Rumen metabolites and enzymatic profiles in crossbred cattle bulls fed on high and low levels of tanniniferous oak (Quercus incana) leaves

V K Paswan and A Sahoo*

Indian Veterinary Research Institute, Campus Mukteswar, Nainital, Uttarakhand, India, 263138
* Animal Nutrition Division, Central Sheep and Goat Research Institute, Avikanagar, Rajasthan-304501, India
sahooarta1@gmail.com

Abstract

The rumen studies were conducted in two male fistulated crossbred cattle fed on three different feeding systems prevalent in high hill Himalayan regions of Uttarakhand involving control (OL0) animals (rumen fistulated crossbred cattle bull, 312 kg BW) fed on grass hay (GH), OL40 on GH + oak leaves (OL) at 60:40 artio and OL60 on GH + OL at 40:60 ratio in a complete switch over design each lasted for 6 weeks. Strained rumen liquor was assayed immediately for pH and then fractioned for ciliate protozoa count, rumen biochemical attributes and enzyme profile.

Increasing the levels of OL enhanced (P<0.001) total DM intake (kg/d) in OL40 (7.35) and OL60 (7.71) compared to control (6.67). Supplemental OL also enhanced intake of other nutrients, notably CP intake was 42.6 and 64.5% higher (P<0.001) in OL40 and OL60 compared to OL0. The contribution of CT from OL was @ 1.25 and 1.80 % of DMI in OL40 and OL60. The ciliate protozoa population (×10⁴) ranged from 8.87 (OL40) to 9.39 (OL60) and the difference between the groups was non-significant. The holotrichs and entodiniomorphs population was also non-significantly different between the groups, but the later predominated (>10 times) in all the feeding systems. Rumen pH was comparable in the three dietary groups (6.92-7.06). The N fractions (total N, ammonia N, non-protein N) except trichloroacetic acid precipitable N increased linearly (P < 0.05) with the increased levels of OL feeding. The volatile fatty acid (VFA) profile showed decreased (P < 0.01) acetate and butyrate concentration with increased levels of OL feeding resulting in non-significantly narrower (P = 0.155) acetate: propionate ratio. There was a significant decline (P = 0.020) in total VFA concentration in OL60 (54.20 mM/L) compared to other dietary groups (OL0, 77.11 and OL40, 72.66 mM/L). Carboxymethyl cellulase and xylanase activities showed an increasing trend (P < 0.01) with increased in OL, but protease activity remained unaffected.

The study revealed an increased feed and nutrient intake and positive shift in rumen fermentation pattern with increased level of OL, which needs to be validated based on production performance prior to recommending an optimized level in the feeding system at high hill Himalayan region.

Key words: Cattle, high hill Himalaya, oak leaves, rumen fermentation

Introduction

Tree forages, which are generally considered as emergency fodders for livestock, form an integral part of ruminant feeds for most part of the year in high altitude Himalayan regions of India (Uttaranchal, U.P., H.P., J&K and North-Eastern States). In these hilly areas, because of feed habit, the ability of ruminant livestock to utilize nutrients from the feed is supposed to be high and also the composition of their gut microbial environment is expected to be different from the ruminants reared in plane and thus may have enhanced capacity to digest and utilize nutrients from local feed resources including tanniniferous tree leaves (Sahoo 2008). Oak leaves contain a significant amount of tannins (Sharma et al 2008; Paswan et al 2008). Although tannins are generally regarded as antinutritional, condensed tannins (CT) at low concentrations alter ruminal fermentation (Bhatta et al 2002) and may aid microbial protein synthesis (Bhatta et al 2001). The populations of rumen microorganisms are affected by several factors like quality and quantity of feed, feeding frequency and feed additives (Hungate 1966; Varga and Kolver 1997; Vercoe et al 2009). Similarly, the rumen metabolites concentration viz., volatile fatty acids and different nitrogen fractions are affected by nature and quality of feed (Chaudhary et al 1995; Santra and Karim 2001; Samanta et al 2003). Therefore, the prevalent feeding systems at high hill Himalaya based on local grass hay (GH), and with high and low levels of oak leaves (OL) was evaluated by assessing the rumen environment (protozoal population, ruminal metabolites, enzymatic profile) fistulated crossbred bulls.

