J Aufre `re

M Dudilieu

D Andueza

C Poncet

R Baumont

Mixing sainfoin and lucerne to improve the feed value of legumes fed to sheep by the effect of condensed tannins

Keywords: ruminants, in vivo, condensed tannins, sainfoin-lucerne mixtures. Implications

The aim of this study was to investigate whether the use of sainfoin-based condensed tannins (CT) enhances feed value when given with tannin-free legumes (lucerne) to sheep. The experiments were conducted with fresh sainfoin and lucerne harvested at two stages (vegetative stage as compared with early flowering) in the first growth cycle. Fresh sainfoin and lucerne forages were combined in ratios of 100 : 0, 75 : 25, 25 : 75 and 0 : 100 (denoted S100, S75, S25 and S0, respectively). Voluntary intake, organic matter digestibility (OMD) and nitrogen (N) retention were measured in sheep fed the different sainfoin and lucerne mixtures. Loss of dry matter (DM) and N from polyester bags suspended in the rumen, abomasum and small intestine (SI) was also measured using rumen-fistulated sheep and intestinally fistulated sheep. The CT content in sainfoin (S100) decreased with increasing percentage of lucerne in the mixture (mean value from 58 g/kg DM for S100 to 18 g/kg DM for S25) and with growth stage (S100: 64 to 52 g/kg DM). OMD did not differ between different sainfoin/lucerne mixture ratios. Sainfoin and lucerne had an associative effect (significant quadratic contrast) on voluntary intake, N intake, total-tract N digestibility, N in faeces and urine (g/g N intake) and N retained (g/g N intake). Compared with lucerne mixtures (S0 and S25), high-sainfoin-content mixtures (S100 and S75) increased the in situ estimates of forage N escaping from the rumen (from 0.162, 0.188 for S0 and S25 to 0.257, 0.287 for S75 and S100) but decreased forage N intestinal digestibility (from 0.496, 0.446 for S0 and S25 to 0.469, 0.335 for S75 and S100). The amount of forage N disappearing from the bags in the SI (per g forage N) was the highest for high-sainfoin mixtures (from 0.082, 0.108 for S100 and S75 to 0.056, 0.058 for S25 and S0, P , 0.001). Rumen juice total N (tN) and ammonia N (NH3-N) values were the lowest in the high-sainfoin diet (mean tN 0.166 mg/g in S100 as compared with 0.514 mg/g in S0; mean NH3-N 0.104 mg/g in S100 as compared with 0.333 mg/g in S0, P , 0.001).

Introduction

Lucerne is one of the most nutritious forages available, and is used widely in diets for ruminants. Lucerne proteins are often poorly utilised by ruminants as they are quickly and extensively degraded in the rumen, which leads to nitrogen (N) losses via urinary urea excretion [START_REF] Min | The effect of condensed tannins on the nutrition and health of ruminants fed fresh temperate forages[END_REF] because energy is not sufficient. Technological processes can be proposed to overcome the problem of excessive solubility such as the production of dehydrated lucerne as a source of high-dietary-value proteins but it is energy consuming. Haymaking and barn drying provide an intermediate degree of protein protection. Other solutions included the use of forages containing condensed tannins (CT). CT are a group of plant secondary compounds that bind protein and inhibit its ruminal degradation [START_REF] Barry | The implications of condensed tannins on the nutritive value of temperate forages fed to ruminants[END_REF]. [START_REF] Mcmahon | A review of the effects of forage condensed tannins on ruminal fermentation and bloat in grazing cattle[END_REF] and [START_REF] Julier | Quelles le ´gumineuses fourrage `res (espe `ces et varie ´te ´s) et quelles conduites pour ame ´liorer l'autonomie prote ´ique des e ´levages herbivores[END_REF] suggested that -E-mail: jocelyne.aufrere@clermont.inra.fr the utilisation of transgenic lucerne cultivars able to produce CT could be an alternative way to reduce protein losses. Another more realistic solution is the use of a mixture of forages with and without CT, which leads to protein degradability in the rumen of the mixture being lower than the average of the two initial fodders as has been demonstrated in in vitro conditions by [START_REF] Julier | Effect of condensed tannin content on protein solubility in legume forages[END_REF] for a mixture of lucerne and birdsfoot trefoil. Previous in vitro results [START_REF] Waghorn | Effect of condensed tannins in Lotus corniculatus on the nutritive value of pasture for sheep[END_REF] have shown that CT from Lotus corniculatus are able to bind with and precipitate protein from a ryegrass/clover pasture, but when these forages were fed together to sheep, the CT had only low effects on digestion and animal performances. Aufre `re et al. (2005) showed that surplus CT in sainfoin can efficiently reduce the in vitro N solubility of lucerne when well mixed with it. The aim of the study reported here was to test this hypothesis in in vivo conditions.

This study was designed to measure the effects of CT in sainfoin co-fed with lucerne to sheep. The trial was conducted indoors, with sheep fed individually in order to fully control the proportions of sainfoin and lucerne provided. The effects of the proportion of sainfoin in the mixture and of the growth stages of the two plants on voluntary intake, digestibility and N balance were investigated in vivo. The digestive characteristics of the mixtures were assessed by in situ measurements in the rumen and the small intestine.

Material and methods

Forages

Sainfoin (Onobrychis viciifolia L., cv. Zeus) and lucerne (Medicago sativa L., cv Aubigny) swards were sown in spring 2005 on the INRA's Clermont-Ferrand/Theix (France) site at an altitude of 850 m on a deep silt loam soil. Mineral fertilisation was supplied in spring 2006 at 37 kg P and 56 kg K/ha.

The experiment was conducted in 2006 using fresh sainfoin and lucerne harvested at two dates (stages 1 and 2) in the first growth cycle. Both forages were harvested on the same date (3rd week of May and 2nd week of June). The sum of temperatures from 1st February was 5868C for stage 1 and 8028C for stage 2. For sainfoin, stage 1 was defined as the vegetative stage (30 to 40 cm height) and stage 2 was defined as the early flowering stage (5% to 10% of stems in a metre row observed with at least one open flower). Lucerne was cut at the vegetative stage and the budding stage for stages 1 and 2, respectively.

Animals and experimental design

The study was carried out indoors at the INRA-UE-RT experimental farm in France (45842 0 N, 038304 0 E). The sheep were handled by specialist personnel trained in animal care and welfare according to European Union Directive No. 609/186, under agreement No. A63 565.

Fresh sainfoin and lucerne forages were combined in ratios of 100 : 0, 75 : 25, 25 : 75 and 0 : 100 (as fed) denoted S100, S75, S25 and S0, respectively. A total of four groups of six Texel sheep (12 months old, 60 6 3 kg live weight) were used simultaneously for measurements of voluntary intake, digestibility and N balance on each mixture.

Concurrently, four groups of three adult Texel sheep, fitted with a large rumen cannula, were used to measure the in-rumen degradability of the different mixtures using the nylon bag technique. Rumen fluid samples were collected in kinetics on these sheep.

Another group of four adult Texel sheep fitted with duodenal and ileal T-type cannulae were used to determine intestinal digestibility using the mobile nylon bag technique according to [START_REF] Aufre `re | In vivo and in situ measurements of the digestive characteristics of sainfoin in comparison with lucerne fed to sheep as fresh forages at two growth stages and as hay[END_REF].

All the sheep were housed indoors in individual pens for in situ measurements or in individual metabolic crates for in vivo measurements. They were allowed ad libitum access to water and salt (NaCl) block. Before starting the experiments, the sheep were drenched with an anthelmintic (Ivomec, Merial, Lyon, France) to eliminate internal and external parasites. The animals were fed ad libitum (10% refusal) at 0900 h and 1700 h every day. The actual refusal proportion was on average 9.9% dry matter (DM). Forages were cut daily using a sickle bar mower at about 5-cm cutting height, and then chopped into 5-to 10-cm pieces with a chaff cutter to prevent wastage by the animal. Forages formed of the two legumes were mixed well in the different proportional ratios and were immediately distributed in the morning. The mixtures were then conserved at 48C for the evening meal. The chemical composition (N and NDF of refusals) did not reveal the dietary preference of the sheep (N: 28, 16 (S100), 27, 18 (S75), 28, 24 (S25), 31, 27, (S0) at stages 1 and 2, respectively; NDF: 470, 525 (S100), 471, 546 (S75), 474, 512 (S25), 446, 496, (S0) at stages 1 and 2, respectively). However, there may be a selection of different parts of the plant which was explained by differences in N, NDF contents between forages offered and refusals (N: 31, 22 (S100), 32, 24 (S75), 35, 29 (S25), 36, 32, (S0) at stages 1 and 2, respectively; NDF: 443, 432 (S100), 419, 414 (S75), 371, 380 (S25), 347, 363, (S0) at stages 1 and 2, respectively).

The sheep were allowed to adapt to the diet and to the crate during the first 2 weeks of each period. Measurements were performed in the 3rd week.

In vivo measurements and sampling Voluntary intake, digestibility and N balance measurements were performed on 6 consecutive days [START_REF] Demarquilly | Pertes fe ´cales et digestibilite ´des aliments et des rations[END_REF]. The sheep were housed in metabolic crates equipped for daily collection of total faeces and urine. Loss of ammonia from urine was prevented by the daily addition of 50 ml of 30% (w/v) sulphuric acid to the collection flask. Daily aliquots of faeces and urine were pooled per sheep for the week of measurements. During this week of measurement, the forages offered were sampled every day, and then pooled over the week. Samples of fresh forages, refusals and faeces were dried in a forced-air oven at 608C for 72 h before being pooled. All samples were ground through a 1-mm sieve before analysis. Pooled samples of urine were conserved at 2208C for the analysis of total N (tN).

Feed value of sainfoin-lucerne mixture

In situ degradation in the rumen Nitrogen degradability was measured via the nylon bag procedure using Dacron bags (pore size 53 6 15 mm; Ankom Co., Fairport, NY, USA) with an internal surface of 5 3 11 cm. Fresh forages sampled mid-week during the in vivo measurements were shredded before bagging. The degradability of the four mixtures (S0, S25, S75, S100) was measured on a group of three fistulated sheep fed with the same mixture.

Bags were filled with a quantity of fresh forage equivalent to 2.5 g DM, and then sealed with two stitches and quickly frozen in liquid nitrogen for storage at 2208C until analysis. Incubation periods were 2, 4, 8, 16, 24 and 48 h, with two bags for 2-, 4-and 8-h measurements, six bags for 16 h measurements and three bags for the 24-and 48-h measurements. Samples of a common hay were incubated (8 h) daily in duplicate in the rumen of each animal to detect any changes in degradation levels during the experiment. After removal from the rumen, the bags were washed and kept at 2208C until analysis. Before analysis, the bags were thawed and then washed in a washing machine (3 3 10 min) with cold water until the rinse water was clear. The bags were then beaten for 7 min in a 'stomacher', then re-washed (2 3 10 min), dried at 608C for 48 h and ground through a 1-mm screen. In order to take into account adherent bacteria not detached by this treatment, a correction was performed according to [START_REF] My | Estimation of the extent of bacterial contamination in bag residues and its influence on in sacco measurements of forage nitrogen degradation in rumen[END_REF]. The soluble N fraction was determined by soaking the bags containing the samples in warm water (408C) for 1.5 h, and was considered as the proportion of forage N that had disappeared from the bags at 0 h.

