Self-selection of plant bioactive compounds by sheep in response to challenge infection with Haemonchus contortus

Physiology & Behavior 194 (2018) 302–310

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Self-selection of plant bioactive compounds by sheep in response to challenge infection with Haemonchus contortus

T

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Cesar H.E.C. Polia, , Kara J. Thornton-Kurthb, Jerrad F. Legakoc, Carolina Bremma, Viviane S. Hampela, Jeffery Hallb, Ignacio R. Ipharraguerred, Juan J. Villalbab a Programa de Pós-Graduação em Zootecnia, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 7712, Porto Alegre, RS 91450000, Brazil b Utah State University, Old Main Hill, Logan, UT 84322, USA c Texas Tech, 2500 Broadway, Lubbock, TX 79409, USA d Institute of Human Nutrition and Food Science, Christian-Albrechts-University, Herrmann Rodewald Str. 6, D-24118 Kiel, Germany

A R T I C LE I N FO

A B S T R A C T

Keywords: Ingestive behavior Preference Feed efficiency Lamb Gastrointestinal parasite Plant secondary compound

Plant bioactives can potentially benefit herbivores through their effects on health and nutrition. The objective of this study was to determine the importance of polyphenols and terpenes on the ability of lambs to self-select these compounds when challenged by a parasitic infection and the subsequent impact on their health and productivity. Thirty-five lambs were housed in individual pens and assigned to five treatment groups (7 animals/ group), where they received: 1) A basal diet of beet pulp:soybean meal (90:10) (CONTROL); 2) The same diet, but containing 0.3% of bioactive natural plant compounds extracted from grape, olive and pomegranate (BNP); 3) A simultaneous offer of the diets offered to the Control and BNP groups (Choice-Parasitized; CHP-1); 4) The Control diet, and when lambs developed a parasitic infection, the choice described for CHP-1 (CHP-2); and 5) The same choice as CHP-1, but animals did not experience a parasitic burden (Choice-Non-Parasitized; CHNP). Lambs, except CHNP, were dosed with 10,000 L3 stage larvae of Haemonchus contortus. Infected lambs under choice treatments (CHP-1 and CHP-2) modified their feeding behavior in relation to the CHNP group as they increased their preference for the feed containing polyphenols and terpenes, interpreted as a behavior aimed at increasing the likelihood of encountering medicinal compounds and nutrients in the environment that restore health. This change in behavior corresponded with an improvement in feed conversion efficiency. However, an increased preference for the diet with added plant bioactives did not have an effect on parasitic burdens, hematological parameters, blood oxidation, or serum concentration of IgE.

1. Introduction Bioactives in plant tissues represent a significant benefit for the nutrition, health and welfare of ruminants grazing diverse plant communities. There are multiple studies showing the positive effects of plant secondary compounds on different aspects of sheep production such as growth [1–3], gastrointestinal parasite control [4], enteric methane production [5] and meat quality [6]. In addition, there is evidence suggesting that herbivores modify their food selection and preference as a function of their physiological needs [7–9] and biochemical characteristics of the diet [1]. For instance, herbivores increase their preference for bioactive-containing plants and rations in order to improve their health [10, 11]. Self-medication stemming from an integration of individual needs with the chemical characteristics of

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food is an adaptive behavior that contributes to improve the fitness of livestock challenged by disease [12, 13]. Condensed tannins have been used as a model to explore selfmedicative behaviors in ruminants [14–16] given that these compounds have direct negative impacts on the parasite, mainly through: (1) lower establishment of the infective third-stage larvae (L3) in the host, (2) lower excretion of eggs by adult worms and (3) impaired development of eggs into L3 [17]. In addition to the direct negative impacts of condensed tannins on endoparasites, other plant secondary compounds may provide direct and/or indirect medicinal effects to the host through their antioxidant or immune enhancing properties. This is because parasitic infections increase reactive oxygen species in the host, causing lipoperoxidation, cellular damage and inflammation [18–20]. Thus, self-selection of bioactive compounds other than condensed tannins

Corresponding author. E-mail addresses: [email protected] (C.H.E.C. Poli), [email protected] (K.J. Thornton-Kurth), [email protected] (J.F. Legako), jeff[email protected] (J. Hall), [email protected] (I.R. Ipharraguerre), [email protected] (J.J. Villalba). https://doi.org/10.1016/j.physbeh.2018.06.013 Received 11 December 2017; Received in revised form 2 April 2018; Accepted 11 June 2018 Available online 12 June 2018 0031-9384/ © 2018 Elsevier Inc. All rights reserved.

