Effects of Chitosan on Enteric Methane Production and Nutrient Digestibility of Beef Heifers

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1 Effects of Chitosan on Enteric Methane Production and Nutrient Digestibility of Beef Heifers D. D. Henry, F. M. Ciriaco, M. Ruiz-Moreno, V. R. G. Mercadante, T. M. Schulmeister, G. C. Lamb, N. DiLorenzo Synopsis Global impact of cattle production is a growing concern for policy makers and the general public and chitosan has the potential of reducing the carbon footprint of beef production by enhancing total tract digestibility of high-roughage diets. Summary A study was conducted to evaluate the effects of chitosan on enteric methane (CH 4) and total tract nutrient digestibility of beef heifers consuming either a high-concentrate (HC; 85% concentrate) or a low-concentrate (LC; 36% concentrate) diet. Heifers were provided their respective diets ad libitum along with one of three dietary inclusion rates of chitosan: 1) CTRL=0.0% of chitosan in the diet dry matter (DM); 2) CHI-0.5=0.5% of chitosan in the diet DM; and 3) CHI-1=1.0% of chitosan in the diet DM. Twenty-four crossbred heifers were housed in the University of Florida Feed Efficiency Facility (FEF) at the North Florida Research and Education Center (NFREC) for two 22-d periods where daily intake was monitored using the GrowSafe system. Neither chitosan nor diet had any effect on dry matter intake (DMI). Average daily gain (ADG) was not affected by chitosan; however, as expected, heifers consuming HC had greater gains compared to those consuming LC. Enteric CH 4 production was similar among chitosan treatments. When comparing CH 4 production between HC and LC, heifers consuming LC produced nearly three times as much CH 4 per day compared to HC. There was a chitosan diet interaction for digestibility of DM, organic matter (OM), neutral detergent fiber (NDF) and acid detergent fiber (ADF). Heifers consuming LC with CHI-1 had greater digestibility of DM, OM and ADF with a tendency for enhanced digestibility of NDF. Chitosan did not have an effect on enteric production of CH 4; however, total tract digestibility of nutrients was improved when heifers received 1.0% of chitosan in the diet DM along with a high-roughage diet. Introduction American agriculture produces approximately 6% of total U.S. greenhouse gas emissions (EPA, 2012). Livestock production accounts for 3.2% of the approximately 430 teragrams (Tg) CO 2eq produced by agriculture in the US each year. Enteric CH 4 productions is responsible for nearly 55% of livestock s contribution to greenhouse gas emissions (Pitesky et al., 2009). Production of CH 4 warranted of review not only because it has about 25 times the global warming potential of CO 2, but also due to its effects on energy expenditures. In cattle, CH 4 can account for up to 12% of gross energy losses; thereby, it is possible that for every 100 calories taken in, 12 calories could be lost to CH 4 production (Johnson and Johnson, 1995). For several years, researchers have sought strategies to mitigate enteric CH 4 production in ruminants through various approaches, such as lipid supplementation (Beauchemin et al., 2007), alternative hydrogen sinks (e.g., nitrates and sulfates; van Zijderveld et al., 2010) and ionophores (Appuhamy et al., 2013). It is crucial to continue investigating current strategies as well as novel techniques of CH 4 emission abatement. North Florida Research and Education Center, University of Florida, Marianna, FL

