Effect of casein and propionate supply on whole body protein metabolism in lactating dairy cows

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1 Effect of casein and propionate supply on whole body protein metabolism in lactating dairy cows G. Raggio 1, G. E. Lobley 2, S. Lemosquet 3, H. Rulquin 3, and H. Lapierre 4,5 1 Department of Animal Science, Université Laval, Ste-Foy, Quebec, Canada G1K 7P4; 2 Rowett Research Institute, Aberdeen, UK, AB21 9SB; 3 INRA, UMR Production du Lait, Saint-Gilles, France; 4 Dairy and Swine Research & Development Centre, Agriculture and Agri-Food Canada, Lennoxville, Quebec, Canada J1M 1Z3. Received 21 June 2005, accepted 14 November Raggio, G., Lobley, G. E., Lemosquet, S., Rulquin, H. and Lapierre, H Effect of casein and propionate supply on whole body protein metabolism in lactating dairy cows. Can. J. Anim. Sci. 86: The effects of Casein (Cas) and propionate (C3) on whole body (WB) leucine (Leu) metabolism were determined in three multiparous Holstein cows, fitted with both duodenum and rumen cannulas, used in a Youden replicated square with 14-d periods. Cows were fed a grass silage-based diet estimated to supply MJ d 1 of NE L and 1593 g d 1 of protein digested in the intestine. Cas (743 g d 1 in the duodenum) and C3 (1041 g d 1 in the rumen) infusions were tested in a factorial arrangement. For each period, on day 11, L[1 13 C] leucine (4.3 mmol h 1 ) and on day 13, [ 13 C] sodium bicarbonate (4.2 mmol h 1 ) were infused into a jugular vein. Blood samples were taken from a carotid artery to measure enrichments of 13 CO 2 (days 11 and 13) and of 13 C[4-methyl 2-oxopentanoate] (MOP, day 11). Both Cas and C3 treatments separately increased milk protein concentration and yield, but only Cas treatments increased milk yield (18%). The overall increments in Leu irreversible loss rate (ILR) (26%), oxidation (146%), protein synthesis (15%), and output in milk protein (21%) suggest a general response in protein turnover to Cas treatments. C3 treatments tended to increase Leu WB ILR (5%), protein synthesis (7%) and in milk (7%), with a tendency for a Cas C3 interaction on WB Leu oxidation. The latter suggests that the impact of energy on protein metabolism depends on the level of protein supply. Key words: Dairy cow, leucine, casein, propionate, whole body irreversible loss rate. Raggio, G., Lobley, G. E., Lemosquet, S., Rulquin, H. et Lapierre, H Effet de la caséine et du propionate sur la métabolism proteique corporel de la vache laitière. Can. J. Anim. Sci. 86: Les effets de la caséine (Cas) et du propionate (C3) sur le métabolisme corporel de la leucine (Leu) ont été étudiés sur trois vaches Holstein multipares, possédant des canules ruminale et duodénale, selon un double carré de Youden avec des périodes expérimentales de 14 jours. Les vaches recevaient une ration à base de balles rondes enrubannées, apportant MJ jour 1 EN L et 1593 g jour 1 de protéines digestibles dans l intestin. Les effets d infusions de Cas (743 g jour 1 au duodénum) et de C3 (1041 g jour 1 au rumen) ont été étudiés selon un plan factoriel. À chaque période, au jour 11, de la L[1-13 C]Leu (4.3 mmol h 1 ) et au jour 13, du [ 13 C]sodium bicarbonate (4.2 mmol h 1 ) ont été perfusés dans une veine jugulaire. Des prélèvements sanguins carotidiens ont été effectués pour mesurer l enrichissement isotopique du 13 C[4-methyl 2-oxopentanoate] (MOP, jour 11) et du 13 CO 2 (jour 11 & 13). Les traitements de Cas et de C3 ont augmenté la concentration et le rendement en protéines du lait, mais seuls les traitements de Cas ont augmenté le volume de lait (18%). L augmentation du flux corporel de la Leu (26%), de son oxydation (146%), de son utilisation pour la synthèse protéique (15%) et sa sécrétion dans la protéine du lait (21%) suggère une stimulation générale du métabolisme protéique par les traitements Cas. Les traitements C3 tendent à augmenter le flux corporel de Leu (5%), la synthèse protéique (7%) et la Leu sécrétée dans le lait (7%), avec cependant une tendance à une interaction entre Cas et C3 sur l oxydation corporelle de la Leu. Ceci suggère que l impact d une supplémentation en protéines sur le métabolisme protéique est tributaire du niveau d énergie fourni à l animal. In dairy cows, milk protein yield can be manipulated by varying either energy or protein intakes (de Peters and Cant 1992). These responses may reflect changes in whole body (WB) protein metabolism (including synthesis and oxidation), as observed when total food supply is altered in growing cattle (Lobley et al. 1987; Lapierre et al Of more interest is to know whether, and how, individual macronutrients, particularly energy or protein, might impact on protein metabolism and animal performance. This is important because, in mechanistic terms, either protein/amino acids 5 To whom correspondence should be addressed ( lapierreh@agr.gc.ca). Mots clés: Vache laitière, leucine, caséine, propionate, flux corporel. 81 (AA) or energy (glucose or precursors such as propionate) may act differently. In the non-lactating state, there appears to be consistent responses in WB metabolism to increased protein supply across a wide range of species. For example, in sheep, infusion of casein into the abomasum ( Liu et al. 1995) elevat- Abbreviations: AA, amino acid; C3, propionate; CER, carbon dioxide entry rate; Cas, casein; IE, isotopic enrichment, ILR, irreversible loss rate; FO, fractional oxidation; MOP, 4-methyl 2-oxopentanoate; PS, protein synthesis; WB, whole body

