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A tannin-blocking agent does not modify the preference of sheep towards tannin-containing plants
PHB-10805; No of Pages 6 Physiology & Behavior xxx (2015) xxx–xxx
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Keywords: Selectivity Plant secondary compounds Fiber Small ruminants Intake
Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Yucatán, Apdo, 4-116 Itzimná, 97100, Km 15.5 Carretera Mérida-Xmatkuil, Mérida, Yucatán, Mexico Centro de Enseñanza Investigación y Extensión en Ganadería Tropical, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Km. 5.5 Carretera Federal Tlapacoyan-Martínez de la Torre, C.P. 93600 Veracruz, Mexico
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Article history: Received 10 March 2014 Received in revised form 27 March 2015 Accepted 2 April 2015 Available online xxxx
Sheep have been suggested to use their senses to perceive plant properties and associate their intake with consequences after ingestion. However, sheep with browsing experience do not seem to select against tannin-rich browsing materials in cafeteria trials. Thus, the objective of the present study was to evaluate the relationship between the chemical composition, selectivity index (SI), preference and intake rate (IR) of tannin-containing forage trees offered to sheep in cafeteria experiments. Four trees were selected for their condensed tannin content and their varying biological activities. Havardia albicans (high biological activity), Leucaena leucocephala (medium biological activity), Acacia gaumeri (low biological activity) and Brosimum alicastrum (very low biological activity) were used in this study. Ten hair sheep (23.7 kg ± 1.43 LW) with eight months of browsing experience in native vegetation were used in this study. Polyethylene glycol (PEG 3600 MW) was administered to five sheep during all experiments. In experiment 1, fresh foliage from all trees was offered ad libitum for 4 h. In experiment 2, B. alicastrum was withdrawn and the preference was determined again. The forage preference in experiment 1 was A. gaumeri (14.77 g DM/kg LW) N B. alicastrum (11.77 g DM/kg LW) N H. albicans (3.71 g DM/kg LW) = L. leucocephala (1.87 g DM/kg LW) (P b 0.05). The preference in experiment 2 was A. gaumeri N H. albicans = L. leucocephala. PEG administration had no effect on the preference or IR. The intake rate seemed to have been affected by the plant density. Moreover, fiber compounds were found to be better predictors of DM intake than polyphenolic compounds at levels typically found in the evaluated forages. It was concluded that tannins and PEG did not modify the preferences of sheep in cafeteria trials. Thus, tannins are not involved in the preference regulation of animals with browsing experience. © 2015 Published by Elsevier Inc.
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also found by Rogosic et al. [26]. In addition, Favreau et al. [12] suggested that unless the sheep were conditioned, they preferred umami tastes and were indifferent to bitter tastes. The umami taste can be associated with protein content, while the bitter taste can be associated with tannin content. Hence, we hypothesized that the CT content of browse legumes was not a limiting factor in the feed preference of sheep and goats. Thus, adding the tannin-blocking agent PEG should not affect animal preference and selectivity. Therefore, the objective of the present study was to evaluate the relationship between the chemical composition, the selectivity index (SI), the preference and the intake rate (IR) of tannin-containing forage trees offered to sheep in cafeteria experiments.
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2. Materials and methods
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2.1. Study area
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G. Hernández-Orduño a, J.F.J. Torres-Acosta a, C.A. Sandoval-Castro a,⁎, C.M. Capetillo-Leal a, A.J. Aguilar-Caballero a, M.A. Alonso-Díaz b
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A tannin-blocking agent does not modify the preference of sheep towards tannin-containing plants
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1. Introduction
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Most tropical trees and shrubs have relatively high levels of plant secondary metabolites (PSM) [21,27]. Those PSMs, specifically condensed tannins (CT), have been reported as modulators of the intake of single feed under controlled conditions because large quantities of CTs seem to reduce intake (see review [16]). The role of condensed tannins in the feed preference during short term cafeteria experiments has been challenged in goats and sheep with browsing experience [1,3]. Both the fiber components and foliage density have been suggested to be involved in the regulation of IR [1,3]. Although some studies have found that adding a tannin blocking agent (polyethylene glycol, PEG) modifies shrub preference in goats and sheep [26,31]. A recent experiment with goats [14] showed that adding PEG to the tannin-rich browse offered in a cafeteria trial did not modify the animals' preferences. This result was
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⁎ Corresponding author. E-mail address: [email protected] (C.A. Sandoval-Castro).
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This work was carried out at the Faculty of Veterinary Medicine and 72 Animal Science, Universidad Autonoma de Yucatan (FMVZ-UADY). The 73
http://dx.doi.org/10.1016/j.physbeh.2015.04.006 0031-9384/© 2015 Published by Elsevier Inc.
Please cite this article as: G. Hernández-Orduño, et al., A tannin-blocking agent does not modify the preference of sheep towards tannincontaining plants, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.04.006
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2.2. Experimental forages
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The fresh leaves of the legumes Havardia albicans (HA), Acacia gaumeri (AG), and Leucaena Leucocephala (LL) have been reported as being rich in polyphenolic compounds [9,15]. Brosimum alicastrum (BA) has been reported as being preferred by cattle [28] and sheep [37]. Because BA has a low concentration of condensed tannins, this species was also included in the present study.
2.3. Experimental animals
85 86
Ten female Pelibuey hair sheep (initial average weight 23.7 kg ± 1.43, 10–11 months old) were used. All of the animals had eight months of browsing experience in the native deciduous tropical forest of the area. The animals were drenched with an effective wide spectrum anthelmintic three days prior to the first adaptation period.
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The animals were divided in two groups (n = 5): with and without addition of 25 g polyethylene glycol (PEG, MW 3600, Sigma Co.). PEG was diluted in water (1:1 w/v) and was dosed directly into the mouth before and 30 min after fresh leaves were offered [32]. The animals were allocated into individual pens (3 m × 3 m). Each animal was fed fresh grass (Pennisetum purpureum) ad libitum and 200 g of a grain-based concentrate (wheat bran, soybean meal and sorghum grain) every day. Fresh leaves (50 g) of each plant were offered together on a daily basis during a five-day adaptation period. The amount offered (50 g each plant) was selected to ensure their total ingestion and that the animals had previous knowledge of the plants offered (no refusals were observed). Then, the preferences of the animals were measured using a multiple Latin square design [6]. The animals were offered fresh leaves of each plant ad libitum in individual plastic feeders for 4 h periods. The positions of the feeders were changed daily to avoid conditioned learning (association) between the feeder positions and the forage species. Based on the preliminary observations of the container capacity, at least 200 g fresh leaves were always available. Refusal was measured every hour to obtain the cumulative intake and the containers were replenished with the measured amounts. After the 4 h period, the tree forages were withdrawn and the animals received concentrate feed (200 g) and grass (ad libitum). The food was offered for 15 h only. The remaining 5 h animals were maintained without feed. Daily feed samples were collected, dried (60 °C), milled (1 mm sieve) and kept in airtight containers for later analysis.
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2.5. Preference experiment 2
118 119
In this experiment, the choice of forage was restricted to the three tannin-containing plants (HA, AG, LL). This experiment was managed in a similar manner to experiment 1 but was preceded by a 4-day adaptation period. The feeding and sampling procedures were performed as described above. The animals in both experiments adapted well to the experimental conditions. The pens were constructed with wire mesh, which allowed for visibility among the animals. The animals had apparently normal behavior and none of them were removed from the experiment due to abnormal behavior or disease.