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batches that were fed to animals during periods of 7 to 10 days, when new batches were made. The plant bioactives added to the diet were stable during storage [26]. There are no detected levels of polyphenols or terpenes in the ingredients of the basal diet [28]. Animals received the same feed as CONTROL before being infected. However, when they developed a parasitic infection, these animals had the same choice described for CHP-1. A fifth treatment group, Choice-Non-Parasitized (CHNP), received the same choice as CHP-1, but animals did not experience a parasitic burden. Lambs were randomly distributed across treatments (7 lambs/ treatment) and pens, considering the variation of gender (female, ram lambs and wethers) and weight, resulting in a uniform distribution of animals within each treatment group. After 22 days of consuming their respective rations, lambs in groups CONTROL, BNP, CHP-1 and CHP-2 were infected with an oral dose that delivered an inoculum of 10,000 infective larvae (L3) of Haemonchus contortus. All groups continued ingesting the respective rations described above. After three weeks, when animals developed a parasitic infection, group CHP-2 had the same choice as CHP-1 and CHNP. Subsequently, all animals received their respective rations for 18 more days. Thus, this design aimed at exploring the effect of bioactive compounds (BNP vs. CONTROL) on parasitic burdens in sheep as well as the behavior patterns of sheep when given an opportunity to select a bioactive-containing food with the potential to improve their health. The design also allowed to determine the self-selection of plant bioactive compounds by sheep when experiencing (CHP-1) a parasitic infection vs. those animals that were parasite-free (CHNP). Finally, the study determined the benefit and selection of plant bioactives before (CHP-1) or after (CHP-2) lambs experienced a parasitic infection. Lambs were fed once a day at 0800 and they received their rations in ad libitum amounts. The amounts offered were adjusted every morning so that animals had at least 10% of refusal in their feeders on the ensuing day at 0730. If the animal had < 10% left from the previous day, an additional 500 g of feed was added to the feeders.

may provide a significant mechanism leading to protection against the oxidative stress or inflammation caused by endoparasites. Previous research demonstrates that provision of an antioxidant (i.e., vitamin E) to lambs infected with Haemonchus contortus led to a decrease in parasitic burdens when compared to control (unsupplemented) animals [21]. Several plant secondary compounds like polyphenols and terpenoids have established antioxidative, anti-inflammatory and gene regulatory properties [22–24] with the potential to attenuate parasitic infections in herbivores. Moreover, antioxidant compounds like polyphenols and triterpenic acids have been implicated in gut health and growth performance [25, 26]. Nevertheless, the concept of “nutraceutical” selfmedication has not been applied to these bioactive natural plant compounds in mammalian herbivores. Parasitism induce negative internal states, which may trigger changes in foraging behavior that enhance the likelihood of finding prophylactic plant secondary compounds in the environment [11, 27]. However, it is unknown whether parasitized ruminants are more likely to increase their preference for the novel orosensorial dimensions promoted by the presence of polyphenols and terpenes in a feed. We hypothesized that gastrointestinal parasitism in sheep causes deviations from homeostasis, which enhance intake of feeds containing bioactive compounds and result in health and productivity improvements. Thus, the objective of this study was to determine the importance of bioactive polyphenols and terpenes on parasitized lamb performance, ingestive behavior and gastrointestinal parasite burdens. 2. Material and methods The study was carried out at the Green Canyon Ecology Center, Utah State University, located in Logan, Utah (41° 44′ 76″ N; -111° 50′ 3.80″ W) between June 22nd and August 23rd, 2016. 2.1. Animals Thirty-five lambs (2–3 months of age) of 42 ± 4.0 Kg BW were used during the study. Before the study began, all animals were orally dosed against gastrointestinal parasites with Levamisole and Albendazole at 7.5 mg/kg body weight in order to eliminate the presence of gastrointestinal parasites in their digestive tract. Animals were housed in individual pens measuring 2.4 × 3.6 m under a protective roof. Pens were made of a metallic grid and fixed with metallic wires without sharp edges. Each pen had a wooden feeder. A water line with automatic nipples provided free access to drinking water. The lambs also had free access to trace salt mineral blocks. The animals were housed in individual cages in a way that they had visual contact with their peers so they did not experience stress due to isolation. The study was conducted according to procedures approved by the Utah State University Institutional Animal Care and Use Committee (approval # 2618).

2.3. Measurements 2.3.1. Intake Daily ration intake was measured by the difference between the amounts offered and refused. Preference for groups offered a choice between the basal diet and the bioactive containing diet was estimated as intake of Polyphenols+Terpenes/total food intake. 2.3.2. Diet quality The rations were sampled according to the batches that were mixed during the study: end of June (before starting the experiment), beginning of July, end of July, beginning of August and end of August (before finishing the experiment). The samples were analyzed for dry matter (DM), crude protein (CP) according to AOAC [29], neutral detergent fiber (NDF) according to Van Soest et al. [30], and acid detergent fiber (ADF) according to Goering and Van Soest [31]. The total digestible nutrients (TDN) and the metabolizable energy (ME) were calculated according to the information in the NRC [28] feed composition tables. There were no significant differences in diet quality composition between different batches, and means are shown in Table 1.

2.2. Treatments and experimental design Parasitized lambs were assigned to four treatment groups and offered ad libitum amounts of feeds that differed in the presence of bioactive natural plant compounds: 1) CONTROL, a basal diet (Basal) of beet pulp:soybean meal (90:10) balanced for an average live weight gain of 200 g/day according to NRC [21]; 2) Added bioactive natural plant compounds (BNP), the same ration but containing 0.3% of natural plant compounds (Polyphenols+Terpenes) extracted from grape, olive and pomegranate (GOPE, GOPE-40 ProNutra Solutions S.L., Spain) standardized in polyphenols - 30% proanthocyanidins, 8% ellagic acid, 1,5% gallic acid, 1% hydroxytyrosol, and 6% triterpenic acids (maslinic and oleanolic acid); 3) Choice-Parasitized (CHP-1), a simultaneous offer of Basal and Polyphenols+Terpenes feeds; and 4) CHP-2. The diets were prepared at the research facility by mixing the ingredients into

