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Nitrogen and mineral balance of lambs artificially infected with Haemonchus contortus and fed tanniferous sainfoin (Onobrychis viciifolia)¹

A. Scharenberg^*,1, F. Heckendorn, Y. Arrigo^*, H. Hertzberg, A. Gutzwiller^*, H. D. Hess^*, M. Kreuzer and F. Dohme^*,2

^* Agroscope Liebefeld-Posieux, Research Station ALP, Tioleyre 4, CH-1725 Posieux, Switzerland and Research Institute for Organic Farming (FiBL), Ackerstrasse, CH-5070 Frick, Switzerland ETH Zurich, Institute of Animal Science, CH-8092 Zurich, Switzerland

	Abstract

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Tanniferous temperate legumes are assumed to possess anthelmintic properties, but it is unclear whether this is the direct result of condensed tannins (CT) or is mediated indirectly via an improved metabolic protein supply. A metabolism experiment was conducted to differentiate between these factors by feeding the CT plant sainfoin (19.7% CP in DM) to lambs infected with the abomasal blood-sucking nematode Hemonchus contortus. A total of 18 infected lambs were fed sainfoin either untreated or treated with polyethylene glycol, a CT-inactivating agent, or a grass-clover mixture (13.2% CP in DM) over 3 wk (n = 6). Six uninfected lambs received the grass-clover mixture as a control. In addition to indicators of infection (fecal egg count, packed-cell volume, abomasal worm burden, and serum protein), nutrient digestibility, the balance of N and selected minerals, ruminal fluid characteristics, and plasma AA levels were determined mostly in the final experimental week. The specific effects of the sainfoin CT, the extra CP with sainfoin, and the infection were statistically evaluated by contrast analysis. The sainfoin CT exerted no beneficial effects on resilience to nematode infection and exerted only minor effects on ruminal ammonia or blood urea concentrations and the excretory pattern of N. Plasma alanine, aspartate, and proline concentrations tended to be greater (P 0.09) because of the sainfoin CT, whereas the other AA remained unaffected. Intake of the mineral supplement was lower (P < 0.001) for lambs fed sainfoin compared with those fed sainfoin treated with polyethylene glycol. Feeding the high-protein sainfoin instead of the grass-clover mixture increased (P < 0.001) N retention and apparent OM digestibility, whereas digestibility of NDF and ADF were decreased (P < 0.001). Feeding sainfoin also decreased (P 0.04) plasma alanine, glycine, isoleucine, and total nonessential AA compared with the grass-clover mixture. Although fecal egg count, worm burden, and packed cell volume were not affected by the greater CP supply associated with sainfoin feeding, serum albumin level was increased (P = 0.008). The lack of effects of sainfoin on resilience to nematode infection might have been the result of the unexpectedly low CT content (3.6% in DM) of the material used. It cannot be excluded that longer term feeding of this batch of sainfoin might have been effective.

Key Words: Haemonchus contortus • nitrogen • polyethylene glycol • ruminant • sainfoin • sheep

	INTRODUCTION

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There is increased interest in alternative methods of controlling gastrointestinal nematodes because of increased anthelmintic resistance. One promising option seems to be the use of condensed tannin (CT)- containing forage plants with anthelmintic properties. Sainfoin (Onobrychis viciifolia) is a temperate legume with significant amounts of CT (3 to 10% in DM) that has been demonstrated to have anthelmintic properties in sheep or goats infected with intestinal or abomasal nematodes (Paolini et al., 2005; Heckendorn et al., 2007). Sainfoin also has a relatively high nutritional value (Egan and Ulyatt, 1980; Scharenberg et al., 2007b). In infected sheep, consumption of CT plants may enhance resilience (i.e., maintaining productivity during infection). They could reduce the worm burden directly (Hoste et al., 2006) or could improve the metabolic protein supply, thus enforcing resilience by equilibrating the parasite-caused metabolic protein loss (Hoste, 2001). This extra metabolic protein may come from the elevated CP contents of the legumes and also from CT decreasing ruminal proteolysis and thus possibly increasing the AA amounts arriving at, and being absorbed from, the intestine (Waghorn et al., 1994b). The extent to which the individual factors contribute to the anthelmintic properties of CT-containing plants is still unclear.