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2.6.3. Selectivity index (SI) 143 The SI was calculated for each sheep daily during the experimental 144 periods [10]: 145 Q6 For Experiment 1,
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For Experiment 2, h i SI ¼ ð1=3−Pi Þ∧ 2 þ 1=3−P j ∧ 2 þ ð1=3−Pk Þ∧ 2 =ð2=3Þ; 150
where Pi, Pj, Pk and Pl are the proportions of the food consumed per day. An animal was qualified as either completely selective when only one 151 plant was consumed (index = 1) or as completely unselective when 152 the same proportions of each plant species were consumed (index = 0) 153
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94 95
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131 132
h i SI ¼ ð1=4−Pi Þ∧ 2 þ 1=4−P j ∧ 2 þ ð1=4−Pk Þ∧ 2 þ ð1=4−Pl Þ∧ 2 =ð3=4Þ;
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2.6.2. Foliage density To assess the effects of foliage structure on the DM intake, the foliage density of each plant was determined by placing 200 g of fresh foliage from each plant in containers. No attempt was made to press the foliage into the container. Then, the volume (cm3) occupied by the foliage was measured. The density (w/v) was measured three times for each plant.
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2.6.1. Total foliage intake (TFI) and intake rate (IR) TFI was recorded as the difference between the weight of the feed offered and the weight of the foliage remaining after the 4 h of consumption. For each animal, the time spent feeding (minutes) on each type of foliage during hours 1 and 4 was recorded. The IR was calculated as the amount of feed consumed (DM) per effective minute spent eating each type of feed. The average IR was calculated by pooling the data from hours 1 and 4.
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2.6. Variables
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climate of the area is tropical and sub-humid, with summer rainfalls. The average temperature varies from 26 to 27.8 °C and the annual rainfall ranges from 940 to 1100 mm.
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2.7. Laboratory analysis
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The samples were analyzed for their DM, N and ash contents (7.007, 2.057 and 7.009, respectively) according to A.O.A.C. [4]. NDF and ADF were uncorrected for residual ash, and NDF was determined using sodium sulfite without alpha amylase. The lignin content was also determined [33]. In addition, the cellulose (Cel) (calculated by the difference between ADF and Lignin), hemicellulose (Hem) (calculated by the difference between NDF and ADF) and cellulose + hemicellulose (Cel + Hem) contents were estimated. The foliage samples were oven-dried at 50 °C, ground and extracted using acetone:water (70:30 v/v). The samples were sonicated for 20 min and were then centrifuged for 10 min. The supernatants were used to determine the presence of phenolic compounds, including the total phenols (TP) (using gallic acid as standard) [24] and total tannins (TT) (Folin–Ciocalteu + PVPP method, [19]) using tannic acid as a standard. The condensed tannins (CT) were determined by butanol HCL (anthocyanidin equivalent, [23]) and the vanillin assay (catechin as standard, [25]). The biological activity was determined by a radial diffusion method [13], as described by Díaz-Ortega et al. [9]. The protein precipitating power (units of astringency) was expressed as the area of precipitation around the plant extract wells relative to the precipitation of the standard. 4 replicates of each extract were assayed.
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2.8. Statistical analysis
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In all experiments, the preference was measured as the intake (g DM), intake time (min) and IR (g DM/min) at hours 1 and 4 and in total. The preference was analyzed based on the multiple Latin square design, in which each square = an animal, the column = the feeder position, the rows = the days of trial, and the treatments = each
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Please cite this article as: G. Hernández-Orduño, et al., A tannin-blocking agent does not modify the preference of sheep towards tannincontaining plants, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.04.006
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G. Hernández-Orduño et al. / Physiology & Behavior xxx (2015) xxx–xxx
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The chemical compositions of the foliage are described in Table 1. The CP content of the plants ranged from 14 to 30%. In both experiments, LL had the highest CP content, while HA had the highest Lignin and ADF content. BA had the highest NDF content. HA had the highest TP, TT and CT contents, while BA had the lowest. The foliage densities of AG, HA, and LL were similar (0.035, 0.041 and 0.041, respectively), and all were more dense than BA (0.019; P b 0.0001). The biological activities of AG, LL, HA, and BA were 1.70 ± 0.64, 3.52 ± 1.62, 3.77 ± 0.78 and 1.03 ± 0.05 units, respectively.
t1:1 t1:2
Table 1 Chemical composition (g/100 g DM) of the plants offered to the sheep.
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t1:3
CP
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20
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Experiment 1 A. gaumeri H. albicans L. leucocephala B. alicastrum P. purpureum Concentrate Experiment 2 A. gaumeri H. albicans L. leucocephala P. purpureum Concentrate
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Lig
ADF
NDF
Cel
3.3. Sheep intake and preference
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When the four plants were offered, the foliage consumption and intake time of all of the plants were higher during the first hour compared to the fourth hour. This was similar when BA was withdrawn from the trial. The IRs during the first hour were higher than the IRs during the fourth hour. In experiment 1, the average IRs of AG, HA and LL were similar, while the average IR was lower for BA. In experiment 2, the average IRs of the plants were similar (Tables 3 and 4). When all plants were offered, the preferences of the sheep (intake) were as follows: A. gaumeri N B. alicastrum N H. albicans = L. leucocephala (P = 0.0001) (Tables 2 and 3). A negative correlation was found between the intake of CP (r = − 0.330, P = 0.003) and the intake of lignin (r = − 0.485, P = 0.000). Positive correlations were found between the intake of NDF (r = 0.460, P = 0.000), Cel (r = 0.319, P = 0.004), hemicellulose (r = 0.496, P = 0.000) and cellulose + hemicellulose (r = 0.493, P = 0.000) (Table 4). Negative relationships were found between the polyphenolic compound contents and the intake: TP (r = −0.515, P = 0.000), TT (r = −0.563, P = 0.000), CT (butanol–HCl, r = −0.424, P = 0.000; and vanillin, r = − 0.565, P = 0.000) and biological activity (r = − 0.669, P = 0.000) (Table 4). However, no correlation was found between the PEG supplementation and the intake (P N 0.05). When only tannin-containing plants were offered, the sheep preferences were as follows: A. gaumeri N H. albicans = L. leucocephala (P = 0.0001). A positive correlation was found between the intake and NDF (r = 0.737, P = 0.000), Hem (r = 0.490, P = 0.001), and Hem + Cel (r = 0.506, P = 0.000). Negative correlations were found between the polyphenolic compound contents and the intake: TP (r = − 0.546, P b 0.000), TT (r = −0.562, P b 0.000), CT (butanol–HCl, r = −0.460, P b 0.001; and vanillin, r = −0.536, P b 0.000), and biological activity (r = −0.486, P b 0.001) (Table 4). No significant effect of PEG supplementation was found (P N 0.05). When BA was removed, TFI and FDMI (g/kg LW) diminished (32.12 vs 21.52 for TFI and 47.91 vs. 36.86 for FDMI). However, the consumption of CT (g/kg LW) seemed to decrease from 0.91 (butanol–HCl) or 10.8 (vanillin assay) to 0.59 or 0.98, respectively (Table 5).
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When all plants were offered in experiment 1, the SI was 0.20 with PEG and 0.18 ± 0.018 without PEG. In experiment 2, the SI increased (P b 0.001) when BA was removed (0.26 with PEG and 0.36 ± 0.0341 without PEG). Though PEG had no effect on the SI (P N 0.05), the presence of BA had an effect on the SI (P b 0.0001).
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3.2. Selectivity (SI)
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individual plant. To analyze the effects of PEG, the animals were used as nominal factors, the PEG treatments were used as blocks, and the PEG × plant interaction was included in the model. In addition, the proportions (% eaten of the offered forage) were analyzed using a generalized linear mixed model (Proc Glimmix, SAS, [30]) using a beta distribution, a logit link and a Laplace estimation. The model included the fixed effects of feeder position, experimental day, treatment (tree leaves) and day × treatment interaction. Different animals (pen) were used as the random effects blocking factor. The results obtained were similar to those found in the multiple Latin square analysis proposed by Borman et al. [6] and are therefore not presented. SI was compared between experiments. Thus, the data from both experiments were used in general linear models that included the effects of PEG and B. alicastrum as well as their interaction in a 2 (with/without PEG) × 2 (with/without B. alicastrum) factorial design. The density (g/cm3) and biological activity (units of astringency) of each different foliage type were compared using a completely randomized design. The differences among the means with P b 0.05 were accepted as statistically significant. When significant differences between means were found, Tukey post-hoc analyses were used. Pearson correlation analyses were completed between the chemical compositions of the foliage types and the animals' preferences (DMI). The probability value indicative of statistical significance was P b 0.05. The stepwise selection technique of the stepwise procedure [29] was used to determine the best predictor for the preference (DMI) in each experiment. The concentrate supplement was a fixed amount for all groups. Thus, the concentrate intake data were not subjected to any type of analyses. In addition, PEG supplementation had no significant interaction with any other main effect. Therefore, only the main effects are described in the results and discussion sections.