2.3.3. Animal weight gain Animals were weighed, with a previous liquid and solid fast period of 12 h. The animals were weighed for the first time on the first day of the experiment. After this day, animals were weighed every three weeks. 2.3.4. Animal feed efficiency Feed Efficiency was calculated by dividing the animal average daily weight gain by the respective daily food intake. This represents the animal gain/kg of ration consumed. 303

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2.3.6.5. Serum haptoglobin concentration. Haptoglobin concentration in serum was measured using the PHASE™ Haptoglobin assay (TP-801, Tridelta Development, Maynooth, Ireland) following the manufacturer's protocol. Quantification was performed on a BioTek Synergy H2 plate reader (BioTek, Winooski, VT) colorimetrically at 630 nm. Serum haptoglobin concentration is reported as mg/mL of serum.

Table 1 Mean nutritional composition (on a dry matter basis) of the feed used during the study. Parameters

MEAN

SEb

Pc

Crude protein (%) Acid detergent fiber (ADF) (%) Neutral detergent fiber (NDF) (%) ADF/NDF Total digestible nutrients (%)a Metabolizable energy (Mcal/Kg)a

13 23 37 63 76 2,7

0.34 0.20 0.43 0.67

0.5288 0.4233 0.4991 0.2451

2.4. Experimental design and statistical analysis Results were analyzed as a completely randomized design with seven replications per treatment. Individual animal was considered the experimental unit. Statistical analyses were performed using mixed models with fixed effects of treatment, period and their interaction. Animal was included as a random effect in the model. Period was considered to be the repeated measure over time, where each period represented the measurement of the same experimental unit under a different condition. In order to adjust the temporal autocorrelation observed in data measured on time, covariance structures were tested to fit the models, based on Akaike's Information Criterion (AIC), where the smallest value represents the best model adjusted. Two different models were performed in order to compare different periods (group of different days) to clarify the effects of parasite burdens across time: a) two periods before and after infection; b) three periods of 18 days: before the animals were infected, during the development of infection, and after the animals were infected. For comparing the ‘Period’ effect of 18 days, it was necessary to adjust the effect of day within each period. This adjustment was achieved with the inclusion of the ‘Day’ in the model as random effect. In this case, the statistical model had the fixed effects for treatment, period and their interaction, and the random effects included animal effect within treatment and day. The normal distribution of data was tested by Kolmogorov–Smirnov test (P > .05). Data were transformed when necessary by the logaritmic function. The statistical analyses were performed using the Mixed procedure of SAS program (SAS® 9.4 Foundation for Microsoft Windows for ×64, Cary, NC: SAS Institute Inc.), and the averages were compared by Contrasts Test analysis at a 95% confidence level (P < .05).

a

Calculated according to the values presented in NRC (2007). Standard error. c Probability of difference when comparing the different diets in the study across different food batches mixed along the study. b

2.3.5. Fecal egg counts Fecal samples were taken at 0800 am from the rectum of each animal, stored in an ice chest and analyzed the same day for fecal egg counts using the McMaster egg counting procedure for quantifying nematode eggs. This technique is an indirect estimate of parasitic burdens. Samples were taken before infecting the animals, and each week after the infection (August 5th, 12th and 22nd). 2.3.6. Blood analyses Blood samples (with and without EDTA added; Becton Dickinson Vacutainer System; Beckton Dickinson and Company, Franklin Lakes, NJ; 10 mL tubes) were collected via jugular venous puncture in the morning, after a 12 h of fasting. 2.3.6.1. Haemogram. Samples with EDTA were immediately submitted to the Utah Veterinary Diagnostic Laboratory (Logan, UT) for total blood cell count (Advia 120 Hematology Analyzer; Siemens Healthcare Diagnostics, Tarrytown, NY). Blood samples for an haemogram were collected before starting the experiment (June 15), the day of infection (July 14th), after being infected (August 15th), and at the end of the experiment (August 23rd). 2.3.6.2. Serum collection. Serum was extracted from samples without EDTA for analysis of lipid peroxidation. Serum was isolated after the samples were allowed to clot by centrifugation at 1500g for 10 min at 20 °C. A total of 5 mL of serum was collected and stored at -80 °C for subsequent analysis. Blood for extracting serum was collected before the beginning of the experiment (June 15th), before the animals were infected (June 30th), during the day of infection (July 14th), at the beginning of the infection period (August 3rd), after being infected (August 15th) and at the end of the experiment (August 23rd).

3. Results 3.1. Diet quality Results for the average composition of the rations used in the study across different periods are presented in Table 1.There was no difference in diet quality during the different periods of the study between the basal diet and the basal diet with the addition of polyphenols and terpenes.

2.3.6.3. Serum oxidation (TBARS). Lipid peroxidation was measured in serum using a commercially available kit (10,009,055, Cayman Chemical, Ann Arbor, MI) as per the manufacturer's protocol. This assay has been used to successfully measure Thiobarbituric acid reactive substances (TBARS) in serum from sheep in previously published research [32]. Relative concentration of TBARS was quantified using fluorimetric detection (excitation 530 nm, and emission 550 nm) of duplicate samples on a BioTek Synergy H2 plate reader (BioTek, Winooski, VT) and was based on comparison with standard curves. Lipid peroxidation is reported as μM of malondialdehyde (MDA).