To differentiate among these factors, the objective of the present study was to evaluate the effect of sainfoin, compared with a low-CT grass-clover mixture, on infection parameters in lambs artificially infected with Hemonchus contortus, a blood-sucking abomasal nematode. Furthermore, specific focus was placed on N and mineral balances and traits reflecting digestion and metabolic AA supply to evaluate other effects of sainfoin feeding.

	MATERIALS AND METHODS

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Experimental Forages

The forages were grown in Posieux, Switzerland (latitude: 46°46 ' N; longitude: 07°06' E; altitude: 650 m). Sainfoin was harvested in spring 2005 from the first cut of a plot sown the year before. At harvest, it constituted 81.3% sainfoin, 8.7% dandelion (Taraxacum officinale), and 6.3% grasses (fresh matter basis). Drying of the freshly cut sainfoin was carried out by using a special device (Physitech, Wabern, Switzerland) whereby 30°C air was forced in a closed system through the forages and the forages were dried in a condenser and warmed again. This procedure was chosen to minimize leaf losses and to obtain a material with a high nutritional value (Scharenberg et al., 2007a). The grass-clover mixture was harvested from a pasture after 56 d of regrowth (second cut) and was proportionately composed (fresh matter basis) of 49.2% grasses (54.0% Lolium perenne, 26.5% Phleum pratense), 27.1% clover (84.6% Trifolium repens), and 23.7% forbs (98.9% dandelion). The grass-clover mixture was wilted on the field for 1 d and subsequently subjected to forced warm-air drying in the barn.

Animals and Experimental Design

The experiment was conducted according to the Swiss Guidelines of Animal Welfare, and the experimental protocol was approved by the Veterinary Department of Fribourg, Switzerland.

The experiment was carried out with 36 female White Alpine Sheep lambs, which were allocated according to their BW to 3 groups of 12 lambs. Before beginning the experiment, all lambs were dewormed (10 mg of tric-labendazol/kg of BW; 7.5 mg of levamisole/kg of BW; Endex, Novartis AG, Bale, Switzerland; 3.8 mg of albendazole/kg of BW; Albazol Suspension, Dr E. Gräub AG, Bern, Switzerland) and received an i.m. injection of 75 mg of vitamin E and 3 mg of Se (Selen-E Vetag, Veterinaria AG, Zürich, Switzerland). Subsequently, each group was used in 1 of the 3 experimental series lasting 7 wk each. The average BW of the lambs in the 3 groups at the beginning of each experimental series was similar (32.5 ± 1.1 kg, 30.9 ± 1.5 kg, and 32.5 ± 1.1 kg). The lambs of the 3 groups were 108 ± 4.8 d, 117 ± 10.5 d, and 122 ± 15.7 d old, respectively. Lambs of this age were chosen because it is likely that their immune competence against H. contortus was not yet established (Manton et al., 1962). At the beginning of each series, 7,000 infective larvae of H. contortus per animal were administered in 9 of the 12 lambs with a flexible stomach tube. During the first 4 wk of the experiment, infected and uninfected sheep were housed separately in groups in straw-bedded boxes and had ad libitum access to the grass-clover mixture. Additionally, twice daily 10 g each of a commercial mineralized salt mix (UFA 998, UFA, Herzogenbuchsee, Switzerland) was supplied. To avoid reinfection, straw of the infected sheep was changed weekly.

After 4 wk, when the infection was established, 6 of the 9 infected lambs from each series were selected and grouped by BW and fecal egg counts (FEC). These lambs were allocated to 3 treatments that were applied for the next 3 wk. The treatments consisted of dried sainfoin (SI), dried sainfoin treated with polyethylene glycol (PEG, a CT-binding agent; SIPEG), or a grassclover mixture poor in CT (CI). For the SIPEG treatment, 200 mL of an aqueous 25% (wt/vol) PEG solution (Polyethylene glycol 4000, Roth, Karlsruhe, Germany) was poured over each meal at least 9 h before feeding. Finally, 2 of the 3 uninfected lambs per series were selected by BW (best fitting to the infected lambs) as control animals and were offered the grass-clover mixture (CU). The experimental diets, fed to the animals from wk 5 to 7, were calculated to supply 66 g of OM/kg of metabolic BW (BW^0.75) daily from the forage. This level was chosen to meet the requirements of the lambs (Agroscope Liebefeld-Posieux, 2006) and, at the same time, to ensure a similar and complete intake in all lambs in all series. Feeding levels were adjusted when weekly BW were taken. The feeding took place at 0730 and 1630 h. A quantity of 20 g/d of the same mineralized salt mix as that used in the first 4 wk was offered for 30 min, always before the forage feeding. During the entire experiment, sheep had free access to fresh water.