2 Chitosan (N-acetyl-D-glucosamine polymer) is a natural biopolymer formed from the deacetylation of chitin. Chitin, the second most abundant organic compound on earth, can be found in the cell walls of lower plants and the exoskeletons of some arthropods and crustaceans (e.g., crab and shrimp). Chitosan has been studied for various applications in food preservation and medicine due to its antimicrobial actions (Cuero, 1999; Shahidi et al., 1999). In the U.S., chitosan has been deemed generally recognized as safe (GRAS) status, which ultimately allows for chitosan to be used as an alternative to antibiotics. Chitosan should not be considered a single compound, but rather a series of compounds with differing levels of deacetylation and other physic-chemical characteristics (Goiri et al., 2009). Chitosan, as an in vivo CH 4 inhibitor, is a novel product for ruminants; however, there have been studies with monogastrics (i.e., poultry and swine) showing it can alter protein fermentation in the lower gastrointestinal tract (O Shea et al., 2011; Han et al., 2013). The objective of this study was to determine the effects of increasing inclusion rates of chitosan on enteric CH 4 production and total tract digestibility of nutrients in beef heifers. Materials and Methods The study was conducted at the FEF in Marianna, FL. Chitosan was provided by PharmaNutrients Inc. (Lake Forest, IL). A total of 24 crossbred heifers (838±68 lb; average body weight ± standard deviation) were used in a randomized block design with a 2 3 factorial arrangement of treatments. Factors included diet, a HC (85% concentrate) and LC (36% concentrate), and chitosan inclusion rate, 0.0, 0.5, and 1.0% of diet DM. On d 0, heifers were weighed after 16-h feed withdrawal and stratified by initial body weight (BW), and randomly assigned to one of three inclusion rates of chitosan (DM basis): CTRL = no supplementation CHI-0.5 = 1 lb/d of the 50:50 molasses and crude glycerol mixture CHI-1.0 = 3 lb/d of the 50:50 molasses and crude glycerol mixture Heifers were housed two per pen for 22 d and had ad libitum access to water and their respective diets (Table 1). After the 14-d adaptation to diets and facility, heifers were weighed and collection of daily feed intake data began. Each pen at the FEF was equipped with two GrowSafe feed bunks (GrowSafe System Ltd., Airdrie, Alberta, Canada) to record intake by weight change measured to the nearest gram. Heifers were adapted to CH 4 collection canisters (Figure 1) for three days prior to collection. Collection and analysis of CH 4 was performed for five consecutive days (d 13 to 18). Beginning on d 17 and d 18, feed and fecal samples were collected, respectively, for four consecutive days to determine apparent total tract digestibility of DM, OM, CP, NDF, and ADF. Fecal samples were collected twice daily at 0800 and 1600 h via rectal grab. Feed samples were pooled within pen and fecal samples were pooled within heifer. Indigestible NDF (indf) was used as an internal indigestible marker. On d 22 heifers were weighed again after withholding feed for 16 h, for evaluation of performance during the entire period. Concentrations of indf in feed and feces were determined as described by Cole et al. (2011) with the following modification: in vitro incubations were conducted for 288 h instead of 96 h using the Daisy II incubator (Ankom Technology, Macedon, NY) to ensure complete digestion of potentially digestible NDF in hay as reported by Krizsan and Huhtanen (2013).

3 Data were analyzed as a randomized block design with a 2 3 factorial arrangement using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC) with heifer as the experimental unit. The model included the fixed effects of diet and chitosan inclusion rate and the random effect of period (block). Orthogonal polynomial contrasts were conducted to determine linear and quadratic effects of chitosan inclusion rate. Significance was declared at P Results Inclusion of chitosan had no effect (P>0.05) on ADG (data not shown) or DMI. No diet chitosan interactions occurred for CH 4 production (Table 2). There was no effect of chitosan on CH 4 production as g/d (P=0.71), g/kg of DMI (P=0.65), g/kg of DM digested (P=0.77), or g/kg of metabolic BW (P=0.79). However, diet affected (P<0.01) enteric CH 4 production where animals consuming LC produced nearly three times more CH 4 than animals consuming HC. A diet chitosan interaction was observed for nutrient digestibility of DM (P=0.05), OM (P=0.03) and ADF (P=0.05) with a tendency for an interaction on NDF (P=0.08) digestibility (Table 3). When chitosan was included up to 1.0% of the diet DM in the LC diet, total tract digestibility of DM and OM was increased by 21 and 19%, respectively. The increase on DM and OM digestibility was likely due to a 21% increase in ADF digestibility and a tendency for a 21% increase in NDF digestibility. With the results observed from this study, it appears that chitosan may alter ruminal fermentation to improve total tract digestibility of low-quality diets. It may be beneficial to provide cattle on low-quality pastures, such as cow-calf systems in the SE U.S., with chitosan to improve digestibility of the forage; however, further research is needed to test this hypothesis. Literature Cited Appuhamy, J. A. D. R. N., et al J. Dairy Sci. 96: Beauchemin, K. A., et al Can. J. Anim. Sci. 87: Cuero, R. G Chitin and Chitinases EPA Goiri, I., et al Anim. Feed Sci. Technol. 148: Han, W., et al J. Anim. Sci. 91: Johnson, K. A. and D. E. Johnson J. Anim. Sci. 73: O Shea, C. J., et al J. Anim. Sci. 89: Pitesky, M. E., et al Clearing the Air. Livestock s Contribution to Climate Change. Shahidi, F. J., et al Trends Food Sci. Technol. 10:37-51.