2 82 CANADIAN JOURNAL OF ANIMAL SCIENCE ed WB protein turnover, as did increased protein intake in humans (Garlick et al. 1991). In such cases, the increased WB protein turnover was the result of a combined increment in both oxidation and protein synthesis. In contrast, for the lactating dairy cow, changes in WB protein metabolism appear to be less consistent. For example, WB leucine kinetics can be unaffected by an increment in crude protein supply (Bequette et al. 1996a), numerically increased with a caseinate infusion (Oldham et al. 1980) or significantly improved with additional metabolisable protein supply (Lapierre et al. 2002). The reasons for these different responses are unclear and need to be understood in order to build better predictive models of dairy cow metabolism. The effect of energy on WB protein metabolism is even more unclear. Compared with the effect of additional protein supply, supplementation of pigs with non-protein energy sources increased WB protein synthesis to a lesser extent, but reduced leucine catabolism (Reeds et al. 1981). Similarly, in humans, energy also had a smaller effect on WB protein turnover than did protein supply (Garlick et al. 1991), while in sheep, Abdul-Razzaq and Bickerstaffe (1989) observed a reduced protein turnover with infusion of propionic acid, linked with decreased protein breakdown. These data in other species show that although energy supply can potentially impact protein metabolism, the exact mechanisms may vary with experimental conditions. Although no data are available on the effect of energy supply on protein kinetics in dairy cows, milk represents an important export protein and is probably associated with increased rates of protein synthesis, at least for the mammary gland. Increasing energy supply either through infusions of propionic acid into the rumen or glucose into the duodenum can elevate milk protein output (Hurtaud et al. 1998a). Such responses must involve more efficient use of dietary AA supply, i.e. decreased oxidation. In ruminants, the major glucogenic source is propionate (McBride et al. 1998). Besides acting as a precursor for glucose, in vitro studies demonstrated that this nutrient can be a potential inhibitor of the other glucogenic pathways (Demigné et al. 1991) leading to sparing effect on AA for a potential increase in milk protein output. This mechanism, that should be linked to decreased ureagenesis, has not been convincingly demonstrated in vivo in sheep (Kim et al. 1999). In terms of interactions, none were observed between glucose and protein supply on protein milk yield (Clark et al. 1977; Vanhatalo et al. 2003) and, similarly, a combination of glucose and histidine showed additive responses (Huhtanen et al. 2002). Surprisingly, the interaction between the main glucose precursor, propionate, and protein has not been tested. In an attempt to resolve some of the current uncertainties about the separate and interactive effects of protein and propionate on metabolism in the lactating cow, the current study was designed to examine the impact of casein and propionic acid supply on milk production, and on mammary and WB kinetics of leucine and glucose. This research describes the findings that relate to WB leucine metabolism. MATERIALS AND MEtHODS Animals Three multiparous Holstein cows, averaging 615 ± 24 kg BW and 65 ± 4 d in milk at the beginning of the study, had been fitted, before calving, with both a proximal T-shaped duodenal cannula, placed 10 to 15 cm from the pylorus, and a ruminal cannula (Hurtaud et al. 1998b). One month before the beginning of the experiment, the cows were further prepared with two permanent catheters (Polyurethane catheter tubing, i.d. 1.0 mm, o.d. 1.7 mm, UNO, Roestvaststaal BV, Holland), one inserted in the right carotid artery and the other in the right subcutaneous abdominal vein as described previously (Guinard et al. 1994). These two permanent catheters were protected in a silicone rubber tubing (Silclear TM grade medical silicone tubing, i.d. 1.57, o.d mm, VWR International SAS, Briare, France). One ring of Dacron (Mersutures, TS53, Ethicon, France) was placed around the catheters at the point of exteriorisation to prevent infection. During the experiment, problems were encountered with one arterial catheter and a replacement was inserted in the left carotid artery. For the tracer infusions, a silicone rubber catheter (length, 21 cm; i.d., 1.02 mm; o.d., 2.16 