During the adaptation period to the experimental diets (wk 5 and 6), sheep were housed in individual boxes on sawdust. During wk 7 of the experiment (collection period), sheep were kept in metabolic crates that allowed the complete and separate collection of urine and feces. Before the beginning of the collection period, the animals had been accustomed to this procedure by putting them into the crates for 2 d. At the end of the collection period, lambs were slaughtered to determine the H. contortus burden in the abomasum. Twelve lambs were excluded from the experiment.

Sampling Procedure

Beginning on the day of the infection, samples of blood and feces were taken once weekly at 2 h after the morning feeding to monitor the course of the infection by determining the packed cell volume (PCV) and the FEC. Furthermore, blood was sampled at the same time just before the infection and before and after the collection period. The samples were taken from the vena jugularis and collected in vacuum tubes with Z Serum Clot Activator (Greiner Bio-One, Solingen, Germany) and stored at room temperature or in vacuum tubes containing K₃EDTA (Greiner Bio-One) and cooled on ice. Samples were centrifuged at 1,500 x g for 15 min. Before infection, and before and immediately after the collection period, rumen fluid was taken with a flexible stomach tube 2 h after the morning feeding and strained through 2 layers of cheesecloth. For ammonia determination, 5 mL of ruminal fluid was mixed with 0.1 mL of 5% (wt/vol) trichloroacetic acid. For later VFA analysis, 10 mL of ruminal fluid was mixed with 0.2 mL of 25% (wt/vol) sulfuric acid solution. Blood and ruminal fluid samples were stored at –20°C. For the determination of protozoal and bacterial counts, 200 and 20 µL of strained ruminal fluid were diluted with 800 and 1,980 µL of Hayem solution (HgCl₂, 25 g/L; Na₂SO₄, 25.0 g/L; NaCl, 5.0 g/L), respectively. Feed samples were taken daily and pooled every second week until the adaptation period began. During the adaptation and collection periods, daily samples were pooled into weekly samples. During the collection period, feces, urine, and refusals of forage were collected quantitatively once daily and water intake was recorded. Refusals of the mineral mix were sampled over the collection period, dried at 60°C to avoid interference with saliva, and weighed. An aliquot of the urine was acidified with 3 M sulfuric acid to prevent gaseous N losses. Feces and urine samples were stored at –20°C. At the end of the collection period, samples of feces, urine, and forage refusals were pooled across day for every lamb.

Immediately after slaughter, the abomasum of the animals was removed and ligated. Subsequently, it was opened, thoroughly washed to collect the luminal contents, and preserved in 10% (vol/vol) formalin.