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3
Hem
C+H
Ash
TP
TT
CT Bu
Va
22.31 16.64 29.42 14.82 7.16 21.73
8.68 15.14 9.46 6.77 9.25 1.42
20.79 28.56 22.97 29.72 48.35 7.7
42.41 38.99 42.27 51.88 78.24 26.58
12.11 13.42 13.51 22.95 39.1 6.28
21.62 10.43 19.3 22.16 29.89 18.88
33.73 23.85 32.81 45.11 68.99 25.16
8.31 4.63 6.78 11.17 5.77 5.18
4.7 7.4 5.2 3.8 – –
1.18 5.85 3.02 1.79 – –
4.16 4.68 4.11 0.42 – –
2.79 11.66 5.63 1.08 – –
20.64 16.71 30.26 7.37 26.73
10.78 13.57 8.82 9.4 1.78
23.84 27.2 21.83 48.98 9.41
45.2 40.4 41.1 77.72 24.95
13.06 13.63 13.01 39.58 7.63
21.36 13.2 19.27 28.74 15.54
34.42 26.83 32.28 68.32 23.17
8.35 4.59 8.2 6.9 14.84
3.3 12.8 6.6 – –
0.37 11.31 4.51 – –
1.91 5.49 2.65 – –
2.30 11.33 5.31 – –
CP, crude protein; L, lignin; ADF, acid detergent fiber; NDF, neutral detergent fiber; Cel, cellulose; Hem, hemicellulose; C + H, cellulose + hemicellulose; TP, total polyphenols; TT, total tannins; CT, condensed tannins, Bu, butanol–HCl; Va, vanillin.
Please cite this article as: G. Hernández-Orduño, et al., A tannin-blocking agent does not modify the preference of sheep towards tannincontaining plants, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.04.006
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G. Hernández-Orduño et al. / Physiology & Behavior xxx (2015) xxx–xxx
Table 2 Intake (I), time spent eating (T) and intake rate (IR) during hours 1 and 4, respectively. Experiment
Tree
t2:4 t2:5 Q1 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14
I1
I4
T1
T4
IR1
IR4
g DM
g DM
min
min
g DM/min
g DM/min
1
A. gaumeri H. albicans L. leucocephala B. alicastrum SEM
220.86a 46.36b 22.13b 167.79c 9.58
19.19a 4.68b 2.79b 19.30a 2.10
17.53a 4.52b 4.06b 22.64c 0.97
3.52ª 0.92b 1.38b 6.03c 0.56
13.41a 11.93ac 5.13b 8.44bc 0.95
4.29a 4.05ª 1.73ª 5.36a 0.99
2
A. gaumeri H. albicans L. leucocephala SEM
225.21a 53.17b 39.91b 11.45
22.89ª 11.60b 7.10b 2.83
29.57ª 7.90b 6.00b 1.05
5.67ª 2.07b 2.10b 0.55
7.59ª 6.46ª 7.32a 0.75
3.88ª 6.05ª 3.60a 1.41
I1 = intake during the first hour; I4 = intake during the fourth hour; T1 = effective minutes eating during the first hour; T4 = effective minutes eating during the fourth hour; IR1 =
t2:16 t2:17
intake rate during the first hour; IR4 = intake rate during the fourth hour. For each variable, the means are compared only within and not between experiments.
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4. Discussion
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4.1. Chemical composition
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During the four experiments, the chemical compositions and polyphenolic contents of the foliage types were within the ranges commonly reported in the region [1,28]. As expected, the BA foliage had a negligible level of CT.
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4.2. PEG effect
273 274
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No clear patterns explaining the preferences or intake regulation were found in relation to the presence of polyphenolic compounds in the browse materials. The presence of these compounds and the presence of fiber components had similar associations with the animals' preferences. Furthermore, dosing animals with PEG did not have an effect on their preferences or change the animals' selectivity (P N 0.05). Thus, these results confirmed the limited role of these compounds in the short-term preferences of animals that are able to choose their diets [1]. It is likely that a different response would be obtained if the animals were restricted to single tannin-containing forages. However, this is contrary to previous results with goats [14], which showed that the CT intake did not increase when B. alicastrum was removed. Sheep are less adapted than goats to foraging–browsing behavior [5,36], and their limited ingestion or avoidance of PSMs should be considered to be behavioral strategies that are integrated with their physiological abilities to either detoxify and excrete PSMs or to endure and overcome their effects [17,20]. Similarly, Rogosic et al. [26] found that PEG supplementation had no effects on the shrub intake of goats. By contrast, sheep tended to increase their shrub intake when PEG was given as a supplement and the shrub availability was reduced from six to three. A major
t3:1 t3:2
Table 3 Intake, total time, average intake rate (IR), and total forage intake (FDMI, foliage + grass).
286 287 288 289 290 291
t3:3 t3:4 t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13 t3:14 t3:15 t3:16 t3:17 t3:18
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4.3. Foliage preferences
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difference between previous reports that used Mediterranean shrubs and the present study is that the shrubs differ in protein content between the two sets of studies. As discussed by Alonso-Díaz et al. [2], the shrubs available in tropical areas such as Yucatán, Mexico, are higher in protein content, and a proportion of the protein might be consist of tannin-binding proteins that mimic the effects of PEG, thereby reducing its effectiveness.
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When four plants were available (experiments 1), the sheep preferred AG, and their preferences were apparently influenced by the CP content of the plants (Table 5). Preference was also more strongly correlated with the fiber fraction (lignin, ADF, NDF, Cel, Hem + Cel) than with either the polyphenolic compounds (TP, TT, CT) or the biological activities. There was a negative relationship between the intake and the lignin content (r = −0.330, P b 0.003). The animals preferred plants with lower lignin contents, such as BA and AG, over trees with higher lignin contents, such as HA and LL. A negative association between intake and the lignin content has been found previously in cafeteria [5,14] and non-cafeteria trials [7]. Because lignin has been associated with the potential digestibility of the cell wall, a high lignin content in the foliage can limit intake [8,34]. It has been hypothesized that the negative relationship found between preference and lignin suggests that the latter may be detected orally as an indicator of a hard material. Thus, by selecting softer forages, animals also select highly and rapidly digestible materials [28]. Similarly to previous results [1,3] it is possible that the animals selected foraging materials based on their potential digestibility or macronutrient
T
C
E
R
284 285 Q7
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282 283
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280 281
C
279
N
277 278
Experiment Plant 1
2
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275 276
F
t2:15
Experiment 1
21.05 a 5.44 b 5.44 b 28.67 c 1.02
8.85 a 7.99 a 3.43 b 6.90 a 0.66
A. gaumeri 338.03 a H. albicans 104.94 b L. leucocephala 66.61 b SEM 16.64
35.23 a 9.97 b 8.10 b 1.23
5.74 a 6.26 a 5.46 a 0.75
1130–47.91
870–36.86
Total time = time spent eating during the first + fourth hours; Average IR = intake rate during the first and fourth hours. Different letters between the means for the same variable within the same experiment indicate significant differences. For each variable, the means are compared only within and not between experiments.