3.2. Fecal egg counts and animal weights All animals had no Haemonchus contortus eggs in their feces before the infection. After the infection (third period), no differences in fecal egg counts (EPG) were detected among treatments that were parasitized (P = .9811) (Table 2). No differences were observed among treatments in relation to animal BW (P = .3193; Table 2), but a period effect (P < .0001) was detected, showing as expected, a positive evolution of BW across time. 3.3. Feed intake and preference

2.3.6.4. Serum IgE concentration. Concentration of serum IgE was measured using a commercially available quantitative competitive immunoassay (SI0044, ABclonal, Woburn, MA) as per the manufacturer's protocol. Quantification was performed on a BioTek Synergy H2 plate reader (BioTek, Winooski, VT) colorimetrically at 450 nm. Serum IgE concentration is reported as μg/mL of serum.

During Period 1, when animals were not infected and received a choice between the basal and the bioactive containing diets (CHNP and CHP-1), they ate more of the former than of the latter feed (P < .0001) and showed similar low values of preference for the bioactive304

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Table 2 Number of eggs per gram of feces (EPG), body weight (BW), average daily gain (ADG) and feed efficiency before and after a parasitic infection (Haemonchus contortus) while animals were subjected to different dietary treatments: CONTROL: A BASAL diet without plant bioactives; POLYPHENOLS+TERPENES: BASAL+ polyphenols and terpenes; CHP-1: Choice between BASAL and POLYPHENOLS+TERPENES fed from the beginning of the experiment; CHP-2: The same as in CHP-1 but only after infection. CHNP: The same as in CHP-1 but the animals were not infected. Parameters

CONTROL

POLYPHENOLS+TERPENES

CHP-1

CHP-2

CHNP

SE3

P4

EPG BW (kg) ADG (kg)

2423 48 0.229

2831 49 0.230

2340 50 0.263

2972 50 0.253

02 52 0.278

683.0 1.8 0.0186

0.9811 0.6725 0.3193

Feed efficiency1 Before Infection After Infection

0.1275BC 0.1237AB

0.1470ABa 0.1029Bb

0.1285BCb 0.1408Aa

0.1158Cb 0.1527Aa

0.1604Aa 0.1396Ab

0.01041 0.11090

< 0.0001

1

Average daily gain divided by the corresponding daily feed intake. Not included in the statistical analysis. 3 Standard error. 4 Probability of difference among treatments and the interaction treatment x period. Means in a row with different upper case letter superscripts differ within each period, according to LSMEANS test analysis (P < .05). Means in a column with different lower case superscripts differ, according to a orthogonal contrast test analysis (P < .05). 2

(P < .05). The CONTROL treatment showed intermediate values, which did not differ (P > .05) from either the choice treatments or the BNP treatment.

containing feed (intake of Polyphenols+Terpenes/total food intake) (Fig. 1a, b, c). No differences in total feed intake or preference were detected among treatments in period 1 (P > .05; Fig. 2a,b,c). During the second period of the study, when animals in CHP-1, CHP-2, Control and BNP were developing a parasitic infection, feed intake was lower for animals under CHP-1 than for non-parasitized animals (CHNP), but intake of the Polyphenols+Terpenes feed did not differ between treatments (P > .05; Fig. 1, a,b,c). When comparing total feed intake across animals that had (CHNP and CHP-1) or did not (Control, BNP, CHP-2) have a choice during this period, no differences were detected among treatments (P > .05; Fig. 2b, c). During the third period of the study, after animals in CHP-1, CHP-2, Control and BNP were infected, there was a notable difference on food intake among treatments that received a choice. Lambs in CHP-1 and CHP-2 showed greater intake of the Polyphenols+Terpenes feed, and lower intake of the Basal feed than non-parasitized animals (CHNP) (Fig. 1, a,b,c; contrast test between CHNP and CHP1 for Polyphenols +Terpenes feed intake P = .002, and Basal feed intake P = .0062). In this third period, lambs consumed on average 888 ± 72, 761 ± 77 and 609 ± 72 g/day of the Polyphenols+Terpenes feed, and they ingested 1246 ± 72, 1564 ± 78, 1721 ± 72, and g/day of the Basal feed for the CHP-1, CHP-2 and CHNP treatments, respectively. When comparing preference for the bioactive-containing diet (intake of Polyphenols+Terpenes diet relative to total feed intake) for the two treatments that had a choice from the beginning of the experiment (CHNP and CHP1), a significant treatment by period interaction (P = .0003) was observed. Lambs in CHP-1 showed a greater preference for the bioactive-containing feed than non-parasitized lambs during period 3, when animals in CHP1 were infected (42 vs 27 ± 3.6%; P = .0008; Fig. 2a). Lambs that received a choice only after experiencing the parasitic infection (CHP-2) did not differ in their preference for the bioactive containing feed from CHNP (33 ± 3.2%; P = .1028), and they showed lower preference for this feed than the CHP1 treatment (P = .0425).