mm; Silclear tubing, Degania, Israel) was inserted in the left jugular vein before the beginning of the experiment. Catheter maintenance was performed as described previously (Guinard et al. 1994). The experimental procedures were reviewed and approved by the Animal Care Committee of the French Ministry of Agriculture. Feeding and Treatments The diets were formulated with the INRA model (Institut National de la Recherche Agronomique 1989). The same diet, 55% grass silage and 45% concentrate (on DM basis: 54% peas; 30% dried sugar beet pulp; 1% cane molasses; 4% soybean oil; 4% NaHCO 3 ; 3% CaCO 3 ; 3 % CaHPO 4, 1% mineral and vitamin premix), was fed to all the cows (Table 1). The basal diet supplied 124 MJ d 1 NE L and 1593 g d 1 of protein digested in the intestine (PDI: INRA 1989) to the cows on the control treatment, equivalent to 97% of energy and protein requirements. When estimated using NRC (2001), the supply on the basal diet was 119 MJ d 1 NE L and 1624 g d 1 metabolizable protein. This diet, the same as that used by Rigout et al (2003), was estimated to supply little intestinal glucose. The quantity of wet feed offered was adjusted every day to insure the same delivery of DM on each experimental day, after determination of DM content of the grass silage. Superimposed on the basal diet, four treatments were tested in a factorial arrangement: duodenal infusion of calcium caseinate (743 ± 7 g d 1 : 687 g d 1 PDI) estimated to provide 7.9 MJ d 1 of NE L (Guinard et al. 1994) and/or ruminal infusions of propionic acid (1042 ± 8 g d 1 ) estimated to provide 15.6 MJ d 1 of NE L, as used in Rigout et al. (2003). Therefore, treatments were: (1) control (Ctrl), (2) casein (Cas), (3) propionic acid (C3) and (4) the casein and propionic acid combined (Cas + C3). The infusion dose of casein was the highest dose used by Guinard et al. (1994), but within the range of protein supply for a dairy ration. Propionate was chosen as the energy source as it is the primary glucose precursor in dairy

3 RAGGIO ET AL. CASEIN AND PROPIONATE ON PROTEIN METABOLISM IN DAIRY COWS 83 Table 1. Chemical composition of the feed ingredients Analysis (g kg DM 1 ) Grass silage Concentrate CP NDF ADF Lignin Fat Organic matter NDF-N z ADF-N y NE L (MJ kg DM 1 ) z Neutral detergent fiber insoluble nitrogen. y Acid detergent fiber insoluble nitrogen. cow diets and should have the strongest effect on sparing AA for gluconeogenesis. It is also less metabolically characterized than glucose in dairy cows. Propionate was infused at the dose that yielded the maximal effect on milk protein observed by Rigout et al. (2003). The original design was a 4 4 Latin square, but the loss of one cow led to a re-design of the experiment as two incomplete 4 3 Youden squares with three periods each for a total of 18 observations distributed as follows: Ctrl, n = 5; Cas, n = 4; C3, n = 5; Cas + C3, n = 4. One cow had problems with the arterial catheter during her third period (Cas treatment), therefore, n = 3 for Cas for the blood measurements. Each treatment period lasted 14 d, and the first 2 d of each period included transition for the infusions (the cows received on day 1, 0.33, and on day 2, 0.66 of the total subsequent infusion). The casein infusate (Armor Proteins, St Brice en Cobles, France) was prepared daily at 30 C in 15 kg of tap water. The dose of C3 (Langlois S.A., Saint Jacques de la Lande, France) was dissolved daily in 40 kg of tap water. A buffer solution (540 ± 4 g of NaHCO 3 plus 280 ± 2 g of KHCO 3 ) was prepared daily in 10 kg of tap water. This buffer was used to limit ruminal ph decrease with C3 to avoid acidosis and the buffer was infused in all cows to maintain constant between treatments the anion-cation balance. Urea (average daily supply of 99 g) was added to this buffer solution as a dietary supplement. All the cows in all treatments received a similar volume in the rumen (total of 49.5 ± 0.6 kg including 9.95 ± 0.07 kg of buffer and 39.5 ± 0.7 kg of water or propionic acid solution) and in the duodenum (14.8 ± 0.14 kg of water or casein solution). These solutions were infused continuously by means of peristaltic pumps. The concentrate was supplied every 3 h in equal portions from automatic feeders, starting at Grass silage was fed three times per day: 25% at 0715, 25% at 1315, 50% between 1715 and 1915, except during the kinetic days (days 11 and 13) when the silage was fed four time per day: 12.5% at 0715, 1015, 1315 and 1615 and then 50% at Throughout the whole study, access to the diet was limited to 1 h after the concentrate distribution times. Cows had free access to water and were housed in individual tie stalls. Every day before 0715, any feed refusals were weighed. Sampling and Laboratory Analyses Cows were milked twice daily (0630 and 1830) with yield recorded at each milking, and each sample was assayed for fat and true protein compositions by infrared analysis (Milkoscan, Foss Electric, Hillerød, Denmark). During