Effect of supplementing tanniferous tree leaves mixture on immune response and GI nematodes in kids

N Dutta, M Dubey*, P S Banerjee, A K Pattanaik, K Sharma**, P Kumar and A Narang

Centre of Advanced Faculty Training in Animal Nutrition,
Indian Veterinary Research Institute, Izatnagar-243 122, India
dutta65@gmail.com
* College of Veterinary Science and Animal Husbandry, Jabalpur- 482 001, India
** Indian Council of Agricultural Research, New Delhi-110 012, India

Abstract

The aim of present study was to ascertain the effect of condensed tannins (CT) from tropical tanniferous tree leaves on growth, immune response and GI nematodes in growing kids. Eighteen indigenous kids were randomly divided into three groups of six each in a completely randomized design and fed three iso-nitrogenous diets to contain 0 (CT-0), 1.0 (CT-1) and 2.0 (CT-2) percent CT through a dried and ground leaf meal mixture of Ficus infectoria, Psidium guajava and Ficus bengalensis. The respective diets were fed to kids on a basal diet of wheat straw as per requirements for 180 days. Blood-biochemical parameters and erythrocytic antioxidant status was monitored at 45-days intervals.

Results indicated that the final body weight, total body weight gain and average daily gain were significantly (P<0.05) higher in CT supplemented groups (CT-1 & CT-2) than control (CT-0) group. Feed conversion ratio was significantly (P<0.05) higher in treatment CT-2 followed by CT-1 and CT-0, respectively. Feeding of both the CT-containing diets significantly (P<0.01) decreased the faecal egg counts (FECs); however, FECs were increased significantly in control group. Serum glucose, total protein were similar among the dietary treatments except for a significant (P<0.01) reduction in serum urea level in kids fed CT-containing diets. There was significant (P<0.05) improvement in the erythrocytic antioxidant status and humoral immune response in kids fed CT diets. It is concluded that a noticeable encouraging impact was apparent on growth, FCR, antioxidant status, humoral immune response and GI nematodes status in kids given diet containing 1-2% CT from a leafmeal mixture.

Keywords: Condensed tannins, Ficus bengalensis, Ficus infectoria, growth, Psidium guajava

Introduction

Goats have been recognized as the most valuable livestock for promoting economic health of marginal and landless farmers in many developing countries of the world. However, the developments of proper feeding strategies for goats are of paramount importance to help them grow and produce well even in intensively cultivated areas. Poor nutrition results in lowrates of production, reproduction as well as increased susceptibility to various diseases. Gastrointestinal (GI) nematodes infections are widespread in the tropics, and result in poor performance of ruminants. Animals infected with these nematodes exhibited higher requirements for protein and minerals due to the loss of endogenous nitrogen (blood, plasma, mucin and sloughed cells) and lowered phosphorus absorption (Poppiet al 1985, Kahn and Diaz-Hernandez 2000). Drug resistance has become an important issue in small ruminant husbandry when anthelmintics are applied frequently at high levels. The use of natural substances is becoming preferable and may offer better control than using chemical compounds to treat parasites (Chandrawathaniet al 2000).Condensed tannins have been shown to influence worm burden in sheep under experimental conditions (Athanasiadouet al 2000). Tannins enhance protein metabolism and absorption in the gut (Sykes and Coop 2001) and may also have a direct killing effect on gut parasites (Athanasiadouet al 2000, Cresswellet al 2004). Free radicals and reactive oxygen species, generated due to aerobic metabolism, can be extremely damaging to biological systems (Padh 1991). Polyphenolics have been shown to possess free radical scavenging and metal chelating activity in addition to their reported anticarcinogenic properties (Middleton 1998) and, it is hypothesized that dietary CT may improve the antioxidant system thereby improving health. Keeping this background in view, the present experiment was undertaken to study the effect of CT from a mixture of tanniferous tree leaves (Ficus infectoria, Psidium guajava and Ficus bengalensis) on growth, immune response and GI nematodes in kids.

Materials and methods

Animals, management and treatments  

The experiment that lasted for 180 days was conducted at the Animal Nutrition Research Sheds of the Indian Veterinary Research Institute (IVRI), Izatnagar in Uttar Pradesh province of India.  Eighteen 6-months-old indigenous kids (11.33±1.52 kg initial BW), were randomly allocated to three dietary treatments, 6 each in a completely randomized design and assigned to 3 dietary treatments CT-0, CT-1, and CT-2 containing 0, 1.0, and 2.0 percent CT of diet, respectively. Prior to start of the trial the kids were treated with broad-spectrum anthelmintic (Albendazole suspension; Smith Kline Pharmaceuticals Limited, India, at 8 mg/kg BW). The kids were treated for ectoparasites [Butox liquid (Deltamethrin, Hoechst India Limited, Mumbai, India) diluted @ 2 ml in 1 litre of water and sprayed on the body surface]. The kids were penned individually with free access to fresh water in ventilated sheds and allowed exercise out-doors in an adjacent dry paddock daily between 08:30 and 09:30 h. 

Diets

All the experimental kids were offered a basal diet of wheat straw (ad libitum) along with required amount of concentrate mixture. The required quantity of dried and ground leaf meal mixture was added in the concentrate mixture of kids in group CT-1 and CT-2 in the morning to bring CT content to 1.0 and 2.0 percent of total diet. The leaf meal mixture was prepared by mixing Ficus infectoria, Psidium guajava and Ficus bengalensis in the ratio of 70:20:10. The leaves harvested in one lot in the month of July from the IVRI campus were dried and ground in an electric grinder before mixing in the supplement. The daily allowance of the supplements (maize: 30.0, deoiled groundnut cake: 37.0, wheat bran: 30.0, mineral mixture: 2.0 and common salt: 1.0 percent) was offered in single meals (at 09:30 h) in the morning; green fodder (100 g) and wheat straw was then offered ad libitum, when all the kids had consumed the concentrate. All goats were fed so as to meet their requirements (Kearl 1982) for maintenance and a growth of 50 g/day.  The amount of supplement offered to individual kids was adjusted fortnightly as per the BW changes of each animal to meet the nutrient requirement.

Measurements and sampling

Straw residues remaining were weighed 24 h post-feeding to ascertain daily feed consumption. Faecal samples were collected per rectum from all kids every fortnight for faecal egg count. Daily DM intake and fortnightly BW of all the kids were recorded before feeding in the morning throughout the study.

Blood samples were collected via jugular vein puncture in the morning before feeding at 45 day intervals. Serum was separated and preserved at −20 ⁰C for glucose, proteins and urea analysis. For antioxidant enzymes estimation the blood samples were collected in 15 ml calibrated tube with anticoagulant acid citrate dextrose @ 1.5ml/10 ml blood and centrifuged at 2000 rpm for 15 min at 4⁰C, followed by separation of plasma and buffy coat. The resulting erythrocyte pellet was washed thrice with 250 mosm/L (pH-7.4) phosphate buffered saline (PBS) as per Yagi et al (1989). The erythrocyte pellet (packed RBC) thus obtained was mixed with an equal volume of PBS to form RBC suspension. 0.5 ml RBC suspension was mixed with 4.5 ml stabilizing solution (EDTA, 2.7 mM and 0.7 mM, marcaptoethanol) to prepare a haemolysate of 1:20 dilution.

The humoral immune response was ascertained by assessing the antibody response to chicken-RBC (CRBC). Accordingly, 0.1 ml CRBC mixed with equal volume of Freund’s incomplete adjuvant was inoculated intramuscular to all the kids under different dietary treatments. Serum samples collected at 0, 7, 14, 21 days post- inoculation was assayed for antibody titre using heamagglutination test.

Laboratory analyses

Samples of feeds, residues and faeces were milled to pass through a 1mm sieve and then analyzed following the methods of AOAC (1995) to determine DM by the oven drying method (934.01), organic matter (OM) by muffle furnace incineration (967.05), crude protein (CP) by kjeldahl method (984.13)  N×6.25), ether extract (EE; 920.39), ash (942.05). Neutral detergent fibre (NDF) and acid detergent fibre (ADF) were essentially determined by the methods of Van Soest et al (1991). NDF was assayed with sodium sulphite in the NDF reagent without α-amylase and the results were expressed with residual ash. The CT content in leaf meal mixture was estimated by Butanol-HCl method (Makkar 2000).  Serum glucose concentration was determined colorimetrically (Hultmann 1959). The serum total protein and urea content were measured as per Wotton (1964) and (Rahmatulla and Boyde 1980), respectively. The GSH and catalase were estimated by dithio-bis-2 nitro benzoic acid (DTNB) method of Prins and Loos (1969) and Bergmeyer (1983), respectively. The superoxide dismutase (SOD) activity was estimated as per the method described by Madesh and Bal Subramanian (1998). The lipid peroxides (LPO) level was determined by estimating the concentration of malonaldialdehyde (MDA) in nmol/ml of RBC haemolysate (Placer et al 1966). Faecal egg counts were made using modified McMaster technique (Anonymous 1984). One g of faeces was weighed, mixed thoroughly with 14 mol saturated salt solution and charged in one chamber of McMaster slide having capacity of 0.3 ml. Total number of eggs counted in the chamber was multiplied by 50 to get the number of eggs present in 1.0 g of faeces.

Statistical analyses

The data obtained were subjected to analysis of variance using SPSS 11.0 software and treatment means were ranked using Duncan’s multiple range tests. Significance of treatments with respect to different characters was declared at P<0.05 unless otherwise stated. All the statistical analysis procedures were done as per Snedecor and Cochran (1994).

Results and discussion

Chemical composition

The chemical composition of wheat straw, concentrate mixture and tree leaves mixture is given in Table 1. The chemical composition of concentrate mixture and wheat straw was within normal range and comparable to values reported by earlier workers (Patraet al 2006, Deyet al 2008). The concentration of NDF and ADF was higher in leaf meal mixture as compared to concentrate; this could be attributed to the high cell-wall constituents usually present in leaf meal (Patraet al 2003, Anbarasuet al 2004, Deyet al 2008).   The condensed tannins content of leaf meal mixture was 13.3 percent.

Voluntary feed intake

The mean DM intake through wheat straw was higher (P=0.051) in CT-1 as compared to CT-0, while that in CT-2 was comparable to both. However, total feed (DM) consumption (g/d) was significantly (P<0.05) improved in both the CT-supplemented groups as compared to the control group (CT-0) (Table 2). The results indicated that supplementation of CT through leaf meal mixture stimulated voluntary feed intake through higher consumption of wheat straw. The DM intake (g/kgW^0.75) over the entire feeding period is given in Figure 1. Interestingly, perusal of the fortnightly pattern of feed consumption revealed that the intakes of concentrate, wheat straw and, consequently the total DM were improved in kids supplemented with tanniferous leaf meal mixture (1-2 % CT of diet). The average intake of DM (342-408 g/d) by kids was within the normal range (Kearl 1982) and this clearly indicates that all the experimental diets were palatable. Similar to the present study, higher voluntary intake on moderate (1-4%) CT containing diets was reported by many workers (Terrill et al 1992, Ramirez-Restrepo et al 2004, Dey et al 2008).

Figure 1. Effect of condensed tannins supplementation on DMI (g/kgW0.75) by kids

Growth and feed conversion ratio

Live weight changes and animal performance

Initial BW (kg) in kids did not differ significantly (P<0.05) among dietary treatments; however, final BW, total BW gain (kg) and average daily gain (ADG, g) for the period of 180 days were significantly (P<0.05) higher in  both the CT-supplemented groups (CT-1 and CT-2) relative to control group (CT-0). Feed conversion ratio (FCR) by kids (kg DMI kg^-1 gain) was also significantly (P<0.05) better in CT supplemented groups as compared to control. The encouraging results of ADG and FCR at 1-2% level of CT in the diets gives an indication that the binding effect of tannins was pronounced at this level. At appropriate concentration, the CT reduced the degradation of sulphur amino acids in the rumen, increases the irreversible loss of cystine from plasma and increased the flow of cystine to body synthetic reaction (Wang et al 1994, Deyet al 2008) and thereby improves the performance of kids.

Effect of CT on faecal egg counts

Mean faecal egg counts (FECs) (per gram) were significantly (P<0.05) decreased with CT supplementation as compared to control (CT-0). The FECs in CT-0 were significantly (P<0.05) increased after two months of feeding, whereas, in CT-supplemented groups (CT-1 and CT-2) the FECs remained unchanged throughout the 180 days experimental period (Figure 2). Results for biochemical parameters clearly indicated that supplementation of CT at 1-2% of diet through tanniferous leaf meal mixture had a negative effect on GI nematodes. The present results are in conformity with the previous reports (Barry et al 2001, Min et al 2003, Sokerya and Preston 2003), who showed that dietary supplementation of CT may be used an alternative parasite management strategy.  Dietary supplementation of CT may enhance resistance against GI nematodes through increases in tissue protein supply, which are prioritized for repair and immune response (Barry et al 2001, Niezen et al 2002).  The CT could form a complex with nutrients and inhibit nutrients availability for larval growth or decrease GI nematodes metabolism directly through inhibition of oxidative phosphorylation (Scalbert, 1991), causing larval death (Athanasiadouet al 2001).  Molanet al (2001; 2002) have observed that CT extracted from several forages can disrupt the life cycle of nematodes by preventing their eggs from hatching and by preventing larval development to the infective stage. In the present study, it is not clear whether the reduction in FEC was due to reduced worm load or due to decreased fecundity of GI nematodes. Nevertheless, the present findings have got tremendous implications on the epidemiology of infection. Reduced FEC means less contamination of pasture with infective larvae, which in turn results into less infection in the animals grazing on the pasture. Moreover, the mean FECs of the treated groups were much lower than the threshold level, which warrants for anthelmintic medication. Thus the frequency of medication can be curtailed in the CT supplemented animals. This is especially important in hot and humid climatic conditions like India where frequent medication is the only way to get rid of this menace.

No appreciable difference in FECs was noticed up to 4th period of experimentation in the three groups of animals. The probable reason was that the experiment was started in the month of December- January, which is not considered as a favorable season for GI nematode infection due to adverse environmental conditions. Additionally, persistent effect of the anthelmintic medication of the kids prior to start of the experiment might have played some role in preventing the establishment of the worms.  GI parasitism increases the amino acid demand of gastrointestinal tissues. Peripheral tissues are consequently denied the nutrients required for optimal growth. Normal animal performance in the face of larval challenge may be possible if protein supply is increased and this is possible by supplementation of CT in the feed as revealed in this study.

Figure 2. Effect of condensed tannins supplementation on faecal egg counts by kids

Blood-biochemical parameters and erythrocytic antioxidant status

The data on blood parameters and erythrocytic antioxidants are presented in Table 3. Mean values of serum glucose and total protein were comparable among the groups and, within the suggested physiological range for goats (Kaneko 1997). The comparable levels of glucose and total protein indicated normal physiological condition of all the experimental kids. On the contrary, mean serum urea level was significantly lower (P<0.01) in kids in CT fed groups (CT-1 and CT-2), which may be attributed to the reduced rumen protein breakdown and increased essential amino acids (EAAs) absorption (Deyet al 2008). The influence of dietary supplementation of CT on antioxidant status of the kids was assessed through estimation of various antioxidant enzymes. There was significant (P<0.05) improvement in the erythrocytic antioxidant status as could be ascertained from an increased concentrations of SOD, catalase and total thiols concomitant to a reduction in LPO in the CT supplemented groups as compared to the control (Table 3).  The  increased activity of SOD are in agreement with the findings of Ho et al (1999), who reported that proanthocyandins A-1 component of tannins from Vacciniumvitis-idaea L had strong SOD activity. Lipid peroxidation is used as an indicator of oxidative stress in cells and tissues. Since, polyunsaturated fatty acids generate malondialdehyde and 4-hydroxyalkenals, the measurement of these compounds could be used as indicators of lipid peroxidation. The supplementation of CT in the diet of kids in the present study was found to decrease lipid peroxidation which is in consistency with the observations of Lin et al (2001).  Besides, the increased level of total thiol groups could also partially explain the reduced lipid peroxidation, as these are known to play greater protective role in preventing lipid peroxidation of membranes.

Immune response

The humoral immune response was ascertained by assessing the antibody response to CRBC. The results revealed that the kids under treatment CT-1 exhibited greater titre value indicating of improved humoral response in comparison to control (CT-0). The response was diminished in CT-2 group (Figure 3), although, it was better in comparison to CT-0. Hence, it can be deduced that CT supplementation at 1% level induces better humoral immune response in kids. Similar to present results Kumar et al (2007) observed that the birds given tannins containing raw red sorghum exhibited higher humoral immune response assessed through HA titre. By making the protein unavailable for digestion and absorption until it reaches the more acidic abomasum, tannins also enhance nutrition by providing high-quality protein to the small intestines (Barry et al 2001). This high-quality protein bypass effect has the potential to enhance the immune response and increase resistance to GI nematodes (Min et al 2004). The positive response of the immune system to protein intake has been shown in metabolism studies with ewes (Houdijk et al 2000) and lambs (Abbott et al 1988). Bypassing amino acids like arginine, glutamine and cysteine can enhance immune responses as these amino acids regulate activation of T and B lymphocytes, natural killer cells and macrophages, gene expression and lymphocyte proliferation, and the production of antibodies, cytokines and other cytotoxic substances (Li et al 2007). Parasitized Angora does grazing forages containing CT (i.e., Sericea lespedeza) had enhanced immune responses (Min et al 2005), and lambs grazing CT containing sulla (Hedysarum coronarium) had higher antibody titers against secretory-excretory antigens to O. circumcincta and to T. colubriformis (Niezen et al 2002). The combined effects of improved antioxidant status and possible increase in availability of EAAs might have contributed to the improved humoral immune response observed in present study.

Figure 3. Effect of condensed tannins supplementation on humoral immune response by kids

Conclusions

• Condensed tannins from tropical tanniferous tree leaves mixture (F. infectoria, P. guajava and F.bengalensis) can be used as functional feed supplement in the diet of growing kids.

  
  

• A noticeable encouraging impact was apparent on growth, FCR, antioxidant status, humoral immune response in kids given diet containing 1-2% CT from a leaf meal mixture. 

  
  

• Dietary supplementation of CT decreased the GI nematodes in kids.

  
  

• On the basis of results it is deduced that supplementation of CT from tanniferous tree leaves mixture at 1-2% of diet may be beneficial for kids and can be integrated in small holder farming system.

Acknowledgement

This study was financially supported by funds provided by Indian Council of Agricultural Research (ICAR), New Delhi, India.

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Received 2 October 2011; Accepted 9 January 2012; Published 7 February 2012

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