Ruminal fluid sampling

During the experimental weeks, rumen fluid was taken from the 12 sheep fitted with a rumen cannula that were fed the four mixtures. Samples were collected before the morning meal (time after the meal (T) 0 h), and at 1, 2, 3 and 7 h after feeding. For each sample, 150 ml of rumen fluid was collected, muslin-filtered and centrifuged for 5 min at 120 3 g at 48C to remove large dietary particles and protozoa. The supernatant was centrifuged at 27 000 3 g for 20 min at 148C to remove small dietary particles and bacteria. Proteins were then precipitated by adding trichloroacetic acid (125 g/l) and separated after centrifuging (at 20 000 3 g for 10 min).

In situ intestinal digestibility In situ intestinal N digestibility of sainfoin/lucerne mixtures was subsequently measured in a separate experiment using the mobile nylon bag technique on sheep fitted with duodenal and ileal cannulae. The residues recovered after 16 h of rumen incubation were considered representative of the forage residues escaping from the rumen, and were thus used to fill the intestinal nylon bags (18 mm 3 18 mm, 48 mm pore size, 100 mg/bag). The bags were closed by heat sealing. The residues recovered after 16 h of rumen incubation were dried at 608C and ground through a 1 mm sieve before filling the bags. The bags were then incubated for 1.5 h at 398C in an HCl solution (0.01 N, pH 2.7) containing pepsin (2 g/l, Merck 2000 FIP-UIG, Darmstadt, Germany) to simulate abomasal digestion. After the morning meal, five bags per sheep were introduced into the duodenum at a rate of one bag every 30 min, that is, 5 3 4 bags for each growth stage of each fresh forage of each mixture; three hours after placing the Dacron bags in the duodenum, the ileal cannula was opened for bag recovery every 30 min. The recovered bags were roughly rinsed under cold tap water and stored at 2208C. After thawing, all the bags were washed (5 3 2 min) using a standardised procedure involving shaking in 1 l flasks containing 500 ml of warm water (398C), and then dried in a forced-air oven at 608C for 48 h. The results are presented as digestibility in the small intestine (SI), although they also included the disappearance of forage components from the HCL-pepsin treatment to simulate digestion in the abomasum.

Laboratory analyses

The total N content in forages, refusals, faeces and urines was determined using the Kjeldhal method. N in samples from in situ measurements was analysed using the Dumas method.

NDF, ADF and ADL were analysed according to [START_REF] Soest | Use of detergents in the analysis of fibrous feeds. IV Determination of plant cell-wall constituents[END_REF] as modified [START_REF] Pagan | Effect of drying method and assay methodology on detergent fiber analysis in plants containing condensed tannins[END_REF].

Pepsin-cellulase digestibility (Dcellms) was determined according to Aufre `re and [START_REF] Aufre Re | In vivo digestibility and prediction of digestibility of some by-products[END_REF]. The CT content was determined on freeze-dried samples using the HCl-butanol technique [START_REF] Terrill | Determination of extractable and bound condensed tannin concentrations in forage plants, protein concentrate meals and cereal grains[END_REF].

Ammonia N (NH3-N) in rumen fluid was determined using the Weatherburn method (1967).

Calculations and statistical analysis

The in situ forage N disappearance curves in the rumen were fitted to the model of [START_REF] Ørskov | The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage[END_REF] using a non-linear regression procedure. The proportion of N degraded at time (t) was calculated according to the equation N degraded 5 a 1 b (1 2 exp 2ct ), where 'a' is the rapidly degradable fraction, 'b' is the slowly degradable fraction and 'c' is the degradation rate of b. (100 2 a 2 b) was considered as the undegradable fraction. The effective degradability of N (DegN) was calculated according to the equation: DegN 5 a 1[(b 3 c)/(c 1 kp]), with the fractional passage rate kp assumed to be 0.06/h.

The same model was used to calculate the effective degradability of DM (DegDM).

In situ estimated forage N intestinal digestion was calculated as the fraction of initial forage N disappearing from mobile bags in the abomasum and the small intestine.

The different mixtures were called diet (D). Data from in vivo and in situ measurements were subjected to analysis of variance according to the repeated measurement model for testing diet (D) and growth stage (G) effects. Growth stage (G) was considered as the repeated variable, with animal as a random variable.

For the NH3-N and N contents in the rumen juice, D, T and G effects were subjected to analysis of variance according to a repeated measurement model. Time was considered as the repeated variable, with animal as a random variable.

Linear and quadratic contrasts were established to identify the effects related to the dietary proportions of the CT-containing legumes (sainfoin) in the mixtures. The mean differences between sainfoin-lucerne ratios were evaluated using the Tukey test and were considered significant when P , 0.05.

All analyses were performed using the Mixed procedure of the SAS software package (Statistical Analysis Systems Institute, 2000).

Results

Chemical composition

The N content was lower for sainfoin (S100) than for lucerne (S0). NDF, ADF and ADL (mean values) decreased when the percentage of lucerne increased in the mixture and had lower values for the mixtures (S0 and S25; Table 1). As expected, within each species, the N content decreased and ADL increased with plant maturity (Table 1). The N content and Dcellms of mixtures increased progressively with the increase in the percent lucerne content in the mixture (Table 1). The decrease in the N content between stage 1 and stage 2 was stronger for sainfoin than for lucerne (28.6 g/kg DM as compared 24.8 g/kg DM). This decrease remained high for S75 (7.8 points), whereas it ranged from 5.8 to 4.8 points for the other mixtures.

Moreover, Dcellms was closely linked to organic matter digestibility (OMD; R 2 5 0.78) for the mixtures. The CT content of sainfoin decreased with the growth stage (64 to 52 g/kg DM) and when the percentage of lucerne increased (S25: 19.8 and 16 g/kg DM).

Voluntary intake, digestibility and N balance (Table 2) The voluntary DM and N intake increased significantly with the proportion of lucerne, showing a quadratic effect (P , 0.01 for quadratic contrast), and the highest DM and N intake values were obtained with diets containing 25%, 75% or 100% lucerne (i.e. S75, S25 and S0). Voluntary DM intake did not appear to decrease between the vegetative and the early flowering stage, whereas N intake decreased at the early flowering stage (P , 0.001).

Table 2 Voluntary intake (g/kg W 0.75 ), N intake (g/day per sheep), OM and N digestibility in the total tract and N retention variables measured on four groups of six sheep simultaneously fed ad libitum on different mixtures of sainfoin and lucerne (S100, S25, S75, S0) as fresh forage at the vegetative stage (stage 1) and at the early flowering stage (stage 2) Feed value of sainfoin-lucerne mixture OMD of the forages remained similar for sainfoin and lucerne and was unaffected by plant mixture ratios (P . 0.05). However, OMD decreased significantly with the growth stage (P , 0.001). This decrease was stronger for lucerne (S0: 0.77) than for sainfoin (S100: 0.41), which consequently led to an interaction between diet and growth stage.

Faecal N output (g/g N intake) increased significantly via a quadratic effect as the proportion of sainfoin in the diet increased. Conversely, urinary N output was higher with lucerne (S0) and decreased via a quadratic effect with increasing proportions of sainfoin in the diet. No significant differences were found between S75 and S100. Total-tract N digestibility followed the same pattern as urinary N.

The N retained (g/g N intake) was significantly affected by the diet, although there were no differences between the two plants fed alone (S0 and S100). Interestingly, the amount of N retained increased significantly (P , 0.01 for the quadratic effect without a linear effect) for the two mixtures (S25 and S75). When N retained was expressed in g/day per sheep, the highest values were again obtained for the same mixtures and also for S0.

Nitrogen output in faeces and urine increased significantly with plant maturity, whereas the N content of the forages decreased and thus N digestibility and N retained by the animals decreased, without interaction with the diet effect. The observed decrease with plant maturity did not modify the classification.

In situ DM and N degradability in the rumen The DegDM of sainfoin (S100) was close to that of lucerne (S0) at stage 1 but decreased more markedly with forage maturity than for lucerne (0.18 units as compared with 0.05 units). Consequently, the interaction between diet and growth stage has a significant effect on DegDM (D 3 G, P , 0.001). The DegDM of the mixtures (S75 and S25) was linearly affected by the proportion of the two plants.

At stage 1, the DegN of sainfoin (S100) and the S75 mixture was lower than the DegN of S25 and S0 (mean of 0.765 as compared as 0.835; P , 0.001). The decrease in DegN with forage maturity was significantly stronger for sainfoin (0.10 units) than for lucerne (0.04 units; D 3 G, P , 0.001). As for DegDM, the DegN was linearly affected by the proportion of the two plants in the mixture. At both vegetation stages, the rapidly degradable N fraction 'a' was much smaller for sainfoin (S100) than for lucerne (S0). This fraction increased linearly with increasing proportions of lucerne in the legume mixture (P , 0.001). The opposite pattern was observed for the slowly degradable fraction 'b' (P , 0.001; Table 3).

Rumen fluid composition (tN (Figure 1) and NH3-N (Figure 2)). There were significant differences in the tN and NH3-N content in the rumen fluid with diet, with time following the meal, and with the D 3 T interaction. Both total tN and NH3-N decreased significantly with the growth stage (P , 0.01 and P , 0.001, respectively).

The total N and NH3-N content in the rumen fluid increased for all diets to peak at 1 or 2 h after feeding, but had decreased Aufre `re, Dudilieu, Andueza, Poncet and Baumont back to the pre-feeding values at 7 h after feeding. The peak values were higher for lucerne than sainfoin (0.821 as compared with 0.220 mg/g for tN and 0.456 as compared with 0.145 mg/g for NH3-N at 2 h post-feeding, respectively; P , 0.001). N in NH3-N concentrations decreased linearly from S0 to S100 as the proportion of sainfoin in the mixtures increased according to the following model: Y 5 20.0023x 1 0.3335; R 2 5 0.99, r.s.d. 5 0.004, where Y is the NH3-N concentration and x is the proportion of sainfoin in the mixture expressed as percentage.

In situ N digestion in the SI Consistent with the results measured for DegN, the fraction of forage N remaining in the nylon bags after a 16-h incubation in the rumen was much higher for sainfoin (S100, S75) than for lucerne (S25, S0), with the highest value being measured for stage-2 S100 (P , 0.001), in agreement with its lowest DegN (Table 4).

The fraction of forage N remaining in the bags recovered at the ileum was almost 2 times higher for sainfoin (S100, S75) than for lucerne (S25, S0; D, G, D 3 G all P , 0.001; quadratic contrasts). Finally, although N digestibility in the SI was significantly lower for S100 than for S0, S75 and S25, the quantity of N that disappeared in the intestine expressed in g/g of initial forage N or DM was higher for the S75 mixture than for sainfoin (S100) and for lucerne (S0) and the S25 mixture, with a significant quadratic effect (P , 0.001; Table 4).

Discussion

The reported associative effects between forages vary broadly among studies [START_REF] Niderkorn | Associative effects between forages on feed intake and digestion in ruminants[END_REF], but there are few publications on the effect of mixing a tannincontaining and a tannin-free forage. Many papers conclude Feed value of sainfoin-lucerne mixture that there is an improvement in the N value in mixtures compared with pure forage, but without specifying whether the improvement is due to an associative effect (non-linear contrast) or not (linear contrast).

