Manipulation of rumen ecology by dietary lemongrass (Cymbopogon citratus Stapf.) powder supplementation 1

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1 Manipulation of rumen ecology by dietary lemongrass (Cymbopogon citratus Stapf.) powder supplementation 1 M. Wanapat,* 2 A. Cherdthong,* P. Pakdee,* and S. Wanapat *Tropical Feed Resources Research and Development Center (TROFREC), Faculty of Agriculture, Khon Kaen University, Khon Kaen, 40002, Thailand; and Department of Plant Science and Natural Resources, Faculty of Agriculture, Khon Kaen University, Khon Kaen, 40002, Thailand ABSTRACT: This experiment was conducted to investigate the effect of lemongrass [Cymbopogon citratus (DC.) Stapf.] powder (LGP) on rumen ecology, rumen microorganisms, and digestibility of nutrients. Four ruminally fistulated crossbred (Brahman native) beef cattle were randomly assigned according to a 4 4 Latin square design. The dietary treatments were LGP supplementation at 0, 100, 200, and 300 g/d with ureatreated rice straw (5%) fed to allow ad libitum intake. Digestibilities of DM, ether extract, and NDF were significantly different among treatments and were greatest at 100 g/d of supplementation. However, digestibility of CP was decreased with LGP supplementation (P < 0.05), whereas ruminal NH 3 -N and plasma urea N were decreased with incremental additions of LGP (P < 0.05). Ruminal VFA concentrations were similar among supplementation concentrations (P > 0.05). Total viable bacteria, amylolytic bacteria, and cellulolytic bacteria were significantly different among treatments and were greatest at 100 g/d of supplementation ( , , and cfu/ml, respectively). Protozoal populations were significantly decreased by LGP supplementation. In addition, efficiency of rumen microbial N synthesis based on OM truly digested in the rumen was enriched by LGP supplementation, especially at 100 g/d (34.2 g of N/kg of OM truly digested in the rumen). Based on this study, it could be concluded that supplementation of LGP at 100 g/d improved digestibilities of nutrients, rumen microbial population, and microbial protein synthesis efficiency, thus improving rumen ecology in beef cattle. Key words: lemongrass powder, manipulation, microbial protein synthesis, rumen ecology, rumen fermentation, supplementation 2008 American Society of Animal Science. All rights reserved. J. Anim. Sci : doi: /jas INTRODUCTION In the last few years, there has been an increasing interest in exploiting natural products as feed additives to solve problems in animal nutrition and livestock production (Wallace et al., 2002). Herbs have been evaluated for their ability to alter ruminal fermentation and improve nutrient utilization in ruminants (Wang et al., 2000; Greathead, 2003). In addition, supplementation of herbs to ruminants could possibly decrease stress to the animals (Hosoda et al., 2006). When dried herbs were fed to lactating cows, the characteristic smell of 1 We express our sincere thanks to the Tropical Feed Resources Research and Development Center (TROFREC), Khon Kaen University, Thailand Research Fund (TRF) through the Royal Golden Jubilee PhD Program for providing financial support for the research and use of the research facilities. 2 Corresponding author: metha@kku.ac.th Received January 19, Accepted August 8, cow milk was suppressed due to the transmission of components peculiar to such herbs into the milk of cows (Ando et al., 2001). Lemongrass [Cymbopogon citratus (DC.) Stapf.] is one herb of interest, and it is widely used in tropical countries, especially in Southeast Asia in human foods. Citral is a key component of the essential oils extracted from lemongrass that is necessary for vitamin A synthesis. More recently, essential oils have attracted attention for their potential as alternatives to feed antibiotics and growth promoters in livestock (Wallace, 2004). Essential oils from a variety of sources have been shown to alter growth and metabolism of several types of bacteria, including rumen bacteria. However, many of the investigations conducted to date on essential oils have been laboratory-based (i.e., in vitro incubations) and of a short-term nature (McIntosh et al., 2003; Newbold et al., 2004; Castillejos et al., 2005; Benchaar et al., 2007). Effects of lemongrass on antibacterial (Valero and Salmeroìn, 2003), antioxidant (Cheel et al., 2005), antinociceptive (Viana et al., 2000), and antihy- 3497

