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Relationship between intake of tannin-containing tropical tree forage, PEG supplementation, and salivary haze development in hair sheep and goats
Biochemical Systematics and Ecology 68 (2016) 101e108
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Relationship between intake of tannin-containing tropical tree forage, PEG supplementation, and salivary haze development in hair sheep and goats A.A. Pech-Cervantes, J. Ventura-Cordero, C.M. Capetillo-Leal, J.F.J. Torres-Acosta, C.A. Sandoval-Castro* n, Km. 15.5 Carretera M noma de Yucata Facultad de Medicina Veterinaria y Zootecnia, Universidad Auto erida-Xmatkuil, Apartado Postal n, 97100, Mexico 4-116, Itzimna, M erida, Yucata
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 February 2016 Received in revised form 28 June 2016 Accepted 3 July 2016
The objective of this study was to estimate the relationship between tannin binding salivary protein (TBSP) and condensed tannins (CT) intake in hair sheep and creole goats. Foliage was obtained from trees with different levels of CT content; animals were offered foliage ad libitum, with or without polyethylene glycol (PEG). Saliva haze development (SHD) was evaluated as evidence for TBSP. PEG consumption did not affect dry matter intake (DMI) (P > 0.05). Lignin (R ¼ 0.714, P < 0.001) and Crude Protein (CP) (R ¼ 0.622, P < 0.001) contents had a stronger association with DMI than CT (R ¼ 0.622, P < 0.011) in sheep; no significant association was found in goats. The positive relationship between tannin intake and SHD (P < 0.05) was not confirmed after PEG supplementation in sheep (P > 0.09), but remained significant for goats (P < 0.01), except for those fed Lysiloma latisiliquum (P ¼ 0.07). Foliage lignin or CP contents are better predictors of foliage intake than CT. Sheep and goats fed with tropical tree forages containing different levels of tannins exhibited differences in intake behavior; moreover, individual variations in TBSP expression helps explaining foliage DMI. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Condensed tannins Feed consumption Salivary protein Haze development
1. Introduction Several tropical forage trees are commonly used as a source of macronutrients for ruminants in spite of their secondary metabolite content. Consumption of plant materials containing compounds (e.g., tannins) may have a detrimental effect on the animals such as reduction of food intake and digestibility (Alonso-Díaz et al., 2010). Some animal species evolved defense mechanisms for such compounds; those are salivary proteins commonly named tannin binding salivary proteins (TBSP), which have a high affinity for tannins (Skopec et al., 2004; Shimada, 2006). The role of TBSP is to reduce the interaction between tannins and dietary proteins, improving the availability of the latter for the animal (Salem et al., 2007), and possibly increasing the intake of tannin-rich forages. In some animal species, the presence of tannins in the diet may stimulate TBSP synthesis as an adaptive response (McArthur et al., 1995; Clauss et al., 2005; Shimada et al., 2006). However, in small ruminants (sheep and goats) the evidence is contradictory. In Mediterranean goats, tannin consumption failed to increase TBSP
* Corresponding author. E-mail address: [email protected] (C.A. Sandoval-Castro). http://dx.doi.org/10.1016/j.bse.2016.07.003 0305-1978/© 2016 Elsevier Ltd. All rights reserved.
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production even after long exposure to tannin-rich plants (Hanovice-Ziony et al., 2010). On the other hand, sheep and goats fed for several years with tannin-rich forages, derived from the native vegetation, showed evidence of TBSP expression (Alonso-Diaz et al., 2012). Further studies in Mexico showed that lambs with no prior contact with tannin-rich materials had elevated TBSP (measured as increased haze development) after a short period of exposure to tannin-rich forages (Vargas~ a et al., 2013). On the other hands, goat kids without prior exposure to tannin-rich forages seemed to have develMagan oped TBSP as an innate feature and were unable to show a further increase in haze development after exposure to tannin-rich forages (Ventura-Cordero et al., 2015). The presence of TBSP in small ruminants from the Yucatan Peninsula has also been postulated as these studies showed a lack of increase in the intake of tannin-rich forages after polyethylene glycol (PEG) was included in the diet with the aim to block the negative effects of tannins (either astringency or post-ingestive effects) ~ o et al., 2012, 2015; Revaud et al., 2014). In spite of previous evidence suggesting a relationship between (Hernandez-Ordun ~ a et al., 2013), there is no clear explanation on why tannin-rich forage consumption and TBSP in tropical sheep (Vargas-Magan tannins do not limit tannin-rich forage intake. Moreover, it is important to confirm the effect of PEG supplementation on forage intake and TBSP expression. Therefore, we hypothesized that tannins contained in foliage would stimulate TBSP production and this response would allow sheep and goats to ingest a larger amount of foliage. Thus, the aim of this study was to evaluate the relationship between the intake of tropical forage trees with different tannin levels and the TBSP content in hair sheep and creole goats. 2. Materials and methods 2.1. Location of the study The experiments were carried out in the small ruminant farm at the Faculty of Veterinary Medicine and Animal Science, noma de Yucata n, Me xico. The weather of the area is classified as AW0 (hot, subhumid, and with summer Universidad Auto rainfall). Mean annual temperature and precipitation range from 26 to 27 C and from 940 to 1110 mm, respectively. 2.2. Forage trees Fresh foliage of Piscidia piscipula, Lysiloma latisiliquum, and Brosimum alicastrum leaves was used for the experiment. To minimize variability in forage chemical composition, leaves were harvested every day as a mixture from at least fifteen individual trees with more than ten years of age. The foliage was homogenized before being offered to the experimental animals. The plants for the trial were selected based on the following criteria: (a) the plants are commonly available in the region; (b) the foliage of those plants is commonly used to feed ruminants in the study area; (c) the knowledge about the condensed tannins (CT) content of the foliage. The foliage of P. piscipula and L. latisiliquum is known to have medium and high ~ o et al., 2005; Alonso-Díaz et al., 2008). On the other hand, the foliage of B. aliCT content, respectively (Monforte-Bricen castrum is highly palatable and with very low to null content of CT (Alonso-Diaz et al., 2009). Therefore, the latter was included as control foliage. 2.3. Experimental animals Sheep experiment. Nine Pelibuey hair sheep with an average live weight (LW) of 27 ± 2.5 kg, and an age of eight months were used. Animals had previous experience with tannin-rich forage intake and therefore, tannin consumption (Vargas~ a et al., 2013). Magan Goat experiment. Twelve Creole goats with an average LW of 24 ± 2.5 kg, and an age of eight months were used. Animals had previous experience with tannin-rich forage intake and hence, tannin consumption (Ventura-Cordero et al., 2015). The experimental sheep and goats were raised nematode-free by maintaining them on concrete floor pens and were fed a diet free of gastrointestinal nematode infective larvae. Before the experiments, sheep and goats were trained to consume PEG, by feeding the foliage with and without PEG for five consecutive days to ensure feed acceptance. All the animals were weighted before the beginning of each experimental period. 2.4. Feed intake Animals were fed daily with a grain-based concentrated feed (500 g FB) in the morning (8:00 h). At 13:00 h animals were fed fresh tree foliage ad libitum. Each animal received foliage of one of the tree species during each period with or without PEG according to each treatment. At 17:00 h animals were offered P. purpureum grass ad libitum. Before the respective feeds were offered, orts were collected, weighed, and recorded. Feeds and orts samples were collected and stored at 4 C until further analysis. 2.5. Tannin binding salivary protein (TBSP) in saliva samples Saliva samples were obtained on the fifteenth day of each experimental period. Animals were sampled during the morning before feeding time. A vacuum pump was connected to Corning® PP centrifuge tubes (catalogue #430290, Corning Mexicana