Chemical composition

The differences in the N and cell wall contents between sainfoin and lucerne at the same growth stage were in agreement with the literature data. The N content decreased faster with the growth stage in sainfoin than in lucerne, probably due to a more rapid change in the leaf-to-stem ratio from the beginning of flowering [START_REF] Borreani | Evolution of yield and quality of sainfoin (Onobrychis vicii folia Scop) in the spring growth cycle[END_REF][START_REF] Bal | Nutritive value of sainfoin (Onobrychis viciifolia) harvested at different maturity stages[END_REF] in sainfoin than in lucerne [START_REF] Theodoridou | Effect of plant development during first and second growth cycle on chemical composition, condensed tannins and nutritive value of three sainfoin (Onobrychis viciifolia) varieties and lucerne[END_REF].

In vivo digestibility and voluntary intake

In agreement with our pepsin-cellulase digestibility results [START_REF] Aufre `re | Valeur alimentaire chez les ruminants des le ´gumineuses contenant des tannins condense ´s en milieux tempe ´re ´s[END_REF], OMD was very similar between the two forages (S100 and S0). [START_REF] Kraiem | Influence of method of forage preservation on fibre and protein digestion in cattle given lucerne, birdsfoot trefoil and sainfoin[END_REF] reported no differences between lucerne, birdsfoot trefoil and sainfoin when compared with hays or silage harvested at early flowering stages. These results were consistent with our findings on DegDM and on the in vivo digestibility of sainfoin compared with lucerne harvested at two growth stages [START_REF] Aufre `re | In vivo and in situ measurements of the digestive characteristics of sainfoin in comparison with lucerne fed to sheep as fresh forages at two growth stages and as hay[END_REF].

In line with the literature data, advancing stages of maturity resulted in a decrease in OMD according to the increase in the cell wall contents (NDF, ADF and ADL) for lucerne (D 3 G , 0.05), whereas the cell wall content of sainfoin remained almost unchanged. Although the cell wall content was higher in sainfoin (S100) than in lucerne (S0), we found no significant effects on OMD among the sainfoin-lucerne mixtures. In contrast to the present findings, [START_REF] Turne | Quebracho tannin influence on nitrogen balance in small ruminants and in-vitro parameters when utilizing alfalfa forages[END_REF] observed an effect of level of quebracho condensed tannin supplementation on lucerne organic matter and fibre digestibility. [START_REF] Wang | Effects of mixing sainfoin with alfalfa on ensiling ruminal fermentation and total tract digestion of silage[END_REF] showed that the OMD of alfalfa-sainfoin mixtures preserved as hay or silage was improved compared with alfalfa preserved alone.

The lower voluntary intake of sainfoin (S100) than lucerne in our results has previously been related to NDF and ADF contents ( [START_REF] Van Soest | Nutritional ecology of the ruminant[END_REF]. Moreover, it has been reported that high levels of CT inhibit feed intake due to their astringent effects on eating and their inhibition of ruminal digestion (Waghorn et al., 1994a;[START_REF] Reed | Nutritional toxicology of tannins and related polyphenols in forage legumes[END_REF]. In our study, the CT contents measured using the HCl-butanol method may have negatively affected voluntary dry matter intake (DMI). [START_REF] Willman | Physical structure of twelve forage species in relation to rate of intake by sheep[END_REF] compared with the physical structure of sainfoin and lucerne forages and their voluntary intake by ruminants, and showed that sainfoin is not fragmented to the same extent as lucerne. [START_REF] Lees | Mechanical disruption of leaf tissues and cells in some bloat-causing and bloat-safe legumes[END_REF] attributed the resistance of sainfoin to mechanical disruption to its greater cell wall and tissue strength relative to lucerne. These studies demonstrate the inherent structural differences between sainfoin and lucerne, in addition to the presence of CT.

We found a positive associative effect of sainfoin and lucerne on voluntary intake, as revealed by the significant quadratic contrast, with the highest voluntary intake being observed with the S25 mixture. [START_REF] Cortes | Ewes increase their intake when offered a choice of herbage species at pasture[END_REF] demonstrated that dry ewes offered a choice of herbage species at pasture (Lolium perenne and Festuca arundinacea) showed an increase in their overall intake, and that this pattern was mediated via an increase in the grazing time rather than an increase in the intake rate. [START_REF] Ginane | Feeding behaviour and intake of heifers fed on hays of various quality, offered alone or in choice situation[END_REF] observed that DMI was higher by at least 10% when heifers were offered a choice between two hays than when the same forages were offered alone. It has been suggested that food diversity may provide animals a positive stimulus Table 4 Effect of sainfoin-lucerne ratios on N remaining in bags after 16-h incubation in the rumen and after passage through the abomasum and SI, digestibility in the SI of 16-h rumen residual forage N, and forage N disappearing from mobile nylon bags in the SI, at two phenological stages Different superscripts in a same line correspond to a significant difference (P , 0.05) between diets.

All results were expressed in gN or N 3 6.25/g initial forage N, except digestibility in the SI.

that increases their motivation to eat and therefore their intake levels.

N utilisation

In line with our findings here, other studies with legume forages have found lower N in vivo digestibility for sainfoin than lucerne, whether fresh or preserved as hay [START_REF] Aufre `re | In vivo and in situ measurements of the digestive characteristics of sainfoin in comparison with lucerne fed to sheep as fresh forages at two growth stages and as hay[END_REF] or silage [START_REF] Fraser | Voluntary intake, digestibility and nitrogen utilization by sheep fed ensiled forage legumes[END_REF]. The differences between S100 and the mixtures in terms of the amounts of N intake and N digested arise from a combination of differences between sainfoin and lucerne in terms of voluntary intake and N content. This decrease in N digestibility was also attributed to the presence of CT in sainfoin. The decrease in ruminal protein degradation with CT results from the ability of the CT to either bind feed protein or suppress proteolytic protozoa. The higher faecal N excretion in S100 is largely counterbalanced by a lower urinary N excretion due to the lower ruminal degradability of nitrogenous compounds. Conversely, the high urinary N excretion for lucerne (S0) results from the higher digestible N content and the higher ruminal N degradability of lucerne compared with sainfoin (quadratic contrast, Figure 3). Getachew et al. (2008a) observed higher faecal N as the tannic acid level on lucerne hay was increased. [START_REF] Tiemann | In vitro ruminal fermentation of tanniniferous tropical plants: plant-specific tannin effects and counteracting efficiency of PEG[END_REF] studied the effect of using two tannin-rich shrub legume species as replacements for a herbaceous high-quality legume (Vigna unguiculata) on the nutritive value of a diet composed of tropical grasses (Brachiaria brizantha and V. unguiculata). They found that faecal N losses increased linearly relative to N intake with increasing proportion of CT-rich legumes in the diet, while the proportionate urinary N losses decreased for both CT-rich legume shrubs. These results suggested that the tannin-protein complexes formed in the rumen may not be fully dissociated post-ruminally [START_REF] Barry | The implications of condensed tannins on the nutritive value of temperate forages fed to ruminants[END_REF]. Moreover, according to [START_REF] Mcmahon | Effect of sainfoin on in vitro digestion of fresh alfalfa and bloat in steers[END_REF], although sainfoin did not completely eliminate bloat in cattle grazing alfalfa-based pastures, there is evidence that sainfoin will reduce the incidence of bloat in grazing cattle if it accounts for at least 15% DM in the pasture.

The relationship between in vivo N digestibility and sainfoin content in the mixture is quadratic and was closed (R 2 5 0.93, P , 0.001), indicating an interaction (associative effect) between sainfoin CT and lucerne. These results are in contrast to those obtained by [START_REF] Waghorn | Effect of condensed tannins in Lotus corniculatus on the nutritive value of pasture for sheep[END_REF] on L. corniculatus in a grass-legume pasture. Their study suggests that the 1% CT from L. corniculatus may be insufficient to affect the nutritive value of fresh forages for sheep. A similar trial conducted previously with ryegrass and Lotus pedunculatus had shown a substantial reduction in N digestibility due to the CT in the Lotus (Waghorn et al., 1994b).

Finally, our results found that the ratio of N retained to N intake was on average the same for S100 and S0 and higher for S75 and S25 (quadratic contrast).

Sainfoin-lucerne mixtures can be fed to ruminants to alter the form of excreted N and to reduce environmental N pollution because faecal N output is considered to be an environmentally less harmful N form than urinary N.

Rumen degradability

DegN was lower in the high-sainfoin mixtures (S100 and S75) than the low-sainfoin mixtures (S25 and S0; mean 5 0.728 as compared with 0.827, respectively). The relation between DegN and sainfoin content in the mixture did not show an associative effect. Figure 3 Plots of faeces N/N intake (Figure 3a) and urinary N/N intake (Figure 3b) against the percentage of sainfoin in the sainfoin-lucerne mixture (S0, S25, S75, S100) studied on fresh forages at the vegetative stage (stage 1) and the early flowering stage (stage 2). N 5 nitrogen.

Feed value of sainfoin-lucerne mixture This lower DegN for sainfoin was expected, as it is well documented that CT reduces ruminal protein degradation by complexing proteins and making them less degradable by ruminal microbes [START_REF] Reed | Nutritional toxicology of tannins and related polyphenols in forage legumes[END_REF]. We found a less significant difference in DegN between sainfoin and lucerne (0.10 units) than that in other studies [START_REF] Aufre `re | In vivo and in situ measurements of the digestive characteristics of sainfoin in comparison with lucerne fed to sheep as fresh forages at two growth stages and as hay[END_REF]0.20 units). The differences in DegN may be explained by differences in the N content between the two forages and differences in the sainfoin CT content or structure between the two experiments.

The results of [START_REF] Hervas | Effect of different doses of quebracho tannins extract on rumen fermentation in ewes[END_REF] suggest that the effect of quebracho CT extract on ruminal fermentation measured by gas production and the bag technique was clearly dosedependent. Getachew et al. (2008b) determined the effects of different types and levels of tannins, that is, gallic acid, tannic acid and quebracho, on the in vitro rumen gas production of lucerne, and concluded that the effect of tannins on rumen fermentation and protein degradation varied with the type and the level of tannins.

Intestinal digestibility

The SI digestibility of residual N from lucerne, estimated using the mobile bag technique, was lower than the published values for tannin-free forages [START_REF] Hvelplund | Estimation of the true digestibility of rumen undegraded dietary protein in the small intestine of Feed value of sainfoin-lucerne mixture ruminants by the mobile bag technique[END_REF], but residual N at the ileum was similar to published values [START_REF] Nozie | La valeur azote ´e des fourrages. Nouvelles estimations de la de ´gradabilite ´des prote ´ines (DT) dans le rumen et de la digestibilite ´re ´elle des prote ´ines alimentaires dans l'intestin gre ˆle: conse ´quences sur les valeurs PDI[END_REF][START_REF] Aufre `re | In vivo and in situ measurements of the digestive characteristics of sainfoin in comparison with lucerne fed to sheep as fresh forages at two growth stages and as hay[END_REF]. The present study was designed to eliminate any microbial N interference as much as possible, and digestion of residual feed N in the large intestine was ruled out by the ileal recovery of intestinal bags. These measurement conditions almost certainly contributed towards the low level of N digestibility obtained.