2 3498 Wanapat et al. Table 1. Ingredients and chemical compositions of concentrate, urea-treated rice straw (UTS), and lemongrass powder (LGP) Ratio Concentrate UTS LGP Ingredients % Cassava chip 70.0 Palm meal 5.0 Brewers grain 3.0 Coconut meal 3.0 Urea 3.0 Rice bran 7.0 Molasses 5.1 Salt 1.0 Mineral mix 2.5 Sulfur 0.5 Chemical composition, % of DM DM, % OM, % Ether extract, % NDF, % ADF, % CP, % Ca, % 0.6 P, % 0.3 ME, 1 MJ/kg TDN Price, US$/kg Calculated values. per-nh 3 -producing ruminal bacterial (McIntosh et al., 2003) activities have been studied. In addition, Hosoda et al. (2006) investigated the effects of the supplementation in the diet of Holstein steers with 3 herbs, especially lemongrass leaf, on blood metabolites and rumen fermentation, but the effects on rumen microbes have not yet been clearly investigated. The objective of this experiment was to study effects of dried lemongrass [C. citratus (DC.) Stapf.] powder (LGP) supplementation on rumen ecology, rumen microorganisms, microbial protein synthesis, and digestibility of nutrients in beef cattle. MATERIALS AND METHODS All procedures involving animals were approved by the Ethical Principles for the Use of Animals for Scientific Purposes of the National Research Council of Thailand. Animals, Treatments, and Experimental Design Four ruminally fistulated crossbred (Brahman native) beef cattle steers with initial BW of 200 ± 50 kg were randomly assigned according to a 4 4 Latin square design to investigate the effect of LGP with urea-treated rice straw (UTS; 5% urea, after method of Wanapat, 1999) as a roughage source on rumen ecology, rumen microorganisms, microbial protein synthesis, and digestibility of nutrients. The dietary treatments were as follows: supplementation with LGP at 0, 100, 200, and 300 g/d. Concentrates containing 13% CP and 75% TDN were offered at 0.5% of BW/d, and UTS was provided for ad libitum intake. The LGP was prepared from whole fresh lemongrass (C. citratus (DC.) Stapf.; 10 to 12 mo], chopped and dried at 60 C for 2 d, ground to pass a 1-mm screen using a Cyclotech Mill (Tecator, Höganäs, Sweden), and then mixed in concentrate. All animals were kept in individual pens, and water was available for ad libitum consumption. The experiment was conducted for 4 periods, and each period lasted 21 d. During the first 14 d, all animals were fed respective diets for ad libitum intake, whereas during the last 7 d, the animals were moved to metabolism crates for total collection during which time they were restricted to 90% of the previous voluntary feed intake of straw and supplemented with concentrate at 0.5% of BW daily to ensure total feed intake. Chemical composition of concentrate, LGP, and UTS are shown in Table 1. Data Collection and Sampling Procedures Feeds were sampled and fecal samples were collected from the total collection of each individual steer on each treatment during the last 7 d of each period at morning and afternoon feeding. Composited samples were dried at 60 C, ground (1-mm screen using Cyclotech Mill, Tecator), and then analyzed for DM, ether extract, ash, CP content (AOAC, 1985), and NDF and ADF (Goer-