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Table 4 t4:1 Pearson correlation coefficients between chemical components and intake in cafeteria t4:2 trials. t4:3
Intake Total time Average IR FDMI (g DM/4 h) (min) (g DM/min) (g/d–g/kg LW)
A. gaumeri 349.64 a H. albicans 87.88 b L. leucocephala 44.34 b B. alicastrum 278.79 c SEM 15.67
295 296
Protein Lignin ADF NDF Cel Hem Cel + Hem Total polyphenols Total tannins Condensed tannins Butanol–HCl Vanillin Biological activity
t4:4
Experiment 2
r
P
r
P
t4:5
−0.330 −0.485 −0.034 0.460 0.319 0.496 0.493 −0.515 −0.563
0.003 0.000 0.767 0.000 0.004 0.000 0.000 0.000 0.000
−0.222 −0.029 −0.061 0.737 −0.285 0.490 0.506 −0.546 −0.562
0.142 0.852 0.689 0.000 0.058 0.001 0.000 0.000 0.000
−0.424 −0.565 −0.669
0.000 0.000 0.000
−0.460 −0.536 −0.486
0.001 0.000 0.001
t4:6 t4:7 t4:8 t4:9 t4:10 t4:11 t4:12 t4:13 t4:14 t4:15 t4:16 t4:17 t4:18
CP, crude protein; L, lignin; ADF, acid detergent fiber; NDF, neutral detergent fiber; Cel, cel- t4:19 lulose; Hem, hemicellulose. t4:20
Please cite this article as: G. Hernández-Orduño, et al., A tannin-blocking agent does not modify the preference of sheep towards tannincontaining plants, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.04.006
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Table 5 Intake (total g/d, as g/100 g DMI and g/kg LW) of the chemical components and secondary compounds from grass and browsed tree foliage.
t5:3
CP
Lig
ADF
NDF
Cel
Hem
Ash
TP
TT
t5:4 t5:5 t5:6 t5:7 t5:8 t5:9 t5:10 t5:11 t5:12 t5:13
CT Bu
Va
Experiment 1 g/d g/100 g DMI g/kg LW
200.2 17.65 8.45
87.1 7.68 3.68
297.8 26.25 12.57
544.6 48.00 23.00
210.7 18.57 8.90
246.8 21.76 10.42
87.8 7.74 3.71
35.8 3.16 1.51
15.6 1.37 0.66
21.7 1.91 0.91
25.5 2.25 1.08
Experiment 2 g/d g/100 g DMI g/kg LW
141.0 16.16 5.96
75.1 8.61 3.17
220.4 25.25 9.31
382.4 43.81 16.15
145.2 16.64 6.13
162.0 18.56 6.84
52.8 6.05 2.23
29.0 3.32 1.22
16.1 1.85 0.68
14.0 1.60 0.59
23.2 2.66 0.98
DMI, dry matter intake; LW, live weight; CP, crude protein; L, lignin; ADF, acid detergent fiber; NDF, neutral detergent fiber; Cel, cellulose; Hem, hemicellulose; TP, total polyphenols; TT, total tannins; CT, condensed tannins; Bu, butanol–HCl; Va, vanillin.
320 321
340 341
contents. This hypothesis was reinforced by the absence of an effect of the PEG supplementation and by the results of a recent study that tested umami and bitter tastes [12]. When BA was excluded (experiment 2), the sheep preferred AG. A positive relationship was found between the animals' preference and the NDF content (r = 0.737, P b 0.000). These data support the hypothesis that preference is driven by the search for digestible material rather than the avoidance of tannins. When BA was unavailable, the total lignin intake (foliage + grass) remained at its previous level (3.68 vs. 3.17 g/kg LW) while the Hem + Cel intake was reduced (19.32 vs. 12.97 g/kg LW), indicating possible rumen fill constraints, as the rumen fill and transit of material through the rumen are regulated by the rate of particle size reduction and the digestibility rate [11]. Similar results were found with goats [14]. The results found in the present study and in previous reports suggest that the rate of particle size reduction is associated with lignin content, while the digestibility rate is associated with digestible fiber. Both factors may be responsible for the intake and regulation of preference. Furthermore, recent evidence has suggested that cows may select digestible NDF from the indigestible NDF, which can result in a higher digestibility of fiber [18]. Although this has not been demonstrated for small ruminants, these animals may have developed a similar strategy due to their smaller rumen sizes and faster outflow rates [34].
342
4.4. Selectivity index
343 344
358 Q9 359
Animals selecting a mixed diet ingest a mixture of secondary plant compounds that are usually eliminated via different metabolic pathways. If animals restrict their diets to plants rich in a single PSM, a single pathway might become saturated. Thus, the strategy of a mixed diet suggests the existence of a trade-off between ingesting nutritious feed and spreading the risks of secondary compound ingestion [2]. Sheep seems to be selective (SI index), focusing their search for digestible materials (Cel + Hem and CP) without specifically avoiding CT consumption. As grazers, sheep do not seem to incorporate a “spread the risk” strategy while foraging different foliage types. On the other hand, goats, as browsers, do seem to incorporate the “spread the risk” strategy [14]. As a result, when BA was available, the sheep preferred AG and BA to the near-total neglect of the remaining foliage types. When BA was not available, the sheep preferred AG. It has been reported that sheep and goats show different selection patterns when they have access to similar types of diet in order to maximize their nutrient intakes [1,3,5, 36].
360
4.5. Intake rate and foliage density
361
BA leaves are bigger (leaf size: 4 to 18 cm in length, 2 to 7.5 cm in width) than the tanniniferous plant leaves offered in these experiments (linear foliole of 8–15 mm). Thus, the BA leaf density (0.019) was lower than the leaf densities of AG, LL and HA (0.035, 0.041, and 0.042, respectively). As a result, the animals had to manipulate the BA leaves one by one, which was probably due to the mouth-to-leaf ratio. Thus, the
338 339
345 346 347 348 349 350 351 352 353 354 355 356 357
362 363 364 365 366
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P
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E
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animals spent more time foraging BA plants, resulting in a lower IR and the association of foliage density with IR. It has been accepted that plant morphology and structure can influence the intake of forages by ruminants [7,35]. Although BA is a highly digestible fodder [1,28], it is not considered an optimal source of total nutrition due to its low IR. BA is not an apparent “best” single plant, as it incurs a higher harvesting cost for the animal. Though the pattern of intake was similar during the 4 h period (see intakes for hours 1 and 4), by the 4th hour, there was no difference in the DMI observed between the different foliage types due to the lower intake. This was probably due to fill/satiety, which resulted in a smaller IR. Similar trends for IR [14,22] and similar effects of the foliage density on the IR [14] have also been reported in goats.
T
334 335
C
332 333
E
330 331
R
328 329
R
326 327
N C O
324 325
U
322 323
F
t5:14 t5:15
367 368 369 370 371 372 373 374 375 376 377 378 379
5. Conclusion
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When faced with a variety of foliage types as in the present cafeteria experiments, sheep consume plants containing PSMs such as tannins. However, tannins did not have an influence on the preference, as the sheep seemed to select foliage depending on their potential macronutrient supply. The use of PEG did not modify the preference of sheep in these cafeteria trials. The present results support the hypothesis that feed tannin content is not the main regulator of preference in sheep with browsing experience. Finally, these results encourage the use of tropical fodder trees containing CT as nutraceuticals.
381
Acknowledgments
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G. Hernández-Orduño received a scholarship from CONACYTMexico to undergo her MSc studies. This work was supported by project CONACYT-SAGARPA-COFUPRO No. 12441. We thank Victor Parra-Tabla for advice and help with the GLIMMIX procedure and Humberto Antonio Peralta-Pino, Adrián Tun and Hilda Lorena Canul-Ku for the technical assistance.