3.5. Blood 3.5.1. Hemogram Results for the haemograms conducted during the study are presented in Table 3. There were several significant effects on blood parameters after the infection (i.e., period effect). On average, lambs had greater white blood (8.0 vs 6.8 ± 0.42 × 1000/μL) and lower red blood cell counts (12.5 vs. 11.2 ± 0.18 106/μL) before than after infection (P < .05). Lambs also showed lower mean corpuscular volume (26.3 vs 28.6 ± 0.46 fL), and mean corpuscular haemoglobin (9.7 vs 10.4 ± 0.14) before than after infection (P < .05). In addition, nonparasitized animals showed greater numbers of white and red blood cells, haemoglobin concentration, haematocrit and packed cell volume, with lower red cell distribution width and numbers of platelets than infected animals (P < .05). There was no significant effect of treatment, regardless of period, on any hemogram parameters. 3.5.2. Blood oxidation – TBARS analysis Averaged across periods, no differences were observed in blood oxidation among the different treatments (23.6 ± 2.31, 23.2 ± 2.31, 22.8 ± 2.42, 22.6 ± 2.57 and 21.2 ± 2.31 μM MDA for CHP1,CHNP, CHP-2, CONTROL and BNP treatments, respectively, P = .9262) and periods (22.7, 22.9, 22.4 ± 2.84 μM MDA for periods 1, 2 and 3, respectively, P = .9816). 3.5.3. IgE concentration There were no significant differences among treatments regarding the concentration of IgE (220.1 ± 38.09, 205.1 ± 38.09, 195.3 ± 38.09, 184.03 ± 45.07 and 171.9 ± 41.14 μg/mL for CHP1,CHNP, BNP, CONTROL and CHP-2 treatments, respectively, P = .9262). The overall average of IgE concentration across different treatments and periods was 196.7 ± 12.24 μg/mL. However, there was a significant reduction of the IgE concentration during the second period of the study, independent of the treatments (208,9, 145.2, 231.7 ± 21.46 μg/mL for the periods 1, 2 and 3, respectively P = .0002).

3.4. Feed efficiency Treatment groups showed different feed efficiencies (P < .001) before and after infection (Table 2). The infected treatments with a choice (CHP-1, CHP-2) were more efficient after than before the infection. In contrast, treatments without a choice (BNP and CONTROL) and without a parasitic infection (CHNP) showed either greater efficiency before than after being infected (BNP and CHNP), or no significant differences (CONTROL). When comparing feed efficiencies of the treatments after the infection, the BNP treatment showed the lowest efficiency, whereas the choice treatments evidenced greater values

3.5.4. Haptoglobin concentration Serum haptoglobin concentration was not affected by treatment (0.73 ± 0.173, 0.71 ± 0.204, 0.68 ± 0.173, 0.61 ± 0.173 and 0.44 ± 0.187 μg/mL for CHNP,CONTROL, CHP-1, BNP and CHP-2 treatments, respectively, P = .7991) or by period (0.54, 0.60, 305

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Fig. 1. Daily intake of feeds by different groups of lambs during the study. Intake was determined before and after a parasitic infection while animals were subjected to different dietary treatments: Panel a: CHNP- Choice between a basal diet without bioactive natural compounds (CONTROL) and the same diet containing bioactive natural compounds (polyphenols and terpenes; BNP) from the beginning of the experiment. Lambs in this treatment were not infected (SEM for CONTROL = 25.9 g; SEM for BNP = 25.07 g). Panel b: CHP-1: The same as in CHNP but the animals were infected (SEM for CONTROL = 18.16 g; SEM for BNP = 20.83 g). Panel c: CHP-2, The same as in CHP-1 but only after infection (SEM for CONTROL = 31.7 g; SEM for BNP = 54.3 g). Values are means for 7 lambs in each group. The arrows represent the day the lambs (except CHNP) were dosed orally with infective third-stage (L3) larvae of Haemonchus contortus, and the day that an infection was observed through fecal egg counts.

Fig. 2. Panel a. Preference (% of intake of the diet in relation to the total daily intake) for a diet containing bioactive natural compounds (polyphenols and terpenes; BNP) when offered in a choice with the same diet without bioactive natural compounds by three groups of lambs: 1)CHNP: Choice offered from the beginning of the experiment. Lambs in this group were not infected (SEM: 1.0%). 2)CHP-1: The same as in CHNP but the animals were infected (SEM = 0.88%), and 3)CHP-2: The same as in CHP-1 but only after infection (SEM = 2.30%). Panel b. Total daily intake by lambs in five groups: 1)CHNP (SEM: 25.0 g), 2)CHP-1 (SEM: 22.5 g), 3)CHP-2 (SEM = 32.2), 4)BNP: Infected lambs received just the basal diet with polyphenols and terpenes (SEM = 22.8 g), and 5)CONTROL: Infected lambs received the basal diet without polyphenols or terpenes (SEM = 23.9 g). Panel c. Total daily intake as a percentage of animal liveweight (LW) by lambs in five groups: 1)CHNP (SEM = 0.07%), 2)CHP-1 (SEM = 0.05%), 3)CHP-2 (SEM = 0.06%), 4)BNP (SEM = 0.05%), and 5)CONTROL (SEM = 0.07%). Values are means for 7 lambs in each group. The arrows represent the day lambs (except CHNP) were dosed orally with infective third-stage (L3) larvae of Haemonchus contortus, and the day that an infection was observed through fecal egg counts.