3 Lemongrass supplementation on rumen ecology 3499 ing and Van Soest, 1970). At the end of each period, rumen fluid and jugular blood samples were collected at 0, 2, 4, and 6 h after feeding. At the time of sampling, 10 ml of blood was drawn into each of the tubes. Each tube contained 12 mg of EDTA. Approximately 200 ml of rumen fluid was taken at each time from the middle part of the rumen using a 60-mL hand syringe. Temperature and ph of rumen fluid were measured using a portable ph and temperature meter (Hanna Instruments HI 8424 microcomputer, Singapore). Rumen fluid samples were then filtered through 4 layers of cheesecloth. Samples were divided into 3 portions; 1 portion was used for NH 3 -N analysis with 5 ml of 1 M H 2 SO 4 added to 50 ml of rumen fluid. The mixture was centrifuged at 16,000 g for 15 min, and the supernatant was stored at 20 C before NH 3 -N analysis using the micro-kjeldahl methods (AOAC, 1985) and VFA analysis using HPLC (Samuel et al., 1997). A second portion was fixed with 10% formalin solution in sterilized 0.9% saline solution. The total direct count of bacteria, protozoa, and fungal zoospores were made by the methods of Galyean (1989) based on the use of a hemocytometer (Boeco, Hamburg, Germany). Another portion was cultured for groups of bacteria using a rolltube technique (Hungate, 1969) for identifying bacteria groups (cellulolytic, proteolytic, amylolytic, and total viable count bacteria). A blood sample (about 10 ml) was collected from a jugular vein (at the same time as rumen fluid sampling) into tubes containing 12 mg of EDTA, and plasma was separated by centrifugation at 500 g for 10 min and stored at 20 C until analysis of plasma urea N according to the method of Crocker (1967). Urine samples were analyzed for total N (IAEA, 1997), and allantoin in urine was determined by HPLC as described by Chen and Gomes (1995). The amount of microbial purines absorbed was calculated from purine derivative excretion based on the relationship derived by Chen and Gomes (1995). Statistical Analysis Statistical analyses were performed using the GLM procedure (SAS Inst. Inc., Cary, NC). Data were analyzed using the model Y ijk = μ + M i + A j + P k + ε ijk, where Y ijk = observation from animal j, receiving diet i, in period k; μ = the overall of mean; M i = the mean effect of LGP concentration (i = 1, 2, 3, 4); A j = the effect of animal (j = 1, 2, 3, 4); P k = the effect of period (k = 1, 2, 3, 4); and ε ijk = the residual error. Treatment means were statistically compared by the new multiple range test of Duncan (Steel and Torrie, 1980). RESULTS AND DISCUSSION Chemical Composition of Feeds The chemical compositions of concentrate, roughage, and LGP are presented in Table 1. Concentrate diets contained 13.1% CP and 75% TDN on a DM basis. The concentrate contained 70% cassava chip and, therefore, low NDF. The UTS contained 58.4% DM and 8.4% CP on a DM basis and was similar to that reported by Wanapat (2000). Furthermore, ether extract content of LGP was 1.57% on a DM basis. Effect on Feed Intake and Digestibility The effects of LGP concentration on feed intake of beef cattle are presented in Table 2. Total DMI was not significantly affected (P < 0.05) by LGP supplementation concentration. These results were similar to previous work by Hosoda et al. (2006), which supplemented lemongrass leaf to dairy steers. Similarly, Benchaar et al. (2007) observed no change in DMI when dairy cows were fed a mixture of essential oil compounds at 750 mg/d. The CP digestibility was significantly different (P < 0.05) and was greatest in control group (77.9%). Apparent digestibilities of DM, ether extract, and NDF were significantly different (P < 0.05) among treatments with the greatest values for beef cattle fed LGP at 100 g/d (Table 2). Ando et al. (2003) reported that supplementation of essential oil (peppermint source) could increase nutrient digestibilities significantly. However, in our study, slightly less nutrient digestibilities were found when increasing concentration of LGP. This result showed that supplementation of LGP at 100 g/d was most suitable. Characteristics of Ruminal Fermentation and Blood Metabolites in Cattle Measured rumen variables included temperature, ph, NH 3 -N, and VFA. Plasma urea N was also determined to investigate the relationship with rumen NH 3 -N and protein utilization. The pattern of ruminal fermentation and overall means are presented in Table 3. Rumen fluid ph and temperature were not altered among treatments, and the values were stable at ph 6.4 to 6.5 and temperature of 38.7 to 