the second week of the experimental period, cows were milked from each half udder separately and milk samples (each half udder) were assayed for fat and protein composition by infrared analysis. Samples of the grass silage and concentrate mixture were taken every day. The DM of the grass silage was determined on daily samples, while the DM of the concentrate was determined every week on a pooled sample by oven drying at 80ºC for 48 h. Feed samples for other analyses were stored at 20ºC until analysis, where they were pooled by period. On day 11, L[1-13 C] leucine (Cambridge Isotope Laboratories, Andover, USA. 99 ape) was infused at 4.3 mmol h 1 (14.20 mg ml 1 dissolved in sterile 0.9% NaCl; 0.66 g solution min 1 ) for 7.5 h (starting 3.5 h after the morning milking), preceded by a priming dose of 4.3 mmol, equivalent to an hour of infusion (Wolfe 1984). On day 13, a mixture (in sterile 0.9% NaCl) of [1-13 C] bicarbonate (Cambridge Isotope Laboratories, Andover, USA. 99 ape; 4.7 mg ml 1 ) and [6.6-2 H 2 ] glucose (Cambridge Isotope Laboratories, Andover, USA. 99 ape; 60 mg ml 1 ) was infused. Within this mixture the rate of [1-13 C] bicarbonate infusion was 4.2 mmol h 1 (1.26 g solution min 1 ) for 4 h between 1200 and 1600, preceded by a priming dose equivalent to 3.2 mmol. Glucose kinetics will be described in a separate paper. On both days, cows were made to stand for at least 15 min before blood sampling. On day 11, hourly samples were taken from the carotid artery starting 3 h after the initiation of the infusion at 1000 (1300, 1400, 1500, 1600 and 1700) and were analyzed for isotopic enrichment (IE) of leucine, 4-methyl 2-oxopentanoate (MOP) and of CO 2. On the same day, samples for AA analysis were taken at 0700, 0900, 1100, 1300, 1400, 1500, 1600 and 1700 and for urea at 0700, 0900, 1100, 1300, 1500 and On day 13, samples were collected starting 1.45 h after the initiation of the infusion at 1200 (1345, 1430, 1515 and 1600) and were analyzed to determine the IE of CO 2. On both days 11 and 13, samples were also collected prior to the initiation of stable isotope infusion to determine natural abundance of leucine, MOP (two samples: 0700 and 0900) and CO 2 (three samples: 0800, 0900 and 1000). Blood samples (7.5 ml) for AA concentrations were collected in heparinized syringes and kept on ice. Plasma was prepared by centrifugation (2000 g at 4ºC for 15 min), 0.7 g plasma was weighed, then 0.4 g of the internal standard solution was added and the mixture was stored at 80 C. The internal standard solution was prepared at the following concentrations: 501 nmol g 1 [5-15 N] Gln, 252 nmol g 1 [indole- 15 N] Trp, 260 nmol g 1 [1-13 C]Cys, 8.8 µmol g 1 [ 15 N 2 ] Urea, plus a U- 13 C Algal hydrolysate. This follows the methodology for determination of AA concentration by isotope dilution as previously described (Calder et al. 1999). Plasma samples for urea were pooled per cow and period and stored at 20ºC until analysis on a multiparameter analyzer (KONE Instruments Corporation, Espoo, Finland) using an enzymatic kit (KONE, urea UV kinetic kit no , KONE diagnostics, KONA Instruments S.A., Evry, France)

4 84 CANADIAN JOURNAL OF ANIMAL SCIENCE For MOP and [1-13 C]leucine IE (expressed as molar percent excess, mpe), 0.7 g plasma was weighed and 0.3 g of an oxohexanoic acid solution (33.3 nmol g 1 ) added, and the mixture was stored at 80ºC until analysis. Determinations of MOP and leucine IE were as described by Calder and Smith (1988). For CO 2 IE, triplicate 1 ml of blood samples were injected into evacuated Vacutainers containing 1 ml of lactic acid, immediately mixed and kept at room temperature until analysis was performed. Determination of IE (mpe) was as previously described (Lobley et al. 2003) based on Read et al. (1984) and Reeds and Hutchens (1994). Calculations In all equations, WB irreversible lost rate (ILR) and infusion rates are expressed in mmol h 1 and IE in mpe. Tracer refers to 13 C-leucine or 13 C-bicarbonate and tracee is the unlabelled leucine and bicarbonate. Whole body leucine ILR or carbon dioxide entry rate (CER) was calculated as follows: WB-ILR tracee or CER tracee = (IE inf / IE a 1) Inf where IE inf and IE a are the IE of the infusate and the mean enrichment of arterial plasma MOP, leucine or 13 CO 2, respectively and Inf is the infusion rate of the 13 C-leucine or 13 C-bicarbonate. Leucine tracer oxidation (mmol h 1 ) was calculated as: Leu tracer oxidation = CER IE a 13 CO 2 -Leu Where CER is the CO 2 entry rate measured during bicarbonate infusion (described above) and IE 13 CO 2 -Leu is the mean arterial enrichment of 13 CO 2 during leucine infusion. For this calculation, it is assumed the CER production during the bicarbonate infusion day is the same as during the day of leucine infusion. The fractional oxidation (FO) was calculated as: FO = Leu tracer oxidation/(inf 13 C-Leu IE inf ) For tracer kinetics, it is assumed that the animal does not distinguish between 13 C- and 