0.76 ± 0.143 μg/mL for the periods 1, 2 and 3, respectively, P = .5380). The overall average of haptoglobin concentration across different treatments and periods was 0.63 ± 0.080 μg/mL. 4. Discussion We hypothesized that parasitized lambs would self-select rations containing polyphenols and terpenes, as nematodes may induce the expression of oxidative processes in the host [33] and antioxidants have been implicated in anthelmintic activity [34]. Thus, we predicted that parasitized sheep would display greater preferences for bioactive-containing rations than non-parasitized sheep. We also predicted that the time of exposure to the bioactive compounds (i.e., before [animals in CHP-1] or after [animals in CHP-2] the development of a parasitic infection) would affect food selection because herbivores are more likely

to learn about the benefits of a medicine when they experience illness and then ingest a medicine that leads to recovery [35]. Finally, we predicted that the consequences of the self-medicative behavior would be beneficial to the animal, evidenced by a decline in parasitic burdens (estimated by EPG) and hematological parameters indicative of disease (e.g., anemia). In support of our predictions, parasitized lambs modified their feeding behavior and they increased their preference for the feed 306

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Table 3 Blood parameters in 5 groups of lambs (n = 7) before and after a parasitic infection (Haemonchus contortus) while animals were subjected to different dietary treatments: CONTROL: A basal diet without bioactive compounds; BNP: CONTROL+ Polyphenols and Terpenes; CHP-1: Choice between CONTROL and BNP since the beginning of the experiment; CHP-2, Like CHP-1 but only after infection. CHNP- Like CHP-1 but the animals were not infected. PERIODS Before infection Parameter White Blood Cells (×1000/μL) Red Blood Cells (106/μL) Haemoglobin level (g/dL) Haematocrit (%) Packed Cell Volume (%) Mean Corpuscular Volume (fL) Mean Corpuscular Haemoglobin Mean Corpuscular Haemoglobin Concentration (g/dL) Cell Haemoglobin Concentration Mean (g/dL) Red Cell Distribution Width Platelets (103/μL) Mean Platelet Volumen (fL) Plasma Protein (g/dL)

CON 7.0 12.9 12.2 33.0 35.7 25.8 9.5 37.1 35.8 18.9 545ab 4.0 6.5

BNP 8.2 12.7 12.0 32.8 35.3 25.9 9.4 36.4 35.0 18.6 594ab 4.0 6.7

SE

P valuea

0.8 0.4 0.3 0.8 0.8 0.8 0.3 0.4 0.4 0.6 76 0.3 0.2

0.7236 0.0055 0.0315 0.0815 0.0223 0.0166 0.0248 0.3509 0.0670 0.2558 0.0165 0.9318 0.4283

After infection CP1 8.0 12.2 12.0 32.7 34.8 26.9 9.9 36.8 35.5 19.1 678a 4.3 6.3

CP2 7.3 12.5 12.2 32.7 34.9 26.0 9.7 37.2 35.8 18.6 588ab 3.9 6.3

CNP 9.4 12.3 12.0 33.0 34.9 26.9 9.7 36.2 34.8 18.7 531b 4.3 6.5

CON b

6.2 11.6ab 11.9ab 32.3ab 34.4ab 28.5 10.5 37.1 35.3 19.6ab 615a 4.3 6.7

BNP ab

7.2 11.0ab 11.2b 31.2b 32.9b 28.5 10.3 36.1 34.7 19.7ab 559a 4.7 6.9

CP1 ab

6.3 10.7b 11.4b 31.3b 33.1b 29.5 10.7 36.5 35.0 20.9a 657a 4.9 6.5

CP2 b

6.1 10.8b 11.2b 30.3b 32.0b 28.2 10.4 36.9 35.5 19.6ab 551ab 4.5 6.4

CNP 8.0a 12.1a 12.3a 33.8a 35.9a 28.1 10.2 36.2 34.9 18.8b 435b 5.0 6.9

Means in a row with different superscripts differ within each period by contrast test analysis (before and after infection; P < .05). a Treatment by Period interaction.