The crucial question was whether or not the plant protein protected from ruminal degradation was digested and absorbed in the SI. Although the residual N content at the end of the SI was higher for the high-sainfoin-content mixtures (S100 and S75), the amount of forage N digested in the SI was higher with the presence of lucerne. Thus, the higher N disappearance in the SI (in g/g initial forage N) under the S100 (mean 5 0.0822) and mainly S75 (0.1085) diets compared with the S25 (0.056) and S0 (0.058) diets resulted from greater higher N escape from the rumen that was not counterbalanced by a lower forage N digestibility in the intestine. These results suggested a positive effect of CT on N utilisation at high-sainfoin-content mixtures. The impact of CT on intestinal function in ruminants is still not well understood. Studies suggest that CT inhibit the ability of endogenous enzymes to cleave proteins into peptides and amino acids and also inhibit their absorption, whereas other studies found that absorption was increased or remained unchanged [START_REF] Waghorn | Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat production -progress and challenges[END_REF].

Evolution in the rumen fluid It is important to underline the concordance between the tN content kinetics and the NH3-N content kinetics. The lower tN and NH3-N concentrations measured in the rumen for the mixtures (S25, S75, S100) may have been obtained because of several reasons. First, the initial N intake was lower for sainfoin (S100 and S25) than for lucerne (S25 and S0). commonly reported effect of tannin-rich forages on protein metabolism in the rumen [START_REF] Min | The effect of condensed tannins on the nutrition and health of ruminants fed fresh temperate forages[END_REF][START_REF] Barry | The implications of condensed tannins on the nutritive value of temperate forages fed to ruminants[END_REF]. The tN and NH3-N values in our results were higher for stage 2 than for stage 1, whereas N intake was lower in stage 2 than stage 1. This contrast may be explained by the lower CT content in stage 2 than in stage 1. Wang et al. (2006) found that cattle grazing lucerne/ sainfoin mixtures containing as little as 85 g sainfoin/kg DM had lower ruminal NH3-N concentrations than those grazing pure lucerne pasture. [START_REF] Kraiem | Influence of method of forage preservation on fibre and protein digestion in cattle given lucerne, birdsfoot trefoil and sainfoin[END_REF] reported lower ruminal NH3-N concentrations in cattle fed diets containing sainfoin as compared with lucerne, whether as hay or as silage.

The ruminal concentration of NH3-N represents an equilibrium in production between utilisation and absorption. It is well-known that CT reduces protein degradation and subsequent deamination in the rumen. Thus, the reduction in the NH3-N concentration likely resulted from decreased ammonia production, which is also consistent with the relatively lower intracellular and extracellular proteolytic activity in ruminal fluid [START_REF] Jones | Effect of sainfoin (Onobrychis viciifolia Scop.) condensed tannins on growth and proteolysis four strains on ruminal bacteria[END_REF][START_REF] Wang | Comparison of alfalfa and mixed alfalfa-sainfoin pastures for grazing cattle: effects on incidence of bloat, ruminal fermentation, and feed intake[END_REF]. The main effect of tannin was to decrease the solubilisation of plant protein.

The effects of the diets composed by different ratios of sainfoin and lucerne on digestive parameters in ruminants are summarised in Figure 4. Our results showed that low levels of tannins (CT,72 g DM intake/day) did not impair the voluntary intake (S75 5 S25 5 S0) or OMD of the mixtures (S100 5 S75 5 S25 5 S0). Ruminal DTN was lower when fresh sainfoin was fed (S100, S75) compared with fresh lucerne (S25, S0). In the rumen, CT (S100, S75) tended to increase the flow of non-ammonia N to the small intestine. The decrease in N digestibility in the total digestive tract can be attributed to the presence of CT in sainfoin (Total-tract N digestibility S100 , S75 , S25 , S0). The higher faecal N excretion for sainfoin (S100, S75) resulted from the lower ruminal N degradability, whereas the lower urinary N excretion resulted from the decrease of rumen NH3-N content. The disappearance of N from the intestine was higher with S75 (S75 . S100 . S25 5 S0). Other beneficial effects of feeding sainfoin forage in association with lucerne, but not studied in this experiment, include a reduction in incidences of bloating [START_REF] Mcmahon | Effect of sainfoin on in vitro digestion of fresh alfalfa and bloat in steers[END_REF]. More research should be carried out in order to identify other factors related to CT that could lead to variations in the effect of CT on the feed value of forage mixtures (content, structure and biological activity of CT, leaf/stem ratio, variety, preservation method and environmental conditions; [START_REF] Theodoridou | Effect of plant development during first and second growth cycle on chemical composition, condensed tannins and nutritive value of three sainfoin (Onobrychis viciifolia) varieties and lucerne[END_REF]2012).

Conclusion

This study shows an associative effect of sainfoin and lucerne (quadratic contrast). The effects of CT of sainfoin on the feed value of sainfoin-lucerne mixtures were the highest for high-sainfoin-content mixtures (S75; CT mean value 5 72 g DM intake/day). At this proportion, voluntary intake, N retention and disappearance of N in SI were maximum and N rumen degradability showed the lowest values. Further experiments are needed to determine whether other secondary compounds are involved in the associative effects of sainfoin-lucerne mixtures.

  rumen DegDM and DegN in mixtures of sainfoin and lucerne studied as fresh forages at the vegetative stage (stage 1) and at the early flowering stage (stage 2degradability of DM or N calculated with a passage rate of 0.06/h. Probability of significant effects due to composition of the experimental diet (D), growth stage (G) and their interaction (D 3 G) for fresh forages (*P , 0.05, **P , 0.01, ***P , 0.001). Different superscripts in a same line correspond to a significant difference (P , 0.05) between diets (Stage 1: lower character, Stage 2: upper character).

Figure 1 Figure 2

 12 Figure1Evolution of the concentration (mg/g) of total nitrogen (tN) measured in rumen fluid in four groups of three sheep simultaneously fed different mixtures of sainfoin and lucerne (S100, S25, S75, S0) as fresh forage at the vegetative stage (stage 1, Figure1a) and at the early flowering stage (stage 2, Figure1b).

  ; SI 5 small intestine; DM 5 dry matter; L 5 linear contrast; Q5quadratic contrast. Probability of significant effects due to composition of the experimental diet (D), growth stage (G) and their interaction (D 3 G) for fresh forages (*P , 0.05, **P , 0.01, ***P , 0.001).

FurthermoreFigure 4

 4 Figure4Schematic diagram of the effects of condensed tannins on digestive parameters in ruminants for sainfoin-lucerne mixtures (S100, S25, S75, S0; from the present results andMcMahon et al., 1999 results). CT 5 condensed tannins; DM 5 dry matter; N 5 nitrogen; NH3-N 5 ammonia N; CH4 5 methane.

Table 1

 1

		S100 stage 1	S100 stage 2	S75 stage 1	S75 stage 2	S25 stage 1	S25 stage 2	S0 stage 1	S0 stage 2
	N	3 1	2 2	3 2	2 4	3 5	2 9	3 6	3 2
	NDF	443	432	419	414	371	380	347	363
	ADF	306	313	290	299	294	273	222	259
	ADL	74	114	69	102	59	74	55	65
	Dcellms	708	594	716	609	729	635	735	647
	CT	64	52	46	37	14	12	nd	nd

Chemical composition, Dcellms and CT content (g/kg DM) of mixtures of sainfoin and lucerne (S100, S75, S25, S0) studied as fresh forages at the vegetative stage and at the early flowering stage (stage 1 and stage 2, respectively) Dcellms 5 pepsin-cellulase digestibility; CT 5 condensed tannins; DM 5 dry matter; N 5 nitrogen.

Table 3

 3 In situ

Acknowledgements

The authors thank the staff at the 'Annexe Elevage' experimental farm for their diligent care of the experimental animals, Bernard Debote for his advice on establishing sainfoin swards and Eric Villeneuve and his staff for growing and cropping the forages.


Benoit Sotton
email: benoit.sotton@thonon.inra.fr

Jean Guillard

Sylvie Bony

Alain Devaux

Isabelle Domaizon

Nicolas Givaudan

François Crespeau

Hélène Huet

Anneville Orlane

Benoı ˆt Sotton

He

` Ne Huet

Orlane Anneville

Impact of Toxic Cyanobacterial Blooms on Eurasian Perch (Perca fluviatilis): Experimental Study and In Situ Observations in a Peri-Alpine Lake

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

During the past century, toxic cyanobacterial blooms have appeared worldwide as a result of the eutrophication of freshwater ecosystems. Toxic cyanobacteria produce a wide variety of metabolic compounds that are toxic for many aquatic organisms and humans [START_REF] Carmichael | Cyanobacteria secondary metabolites-the cyanotoxins[END_REF][START_REF] Azevedo | Human intoxication by microcystins during renal dialysis treatment in Caruaru-Brazil[END_REF][START_REF] Mankiewicz-Boczek | Effects of microcystins-containing cyanobacteria from a temperate ecosystem on human lymphocytes culture and their potential for adverse human health effects[END_REF]. In the aquatic sciences, the most studied toxic compounds produced by cyanobacteria are the microcystins (MCs), of which more than 80 variants have been characterised [START_REF] Codd | Cyanobacterial toxins: risk management for health protection[END_REF][START_REF] Welker | Cyanobacterial peptides-nature's own combinatorial biosynthesis[END_REF]. MCs are known to inhibit protein phosphatases 1 and 2A (PP1 and PP2A) by, an initial non-covalent and reversible binding followed by a final covalent and irreversible linkage step [START_REF] Smith | Possible mechanism for the foodweb transfer of covalently bound microcystins[END_REF] and thereby cause oxidative stress, apoptosis and the disruption of many cellular functions. MCs may act as tumour promoters and as genotoxicants, as shown recently in whitefish [START_REF] Campos | Molecular Mechanisms of Microcystin Toxicity in Animal Cells[END_REF]. Deleterious effects of MCs on the physiology and behaviour of various fish species have been observed [START_REF] Baganz | Comparative study of microcystin-LR-induced behavioral changes of two fish species, Danio rerio andLeucaspius delineatus[END_REF][START_REF] Ernst | Physiological stress and pathology in European whitefish (Coregonus lavaretus) induced by subchronic exposure to environmentally relevant densities of Planktothrix rubescens[END_REF]. Histological disturbances, particularly in the liver, kidney, gills and intestine, have been observed in fish exposed to cyanobacteria and their toxins [START_REF] Carbis | The histopathology of carp, Cyprinus carpio L, exposed to microcystins by gavage, immersion and intraperitoneal administration[END_REF][START_REF] Ernst | Oral toxicity of the microcystin-containing cyanobacterium Planktothrix rubescens in European whitefish (Coregonus lavaretus)[END_REF][START_REF] Trinchet | Pathological modifications following sub-chronic exposure of medaka fish (Oryzias latipes) to microcystin-LR[END_REF].