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Contents lists available at ScienceDirect
Physiology & Behavior journal homepage: www.elsevier.com/locate/phb
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Keywords: Selectivity Plant secondary compounds Fiber Small ruminants Intake
Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Yucatán, Apdo, 4-116 Itzimná, 97100, Km 15.5 Carretera Mérida-Xmatkuil, Mérida, Yucatán, Mexico Centro de Enseñanza Investigación y Extensión en Ganadería Tropical, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Km. 5.5 Carretera Federal Tlapacoyan-Martínez de la Torre, C.P. 93600 Veracruz, Mexico
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Article history: Received 10 March 2014 Received in revised form 27 March 2015 Accepted 2 April 2015 Available online xxxx
Sheep have been suggested to use their senses to perceive plant properties and associate their intake with consequences after ingestion. However, sheep with browsing experience do not seem to select against tannin-rich browsing materials in cafeteria trials. Thus, the objective of the present study was to evaluate the relationship between the chemical composition, selectivity index (SI), preference and intake rate (IR) of tannin-containing forage trees offered to sheep in cafeteria experiments. Four trees were selected for their condensed tannin content and their varying biological activities. Havardia albicans (high biological activity), Leucaena leucocephala (medium biological activity), Acacia gaumeri (low biological activity) and Brosimum alicastrum (very low biological activity) were used in this study. Ten hair sheep (23.7 kg ± 1.43 LW) with eight months of browsing experience in native vegetation were used in this study. Polyethylene glycol (PEG 3600 MW) was administered to five sheep during all experiments. In experiment 1, fresh foliage from all trees was offered ad libitum for 4 h. In experiment 2, B. alicastrum was withdrawn and the preference was determined again. The forage preference in experiment 1 was A. gaumeri (14.77 g DM/kg LW) N B. alicastrum (11.77 g DM/kg LW) N H. albicans (3.71 g DM/kg LW) = L. leucocephala (1.87 g DM/kg LW) (P b 0.05). The preference in experiment 2 was A. gaumeri N H. albicans = L. leucocephala. PEG administration had no effect on the preference or IR. The intake rate seemed to have been affected by the plant density. Moreover, fiber compounds were found to be better predictors of DM intake than polyphenolic compounds at levels typically found in the evaluated forages. It was concluded that tannins and PEG did not modify the preferences of sheep in cafeteria trials. Thus, tannins are not involved in the preference regulation of animals with browsing experience. © 2015 Published by Elsevier Inc.
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 Q4 39
also found by Rogosic et al. [26]. In addition, Favreau et al. [12] suggested that unless the sheep were conditioned, they preferred umami tastes and were indifferent to bitter tastes. The umami taste can be associated with protein content, while the bitter taste can be associated with tannin content. Hence, we hypothesized that the CT content of browse legumes was not a limiting factor in the feed preference of sheep and goats. Thus, adding the tannin-blocking agent PEG should not affect animal preference and selectivity. Therefore, the objective of the present study was to evaluate the relationship between the chemical composition, the selectivity index (SI), the preference and the intake rate (IR) of tannin-containing forage trees offered to sheep in cafeteria experiments.
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2. Materials and methods
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2.1. Study area
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G. Hernández-Orduño a, J.F.J. Torres-Acosta a, C.A. Sandoval-Castro a,⁎, C.M. Capetillo-Leal a, A.J. Aguilar-Caballero a, M.A. Alonso-Díaz b
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A tannin-blocking agent does not modify the preference of sheep towards tannin-containing plants
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1. Introduction
46
Most tropical trees and shrubs have relatively high levels of plant secondary metabolites (PSM) [21,27]. Those PSMs, specifically condensed tannins (CT), have been reported as modulators of the intake of single feed under controlled conditions because large quantities of CTs seem to reduce intake (see review [16]). The role of condensed tannins in the feed preference during short term cafeteria experiments has been challenged in goats and sheep with browsing experience [1,3]. Both the fiber components and foliage density have been suggested to be involved in the regulation of IR [1,3]. Although some studies have found that adding a tannin blocking agent (polyethylene glycol, PEG) modifies shrub preference in goats and sheep [26,31]. A recent experiment with goats [14] showed that adding PEG to the tannin-rich browse offered in a cafeteria trial did not modify the animals' preferences. This result was
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⁎ Corresponding author. E-mail address: [email protected] (C.A. Sandoval-Castro).
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This work was carried out at the Faculty of Veterinary Medicine and 72 Animal Science, Universidad Autonoma de Yucatan (FMVZ-UADY). The 73
http://dx.doi.org/10.1016/j.physbeh.2015.04.006 0031-9384/© 2015 Published by Elsevier Inc.
Please cite this article as: G. Hernández-Orduño, et al., A tannin-blocking agent does not modify the preference of sheep towards tannincontaining plants, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.04.006
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2.2. Experimental forages
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The fresh leaves of the legumes Havardia albicans (HA), Acacia gaumeri (AG), and Leucaena Leucocephala (LL) have been reported as being rich in polyphenolic compounds [9,15]. Brosimum alicastrum (BA) has been reported as being preferred by cattle [28] and sheep [37]. Because BA has a low concentration of condensed tannins, this species was also included in the present study.
2.3. Experimental animals
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Ten female Pelibuey hair sheep (initial average weight 23.7 kg ± 1.43, 10–11 months old) were used. All of the animals had eight months of browsing experience in the native deciduous tropical forest of the area. The animals were drenched with an effective wide spectrum anthelmintic three days prior to the first adaptation period.
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The animals were divided in two groups (n = 5): with and without addition of 25 g polyethylene glycol (PEG, MW 3600, Sigma Co.). PEG was diluted in water (1:1 w/v) and was dosed directly into the mouth before and 30 min after fresh leaves were offered [32]. The animals were allocated into individual pens (3 m × 3 m). Each animal was fed fresh grass (Pennisetum purpureum) ad libitum and 200 g of a grain-based concentrate (wheat bran, soybean meal and sorghum grain) every day. Fresh leaves (50 g) of each plant were offered together on a daily basis during a five-day adaptation period. The amount offered (50 g each plant) was selected to ensure their total ingestion and that the animals had previous knowledge of the plants offered (no refusals were observed). Then, the preferences of the animals were measured using a multiple Latin square design [6]. The animals were offered fresh leaves of each plant ad libitum in individual plastic feeders for 4 h periods. The positions of the feeders were changed daily to avoid conditioned learning (association) between the feeder positions and the forage species. Based on the preliminary observations of the container capacity, at least 200 g fresh leaves were always available. Refusal was measured every hour to obtain the cumulative intake and the containers were replenished with the measured amounts. After the 4 h period, the tree forages were withdrawn and the animals received concentrate feed (200 g) and grass (ad libitum). The food was offered for 15 h only. The remaining 5 h animals were maintained without feed. Daily feed samples were collected, dried (60 °C), milled (1 mm sieve) and kept in airtight containers for later analysis.
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2.5. Preference experiment 2
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In this experiment, the choice of forage was restricted to the three tannin-containing plants (HA, AG, LL). This experiment was managed in a similar manner to experiment 1 but was preceded by a 4-day adaptation period. The feeding and sampling procedures were performed as described above. The animals in both experiments adapted well to the experimental conditions. The pens were constructed with wire mesh, which allowed for visibility among the animals. The animals had apparently normal behavior and none of them were removed from the experiment due to abnormal behavior or disease.
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2.6.3. Selectivity index (SI) 143 The SI was calculated for each sheep daily during the experimental 144 periods [10]: 145 Q6 For Experiment 1,
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For Experiment 2, h i SI ¼ ð1=3−Pi Þ∧ 2 þ 1=3−P j ∧ 2 þ ð1=3−Pk Þ∧ 2 =ð2=3Þ; 150
where Pi, Pj, Pk and Pl are the proportions of the food consumed per day. An animal was qualified as either completely selective when only one 151 plant was consumed (index = 1) or as completely unselective when 152 the same proportions of each plant species were consumed (index = 0) 153
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h i SI ¼ ð1=4−Pi Þ∧ 2 þ 1=4−P j ∧ 2 þ ð1=4−Pk Þ∧ 2 þ ð1=4−Pl Þ∧ 2 =ð3=4Þ;
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2.6.2. Foliage density To assess the effects of foliage structure on the DM intake, the foliage density of each plant was determined by placing 200 g of fresh foliage from each plant in containers. No attempt was made to press the foliage into the container. Then, the volume (cm3) occupied by the foliage was measured. The density (w/v) was measured three times for each plant.