4 van Zijderveld, S. M., et al J. Dairy Sci. 93: Table 1. Ingredients and chemical composition of the basal diets Item 36% concentrate (LC) 85% concentrate (HC) Ingredient composition, % DM Corn gluten feed, pelleted Soybean hulls, pelleted Peanut hulls Liquid supplement Meal supplement Chitosan supplement Chemical composition DM, % OM, % DM CP, % DM NDF, % DM ADF, % DM Ether extract, % DM Calcium, % DM Phosphorus, % DM Westway Converter SR Southeast 32 (Westway Feed Products Inc., New Orleans, LA). Chemical composition of the supplement (as fed basis): 62% DM; 32.0% CP; 0.1% fat; contained trace minerals (cobalt, copper, iron, manganese, zinc, iodine, and selenium). 2 Supplied vitamins (A, D) and minerals (Beef Four Plus, W.B. Fleming Co. Tifton, GA). 3 Chitosan supplement was comprised of (DM basis) 100% soybean meal for control heifers, 75% soybean meal and 25% chitosan for 0.5% chitosan inclusion rate, and 50% soybean meal and 50% chitosan for 1.0% chitosan inclusion rate.

5 Table 2. Effect of diet and inclusion level of chitosan on DMI and CH 4 emissions in beef heifers HC diet 1 LC diet 2 Chitosan inclusion, % of diet DM Chitosan inclusion, % of diet DM P-value 3 Item SEM 4 DIET CHIT CHIT DIET DMI, lb/d CH 4 emissions g/d < g/kg DMI < g/kg DM digested < g/kg MBW < Comprised of 40% corn gluten feed pellets, 39% soybean hulls pellets, 15% peanut hulls, and 6% vitamins and 2 Comprised of 64% peanut hulls,15% corn gluten feed pellets, 15% soybean hulls pellets, and 6% vitamins and 3 Observed significance levels for the main effects of: DIET = diet (n = 24 heifers/mean), CHIT = chitosan inclusion level (n = 16 heifers/treatment), CHIT x DIET = interaction between chitosan inclusion level and diet. 4 Standard error of the mean, n = 8 heifers/treatment. 5 DMI = Dry matter intake average during the methane collection period. 6 Apparent total tract digestibility measured using indf as indigestible marker. 7 MBW = Metabolic body weight.

6 Table 3. Effect of diet and inclusion level of chitosan on nutrient digestibility when indf was utilized as an internal marker HC diet 1 LC diet 2 Chitosan inclusion, % of diet DM Chitosan inclusion, % of diet DM P-value 3 Item SEM 4 DIET CHIT CHIT DIET Digestibility, % DM 62.1 c 62.8 c 60.9 c 33.2 a 35.7 ab 40.3 b 1.73 < OM 63.0 c 63.5 c 61.9 c 34.5 a 37.7 ab 41.1 b 1.46 < CP < NDF 46.3 c 46.7 c 43.7 c 21.3 a 23.8 ab 25.7 b 1.53 < ADF 41.0 bc 41.3 c 36.3 b 18.4 a 21.6 a 22.3 a 1.68 < a,b,c Within a row, means with different superscripts differ, P < Comprised of 40% corn gluten feed pellets, 39% soybean hulls pellets, 15% peanut hulls, and 6% vitamins and 2 Comprised of 64% peanut hulls,15% corn gluten feed pellets, 15% soybean hulls pellets, and 6% vitamins and 3 Observed significance levels for the main effects of: DIET = diet (n = 24 heifers/mean), CHIT = chitosan inclusion level (n = 16 heifers/treatment), CHIT x DIET = interaction between chitosan inclusion level and diet. 4 Standard error of the mean, n = 8 heifers/treatment.