Material and methods 

Animals, feed and feeding

Two adult male crossbred cattle (312 kg average BW) fitted with permanent rumen fistula were taken for the experimentation. The animals were housed in well ventilated shed with facilities for individual feeding under uniform hygienic and managemental conditions. Adequate clean and fresh drinking water was made available daily. All the animals were de-wormed with broad spectrum anthelmintic before the start of the experiment. The dietary treatments simulated three different feeding systems prevalent in the region, viz. control (OL0) fed on GH alone and OL40 and OL60 fed on GH plus OL at 40 and 60 %, respectively. Animals were maintained on the three treatments that extended over three consecutive experimental feeding periods of 6 weeks by following the complete switch-over design. Feed intake from GH and OL was recorded during each switch-over trial. 

Sampling and analysis of rumen fluid

The rumen contents were collected 3 hour post feeding from the animals for two consecutive days at the end of each of the switch-over feeding trial into a pre-warmed flask, immediately measured for pH and then transported to laboratory. About 50g of the contents were stored frozen for studying the enzyme parameters. The contents were then squeezed through four layers of cheese cloth and sampled for protozoa (1 ml, preserved and stained with methyl green formal saline solution), volatile fatty acids (1ml, preserved frozen with metaphosphoric acid) and other biochemical parameters related to N fractions (40 ml, preserved after acidification with few drops of 10N H₂SO₄).

Rumen metabolites

Estimation of volatile fatty acids (VFA) was done using Nucon-5765 gas chromatograph (AIMIL, New Delhi, India) equipped with a double flame ionization detector as per the method described by Cottyn and Boucque (1968). The rumen liquor samples were analysed for ammonia N (NH₃N), total N (TN) and TCA precipitable N (TCAPN) by Kjeltech Auto-analyzer (Tecator, Sweden) as per AOAC (1984). The non protein nitrogen (NPN) was calculated as the difference between TN and TCAPN.

Rumen enzymes

The rumen content was extracted for microbial enzymes as per method described by Hristov et al (1999) and Sahoo et al (2005). The carboxymethylcellulase (CMC) activity was estimated by measuring the amount of reducing sugar released from carboxymethyl cellulose under the assay conditions (Miller 1959). Similarly, the xylanase activity was estimated by measuring the amount of reducing sugar (xylose) released from oat-spelt xylan (Sigma Chemical Co., St. Louis, USA). The activity of proteases was determined by measuring the amount of proteins hydrolysed (Lowry et al 1951) during incubation of the substrate (azocaesin) with the enzyme. The protein content of the enzyme samples was estimated (Lowry et al 1951) and the enzyme activity was expressed in terms of international unit (U, the amount of enzyme that produced one unit of reducing/hudrolysing product per ml per min under the assay conditions) or specific activity (SpA, U/mg protein in the extract).

Ciliate protozoa

The number of protozoa was counted in a known volume of medium and the total number was calculated as per Sahoo (1995).

Chemical analysis

Nitrogen content of the feed samples was determined as described above by the standard Kjeldahl method (AOAC 1984) while neutral detergent and acid detergent fiber were determined as per the procedure of Van Soest et al (1991).

Statistical analysis

The data were analysed using the statistical software SPSS (version 11.0). Differences among treatments were analysed by fixed effects (Model I) ANOVA (Snedecor and Cochran 1989). Polynomial contrasts with linear and quadratic functions were applied to explain any trend at different levels of OL feeding and differences between the treatments were considered at P<0.05.

Results and discussion 

Chemical composition of feeds

The chemical composition (% on DM basis) of GH and OL fed to the animals during the feeding trial is presented in Table 1.

The GH contained OM 92.4, CP 5.9, NDF 81.5 and ADL 10.9. The DM content of OL was 65.4% and it contained CP 10.1, EE 6.0, NDF 63.1 and ADL 21.6. The concentration (% on DM basis) of total phenolics in OL was 6.87 having CT 3.06 and HT 3.18. The GH contained a higher percentage of AIA (4.76) and had moderate levels of Ca (0.58%) and P (0.19%). The OL had a wider Ca: P ratio (16) as compared to grass hay (3). The compositional values of OL with respect to tannin and fiber composition were in conformation with the earlier reports (Makkar and Singh 1991; Paswan et al 2008).

Feed and nutrient intake

Feeding of OL in OL40 and OL60 at 41.0 and 58.8 % of DMI significantly (P<0.001) enhanced feed intake by 10.2 and 15.6 %, respectively (Table 2).