39.6 C, and the ph was within the range considered optimal for microbial digestion of fiber and protein [6.0 to 7.0; Hoover (1986)]. Supplementation with LGP decreased (P < 0.05) ruminal NH 3 -N and plasma urea N. Similarly, supplementation of essential oil from peppermint herb or high essential oil at 3,000 mg/l significantly decreased NH 3 -N concentration (Ando et al., 2003; Busquet et al., 2006). Ruminal NH 3 -N is a major source of N for microbial protein synthesis (Bryant, 1974; Erdman et al., 1986). Ruminal NH 3 -N concentrations were 15.7 to 19.1 mg/dl and were close to those previously reported by Church and Santos (1981) and Wanapat and Pimpa (1999). Decreasing rumen NH 3 -N concentrations also resulted in decreasing concentrations of plasma urea N. Concentrations of plasma urea N are highly correlated to the concentration of NH 3 production in the rumen (Preston et al., 1965; Lewis, 1975). There is considerable evidence that essential oils may affect rumen microbial

4 3500 Wanapat et al. Table 2. Feed intakes and apparent digestibility of beef cattle fed different concentrations of lemongrass powder (LGP) Concentration of LGP supplementation, g/d DMI, % of BW daily UTS Concentrate 0.6 a 0.7 ab 0.8 ab 0.9 b Total Apparent digestibility, % DM 64.7 a 74.8 b 66.8 a 62.0 a OM CP 77.9 a 74.0 b 70.8 bc 69.7 c Ether extract 83.5 ab 86.3 a 84.1 ab 80.0 b NDF 71.0 ab 77.5 a 71.9 ab 66.6 b ADF a c Values within the same row not bearing a common superscript differ (P < 0.05). 2 UTS = urea-treated straw. fermentation (McIntosh et al., 2000; McEwan et al., 2002). Wallace et al. (2002) suggested that the main mechanism of action of essential oils was the inhibition of bacterial attachment to feed particles, and subsequently, NH 3 production (deamination) from AA was decreased. This effect was mediated by the effects of essential oils particularly on hyper-nh 3 -producing bacteria. McEwan et al. (2002) reported that addition of essential oils resulted in a reduction in the number and diversity of hyper-nh 3 -producing bacteria, resulting in decreased rate of NH 3 production from AA. Reduction in protein degradation suggests that at least part of the effects of essential oils can be attributed to decreased proteolysis (Molero et al., 2004). In contrast, Hosoda et al. (2006) reported that urea N concentrations were increased significantly (5.0 to 5.4 mg/dl) by lemongrass leaf supplementation. The production of total VFA, acetate acid, propionic acid, and butyric acid proportions, acetic:propionic ratio, and acetic plus butyric:propionic ratio are shown in Table 3. There were no significant differences (P > 0.05) in VFA concentrations. Total VFA concentrations in all treatments ranged from to mm and were similar to those reported by France and Siddons (1993). Moreover, LGP supplementation had no effects on other VFA variables. These results were in agreement with Hosoda et al. (2006), who reported that supplementation of lemongrass leaf at 5% of the diet did not alter VFA concentration in the rumen in dairy steers. Rumen Microorganism Population Table 4 presents the rumen microorganism population data. Ruminal microbial counts and variable bacteria were significantly different (P < 0.01) among treatments; bacteria, zoospores, total variable bacteria, amylolytic bacteria, and cellulolytic bacteria were greatest when LGP was supplemented at 100 g/d and decreased when supplemented at 200 and 300 g/d, which correlates with the greatest DM and NDF digestibility when LGP was supplemented at 100 g/d. These results Table 3. Rumen temperature, NH 3 -N, plasma urea N, and VFA concentration as affected by feeding different concentrations of lemongrass powder (LGP) in beef cattle Concentration of LGP supplementation, g/d Ruminal ph Ruminal temperature, C NH 3 -N, mg/dl 19.1 a 17.5 b 16.7 bc 15.7 c 0.43 < Plasma urea N, mg/dl 13.5 a 11.1 b 10.8 b 12.3 ab Total VFA, mm VFA, mol/100 mol Acetate (C2) Propionate (C3) Butyrate (C4) C2:C C2 + C4:C a c Values within the same row not bearing a common superscript differ (P < 0.05).

5 Lemongrass supplementation on rumen ecology 3501 Table 4. Effect of concentration of lemongrass powder (LGP) supplementation in concentrate on ruminal microbes and viable bacteria in beef cattle Concentrations of LGP supplementation, g/d Ruminal microbes, cells/g Bacteria, a 6.5 b 5.2 c 4.3 a 0.07 < Protozoa, a 4.8 b 4.5 b 4.6 b Fungal zoospore, a 1.1 b 0.8 c 0.7 a < Viable bacteria, cfu/ml Total, a 4.5 b 3.1 a 3.0 a Amylolytic, a 1.7 b 1.3 c 0.9 d Proteolytic, a 1.1 b 0.9 c 0.7 d Cellulolytic, a 2.0 b 1.4 c 1.0 bc a d Values within the same row not bearing a common superscript differ (P < 0.05). showed effects of essential oil from LGP, which changed diversity of rumen microorganism. Many essential oils have dose-dependent effects on bacteria, protozoa, and fungi (Greathead, 2003). In general, gram-positive bacteria appeared to be more susceptible to inhibition by plant essential oil compounds than did gram-negative bacteria (Davidson and Naidu, 2000). This effect has been related to the presence of an outer membrane on gram-negative organisms, which endows them with a hydrophilic surface that acts as a strong impermeability barrier (Nikaido, 1994). The activity of LGP affects electron transport, ion gradients, protein translocation, phosphorylation steps, and other enzyme-dependent reactions, causing the affected bacteria to lose chemiosmotic control (Ultee et al., 1999). In our study, supplementation of LGP at 200 and 300 g/d decreased bacterial populations relative to 100 g/d of LGP, possibly due to decreases of gram-positive bacteria. Moreover, proteolytic bacterial populations were decreased with increasing concentration of LGP (1.4 to cfu/ml). Similarly, Wallace et al. (2002) reported that hyper-nh 3 -producing bacteria as proteolytic bacteria group were the most sensitive of rumen bacteria to essential oil in pure culture. Ruminobacter amylophilus and Prevotella spp. as hyper-nh 3 -producing bacteria have a great capability to generate NH 3 from AA (Russell et al., 1991) and may be key organisms in mediating these effects. In addition, R. amylophilus was inhibited by essential oil concentrations of at least 200 mg/l (McIntosh et al., 2003). Protozoal populations tended to be decreased with increasing concentration of LGP in the diets. In agreement with these observations, Ando et al. (2003) reported that supplementation of essential oil from peppermint decreased protozoal populations significantly (P < 0.05). N Balance and Efficiency of Microbial Protein Synthesis As shown in Table 5, N intake, excretion of N, and N absorption were not affected by treatments. Nitrogen retention was not affected by 100 g/d of LGP but Table 5. Effect of concentration of lemongrass powder (LGP) supplementation in concentrate on N balance, excretion of urinary purine derivatives (PD), and microbial N supply in cattle Concentration of LGP supplementation, g/d N balance, g/d N intake Fecal N Urinary N N absorption N retention 20.1 a 19.9 a 15.6 b 15.5 b PD, mmol/d Allantoin excretion 37.5 ab 45.7 a 32.6 b 31.3 b Allantoin absorption 27.8 a 35.7 b 24.0 a 20.5 a Microbial protein supply, 2 g of N/d 49.9 ab 57.5 a 45.3 b 43.1 b EMNS, 3 g N/kg of OMDR 32.1 a 34.2 a 23.8 b 22.1 b a,b Values within the same row not bearing a common superscript differ (P < 0.05). 2 Microbial N (g of N/d) = total absorption of PD (Chen et al., 1993). 3 EMNS = efficiency of microbial N synthesis [g of N/kg of OM digested in the rumen (OMDR)], assuming that rumen digestion = 65% of digestion in total tract (ARC, 1990).

6 3502 was decreased when 200 or 300 g/d was supplemented. With regards to N utilization, Owens and Zinn (1988) stated that N excretion and N retention should reflect differences in N metabolism, because N retention was the most important index of the protein nutrition status of ruminants. Supplementation at 100 g/d of LGP resulted in the greatest allantoin absorption. However, efficiency of microbial N synthesis was not affected by 100 g/d of LGP, suggesting that any increase in microbial N synthesis was related to increases in diet digestion. Microbial N supplies from the rumen are summarized in Table 5. The microbial N supply as calculated from purine derivative excretion using the equation of Chen and Gomes (1995) ranged from 43.1 to 57.5 g of N/d. The efficiency of rumen microbial protein synthesis was decreased as concentration of LGP supplementation increased above 100 g/d. 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