Received 3 October 2006, accepted 16 April 2007.

Transcription

1 Adult Trichostrongylus colubriformis infection did not affect protein synthesis rate in whole-body, intestinal, hepatic and skeletal muscle tissues of lambs fed fresh Lucerne (Medicago sativa) E. N. Bermingham 1, W. C. McNabb 1, I. A. Sutherland 2, B. R. Sinclair 1, B. P. Treloar 1, and N. C. Roy 1, 3 1 Food, Metabolism & Microbiology Section, AgResearch Grasslands, Palmerston North 4442, New Zealand; 2 Animal Health Section, AgResearch Hopkirk Research Institute, Palmerston North 4442, New Zealand. Received 3 October 2006, accepted 16 April Bermingham, E. N., McNabb, W. C., Sutherland, I. A., Sinclair, B. R., Treloar, B. P. and Roy, N. C Adult Trichostrongylus colubriformis infection did not affect protein synthesis rate in whole-body, intestinal, hepatic and skeletal muscle tissues of lambs fed fresh Lucerne (Medicago sativa). Can. J. Anim. Sci. 87: The effects of an established Trichostrongylus colubriformis infection on the whole-body and fractional protein synthesis rates in the small intestine, liver, lymphoid tissues, skeletal muscle and skin were determined in lambs fed fresh Lucerne (Medicago sativa; 800 g DM d 1 ) on day 48 post-infection. Lambs were dosed with 6000 L3 T. colubriformis larvae for 6 d (n = 5) or kept as parasite-free controls (n = 6). On day 45, the lambs received a bolus injection of deuterated water to measure the size of the whole-body water pool. On day 48, the lambs were continuously infused with [3, 4-3 H]-valine into the jugular vein and [1-13 C]-valine in the abomasum for 8 h. During the infusion, mesenteric artery blood and terminal tissue samples were collected for measuring the isotopic activity of plasma water, plasma valine, intracellular valine and protein-bound valine. Intestinal worm numbers on day 48 were higher (P < 0.001) in the infected lambs, however, there was no effect (P > 0.10) of parasitic infection on feed intake, liveweight gain, whole-body protein synthesis and fractional protein synthesis of most tissues. Key words: Parasite infection, protein synthesis, lambs Bermingham, E. N., McNabb, W. C., Sutherland, I. A., Sinclair, B. R., Treloar, B. P. et Roy, N. C L infection avec le parasite Trichostrongylus colubriformis n affecte pas le taux de synthèse protéique dans l organisme entier et le petit intestin, le foie et le muscle squelettique chez les agneaux alimentés avec une ration de Lucerne (Medicago sativa) en coupe verte. Can. J. Anim. Sci. 87: Les effets d une infection établie avec le parasite Trichostrongylus colubriformis sur le métabolisme protéique de l organisme entier et la synthèse protéique fractionelle dans le petit intestin, le foie, les tissus lymphoïdes, le muscle squelettique et la peau ont été déterminés chez les agneaux alimentés avec une ration de lucerne (Medicago sativa; 800 g j 1 de matière sèche) en coupe verte 48 jours après infection. Les agneaux ont été dosés à chaque jour avec 6000 larves au stage L3 pour 6 j (n = 5) ou gardés comme contrôles sans parasites (n = 6). Au jour 45, les agneaux ont reçu une injection de deutérium pour mesurer la taille du pool de l eau dans l organisme entier. Au jour 48, les agneaux ont été perfusés avec la [3, 4-3 H]-valine dans la veine jugulaire et la [1 13 C]-valine dans l abomasum pendant 8 heures. Durant les perfusions, le sang de l artère mésentérique et plusieurs tissus ont été collectés pour mesurer l enrichissement isotopique de l eau plasmatique, la valine plasmatique, la valine intracellulaire et la valine liée aux protéines. Au jour 48, le nombre de larves dans le petit intestin était superieur (P < 0.001) chez les agneaux infectés mais il n y avait pas d effet de l infection parasitaire sur la prise alimentaire, le gain de poids, le métabolisme protéique de l organisme entier et la synthèse protéique fractionelle de plusieur tissus. Trichostrongylus colubriformis, the major sheep parasite in New Zealand, imposes a severe financial cost on farmers, due in part to its negative effects on liveweight gain (van Houtert et al. 1995) and wool production (Steel et al. 1980, 1982). Protein is the major constituent of muscle and wool. It is also a large component of the gastrointestinal tract where T. colubriformis comes into contact with the epithelium of the small intestine. Serum protein leakage, intestinal mucus production and intestinal cell exfoliation are some of the consequences of parasitic infections (Colditz 2003). 3 To whom correspondence should be addressed ( nicole.roy@agresearch.co.nz) Mots clés: Infection parasitaire, synthèse protéique, agneaux 315 These protein losses are likely to affect tissue protein turnover, particularly at the site of infection, and thus alter the amino acid (AA) requirements of the small intestine. Abbreviations: AA, amino acid; DM, dry matter; FSR, fractional protein synthesis rate; FSR I, fractional protein synthesis rate based on intracellular pool; FSR P, fractional protein synthesis rate based on plasma pool; ILR, irreversible loss rate; SRA, specific radioactivity; SRA B, specific radioactivity in protein bound pool; SRA I, specific radioactivity in tissue intracellular pool; SRA MAX, plateau SRA in plasma; SRA P, specific radioactivity in plasma pool.