12 C-AA and it is further assumed that all leucine tracer infused is in excess and that a corresponding amount of leucine will be oxidized by the animal (Lobley et al. 2003). Therefore to calculate the WB leucine oxidation (WB-LO) WB-LO = FO (ILR tracee + Inf ) Inf Leucine used for WB protein synthesis was calculated as the difference between WB leucine ILR and leucine oxidation. The conversion into protein (g d 1 ) was based on 63 g of leucine kg 1 of synthesized tissue protein (Lobley et al. 1980) and 98 g of leucine kg 1 of CP in milk (Swaisgood 1995). Statistical Analyses Analyses of variance were made using the MIXED procedure of SAS (SAS Institute, Inc. 1999) according to the experimental plan using the following statistical model: y ijkl = µ + SQUARE i + PERIOD j (SQUARE i ) + COW k + TREAT l + e iikl Differences among treatments were compared using orthogonal contrasts according to the factorial arrangement. Results are expressed as least square means with the highest standard error of mean (SEM). RESULTS Dry matter intake, milk production and composition reported are the average of the second week of experimental periods (Table 2). The DMI was not affected by infusions. There was no interaction between Cas and C3 on milk parameters. Milk yield increased with Cas treatments (P < 0.01). Milk fat concentration (P = 0.04) and yield (P = 0.02) were reduced with C3 infusions, while fat yield increased with Cas infusions (P = 0.001). True protein concentration increased with Cas (P < 0.01) and C3 (P = 0.01), as did true protein yield with Cas (P < 0.001) and C3 infusions (P = 0.02). Similar results for protein concentration (data not shown) and yield (see leucine in milk, Table 3) were obtained on the day of WB leucine kinetics. The WB ILR (Table 3), using arterial MOP IE as representative of the precursor pool, increased with Cas treatments (P < 0.001) and tended to increase with C3 (P = 0.06). With arterial leucine IE selected as precursor, only Cas treatments remained significant (P < 0.001). Absolute ILR values were, on average, 25% lower with arterial leucine than MOP as precursor, due to the higher IE of leucine. There was a tendency for an interaction (P = 0.09) between Cas and C3 on leucine oxidation, with a slight increase when C3 was added to the Ctrl diet, but a decrease by 14% when C3 was added to the Cas infusion. There was a similar trend (P = 0.07) for fractional oxidation. Leucine used for WB protein synthesis increased with Cas treatments (P < 0.01), both for milk protein (P < 0.001) and non-milk protein synthesis (tissues; P = 0.02). Leucine used for total protein synthesis tended (P = 0.08) to increase with C3 treatments, but the effect was due to changes in leucine for milk protein (P = 0.06). Whole body protein synthesis followed the same trend as leucine used for protein synthesis and averaged 4.1, 4.6, 4.2 and 5.1 kg d 1 for Ctrl, Cas, C3 and Cas + C3, respectively. Data for blood bicarbonate IE during the [1-13 C] leucine and [NaH 13 CO 3 ] infusion and the CER are presented in Table 4. The blood 13 CO 2 IE during the NaH 13 CO 3 infusion was reduced with both Cas (P = 0.02) and C3 (P = 0.02) supplementation. In consequence, the CER increased with Cas (P < 0.01) and C3 (P < 0.01) treatments. In contrast, the 13 CO 2 enrichment measured during [1-13 C] leucine administration increased with Cas infusions (P < 0.01) with a tendency (Cas ( C3 interaction, P = 0.08) for a smaller increase when Cas was added to C3 infusion. Amino Acid and Urea Concentrations Arterial AA concentrations are presented in Table 5. With Cas infusions arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine increased (P < 0.05), while gluta-

5 RAGGIO ET AL. CASEIN AND PROPIONATE ON PROTEIN METABOLISM IN DAIRY COWS 85 Table 2. Effect of casein (Cas) and propionate (C3) supply on DMI, milk yield and composition Treatment Ctrl Cas C3 Cas + C3 SEM z Cas C3 Cas C3 DMI (kg d 1 ) Milk (kg d 1 ) < Fat (g kg 1 ) (g d 1 ) < True protein (g kg 1 ) <0.01 < (g d 1 ) < z Least square means presented with pooled SEM, given for n = 4; based on the second week from the total mammary gland (18 observations; Ctrl = 5, Cas = 4, C3 = 5, Cas + C3 = 4). y Probability corresponding to the null hypothesis with Cas, C3 and Cas C3 contrasts. P y Table 3. Effect of casein (Cas) and propionate (C3) supply on whole body (WB) leucine kinetics Treatment Leucine (mmol h 1 ) Ctrl Cas C3 Cas + C3 SEM z Cas C3 Cas C3 From the infusion x WB ILR MOP w < WB ILR LEU w < Oxidation < WB protein synthesis, PS < Milk v < WB non-milk PS u Fractional oxidation t < z Least square means presented with pooled SEM, given for n = 3; 17 observations: Ctrl = 5, Cas = 3, C3 = 5, Cas + C 3 = 4. y Probability corresponding to the null hypothesis with Cas, C3 and Cas C3 contrasts. x Leucine from the infusion calculated as 743 g casein purity digestibility (purity as 99%, digestibility as 90%). w ILR, irreversible loss rate, calculated with the arterial