non-parasitized subjects. Intake of this food by parasitized lambs increased to levels comparable to those recorded for the Basal food, showing that in contrast to healthy individuals, they no longer avoided these bioactive compounds in the choice test. When homeostasis is challenged by disease, consumers may respond by modifying their feeding behavior [11], a response that enhances the likelihood of encountering in diverse plant communities those medicinal compounds and nutrients that restore health [10, 27]. Thus, these results suggest that the negative effects of parasitism have the potential to alter diet selection, enhancing the likelihood of finding medicinal foods that rectify the physiological unbalance caused by disease. However, and in contrast to previous studies (e.g. [15, 50],), the increase in preference for the bioactive-containing food did not result in a reduction of the parasitic infection in the parasitized subjects, as evidenced by the lack of reductions in EPG or improvements in hematological parameters indicative of anemia, enhanced immune response (IgE), antioxidant activity (TBARS), or reduced stress (haptoglobin). In our study, despite the fact that consuming the bioactivecontaining food did not have a positive effect on parasitic burdens, lambs continued to incorporate this food into their diets, and at high rates through the end of the study. Increments in the selection of innocuous flavors in familiar foods typically extinguishes with a prolonged exposure, because animals in an unbalanced physiological state do not experience any benefits from including such flavors into their diets, relative to ingesting the same feed without the flavor [51]. Thus, it is likely that lambs in the present study experienced benefits like an attenuation of their parasitc burdens, a systemic improvement in the immune or redox homeostasis (e.g., local alterations at the gut level not measured in this study), or increased feed efficiencies. The latter was the most likely benefit experienced by lambs given the similar levels of fecal egg counts, indicators of anemia, or immunoglobulin concentrations observed between control and treatment animals. Changes in feeding behavior by sheep are well documented [8, 27, 52–55], and they have been interpreted by different reasons (e.g., endoparasitism, prior exposure to the diet, physiological state, time of day, group size). Additionally, feed preferences in generalist herbivores are not absolute and Newman et al. [54] have referred to this behavior as “partial preference.” In fact, our study demonstrates that this partial preference is dynamic as lambs that were offered a food choice only after experiencing a parasitic burden (CHP-2) displayed an intermediate level of intake of the bioactive-containing food, in-between healthy individuals (CHNP) and subjects which received the choice

containing polyphenols and terpenes relative to a period when they were not infected and in contrast to their non-infected counterparts. This change in behavior was associated with improvements in feed efficiencies, but no treatment effects were observed on parasitic burdens or on the hematological parameters assessed during the study. 4.1. Parasitism, plant bioactives and preference The bioactive natural plant products added to the rations in the present study consisted of a mix of polyphenols (proanthocyanidins, ellagic acid, gallic acid, hydroxytyrosol) and triterpenic acids (maslinic and oleanolic acid) extracted from olives, grapes and pomegranate with significant antioxidant and anti-inflammatory effects in animal models [37–40]. Nevertheless, the lower intake of the bioactive-containing feed in non-infected lambs suggests an avoidance to the flavor of polyphenols and terpenes since, with the exception of these chemicals, rations in the choice tests had the same ingredients and proportions. This behavior can be understood by the fact that herbivores typically reduce intake of familiar foods with added novel flavors (i.e., they are neophobic) in order to reduce the likelihood of poisoning [9]. Wariness of the unfamiliar confers consumers a substantial survival value, such as reducing the likelihood of ingesting harmful plants [41]. In addition, even when experience to the added plant bioactives increased across days of testing, non-parasitized lambs continued to eat more basal diet than the bioactive containing feed through the end of the study. It is likely that such prolonged avoidance of the bioactive-containing feed was a consequence of the flavonoids present in the mix of plant bioactives as phenolic compounds may cause astringency [42–45]. According to Bordenave et al. [46] astringency can be a result of the interactions between flavonoids (such as the proanthocyanidins present in the bioactive-containing feed) and salivary proteins. This reaction can result in loss of solubility and lubrication in the mouth [47]. In addition, there is evidence that proanthocyanidins can promote reductions in food intake due to aversive postingestive feedback [9, 48, 49]. Lambs in the parasitized group CHP-1 displayed similar levels of intake of the bioactive-containing diet as Control animals before being infected (Period 1) or during the period of larval growth after infection (Period 2). Nevertheless, once lambs were parasitized during the third period of the study (evidenced by the significant increases in EPG and by changes in hematological parameters), there was a notable increase in preference for and intake of the bioactive-containing food relative to 307

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likely enhanced the effectiveness of the polyphenols and terpenes at improving the immunometabolic response and thus animal performance. In contrast to infected animals, feed efficiencies in non-parasitized lambs (CHNP) declined from the beginning (Before Infection) to the end (After infection) of the study, a pattern that could be explained by the much lower ingestion of bioactive compounds observed in the CHNP group relative to parasitized lambs, which likely prevented a positive effect of these chemicals on nutrient utilization. Thus, individual differences in metabolism and detoxification capabilities of consumers [69], as well as the specific amounts of bioactives consumed in a diet may dictate the potential benefit of these chemicals on animal fitness. Low to excessive concentrations may not have a positive effect, or they may even turn to be detrimental, with amounts of ingested polyphenols and terpenes that may vary depending on the animal's physiological state and the availability of food alternatives.

even prior to being infected (CHP-1). In fact, parasitized animals in CHP-2 showed a dramatic increase in the intake of the bioactive-containing feed right from the beginning of the period when choice tests were offered, providing another line of compelling evidence that lambs change their dietary preferences in response to infection. The reduced time of exposure to the bioactive-containing likely modulated its consumption to a level below that observed for lambs under CHP-1. Thus, timing and exposure to the novel flavor, as well as the lambs' physiological state influenced the extent to which lambs incorporated bioactive natural compounds into their diets. Our results suggest that the earlier the exposure to the bioactive-containing food while the animal is being infected, the greater the likelihood of incorporating greater amounts of this food into the diet. 4.2. Parasitism, antioxidants and animal growth