2 316 CANADIAN JOURNAL OF ANIMAL SCIENCE Increased fractional protein synthesis rate (FSR; proportion of the tissue protein pool re-synthesized per day) was observed in intestinal tissues during T. colubriformis infections (Symons and Jones 1983; Bermingham et al. 2006). Additionally, parasitic infection has increased the oxidation of leucine in the gastrointestinal tract of sheep by 20 40%, which, together with increased leucine sequestration to these tissues (Yu et al. 2000), had the net effect of decreasing its availability to other tissues including the liver. Despite reduced inflow of AA to the liver, increased FSR in the liver has been observed during a T. colubriformis infection in other studies (Jones and Symons 1982; Bermingham et al. 2006). More skeletal muscle protein may need to be degraded and mobilized to supply increased AA demands of the intestine and liver in sheep infected with T. colubriformis larvae (MacRae and Lobley 1991). Parasitic infection often reduces dietary intake (Sykes et al. 1988), which will further exacerbate the higher AA requirements and consequent AA repartitioning from the skeletal muscle as there will be a reduction in the absorption of AA from the digestive tract of the animal. Only one study has reported data on protein synthesis of the whole-body and FSR of key tissues involved in responding to an adult T. colubriformis infection in lambs at similar intakes of fresh forage (Bermingham et al. 2006). However, Bermingham et al. (2006) fed Sulla, which contains condensed tannins (CT), which can have anti-nutritional effects (Waghorn et al. 1994) as well as impact directly on parasite larvae (e.g., Molan et al. 2002). The hypothesis of this study was that adult T. colubriformis would alter intestinal and hepatic protein synthesis in lambs fed fresh Lucerne, a forage with high nutritive value that does not contain CT. This will be due to the repartitioning of AA between the intestinal tissues, liver and skeletal muscle (to meet the increased demands for AA at the site of infection), rather than because of decreased availability of nutrients from feed. Therefore, the aim of this study was to determine the effects of an adult parasitic infection on valine kinetics in the whole-body and FSR in the small intestine to assess shift in protein synthesis in liver, some lymphoid tissues, skeletal muscle and skin of lambs fed fresh Lucerne. MATERIALS AND METHODS Animals and Feed The experimental procedures for this trial were reviewed and approved by the Crown Research Institute Animal Ethics Committee in Palmerston North, New Zealand, according to the Animals Protection Act (1960) and Animals Protection Regulations (1987) and amendments. At weaning, wether lambs (29 kg) were removed from their dams, shorn, drenched twice with ivermectin (Ivomec Merial, New Zealand Limited) and treated for external parasites using Wipeout (Coopers, Schering- Plough Animal Health Limited, New Zealand). The lambs were maintained on feeding pads for approximately 3 wk, in order to limit exposure of the lambs to larvae present on pasture. The lambs were randomly sampled for faecal egg counts (FEC) during this period in order to ensure that no animals were infected with parasites. One week before surgery, the lambs were brought indoors and housed in individual metabolism crates. During this period the lambs were fed Lucerne pellets (800 g dry matter (DM) d 1 ) and chopped Lucerne hay (200 g DM d 1 ). Three weeks prior to the start of surgery, catheters were prepared using Tygon tubing (Scientific Supplies Limited, Wellington, New Zealand) as described by Huntington et al. (1989). The catheters were sterilized in ethylene oxide at 55ºC for 2.5 h with an 8 h aeration. cycle. Catheters were further aerated for a minimum of 1 wk before being used for surgery. Twelve lambs were fasted for 24 h before surgery. Anaesthesia was initiated with Nembutal (60 mg ml 1 ; 0.5 ml kg 1 body weight; Vetworks, Cambridge, New Zealand) and maintained with isoflurane (1.5%; Vetworks, Cambridge, New Zealand) administered through an endotracheal tube. Permanent indwelling catheters were placed in the mesenteric artery, and the mesenteric, portal and hepatic veins (Huntington et al. 1989) and vena cava (Ortigues and Durand 1995) for blood sampling and infusions. Additional permanent catheters were placed in the mesenteric vein (upstream from the sampling catheter) and abdominal aorta for infusion of p-aminohippuric acid and indocyanin green respectively, to measure plasma flow across the splanchnic tissues and the hind limbs. A permanent Teflon cannula was fitted in the abomasum in preparation for the infusion of [1 13 C]-valine on day 48 post-infection. A temporary catheter was inserted into the jugular vein on day 43 in preparation for a bolus injection of deuterium oxide and infusion of [ 13 C]-sodium bicarbonate on day 45 post-infection and [3, 4-3 H]-valine on day 48. Catheters were maintained as described by Huntington et al. (1989). These catheters were utilised as part of a larger study to measure AA fluxes and kinetics from both arterial and luminal sources 48 d post-infection across the gastrointestinal tract, liver and hind limbs (Bermingham 2004). Once intake had fully recovered after surgery (usually by 3 d post-surgery) lambs were offered approximately 800 g DM d 1 of fresh Lucerne (Medicago sativa) until the conclusion of the trial. Fresh Lucerne was offered rather than the typical ryegrass/white clover-dominant NZ pasture for several reasons; (1) a single-species sward is easier to maintain for similar nutritive value over several weeks, (2) its high nutritive value for sheep, (3) its suitability for cutting and carrying, (4) its relative drought tolerance and (5) it is a legume with similar chemical composition to Sulla (Hedysarum coronarium; Bermingham et al. 2006). The Lucerne was harvested every 2 d with a sickle bar mower by 10:00 am and stored at 4 C. The DM content of the Lucerne was determined daily in order to adjust the amount of wet forage given to maintain DM offered at approximately 800 g d 1. This level of intake was chosen in order minimise differences in feed intake between the control and infected lambs. The lambs were fed at hourly intervals from overhead feeders and water was available ad libitum. The lambs were weighed weekly to monitor liveweight change during the experimental period. Treatment and Parasitology One week after surgery (day 1 of the experimental period) six sheep were given 6000 T. colubriformis L3 larvae per

3 BERMINGHAM ET AL. PARASITIC INFECTION AND PROTEIN SYNTHESIS 317 day orally for 6 d (parasite treatment) while the remaining six sheep were drenched with ivermectin (Ivomec Merial, New Zealand Limited) once ensuring no background infection to serve as controls (control treatment). This infection protocol was essentially a single dose, with the larval dose split across 6 d to reduce the possibility of rumen bypass and therefore maximise the establishment of larvae in the small intestine. The single dose infection permits the quantification of the effects of an adult population of parasite on whole-body and tissue AA metabolism without the confounding effects of new larval challenge that a weekly dosing (trickle) brings. Additionally, a single dose reduced the likely higher ethical cost of catheter failure that could have incurred in the multicatherized lambs in our study (Bermingham et al. 2006). The lambs were assigned to either the control or parasitic group according to a completely randomized block design. Due to the time consuming nature of the surgical procedure, a maximum of four lambs were surgically prepared with catheters in any one week and, therefore, the lambs were blocked according to the week that they underwent surgery, referred to as the group effect in the statistical model. FEC from individual sheep were determined every second day from day 20 to day 45 using the modified McMaster method in which one egg counted equates to 50 eggs/gram wet faeces (Whitlock 1948). Total intestinal worm burdens in the proximal 10 m of the small intestine were determined after slaughter (Sutherland et al. 1999b). Infusions and Blood Sampling Radioactive and stable isotopes were purchased from Amersham Life Science (Buckinghamshire, UK) and Mass Trace, Inc. (Woburn, MA) respectively. Forty-five days after infection, the lambs received a bolus injection of deuterium oxide (0.61 ml kg 1 body weight) into the jugular vein to measure the size of the whole-body water pool. The lambs also received an 8 h continuous infusion of [ 13 C]- sodium bicarbonate (99 atom percent, 229 mg h 1 ) into the jugular vein to measure the whole-body production of carbon dioxide (CO 2 ). The full data on whole-body protein synthesis and oxidation of [ 13 C]-valine is not reported as the isotopic enrichment of the 13 CO 2 could not be completed. Blood was withdrawn continuously every 2 h from the mesenteric artery over the 8 h infusion period (10 ml for time 0 to 2 h and time 2 to 4 h; 20 ml for time 4 to 6 h and time 6 to 8 h) using a peristaltic pump (Waterlow Marlow, 302F). The blood was centrifuged (4 C; 3270 g for 15 min) and the plasma harvested and stored at 85 C to measure DM content and deuterium oxide isotopic enrichment. On day 48, the lambs received a continuous 8 h infusion of [3, 4-3 H]-valine (7.6 MBq h 1 ) into the jugular vein. Concurrently, [1-13 C]-valine (99 atom percent; 118 mg h 1 ) was continuously infused into the abomasum. On day 48, blood was continuously withdrawn over 2-h periods from the mesenteric artery (10 ml for time 0 to 2 h and time 2 to 4 h; 30 ml for time 4 to 6 h and time 6 to 8 h) into a syringe using a peristaltic pump (Waterlow Marlow, 302F) for the measurements reported in this paper. Blood was also withdrawn from the mesenteric, portal, and hepatic veins, and the vena cava in a similar fashion for tissue net flux and kinetic measurements (30 ml for time 4 to 6 h and time 6 to 8 h; not reported here). To prevent blood clotting during the continuous sampling on both days 45 and 48, 2000 IU ovine heparin h 1 was infused into the jugular vein of the lambs over the 8 h infusion period (Lobley et al. 1995). Sampling lines and syringes were kept in an ice water bath to minimise the degradation of blood constituents (Lobley et al. 1995). The valine plasma data presented in this paper from both day 45 and 48 represent the average of samples taken from the last two sampling periods from the mesenteric artery (time 4 to 6 and 6 to 8 h). Only the whole-body kinetics relating to the infusion of [3, 4-3 H] and [1-13 C]-valine and the tissue FSR calculated from the [3, 4-3 H]-valine incorporation into their intracellular valine free and protein bound pools are presented in this paper. After each 2-h collection period, whole blood was centrifuged (4 C; 3270 g for 15 min) and the plasma harvested and either processed or stored at 85 C for further analysis as described below. Tissue Sampling After the completion of blood sampling, but while the isotopic infusates were still being administered, the sheep were euthanized by an intravenous overdose of sodium pentobarbitone (300 mg ml 1 ; 0.5 ml kg 1 liveweight). Tissue samples were rapidly collected from the sheep in the following order: skin, muscle (biceps femoris), liver, duodenum, ileum, spleen, mesenteric lymph nodes and thymus as described previously (Bermingham et al. 2006). Sample Processing and Chemical Analysis To determine the concentration of valine in plasma, 0.5 ml of plasma was treated with 80 mmol L 1 dithiothreitol and 3 mmol L 1 norleucine (as an internal standard) in 0.1% phenol and stored at 85 C. The samples were thawed and transferred to a Centrisart filter ( molecular weight cut off) and then centrifuged at approximately g for 60 min, with the filtrate removed and stored at 85 C until analysis. Quantification of plasma valine concentration was determined by reverse phase high performance liquid chromatography separation of phenylisoothiocyanate derivatives, using a Waters Pico-Tag column ( mm, Waters Corporation, Milford, NMA 01757) as modified from Bidlingmeyer et al. (1984) on a Shimadzu HPLC-10/A system (Shimadzu Scientific Instruments Limited, Columbia, MD 21046). In order to measure the partitioning of tritiated label between valine and water, total [ 3 H] radioactivity in infusates and plasma was determined by mixing 50 µl of sample in 2 ml of scintillation mixture (Starcint, INSUS Systems) and counting for 10 min (Packard Tricarb Model 1500 Scintillation counter) as described by Lee et al. (1999). The proportion of total [ 3 H] radioactivity attributed to valine and its breakdown product (water) in these sampled pools was determined by a flow through liquid scintillation counter (Model 2, β-ram, IN/US systems Inc., NJ) coupled to a high performance liquid chromatograph (HPLC4/A, Shimadzu, Kyoto, Japan) as described in Lee et al. (1999).