Laboratory Analyses

The DM of feeds and forage refusals was quantified gravimetrically (3 h at 105°C). The DM content of feces was determined by lyophilization and subsequent drying at 105°C for 3 h. Before the laboratory analyses, all feed samples and forage refusals were dried at 60°C for 12 h and ground to pass a 1-mm screen (Brabender mill, no. 880804, Brabender, Duisburg, Germany). Lyophilized feces were ground with the same equipment. Cell wall constituents were analyzed by using an Ankom 200/220 Fiber Analyzer (Ankom Technology Corporation, Fairport, NY). Acid detergent fiber (procedure 973.18; AOAC, 1995) was determined with correction for residual ash obtained after incineration at 500°C for 1 h, and NDF was assayed with heat-stable amylase and sodium sulfite and with correction for residual ash (Mertens, 2002). Feed, forage refusals, and feces were dry-ashed and solubilized in 10 M nitric acid, and Ca, P, Mg, and Na concentrations were determined with an inductively coupled plasma optical emission spectrometer (ICP-OES, Optima 2000 DV, Perkin-Elmer, Schwerzenbach, Switzerland). Nonacidified urine, solubilized in nitric acid, was analyzed for mineral content by using the same equipment. Total N content of feeds, forage refusals, feces, and urine was analyzed by the micro-Kjeldahl (procedure 988.05; AOAC, 1995). The CP content was calculated as 6.25 x N content. For determination of AA, feed samples were prepared as recommended by the European Union (1998), including digestion with HCl, and analyzed with an HPLC instrument (Alliance 2695, Waters, Milford, MA) following the manufacturer’s manual (Waters AccQ Tag Chemistry Package 052874 TP, rev. 1). Contents of CT in the forages were analyzed by applying the butanol-HCl method, as described by Terrill et al. (1992). Ammonia concentration in ruminal fluid was analyzed colorimetrically with a commercial test kit (Coffret Urea-Kit S 180, bioMérieux, Geneva, Switzerland). Determination of VFA was performed based on the method of Niven et al. (2004) by using an HPLC instrument (System HPLC, Dionex, Sunnyvale, CA) equipped with an integrated column (Nucleogel ION 300 OA, 300 x 7.8 mm, Macherey-Nagel, Düren, Germany). Ciliate protozoa and bacteria in ruminal fluid were enumerated by using 0.1- and 0.02-mm Bürker counting chambers (Blau Brand, Wertheim, Germany), respectively. Feces samples were processed for FEC according to the methods of Schmidt (1971), and the results are expressed as numbers of eggs per gram of dried feces, as described by Heckendorn et al. (2006). The DM content of the feces was determined in a 3-g subsample dried in a forced-air oven at 105°C for 16 h. Adult worm counts and sex identification were performed in a 10% aliquot of the overall abomasal content.

The PCV was measured manually by using a microhematocrit centrifuge (Hawkley, Lancing, Sussex, UK). Albumin and total protein were analyzed in blood serum with the respective test kits (ALB plus, Total Protein, Roche Diagnostics, Basel, Switzerland), and urea and glucose were determined in blood plasma (Urea kinetic UV 250, bioMérieux; Glucoquant, Roche Diagnostics). For the determination of free plasma AA, 2 internal standards [norvaline (No. 7627, Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) and piperidine-4- carboxylic acid (No. 80650, Fluka-Chemie AG, Buchs, Switzerland)] were added to the plasma samples. Subsequently, the mixture was purified in 3 centrifugation steps (15,600 x g for 15 min). The final supernatant was mixed with a stabilizer (buffer Li-S pH 3.3, Bio- Chrom 80-2038-10, BioChrom, Cambridge, UK) and stored at –80°C until analyzed by reverse-phase HPLC (Bütikofer and Ardö, 1998).

Statistical Analyses

Data were analyzed by using the GLM procedure (SAS Inst. Inc., Cary, NC), with treatment, series, and their interaction as fixed factors. Contrasts were used to separate treatment effects: 1) the CT effect, by comparing the sainfoin treatments with and without PEG (SI vs. SIPEG); 2) the effect of protein intake, by comparing infected sheep receiving sainfoin with PEG and those receiving the grass-clover mixture (SIPEG vs. CI; this comparison was limited by the associated changes of forage type); and 3) the effect of infection, by comparing infected with uninfected sheep fed the grass-clover mixture (CI vs. CU). For the analysis of blood data (except PCV) and ruminal fluid parameters, means were calculated from the data obtained from the days directly before and immediately after the collection period. Data were subjected to the same contrast analysis mentioned above but using the data from the samples taken on the day of infection as a covariate. The FEC and worm count data were log (x + 1) transformed before statistical analysis. For FEC [beginning with the first egg excretion found 3 wk postinfection (p.i.) and excluding treatment CU] and PCV, the weekly collected data were analyzed with the MIXED procedure of SAS by using the repeated statement. The model included treatment, week, and their interaction as fixed factors and series as a random factor. P < 0.05 was considered significant, and P < 0.10 was considered a trend. In calculating the intake of the mineral mix and the mineral balances, data on 1 lamb in the SI treatment were excluded, because the lamb refused to consume the mineral mix.