A large array of xenobiotics is known to be metabolised by fish detoxification systems [START_REF] Van Der Oost | Fish bioaccumulation and biomarkers in environmental risk assessment: a review[END_REF]. The detoxification process involves a variety of enzymes that are implicated in the oxidation, reduction, hydrolysis and conjugation of xenobiotics present in organisms. The most studied detoxification enzymes in fish are the glutathione S-transferases (GSTs) that are responsible for the conjugation of MCs with glutathione (GSH) prior to biliary excretion [START_REF] Campos | Molecular Mechanisms of Microcystin Toxicity in Animal Cells[END_REF][START_REF] Sahin | Biliary excretion of biochemically active cyanobacteria (blue-green algae) hepatotoxins in fish[END_REF]. Experimental studies on the detoxification of MCs in various fish species have shown that this ''natural defence'' is species-specific and that the concentrations of MCs in fish are dose dependent [START_REF] Malbrouck | Effects of microcystins on fish[END_REF]. Fish chronically assimilate MCs by the ingestion of cyanobacteria and contaminated zooplankton and the flow of water through their gills, leading generally to the gradual accumulation of toxins in organs [START_REF] Zhang | Transfer, distribution and bioaccumulation of microcystins in the aquatic food web in Lake Taihu, China, with potential risks to human health[END_REF]. However, exposure to MCs may or may not result in MC accumulation depending on the dynamics and intensity of the MCs intake and detoxification process. The measurement of MC levels in fish sampled in situ is essential, and the interpretation of MC concentration requires a good understanding of the dynamics of detoxification for the particular fish species being studied. The histological and genetic impacts of low toxin concentrations are difficult to evaluate in the field, primarily because of the lack of a reference state. Experimental data are therefore needed to facilitate the interpretation of field observations and to accurately assess the potential impact of MCs on fish species. For these reasons, we combined field and experimental approaches to better estimate the transfer of toxins to natural fish populations and to evaluate the potential impact of toxins on individual fish. We studied perch (Perca fluviatilis), a target fish species in recreational and commercial fisheries in peri-alpine lakes that has been commercialised in both its adult and young-of-the-year (YOY) stages.

In Lake Bourget (France), toxic blooms of the cyanobacterium Planktothrix rubescens occur [START_REF] Jacquet | The proliferation of the toxic cyanobacterium Planktothrix rubescens following restoration of the largest natural French lake (Lac du Bourget)[END_REF] and produce primarily two MC variants termed MC-LR and MC-RR [START_REF] Schwob | Occurrence of toxic Planktothrix rubescens blooms in lake Nantua, France[END_REF][START_REF] Briand | Variations in the microcystin production of Planktothrix rubescens (Cyanobacteria) assessed from a four-year survey of Lac du Bourget (France) and from laboratory experiments[END_REF][START_REF] Paulino | Detection of Planktothrix rubescens (Cyanobacteria) associated with microcystin production in a freshwater reservoir[END_REF]. We used an ecosystemic approach to study in situ the exposure of a YOY perch population to P. rubescens by (i) identifying the main routes of fish exposure to MCs, (ii) assessing toxin accumulation in the fish during chronic exposure to a cyanobacterial bloom and (iii) evaluating the subsequent histological effects in the fish. Parallel experimental studies using environmentally relevant doses of MC-LR were performed in the laboratory to (i) characterise the temporal dynamics of intoxication and detoxification and (ii) assess the histological and genotoxic impacts in YOY perch exposed to environmentally relevant doses of MC-LR.

Materials and Methods

Ethical statement

Investigations were conducted according to the international guiding principles for the use and care of laboratory animals, and in compliance with French regulations (Nu B 74 300-4) on animal welfare approved by the French Ministry of Ecology and Sustainable Development. This study was approved by the ethic committee of the Departmental Direction of Veterinary Services of Haute-Savoie (DDSV). In-situ sampling was made on public area with a sampling authorisation (Nu 2012-209) delivered by the Prefecture of Savoie and did not involve protected species.

In-situ fish were captured by pelagic trawl and quickly killed, as experimental fish, by a cranial choc.

Study site

Lake Bourget (45u449N, 5u529E, 231 m altitude) is a mesotrophic, warm, monomictic lake located in eastern France. It has an area of 42 km 2 , a total volume of 3.5610 9 m 3 and maximum and average depths of 145 and 80 m, respectively [START_REF] Briand | Variations in the microcystin production of Planktothrix rubescens (Cyanobacteria) assessed from a four-year survey of Lac du Bourget (France) and from laboratory experiments[END_REF]. Its water is used as a source of drinking water and for recreational activities and fisheries. The cyanobacterium P. rubescens was first reported in the lake in the 1950 s [START_REF] David | Pe ˆche, Pe ˆcheurs professionnels, pollution dans le lac du Bourget[END_REF]. The occurrence and intensity of its blooms have been precisely studied since the 1990 s [START_REF] Jacquet | The proliferation of the toxic cyanobacterium Planktothrix rubescens following restoration of the largest natural French lake (Lac du Bourget)[END_REF]. P. rubescens is present in the metalimnion of the lake during the summer [START_REF] Cuypers | Impact of internal waves on the spatial distribution of Planktothrix rubescens (cyanobacteria) in an alpine lake[END_REF].

In situ sampling

Six daily east-west transects were performed in the northern part of the lake twice a month in August and September 2009 and once in October 2009. Three points were sampled for each transect. A fluoroprobe (BBE, Moldaenke, Germany) specially calibrated to estimate P. rubescens biomass was used at each sampling point [START_REF] Leboulanger | Application of a submersible spectrofluorometer for rapid monitoring of freshwater cyanobacterial blooms: a case study[END_REF] and the thermocline depth was followed in parallel. At each sampling point, 2 L water samples were taken at three depths (6 m, 12 m and the depth of P. rubescens maximum density) for MC analysis. The water samples were passed through a filter (pore size 1 mm, Nucleopore, Whatman) and stored at 220uC until analysis.

The schooling of perch during the day [START_REF] Masson | Diel vertical and horizontal distribution of crustacean zooplankton and young of the year fish in a sub-alpine lake: an approach based on high frequency sampling[END_REF][START_REF] Guillard | Three-dimensional internal spatial structure of young-of-the-year pelagic freshwater fish provides evidence for the identification of fish school species[END_REF] and their position in the water column were studied using hydroacoustic methods [START_REF] Simmonds | Fisheries acoustics: theory and practice[END_REF] as described previously [START_REF] Sotton | Spatial match between Planktothrix rubescens and whitefish in a mesotrophic perialpine lake: Evidence of toxins accumulation[END_REF].

The intake of cyanobacterial toxins may result from the consumption of intoxicated prey [START_REF] Zhang | Transfer, distribution and bioaccumulation of microcystins in the aquatic food web in Lake Taihu, China, with potential risks to human health[END_REF][START_REF] Ibelings | Distribution of microcystins in a lake foodweb: no evidence for biomagnification[END_REF]. Prey items were identified by stomach content analysis. For this purpose, perch were sampled at night using a pelagic trawl [START_REF] Guillard | Hydroacoustic assessment of young-of-year perch, Perca fluviatilis, population dynamics in an oligotrophic lake (Lake Annecy, France)[END_REF] and stocked at 4uC until laboratory analysis. On the date of maximal concentrations of P. rubescens (27 August), zooplankton were sampled by several vertical tows at depths of 0 to 50 m with a 200 mm-mesh conical net and kept alive in cooler boxes until sorting through air bubbling techniques [START_REF] Straskraba | Preliminary results of a new method for the quantitative sorting of freshwater net plankton into main groups[END_REF]. Then, Cladocerans were picked up on the surface of the water bubbled and immediately deep-frozen in liquid nitrogen until laboratory analysis.

Zooplankton and fish sample preparation

In laboratory, Cladocerans samples were defrosted and Daphnia were then sorted out under a binocular microscope, thoroughly rinsed with ultrapure water to remove all attached P. rubescens filaments and then extracted for MCs analyses.

YOY perch were weighed, measured and dissected. Individual gut contents were observed under a microscope (magnification 40X). Ingested prey items were identified and counted at the genus level. Dorsal muscle and liver were sampled for each fish (n = 27) and preserved in formalin for histological studies. For MC analysis at each sampling date, the individual organ samples were pooled to provide 3 liver samples and 3 muscle samples.

Laboratory experimental design

Experiments were performed in triplicate using YOY perch raised from eggs collected in Lake Geneva (France/Switzerland) and reared in tanks (research unit AFPA, Nancy University, France).

The perch were acclimatised for 1 week prior to experiments in outdoor tanks (480 L) supplied with water from Lake Geneva that was free from toxic cyanobacteria and was pumped continuously from a depth of 36 m. For each replicate, 440 fish (mean weight 3.3660.24 g) were starved for 24 hours prior to treatment and were then distributed in cylindrical 20-L cages (n = 22 fish per cage) that were placed in clean large tanks (1140 L) filled with lake water at 20uC. The fish were anesthetised in water containing 300 mL propiscin per L (0.2%). For each replicate, 110 fish were individually force-fed through a catheter connected to a Hamilton syringe with 10 mL of a 5 mg.L 21 MC-LR solution (i.e., approximately 0.05 mg MC-LR per fish) or with 10 mL of a 50 mg.L 21 MC-LR solution (i.e., approximately 0.5 mg MC-LR per fish). MC-LR (Gentaure Corporation, France) was dissolved in water containing 0.04% phenol red to ensure that the forcefeeding was successful. The MC concentrations of the force-fed solutions were confirmed by HPLC-PDA. The chromatographic resolution of MC-LR was achieved using the following mixture of MilliQ-water -0.3% (v/v) trifluoroacetic acid (A) and acetonitrile (B) as the mobile phase, in an elution gradient run on a chromatographic system (Waters) equipped with Waters 600 pump controller, a PDA 996 photodiode-array detector, a C18 PHENOMENEX Luna 5 mm (4.6 mm6250 mm) column and Waters3.2 millennium chromatography manager software. The flow rate was 1 ml/min and MC-LR peak was identified on the basis of their characteristic absorption spectra (maximum absorption at 238 nm) and its retention time [START_REF] Sotton | Spatial match between Planktothrix rubescens and whitefish in a mesotrophic perialpine lake: Evidence of toxins accumulation[END_REF]. As a placebo treatment, 110 fish were force-fed with 10 ml of a 0.04% neutral solution of phenol red to evaluate the effect of phenol red on the measured endpoints. The control treatment consisted of 110 fish that were not force-fed. During each experiment, the fish were not fed and were maintained at a water temperature of 20uC, a dissolved oxygen concentration of 11.760.2 mg.L 21 , a pH of 8.160.3 and a natural light/dark cycle of 16/8 h. At 6, 12, 24, 48 and 96 h after the start of each experiment, 10 fish per treatment were killed, weighed, measured and dissected. Muscle tissues were sampled, pooled and stored at 280uC until analysis. A liver sample from each fish was divided into two equal parts to provide two pools (one for MC analysis and another for the measurement of enzyme activity) and stored at 280uC until analysis. For each treatment, blood from the caudal vein was collected in a heparinised syringe. The blood samples were diluted 100-fold in a cryopreservative buffer (250 mM sucrose, 40 mM trisodium citrate, 5% dimethyl sulphoxide (DMSO) adjusted to pH 7.6 with 1 M citric acid), deep-frozen in liquid nitrogen and stored at 280uC processing. For histological analysis, 2 fish per treatment were dissected and liver and intestine samples were fixed immediately in formalin solution.