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2.6.1. Total foliage intake (TFI) and intake rate (IR) TFI was recorded as the difference between the weight of the feed offered and the weight of the foliage remaining after the 4 h of consumption. For each animal, the time spent feeding (minutes) on each type of foliage during hours 1 and 4 was recorded. The IR was calculated as the amount of feed consumed (DM) per effective minute spent eating each type of feed. The average IR was calculated by pooling the data from hours 1 and 4.
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2.6. Variables
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climate of the area is tropical and sub-humid, with summer rainfalls. The average temperature varies from 26 to 27.8 °C and the annual rainfall ranges from 940 to 1100 mm.
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2.7. Laboratory analysis
154
The samples were analyzed for their DM, N and ash contents (7.007, 2.057 and 7.009, respectively) according to A.O.A.C. [4]. NDF and ADF were uncorrected for residual ash, and NDF was determined using sodium sulfite without alpha amylase. The lignin content was also determined [33]. In addition, the cellulose (Cel) (calculated by the difference between ADF and Lignin), hemicellulose (Hem) (calculated by the difference between NDF and ADF) and cellulose + hemicellulose (Cel + Hem) contents were estimated. The foliage samples were oven-dried at 50 °C, ground and extracted using acetone:water (70:30 v/v). The samples were sonicated for 20 min and were then centrifuged for 10 min. The supernatants were used to determine the presence of phenolic compounds, including the total phenols (TP) (using gallic acid as standard) [24] and total tannins (TT) (Folin–Ciocalteu + PVPP method, [19]) using tannic acid as a standard. The condensed tannins (CT) were determined by butanol HCL (anthocyanidin equivalent, [23]) and the vanillin assay (catechin as standard, [25]). The biological activity was determined by a radial diffusion method [13], as described by Díaz-Ortega et al. [9]. The protein precipitating power (units of astringency) was expressed as the area of precipitation around the plant extract wells relative to the precipitation of the standard. 4 replicates of each extract were assayed.
155
2.8. Statistical analysis
175
In all experiments, the preference was measured as the intake (g DM), intake time (min) and IR (g DM/min) at hours 1 and 4 and in total. The preference was analyzed based on the multiple Latin square design, in which each square = an animal, the column = the feeder position, the rows = the days of trial, and the treatments = each
176 177
Please cite this article as: G. Hernández-Orduño, et al., A tannin-blocking agent does not modify the preference of sheep towards tannincontaining plants, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.04.006
156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174
178 179 180
G. Hernández-Orduño et al. / Physiology & Behavior xxx (2015) xxx–xxx
223
The chemical compositions of the foliage are described in Table 1. The CP content of the plants ranged from 14 to 30%. In both experiments, LL had the highest CP content, while HA had the highest Lignin and ADF content. BA had the highest NDF content. HA had the highest TP, TT and CT contents, while BA had the lowest. The foliage densities of AG, HA, and LL were similar (0.035, 0.041 and 0.041, respectively), and all were more dense than BA (0.019; P b 0.0001). The biological activities of AG, LL, HA, and BA were 1.70 ± 0.64, 3.52 ± 1.62, 3.77 ± 0.78 and 1.03 ± 0.05 units, respectively.
t1:1 t1:2
Table 1 Chemical composition (g/100 g DM) of the plants offered to the sheep.
204 205 206 207 208 209 210 211
217 218 219 220 221 222
t1:3
CP
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20
C
202 203
E
200 201
R
198 199
R
196 197
N C O
194 195
Experiment 1 A. gaumeri H. albicans L. leucocephala B. alicastrum P. purpureum Concentrate Experiment 2 A. gaumeri H. albicans L. leucocephala P. purpureum Concentrate
U
192 193
Lig
ADF
NDF
Cel
3.3. Sheep intake and preference
230
When the four plants were offered, the foliage consumption and intake time of all of the plants were higher during the first hour compared to the fourth hour. This was similar when BA was withdrawn from the trial. The IRs during the first hour were higher than the IRs during the fourth hour. In experiment 1, the average IRs of AG, HA and LL were similar, while the average IR was lower for BA. In experiment 2, the average IRs of the plants were similar (Tables 3 and 4). When all plants were offered, the preferences of the sheep (intake) were as follows: A. gaumeri N B. alicastrum N H. albicans = L. leucocephala (P = 0.0001) (Tables 2 and 3). A negative correlation was found between the intake of CP (r = − 0.330, P = 0.003) and the intake of lignin (r = − 0.485, P = 0.000). Positive correlations were found between the intake of NDF (r = 0.460, P = 0.000), Cel (r = 0.319, P = 0.004), hemicellulose (r = 0.496, P = 0.000) and cellulose + hemicellulose (r = 0.493, P = 0.000) (Table 4). Negative relationships were found between the polyphenolic compound contents and the intake: TP (r = −0.515, P = 0.000), TT (r = −0.563, P = 0.000), CT (butanol–HCl, r = −0.424, P = 0.000; and vanillin, r = − 0.565, P = 0.000) and biological activity (r = − 0.669, P = 0.000) (Table 4). However, no correlation was found between the PEG supplementation and the intake (P N 0.05). When only tannin-containing plants were offered, the sheep preferences were as follows: A. gaumeri N H. albicans = L. leucocephala (P = 0.0001). A positive correlation was found between the intake and NDF (r = 0.737, P = 0.000), Hem (r = 0.490, P = 0.001), and Hem + Cel (r = 0.506, P = 0.000). Negative correlations were found between the polyphenolic compound contents and the intake: TP (r = − 0.546, P b 0.000), TT (r = −0.562, P b 0.000), CT (butanol–HCl, r = −0.460, P b 0.001; and vanillin, r = −0.536, P b 0.000), and biological activity (r = −0.486, P b 0.001) (Table 4). No significant effect of PEG supplementation was found (P N 0.05). When BA was removed, TFI and FDMI (g/kg LW) diminished (32.12 vs 21.52 for TFI and 47.91 vs. 36.86 for FDMI). However, the consumption of CT (g/kg LW) seemed to decrease from 0.91 (butanol–HCl) or 10.8 (vanillin assay) to 0.59 or 0.98, respectively (Table 5).
231 232
F
215 216
190 191
225 226
O
3.1. Chemical composition, foliage density and biological activity
188 189
When all plants were offered in experiment 1, the SI was 0.20 with PEG and 0.18 ± 0.018 without PEG. In experiment 2, the SI increased (P b 0.001) when BA was removed (0.26 with PEG and 0.36 ± 0.0341 without PEG). Though PEG had no effect on the SI (P N 0.05), the presence of BA had an effect on the SI (P b 0.0001).
R O
214
187
224
P
3. Results
185 186
3.2. Selectivity (SI)
D
213
183 184
T
212
individual plant. To analyze the effects of PEG, the animals were used as nominal factors, the PEG treatments were used as blocks, and the PEG × plant interaction was included in the model. In addition, the proportions (% eaten of the offered forage) were analyzed using a generalized linear mixed model (Proc Glimmix, SAS, [30]) using a beta distribution, a logit link and a Laplace estimation. The model included the fixed effects of feeder position, experimental day, treatment (tree leaves) and day × treatment interaction. Different animals (pen) were used as the random effects blocking factor. The results obtained were similar to those found in the multiple Latin square analysis proposed by Borman et al. [6] and are therefore not presented. SI was compared between experiments. Thus, the data from both experiments were used in general linear models that included the effects of PEG and B. alicastrum as well as their interaction in a 2 (with/without PEG) × 2 (with/without B. alicastrum) factorial design. The density (g/cm3) and biological activity (units of astringency) of each different foliage type were compared using a completely randomized design. The differences among the means with P b 0.05 were accepted as statistically significant. When significant differences between means were found, Tukey post-hoc analyses were used. Pearson correlation analyses were completed between the chemical compositions of the foliage types and the animals' preferences (DMI). The probability value indicative of statistical significance was P b 0.05. The stepwise selection technique of the stepwise procedure [29] was used to determine the best predictor for the preference (DMI) in each experiment. The concentrate supplement was a fixed amount for all groups. Thus, the concentrate intake data were not subjected to any type of analyses. In addition, PEG supplementation had no significant interaction with any other main effect. Therefore, only the main effects are described in the results and discussion sections.