	RESULTS

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Chemical Composition of the Feeds

The grass-clover mixture and the sainfoin used in the collection period differed considerably in their contents of CP, NDF, CT, and minerals (Table 1). Sanfoin had more CT, CP, Ca, and Mg than the grass-clover mixture (Table 1), with a lower content of Na and Fe. The proportions of total AA as essential and semiessential AA were similar between the 2 forages. Concerning individual AA, the proportions of aspartate, histidine, lysine, tyrosine, and mainly proline were greater and the proportions of alanine, arginine, glutamate, glycine, isoleucine, leucine, methionine, phenylalanine, serine, threonine, and valine were slightly lower in sainfoin than in the grass-clover mixture. Analyses of the grass-clover mixture fed during wk 1 to 4 were (DM basis): 89.6 ± 0.52% OM, 13.8 ± 0.52% CP, and 46.9 ± 0.89% NDF (data not shown in tables). During the adaptation period (wk 5 and 6), the nutrient contents of the forages analyzed were similar to those observed in the collection period.

In the uninfected lambs, no H. contortus eggs in feces were found throughout the entire experiment. The first eggs in feces of the infected lambs were counted 3 wk p.i. (Figure 1), and the greatest average FEC was observed in wk 4 p.i. (15.9 ± 5.94 x 10³ eggs/g of fecal DM). Afterward, FEC decreased slightly until the end of wk 7. Differences among dietary treatments were not significant (P > 0.10). The worm burden was also similar (P > 0.10) in all infected animals. At slaughter 147 ± 18, 152 ± 65, and 161 ± 54 worms were found in the abomasum of SI, SIPEG, and CI lambs, respectively. The percentages of female (SI: 46.1 ± 4.0; SIPEG: 50.9 ± 4.3; CI: 51.2 ± 4.0) and male worms (SI: 53.9 ± 3.9; SIPEG: 49.1 ± 4.3; CI: 48.8 ± 4.0) of the total worm count did not differ (P > 0.10). The changes in PCV during the experiment are presented in Figure 2. Regardless of treatments, PCV decreased from wk 1 to 3 and then increased until the end of wk 7. At the beginning of the experiment, PCV differed slightly among treatments, but converged in wk 2 p.i. From wk 3 until the end of wk 7 p.i., uninfected lambs had greater (P < 0.001) PCV than infected lambs. No differences (P > 0.10) in PCV were observed among the SI, SIPEG, and CI treatments. Sheep did not show any clinical symptoms of nematode infection.

View larger version (15K): [in this window] [in a new window]   Figure 1. Fecal egg count (FEC) in infected lambs fed different forages beginning with the first egg excretion after 3 wk postinfection (means of original data). •, Sainfoin; , sainfoin supplemented with polyethylene glycol; , grass-clover mixture. Vertical bars represent SEM of the respective week postinfection. The vertical dashed line indicates the beginning of feeding of the experimental forages.

View larger version (19K): [in this window] [in a new window]   Figure 2. Packed cell volume over the experimental period of 7 wk. Infected sheep: •, sainfoin; , sainfoin supplemented with polyethylene glycol; , grass-clover mixture; , Uninfected sheep fed the grass-clover mixture. Vertical bars represent SEM of the respective week postinfection. Within a week, means with different letters are significantly different at P < 0.05. The vertical dashed line indicates the beginning of feeding of the experimental forages.

No differences among treatments were observed (P > 0.10) in total intake of DM, OM, ADF, and water (Table 2), except for a trend toward greater water intakes in SIPEG compared with CI (protein contrast: P = 0.07) and a trend for greater DMI in CU compared with CI (infection contrast: P = 0.08). The CT from sainfoin clearly reduced the intake of the mineral supplement (CT contrast: P = 0.004). Because of the differences in chemical composition between forages, lambs fed the grass-clover mixture had a greater NDF intake (protein contrast: P < 0.001) compared with lambs fed the sainfoin. The apparent NDF and ADF digestibilities were greater in CI than in SIPEG (protein contrast: P < 0.001). The lowest apparent OM digestibility was observed in SI lambs; this was obviously due to the effect of CT (CT contrast: P < 0.001). As a result, CT also decreased the intake of digestible OM (CT contrast: P < 0.001). This was not the effect of sainfoin because OM digestibility and intake of digestible OM were greater in SIPEG than in CI lambs (protein contrast: P = 0.002). The apparent digestibility of OM (infection contrast: P = 0.06) and ADF (P = 0.02) tended to be greater because of the infection.

