Wanders 2002). Niacin is critical to numerous biological processes in the mammalian system as a result of its role as a precursor in the formation of the two essential coenzymes, nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP). Dietary L-carnitine and niacin may spare lysine and methionine used for synthesis of L-carnitine (Borum 1983) and tryptophane used for synthesis of niacin (Horner et al. 1986) and may increase these amino acids availability in the mammary tissues. Effects of dietary carnitine with dietary fat and carnitine with niacin in ration containing high fat on milk yield composition in dairy cows have not been studied extensively. Therefore, the objective of this research was to evaluate the lactational response and milk components of dairy cows to dietary fat, carnitine and niacin and the individual and/or sinergic effect of L-carnitine and niacin. Materials and Methods Two studies were carried out to evaluate the influence of dietary fat, L-carnitine and niacin on milk yield and milk composition in dairy cows. Both studies were carried out in a 4x4 Latin square design; treatments were arranged in a 2x2 factorial arrangement. Each experimental period within the Latin square was 21 day. During the first 14 d of each period, cows were adjusted to experimental diets, and data were collected during the last 7 d of each experimental period. Eight multiparous Holstein cows were assigned to the studies. The cows were housed in a free stall individual paddock sized 5 m x 3 m. The cows milked twice daily at 06:00 and 17:00, using central computerized milking system, and milk yield of individual cows was recorded at each milking. The studies were carried

2 124 Cuban Journal of Agricultural Science, Volume 45, Number 2, out simultaneously in the experimental Farm of the Faculty of Agriculture, the University of Cukurova, Adana-Turkey. Soy oil was used as fat sources in both studies. L-carnitine (Carnipass TM, Lohmann Animal Health Gmbh & Co. KG. Germany) and niacin (nicotinic acid, Ekol Gda tarim Hayvancilik A. S. Turkey) were mixed during the process of concentrate manufacturing. The Carnipass TM contains minimun 18 % of L-carnitine, which was coated with fat. The soy oil, L-carnitine and niacin were supplemented as feed. In the dietary fat and L-carnitine study, treatments were: 1) no dietary fat and no Carnipass (protected L-carnitine), 2) no dietary fat and % carnipass, 3) 3.6% fat and no carnipass, and 4) 3.6% fat and % carnipass. The cows employed in this trial had ±3.12 days in milk, 21.43±0.90 kg/d milk yield, and 467±4.96 kg live weight at the beginning of the experiment. In dietary L-carnitine-niacin study, treatments were 1) no carnipass and no niacin, 2) no carnipass and % niacin, 3) % carnipass and no niacin, and 4) % carnipass and % niacin. The cows averaging 114±10.54 days in milk, yielding 19.6±1.19 kg/day milk and 469±7.37 kg live Ingredients, as fed % No fat weight were used to determine the impact of niacin, L-carnitine and niacin plus L-carnitine on dairy cows fed the diet containing fat. In both studies, the amount of carnitine supplemented to the diets was calculated according to manufacturer s recommendation, considering feed intake and average body weight of the cows. All diets were fed as total mixed ration, and the cows were fed ad libitum. Total mixed rations consisted of 60 % concentrate and 40 % alfalfa hay ground at cm length (tables 1 and 2). The chemical compositions of the diets are presented in table 1 and table 2. Dry matter, crude protein, ether extract and crude ash of experimental diet were analyzed according to standard AOAC (1998) procedures. NDF and ADF content of the diets were analyzed using the methods of Van Soest et al. (1991) in ANKOM fiber analyzer. Metabolizable energy, rumen undegradable protein, Ca and P content of the diets were calculated based on the