Environmentally relevant concentrations of 0.05 and 0.5 mg MC per fish (corresponding to 16.6 and 166 mg/kg body weight, respectively) were chosen for the experiments based on our previous observation that MC concentrations in zooplankton in Lake Bourget range from 0.07 to 3.5 mg/g dry weight (DW) when P. rubescens is present (unpublished data). We found that MC concentrations in cladocerans under experimental conditions reached 1.1 mg/g fresh weight (FW) for organisms fed with cyanobacteria concentrations equivalent to those measured during the summer blooms in Lake Bourget (unpublished data). Kara ˚s and Thoresson [START_REF] Kara ˚s | An application of a bioenergetics model to Eurasian perch (Perca fluviatilis L.)[END_REF] reported a maximum daily food consumption of approximately 15% of fresh body weight for YOY perch. We therefore estimate that fish weighing approximately 3 g could ingest, via zooplankton, 0.05 mg MCs per day at the beginning of the bloom and 0.5 mg MCs per day at the height of the bloom.

MC extraction and analysis

For MC analysis of P. rubescens, the extraction and measurement procedures described by Sotton et al. [START_REF] Sotton | Spatial match between Planktothrix rubescens and whitefish in a mesotrophic perialpine lake: Evidence of toxins accumulation[END_REF] were used. Zooplankton and YOY perch samples were ground, homogenised in 100% methanol, sonicated in an ultrasonic bath (Elma Sonic) for 5 min and then centrifuged (Beckman AVANTI J30 I) (24000 g, 45 min, 25uC) to precipitate the co-extracted proteins [START_REF] Smith | Standardization of microcystin extraction from fish tissues: A novel internal standard as a surrogate for polar and non-polar variants[END_REF]. This extraction procedure was repeated twice. The supernatants from each extraction were collected with a 5-mL syringe and washed with hexane. The methanol layers were filtered through a syringe filter (pore size 0.2 mm; PTFE, Whatman), pooled and evaporated to dryness at 40uC with a SpeedVac device (SpeedVac Plus SC110A, Savant). Each dry extract was dissolved in an adequate volume of Milli-Q water; this volume varied depending on the sample. ELISA analyses were performed using a Microcystins (Adda-specific) Plate Kit (Abraxis LLC) [START_REF] Ernst | Presence of Planktothrix sp. and cyanobacterial toxins in Lake Ammersee, Germany and their impact on whitefish (Coregonus lavaretus L.)[END_REF][START_REF] Ernst | Recovery of MC-LR in fish liver tissue[END_REF] and absorbances at a wavelength of 450 nm were determined using a microplate reader (Dynex MRX II, Dynex Technologies, Inc., USA). The results are presented as ng MC-LR equivalents per g fresh weight (FW). This method for the quantification of free MC concentrations was chosen because of its sensitivity and specificity. However, the method does not allow the discrimination of pure MC-LR from its metabolites and may have led to the overestimation of pure MC-LR concentrations in our samples [START_REF] Metcalf | Immuno-crossreactivity and toxicity assessment of conjugation products of the cyanobacterial toxin, microcystin-LR[END_REF][START_REF] Mcelhiney | Detection of the cyanobacterial hepatotoxins microcystins[END_REF].

GST activity measurement

Liver samples from experimental YOY perch were homogenised in cold phosphate buffered saline (PBS) and centrifuged at 100006g for 15 min at 4uC. The supernatant was collected, and protein content was measured by spectrophotometry at 595 nm (Varian Cary 50 Spectrophotometer, Agilent Technologies, USA) according to the method of Bradford (1976). GST activity was determined by fluorometry using a GST Fluorometric Assay Kit (Biovision, USA) and a microplate reader (Fluoroskan Ascent FL, Thermo Fisher Scientific, France) at excitation wavelength 380 nm and emission wavelength 460 nm. The results are reported as U per mg total protein content, where U equals the quantity of enzyme that catalyses the transformation of 1 mmol substrate per minute.

DNA damage assessment

DNA damage was measured in erythrocytes by the alkaline Comet assay using a slight modification of the procedure of Singh et al. [START_REF] Narendra | A simple technique for quantitation of low levels of DNA damage in individual cells[END_REF]. The blood cells were thawed rapidly just before the assay, the cell density was adjusted to 10 3 cells.mL 21 cold PBS, and the cell viability was checked by the trypan blue exclusion method [START_REF] Boyse | Cytotoxic test for demonstartion of mouse antibody[END_REF]. The cell viability was found to be .90% in all experiments. Microscope slides were immersed in melted normal agarose prepared in PBS (final concentration 0.8%) and dried overnight at room temperature. A 50-mL aliquot of 1% low melting point agarose in PBS mixed with an equal volume of cell suspension was spread on the slide and a coverslip was placed. The agarose was solidified on an ice-chilled plate, a second layer consisting of 90 mL of 0.5% low melting point agarose was spread on the first layer, a coverslip was placed, and the agarose was again solidified by chilling. The subsequent steps were performed under a dim red light to avoid artificial DNA damage. The cells were treated with a lysing solution (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, pH 10, 1% (v/v) Triton X-100) and 10% (v/v) DMSO for 1 h at 4uC, and the DNA was allowed to unwind for 40 min in an electrophoresis buffer (0.3 M NaOH, 1 mM EDTA, pH .13). Electrophoresis was run for 24 min at 20V and 300 mA (0.6 V/cm). The slides were neutralised by Tris buffer (0.4 M Tris, pH 7.5) and dried for 15 min in absolute ethanol. The DNA was stained with 0.05 mM ethidium bromide and scored using an Axioskop epifluorescence microscope (Zeiss, Germany) and the Comet assay IV image analysis system (Perceptive Instruments Ltd., UK). Randomly selected cells from two replicate slides (50 cells per slide) were analysed. Of the Comet parameters, the tail intensity (percentage of tail DNA) is the most relevant [START_REF] Collins | The comet assay for DNA damage and repair[END_REF]. Comet figures that showed an extremely prominent tail and that lacked a clearly identifiable head were excluded from the analysis because such figures may originate from necrotic or apoptotic processes [START_REF] Fairbairn | The comet assay: a comprehensive review[END_REF].

Histopathological analysis

Liver and intestine samples were fixed in buffered 10% formaldehyde solution for 48 hours, placed in a 70% ethanol solution, dehydrated in successive ethanol baths, embedded into blocks of paraffin wax and cut into 3.5-mm sections. The slides were rehydrated in successive 100% and 95% ethanol baths and stained with hematoxylin-eosin-saffron (HES) by a standard procedure. Periodic acid-Schiff (PAS) staining was performed to reveal the glycogen content of liver cells. Staining was performed according to standard histological procedures [START_REF] Brancroft | Theory and practice of histological techniques[END_REF].

Statistical analysis

Comparisons of fish survival rates between treatments at each sampling time were performed using the nonparametric Mann-Whitney test at p,0.05. MC-LR accumulations at different sampling times were compared in liver and muscle and for each dose using the nonparametric Kruskal-Wallis test followed by multiple pairwise comparisons using the Conover-Iman procedure. Comparisons of MC-LR accumulations between treatments at each sampling time were also performed using the nonparametric Mann-Whitney test. Differences were considered significant at the p,0.05 level for all statistical analyses. Comparisons of GST activity between treatments at each sampling time were performed using the nonparametric Mann-Whitney test at p,0.05. Because the distribution of DNA damage measured by the Comet assay does not follow a Gaussian distribution, both the nonparametric Kruskal-Wallis and Mann-Whitney tests were used for data analysis. All statistical analyses were performed using the XLSTAT 2011 software program.

Results

In situ observations

The YOY perch preyed on various zooplankton taxa during the study period. Their diet consisted mainly of Daphnia and Leptodora.

Copepods and other herbivorous cladocerans (Bosmina, Diaphanosoma) were also present in the stomach contents but had a lower abundance and frequency. Bythotrephes and rotifers were found occasionally and had low abundance (Fig. 1).

The relative abundance of these prey taxa fluctuated during the summer. At the beginning of August, Daphnia was the dominant taxon and comprised 80% of the YOY diet (Fig. 2); this percentage declined steadily until it reached a low value of 10%. Meanwhile, the percentage of Leptodora in the diet increased steadily and reached a maximum of 75% in September. The diet was dominated by herbivorous taxa in August but by carnivorous taxa in September.

The YOY perch were located exclusively in the epilimnion during the entire sampling season. At this depth, the MC concentration remained #0.12 mg.L 21 (Fig. 3). The highest MC concentrations were found between 15 and 20 m, which was the depth of maximal P. rubescens abundance as detected by the BBE probe. During the summer, the maximal MC concentrations were found below the thermocline. At the depth of maximal P. rubescens abundance, MC concentrations were highest in August, with average concentrations that ranged from 2.3 to 3.4 mg.L 21 (Fig. 3).

Although no spatial correlation between perch and P. rubescens abundance was observed, MC was detected in perch liver and muscle samples from August to October. The maximal MC concentrations (34 ng.g 21 FW in liver; 14 ng.g 21 FW in muscle) were detected on the date (27 August 2009) that the maximal sestonic MC-LR equivalent concentrations were measured in the water column. Subsequently, a significant (p,0.05) decrease in MC concentration was observed in both organs. Significant positive correlations were observed between the MC concentration in seston and those in liver (R 2 = 0.7221) and in muscle (R 2 = 0.6369) samples from YOY perch. Regardless of the date, MC concentrations were higher in liver than in muscle (Fig. 4). On the 27 August, when the MC concentrations in P. rubescens and seston in the water column were maximal (Fig. 4), the MC-LR equivalent concentration in Daphnia was 422 ng.g 21 FW.

No histological alterations were observed for any sampling date or any of the organs studied.

Experimental study

Microcystin accumulation. The mortality rate never exceeded 10% in the experimental groups. There were no significant differences in mortality between treatments regardless of the sampling time (data not shown).

Regardless of the experimental MC concentration or the sampling time, no MC was detected in either the control or placebo groups. For both of the MC-LR treatments, the toxin was detected in the liver and muscle samples of perch from the first sampling time (6 h) until the end of the experiment (Figs. 5 and6). At 6 h, the MC concentrations detected in both organs were dosedependent, and the concentrations in the liver were higher than those in muscle. In the liver, for the higher experimental MC-LR dose, a significant increase (p,0.05) in MC-LR concentration was detected at 12 h. The concentration was 37 ng.g 21 FW at 12 h and then fell rapidly to reach a significantly (p,0.05) lower value at 24 h (Fig. 5). In the liver, for the lower experimental MC-LR dose, the MC-LR concentration was significantly (p,0.05) lower at 12 h, increased gradually until 48 h when it reached its maximal value and then declined to reach a significantly (p,0.05) lower value at 96 h (Fig. 5). In the muscle, significant increases in toxin concentration were observed for all experimental MC-LR doses (Fig. 6). At the end of the experiment, The MC-LR concentration was approximately 7 ng.g 21 FW for the higher experimental dose and 4 ng.g 21 FW for the lower experimental dose (Fig. 6).