Condensed tannins increased fecal N losses (CT contrast: P = 0.002), tended to decrease urinary N excretion (P = 0.07), and resulted in a lower proportion of urinary N in total excreted N (P = 0.004; Table 4), but body N retention was unaffected by CT. Sainfoin feeding as such increased N intake by more than 50% compared with feeding the grass-clover mixture (protein contrast: P < 0.001), which was associated with a greater urinary and total excretion of N, but also greater N retention (P < 0.001). When expressed as a proportion of N intake, fecal N excretion was lower (protein contrast: P < 0.001) and urinary N excretion (P = 0.006) was greater in SIPEG than in CI lambs. Apart from a trend (infection contrast: P = 0.07) toward a greater urinary N proportion of the total excreted N, the infection had no effect on N balance.

There were no treatment effects (P > 0.10) on plasma glucose levels, which averaged 3.21 ± 0.41 mM across all groups (data not shown). The CT tended (CT contrast: P = 0.05) to increase total serum protein concentration, whereas this was opposite (P = 0.09) for serum albumin (Table 6). Infection reduced serum concentrations of albumin (infection contrast: P < 0.001) and total protein (P = 0.004). Sainfoin with PEG, compared with the grass-clover treatment, slightly elevated (protein contrast: P < 0.008) the serum albumin level and clearly enhanced plasma urea (P < 0.001), a trait not affected by the other treatments. The CT tended to increase plasma levels of alanine (CT contrast: P = 0.07), aspartate (P = 0.09), and proline (P = 0.08) but none of the other AA. Sheep fed sainfoin treated with PEG, compared with those fed the grass-clover mixture, had decreased plasma concentrations of isoleucine (protein contrast: P = 0.03), alanine (P = 0.04), glycine (P = 0.002), and total nonessential AA (P = 0.03). As a consequence, the ratio of essential to nonessential AA increased (protein contrast: P = 0.02). Infection increased plasma concentrations of arginine (infection contrast: P = 0.02) and decreased concentrations of threonine (P = 0.02) and tryptophan (infection contrast: P = 0.005).

	DISCUSSION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The severity of acute infection was comparable across treatments and was strong enough to significantly affect PCV, serum albumin, and total protein. Because maintenance of body protein, and therefore compensation of blood losses and tissue reparations, has a greater partitioning factor than growth, animals grow slower when infected (Coop and Kyriazakis, 2001). Infection with gastrointestinal nematodes can increase the protein requirement in sheep for these reasons and also because additional protein is needed for repairing the affected sites in the gastrointestinal tract (Holmes, 1993).

Infection with abomasal nematodes may also cause hyperplasia of mucous cells and decreased numbers of chief cells in the abomasum, which in turn can increase abomasal pH and reduce the abomasal digestion of nutrients (Scott et al., 1998), thus possibly resulting in decreased nutrient digestibility. Rowe et al. (1988) found a lower apparent digestibility of OM in lambs infected with H. contortus. However, this could not be confirmed by the present study and might depend on the severity of infection, which was greater in the study of Rowe et al. (1988).

The surprising finding of Rowe et al. (1988) that a parasitic infection of the abomasum alters fermentation processes in the rumen could not be confirmed in the present study. Furthermore, no effects of infection with H. contortus on the N balance were found, which is in agreement with the results of Abbott et al. (1984) but inconsistent with those of Rowe et al. (1988). The findings of Abbott et al. (1984) revealed that most of the additional N flowing from the abomasum caused by blood loss was absorbed from the small intestine. This suggests that the infection with H. contortus and loss of blood into the abomasum does not necessarily impair the metabolic protein supply because of compensatory postabomasal processes, unless coinfection of the small intestine is present. This hypothesis was confirmed by the unchanged fecal N losses in the study of Rowe et al. (1988). However, in that study, despite unchanged fecal N losses, more ammonia was found in the abomasum of parasitized sheep. According to Rowe et al. (1988), the greater amount of ammonia in the abomasum led to a greater amount of ammonia absorbed in the intestine and therefore to greater urinary N losses. Conversely, in the present study no changes in the concentrations of ruminal ammonia and blood urea were found, which suggests that the greater proportion of urinary N in infected, compared with uninfected, lambs was rather a result of the greater maintenance requirement for protein, because spent protein is excreted via urine.