enrichment of 4-methyl 2-oxopentanoate (MOP) or leucine (LEU) as representative of the precursor pool. v Leucine in milk was based on milk production on the half-day of leucine infusion. u WB non-milk PS = WB protein synthesis leucine in milk. t Fractional oxidation:leucine Oxidation/WB ILR MOP. Table 4. Effect of casein (Cas) and propionate (C3) supply on 13 CO 2 isotopic enrichment (IE), carbon dioxide entry rate (CER) and carbon balance Treatment P y Ctrl Cas C3 Cas + C3 SEM z Cas C3 Cas C3 13 CO 2 IE, during leucine inf, mpe <0.01 < CO 2 IE, during bicarbonate inf, mpe < CER (mol h 1 ) <0.01 < Extra carbons x Intake carbons (mmol h 1 ) Extra carbons CER (mmol h 1 ) Extra carbons in milk (mmol h 1 ) % accounted z Least square means presented with pooled SEM, given for n = 3; 17 observations; Ctrl = 5, Cas = 3, C3 = 5, Cas + C3 = 4. y Probability corresponding to the null hypothesis with Cas, C3 and Cas C3 contrasts. x Extra carbons was calculated as Cas inf = CasCER CtrlCER or C3inf = C3CER CtrlCER and Cas + C3inf = (CasCER CtrlCER) + (C3CER CtrlCER). P y mate and glycine decreased (P < 0.01). During C3 infusion, glutamine and serine (P 0.01) and threonine, tryptophan and tyrosine (P 0.04) increased but isoleucine, leucine and valine (P < 0.01) decreased, with a similar tendency (P = 0.09) for lysine. There was also a trend (P = 0.10) for an interaction between Cas and C3 on the concentrations of the branched-chain AA with a greater increase when Cas was added to the Ctrl diet compared with Cas + C3 responses. Arterial concentrations of urea increased (P < 0.001) with Cas infusions but decreased (P = 0.03) with C3 (Table 5). DISCUSSION In the present experiment, C3 and Cas infusions had different impacts on milk yield and fat yield but both nutrients increased protein concentration and protein yield, albeit to different extents. The kinetic data obtained allow some of

6 86 CANADIAN JOURNAL OF ANIMAL SCIENCE Table 5. Effect of casein (Cas) and propionate (C3) supply on plasma arterial concentration of amino acids and urea Treatment Amino acids (µm) Ctrl Cas C3 Cas + C3 SEM z Cas C3 Cas C3 Essential Histidine < Isoleucine <0.001 < Leucine <0.001 < Lysine < Methionine < Phenylalanine < Threonine Tryptophan Valine <0.001 < Non-essential Alanine Arginine < Aspartate Cysteine < Glutamine < Glutamate < Glycine < Proline < Serine Tyrosine < Urea (mm) < z Least square means presented with pooled SEM, given for n = 3; 17 observations: Ctrl = 5, Cas = 3, C3 = 5, Cas + C3 = 4. y Probability corresponding to the null hypothesis with Cas, C3 and Cas C3 contrasts. the WB interactions on protein turnover to be considered in the light of these various modifications of milk yield and composition. Milk Production and Composition The increment in milk production with Cas treatments (18%) is at the upper end of numerous previous reports (Clark et al. 1977; Rulquin 1982; Guinard et al. 1994; Choung and Chamberlain 1995; Vanhatalo 2003). As a result of the improved milk yield at similar fat concentrations, milk fat yield increased with Cas infusions, confirming earlier reports (Guinard et al. 1994; Vanhatalo et al. 2003). Milk protein yield increased as previously observed (e.g., Guinard et al. 1994) with Cas infusion, with approximately 25% of the infused casein recovered as milk protein. The observed increments in milk and protein yields indicated that, as designed, the control diet was not optimal to support maximum milk yield. In the present experiment Cas and Cas + C3 treatments provided 121 and 112.5% of protein requirements, compared with 97% for the control diet. The marginal casein recovery (25%) is in the range of the 30% reported by INRA (1989) for such a protein supply. It is also within the range from previous studies (21% ± 15; see Hanigan et al. (1998) with casein infusion, and similar to that observed for increment in metabolisable protein supply from 1922 to 2517 g d 1 (19%; Raggio et al. 2004). Differences in the actual recoveries between studies probably relate to how close the basal rations were to the requirements of the animal. Less energy was transferred into milk lactose and fat from C3 infusions compared with Cas treatments because C3 infusions did not change milk yield and decreased both milk fat concentration and yield. The milk fat concentration response was similar to other experiments when the cows received C3 infusions into the rumen, as summarized by Rigout et al. (2003). This earlier study also reported a modest improvement (0.7 kg d 1 ) in milk yield, with dairy cows receiving 115% of recommended protein allowances. A similar numerical increase was observed in the current experiment when C3 was added to Cas. This occurred despite the fact that in the current study the increased milk yield was seen when the C3 was added to the Ctrl diet, while in Rigout et al. (2003) the response occurred when C3 was substituted for a similar amount of energy infused as volatile fatty acids for the control cows. Protein in milk also increased (6%) with C3 infusions. This increment is smaller than with Cas treatments, but is in accordance with previous experiments (see Rigout et al. 2003). In the present study, there were modest increases in milk protein yield with C3 infusions in cows receiving protein supply below (97%) or above (113%) requirements. This suggests that part of the C3 effect on milk protein yield links to a change in WB protein metabolism rather than major effects on intestinal AA availability. This effect may be due to an increase of mammary blood flow favoring AA uptake as already seen with duodenal glucose infusion (Rulquin et al. 2004). It may also be due to less AA used as glucose precursors with, in consequence, more available for milk protein production. Whole Body Leucine Kinetics The WB ILR increased with both Cas and C3 infusions. Whole body ILR is known to be sensitive to the inflow of P y

7 RAGGIO ET AL. CASEIN AND PROPIONATE ON PROTEIN METABOLISM IN DAIRY COWS 87 Fig. 1. Effect of extra leucine intake on leucine oxidation, leucine in milk and leucine in tissues. Extra leucine intake: leucine intake in Cas and Cas + C3 infusion. Leucine-milk: was calculated as leucine in milk in Cas Ctrl and (Cas + C3) C3 infusion. Leucine-oxidation: was calculated as leucine oxidation in Cas Ctrl and (Cas + C3) C3 infusion. Leucine-tissues: was calculated as (leucine intake) (leucine milk + leucine oxidation). nutrients such as different MP supply in dairy cows (Lapierre et al. 2002) and at different intake levels in both growing beef steers (Lobley et al. 1987; Wessels et al. 1997; Lapierre et al. 1999) and sheep (Liu et al. 1995; Savary- Auzeloux et al. 2003). In terms of overall protein dynamics what do these changes in WB ILR mean? For essential AA, such as leucine, ILR is equal to protein synthesis plus oxidation and also to protein digested plus protein breakdown. So changes in WB ILR must reflect alterations in at least two of these processes. In the current study, the increase in WB ILR with casein infusion exceeded the additional leucine supply, similar to previous reports in dairy cows (Lapierre et al. 2002) and growing pigs (Reeds et al. 1980), indicative of a stimulation in tissue protein metabolism. Thus, despite increased leucine oxidation, there was an increase in protein synthesis, linked to the improved milk protein yield. Similarly, increases in both WB protein synthesis and oxidation in response to casein supply have been observed in goats (Bequette et al. 1996b) and sheep (Liu et al. 1995) with the response hypothesized as mediated via hormonal actions involving insulin and insulin like growth factor-1 (Bequette et al. 2002). However, in a similar study on dairy cows, with the same rate of infusiont (762 g d 1 ) of casein, there was no change in arterial plasma insulin concentrations (Guinard et al. 1994). The impacts of C3 were weaker, with tendencies to increase WB leucine ILR and decrease oxidation when infused with Cas. The pattern of urea concentrations followed that of leucine oxidation, i.e. a large increment from casein infusion, while C3 was effective in reducing oxidation only when the animals were concomitantly infused with Cas. To our knowledge, there are no reports on the impact of C3 on WB IRL of AA in lactating ruminants. However, in sheep, Abdul-Razzaq et al. (1989) observed a reduced leucine oxidation with C3 infusion, but only in comparison with acetate supplementation. In the current study, there was a tendency for WB protein synthesis to be increased by C3, with this trend driven by the significant improvement in milk protein output. Three mechanisms may contribute to this tendency. First, arterial concentrations of branched-chain AA were reduced by C3 infusions, as already observed with C3 and glucose infusions (Clark et al. 1978; Lemosquet et al. 2004). This reduction in the branched-chain AA is often linked to an increase in insulin (Griinari et al. 1997; Gross et al. 1990; Mackle et al. 2000), which could allow diversion of more substrate for milk protein synthesis. Second, C3 effects on milk protein may also be due to less AA used for glucose synthesis with, in consequence, more available for milk protein production. During the C3 infusions, plasma concentration of some glucogenic AA and two essential AA (threonine and tryptophan) increased as previously observed (Lemosquet et al. 2004). Threonine is a glucogenic AA (via serine) and may be spared if other sources of glucose synthesis are available. Substantial quantities of tryptophan are removed across the gut (Lobley et al. 2003), as is propionate to fuel the energy needs of this tissue (Seal and Parker 1994). In steers, intraruminal infusion of propionate (1 mol d 1 ) led to increased tryptophan absorption and higher arterial concentration (Seal and Parker 1996). A third possibility is increased uptake of essential AA as observed with duodenal glucose infusion in dairy cows (Rulquin et al. 2004), a process driven mainly through increased mammary plasma flow (Bequette et al. 2001; Vanhatalo et al. 2003; Rulquin et al. 2004). Overall, although both energy and protein independently altered WB leucine ILR and milk protein output, there were subtle differences in the mechanisms involved. Casein exerted a much stronger impact on WB protein synthesis, with 30 45%