table values published by NRC (2001). Milk samples were taken from individual cows in the morning milking during the last 7 day each period. Milk fat was determined by Gerber method. Milk samples were also analyzed for dry matter, ash, Table 1. Ingredients and chemical composition of the diets in the dietary fat and L-carnitine study No carnitine Carnitine No carnitine Carnitine Corn Barley Soybean meal Wheat bran Soy oil Vitamin-mineral mixture Dicalcium phosphate Limestone Salt L-Carnitine (Carnipass) Alfalfa hay Chemical composition, (DM basis) DM, % CP, % RUP 2, %CP ME 2, MJ/kg Ether extract, % Crude ash, % NDF, % ADF, % Ca 2, % P 2, % Each kg vitamin mineral mixture provides IU Vit. A, IU Vit D3, mg Vit E, mg Mn, mg Zn, mg Fe, mg Cu, 150 mg Co, 800 mg I, 150 mg Se. 2 RUP: Rumen Undegradable Protein. 2 Calculated based on NRC (2001). Fat

3 Cuban Journal of Agricultural Science, Volume 45, Number 2, milk protein, NPN, and casein nitrogen according to AOAC (1998). The rest of the nitrogen fractions, true protein nitrogen, whey protein, Solids-not-fat (SNF), true protein nitrogen to total nitrogen ratio, casein nitrogen to total nitrogen ratio, were calculated. Whey protein N was calculated as the difference between true protein N and casein N. Solids-not-fat (SNF) was calculated as the difference between total solids and fat. Milk yield and feed intake were measured daily. Milk production efficiency was calculated as milk yield/ dry matter intake. Body weights were determined at the beginning of the experiment and at the end of each period to observe the live weight changes. Data obtained in the studies were analyzed by GLM procedure of SAS (1997) and means were separated by Duncan s Multiple Range Test. Differences between means at P < 0.05 were considered to be significant, and differences between means at P > 0.05 and P < 0.15 were considered to indicate a trend toward a significant difference. Results In the fat-l-carnitine study, fat increased milk yield 45th Anniversary 125 (P < 0.05), improved milk production efficiency (P < 0.01), and tended to increase milk NPN content (P = 0.10, table 3). However, milk fat and protein content were decreased (P < 0.05) markedly by dietary fat. Also, Dietary fat reduced ratio of milk protein N to total N (P = 0.07). Similarly, true protein nitrogen and casein nitrogen were also decreased significantly (P < 0.05) by dietary fat. Body weight change, 4 % fat corrected milk, dry matter intake, milk fat and protein yield were not affected by dietary fat. L-carnitine decreased milk and casein yield significantly (P < 0.05). Milk protein (P=0.08) yield had a tendency to be lower by L-carnitine supplemenmtation. These effects were probably a reflection of the increase in milk yield of the cows fed a diet with fat and no L-carnitine. This resulted in an interaction between dietary fat and L-carnitine (P=0.08). An interaction (P < 0.01) existed between fat and carnitine for nonfat solids of milk for the fat-l-carnitine study. Nonfat solids content in milk was increased markedly by L-carnitine in the cows fed the diet without fat, but it was reduced by L-carnitine in the cows fed the diet containing fat. The reason for this interaction may be Table 2. Ingredients and chemical composition of the diets in L-carnitine-niacin study Ingredients, as fed % No carnitine Carnitine No niacin Niacin No niacin Niacin Corn Barley Soybean meal Wheat bran Soy oil Vitamin-mineral mixture Dicalcium phosphate Limestone Salt L-carnitine (Carnipass) Niacin (nicotinic acid) Alfalfa hay Chemical composition, (DM basis) DM, % CP, % RUP 2, %CP ME 2, MJ/kg Ether extract, % Crude ash, % NDF, % ADF, % Ca 2, % P 2, % Each kg vitamin mineral mixture provides IU Vit. A, IU Vit D3, mg Vit E, mg Mn, mg Zn, mg Fe, mg Cu, 150 mg Co, 800 mg I, 150 mg Se. 2 RUP: Rumen Undegradable Protein. Calculated based on NRC (2001).