Effects of Sainfoin CT

Feeding of sainfoin and other CT plants has been repeatedly demonstrated to have anthelmintic effects (reviewed by Hoste et al., 2006; Heckendorn et al., 2006, 2007). Hoste et al. (2006) suggested that exceeding a threshold of 3 to 4% CT in DM is needed for the feed to develop anthelmintic activity. Accordingly, the amount of sainfoin used in the present study was borderline. Additionally, it is likely that not only the concentration of CT in the diet but also the molecular structure of CT may be critical for the effects of tanniferous forages (Aerts et al., 1999).

In a previous study conducted with uninfected lambs (Scharenberg et al., 2007b), the CT present in sainfoin clearly decreased urinary N excretion, increased fecal N excretion, and resulted in greater plasma levels of essential AA. In the present experiment with infected lambs, sainfoin CT affected N excretion in a similar way, but to a much lesser extent, and total N excretion as well as plasma levels of essential AA were not influenced by CT. The greater fecal N losses in the presence of sainfoin CT indicated that the benefit from the (only slightly) reduced ruminal proteolysis was low. Two possible explanations can be given for the differences between the studies. First, the CT content of sainfoin was low in the present study and possibly below the threshold value required for an effective reduction of protein degradation. The sainfoin used in the study of Scharenberg et al. (2007b) had been harvested from the same plot but 1 yr before, and CT content was approximately 2-fold greater (7.70 vs. 3.64% of DM) than that of the sainfoin used in the present study. Min et al. (2003) summarized that for an overall beneficial effect on protein metabolism, CT content in forages should not exceed 5%, which is inconsistent with the results of a previous study with uninfected lambs (Scharenberg et al., 2007b). Again, specific structural properties of CT might play an important role in that respect. Second, the greater expenditure of AA in the infected lambs, as opposed to uninfected lambs, may have prevented elevated plasma levels of essential AA from being expressed.

In the present study, sainfoin CT were found to increase the molar proportion of acetate and decrease the proportions of propionate and n-valerate. The latter might indicate a reduced ruminal proteolyis but this reduction was probably small in magnitude, because ruminal ammonia concentration did not differ. Condensed tannins reduced ruminal celluloytic bacteria (McSweeney et al., 2000), which may cause a decreasing acetate:propionate ratio, as reported by Carulla et al. (2005) in sheep. However, Stürm et al. (2006), when fermenting different tropical tanniferous legumes in vitro, found an increasing acetate:propionate ratio with increasing dietary CT content. Furthermore, Scharenberg et al. (2007b) observed no effect of sainfoin CT either on the VFA profile or on the bacterial count, which confirms the results of a metastudy (Min et al., 2003) showing that the amount of microbial protein does not change with dietary CT content.

Aversions and preferences for specific dietary minerals, including P and Na, have been reported previously (Provenza et al., 2003). There was a very low consumption of the mineral mix by lambs fed untreated sainfoin compared with PEG-treated sainfoin and the grass-clover mixture. Both CT and PEG have the capacity to bind to minerals (Freeland et al., 1985; Waghorn et al., 1994a; Fraser and Smith, 2000) and may therefore decrease mineral absorption from the intestine. This would explain the greater mineral intake with PEG-treated feed. However, Scharenberg et al. (2007b) reported that supplementing PEG to sheep fed on sainfoin may lead to greater retention of P, Ca, and Mg. A negative influence of CT on Fe absorption has previously been demonstrated in monogastrics, including humans (Santos-Buelga and Scalbert, 2000). Even so, the Fe intake was approximately 5-fold greater for the grass-clover mixture than in sainfoin-fed lambs, and Fe retention was negative in all treatments, mainly because of high fecal losses.