E
181 182
3
Hem
C+H
Ash
TP
TT
CT Bu
Va
22.31 16.64 29.42 14.82 7.16 21.73
8.68 15.14 9.46 6.77 9.25 1.42
20.79 28.56 22.97 29.72 48.35 7.7
42.41 38.99 42.27 51.88 78.24 26.58
12.11 13.42 13.51 22.95 39.1 6.28
21.62 10.43 19.3 22.16 29.89 18.88
33.73 23.85 32.81 45.11 68.99 25.16
8.31 4.63 6.78 11.17 5.77 5.18
4.7 7.4 5.2 3.8 – –
1.18 5.85 3.02 1.79 – –
4.16 4.68 4.11 0.42 – –
2.79 11.66 5.63 1.08 – –
20.64 16.71 30.26 7.37 26.73
10.78 13.57 8.82 9.4 1.78
23.84 27.2 21.83 48.98 9.41
45.2 40.4 41.1 77.72 24.95
13.06 13.63 13.01 39.58 7.63
21.36 13.2 19.27 28.74 15.54
34.42 26.83 32.28 68.32 23.17
8.35 4.59 8.2 6.9 14.84
3.3 12.8 6.6 – –
0.37 11.31 4.51 – –
1.91 5.49 2.65 – –
2.30 11.33 5.31 – –
CP, crude protein; L, lignin; ADF, acid detergent fiber; NDF, neutral detergent fiber; Cel, cellulose; Hem, hemicellulose; C + H, cellulose + hemicellulose; TP, total polyphenols; TT, total tannins; CT, condensed tannins, Bu, butanol–HCl; Va, vanillin.
Please cite this article as: G. Hernández-Orduño, et al., A tannin-blocking agent does not modify the preference of sheep towards tannincontaining plants, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.04.006
227 228 229
233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265
4 t2:1 t2:2 t2:3
G. Hernández-Orduño et al. / Physiology & Behavior xxx (2015) xxx–xxx
Table 2 Intake (I), time spent eating (T) and intake rate (IR) during hours 1 and 4, respectively. Experiment
Tree
t2:4 t2:5 Q1 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14
I1
I4
T1
T4
IR1
IR4
g DM
g DM
min
min
g DM/min
g DM/min
1
A. gaumeri H. albicans L. leucocephala B. alicastrum SEM
220.86a 46.36b 22.13b 167.79c 9.58
19.19a 4.68b 2.79b 19.30a 2.10
17.53a 4.52b 4.06b 22.64c 0.97
3.52ª 0.92b 1.38b 6.03c 0.56
13.41a 11.93ac 5.13b 8.44bc 0.95
4.29a 4.05ª 1.73ª 5.36a 0.99
2
A. gaumeri H. albicans L. leucocephala SEM
225.21a 53.17b 39.91b 11.45
22.89ª 11.60b 7.10b 2.83
29.57ª 7.90b 6.00b 1.05
5.67ª 2.07b 2.10b 0.55
7.59ª 6.46ª 7.32a 0.75
3.88ª 6.05ª 3.60a 1.41
I1 = intake during the first hour; I4 = intake during the fourth hour; T1 = effective minutes eating during the first hour; T4 = effective minutes eating during the fourth hour; IR1 =
t2:16 t2:17
intake rate during the first hour; IR4 = intake rate during the fourth hour. For each variable, the means are compared only within and not between experiments.
266
4. Discussion
267
4.1. Chemical composition
268 269 270 271
During the four experiments, the chemical compositions and polyphenolic contents of the foliage types were within the ranges commonly reported in the region [1,28]. As expected, the BA foliage had a negligible level of CT.
272
4.2. PEG effect
273 274
292
No clear patterns explaining the preferences or intake regulation were found in relation to the presence of polyphenolic compounds in the browse materials. The presence of these compounds and the presence of fiber components had similar associations with the animals' preferences. Furthermore, dosing animals with PEG did not have an effect on their preferences or change the animals' selectivity (P N 0.05). Thus, these results confirmed the limited role of these compounds in the short-term preferences of animals that are able to choose their diets [1]. It is likely that a different response would be obtained if the animals were restricted to single tannin-containing forages. However, this is contrary to previous results with goats [14], which showed that the CT intake did not increase when B. alicastrum was removed. Sheep are less adapted than goats to foraging–browsing behavior [5,36], and their limited ingestion or avoidance of PSMs should be considered to be behavioral strategies that are integrated with their physiological abilities to either detoxify and excrete PSMs or to endure and overcome their effects [17,20]. Similarly, Rogosic et al. [26] found that PEG supplementation had no effects on the shrub intake of goats. By contrast, sheep tended to increase their shrub intake when PEG was given as a supplement and the shrub availability was reduced from six to three. A major
t3:1 t3:2
Table 3 Intake, total time, average intake rate (IR), and total forage intake (FDMI, foliage + grass).
286 287 288 289 290 291
t3:3 t3:4 t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13 t3:14 t3:15 t3:16 t3:17 t3:18
O
293 294
4.3. Foliage preferences
300
D
P
R O
difference between previous reports that used Mediterranean shrubs and the present study is that the shrubs differ in protein content between the two sets of studies. As discussed by Alonso-Díaz et al. [2], the shrubs available in tropical areas such as Yucatán, Mexico, are higher in protein content, and a proportion of the protein might be consist of tannin-binding proteins that mimic the effects of PEG, thereby reducing its effectiveness.
E
When four plants were available (experiments 1), the sheep preferred AG, and their preferences were apparently influenced by the CP content of the plants (Table 5). Preference was also more strongly correlated with the fiber fraction (lignin, ADF, NDF, Cel, Hem + Cel) than with either the polyphenolic compounds (TP, TT, CT) or the biological activities. There was a negative relationship between the intake and the lignin content (r = −0.330, P b 0.003). The animals preferred plants with lower lignin contents, such as BA and AG, over trees with higher lignin contents, such as HA and LL. A negative association between intake and the lignin content has been found previously in cafeteria [5,14] and non-cafeteria trials [7]. Because lignin has been associated with the potential digestibility of the cell wall, a high lignin content in the foliage can limit intake [8,34]. It has been hypothesized that the negative relationship found between preference and lignin suggests that the latter may be detected orally as an indicator of a hard material. Thus, by selecting softer forages, animals also select highly and rapidly digestible materials [28]. Similarly to previous results [1,3] it is possible that the animals selected foraging materials based on their potential digestibility or macronutrient
T
C
E
R
284 285 Q7
R
282 283
O
280 281
C
279
N
277 278
Experiment Plant 1
2
U
275 276
F
t2:15
Experiment 1
21.05 a 5.44 b 5.44 b 28.67 c 1.02
8.85 a 7.99 a 3.43 b 6.90 a 0.66
A. gaumeri 338.03 a H. albicans 104.94 b L. leucocephala 66.61 b SEM 16.64
35.23 a 9.97 b 8.10 b 1.23
5.74 a 6.26 a 5.46 a 0.75
1130–47.91
870–36.86
Total time = time spent eating during the first + fourth hours; Average IR = intake rate during the first and fourth hours. Different letters between the means for the same variable within the same experiment indicate significant differences. For each variable, the means are compared only within and not between experiments.