4.3. Antioxidant and antiparasitic effects

The aforementioned sustained changes in intake for the BNP feed by parasitized lambs can be interpreted in relation to specific benefits, other than anti-parasitic action, that increase animal fitness. Our results suggest that feed choice played an important role in enhancing the efficiency of utilization of the diet selected. As reported by different studies [56–58], herbivores perform better when they are exposed to a variety of feeds items or flavors than when they are constrained to a single feed type. The mixed diet has the potential to improve animal performance due to associative effects [59], the increased likelihood of consuming a nutritionally balanced diet [60], improved rumen environment [61], or reduction of food toxicity [62]. Our study suggests that the simultaneous offering of feeds supplemented or not with plant bioactives improved feed efficiency in parasitized animals, since this effect was exclusively observed in infected individuals. Different mechanisms seem to act in concert for improving feed efficiency in rations that share the same nutrient profile but consumed in a diversity of flavors. Lambs offered the same ration but in a choice with a diversity of flavors, consumed more feed and tended to gain more weight that lambs offered rations with just one flavor [58]. Diverse oro-sensorial stimuli restores the motivation to eat [63] by enhancing feed acceptability and reducing sensory-specific satiety [64]. Additionally, flavor diversity induces a more even spread of feed intake throughout the day, reducing rumen pH fluctuations and potentially enhancing the synchrony of nutrient supply to the host [58]. In addition, Hernandez-Sanabria et al. [65] explain that the efficiency of nutrient utilization is determined by the balance of the fermentation products, which ultimately can be controlled by ruminal microorganisms. Alternatively, offering a dietary choice might have allowed animals to ingest an amount of plant bioactives large enough to attenuate intestinal damage by the infection while preventing toxicity from excessive ingestion of these plant secondary compounds. As a result, the metabolic cost of supporting the immune response triggered by the infection and detoxification of xenobiotics triggered by the ingestion of plant secondary compounds was likely minimized, thereby enhancing the diversion of nutrients to growth rather than maintenance (i.e., improved feed efficiency). Alternatively, antioxidants increase feed efficiency in consumers [26], an effect which was not observed in lambs consuming only the bioactive-containing diet (BNP); in fact, these animals showed the lowest values of feed efficiency during the infection period. It is possible that the concentration of antioxidants in the BNP diet was high, and it has been shown that at high dietary concentrations of antioxidants, feed efficiencies decline as a consequence of a reduction in the efficiency of nutrient absorption in the gastrointestinal tract [26]. Additionally, as suggested before, the ingestion of large quantities of plant secondary compounds might have increased the metabolic cost associated with the detoxification of xenobiotics and, as a consequence, the use of nutrients for purposes other than growth. Thus, we also suggest that animals in the choice treatments “diluted” the concentration of plant bioactives in their diet relative to those subjects under the BNP treatment, which

Although prior studies [67, 68] have shown beneficial effect of plant extracts rich in polyphenols on sheep physiology, fecal egg counts and haemogram (Table 3) reveal signs of parasitism (e.g., high numbers of EPG in feces) and infection (e.g., anemia) that were not attenuated by ingestion of the bioactive containing food. The lack of an effect of the diet containing polyphenols and terpenes on blood oxidation might be partly explained by a previous study [69], using 14C-labelled condensed tannins in sheep diets, where it was found that little if any carbon from condensed tannins was absorbed into the animals' tissues. The low recoveries of proanthocyanidins are considered to be a consequence of either molecular conformational changes, or interference from other digesta constituents. More recently, Nudda et al. [70], feeding milk sheep with grape seed, a rich source of proanthocyanidins, did not find any significant effects on milk or in animals' hematological and metabolic parameters. This lack of effect was also observed by the fact that there were no differences in serum IgE levels among the treatments in this study. IgE is considered a significant immunoglobulin to eliminate gastro-intestinal parasites [71]. The hypothesis that food choice induced a state of “positive stress” in lambs [66] which induced hormonal changes that enhanced feed efficiency was not observed in this study. When animals are challenged with solving problems, this effect may be a positive source of stress as long as the animal possesses the skills and resources to effectively solve the problems with which they are presented [66]. However, the choice between two types of food with limited constrains in the present study did not appear to generate stress, as evidenced by the similar concentration of serum haptoglobin observed across different treatments. Collectively, our research shows that lambs modified their feeding behavior during a parasitic burden by incorporating more feed containing an array of polyphenols and triterpenic acids with anti-inflammatory and antioxidant effects. This behavior may be explained as an improved utilization of nutrients by animals exposed to choice tests. However, there was no direct effect of the natural extracts from olives, grapes and pomegranate on Haemonchus contortus EPG, blood oxidation, or IgE levels. Nevertheless, the likelihood of additional beneficial effects like reduced oxidative stress and inflammation at the cellular level, or attenuation of tissue damage at the gut level could not be ruled out [24, 26, 36], given that assessments in the present study provided health indicators at the systemic level. Acknowledgement This research was supported by grants from the Utah Agricultural Experiment Station (UAES-1321), the National Council for Scientific and Technological Development (CNPq-232963/2014-2) of Brazil, and by Coordination for the Improvement of Higher Education Personnel (CAPES-8888.102309/2016-00) of Brazil. This paper is published with the approval of the Director, Utah Agricultural Experiment Station, and 308

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Utah State University, as journal paper number UAES #8954. We thank R. Stott for veterinary services and R. Lira for technical support and ProNutra Solutions S.L. for supplying the antioxidant product. We also thank two anonymous. Cesar H. E. C. Poli is member of the MARCARNE network, funded by CYTED (ref. 116RT0503).

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