Effects of Sainfoin Replacing the Grass- Clover Mixture (Protein Supply)

The ability of sainfoin to reduce nematodes could partly result from the greater protein requirements of infected animals, as discussed above. Because of the increase in fecal N excretion attributable to CT (Kumar and Singh, 1984; Waghorn et al., 1994b; Jansman et al., 1995), feeding sainfoin can be useful for the protein metabolism of ruminants only if the actually digested part of the ingested CP is utilized more efficiently. Legumes such as sainfoin have greater protein contents and might meet the protein requirements of healthy growing lambs, even if the availability of CP is decreased because of the presence of CT. This might not apply for infected lambs. For that reason, comparison of the PEG-treated sainfoin with the grass-clover mixture should reveal the full potential of sainfoin protein, although there is the limitation that other properties differing between the 2 forages might add to the effects as well. However, beneficial effects concerning indicators of the metabolic protein supply (N balance, plasma AA, serum protein and albumin, PCV, metabolic urea load) could be seen only in serum albumin concentration, which was slightly greater compared with that of the other infected lambs. In contrast, Datta et al. (1999) found greater BW gain, greater wool production, greater antibody responses, and lower FEC in lambs infected with H. contortus and Trichostrongylus colubriformis and fed high-protein diets for 9 wk. Furthermore, Wallace et al. (1998) observed significant improvements in resilience (PCV, plasma albumin, mean cell volume) of H. contortus-infected lambs between urea-supplemented (+31 g of MP) and control lambs beginning 4 wk p.i., but they detected no effect on worm burden. This suggests that the experimental period was too short for the greater CP supply to have a positive effect.

As expected from the different CP contents, feeding of PEG-treated sainfoin massively increased ruminal ammonia and plasma urea content compared with sheep fed the grass-clover mixture. With few exceptions, differences in plasma AA levels between sheep fed PEG-treated sainfoin compared with sheep fed the grass-clover mixture mainly reflected the differences in AA composition of the 2 forages. Therefore, an influence of an increased CP supply on plasma AA concentrations could not be demonstrated. Regarding bacterial and protozoal counts in the rumen fluid, differences between the sainfoin and grass-clover mixture treatments could not be shown, which was surprising for the greater and lower CP supply. Although levels of plasma glucose did not vary because of the differing forages, indicating that energy was not limiting in either case, there were some shifts in VFA profile, likely owing to differences in the fiber content of the forages. A greater Fe turnover attributable to the increased protein supply, as was shown by Abbott et al. (1984), was partially confirmed by the greater urinary Fe losses. However, the effects of protein supply on mineral balance were likely masked by the different mineral contents of the 2 forages.

In conclusion, even though sainfoin can be seen as one of the most promising species among temperate, tanniferous forage plants, no beneficial effects of an increased CP supply (sainfoin with PEG) or of CT (sainfoin without PEG) on resilience to nematode infection were observed. This makes it impossible to decide whether the CT or CP supply or both are responsible for the potential anthelmintic effect of sainfoin. Compared with other studies showing such a positive effect, the period of sainfoin feeding was short and the CT content in sainfoin was low in the present study. The latter even caused unexpectedly low effects on ruminal protein metabolism. The reasons for such varying CT contents of sainfoin, especially because CT were low in the second year of establishment compared with the first year, are still unclear but are important to clarify. In future studies, special emphasis must also be placed on the mode of action of sainfoin CT in enhancing resilience in infected sheep. In addition, the time frame and the concentration of CT that are required to counteract parasites effectively must be defined before sound advice can be given on the use of sainfoin as a successful alternative strategy to control gastrointestinal nematodes in sheep flocks.

	Footnotes

 
¹ Anna Scharenberg acknowledges a grant from the Swiss Federal Office for Agricultural (FOAG, Bern). The authors are grateful for the technical support received during the experiment, especially from Marie-Hélène Kolly.

² Corresponding author: frigga.dohme{at}alp.admin.ch

Received for publication July 24, 2007. Accepted for publication March 26, 2008.

	LITERATURE CITED

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 


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