8 88 CANADIAN JOURNAL OF ANIMAL SCIENCE of the increment directed towards extra milk protein. As total mammary gland protein synthesis has been reported to average 1.3 times milk protein output (Bequette et al. 1996a; Thivierge et al. 2002), not only was mammary gland protein synthesis increased by casein supply, but other tissues must also have responded. In contrast, C3 had a smaller effect on protein synthesis and reduced oxidation of leucine only when added to Cas and thus helped increased milk protein yield further. This may be through changes in the hormone milieu, increased energy supply to support mammary gland metabolism or from sparing of AA use for glucogenic purposes. Some of these issues will become clearer from the companion measurements on glucose and mammary gland metabolism. Efficiency of nutrient use can be considered from the leucine net balance (Fig. 1). Any extra AA infused has three main net fates; oxidation, conversion into milk protein and retention in the body (mainly as tissue gain). The infusion of casein increased leucine absorption by 20.9 mmol h 1, with part recovered as extra milk protein (5.1 mmol h 1 with Cas and 5.3 mmol h 1 with Cas + C3 infusions). Most of the remainder was oxidized, 13.9 and 10.4 mmol h 1 for Cas and Cas + C3, respectively. The slight increased transfer into milk with Cas + C3 was more than compensated by less oxidation and, in consequence, net balance increased from 1.9 to 5.1 mmol h 1 (obtained by difference). The actual values, but not the partition, depend on the assumption that plasma MOP truly reflects the precursor for both oxidation and protein synthesis. Carbon Balance Casein and C3 had different impacts on carbon efficiency (i.e., conversion of infused C to milk C). Less of the C3 infused was converted into milk products as milk yield did not increase and milk fat decreased. Thus, the carbon balance showed that only 5% (0.08 mol C h 1 ) of infused (1.74 mol C h 1 ) C3 appeared in milk, while 77% (1.4 mol C h 1 ) appeared as extra CO 2 production. The remaining carbon, 18% (0.31 mol C h 1 ), may be incorporated into body tissues other than mammary epithelial cells (e.g., liver fat) or may represent measurement error. In contrast, for casein infusion alone (0.970 mol C h 1 ), 25% (0.24 mol C h 1 ) of the carbon intake appeared in milk with 73% (0.70 mol C h 1 ) oxidized to CO 2, the remaining 2% may represent measurement error. The incremental ratio milk carbon / intake carbon was 0.12 for Cas + C3, therefore better C efficiency was obtained from Cas supply alone. Across all treatments, most of the infused extra carbon was recovered (90%) either as CO 2 or milk products. CONCLUSION While both Cas and C3 independently increased WB leucine ILR, protein synthesis and leucine exported in milk, the magnitude of the responses differed, being greater with Cas. Furthermore, Cas treatments increased oxidation while C3 decreased leucine oxidation when infused in the presence of casein. Cas increased the arterial concentrations of all the essential AA, whereas C3 elevated concentrations of glucogenic AA. Together, these data suggest that C3 and Cas improve milk protein output by different mechanisms, with an additional energy supply as propionate being also effective when sufficient protein is supplied. ACKNOWLEDGEMENTS The authors gratefully thank, from the UMR PL, INRA, France, Y. Lebreton for surgeries, P. Lamberton and his team for technical support and animal care during the experiment and C. Nahuet, L. Finot, N. Huchet, I. Jicquel, and S. Rigault for their help in laboratory analyses, and from the Rowett, A. G. Calder and S. E. Anderson for mass spectrometry analyses.the authors also wish to acknowledge the financial support of the INRA, the European Union for the award of Marie Curie Training Site funding to the Rowett Research Institute, SEERAD and the National Science and Engineering Research Council of Canada and Agriculture and Agri-Food Canada (Lennoxville Research Centre Contribution number 874). Abdul-Razzaq, H. A. and Bickerstaffe, R The influence of rumen volatile fatty acids on protein metabolism in growing lambs. Br. J. Nutr. 62: Bequette, B. J., Metcalf, J. A., Wray-Cahen, D., Backwell, F. R. C., Sutton, J. D., Lomax, M. A., MacRae, J. C. and Lobley, G. E. 1996a. Leucine and protein metabolism in the lactating dairy cow mammary gland: responses to supplemental dietary crude protein intake. J. Dairy Res. 63: Bequette, B. J., Backwell, F. R., MacRae, J. C., Lobley, G. E., Crompton, L. A., Metcalf, J. A. and Sutton, J. D. 1996b. 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