4 126 Cuban Journal of Agricultural Science, Volume 45, Number 2, related to reduction of milkk fat and protein contents of dietary fat. In the dietary L-carnitine and niacin study, milk yield tended to be reduced (P=0.12) by L-carnitine. L-carnitine did not have significant effect on milk composition. In addition, the results obtained from the L-carnitine and niacin study indicated that milk yield, milk composition, feed intake, body weight change and other parameters measured were not affected by dietary niacin (table 4). of fat in the diet in the present study were a reflection of higher efficiency of energy utilization of fat (Cant et al. 1993b). The milk protein percentage was significantly reduced by dietary fat (P <0.05), which agreed with several reports (Alzahal et al and Duske et al. 2009). The milk protein content was 0.23 percentage units lower for cows fed high fat than for cows fed low fat (3.21 vs %). Distribution of milk N fractions was affected by dietary fat (Table 3). Dietary fats reduced total N, Table 3. The effects of dietary fat and L-carnitine on nutrient intakes, milk yield and milk composition Fat (F) No Yes Effects (P<) SEM L-Carnitine (LC) No Yes No Yes F LC FxLC BWC, kg/day Milk yield, kg/d % FCM, kg/d DMI, kg/d MPE (Milk yield/dmi) Protein yield, kg/d a Fat yield, kg/d Casein yield, kg/d Milk Composition, % Fat Protein SNF Nitrogen fractions Total N, g/l Protein N, g/l Casein N, g/l Whey N, g/l NPN, g/l Protein N/Total N, % Casein N/Total N, % BWC: Body weight change, FCM: Fat corrected milk, DMI: Dry matterintake, MPE: Milk production efficiency (MPE=milk yield/dmi), SNF: Solids non fat; NPN: Non Protein Nitrogen Discussion In the dietary fat-l-carnitine study, dietary fat increased milk yield approximately 1.73 kg/d (18.8 vs kg/d), decreased milk protein percentage (3.44vs %), but not milk protein yield. Cows fed diets containing fat may increase milk production because of increased energy intake or improved efficiency of utilization of energy (Klusmeyer and Clark 1991). In our study, dietary fat increased milk yield and this result was agreement with most studies (Kim et al. 1993, Maiga et al and Alzahal et al. 2008), although some studies have not observed any increase (Beaulieu and Palmquist 1995 and Cervantes et al. 1996) or decrease (Duske et al. 2009) in milk yield with supplemental fat. Increase in milk yield but no changes in DMI and BWC due to use protein N, and casein N. Milk NPN content of the cows fed the diet containing supplemental fat was tended to be higher than the cows fed low dietary fat. Proportion of milk protein N to total N tended to be lower by dietary fat. Dietary fat may reduce the total N content of milk, particularly the casein fractions (Chow et al and Onetti & Grummer 2006). As mentioned above, a depression in milk protein percentage content occurs when fat is supplemented in the diet (Alzahal et al and Duske et al. 2009). Dietary fat may limit essential minoacid supply to the small intestine by decreasing microbial growth as fat was replaced by fermentable carbohydrates (Palmquist et al. 1993) which is the energy source for rumen microorganisms and can affect partitioning of nutrientdd between mamary glands and other tissues by shifting hormonal balance due to dietary carbohydrates

5 Cuban Journal of Agricultural Science, Volume 45, Number 2, Table 4. The effects of L-carnitine and niacin on nutrient intakes, milk yield and milk composition L-Carnitine (LC) No Yes Effects(P<) SEM Niacin (N) No Yes No Yes LC N LCxN BWC, kg/day Milk yield, kg/d % FCM, kg/d DMI, kg/d MPE (Milk yield/dmi) Protein yield, kg/d Fat yield, kg/d Casein yield, kg/d Milk composition, % Fat Protein SNF Nitrogen fractions Total N, g/l Protein N, g/l Casein N, g/l Whey N, g/l NPN, g/l Protein N/Total N, % Casein N/Total N, % BWC: Body weight change, FCM: Fat corrected milk, DMI: Dry matter intake, MPE: Milk production efficiency (MPE=Milk yield/dmi), SNF: Solid non fat, NPN: Non protein nitrogen. and fat leveles (Cant et al. 1993ab) Futhermore, effect (Hoffman et al. 1991) might play a role in this result as milk production increased by increase in dietary fat. Dietary fat decreased milk fat percentage (3.39 vs %), but did not affect milk