There was also linear increase in OM, CP, EE and TCHO intake at higher levels of OL feeding, the reason being higher DM intake in OL40 and OL60 and comparatively higher concentration of these nutrients in OL than GH. A lower NDF and ADF content in OL than GH reduced the gap in nutrient intake to non-significant level in the three dietary groups. The CT from OL contributed 1.25 and 1.80 % of DMI in OL40 and OL60, respectively. A lower concentration of CT in the diet has been reported to have beneficial effect on rumen fermentation, N utilization and animal health and productivity (Barry and McNabb 1999; Waghorn and McNaab 2003; Singh and Sahoo 2004). 

Ciliate protozoa population

The dynamic nature of the rumen microbial ecosystem is mostly related to diet diversity (Hoover and Miller 1991; Russell and Rychlik 2001; Vercoe et al 2009) and hence, alteration due to the systems of feeding based on GH and with different levels of OL may be expected. However, the ciliate protozoa population (×10⁴) and their types (distribution of holotrichs, entodiniomorphs) in different dietary groups were non-significantly different (Table 3).

More significantly, the total number was observed to be one tenth of the population that observed in cattle at plains (Kamra 2005) without tanniniferous feed. Singh and Bhat (2001) discussed the importance of CT on enhanced animal production associated with increased N outflow from the rumen. Thus, CT from OL might have induced a negative effect on protozoa population. Makkar et al (1995) also observed a significant decrease in both types of protozoa with the addition quebracho tannins in a RUSITEC device. Although tannin effects on ruminal protozoa counts are variable in assays carried out in vivo (Makkar 2003), some evidence exists for lower protozoa number in the presence of tannins. Wang et al (1994) also found lower protozoa numbers in the rumen of sheep fed diets containing tannins compared with those receiving an intraruminal infusion of PEG. Entodiniomorphs accounted for around 90% of total protozoa. This finding was in agreement with values observed by other authors (Santra and Karim 2001; Hindrichsen et al 2002).

Rumen metabolites

There was no significant difference in pH (Table 3) between the three groups and its value near neutral is attributed to all roughage diet. The N fractions (TN, NH₃N, NPN except TCAPN; Table 2) increased linearly with the OL feeding. The NH₃N concentration (mg/dl) was lower in OL0 (6.30) and OL40 (6.87) compared to OL60 (8.31). This change is due to higher N (CP) content of OL compared to GH (Table 1) that was being available through dietary sources. But, interestingly, the proportionate concentration with respect to TN was similar in all the three groups (13.26, 12.38 and 13.30 % in OL0, OL40 and OL60, respectively) indicating CT-optimized NH₃N level for better rumen fermentative activity (Singh and Bhat 2001). The total N concentration correlated well with the total N/CP intake of the animals. The TCAPN (mg/dl) was non-significantly (P = 0.299) lower in OL0 (35.8) compared to OL fed groups (41.8, 42.3). There was 15-18% higher TCAPN (an index of microbial protein) in the RL of OL fed animals. These alterations in ruminal N parameters could be attributed to formation of CT protein complexes or selective inhibition of microbes or their enzymes involved in catabolism of protein or increased uptake of NH₃ by microbial cells.

The total volatile fatty acid concentration (Table 3), which was similar in OL0 and OL40, but lower in OL60 was indicative of better energetic efficiency in high OL fed animals, i.e. more of VFA may have been utilized for microbial protein synthesis. The acetate and butyrate concentration showed a linear decrease with the increase in OL in the diet, and it contributed non-significantly (P = 0.155) narrower acetate: propionate ratio and above all, a relatively higher proportion of propionate in the rumen fluid of OL fed groups. Makkar et al (1995) also found higher molar proportions of propionate in a RUSITEC device when ruminal microorganisms were exposed to quebracho tannins.

A non-significant quadratic response for the above parameters was indicative of no adverse effect of high OL (60%) feeding along with GH, rather demonstrated a positive shift.

Rumen enzymes

The activity of fermentative enzymes in rumen contents of the groups receiving oak leaves showed significantly higher CMC activity (U) than the group fed on GH alone, but the specific activity (SpA) was highest (0.072) in the group receiving less OL followed by OL60 (0.063) and OL0 (0.035) (Table 4). But, a quadratic effect was indicative of reduced CMC activity at higher levels of OL.