4 318 CANADIAN JOURNAL OF ANIMAL SCIENCE The isotopic enrichment of water in plasma was determined using a method adapted from Yang et al. (1998). The samples were analysed on a gas chromatograph (Shimadzu GC QP) equipped with a mass selective detector (Shimadzu QP2010) as described in Bermingham et al. (2006). Subsamples (4 5 g) of frozen tissue were pulverized in liquid nitrogen using a modified French Cell press as described by Lee et al. (1993). The pulverized tissue was then stored at 85ºC until further processing. Approximately 1 g of smashed tissue was homogenized in an extraction buffer (20 mmol L 1 Tris ph 7.8; 2.5 mmol L 1 ethylene diamine tetra acetic acid; 0.3% sodium dodecyl sulphate). The homogenate was centrifuged at approximately x g at 4 C for 30 min. The supernatant containing intracellular peptides, AA and soluble protein-bound AA was removed and the resulting pellet (protein-bound fraction) rewashed in extraction buffer and centrifuged for a further 30 min in order to determine the total [ 3 H] counts associated with valine in the protein-bound pool as described earlier. The pellet was freeze dried and stored at room temperature until analyzed for the concentration of AA. Valine concentration in the protein-bound fraction was determined by ion exchange chromatography (Shimadzu Scientific Instruments Limited, Columbia, MD 21046) with a post-column reaction system using ninhydrin as the derivatising agent after an acid hydrolysis of a 50 mg pellet with 6.0 mol L 1 HCl at 110 C for 22 h. The hydrolysates were filtered and rotary evaporated to near dryness, washed in deionized water and rotary evaporated again before being taken up into 0.2 mol L 1 sodium citrate buffer (ph 2.2). Total radioactivity of the hydrolysate and the proportion of radioactivity attributed to valine and its breakdown product (water) was determined as described above for plasma valine. Free pool supernatant (2 ml) was mixed with 1 ml of 0.75% (9 mol L 1 ) sodium dodecyl sulphate/ethylene diamine tetra acetic acid to break up the protein structures in the supernatant, 200 µl of 80 mmol L 1 dithiothreitol (ph 8.0) as an antioxidant and 100 µl of 3 mmol L 1 norleucine as an internal standard in 0.1% phenol. The samples were mixed and left to stand at room temperature for 15 min and then deproteinized with 1 ml of 30% trichloroacetic acid (wt/wt) and centrifuged at 4 C at 3270 g for 15 min. The resulting supernatant containing free pool AA was filtered (0.45 µm, cellulose acetate) and stored at 85 C until analyzed for AA concentration and total radioactivity as described above for plasma valine. Calculations Specific radioactivity (SRA; dpm nmol 1 ) of valine in plasma (SRA P ) tissue free pool (SRA I ) and tissue-bound proteins (SRA B ) was calculated by dividing the total radioactivity of the sampled pool (dpm ml 1 ) by the concentration of valine in the sampled pool (nmol ml 1 ). The rise in plasma valine SRA to plateau was described using the following exponential model (Eq. 1). Valine SRA (aterial) = valine SRA MAX (1 e kt ) (1) The valine SRA MAX in Eq. 1 is the plateau value of valine SRA assuming the radioactivity increase at a rate k over time t (Waterlow et al. 1978). The whole-body model for AA kinetics described by Waterlow et al. (1978) was used to estimate whole-body valine kinetics. Whole-body irreversible loss (ILR) of valine estimated from both the [ 3 H]- valine and [ 13 C]-infusions were determined according to Eq. 2 (Harris et al. 1992). ( ) ( ) ( ) 1 Valine ILR mmol h infusion rate of 3 H 1 -valine dpm h = 1 SRA max dpm mmol In order to calculate the whole-body valine oxidation from the infusion of [ 3 H]-valine, the total amount of [ 3 H]- water produced from valine over the period of infusion was quantified using the equations outlined by Beckett et al. (1992). Following the injection of deuterium, the isotopic enrichment of water follows an exponential decay curve. Therefore, to determine the actual body water volume using a deuterium bolus, an exponential decay curve was plotted with the plasma water isotopic enrichments on the y-axis and time of sampling (0 to 2 h, 2 to 4 h, 4 to 6 h and 6 to 8 h) in relation to the administration of the deuterium on the x-axis. The values extrapolated back to time zero is the isotopic enrichment of water at the time of the injection. The latter is used to calculate the volume of body water. Wholebody valine used for protein synthesis was calculated as the difference between ILR and oxidation and was converted to protein synthesis (g d 1 ) assuming a valine concentration of 36 mg g 1 tissue (MacRae et al. 1993). The traditional equation for the estimation of tissue FSR was not used in this study as this equation requires the estimation of the rate constant describing the rise to plateau of free valine SRA in plasma. This can be achieved by performing frequent intermittent blood samples during the course of a labelled AA infusion. The accurate rise to plateau of valine SRA in plasma could not, therefore, be estimated. Instead the FSR in whole duodenum and ileum, duodenal and ileal smooth muscle, mesenteric lymph node, spleen, liver, thymus, skeletal muscle and skin was determined according to Eq. 3 (Wykes et al. 1996). ( ) 1 FSR % d = valine 100 valine SRA protein bound ( ) SRA ( precursor pool) period of infusion ( d) In Eq. 3, the length of the infusion period equals 8 h or 0.33 d. The SRA of valine in the free pool of each tissue was assumed to reflect the steady state SRA of the true precursor pool, valine-trna, for protein synthesis (FSR I ). These FSR estimates were also compared with the FSR obtained using the SRA of free valine in arterial plasma (mesenteric (2) (3)

5 BERMINGHAM ET AL. PARASITIC INFECTION AND PROTEIN SYNTHESIS 319 artery) as a precursor pool (FSR P ). The FSR estimates calculated using Eq. 3 will underestimate the actual rate of FSR. As isotopic steady state was achieved in arterial plasma between 6 to 8 h of infusion in the current study, this equation assumes that the SRA of plasma valine is at plateau from the start of the labelled AA infusion (which can be achieved giving a priming dose of the same AA). Statistical Analysis Statistical analysis for all variables was performed using a General Linear Model (SAS Institute, Inc. 1999, Version 8) with treatment and group (the week that the lamb underwent surgery) used as sources of variation in the model. Additionally, feed intake, faecal egg counts and liveweight were analysed using Proc Mix repeated measures, with treatment, group and time used as sources of variation in the model. The data was checked for normality and the presence of outliers by plotting residuals versus the predicted residuals. Faecal egg counts were transformed by ln (x + 1) before analysis in order to ensure symmetry in the data and to standardise variances across the treatments (Sutherland et al. 1999a). Probability values lower than 0.05 were considered to indicate a significant difference and values between 0.05 and 0.10 to indicate a trend. Results are presented as least squares means and associated pooled standard deviation. One sheep from the parasite treatment was omitted from all statistical analysis as the Captec TM Alkane CRC capsule (used as a marker for digesta flow as part of a larger study) blocked the reticulo-rumen orifice and affected feed intake 2 d prior to the blood-sampling period. In the current study, only one catheter was found with formed fibrous sheaths at the tip, and consequently 99% success rate of catheter was observed. RESULTS On day 48 post-infection, Trichostrongylus colubriformis burdens in the small intestine were significantly higher (P < 0.001) in the infected lambs (440 vs (SD 1101) worms in the control and parasitized lambs, respectively. Consequently, FEC were significantly increased (P < 0.001) in parasitized lambs over the course of the experiment (Fig. 1). However, the presence of parasites in the small intestine did not have any impact (P > 0.05) on feed intake over the course of the experiment (Fig. 2) averaging 826 and 796 (SD 18) g DM d 1 in the control and parasitized lambs, respectively. Liveweight was lower (P < 0.05; Fig. 2) during the experimental period in parasitized lambs, averaging 36 and 34 (SD 1.4) kg in the control and parasitized lambs, respectively. Average change in liveweight was similar between the treatments (c. 20 g d 1 ). The size of the whole-body water pool in the lambs was unaffected by the parasite infection (Table 1). Whole-body valine ILR and oxidation calculated from the intra-venous 3 H-valine infusion were also similar between treatment groups (Table 1). Whole-body protein synthesis was not affected (P > 0.10) during parasitic infection (Table 1). Whole-body valine oxidation accounted for 12 14% of whole-body valine ILR and was unaffected by infection (Table 1). Similarly, the proportion of whole-body ILR that was used for whole-body protein synthesis was unaffected by the presence of T. colubriformis in the small intestine and averaged 86 88% (Table 1). Whole-body ILR (calculated from the infusion of 13 C-valine into the abomasum) was also similar between the control and parasite lambs [316.9 vs (SD 124.6) mmol d 1 ]. Plasma concentration of valine in mesenteric artery plasma was similar between treatments [203 vs. 235 (SD 64) µmol L 1 in the control and parasite lambs, respectively] as was the SRA of valine [SRA P ; vs (SD 11.0) dpm nmol 1, respectively]. The concentration of valine in the intracellular and protein-bound pools of most tissues (Tables 2, 3 and 4) with the exception of whole duodenal tissue intracellular pool (Table 2) was similar between treatments. With the exception of the spleen, where the SRA I tended to increase (P < 0.10; Table 3) there was no effect of parasitic infection on the SRA I of tissues (Tables 2, 3 and 4). The SRA B for each tissue was unaffected by the presence of parasitic infection (Tables 2, 3 and 4). In whole ileal tissue, FSR I tended to decrease (P < 0.10) from 49 to 36% in the presence of parasite infection (Table 2); however, there was no effect of parasitic infection when arterial valine was used as a precursor pool (FSR P ; Table 2). There was no effect of parasite infection on FSR in any other tissues (Tables 2, 3 and 4). DISCUSSION In the present study, the effects of an established T. colubriformis population in the small intestine were not enough to elicit change in FSR in most of the key tissues usually involved in fighting a parasitic infection. Although T. colubriformis inhabit the proximal small intestine and cause damage to the duodenal tissues by tunnelling into the intestinal crypts and the villi epithelium (Coop and Angus 1975), duodenal FSR estimates were similar between the control and parasitized lambs. This suggests that the level of parasite establishment in the duodenum was insufficient to cause an increase in the rate of protein synthesis of this tissue. Alternatively, it is possible that the damage caused to the intestine occurs with the initial establishment of the larvae and that the adult worm, once established, has little effect on the protein synthesis of this tissue. The FSR I for whole ileal tissue protein indicated that the synthesis of constitutive proteins in this tissue was reduced by 13% during parasitic infection. As the FSR of smooth muscle protein of ileal tissue was unaffected by the presence of parasites, this suggests less mucosal protein was being synthesized daily; however, this was not measured directly in this study. This contradicts Symons and Jones (1983), who observed similar small intestine FSR despite a 24% increase in daily protein synthesis in guinea pigs infected with T. colubriformis. In the current study, the weight and the nitrogen content of the small intestine were not measured and, therefore, the absolute rate of protein synthesis could not be estimated from FSR. Other nematodes like Nippostrongylus brasiliensis, which also inhabit the proximal small intestine, have altered morphology and increased

6 320 CANADIAN JOURNAL OF ANIMAL SCIENCE Fig. 1. Faecal egg count (FEC; eggs g wet faeces 1 ) in lambs infected with or without Trichostrongylus colubriformis and fed fresh Lucerne (Medicago sativa). mucus production in the large intestine (Cheema and Scofield 1982). Our previous study also reports changes in protein metabolism in intestinal tissues distal to the site of infection, namely increased FSR of ileal smooth muscle and the mesenteric lymph nodes (Bermingham et al. 2006). Initiation of a general immune response may account for increased FSR in tissues distal to the sites of infection (Symons 1978). However, reasons for these trends are unclear. The FSR of other tissues (spleen, liver, biceps femoris, skin, thymus) were not affected by the presence of parasitic infection 48 d post-infection. Parasitic infection has increased the rates of synthesis of hepatic constitutive proteins (Jones and Symons 1982; Bermingham et al. 2006). The 4% decrease (P < 0.15) in skin FSR I observed in the current study is consistent with previous studies in parasitized sheep (Symons and Jones 1975; Steel et al. 1980, 1982). Whilst these studies have shown decreased skeletal muscle protein synthesis during parasitic infection, similar infection protocols to the current study showed that an adult T. colubriformis infection did not affect the FSR of the biceps femoris (Bermingham et al. 2006). Values presented in this study, with the exception of skeletal muscle FSR, agree with those reported for the small intestine (21 to 107% d 1 ), liver (16 to 27% d 1 ) and spleen (9 to 38% d 1 ) in ruminants using constant infusion of labelled AA such as leucine, lysine and phenylalanine (Schaefer et al. 1986; Connell et al. 1997). The estimates of skeletal muscle FSR (less than 1%) are lower in this study and our previous study (Bermingham et al. 2006) compared with those of other studies (2 5%; Davis et al. 1981; Schaefer et al. 1986). However, there is a positive relationship between FSR and intake (Oddy et al. 1987; Lobley et al. 1994). Thus the higher feeding level used in Davis et al. (1981) and Schaefer et al. (1986; c g DM d 1 ) compared with the restricted level of feed intake (800 g DM d 1 ) used in the present study may also be cause for these differences. We should, therefore, be cautious when comparing the effect of parasitic infection on skeletal muscle FSR in the current study. The lack of effect of parasitic infection on most tissue FSR is in agreement with similar whole-body AA ILR reported in the current study and other studies (Yu et al. 2000; Bermingham et al. 2006). The whole-body estimates of valine kinetics based on the infusion of 3 H-valine into the jugular vein (c. 100 mmol d 1 ) presented in this study are in agreement with those presented in the literature for other intravenous labelled AA infusions in uninfected animals (c. 48 to 200 mmol d 1, Harris et al. 1992; Connell et al. 1997; Bermingham et al. 2006). Valine ILR estimates based on the

7 BERMINGHAM ET AL. PARASITIC INFECTION AND PROTEIN SYNTHESIS 321 Fig. 2. Feed intake (g DM d 1 ) and liveweight (kg) in lambs infected with or without Trichostrongylus colubriformis and fed fresh Lucerne (Medicago sativa). infusion of 3 H-valine into the jugular vein were 3 times lower than those obtained based on the infusion of 13 C- valine into the abomasum. The valine ILR estimates based on the infusion of 3 H-valine will not include the dilution of the valine tracer by the first pass metabolism through the gastrointestinal tract and this may cause them to be lower than the 13 C-valine estimates. This was also observed in a previous study (Bermingham et al. 2006). Additionally, differences in whole-body estimates of AA ILR due to differences in the metabolism of the labeled isotope have been observed previously (Krempf et al. 1990; Yu et al. 1990). The lack of effects of parasitic infection on whole-body and tissue protein synthesis in this study could be attributable in part to the procedure of larvae infection. This point was covered in detail in Bermingham et al. (2006). Briefly, the majority of the studies in sheep reported in the literature have involved long-term constant (at least weekly) infection procedures (e.g., Steel et al. 1980; Jones and Symons 1982; Yu et al. 2000) and/or larger infection dose (Steel et al. 1980; Jones and Symons 1982). It is also possible that alterations in whole-body and tissue protein synthesis would have been observed at an earlier stage of parasite establishment, such as when the lamb was first infected with T. colubriformis and when faecal egg production was at its highest point during the infection (around 26 d post-infection). It is possible that prior exposure to parasites may have occurred in the lambs, and that this may also have influenced the results due to an enhanced immune system, but random samples of pre-surgery egg counts were in all cases negative. Therefore, it is unlikely that prior exposure played any part in the current study. Bermingham et al. (2006) reported that the presence of an established parasitic infection in the small intestine of lambs increased the FSR in the duodenal and ileal smooth muscle, mesenteric lymph nodes and liver. However, in contrast to the current study where both control and parasitized lambs fed fresh Lucerne gained weight (20 g d 1 ), the parasitized lambs in Bermingham et al. (2006) lost weight ( 50 g d 1 ). This may explain why similar pre-surgical, surgical and infection procedures had more severe impacts on tissue protein metabolism in Bermingham et al. (2006) than the current study. Additionally, while these forages have a similar chemical composition, Sulla contains a low concentration of condensed tannins (CT). The feeding of forages containing low levels of CT has been observed to improve performance in both uninfected (e.g., Min et al. 1998; Burke et al. 2002) and infected animals (Neizen et al. 1998). A direct comparison between Lucerne (no CT; current study) and Sulla (contains CT; Bermingham et al. 2006) and the possible interactions during parasitic infection is not possible as

8 322 CANADIAN JOURNAL OF ANIMAL SCIENCE Table 1. Whole-body valine irreversible loss rate, oxidation and protein synthesis in lambs infected with or without Trichostrongylus colubriformis and fed fresh Lucerne (Medicago sativa) based on the infusion of [ 3 H]-valine into the jugular vein Control Parasite Parameter n = 6 n = 5 Pooled SD P value Irreversible loss rate (mmol d 1 ) Water pool size (L) z Total 3 H 2 O from valine (dpm 10 7 ) Total 3 H-valine infused (dpm 10 7 ) Oxidation (%) (mmol d 1 ) Protein synthesis (mmol d 1 ) (g d 1 ) z Calculated from the infusion of deuterated water on day 45 post-infection. Table 2. Valine concentration and specific radioactivity (SRA) of tissue free pool and protein bound and fractional synthesis rates (FSR) z in the small intestine in lambs infected with or without Trichostrongylus colubriformis and fed fresh Lucerne (Medicago sativa) based on the infusion of [ 3 H]- valine into the jugular vein Control Parasite Tissue Parameter n = 6 n = 5 Pooled SD P value Duodenum smooth muscle Valine I (µmol L 1 ) Valine B (g g 1 DM) SRA I (dpm nmol 1 ) SRA B (dpm nmol 1 ) FSR P (% d 1 ) FSR I (% d 1 ) Whole duodenum Valine I (µmol L 1 ) Valine B (g g 1 DM) SRA I (dpm nmol 1 ) SRA B (dpm nmol 1 ) FSR P (% d 1 ) FSR I (% d 1 ) Ileum smooth muscle Valine I (µmol L 1 ) Valine B (g g 1 DM) SRA I (dpm nmol 1 ) SRA B (dpm nmol 1 ) FSR P (% d 1 ) FSR I (% d 1 ) Whole ileum Valine I (µmol L 1 ) Valine B (g g 1 DM) SRA I (dpm nmol 1 ) SRA B (dpm nmol 1 ) FSR P (% d 1 ) FSR I (% d 1 ) z Where subscripts I and P denote intracellular and plasma precursor pools, respectively, and B denotes the SRA of the protein-bound fraction. these studies were performed independently, and feed intake differed between parasitised lambs fed Lucerne (800 g DM d 1 ) and Sulla (690 g DM d 1 ). However, the control lambs in both experiments had similar feed intakes (830 vs. 770 g DM d 1 in the Lucerne and Sulla experiments, respectively); thus, it is possible to compare the control lambs in both experiments to elucidate possible effects of feeding CT on tissue FSR. In whole duodenum and ileum tissue, the average FSR P and FSR I in the control lambs fed Sulla were generally higher than those fed Lucerne. Lambs fed diets containing 5% quebracho tannin had epithelial degeneration and ulceration in the jejunum and ileum (Wang et al. 1996; Dawson et al. 1999), suggesting a higher FSR in these tissues. However, there were no morphological changes in the duodenum, possibly due to the alkaline environment of the jejunum and ileum (Wang et al. 1996; Dawson et al. 1999). Similar levels of CT present in fresh Lotus pedunculatus (5.5% CT) had no effect on the morphology of the duodenum, jejunum or ileum in lambs (Walton et al. 2001). This suggests that various types of CT may differ in their effects on FSR within the digestive tract as well as on animal production, due to variations in their chemical structure (Aerts et al. 1999). While the FSR P of the liver were similar in the control Lucerne- and Sulla-fed lambs, the mesenteric lymph node FSR I was two to three times greater in the control Sulla-fed lambs. It is possible that the CT present in Sulla resulted in a localised immune response in the GIT due to harmful

9 BERMINGHAM ET AL. PARASITIC INFECTION AND PROTEIN SYNTHESIS 323 Table 3. Valine concentration and specific radioactivity (SRA) of tissue free pool and protein bound and fractional synthesis rates (FSR) z in the liver and lymphoid tissues in lambs infected with or without Trichostrongylus colubriformis and fed fresh Lucerne (Medicago sativa) based on the infusion of [ 3 H]-valine into the jugular vein Control Parasite Tissue Parameter n = 6 n = 5 Pooled SD P value Liver Valine I (µmol L 1 ) Valine B (g g 1 DM) SRA I (dpm nmol 1 ) SRA B (dpm nmol 1 ) FSR P (% d 1 ) FSR I (% d 1 ) Mesenteric lymph nodes Valine I (µmol L 1 ) Valine B (g g 1 DM) SRA I (dpm nmol 1 ) SRA B (dpm nmol 1 ) FSR P (% d 1 ) FSR I (% d 1 ) Spleen Valine I (µmol L 1 ) Valine B (g g 1 DM) SRA I (dpm nmol 1 ) SRA B (dpm nmol 1 ) FSR P (% d 1 ) FSR I (% d 1 ) Thymus Valine I (µmol L 1 ) Valine B (g g 1 DM) SRA I (dpm nmol 1 ) SRA B (dpm nmol 1 ) FSR P (% d 1 ) FSR I (% d 1 ) z Where subscripts I and P denote intracellular and plasma precursor pools, respectively, and B denotes the SRA of the protein-bound fraction. Table 4. Valine concentration and specific radioactivity (SRA) of tissue free pool and protein bound and fractional synthesis rates (FSR) z in the muscle and skin in lambs infected with or without Trichostrongylus colubriformis and fed fresh Lucerne (Medicago sativa) based on the infusion of [ 3 H]- valine into the jugular vein Control Parasite Tissue Parameter n = 6 n = 5 Pooled SD P value Skeletal muscle Valine I (µmol L 1 ) Valine B (g g 1 DM) SRA I (dpm nmol 1 ) SRA B (dpm nmol 1 ) FSR P (% d 1 ) FSR I (% d 1 ) Skin Valine I (µmol L 1 ) Valine B (g g 1 DM) SRA I (dpm nmol 1 ) SRA B (dpm nmol 1 ) FSR P (% d 1 ) FSR I (% d 1 ) z Where subscripts I and P denote intracellular and plasma precursor pools, respectively, and B denotes the SRA of the protein-bound fraction. interactions with the GIT mucosa; however, as the histological evaluation of the intestine has not been determined in this study, this remains speculative. The skin and muscle estimates of protein FSR I (10 to 12% d 1 ) were fairly consistent between the control Lucerne and Sulla-fed lambs. Therefore, it seems unlikely that the improvements in wool growth and liveweight gain observed in CT-fed animals (Min et al. 1998; Burke et al. 2002) are due to an increase in the amount of AA available for production. However, in both studies intake was restricted to approximately maintenance requirements (800 g DM d 1 ) and therefore a larger difference between the two studies may become apparent only at higher intakes. CONCLUSIONS There was no effect of parasitic infection on most intestinal tissues and tissue involved in immune responses (mesenteric lymph nodes, spleen, liver and thymus). The latter suggests

10 324 CANADIAN JOURNAL OF ANIMAL SCIENCE that there was no stimulation of the immune response in these lambs 48 d post-infection, there was no effect of parasitic infection on liveweight gain over the course of the experiment and no effect on skeletal muscle FSR. This suggests that an established parasitic infection did not result in a repartitioning of AA from the skeletal muscle to the intestines, liver and/or other tissues involved in metabolic changes and immune responses. It is likely that alterations in tissue FSR may have been detected at earlier stages of parasite infection, with the most critical periods likely to be the initial infection (days 1 6) and when faecal egg counts were at their highest (day 26 post-infection). ACKNOWLEDGEMENTS Many thanks to Gordon Reynolds and Brett Guthrie (Institute of Food Nutrition and Human Health, Massey University, Palmerston North, New Zealand) for the surgical procedure and Jason Peters (AgResearch Limited, New Zealand) for technical assistance throughout the experimental period. This study was funded by C. Alma Baker Trust, The Todd Foundation, New Zealand Foundation for Research, Science and Technology, the Leonard Condell Trust and Meat New Zealand. E. N. Bermingham thanks Meat New Zealand for funding her PhD Studies. Aerts, R. J., McNabb, W. C., Molan, A., Brand, A., Barry, T. N. and Peters, J. S Condensed tannins from Lotus corniculatus and Lotus pedunculatus exert different effects on the in vitro rumen degradation of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein. J. Sci. Food Agric. 79: Beckett, P. R., Cadenhead, A. and Fuller, M. F Valine oxidation: the synthesis and evaluation of L-[3-3 H]valine as a tracer in vivo. Br. J. 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