297 298 299
301 302 303 304 305 306 307 308 309 310 311 Q8 312 313 314 315 316 317 318 319
Table 4 t4:1 Pearson correlation coefficients between chemical components and intake in cafeteria t4:2 trials. t4:3
Intake Total time Average IR FDMI (g DM/4 h) (min) (g DM/min) (g/d–g/kg LW)
A. gaumeri 349.64 a H. albicans 87.88 b L. leucocephala 44.34 b B. alicastrum 278.79 c SEM 15.67
295 296
Protein Lignin ADF NDF Cel Hem Cel + Hem Total polyphenols Total tannins Condensed tannins Butanol–HCl Vanillin Biological activity
t4:4
Experiment 2
r
P
r
P
t4:5
−0.330 −0.485 −0.034 0.460 0.319 0.496 0.493 −0.515 −0.563
0.003 0.000 0.767 0.000 0.004 0.000 0.000 0.000 0.000
−0.222 −0.029 −0.061 0.737 −0.285 0.490 0.506 −0.546 −0.562
0.142 0.852 0.689 0.000 0.058 0.001 0.000 0.000 0.000
−0.424 −0.565 −0.669
0.000 0.000 0.000
−0.460 −0.536 −0.486
0.001 0.000 0.001
t4:6 t4:7 t4:8 t4:9 t4:10 t4:11 t4:12 t4:13 t4:14 t4:15 t4:16 t4:17 t4:18
CP, crude protein; L, lignin; ADF, acid detergent fiber; NDF, neutral detergent fiber; Cel, cel- t4:19 lulose; Hem, hemicellulose. t4:20
Please cite this article as: G. Hernández-Orduño, et al., A tannin-blocking agent does not modify the preference of sheep towards tannincontaining plants, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.04.006
G. Hernández-Orduño et al. / Physiology & Behavior xxx (2015) xxx–xxx t5:1 t5:2
5
Table 5 Intake (total g/d, as g/100 g DMI and g/kg LW) of the chemical components and secondary compounds from grass and browsed tree foliage.
t5:3
CP
Lig
ADF
NDF
Cel
Hem
Ash
TP
TT
t5:4 t5:5 t5:6 t5:7 t5:8 t5:9 t5:10 t5:11 t5:12 t5:13
CT Bu
Va
Experiment 1 g/d g/100 g DMI g/kg LW
200.2 17.65 8.45
87.1 7.68 3.68
297.8 26.25 12.57
544.6 48.00 23.00
210.7 18.57 8.90
246.8 21.76 10.42
87.8 7.74 3.71
35.8 3.16 1.51
15.6 1.37 0.66
21.7 1.91 0.91
25.5 2.25 1.08
Experiment 2 g/d g/100 g DMI g/kg LW
141.0 16.16 5.96
75.1 8.61 3.17
220.4 25.25 9.31
382.4 43.81 16.15
145.2 16.64 6.13
162.0 18.56 6.84
52.8 6.05 2.23
29.0 3.32 1.22
16.1 1.85 0.68
14.0 1.60 0.59
23.2 2.66 0.98
DMI, dry matter intake; LW, live weight; CP, crude protein; L, lignin; ADF, acid detergent fiber; NDF, neutral detergent fiber; Cel, cellulose; Hem, hemicellulose; TP, total polyphenols; TT, total tannins; CT, condensed tannins; Bu, butanol–HCl; Va, vanillin.
320 321
340 341
contents. This hypothesis was reinforced by the absence of an effect of the PEG supplementation and by the results of a recent study that tested umami and bitter tastes [12]. When BA was excluded (experiment 2), the sheep preferred AG. A positive relationship was found between the animals' preference and the NDF content (r = 0.737, P b 0.000). These data support the hypothesis that preference is driven by the search for digestible material rather than the avoidance of tannins. When BA was unavailable, the total lignin intake (foliage + grass) remained at its previous level (3.68 vs. 3.17 g/kg LW) while the Hem + Cel intake was reduced (19.32 vs. 12.97 g/kg LW), indicating possible rumen fill constraints, as the rumen fill and transit of material through the rumen are regulated by the rate of particle size reduction and the digestibility rate [11]. Similar results were found with goats [14]. The results found in the present study and in previous reports suggest that the rate of particle size reduction is associated with lignin content, while the digestibility rate is associated with digestible fiber. Both factors may be responsible for the intake and regulation of preference. Furthermore, recent evidence has suggested that cows may select digestible NDF from the indigestible NDF, which can result in a higher digestibility of fiber [18]. Although this has not been demonstrated for small ruminants, these animals may have developed a similar strategy due to their smaller rumen sizes and faster outflow rates [34].
342
4.4. Selectivity index
343 344
358 Q9 359
Animals selecting a mixed diet ingest a mixture of secondary plant compounds that are usually eliminated via different metabolic pathways. If animals restrict their diets to plants rich in a single PSM, a single pathway might become saturated. Thus, the strategy of a mixed diet suggests the existence of a trade-off between ingesting nutritious feed and spreading the risks of secondary compound ingestion [2]. Sheep seems to be selective (SI index), focusing their search for digestible materials (Cel + Hem and CP) without specifically avoiding CT consumption. As grazers, sheep do not seem to incorporate a “spread the risk” strategy while foraging different foliage types. On the other hand, goats, as browsers, do seem to incorporate the “spread the risk” strategy [14]. As a result, when BA was available, the sheep preferred AG and BA to the near-total neglect of the remaining foliage types. When BA was not available, the sheep preferred AG. It has been reported that sheep and goats show different selection patterns when they have access to similar types of diet in order to maximize their nutrient intakes [1,3,5, 36].
360
4.5. Intake rate and foliage density
361
BA leaves are bigger (leaf size: 4 to 18 cm in length, 2 to 7.5 cm in width) than the tanniniferous plant leaves offered in these experiments (linear foliole of 8–15 mm). Thus, the BA leaf density (0.019) was lower than the leaf densities of AG, LL and HA (0.035, 0.041, and 0.042, respectively). As a result, the animals had to manipulate the BA leaves one by one, which was probably due to the mouth-to-leaf ratio. Thus, the
338 339
345 346 347 348 349 350 351 352 353 354 355 356 357
362 363 364 365 366
O
R O
P
D
E
336 337
animals spent more time foraging BA plants, resulting in a lower IR and the association of foliage density with IR. It has been accepted that plant morphology and structure can influence the intake of forages by ruminants [7,35]. Although BA is a highly digestible fodder [1,28], it is not considered an optimal source of total nutrition due to its low IR. BA is not an apparent “best” single plant, as it incurs a higher harvesting cost for the animal. Though the pattern of intake was similar during the 4 h period (see intakes for hours 1 and 4), by the 4th hour, there was no difference in the DMI observed between the different foliage types due to the lower intake. This was probably due to fill/satiety, which resulted in a smaller IR. Similar trends for IR [14,22] and similar effects of the foliage density on the IR [14] have also been reported in goats.
T
334 335
C
332 333
E
330 331
R
328 329
R
326 327
N C O
324 325
U
322 323
F
t5:14 t5:15
367 368 369 370 371 372 373 374 375 376 377 378 379
5. Conclusion
380
When faced with a variety of foliage types as in the present cafeteria experiments, sheep consume plants containing PSMs such as tannins. However, tannins did not have an influence on the preference, as the sheep seemed to select foliage depending on their potential macronutrient supply. The use of PEG did not modify the preference of sheep in these cafeteria trials. The present results support the hypothesis that feed tannin content is not the main regulator of preference in sheep with browsing experience. Finally, these results encourage the use of tropical fodder trees containing CT as nutraceuticals.
381
Acknowledgments
390
G. Hernández-Orduño received a scholarship from CONACYTMexico to undergo her MSc studies. This work was supported by project CONACYT-SAGARPA-COFUPRO No. 12441. We thank Victor Parra-Tabla for advice and help with the GLIMMIX procedure and Humberto Antonio Peralta-Pino, Adrián Tun and Hilda Lorena Canul-Ku for the technical assistance.
391 392
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397
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Please cite this article as: G. Hernández-Orduño, et al., A tannin-blocking agent does not modify the preference of sheep towards tannincontaining plants, Physiol Behav (2015), http://dx.doi.org/10.1016/j.physbeh.2015.04.006
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