The xylanase activity (U) was significantly higher in OL40 (0.357) and OL60 (0.396) than the control OL0 (0.302), but when expressed in SpA, the difference was non-significant. The proteases activity was similar in all the three groups. This was despite a higher CP intake in OL40 (142.6%) and OL60 (164.5%), which supposed to induce increased proteolysis but negated/diminished in presence of CT and thus become non-significantly different between the groups. It has been reported that CT diminishes rumen proteolysis, and also limit microbial activity in the rumen (Bae et al 1993; McAllister et al 1994). Many authors suggested that concentrations of CT below 50 g/kg of DM in the diet, as that observed in the present OL feeding groups, do not have adverse effects on ruminal fermentation (Barry and McNabb 1999; Salawu et al 1999).

Conclusion 

Feeding of oak leaves at 40 and 60 % level along with local grass hay showed an increase in feed and nutrient intake and a positive shift on rumen metabolic (propionate-type fermentation, microbial energetic efficiency) and enzymatic (fiber-degrading and proteolytic activity) profile of animals. Thus, oak leaves appear to be a promising forage supplement to low-quality roughage-based diet due to its increase usage and easy on-farm availability at high altitude hilly regions.

References 

AOAC 1984 Official Methods of Analysis. Association of Official Analytical Chemists, 13th ed. Arlington, Virginia, USA.

Bae H D, McAllister T A, Tanke J, Cheng K J and Muir A D 1993 Effect of CT on endoglucanase activity and filter paper digestion by Fibrobacter succinogenes S85. Applied Environmental Microbiology 59: 2132-2138. http://aem.asm.org/cgi/reprint/59/7/2132

Barry T N and McNabb W C 1999 The implications of condensed tannins on the nutritive value of temperate forages fed to ruminants. British Journal of Nutrition 81: 263–272. http://journals.cambridge.org/action/displayFulltext?type=1&fid=931192&jid=BJN&volumeId=81&issueId=04&aid=931180

Bhatta R, Krishnamoorthy U and Mohammed F 2001 Effect of tamarind (Tamarindus indica) seed husk tannins on in vitro rumen fermentation. Animal Feed Science and Technology 90:143–152

Bhatta R, Shinde A K, Vaithiyanathan S, Sankhyan S K and Verma D L 2002 Effect of polyethylene glycol-6000 on nutrient intake, digestion and growth of kids browsing Prosopis cineraria. Animal Feed Science and Technology 101:45–54.

Chaudhary L C,  Srivastava A and Singh K K 1995 Rumen fermentation pattern and digestion of structural carbohydrates in buffalo (Bubalus bubalis) calves as affected by ciliate protozoa. Animal Feed Science Technology 56:111-117.

Cottyn B G and Boucque C V 1968 Rapid method for the gas chromatographic determination of volatile fatty acids in rumen fluid. Journal of Agriculture and Food Chemistry 16:105-107.

Hindrichsen I K, Osuji P O, Odenyo A A, Madsen J and Hvelplund T 2002 Effects of supplementation of a basal diet of maize stover with different amounts of Leucaena diversifolia on intake, digestibility, nitrogen metabolism and rumen parameters in sheep. Animal Feed Science and Technology 98:131–142.

Hoover W H and Miller T K 1991 Rumen digestive physiology and microbial ecology. Veterinary Clinics of North American Food Animal Practice 7:311-325.

Hristov A N, McAllister T A, Van Herk F H, Cheng K J, Newbold C J and Cheeke PR 1999 Effect of Yucca schidigera on ruminal fermentation and nutrient digestion in heifers. Journal of Animal Science 77:2554–2563. http://jas.fass.org/cgi/reprint/77/9/2554

Hungate R E 1966 The Rumen and its Microbes. Academic Press, London. pp 26–36.

Kamra D N 2005 Rumen microbial ecosystem. Current Science 89:124-135.

Lowry O H, Rosebrough N J, Farr A L and Randall R J 1951 Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193:265-275.
 

McAllister T A, Bae H D, Jones G A and Cheng K J 1994 Microbial attachment and feed digestion in the rumen. Journal of Animal Science 72 :3004-3018. http://jas.fass.org/cgi/reprint/72/11/3004

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 Research 49:241–256.

Makkar H P S, Blummel M and Becker K 1995 In vitro effects and interactions of tannins and saponins and fate of tannins in the rumen. Journal of Science Food and Agriculture, 69:481–493.

Makkar H P S and Singh B 1991 Distribution of CT (proanthocyanidins) in various fibre fractions in young and mature leaves of some oak species. Animal Feed Science and Technology 32:253-260.

Miller G L 1959 Use of dinitrosalisylic acid reagent for determination of reducing sugar. Analytical Chemistry 31:426–428.

Paswan V K, Mahapatra R K, Meena H R and Sahoo A 2008 Nutrient Composition and Phenolic Constituents in some feed and fodder samples from Temperate Regions of Kumaon Himalaya. Indian Journal of Animal Nutrition 25:19-24.

Russell J B and Rychlik J L 2001 Factors that alter rumen microbial ecology. Science 292 (5519):1119-22.

Sahoo A 1995 Ciliate protozoa- Microscopic examination, counting and identification.. In: Recent Advances in Bioconversion and Feed Utilisation in Ruminants, S P S Bedi, D N Kamra, R Singh and L C Chaudhary (editors), CAS in Animal Nutrition, IVRI, Izatnagar, India.

Sahoo A 2008 Livestock feeding and nutrition at high altitude temperate regions. In: Compendium of Short Course on “Nutritional strategies for sustainable and green livestock production” September 10-30, 2008, CAS in Animal Nutrition, IVRI, Izatnagar, India.

Sahoo A, Kamra D N and Pathak N N 2005 Pre- and postweaning attributes in faunated and ciliate-free calves fed calf starter with or without fish meal. Journal of Dairy Science 88: 2027–2036. http://www.journalofdairyscience.org/article/PIIS0022030205728794/fulltext

Salawu M B Acamovic T, Stewart C S Hvelplund T and Weisbjerg M R 1999 The use of tannins as silage additives: effects on silage composition and mobile bag disappearance of dry matter and protein. Animal Feed Science and Technology 82:243–259.

Samanta A K, Singh K K, Das M M, Maity S B and Kundu S S 2003 Effect of complete feed block on nutrient utilisation and rumen fermentation in Barbari goats. Small Ruminant Research 48:95–102.

Santra A and Karim S A 2001 Nutrient utilization, rumen fermentation characteristics and ciliate protozoa population in sheep and goat under stall feeding. Indian Journal of Animal Sciences 71:852–856.

Sharma R K, Singh B and Sahoo A 2008 Exploring feeding value of oak (Quercus incana) leaves: nutrient intake and utilization in calves. Livestock Science 118:157-165.

Singh B and Bhat T K 2001 Tannins revisited-changing perceptions of their effects on animal system. Animal Nutrition and Feed Technology 1:3–18.

Singh B and Sahoo A 2004 Newer concepts, implications and approaches in the use of unconventional feeds to improve animal production. In: Proceeding, V Biennial Conference of ANA, 24-26 November, 2004. NIANP, Bangalore, India. pp1-14.

Snedecor G W and Cochran W G 1989 Statistical Methods, 8th edition. Iowa State Univ. Press, Ames, IA.

Varga G A and Kolver E S 1997 Microbial and animal limitations to fiber digestion and utilization. The Journal of Nutrition 127:819S-823S. http://jn.nutrition.org/cgi/reprint/127/5/819S

Vercoe P E, Durmic Z and Revell D K 2009 Rumen microbial ecology: Helping to change landscapes. Options Mediterraneennes A 85:225-236. http://ressources.ciheam.org/om/pdf/a85/00801010.pdf

Van Soest P J and Mason V C 1991 The influence of the maillard reaction upon the nutritive value of fibrous feeds. Animal Feed Science and Technology 32:45-56.

 Waghorn G C and McNabb W C 2003 Consequences of plant phenolic compounds for productivity and health of ruminants. Proceedings of The Nutrition Society 62:383-392. http://journals.cambridge.org/action/displayFulltext?type=1&fid=2060048&jid=PNS&volumeId=62&issueId=02&aid=2060036

Wang Y, Waghorn G C, Douglas G B, Barry T N and Wilson G F 1994 The effects of the condensed tannin in Lotus corniculatus upon nutrient metabolism and upon body and wool growth in grazing sheep. Proceedings of The New Zealand Society of Animal Production 54: 219–222.

Received 5 May 2010; Accepted 29 August 2010; Published 1 November 2010

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