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S.A., Monterrey, Mexico) by means of a flexible plastic tube to extract the saliva sample (at least 60 mL per animal). Corning® tubes were maintained inside a refrigerated cooling box. An aliquot of the saliva sub-sample (30 mL) was employed for the turbidity test (haze development). A second 30 mL aliquot was lyophilized for total protein content measurement (Lowry et al., 1951). 2.6. Turbidity test The 30 mL aliquot were centrifuged at 4000 rpm for 10 min to eliminate solid particles contaminant. The supernatant was recovered for the turbidity test (Horne et al., 2002) using tannic acid (0.1 g/50 mL distilled water) as standard. For the assay, 3.84 mL of distilled water and 0.16 mL of the tannic acid solution were added to each tube. Afterward, 4 mL of saliva was added to each tube and the samples were homogenized using a bench top Vortex (Benchmark BV1000, Benchmark Scientific, Edison, NJ, USA). The saliva solutions were transferred to a cuvette for absorbance measurements at 610 nm, using a spectrometer (Perking Elmer® lambda 25). The readings were made at 0 (baseline) and 90 min after tannic acid addition. Salivary haze development was measured as the change in transmittance (D% SHD) after the saliva was mixed with the tannin standard. A turbidity index (D% SHD) was calculated according to the following formula:
D% SHD ¼ ððt90 t0 Þ=t0 Þ 100 Where:D% SHD represents the percentage change in salivary haze development, t90 the transmittance at 90 min, and t0 the transmittance at baseline. Increased haze development was measured as a reduced transmittance and resulted in a negative value (D% SHD). Haze development was used as evidence of TBSP activity. 2.7. Feed analysis A 200 g sample was obtained for each feedstuff. The samples were oven-dried at 60 C for 48 h, ground, and kept in plastic bags until further analyses using official procedures for dry matter (DM) and ash (A.O.A.C., 1980). Samples were analyzed for total nitrogen using a Leco CN-2000 analyzer (Leco CN series 3740 analyzer, Leco Corporation, Saint Joseph, MI, USA) and crude protein (CP) was calculated as total nitrogen 6.25. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were analyzed using the Ankom200 fiber analyzer (Ankom Technology Corp., Macedon, NY); sodium sulfite was used in the NDF analysis procedure without alpha amylase. Lignin was determined as previously described by Van Soest et al. (1991). Total tannins (TT) and condensed tannins (CT) were also determined using the Folin-Ciocalteu þ PVPP method (Makkar, 2003) and the vanillin assay (catechin equivalent, Price et al., 1978) (Table 1). 2.8. Experimental design and statistical analysis Animals were used in a crossover study with six different treatments: three foliage species (P. piscipula, L. latisiliquum, and B. alicastrum), and two PEG levels (0 and 50 g/kg DM). For sheep, a total of six experimental periods were used allowing nine replicates per treatment. For goats, three experimental periods resulted in nine replicates per treatment. Each experimental period lasted 15 days. Ten days was the time used for foliage adaptation and five days were used for the intake measurements. Additionally, five days were allowed between each experimental period in order to avoid any residual effect on intake. During this interval, animals were fed with concentrate feed and P. purpureum grass only. Feed intake, turbidity (haze development), and saliva protein were analyzed using a mixed procedure which included the fixed effects of Treatment þ Period þ Treatment Period interaction; animal was included as random effect. Table 1 Chemical composition (%DM) of feeds employed during the sheep and goat experiments.
Sheep Concentrate feed P. purpureum L. latisiliquum P. piscipula B. alicastrum Goat Concentrate feed P. purpureum L. latisiliquum P. piscipula B. alicastrum
DM
CP
NDF
ADF
Ash
Lignin
Folin
Folin þ PVPP
Vanillin
89.0 32.1 57.2 57.7 59.0
16.01 3.90 14.08 13.11 16.58
27.10 72.20 50.73 43.85 43.86
16.90 48.90 43.91 32.94 35.58
7.10 6.06 5.82 10.94 9.69
ND 8.75 28.17 14.94 9.16
0 0.32 2.06 0.74 1.39
0 0.32 0.88 0.73 1.46
0 0 6.47 1.40 0
89.0 36.0 59.6 58.5 61.0
15.27 4.66 12.21 11.40 12.45
28.56 73.99 45.05 39.75 38.87
18.25 47.37 35.62 28.75 27.39
6.75 6.27 5.74 12.04 9.58
ND 8.33 23.00 13.47 6.72
0 6.78 12.89 9.95 7.35
0 0 12.25 1.72 1.61
0 0 7.63 1.42 1.14
DM: Dry matter, CP: Crude protein, NDF: neutral detergent fiber, ADF: acid detergent fiber, ND: not determined. Pennisetum purpureum, Lysiloma latisiliquum, Piscidia piscipula, Brosimum alicastrum.
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Correlation analysis was performed between feed intake (g DM/kg LW0.75) and feed chemical composition (CP, FDN, ADF, lignin, and CT). Stepwise and best subsets analyses were also performed with the predictors that showed a significant correlation (Minitab 15). In addition, to assess the influence of tannin intake (g CT/kg LW0.75) on TBSP response (D% SHD), linear regression analyses were performed for each foliage species (GraphPad Prism 5 for Windows).
3. Results 3.1. Feed intake The highest foliage and total DM intake were observed in sheep fed with B. alicastrum (P < 0.05). The lower intakes were observed when animals were fed with L. latisiliquum and P. piscipula. PEG addition did not improve foliage intake (P < 0.05) (Table 2). Highest CT and lignin intake were observed with L. latisiliquum while the highest CP and NDF intake were observed with B. alicastrum (P < 0.05) (Table 2). For what concerns goats, we found differences in foliage consumption depending on plants (P < 0.05) (Table 2), and the lowest intake was observed when animals consumed tannin-rich plants. PEG supplementation did not modify foliage intake and total intake was similar in both PEG levels. For sheep the Pearson correlation coefficients between chemical composition and intake are presented in Table 3. Significant correlations were found between foliage intake and all chemical fractions (CP, NDF, ADF, lignin, and CT) (P < 0.05). Step-wise procedure and best subsets analyses indicated that lignin, followed by CP, was the best predictor for DMI either when animals received PEG or not. The relationships were described by the following equations:
Table 2 Feed intake (g DM/d) and intake of feed fractions (g/d) by sheep and goats fed with tropical forage trees with and without PEG supplementation (50 g/kg DM). Offered foliage
Sheep Without PEG Foliage Grass Concentrate Total DMI Total CT Total Lignin Total NDF Total CP With PEG Foliage Grass Concentrate Total DMI Total CT Total Lignin Total NDF Total CP Goats Without PEG Foliage Grass Concentrate Total DMI Total CT Lignin NDF CP With PEG Foliage Grass Concentrate Total DMI CT Lignin NDF CP
S.E.D
L. latisiliquum
P. piscipula
B. alicastrum
341.64a 216.97a 449.30 1007.91a 22.1a 115.21a 451.73b 128.5b
387.65a 198.75a 449.30 1035.71a 5.41b 76.39b 435.30b 130.51b
776.94b 166.95a 449.30 1393.2b 0c 85.45b 583.07a 207.28a
21.18 10.06 0 25.65 0.71 3.49 12.2 3.16
338.15a 202.33a 449.30 989.79a 21.88a 112.95a 439.39b 127.45b
354.32a 206a 449.30 1009.63a 4.94b 71.94b 425.92 b 126.42b
779.25b 185.92 a 449.30 1414.48b 0c 87.32b 597.78a 208.40a
21.18 10.06 0 25.65 0.71 3.49 12.2 3.16
434.50a 144.08a 445 1023.5a 33.14a 111.90a 429.4a 127.20b
408a 138.43a 445 991.5a 5.80b 66.52b 391.77a 120.47b
617.83b 84.55b 445 1147.33a 7.02b 48.54b 429.79a 149.17a
32.77 4.29 0 34.42 1.62 5.53 9.50 4.02
449.89a 155.64a 445 1050.66a 34.33a 116.42a 444.94a 129.59a
362.9a 145.2a 445 953.16b 5.16b 60.99b 378.79a 115.60b
593.94b 115.34a 445 1154.33a 6.75b 49.50b 443.28a 147.50a
32.77 4.29 0 34.42 1.62 5.53 9.50 4.02
Different letters in the same row indicate significant differences (P < 0.05).
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Table 3 Pearson correlation coefficient between DMI and chemical composition of tropical forage trees in sheep and goats with and without PEG supplementation (50 g/kg DM). Sheep
Lignin Crude Protein Condensed Tannins Neutral Detergent Fiber Acid Detergent Fiber
R P R P R P R P R P
Goat
Without PEG
With PEG
Without PEG
With PEG
DMI
DMI
DMI
DMI
0.714 0.001 0.622 0.001 0.484 0.011 0.424 0.028 0.299 0.130
0.688 0.002 0.718 0.001 0.429 0.025 0.399 0.039 0.306 0.121
0.566 0.140 0.050 0.844 0.061 0.809 0.262 0.293 0.331 0.179
0.378 0.122 0.165 0.512 0.041 0.871 0.044 0.861 0.130 0.608
R: Regression coefficient. P: Probability value.
Without PEG : DMI g=kgLW0:75 ¼ 2 14:7 lignin ð%Þ þ 51:8 CP ð%Þ: R2adjusted ¼ 58:7% With PEG : DMI g=kgLW0:75 ¼ 388 13 ligninð%Þ þ 75:6 CPð%Þ: R2adjusted ¼ 64:9% For goats, the correlations were not significant (P > 0.05) and best subset analyses were not performed. The inclusion of PEG in the diet did not modify the relationship and no significant correlation was found (P > 0.05) (Table 3). 3.2. Turbidity index A relationship was found between tannin intake (g CT/kg LW0.75) and D% SHD in sheep fed L. latisiliquum (P < 0.05, Table 4). The same trend was observed for sheep fed P. piscipula, although this was not significant (P ¼ 0.17). No significant relationship was found between D% SHD and tannin intake in sheep fed B. alicastrum. The relationship between tannin intake and D% SHD was not observed in sheep fed L. latisiliquum and supplemented with PEG (P > 0.05). The trend observed with P. piscipula also disappeared when PEG was added to the sheep diet (P > 0.05) (Table 4). In goats, similar effects were observed: a negative correlation was found between tannin consumption (g CT/kg LW0.75) and turbidity in goats fed L. latisiliquum (P < 0.05, Table 4) and supplemented with both PEG concentrations; no correlation was found for P. piscipula and B. alicastrum. 3.3. Salivary protein The protein content of saliva in sheep and goats is presented in Table 5. The content (mg/g dry saliva) was similar both for sheep and goats; we failed to see the effect of foliage ingested or PEG supplementation (P < 0.05). 4. Discussion 4.1. Feed intake During feed consumption, animals can express behavioral changes related to sensorial experiences, which can be triggered ~ o et al., 2012), its chemical by the physical characteristics of the feed (i.e., shape and density of the foliage; Hernandez-Ordun characteristics (i.e., compounds such as tannins and lignin; Alonso-Díaz et al., 2008, Alonso-Diaz et al., 2009; Provenza et al., 2009) and nutritional value (i.e., digestibility; Sandoval-Castro et al., 2005). Results from the present study revealed that the CT content of different foliage (Table 1) did not cause a reduction in foliage intake (Table 2). Furthermore, PEG supplementation did not increase tannin-rich foliage intake (Table 2). Similar results have been consistently reported both with sheep ~o and goats in other countries (Decandia et al., 2008; Fagundes et al., 2014) as well as in Yucatan, Mexico (Hernandez-Ordun et al., 2012, 2015; Revaud et al., 2014). The fiber and lignin content of the foliage (Table 1) appeared to be the main constraint to DMI rather than the CT content (Table 3). It is well known that fibers contained in feed can be a limiting factor for DMI in ruminants (Leng, 1990; Van Soest et al., 1991). In agreement with previous work done on tropical hair sheep (Alonso-Diaz et al., 2009), it has been shown that lignin and a high CP content can also be negatively associated with DMI. The latter is similar to our results, where lignin (fiber) became the first limiting factor for DMI in both foliage and grass, where lignin content is particularly high. On the other hand, the CP content of foliage was positively associated with DMI. It is possible that animals try to compensate the low CP content of the basal diet (grass) with the high CP content of the foliage. High protein
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Table 4 Relationship between tannin intake (g tannin/kg LW0.75) and saliva turbidity index (% turbidity change) of sheep and goats fed different tropical forage trees with and without PEG supplementation (50 g/kg DM). Plant SHEEP Without PEG Lysiloma latisiliquum Piscidia piscipula Brosimum alicastrum With PEG Lysiloma latisiliquum Piscidia piscipula Brosimum alicastrum GOATS Without PEG Lysiloma latisiliquum Piscidia piscipula Brosimum alicastrum With PEG Lysiloma latisiliquum Piscidia piscipula Brosimum alicastrum
a
a
Equation
R2
P
TI ¼ 4.635 (±1.166) CTI þ 16.14 (±5.613) TI ¼ 15.62 (±3.103) CTI þ 10.56 (±3.342) e
0.693 0.7836 e
0.0054 0.0015 e
TI ¼ 8.611 (±4.44) CTI þ 35.38 (±21.43) TI ¼ 12.03 (±11.18) CTI e 17.77 (±11.47) e
0.3486 0.142 e
0.0941 0.3175 e
TI ¼ 7.140 (±1.005) CTI þ 12.31 (±2.56) TI ¼ 46.02 (±7.92) CTI þ 14.94 (±3.73) TI ¼ 64.27 (±14.63) CTI þ 31.03 (±7.704)
0.9266 0.894 0.8283
0.0021 0.0044 0.0118
TI ¼ 10.76 (±4.433) CTI þ 21.05 (±11.80) TI ¼ 25.06 (±4.958) CTI þ 3.66 (±2.066) TI ¼ 27.81 (±4.391) CTI þ 8.25 (±2.206)
0.5955 0.8646 0.9093
0.0722 0.0072 0.0032
Turbidity index (TI) ¼ slope value condensed tannin intake (CTI) (g/kg LW0.75) þ intercept value. a For sheep fed Brosimum alicastrum foliage, CT was below detection level and therefore regression analysis could not be performed.
Table 5 Salivary protein content of sheep and goats (mg/g dry saliva) in the saliva of sheep fed with tropical forage trees with and without PEG supplementation. Without PEG
With PEG
Salivary protein
L. Latisiliquum
P. piscipula
B. alicastrum
L. Latisiliquum
P. piscipula
B. alicastrum
SED
Goat Sheep
2.77a 2.70a
3.01a 2.64a
2.64a 2.73a
3.60a 3.06a
3.96a 2.61a
3.94a 2.88a
0.50 0.42
Means with similar letters in the same row indicate no significant differences (P < 0.05).
content of foliage can be a limiting factor for DMI when the animal’s CP requirements are surpassed and disposing of excess protein becomes energetically costly.
4.2. Tannin binding salivary protein The working hypothesis was that supplementing a foliage with high tannins content would induce a higher TBSP response (D% SHD), indicative of a physiological response (higher capacity to block tannins), allowing sheep or goats to increase their DMI. However, the protein content of the lyophilized saliva was similar in all treatments, initially suggesting a lack of response to the tannin stimuli measured as protein concentration. Thus, when the DMI corresponding to each plant was analyzed, no correlation was found between foliage tannin content and D% SHD and between D% SHD and DMI. As explained above, it appeared that lignin (fiber) is the main component limiting DMI (Table 3). Nevertheless, we observed a pattern in animals within the same experimental group (L. latisiliquum- and P. piscipula-supplemented groups), which were subsequently analyzed. Indeed, when the relationship between tannin intake and D% SHD was analyzed in sheep or goats fed the same foliage, we observed that animals producing a higher TBSP response had lower feed intake. This was observed in goats fed all three foliage and in sheep fed L. latisiliquum and P. piscipula foliage (Table 4, P < 0.01). The latter suggested that animals with a high TBSP content (measured as D% SHD) might detect the astringency derived from CT better. This effect might be similar to that found in humans where a dry puckering sensation is usually observed during the consumption of unripe fruits, which cause the food to be unpalatable (Gibbins and Carpenter, 2013). The use of PEG supplementation seemed to reduce the astringency caused by tannins in sheep, as no relationship was found when PEG was given as a supplement (P > 0.05, Table 4). On the other hand, the relationship remained for goats fed P. piscipula and B. alicastrum, and a trend (P ¼ 0.07) was observed in goats fed L. latisiliquum (Table 4). These results might explain the lower correlation between CT and foliage intake in contrast to the higher correlation found between lignin or CP and foliage intake. Therefore, the results allow us to hypothesize that the variation in DM intake between animals results from differences in their ability to detect CT (oral astringency leading to increased TBSP) and it is relatively larger than the variation caused by fiber intake (causing increased rumen fill and hence satiety). Hence, a higher TBSP response causes a reduction in DM intake while a lower TBSP response allows higher intake of feeds containing CT, an effect that, since it is averaged across animals, masks the cause-effect relationship between CT content and foliage DMI. On the contrary, the higher rumen filling effect caused by the response to dietary fiber intake might have lower variation between animals and becomes a more consistent constraining factor against DMI. The protein content of
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lyophilized saliva failed to detect this effect as it is not a measurement of its activity (capacity to bind tannins) and the protein concentration in fresh saliva was not accounted for (protein content/mL of fresh saliva). n (Gonza lez-Pech et al., Although sheep and goats are able to graze and browse in the tropical deciduous forests of Yucata 2014), goats are considered generalized browsers in contrast to sheep, which are defined as specialized grazers. Therefore, lez-Pech et al., 2015). This difference in goats would be more suited to ingest CT from the foliage of browsed plants (Gonza feeding behavior between sheep and goats for what concerns foliage containing high quantities of CT could be the reason behind the different TBSP concentration present in sheep and goats. While sheep may increase TBSP production in response ~ a et al., 2013), recent studies suggested that goats have high innate TBSP levels, which imply to tannin intake (Vargas-Magan that it is not possible to increase the salivary response further even with an elevated CT consumption (Ventura-Cordero et al., 2015). As a net outcome, PEG supplementation can partially inhibit the astringency stimuli in sheep, resulting in the modification of tannin rich-foliage, while PEG supplementation does not have any effect on the astringency signal in goats. This might help to explain the differences in the response of PEG supplementation between sheep and goats obtained in the present study. It is important to note that the sheep used in the present study had previous experience with tannincontaining foliage and they exhibited an increased TBSP response after several weeks of L. latisiliquum foliage feeding ~ a et al., 2013). There is evidence of some degree of specificity in the affinity between TBSP and tannins usually (Vargas-Magan contained in the sheep diet (Alonso-Diaz et al., 2012) and other mammals such as Alces alces, Castor canadiensis, and Odocoileus hemionus (Hagerman and Robbins, 1993). Understanding astringency development in the mouth of ruminants could help finding mechanisms to avoid the negative effects of astringency on foliage intake from tannin-rich plants (in a similar fashion as with PEG supplementation). However, it remains to be defined whether goats benefit from a large TBSP response, for example increased feed utilization (i.e., increased organic matter digestibility, volatile fatty acid production, etc.), which might arise from TBSP. It also remains to be ~o defined whether such proteins act as tannin-blocking agents in a similar manner as PEG supplementation (Monforte-Bricen et al., 2005; Salem et al., 2007). If a high TBSP content is associated with a higher foliage intake, this might result in a larger total nutrient intake, which could overcome the anti-nutritional effects of tannins possibly leading to partial nutraceutical effects, such as the anthelmintic effect observed in some tannin-rich foliage (Sandoval-Castro et al., 2012). This area will need to be further addressed in future studies. Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was financially supported by CONACYT-CB/106146. Andres Pech-Cervantes acknowledge the Consejo Nacional xico) for a scholarship to pursue his MSc degree. de Ciencia y Tecnología (CONACYT-Me References Alonso-Díaz, M.A., Torres-Acosta, J.F.J., Sandoval-Castro, C.A., Hoste, H., Aguilar-Caballero, A.J., Capetillo-Leal, C.M., 2008. Is goats’ preference of forage trees affected by their tannin or fiber content when offered in cafeteria experiments? Animal Feed Sci. Technol. 141, 36e48. Alonso-Diaz, M.A., Torres-Acosta, J.F.J., Sandoval-Castro, C.A., Hoste, H., Aguilar-Caballero, A.J., Capetillo-Leal, C.M., 2009. Sheep prefence for different tanniferous tree fodders and its relationship with in vitro gas production and digestibility. Animal Feed Sci. Technol. 15, 175e185. Alonso-Díaz, M.A., Torres-Acosta, J.F.J., Sandoval-Castro, C.A., Hoste, H., 2010. Tannins in tropical tree fodders fed to small ruminants: a friendly foe? Small Ruminant Res. 89, 64e173. Alonso-Diaz, M.A., Torres-Acosta, J.F.J., Sandoval-Castro, C.A., Capetillo-Leal, C.M., 2012. Amino acid profile of the protein from whole saliva of goats and sheep and its interaction with tannic acid and tannins extracted from the fodder of tropical plants. Small Ruminant Res. 103, 69e74. AOAC, 1980. Official Methods of Analysis, thirteenth ed. Association of Official analytical Chemists, Washington, DC, USA. Clauss, M., Gehrke, J., Hatt, J.M., Dierenfeld, E.S., Flach, E.J., Hermes, R., Castell, J., Streich, W.J., Fickel, J., 2005. Tannin-binding salivary proteins in three captive rhinoceros species. Comp. Biochem. Physiol. 140, 67e72. Decandia, M., Cabiddu, A., Sitzia, M., Molle, G., 2008. Polyethylene glycol influences feeding behaviour of dairy goats browsing on bushland with different herbage cover. Livest. Sci. 116, 183e190. Fagundes, G.M., Modesto, E.C., Fonseca, C.E.M., Lima, H.R.P., Muir, J.P., 2014. Intake, digestibility and milk yield in goats fed Flemingia macrophylla with or without polyethylene glycol. Small Ruminant Res. 116, 88e93. Gibbins, H.L., Carpenter, G.H., 2013. Alternative mechanism of astringency- what is role of saliva? J. Texture Stud. 44, 364e375. lez-Pech, P.G., Torres-Acosta, J.F.J., Sandoval-Castro, C.A., 2014. Adapting a bite coding grid for small ruminants browsing a deciduous tropical forest. Gonza Trop. Subtropical Agroecosyst. 17, 63e70. lez-Pech, P.G., Torres-Acosta, J.F.J., Sandoval-Castro, C.A., Tun-Garrido, J., 2015. Feeding behavior of sheep and goats in a deciduous tropical forest Gonza during the dry season: the same menu consumed differently. Small Ruminant Res. 133, 128e134. Hagerman, E., Robbins, C.T., 1993. Specificity of tannin-binding salivary proteins relative to diet selection by mammals. Can. J. Zool. 628e633. Hanovice-Ziony, M., Gollop, N., Landau, S.Y., Ungar, D.E., Muklada, H., Glasser, T.A., Perevolotosky, A., Walker, J.W., 2010. No major role for tannin binding proteins as a defense against dietary tannins in Mediterranean goats. J. Chem. Ecol. 7, 736e743. ~ o, G., Torres-Acosta, J.F.J., Sandoval-Castro, C.A., Aguilar-Caballero, A.J., Capetillo-Leal, C.M., Alonso-Diaz, M., 2012. In cafeteria trials with Hernandez-Ordun tannin rich plants, tannins do not modify foliage preference of goats with browsing experience. Ethol. Ecol. Evol. 24, 332e343. ~ o, G., Torres-Acosta, J.F.J., Sandoval-Castro, C.A., Aguilar-Caballero, A.J., Capetillo-Leal, C.M., Alonso-Diaz, M., 2015. A tannin-blocking agent Hernandez-Ordun does not modify the preference of sheep towards tannin-containing plants. Physiology Behav. 145, 106e111. Horne, J., Hayes, J., Lawless, H.T., 2002. Turbidity as a measure of salivary protein reactions with astringent substances. Chem. Senses 27, 653e659. Leng, R.A., 1990. Factors affecting the utilization of “poor-quality” forages by ruminants particularly under tropical conditions. Nutr. Res. Rev. 3, 277e303. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265e275.
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Makkar, H.P.S., 2003. Effects and fate of tannins in ruminant animals, adaptation to tannins, and strategies to overcome detrimental effects of feeding tannin-rich feeds. Small Ruminant Res. 49, 241e256. McArthur, C., Sanson, G.D., Beal, A.M., 1995. Salivary proline-rich proteins in mammals: roles in oral homeostasis and counteracting dietary tannin. J. Chem. Ecol. 6, 663e691. ~ o, G.E., Sandoval-Castro, C.A., Ramírez-Avile s, L., Capetillo, L.C.M., 2005. Defaunating capacity of tropical fodder trees: effects of polyMonforte-Bricen ethylene glycol and its relationship to in vitro gas production. Animal Feed Sci. Technol. 123, 313e327. Price, M.L., Van Scoyoc, S., Butler, L.G., 1978. A critical evaluation of the vanillin reaction as an assay for tannin in sorghum grain. J. Agric. Food Chem. 26, 1214e1218. Provenza, F.D., Villalba, J.J., Wiedmeier, R.W., Lyman, T., Owens, J., Lisonbee, L., Clemensen, A., Welch, K., Gardner, D., Lee, S., 2009. Value of plant diversity for diet mixing and sequencing in herbivores. J. Range Manag. 31, 45e49. Revaud, M.H.R., Gonz alez-Pech, P.G., Ventura-Cordero, J., Torres-Acosta, J.F.J., Sandoval-Castro, C.A., Hoste, H., 2014. Polyethylenglycol sprayed on the foliage or orally dosage have no influence on the consumption of tannin rich plants in adult goats with browsing experience. Trop. Subtropical Agroecosyst. 17, 354e355. Salem, A.Z.M., Robinson, P.H., El-Adawy, M.M., Hassan, A.A., 2007. In vitro fermentation and microbial protein synthesis of some browse tree leaves with or without addition of polyethylene glycol. Animal Feed Sci. Technol. 138, 318e330. Sandoval-Castro, C.A., Lizarraga-Sanchez, H.L., Solorio-Sanchez, F.J., 2005. Assessment of tree fodder preference by cattle using chemical composition, in vitro gas production and in situ degradability. Animal Feed Sci. Technol. 123, 277e289. rez, J.I., 2012. Using plant bioactive materials to control gastrointestinal tract Sandoval-Castro, C.A., Torres-Acosta, J.F.J., Hoste, H., Salem, A.Z.M., Chan-Pe helminthes in livestock. Animal Feed Sci. Technol. 176, 192e201. Shimada, T., 2006. Salivary proteins as a defense against dietary tannins. J. Chem. Ecol. 32, 1149e1163. Shimada, T., Saitoh, T., Sasaki, E., Nishitani, Y., Osawa, R., 2006. Role of tannin-binding salivary proteins and Tannase-producing bacteria in the acclimation of the japanese wood mouse to acorn tannins. J. Chem. Ecol. 32, 1165e1180. Skopec, M.M., Hagerman, A.E., Karasov, W.H., 2004. Do salivary proline-rich proteins counteract dietary hydrolyzable tannin in laboratory rats? J. Chem. Ecol. 30, 1679e1692. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 10, 3583e3597. ~ a, J., Aguilar-Caballero, A.J., Torres-Acosta, J.F.J., Sandoval-Castro, C.A., Hoste, H., Capetillo-Leal, C.M., 2013. Tropical tannin rich fodder intake Vargas-Magan modifies saliva binding capacity in sheep. Animal 7, 1921e1924. Ventura-Cordero, J., Sandoval-Castro, C.A., Torres-Acosta, J.F.J., Capetillo-Leal, C.M., 2015. Do Goats have a salivary constitutive response to tannins? J. Appl. Animal Res. http://dx.doi.org/10.1080/09712119.2015.1102728 (in press).
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Relationship between intake of tannin-containing tropical tree forage, PEG supplementation, and salivary haze development in hair sheep and goats A.A. Pech-Cervantes, J. Ventura-Cordero, C.M. Capetillo-Leal, J.F.J. Torres-Acosta, C.A. Sandoval-Castro* n, Km. 15.5 Carretera M noma de Yucata Facultad de Medicina Veterinaria y Zootecnia, Universidad Auto erida-Xmatkuil, Apartado Postal n, 97100, Mexico 4-116, Itzimna, M erida, Yucata
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 February 2016 Received in revised form 28 June 2016 Accepted 3 July 2016
The objective of this study was to estimate the relationship between tannin binding salivary protein (TBSP) and condensed tannins (CT) intake in hair sheep and creole goats. Foliage was obtained from trees with different levels of CT content; animals were offered foliage ad libitum, with or without polyethylene glycol (PEG). Saliva haze development (SHD) was evaluated as evidence for TBSP. PEG consumption did not affect dry matter intake (DMI) (P > 0.05). Lignin (R ¼ 0.714, P < 0.001) and Crude Protein (CP) (R ¼ 0.622, P < 0.001) contents had a stronger association with DMI than CT (R ¼ 0.622, P < 0.011) in sheep; no significant association was found in goats. The positive relationship between tannin intake and SHD (P < 0.05) was not confirmed after PEG supplementation in sheep (P > 0.09), but remained significant for goats (P < 0.01), except for those fed Lysiloma latisiliquum (P ¼ 0.07). Foliage lignin or CP contents are better predictors of foliage intake than CT. Sheep and goats fed with tropical tree forages containing different levels of tannins exhibited differences in intake behavior; moreover, individual variations in TBSP expression helps explaining foliage DMI. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Condensed tannins Feed consumption Salivary protein Haze development
1. Introduction Several tropical forage trees are commonly used as a source of macronutrients for ruminants in spite of their secondary metabolite content. Consumption of plant materials containing compounds (e.g., tannins) may have a detrimental effect on the animals such as reduction of food intake and digestibility (Alonso-Díaz et al., 2010). Some animal species evolved defense mechanisms for such compounds; those are salivary proteins commonly named tannin binding salivary proteins (TBSP), which have a high affinity for tannins (Skopec et al., 2004; Shimada, 2006). The role of TBSP is to reduce the interaction between tannins and dietary proteins, improving the availability of the latter for the animal (Salem et al., 2007), and possibly increasing the intake of tannin-rich forages. In some animal species, the presence of tannins in the diet may stimulate TBSP synthesis as an adaptive response (McArthur et al., 1995; Clauss et al., 2005; Shimada et al., 2006). However, in small ruminants (sheep and goats) the evidence is contradictory. In Mediterranean goats, tannin consumption failed to increase TBSP
* Corresponding author. E-mail address: [email protected] (C.A. Sandoval-Castro). http://dx.doi.org/10.1016/j.bse.2016.07.003 0305-1978/© 2016 Elsevier Ltd. All rights reserved.
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production even after long exposure to tannin-rich plants (Hanovice-Ziony et al., 2010). On the other hand, sheep and goats fed for several years with tannin-rich forages, derived from the native vegetation, showed evidence of TBSP expression (Alonso-Diaz et al., 2012). Further studies in Mexico showed that lambs with no prior contact with tannin-rich materials had elevated TBSP (measured as increased haze development) after a short period of exposure to tannin-rich forages (Vargas~ a et al., 2013). On the other hands, goat kids without prior exposure to tannin-rich forages seemed to have develMagan oped TBSP as an innate feature and were unable to show a further increase in haze development after exposure to tannin-rich forages (Ventura-Cordero et al., 2015). The presence of TBSP in small ruminants from the Yucatan Peninsula has also been postulated as these studies showed a lack of increase in the intake of tannin-rich forages after polyethylene glycol (PEG) was included in the diet with the aim to block the negative effects of tannins (either astringency or post-ingestive effects) ~ o et al., 2012, 2015; Revaud et al., 2014). In spite of previous evidence suggesting a relationship between (Hernandez-Ordun ~ a et al., 2013), there is no clear explanation on why tannin-rich forage consumption and TBSP in tropical sheep (Vargas-Magan tannins do not limit tannin-rich forage intake. Moreover, it is important to confirm the effect of PEG supplementation on forage intake and TBSP expression. Therefore, we hypothesized that tannins contained in foliage would stimulate TBSP production and this response would allow sheep and goats to ingest a larger amount of foliage. Thus, the aim of this study was to evaluate the relationship between the intake of tropical forage trees with different tannin levels and the TBSP content in hair sheep and creole goats. 2. Materials and methods 2.1. Location of the study The experiments were carried out in the small ruminant farm at the Faculty of Veterinary Medicine and Animal Science, noma de Yucata n, Me xico. The weather of the area is classified as AW0 (hot, subhumid, and with summer Universidad Auto rainfall). Mean annual temperature and precipitation range from 26 to 27 C and from 940 to 1110 mm, respectively. 2.2. Forage trees Fresh foliage of Piscidia piscipula, Lysiloma latisiliquum, and Brosimum alicastrum leaves was used for the experiment. To minimize variability in forage chemical composition, leaves were harvested every day as a mixture from at least fifteen individual trees with more than ten years of age. The foliage was homogenized before being offered to the experimental animals. The plants for the trial were selected based on the following criteria: (a) the plants are commonly available in the region; (b) the foliage of those plants is commonly used to feed ruminants in the study area; (c) the knowledge about the condensed tannins (CT) content of the foliage. The foliage of P. piscipula and L. latisiliquum is known to have medium and high ~ o et al., 2005; Alonso-Díaz et al., 2008). On the other hand, the foliage of B. aliCT content, respectively (Monforte-Bricen castrum is highly palatable and with very low to null content of CT (Alonso-Diaz et al., 2009). Therefore, the latter was included as control foliage. 2.3. Experimental animals Sheep experiment. Nine Pelibuey hair sheep with an average live weight (LW) of 27 ± 2.5 kg, and an age of eight months were used. Animals had previous experience with tannin-rich forage intake and therefore, tannin consumption (Vargas~ a et al., 2013). Magan Goat experiment. Twelve Creole goats with an average LW of 24 ± 2.5 kg, and an age of eight months were used. Animals had previous experience with tannin-rich forage intake and hence, tannin consumption (Ventura-Cordero et al., 2015). The experimental sheep and goats were raised nematode-free by maintaining them on concrete floor pens and were fed a diet free of gastrointestinal nematode infective larvae. Before the experiments, sheep and goats were trained to consume PEG, by feeding the foliage with and without PEG for five consecutive days to ensure feed acceptance. All the animals were weighted before the beginning of each experimental period. 2.4. Feed intake Animals were fed daily with a grain-based concentrated feed (500 g FB) in the morning (8:00 h). At 13:00 h animals were fed fresh tree foliage ad libitum. Each animal received foliage of one of the tree species during each period with or without PEG according to each treatment. At 17:00 h animals were offered P. purpureum grass ad libitum. Before the respective feeds were offered, orts were collected, weighed, and recorded. Feeds and orts samples were collected and stored at 4 C until further analysis. 2.5. Tannin binding salivary protein (TBSP) in saliva samples Saliva samples were obtained on the fifteenth day of each experimental period. Animals were sampled during the morning before feeding time. A vacuum pump was connected to Corning® PP centrifuge tubes (catalogue #430290, Corning Mexicana
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S.A., Monterrey, Mexico) by means of a flexible plastic tube to extract the saliva sample (at least 60 mL per animal). Corning® tubes were maintained inside a refrigerated cooling box. An aliquot of the saliva sub-sample (30 mL) was employed for the turbidity test (haze development). A second 30 mL aliquot was lyophilized for total protein content measurement (Lowry et al., 1951). 2.6. Turbidity test The 30 mL aliquot were centrifuged at 4000 rpm for 10 min to eliminate solid particles contaminant. The supernatant was recovered for the turbidity test (Horne et al., 2002) using tannic acid (0.1 g/50 mL distilled water) as standard. For the assay, 3.84 mL of distilled water and 0.16 mL of the tannic acid solution were added to each tube. Afterward, 4 mL of saliva was added to each tube and the samples were homogenized using a bench top Vortex (Benchmark BV1000, Benchmark Scientific, Edison, NJ, USA). The saliva solutions were transferred to a cuvette for absorbance measurements at 610 nm, using a spectrometer (Perking Elmer® lambda 25). The readings were made at 0 (baseline) and 90 min after tannic acid addition. Salivary haze development was measured as the change in transmittance (D% SHD) after the saliva was mixed with the tannin standard. A turbidity index (D% SHD) was calculated according to the following formula:
D% SHD ¼ ððt90 t0 Þ=t0 Þ 100 Where:D% SHD represents the percentage change in salivary haze development, t90 the transmittance at 90 min, and t0 the transmittance at baseline. Increased haze development was measured as a reduced transmittance and resulted in a negative value (D% SHD). Haze development was used as evidence of TBSP activity. 2.7. Feed analysis A 200 g sample was obtained for each feedstuff. The samples were oven-dried at 60 C for 48 h, ground, and kept in plastic bags until further analyses using official procedures for dry matter (DM) and ash (A.O.A.C., 1980). Samples were analyzed for total nitrogen using a Leco CN-2000 analyzer (Leco CN series 3740 analyzer, Leco Corporation, Saint Joseph, MI, USA) and crude protein (CP) was calculated as total nitrogen 6.25. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were analyzed using the Ankom200 fiber analyzer (Ankom Technology Corp., Macedon, NY); sodium sulfite was used in the NDF analysis procedure without alpha amylase. Lignin was determined as previously described by Van Soest et al. (1991). Total tannins (TT) and condensed tannins (CT) were also determined using the Folin-Ciocalteu þ PVPP method (Makkar, 2003) and the vanillin assay (catechin equivalent, Price et al., 1978) (Table 1). 2.8. Experimental design and statistical analysis Animals were used in a crossover study with six different treatments: three foliage species (P. piscipula, L. latisiliquum, and B. alicastrum), and two PEG levels (0 and 50 g/kg DM). For sheep, a total of six experimental periods were used allowing nine replicates per treatment. For goats, three experimental periods resulted in nine replicates per treatment. Each experimental period lasted 15 days. Ten days was the time used for foliage adaptation and five days were used for the intake measurements. Additionally, five days were allowed between each experimental period in order to avoid any residual effect on intake. During this interval, animals were fed with concentrate feed and P. purpureum grass only. Feed intake, turbidity (haze development), and saliva protein were analyzed using a mixed procedure which included the fixed effects of Treatment þ Period þ Treatment Period interaction; animal was included as random effect. Table 1 Chemical composition (%DM) of feeds employed during the sheep and goat experiments.
Sheep Concentrate feed P. purpureum L. latisiliquum P. piscipula B. alicastrum Goat Concentrate feed P. purpureum L. latisiliquum P. piscipula B. alicastrum
DM
CP
NDF
ADF
Ash
Lignin
Folin
Folin þ PVPP
Vanillin
89.0 32.1 57.2 57.7 59.0
16.01 3.90 14.08 13.11 16.58
27.10 72.20 50.73 43.85 43.86
16.90 48.90 43.91 32.94 35.58
7.10 6.06 5.82 10.94 9.69
ND 8.75 28.17 14.94 9.16
0 0.32 2.06 0.74 1.39
0 0.32 0.88 0.73 1.46
0 0 6.47 1.40 0
89.0 36.0 59.6 58.5 61.0
15.27 4.66 12.21 11.40 12.45
28.56 73.99 45.05 39.75 38.87
18.25 47.37 35.62 28.75 27.39
6.75 6.27 5.74 12.04 9.58
ND 8.33 23.00 13.47 6.72
0 6.78 12.89 9.95 7.35
0 0 12.25 1.72 1.61
0 0 7.63 1.42 1.14
DM: Dry matter, CP: Crude protein, NDF: neutral detergent fiber, ADF: acid detergent fiber, ND: not determined. Pennisetum purpureum, Lysiloma latisiliquum, Piscidia piscipula, Brosimum alicastrum.
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Correlation analysis was performed between feed intake (g DM/kg LW0.75) and feed chemical composition (CP, FDN, ADF, lignin, and CT). Stepwise and best subsets analyses were also performed with the predictors that showed a significant correlation (Minitab 15). In addition, to assess the influence of tannin intake (g CT/kg LW0.75) on TBSP response (D% SHD), linear regression analyses were performed for each foliage species (GraphPad Prism 5 for Windows).
3. Results 3.1. Feed intake The highest foliage and total DM intake were observed in sheep fed with B. alicastrum (P < 0.05). The lower intakes were observed when animals were fed with L. latisiliquum and P. piscipula. PEG addition did not improve foliage intake (P < 0.05) (Table 2). Highest CT and lignin intake were observed with L. latisiliquum while the highest CP and NDF intake were observed with B. alicastrum (P < 0.05) (Table 2). For what concerns goats, we found differences in foliage consumption depending on plants (P < 0.05) (Table 2), and the lowest intake was observed when animals consumed tannin-rich plants. PEG supplementation did not modify foliage intake and total intake was similar in both PEG levels. For sheep the Pearson correlation coefficients between chemical composition and intake are presented in Table 3. Significant correlations were found between foliage intake and all chemical fractions (CP, NDF, ADF, lignin, and CT) (P < 0.05). Step-wise procedure and best subsets analyses indicated that lignin, followed by CP, was the best predictor for DMI either when animals received PEG or not. The relationships were described by the following equations:
Table 2 Feed intake (g DM/d) and intake of feed fractions (g/d) by sheep and goats fed with tropical forage trees with and without PEG supplementation (50 g/kg DM). Offered foliage
Sheep Without PEG Foliage Grass Concentrate Total DMI Total CT Total Lignin Total NDF Total CP With PEG Foliage Grass Concentrate Total DMI Total CT Total Lignin Total NDF Total CP Goats Without PEG Foliage Grass Concentrate Total DMI Total CT Lignin NDF CP With PEG Foliage Grass Concentrate Total DMI CT Lignin NDF CP
S.E.D
L. latisiliquum
P. piscipula
B. alicastrum
341.64a 216.97a 449.30 1007.91a 22.1a 115.21a 451.73b 128.5b
387.65a 198.75a 449.30 1035.71a 5.41b 76.39b 435.30b 130.51b
776.94b 166.95a 449.30 1393.2b 0c 85.45b 583.07a 207.28a
21.18 10.06 0 25.65 0.71 3.49 12.2 3.16
338.15a 202.33a 449.30 989.79a 21.88a 112.95a 439.39b 127.45b
354.32a 206a 449.30 1009.63a 4.94b 71.94b 425.92 b 126.42b
779.25b 185.92 a 449.30 1414.48b 0c 87.32b 597.78a 208.40a
21.18 10.06 0 25.65 0.71 3.49 12.2 3.16
434.50a 144.08a 445 1023.5a 33.14a 111.90a 429.4a 127.20b
408a 138.43a 445 991.5a 5.80b 66.52b 391.77a 120.47b
617.83b 84.55b 445 1147.33a 7.02b 48.54b 429.79a 149.17a
32.77 4.29 0 34.42 1.62 5.53 9.50 4.02
449.89a 155.64a 445 1050.66a 34.33a 116.42a 444.94a 129.59a
362.9a 145.2a 445 953.16b 5.16b 60.99b 378.79a 115.60b
593.94b 115.34a 445 1154.33a 6.75b 49.50b 443.28a 147.50a
32.77 4.29 0 34.42 1.62 5.53 9.50 4.02
Different letters in the same row indicate significant differences (P < 0.05).
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Table 3 Pearson correlation coefficient between DMI and chemical composition of tropical forage trees in sheep and goats with and without PEG supplementation (50 g/kg DM). Sheep
Lignin Crude Protein Condensed Tannins Neutral Detergent Fiber Acid Detergent Fiber
R P R P R P R P R P
Goat
Without PEG
With PEG
Without PEG
With PEG
DMI
DMI
DMI
DMI
0.714 0.001 0.622 0.001 0.484 0.011 0.424 0.028 0.299 0.130
0.688 0.002 0.718 0.001 0.429 0.025 0.399 0.039 0.306 0.121
0.566 0.140 0.050 0.844 0.061 0.809 0.262 0.293 0.331 0.179
0.378 0.122 0.165 0.512 0.041 0.871 0.044 0.861 0.130 0.608
R: Regression coefficient. P: Probability value.
Without PEG : DMI g=kgLW0:75 ¼ 2 14:7 lignin ð%Þ þ 51:8 CP ð%Þ: R2adjusted ¼ 58:7% With PEG : DMI g=kgLW0:75 ¼ 388 13 ligninð%Þ þ 75:6 CPð%Þ: R2adjusted ¼ 64:9% For goats, the correlations were not significant (P > 0.05) and best subset analyses were not performed. The inclusion of PEG in the diet did not modify the relationship and no significant correlation was found (P > 0.05) (Table 3). 3.2. Turbidity index A relationship was found between tannin intake (g CT/kg LW0.75) and D% SHD in sheep fed L. latisiliquum (P < 0.05, Table 4). The same trend was observed for sheep fed P. piscipula, although this was not significant (P ¼ 0.17). No significant relationship was found between D% SHD and tannin intake in sheep fed B. alicastrum. The relationship between tannin intake and D% SHD was not observed in sheep fed L. latisiliquum and supplemented with PEG (P > 0.05). The trend observed with P. piscipula also disappeared when PEG was added to the sheep diet (P > 0.05) (Table 4). In goats, similar effects were observed: a negative correlation was found between tannin consumption (g CT/kg LW0.75) and turbidity in goats fed L. latisiliquum (P < 0.05, Table 4) and supplemented with both PEG concentrations; no correlation was found for P. piscipula and B. alicastrum. 3.3. Salivary protein The protein content of saliva in sheep and goats is presented in Table 5. The content (mg/g dry saliva) was similar both for sheep and goats; we failed to see the effect of foliage ingested or PEG supplementation (P < 0.05). 4. Discussion 4.1. Feed intake During feed consumption, animals can express behavioral changes related to sensorial experiences, which can be triggered ~ o et al., 2012), its chemical by the physical characteristics of the feed (i.e., shape and density of the foliage; Hernandez-Ordun characteristics (i.e., compounds such as tannins and lignin; Alonso-Díaz et al., 2008, Alonso-Diaz et al., 2009; Provenza et al., 2009) and nutritional value (i.e., digestibility; Sandoval-Castro et al., 2005). Results from the present study revealed that the CT content of different foliage (Table 1) did not cause a reduction in foliage intake (Table 2). Furthermore, PEG supplementation did not increase tannin-rich foliage intake (Table 2). Similar results have been consistently reported both with sheep ~o and goats in other countries (Decandia et al., 2008; Fagundes et al., 2014) as well as in Yucatan, Mexico (Hernandez-Ordun et al., 2012, 2015; Revaud et al., 2014). The fiber and lignin content of the foliage (Table 1) appeared to be the main constraint to DMI rather than the CT content (Table 3). It is well known that fibers contained in feed can be a limiting factor for DMI in ruminants (Leng, 1990; Van Soest et al., 1991). In agreement with previous work done on tropical hair sheep (Alonso-Diaz et al., 2009), it has been shown that lignin and a high CP content can also be negatively associated with DMI. The latter is similar to our results, where lignin (fiber) became the first limiting factor for DMI in both foliage and grass, where lignin content is particularly high. On the other hand, the CP content of foliage was positively associated with DMI. It is possible that animals try to compensate the low CP content of the basal diet (grass) with the high CP content of the foliage. High protein
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Table 4 Relationship between tannin intake (g tannin/kg LW0.75) and saliva turbidity index (% turbidity change) of sheep and goats fed different tropical forage trees with and without PEG supplementation (50 g/kg DM). Plant SHEEP Without PEG Lysiloma latisiliquum Piscidia piscipula Brosimum alicastrum With PEG Lysiloma latisiliquum Piscidia piscipula Brosimum alicastrum GOATS Without PEG Lysiloma latisiliquum Piscidia piscipula Brosimum alicastrum With PEG Lysiloma latisiliquum Piscidia piscipula Brosimum alicastrum
a
a
Equation
R2
P
TI ¼ 4.635 (±1.166) CTI þ 16.14 (±5.613) TI ¼ 15.62 (±3.103) CTI þ 10.56 (±3.342) e
0.693 0.7836 e
0.0054 0.0015 e
TI ¼ 8.611 (±4.44) CTI þ 35.38 (±21.43) TI ¼ 12.03 (±11.18) CTI e 17.77 (±11.47) e
0.3486 0.142 e
0.0941 0.3175 e
TI ¼ 7.140 (±1.005) CTI þ 12.31 (±2.56) TI ¼ 46.02 (±7.92) CTI þ 14.94 (±3.73) TI ¼ 64.27 (±14.63) CTI þ 31.03 (±7.704)
0.9266 0.894 0.8283
0.0021 0.0044 0.0118
TI ¼ 10.76 (±4.433) CTI þ 21.05 (±11.80) TI ¼ 25.06 (±4.958) CTI þ 3.66 (±2.066) TI ¼ 27.81 (±4.391) CTI þ 8.25 (±2.206)
0.5955 0.8646 0.9093
0.0722 0.0072 0.0032
Turbidity index (TI) ¼ slope value condensed tannin intake (CTI) (g/kg LW0.75) þ intercept value. a For sheep fed Brosimum alicastrum foliage, CT was below detection level and therefore regression analysis could not be performed.
Table 5 Salivary protein content of sheep and goats (mg/g dry saliva) in the saliva of sheep fed with tropical forage trees with and without PEG supplementation. Without PEG
With PEG
Salivary protein
L. Latisiliquum
P. piscipula
B. alicastrum
L. Latisiliquum
P. piscipula
B. alicastrum
SED
Goat Sheep
2.77a 2.70a
3.01a 2.64a
2.64a 2.73a
3.60a 3.06a
3.96a 2.61a
3.94a 2.88a
0.50 0.42
Means with similar letters in the same row indicate no significant differences (P < 0.05).
content of foliage can be a limiting factor for DMI when the animal’s CP requirements are surpassed and disposing of excess protein becomes energetically costly.
4.2. Tannin binding salivary protein The working hypothesis was that supplementing a foliage with high tannins content would induce a higher TBSP response (D% SHD), indicative of a physiological response (higher capacity to block tannins), allowing sheep or goats to increase their DMI. However, the protein content of the lyophilized saliva was similar in all treatments, initially suggesting a lack of response to the tannin stimuli measured as protein concentration. Thus, when the DMI corresponding to each plant was analyzed, no correlation was found between foliage tannin content and D% SHD and between D% SHD and DMI. As explained above, it appeared that lignin (fiber) is the main component limiting DMI (Table 3). Nevertheless, we observed a pattern in animals within the same experimental group (L. latisiliquum- and P. piscipula-supplemented groups), which were subsequently analyzed. Indeed, when the relationship between tannin intake and D% SHD was analyzed in sheep or goats fed the same foliage, we observed that animals producing a higher TBSP response had lower feed intake. This was observed in goats fed all three foliage and in sheep fed L. latisiliquum and P. piscipula foliage (Table 4, P < 0.01). The latter suggested that animals with a high TBSP content (measured as D% SHD) might detect the astringency derived from CT better. This effect might be similar to that found in humans where a dry puckering sensation is usually observed during the consumption of unripe fruits, which cause the food to be unpalatable (Gibbins and Carpenter, 2013). The use of PEG supplementation seemed to reduce the astringency caused by tannins in sheep, as no relationship was found when PEG was given as a supplement (P > 0.05, Table 4). On the other hand, the relationship remained for goats fed P. piscipula and B. alicastrum, and a trend (P ¼ 0.07) was observed in goats fed L. latisiliquum (Table 4). These results might explain the lower correlation between CT and foliage intake in contrast to the higher correlation found between lignin or CP and foliage intake. Therefore, the results allow us to hypothesize that the variation in DM intake between animals results from differences in their ability to detect CT (oral astringency leading to increased TBSP) and it is relatively larger than the variation caused by fiber intake (causing increased rumen fill and hence satiety). Hence, a higher TBSP response causes a reduction in DM intake while a lower TBSP response allows higher intake of feeds containing CT, an effect that, since it is averaged across animals, masks the cause-effect relationship between CT content and foliage DMI. On the contrary, the higher rumen filling effect caused by the response to dietary fiber intake might have lower variation between animals and becomes a more consistent constraining factor against DMI. The protein content of
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107
lyophilized saliva failed to detect this effect as it is not a measurement of its activity (capacity to bind tannins) and the protein concentration in fresh saliva was not accounted for (protein content/mL of fresh saliva). n (Gonza lez-Pech et al., Although sheep and goats are able to graze and browse in the tropical deciduous forests of Yucata 2014), goats are considered generalized browsers in contrast to sheep, which are defined as specialized grazers. Therefore, lez-Pech et al., 2015). This difference in goats would be more suited to ingest CT from the foliage of browsed plants (Gonza feeding behavior between sheep and goats for what concerns foliage containing high quantities of CT could be the reason behind the different TBSP concentration present in sheep and goats. While sheep may increase TBSP production in response ~ a et al., 2013), recent studies suggested that goats have high innate TBSP levels, which imply to tannin intake (Vargas-Magan that it is not possible to increase the salivary response further even with an elevated CT consumption (Ventura-Cordero et al., 2015). As a net outcome, PEG supplementation can partially inhibit the astringency stimuli in sheep, resulting in the modification of tannin rich-foliage, while PEG supplementation does not have any effect on the astringency signal in goats. This might help to explain the differences in the response of PEG supplementation between sheep and goats obtained in the present study. It is important to note that the sheep used in the present study had previous experience with tannincontaining foliage and they exhibited an increased TBSP response after several weeks of L. latisiliquum foliage feeding ~ a et al., 2013). There is evidence of some degree of specificity in the affinity between TBSP and tannins usually (Vargas-Magan contained in the sheep diet (Alonso-Diaz et al., 2012) and other mammals such as Alces alces, Castor canadiensis, and Odocoileus hemionus (Hagerman and Robbins, 1993). Understanding astringency development in the mouth of ruminants could help finding mechanisms to avoid the negative effects of astringency on foliage intake from tannin-rich plants (in a similar fashion as with PEG supplementation). However, it remains to be defined whether goats benefit from a large TBSP response, for example increased feed utilization (i.e., increased organic matter digestibility, volatile fatty acid production, etc.), which might arise from TBSP. It also remains to be ~o defined whether such proteins act as tannin-blocking agents in a similar manner as PEG supplementation (Monforte-Bricen et al., 2005; Salem et al., 2007). If a high TBSP content is associated with a higher foliage intake, this might result in a larger total nutrient intake, which could overcome the anti-nutritional effects of tannins possibly leading to partial nutraceutical effects, such as the anthelmintic effect observed in some tannin-rich foliage (Sandoval-Castro et al., 2012). This area will need to be further addressed in future studies. Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was financially supported by CONACYT-CB/106146. Andres Pech-Cervantes acknowledge the Consejo Nacional xico) for a scholarship to pursue his MSc degree. de Ciencia y Tecnología (CONACYT-Me References Alonso-Díaz, M.A., Torres-Acosta, J.F.J., Sandoval-Castro, C.A., Hoste, H., Aguilar-Caballero, A.J., Capetillo-Leal, C.M., 2008. Is goats’ preference of forage trees affected by their tannin or fiber content when offered in cafeteria experiments? Animal Feed Sci. Technol. 141, 36e48. 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