GST activity. Regardless of the sampling time, no significant difference in GST activity was found in the control vs. placebo groups or in the control vs. experimental groups (Fig. 7). There was high variability among the replicates.

Histology. Regardless of the sampling time or the organ studied (liver vs. intestine), no significant histological differences were observed among the placebo, control and experimental groups (treated with 0.5 or 0.05 mg MC-LR/fish) (Fig. 8). Regardless of the sampling time, PAS staining did not reveal any differences in glycogen storage or consumption in the livers of the control vs. experimental groups (Fig. 9).

DNA damage. Significant DNA damage, expressed as % tail intensity, was observed in the erythrocytes of the experimental group (Fig. 10). Basal DNA damage in the placebo and control groups remained in the range of 3 to 20% tail intensity throughout the study. A 3-fold increase in DNA damage was observed 12 hours after the initial force-feeding for the higher MC-LR dose. A clear trend of decreasing DNA damage with time was observed in the group treated with 0.5 mg MC-LR, although DNA damage after 48 h was still significantly greater in comparison with the control group. The induction of DNA damage in the group that received the lower MC-LR dose was delayed relative to the group that received the higher dose in that the damage was significantly increased (5-fold) in comparison with the control group only after 48 h. Regardless of the MC-LR dose, none of the experimental groups displayed evidence of genotoxicity greater than that of the control group after 96 h.

Discussion

Two points regarding the MC concentrations measured in our study must be discussed. Firstly, only free MCs were measured. MCs undergo a two-step interaction with phosphatase proteins 1 and 2A [START_REF] Craig | Molecular mechanisms underlying the interaction of motuporin and microcystins with type-1 and type-2A protein phosphatases[END_REF]: (i) a rapid and reversible binding with the phosphatases and (ii) a covalent linkage between the Nmethyldehydroalanine residue (Mdha) of the MCs and a nucleophilic site on the phosphatases, leading to irreversible inactivation [START_REF] Mackintosh | The cyanobacterial toxin microcystin binds covalently to cysteine-273 on protein phosphatase 1[END_REF][START_REF] Dawson | The toxicology of microcystins[END_REF]. To measure the total MC concentration, the free and the covalently bound MCs must be extracted from the sample by two different methods [START_REF] Neffling | Detection of free and covalently bound microcystins in animal tissues by liquid chromatography-tandem mass spectrometry[END_REF]. It has been estimated that 38-99% of MCs [START_REF] Ibelings | Distribution of microcystins in a lake foodweb: no evidence for biomagnification[END_REF][START_REF] Williams | 14Clabelled microcystin-LR administered to Atlantic salmon via intraperitoneal injection provides in vivo evidence for covalent binding of microcystin-LR in salmon livers[END_REF][START_REF] Williams | Evidence for a Covalently Bound Form of Microcystin-LR in Salmon Liver and Dungeness Crab Larvae[END_REF] may be covalently linked to phosphatases and thereby unextractable by the organic solvents that are used for free MCs. The measurement of only free MCs may therefore lead to an underestimation of total MC concentra-tions in samples. Secondly, the free MC concentrations measured in liver and muscle samples from YOY perch are most likely greater than the real concentrations of pure free MC-LR that are present in these two organs. Our ELISA method, which targets molecules that possess an unusual b-amino acid (ADDA; 4E, 6E 3amino-9-methoxy-2, 6, 8-trimethyl-10-phenyldeca-4, 6-dienoic acid), quantifies all molecules that possess ADDA as pure MCs but also so quantifies the detoxification products of MCs, which are less toxic [START_REF] Metcalf | Immuno-crossreactivity and toxicity assessment of conjugation products of the cyanobacterial toxin, microcystin-LR[END_REF]. Our method may therefore lead to an overestimation of free MC levels in YOY perch; i.e., the toxin values presented here may underestimate the total MC concentrations in samples as a result of the extraction method that targets only free MCs while the pure MC-LR concentrations of samples may be overestimated as a result of the ELISA measurements that quantify both pure MCs and their metabolites. Previous studies have revealed differences between fish species in terms of their sensitivity to cyanobacterial toxins [START_REF] Malbrouck | Effects of microcystins on fish[END_REF]. The number of feeding experiments on fish using MCs or cyanobac-teria has greatly increased in recent years. However, few studies have focused on fish species endemic to the peri-alpine lakes in which P. rubescens blooms have occurred repeatedly during the past 10 years [START_REF] Jacquet | The proliferation of the toxic cyanobacterium Planktothrix rubescens following restoration of the largest natural French lake (Lac du Bourget)[END_REF][START_REF] Anneville | Seasonal and inter-annual scales of variability in phytoplankton assemblages: comparison of phytoplankton dynamics in three peri-alpine lakes over a period of 28 years[END_REF][START_REF] Gandolfi | Planktothrix populations in subalpine lakes: selection for strains with strong gas vesicles as a function of lake depth, morphometry and circulation[END_REF]. In particular, there have been no studies on the Eurasian perch, which is important both commercially and for sport fishing, is widespread throughout Europe and Asia and has been successfully introduced in other continents.

The mortality rates for YOY perch observed following forcefeeding with environmentally relevant MC concentrations was low in the present study. Fish weighing 3 g were treated with 0.5 mg and 0.05 mg of MC-LR per fish; these doses correspond to approximately 166 mg/kg and 16.6 mg/kg body weight, respectively. The MC-LR concentrations used were well below the median lethal concentration values (LC50) obtained after injection of 550 mg MC-LR/kg body weight for common carp (Cyprinus Figure 6. MC-LR accumulation in muscle of YOY perch exposed to two relevant doses, 0.5 mg/fish and 0.05 mg/fish. * corresponds to a significant difference observed between microcystin treatments (Mann-Whitney, p,0.05). MC-LR equivalent concentrations are expressed as ng.g 21 fresh weight. Different letters correspond to a significant difference between sampling times (Mann-Whitney, p,0.05). doi:10.1371/journal.pone.0052243.g006 Figure 7. Glutathion S-transferase (GST) activity in liver of YOY perch force-fed with two doses of MC-LR (0.05 mg and 0.5 mg/fish). GST activity is expressed as U.mg 21 proteins. doi:10.1371/journal.pone.0052243.g007 carpio) and rainbow trout (Oncorhynchus mykiss) [START_REF] Ra ˚bergh | Histopathological effects of microcystin-LR, a cyclic peptide toxin from the cyanobacterium (blue-green alga) Microcystis aeruginosa on common carp (Cyprinus carpio L.)[END_REF][START_REF] Tencalla | Toxicity of Microcystis aeruginosa peptide toxin to yearling rainbow trout (Oncorhynchus mykiss)[END_REF]. This fact may explain the low mortality rates observed in our study. MC-LR was administered by force-feeding in the present study in contrast to the injection method using in the carp and trout studies. The route of exposure is presumed to be an important factor affecting toxicity. Tencalla et al. [START_REF] Tencalla | Toxicity of Microcystis aeruginosa peptide toxin to yearling rainbow trout (Oncorhynchus mykiss)[END_REF] demonstrated a higher toxicity of MC-LR administered by injection than by force-feeding in rainbow trout.

During the present study, MC-LR equivalent concentrations were detected in liver and muscle from 6 hours until 96 hours after treatment. In both of these organs, a rapid accumulation was observed for both the high and low MC-LR doses. In muscle, the MC-LR equivalent concentration increased for both doses in all of the experiments, and the highest concentrations found for the higher dose. In liver, the maximal MC-LR equivalent concentration was detected after 12 hours for the higher MC-LR dose and 6 hours for the lower dose. Following the peak, a decline in the MC-LR equivalent concentration was observed for both doses. These findings suggest that MC-LR may have been bound to PPase and thus undetected by our measurement method (ELISA) targeting only free MCs or excreted from the liver during the initial hours of the experiments; such processes may also occur in the field and influence the measurements. Similar results were observed previously on fish force-fed with toxic cyanobacteria [START_REF] Sahin | Biliary excretion of biochemically active cyanobacteria (blue-green algae) hepatotoxins in fish[END_REF][START_REF] Tencalla | Biochemical characterization of microcystin toxicity in rainbow trout (Oncorhynchus mykiss)[END_REF]. Before its excretion, MC-LR undergoes a complex detoxification process that involves various enzymes of which the most studied are the glutathione S-transferases (GSTs) which conjugate MCs to GSH [START_REF] Campos | Molecular Mechanisms of Microcystin Toxicity in Animal Cells[END_REF]. In the present study, no significant difference in GST activity was observed between the control group and the experimental groups that were force-fed with MC-LR. This finding suggests that MC-LR ingested by YOY perch via zooplankton consumption does not induce an increase in the GST activity, which is consistent with other study [START_REF] Li | Responses of antioxidant systems in the hepatocytes of common carp (Cyprinus carpio L.) to the toxicity of microcystin-LR[END_REF][START_REF] Malbrouck | Hepatic accumulation and effects of microcystin-LR on juvenile goldfish Carassius auratus L[END_REF]. However in our study, MC-LR concentrations in the livers of YOY perch declined even though there was no change in GST activity. This observation could suggest that the basal level of GST activity in YOY perch was sufficient to detoxify the MCs concentrations that were administered in our fish. Furthermore, it could not be exclude the possible existence of another important MC-LR detoxification pathway involving conjugation of the toxin to molecules containing cystein residues (e.g. polypeptides, PPases) [START_REF] Zhang | The role of cysteine conjugation in the detoxification of microcystin-LR in liver of bighead carp (Aristichthys nobilis): a field and laboratory study[END_REF].

In the present study, no histological alterations were observed in the experimental groups for any of the organs studied. This finding is not surprising in view of other reports on this topic. Most histological alterations have been observed following immersion, intraperitoneal injection or even force-feeding but with acute doses of MCs [START_REF] Carbis | The histopathology of carp, Cyprinus carpio L, exposed to microcystins by gavage, immersion and intraperitoneal administration[END_REF][START_REF] Fischer | Pathological and Biochemical Characterization of Microcystin-Induced Hepatopancreas and Kidney Damage in Carp (Cyprinus carpio)[END_REF]. The liver, kidneys, gills and intestine are the organs most affected histologically by exposure to MCs. Histological alterations have been observed frequently following exposure to high MC concentrations or to a high biomass of cyanobacteria [START_REF] Malbrouck | Effects of microcystins on fish[END_REF]. Thus, in both in situ and experimental studies, MC-LR concentrations are typically too low to induce histological damage in YOY perch. Differences in the administration routes of toxins and in the sensitivity of various fish species to cyanobacterial toxins are additional factors that may account for the differences in results among studies [START_REF] Malbrouck | Effects of microcystins on fish[END_REF].