fat yield in dietary fat-l-carnitine study (table 3). Similar results were reported by Onetti and Grummer (2006) and Alzahal et al. (2008). Milk fat depression could be also due to the accumulation of trans fatty acids in the rumen during the hydrogenation of unsaturated long-chain fatty acids (Grummer 1991), and inhibition of milk fat synthesis in the mammary gland (Onetti et al. 2002). In the present studies, unprotected soybean oil were used as a fat source, and these reports, related to milk yield, milk fat and protein content, support the findings in the dietary fat-carnitine study. The results of both experiments indicated that L-carnitine supplementation to the diets of midlactation cows decreased milk yield, but did not affect milk composition significantly. Carnitine had no effect on the distribution of N in milk. However, proportion of casein N to total N had a tendency to be higher in the L-carnitine and niacin study, and this could be resulted from the sparing effects of supplemental L-carnitine for lysine and methionine as they used for de novo carnitine synthesis (Borum 1983). It is well known that lysine (Chow et al. 1990) and methionine (Chow et al and Kowalski et al. 2003) are the most limiting amino acids for milk protein synthesis and milk yield. The decreases in milk yields in both experiments may be resulted from negative effect of L-carnitine on apparent digestibility of dry matter, organic matter, crude protein, ADF, NDF, energy and fatty acid digestibility (LaCount et al. 1996a, b). The efficacy of carnitine supplementation for ruminants is not clear within the literature. A brief example would be the results of LaCount et al. (1995, 1996 a, b). LaCount et al. (1995) and Carlson et al. (2006) reported that carnitine supplementation did not alter milk yield. LaCount et al. (1996a, b) were, however, unable to obtain a beneficial response in nutrient digestibilities when carnitine was supplemented in their studies. Our results supported findings of Carlson et al. (2007). They showed that dairy cow received 100 g carnitine d -1 reduced dry matter intake and milk yield. Postruminal L-Carnitine infusion increased milk yield of dairy cows only under restricted feeding condition (Carlson et al. 2006). This increase is stated by decrease in liver total lipid concentration and triglyceride accumulation. The results in the present study were not in agreement with the literature totally, and the contrasting result could be more probably resulted from the form of carnitine (carnipass) used in the present study, which is, embedded to fat to protect from rumen ph and microbial activity. Some factors have been reported that might affect the response of dairy cows to carnitine. LaCount et al.

6 128 Cuban Journal of Agricultural Science, Volume 45, Number 2, (1996a, b) and Carlson et al. (2006) addressed ruminal degradation of carnitine. They observed that the de degradation of carnitine increased when ruminal fluid was from cows that had been adapted to carnitine. La Count et al. (1996b) reportet that degradation fo carnitine increased after carnitine had been provided in the diet for more than two weeks. However, Citil et al. (2009) reported that oral carnitive treatment affected serum triglycerides, cholesterol, urea and glucose concentration of lactating ewes at 3 weeks after the initial carnitive treatment. Additionally, La Count et al. (1996b) reported that diet affected carnitine degradation rate. Carnitine was degraded faster in ruminal fluid from cows that had received high-forage diets (50 %) than in fluid obtained from cows fed in high-grain diet (75 %). The interaction between fat and carnitine were significant for milk SNF only. Milk SNF content the cows fed the diet without fat was increased (8.19 vs %) by L-carnitine, but it was decreased (8.44 vs %) by L-carnitine in the cows fed the diet with fat. This interaction effect on SNF is attributable to changes in milk fat and protein content. Similarly LaCount et al. (1995) found that SNF content was increased by L-carnitine supplementation. In the carnitine-niacin study, niacin did not significantly affect milk yield, milk composition and other parameters of dairy cows in mid lactation. However casein nitrogen to total nitrogen ratio of the cow fed the diet containing niacin was slightly higher than those receiving no niacin. This trend may be related to the sparing effects of niacin for tryptophan used for synthesis of niacin (Horner et al. 1986). Niacin supplementation for lactating cows has increased milk production (Jaster et al and Muller et al. 1986) and alleviated the milk protein depression induced by added dietary fat (Horner et al and Erickson et al. 1992). However some other studies reported no improvement in milk yield and milk composition with niacin (Bernard et al and Taşdemir and Görgülü 1998 and Zimbelman et al. 2010). The DMI were similar for cows fed all diets (Table 4). Some studies have reported similar dry matter intake when carnitine (LaCount et al. 1995, 1996a, b) or niacin (Erickson et al. 1992, Horner et al and Martínez et al. 1991) were fed. Furthermore, NRC (2001) evaluated 30 lactation studies with niacin and reported only one positive response to niacin and no response in the others. Similarly Schwab et al. (2005) investigated niacin supplementation in dairy cow with meta-analysis and they reported that there is a high variability in response to supplemental niacin. Reasons for the highly variable results when niacin was fed to dairy cows might include differences in the composition of the diets, production level of cows, experimental design (long-term, continuous feeding vs. short-term, Latin square design), season of year in which the experiment was conducted as affected by heat stress, type of fat fed, source and amount of protein in the diet, amount of degradable and undegradable crude protein in the diet, and amount of structural, nonstructural carbohydrate in the diet (Christensen et al. 1996) and source of niacin (Jaster and Ward 1990). The results obtained in the studies indicated that dietary fat increased milk yield and milk NPN but decreased milk fat, protein, true protein nitrogen and casein nitrogen in midlactation dairy cows. L-carnitine decreased milk yield significantly and did not prevent the decrease in milk fat test due to use of fat in the diet containing 60% concentrate and 40% of alfalfa hay. Further studies carried out with long term and high number of animal are needed to investigate the effects of L-carnitine supplementation on performance of dairy cows. Acknowledgments The authors wish to thank to Cukurova University Research Fund for financial support and Dr. S. Jacobs, Lohmann Animal Health, Cuxhaven, Germany, for the gift of Carnipass (Protected L-carnitine) and Dr. O. Yucelt, Ekol Gıda Tarim Hayvancilik A.S-Turkey for the gift of niacin (nicotinic acid). References Alzahal, O., Odongo, N.E., Mutsvangwa, T., Or-Rashid, M.M., Duffield, T.F., Bagg, R., Dick, P., Vessie, G. & McBride, B.W Effects of monensin and dietary soybean oil on milk fat percentage and milk fatty acid profile in lactating dairy cows. J. Dairy Sci. 91: 1166 AOAC Official Methods of Analysis. 16th Ed., 4th Ass. Off. Anal. Chem. Washington, D.C. Beaulieu, A.D. & Palmquist, D.L Differential effects of high fat diets on fatty acid composition in milk of Jersey and Holstein cows. J. 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7 Cuban Journal of Agricultural Science, Volume 45, Number 2, when diets high in fat or concentrate are fed. J. Dairy Sci. 73:1051 Christensen, R.A., Overton, T.R., Clark, J.H., Drackley, J.K., Nelson, D.R. & Blum, S.A Effect of dietary fat with or without nicotinic acid on nutrient flow to duodenum of dairy cows. J. Dairy Sci. 79:1410 Citil, M., Karapehlivan, M., Erdogan, H. E., Yucayurt, R., Atakisi, E. & Atakisi, O Effect of orally administered L-carnitine on selected biochemical indicators of lactating Tuj-ewes Small Ruminants. Res. 81:174 DePeters, E.J., Taylor, S.J. & Baldwin, R.L Effect of dietary fat in isocaloric rations on the nitrogen content of milk from holstein cows. J. Dairy Sci. 72:2949 Duske, K., Hammon, H.M., Langhof, A.K., Bellmann, O., Losand, B., Nürnberg, K., Nürnberg, G., Sauerwein, H., Seyfert, H.M. & Metges, C.C Metabolism and lactation performance in dairy cows fed a diet containing rumen-protected fat during the last twelwe weeks of gestation. J. 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