A transient increase in DNA damage was observed in perch erythrocytes following treatment with MC-LR, after 12 h and 48 h for the higher and the lower MC-LR dose, respectively. Reactive oxygen species are known to be produced following the exposure of fish to MCs [START_REF] Jos | Toxic cyanobacterial cells containing microcystins induce oxidative stress in exposed tilapia fish (Oreochromis sp.) under laboratory conditions[END_REF][START_REF] Prieto | Differential oxidative stress responses to microcystins LR and RR in intraperitoneally exposed tilapia fish (Oreochromis sp.)[END_REF]. This can partially explain the genotoxicity observed after perch exposure to MC-LR. The possibility cannot be ruled out that the DNA damage observed in perch erythrocytes arises from other pathways such as the production of instable DNA adducts that could be revealed through the comet assay. However, such underlying mechanisms of toxicity remain to be confirmed in fish. Regardless of the MC-LR dose, DNA damage levels returned to those of the control group 4 days after treatment. Such decreases in DNA damage may be explained by DNA repair and/or erythrocyte turnover; it is not possible to take into account the formation of apoptotic erythrocytes during Comet assay image analysis because of the huge number of DNA strand breaks in apoptotic cells [START_REF] Olive | Radiation-Induced Apoptosis Measured in TK6 Human B Lymphoblast Cells Using the Comet Assay[END_REF]. Genotoxic stress was thus observed in perch exposed to environmentally relevant MC-LR doses in terms of the toxin concentration ranges analysed in seston. YOY perch may therefore suffer drastic effects from the major algal blooms observed in peri-alpine lakes. This idea is consistent with the results of a previous study of another lake fish species, the European whitefish (Coregonus lavaretus) [START_REF] Sotton | Shortterm uptake of microcystin-LR by Coregonus lavaretus: GST activity and genotoxicity[END_REF].

P. rubescens, a MC-producing cyanobacterium, is found frequently in the metalimnion of peri-alpine lakes during the summer [START_REF] Jacquet | The proliferation of the toxic cyanobacterium Planktothrix rubescens following restoration of the largest natural French lake (Lac du Bourget)[END_REF][START_REF] Schwob | Occurrence of toxic Planktothrix rubescens blooms in lake Nantua, France[END_REF][START_REF] Ernst | Presence of Planktothrix sp. and cyanobacterial toxins in Lake Ammersee, Germany and their impact on whitefish (Coregonus lavaretus L.)[END_REF]. The development of P. rubescens in this water layer is facilitated by various specialised features such as gas vesicles that adjust the position of the organism in the water column in response to the vertical gradient of irradiance [START_REF] Walsby | The Burgundy-blood phenomenon: a model of buoyancy change explains autumnal waterblooms by Planktothrix rubescens in Lake Zu ¨rich[END_REF], photosynthetic pigments rich in phycoerythrin protein that absorb the entire spectrum of photosynthetic active radiation (PAR) [START_REF] Feuillade | The cyanobacterium (blue-green algae) Oscillatoria rubescens D.C[END_REF] and the excretion of alkaline phosphatases that utilise the organic forms of phosphorus when inorganic phosphate concentrations are low [START_REF] Feuillade | The cyanobacterium (blue-green algae) Oscillatoria rubescens D.C[END_REF]. In the present study, high biomasses of toxic P. rubescens were observed in the metalimnion during the summer. However, data concerning the interactions of P. rubescens and MCs with fish species in Lake Bourget are still scarce.

In this study, schools of YOY perch were observed in the epilimnion of the pelagic area above the P. rubescens blooms. This vertical distribution is consistent with observations in other studies [START_REF] Guillard | Three-dimensional internal spatial structure of young-of-the-year pelagic freshwater fish provides evidence for the identification of fish school species[END_REF][START_REF] Guillard | Hydroacoustic assessment of young-of-year perch, Perca fluviatilis, population dynamics in an oligotrophic lake (Lake Annecy, France)[END_REF][START_REF] Probst | Hydroacoustic observations of surface shoaling behaviour of young-of-the-year perch Perca fluviatilis (Linnaeus, 1758) with a towed upward-facing transducer[END_REF]. YOY perch are present in the epilimnion because of their thermal preferendum for the 16-26uC range [START_REF] Wang | Distribution of perch (Perca fluviatilis L.) during their first year of life in Lake Constance[END_REF]. Thus, YOY perch are spatially separated and have no direct contact with P. rubescens and their toxins. However, MCs were detected in the liver and muscles of YOY perch, and the maximal MC concentrations for both organs were found in August when the maximal MC concentrations in seston were also observed. MCs accumulation in perch has already reported in bloom conditions by several authors before [START_REF] Ibelings | Distribution of microcystins in a lake foodweb: no evidence for biomagnification[END_REF][START_REF] Wilson | Evaluation of the human health threat associated with the hepatotoxin microcystin in the muscle and liver tissues of yellow perch (Perca flavescens)[END_REF][START_REF] Gkelis | The presence of microcystins and other cyanobacterial bioactive peptides in aquatic fauna collected from Greek freshwaters[END_REF]. The MCs found in the fish are derived principally from their food sources. Daphnia are a major component of the perch diet and are known to be contaminated by MCs; i.e., Daphnia ingest filamentous cyanobacteria such as P. rubescens and consequently accumulate MCs [START_REF] Repka | Effects of food type on the life history of Daphnia clones from lakes differing in trophic state. I. Daphnia galeata feeding on Scenedesmus and Oscillatoria*[END_REF][START_REF] Oberhaus | Comparative effects of the quality and quantity of light and temperature on the growth of Planktothrix agardhii and P. rubescens1[END_REF]. In Lake Bourget, Daphnia therefore appear to be a major vector of MCs to zooplanktivorous fish. The diel vertical migration (DVM) characteristic of Daphnia species [START_REF] Hays | A review of the adaptive significance and ecosystem consequences of zooplankton diel vertical migrations[END_REF] is an important factor leading to the ingestion of P. rubescens filaments. While passing through the metalimnion, Daphnia ingest P. rubescens filaments, accumulate MCs and are subsequently eaten by YOY perch in the epilimnion. Thus, the YOY perch in the lake are chronically exposed to MC via their food sources leading to contamination of fish by MCs. However, our experiment has shown that perch are able to detoxify and excrete a portion of ingested MC and thereby limit their toxin concentration. Such detoxification and excretion processes are active during chronic MC exposure, as indicated by the absence of a continuous increase in MC concentration. However, they do not allow the reduction in MC concentration as observed in our experiments and a significant correlation between MC concentrations in the YOY perch and in seston was observed during the in situ study. Our experimental results suggested that the tested MC concentrations did not induce histological damage and our field observation showed that even with chronic exposure the MC concentrations recorded in Lake Bourget did not induce histological damage in YOY perch. Genotoxic impacts were not assessed in situ because of the complexity of measuring the effect of a targeted molecule on these parameters under in situ conditions. Other substances may well affect these parameters and thereby confound the real impact of MCs on perch. Our laboratory experiments under controlled conditions allowed us to assess the negative genotoxic impact (i.e., DNA damage) of MC-LR on YOY perch and to conclude that similar effects on YOY perch may occur during P. rubescens bloom conditions.

Finally, perch are highly commercialized for human consumption and YOY are used to be eaten fry and completely. As a consequence, the results of that study raise questions about the possible human threat linked to the consumption of wild perch.

Concentrations in muscle of YOY perch raise 14.2 ng.g 21 fresh weight (FW) in August during the bloom of P. rubescens. The WHO has established recommendations [START_REF] Chorus | Toxic cyanobacteria in water: a guide to their public health consequences, monitoring and management[END_REF] concerning the amount of cyanotoxin that an individual can consume per day for a lifetime, the microcystin total daily intake threshold (TDI) (0.04 mg of microcystin/kg of body weight/day). Thus, with concentrations found in our study in August, a man weighting 80 kg and consuming around 150 g of perch fillets (a typical restaurant portion) absorbs 2.13 mg of microcystin per day which is below the TDI (3.2 mg/day for a man weighting 80 kg). Consequently, the consumption of perch fillets coming from the Lake Bourget seems to be safety for people, even during severe P.rubescens bloom as it occurred in 2009 compared to previous years. However, as previously discussed, the methodological biases do not allow a true estimation of the MCY concentrations in the fish tissues analysed. A as consequence, our results clearly underline the human exposure to MCs through the consumption of fish caught during P. rubescens blooms but more studies are needed to estimate the real risk link to the consumption of that fish.

Conclusion

During the P. rubescens bloom in Lake Bourget in 2009, YOY perch populations were located in the epilimnion because of their thermal preferendum. The YOY perch were therefore never in direct contact with the P. rubescens bloom, which was located in the metalimnion. However, the YOY perch were exposed to MCs through the consumption of Daphnia. The MC concentrations ingested by YOY perch in the lake during 2009 did not result in an accumulation of the toxin sufficient to be unsafely for the human consumption or to induce lethal histological damages in fish. Indeed, YOY perch are able to detoxified and excreted rapidly MCs to avoid most of their negative impacts. However, genotoxic damage could occur even at low concentrations of MCs ingested possibly leading long term negative impacts on perch populations.

Figure 1 .

 1 Figure 1. Average prey preference of YOY perch during the studied period. Average prey specific abundance observed in the stomach content (X axis) plotted against the average frequency of occurrence of the prey in the diet (Y axis). doi:10.1371/journal.pone.0052243.g001

Figure 2 .Figure 3 .

 23 Figure 2. Temporal changes of the relative abundance of the zooplankton prey taxa observed in stomach contents. The number of analysed stomach contents is indicated above the bars. doi:10.1371/journal.pone.0052243.g002

Figure 4 .Figure 5 .

 45 Figure 4. Microcystin concentrations in liver and muscle of YOY perch and in seston during summer 2009 in Lake Bourget. Sestonic MC-LR equivalent concentrations are expressed as mg.L 21 and correspond to the sum of average concentrations found at the three depths sampled (6 m, 12 m, P. rubescens peak depth). MC-LR equivalent concentrations measured in organs are expressed as ng.g 21 fresh weight. doi:10.1371/journal.pone.0052243.g004

Figure 8 .Figure 9 .

 89 Figure 8. Light microscopy transverse sections of liver (A-B) and intestine (C-D) of control (A-C) and treated (0.5 mg of MC-LR) (B-D) YOY perch, stained with hematoxylin-eosin-saffron. Scale bar: 50 mm for liver and 100 mm for intestine. doi:10.1371/journal.pone.0052243.g008

Figure 10 .

 10 Figure 10. DNA damage in YOY perch erythrocytes measured through the comet essay. Star indicates significant differences between the different treatments and control group values (Mann-Whitney, p-value,0.05). doi:10.1371/journal.pone.0052243.g010

PLOS ONE | www.plosone.org 1 December 2012 | Volume 7 | Issue 12 | e52243

PLOS ONE | www.plosone.org

December 2012 | Volume 7 | Issue 12 | e52243

Acknowledgments

The authors thank Jean-Christophe Hustache, Michel Colon, Pascal Perney and Raphae ¨l D'Elbee for their technical help during field sampling, Se ´bastien Cachera (CISALB) and Ge ´rard Paolini (CALB) for their logistical support during field sampling and Leslie Laine ´and Jean-Paul Dubois for identification of zooplankton and measurement of fishes. We are grateful to Professor Pascal Fontaine (UR AFPA, Nancy) for providing experimental YOY perch.

B.S.'s PhD is a CIBLE project funded by the Rho ˆne-Alpes region. N.G.'s CDD and experiments were financed by ONEMA (Office National de l'Eau et des Milieux Aquatiques). The in-situ sampling and data analyses were part of ''Projet innovant'' funded by the department ''Ecologie des fore ˆts, Prairies et milieux Aquatiques'' (EFPA) of the ''Institut National de la Recherche agronomique'' (INRA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions