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Animal Feed Science and Technology 147 (2008) 279–291 Available online at www.sciencedirect.com Effect of monensin on performance in growing ruminants reared under different environmental temperatures Ma´rcia Saladini Vieira Salles a,∗, Marcus Antonio Zanetti b, Evaldo Antonio Lencioni Titto b, Renata Maria Consentino Conti c a Ageˆncia Paulista de Tecnologia dos Agronego´cios, Avenida Bandeirantes 2419, CEP: 14030-670 Ribeira˜o Preto, SP, Brazil b Faculdade de Zootecnia e Engenharia de Alimentos-USP, Brazil c Anhanguera Educacional, Brazil Received 3 May 2007; received in revised form 18 January 2008; accepted 30 January 2008 Abstract To evaluate the effect of monensin on the performance of growing cattle under different environ- mental temperatures, 24 male calves (81.9 ± 7.7 kg mean weight and 100 days old) were distributed in a 2 × 2 factorial arrangement, contrasting 0 or 85 mg monensin/animal per day at 24.3 or 33.2 ◦C (environmental temperatures). Monensin supplementation increased weight gain (P=0.036), improved feed efficiency (P=0.040), increased ruminal concentrations of volatile fatty acids (VFA; P=0.003) and decreased the molar proportion of butyrate (P=0.034); all effects irrespective of environmental temperatures. A temperature-dependent monensin effect was detected on nitrogen retention (P=0.018) and N retained:N absorbed ratio (P=0.012). Animals fed monensin retained higher N amounts than those of the non-supplemented ones when the environmental temperature was 33.2 ◦C. Environmental temperature and monensin supplementation showed an interaction effect on urine N concentration (P=0.003). Temperature did not affect N excretion in monensin-fed animals, but increased N excretion in the non-supplemented ones. Monensin increased the crude protein (CP) digestibility (P=0.094) for Abbreviations: CP, crude protein; VFA, volatile fatty acids; NH3-N, ammoniacal nitrogen; DM, dry matter; EE, ether extract; NDF, neutral detergent fiber; GE, gross energy; DMD, dry matter digestibility; CPD, CP digestibility; ADFD, ADF digestibility; EED, EE digestibility; CED, crude energy digestibility; DEI, digestible energy intake; T3, triiodothyronine. ∗ Corresponding author. Tel.: +55 16 36371849. E-mail address: marciasalles@apta.sp.gov.br (M.S.V. Salles). 0377-8401/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2008.01.008 280 M.S.V. Salles et al. / Animal Feed Science and Technology 147 (2008) 279–291 animals at both temperatures. In conclusion, monensin changes the metabolism of the heat-stressed animals by increasing rumen VFA concentration, digestibility and protein retention, thus improving food use and weight gain. © 2008 Elsevier B.V. All rights reserved. Keywords: Ammoniacal nitrogen; Digestibility; Feed efficiency; Nutrition; Volatile fatty acids; Weight gain 1. Introduction Animals from tropical and subtropical areas are under heat stress most of the year, a condition that impairs productivity. Nutritional management has been proposed to decrease the effects of heat stress. Monensin-supplemented food improves rumi- nant performance by enhancing feed efficiency (Goodrich et al., 1984; Rumsey, 1984; Schelling, 1984). Thus, unraveling monensin action on heat-stressed animals is impor- tant for improve performance of growing ruminants in the tropical and subtropical areas. Ruminant rearing aims at the best conversion of forage and grains into meat and milk for human consumption, and economically important task for producers (Johnson, 1987). As such feed conversion depends on environmental conditions, the search for new feed additives that may reduce heat stress effects in these animals is a relevant task for improving efficiency and performance in ruminants. Management of rumen fermentation efficiency has been achieved by increasing propi- onate production and decreasing methanogenesis and proteolysis. The first studies toward this goal manipulated diet, but in recent decades feed additives have been investigated and used in animal feeding (Bergen and Bates, 1984). Ionophores promote competition that benefits certain rumen microorganisms while harming others. Metabolic energy availability is improved because propionate pro- duction in rumen increases and methane production declines (McGuffey et al., 2001). Another important effect of monensin is the decreased protein and peptide degrada- tion by rumen microorganisms (Wallace et al., 1990). The decreased microbial protein synthesis is compensated by increased dietary protein, which reaches the intestine with- out changing the total amount of absorbed amino acids (NRC, 1989). This ionophore action consists of decreasing the number of monensin-sensitive bacteria, which are ammo- nia producers and require energy sources other than carbohydrates (Yang and Russel, 1993). Goodrich et al. (1984) suggested that monensin improves dry matter digestibility, decreases heat production in fasting animals and increases net energy for enhanced per- formance. Since performance is affected by monensin and environmental temperature, the question is whether environmental temperature modulates monensin action on per- formance. Thus, this study investigated the hypothesis that heat stressor affects monensin supplementation action on performance, rumen parameters and feed digestibility in Holstein calves. M.S.V. Salles et al. / Animal Feed Science and Technology 147 (2008) 279–291 281 2. Materials and methods 2.1. Animals, experimental design and housing Twenty-four male Holstein calves (81.9 ± 7.7 kg mean weight and 100 days old) were blocked by weight (Block 1: 67.0–80.0 kg; Block 2: 82–95 kg) and randomly assigned to one of four groups: without monensin at 24.3 or at 33.2 ◦C; fed 85 mg monensin/animal per day at 24.3 or at 33.2 ◦C. A heat stressor (33.2 ◦C) was imposed on 12 animals in a climatic chamber with controlled air heating. Circadian temperature variation was simulated by maintaining higher temperatures from 11:00 to 24:00 h and the lower temperatures from midnight to dawn. The remaining 12 animals were maintained in a covered area next to the cli- matic chamber. Air temperature and humidity were recorded in both environments, three times a day (7:00, 13:00 and 17:00 h), using a SATO thermohygrometer (battery powered, graphic display, resolution of 0.1 ◦C, and 1% relative air humidity) (mean values are in Table 1). The animals were kept in individual rubber-covered iron cages. The experimental period lasted 38 days (10 days for temperature and cage adaptation and monensin addition, and 28 days for data collection). 2.2. Diet and feeding schedule Diet consisted of total mixed ration offered ad libitum twice a day (Table 2). The source of the monensin was Rumensin® (Eli Lilly-Elanco), which contains 100 g active monensin/kg. The monensin was individually administered in soft gels capsules containing 0.85 g of Rumensin® (85 mg monensin/animal per day, a dose providing 33 mg monensin/kg DM) immediately after feeding by inserting the capsules into the esophagus with a syringe. This procedure ensured the same ionophore dose for the animals, because heat stress is expected to decrease feeding. Table 1 Weekly means of environmental temperature and relative humidity at three different times of the day inside and outside the climate chamber where the animals were held Weeks Temperature (◦C) Relative air humidity (%) 7:00 13:00 17:00 Mean ± S.E.M. 7:00 13:00 17:00 Mean ± S.E.M. Inside the climate chamber 1st 28.6 34.7 35.1 32.8 ± 0.1 74.8 62.7 60.3 65.9 ± 0.4 2nd 29.5 36.4 34.5 33.5 ± 0.2 78.7 56.6 63.6 66.3 ± 0.5 3rd 30.3 36.5 33.0 33.2 ± 0.1 75.1 54.7 49.3 62.8 ± 0.5 4th 29.6 36.9 32.9 33.1 ± 0.2 74.7 55.0 57.9 63.0 ± 0.6 Outside the climate chamber 1st 14.8 22.6 23.2 20.2 ± 0.3 82.4 58.4 59.4 66.8 ± 1.0 2nd 16.6 24.3 23.9 21.6 ± 0.2 90.1 60.7 58.9 69.9 ± 0.9 3rd 17.6 29.1 24.1 23.6 ± 0.3 73.7 42.3 39.6 51.9 ± 0.8 4th 17.7 29.2 27.8 24.9 ± 0.4 72.8 40.5 37.7 50.3 ± 1.2 282 M.S.V. Salles et al. / Animal Feed Science and Technology 147 (2008) 279–291 Table 2 Ingredients ratio and chemical composition of experimental diet Ingredients (g/kg) Coast-cross haya 300 Soy meal 200 Corn meal 320 Wheat meal 125 Mineral supplementation 15 Kaolin 40 Chemical composition (g/kg DM) Dry matter (g/kg) 891 Crude protein 148 Neutral detergent fiber 108 Ash 25 Ether extract 42 Ca 3.9 P 3.8 Mg 2.2 Na 1.5 K 6.6 Cu (mg/kg) 21 Zn (mg/kg) 61 a Crushed to 1.5-cm long. 2.3. Sample collection and chemical analysis Chemical analyses of the rations and feces were based on the AOAC Official Method (1995) number 930.15 for DM, 984.13 for CP, and 920.39 for EE. NDF was analyzed according to Van Soest et al. (1991) and assayed without a heat-stable amylase and expressed inclusive of residual ash. Silva and Queiroz (2002) methodology was used to determine gross energy (GE) in the food and feces using a PARR® bomb calorimeter (Parr Instrument Company, Moline, IL, USA). The animals were weighed at the beginning and at the end of the experiment, always after a 24-h period of food and water deprivation. Rumen liquid was collected by esophageal catheter every Wednesday at 16:00 h. The pH was measured as the liquid was removed, and the remaining material was filtered through several gauze layers. A portion of 1.0 mL formic acid was added to 5.0 mL of the liquid collected, and this mixture was stored in a glass container and frozen at −20 ◦C for further VFA analyses. Another 2.0 mL volume of rumen liquid was placed in a glass container with 1.0 mL sulfuric acid 1N for ammoniacal nitrogen (NH3-N) analysis. The samples were thawed at room temperature and centrifuged for 15 min at 4 ◦C and 15,000 rpm. VFA concentrations were measured by gas chromatography (Varian Star 3600 CX) and ammoniacal nitrogen (NH3-N) by the colorimetric method proposed by Kulasek (1972) and adapted by Foldager (1977). Nitrogen balance was determined for 5 consecutive days and all the urine and feces were collected during this period. Apparent digestibility was also determined in this period and for the next 5 consecutive days (total of 10 days) in all the feces samples. M.S.V. Salles et al. / Animal Feed Science and Technology 147 (2008) 279–291 283 To collect feces, plastic-covered cloth bags were tied to the calves using soft wide cloth belts that caused no injury to the animals. The feces were sampled daily and 100 g/kg was frozen in plastic bags for further analyses. Urine was collected in graduated buck- ets attached to the cages; after the volume was recorded, 50 mL/L of the urine was frozen in plastic bottles for further analyses. To avoid nitrogen volatilization, 200 mL of a solution containing 100 mL/L of sulfuric acid was added daily to the urine collec- tion buckets. Ration samples were also collected daily during nitrogen balance and the apparent digestibility assay and frozen in plastic bags. The balance was carried out accord- ing to Zanetti et al. (1987). The thawed and homogenized feces and urine and ration samples were analyzed for N content based on AOAC Official Method (1995) number 984.13. To assess heat stress in the animals, rectal temperature was measured every morning with a mercury thermometer and radiant body heat with a SATO infrared thermometer; hormone analyses of the animals’ blood were also performed. Blood was sampled by punction of the jugular vein every Tuesdays at 15:00 h; four samples (one mean value) were collected from each animal; the serum was separated by centrifugation, stored in Eppendorf tubes and frozen at −20 ◦C. T3 and cortisol plasma levels were analyzed by immunoenzymatic kits (ELISA tests). 2.4. Statistical analysis and calculations The data were analyzed in a randomized block design (calves were assigned to one of two weight blocks), in a 2 × 2 factorial structure (with or without monensin addiction versus two environmental temperatures), by analysis of variance run in the PROC GLM of SAS (SAS Institute Inc., 1985) with the main effects and interactions contrasting the degrees of freedom (F test). Blood cortisol concentration data were normalized by logarithm transformation. The means presented here correspond to non-transformed data, but S.E.M. and P values were derived from log-transformed data. The results are presented as mean values and standard error of the means. A prob- ability of P<0.10 was accepted as significant. Variables associated to nitrogen balance include: intake, urine and feces concentration, absorbed and retained amount (all vari- ables as g/day) and apparent absorption index (as a fraction), apparent retention index (as a fraction) and index of the relation between nitrogen retention and absorption (as a fraction). The absorbed amount was calculated by subtracting the amount of nutrients excreted in the feces from that ingestion. The absorption index was obtained by dividing the amount of absorbed nutrient by the amount of ingested nutrient. The retained amount was obtained by subtracting the amount of nutrient excreted in both feces and urine from that ingestion. The retention index was calculated by dividing the amount of retained nitro- gen by the amount of ingested nitrogen, and the index of the relation between nitrogen retention and absorption was obtained by dividing the amount retained by the amount absorbed. Performance was measure as feed efficiency, that is, the relation between weight gain (kg) and dry matter (kg) ingested by the animals. The concentration of total fatty acids is the sum of acetic, propionic and butyric acids (mM), and the molar proportion (mol/100 mol) is determined by the amount of total volatile fatty acids. 284 M.S.V. Salles et al. / Animal Feed Science and Technology 147 (2008) 279–291 Table 3 Performance, rectal temperature, radiant body temperature and T3 hormones in growing ruminants supplemented with monensin (M) at different environmental temperatures (T) Monensin P 0 mg 85 mg S.E.M. M T M×T 24.3 ◦C 33.2 ◦C 24.3 ◦C 33.2 ◦C n 6 6 6 6 Feed ingestion (kg DM/day) 2.99 2.14 3.35 2.01 0.15 ns <0.001 ns Weight gain (kg/day) 0.58 0.00 0.74 0.13 0.06 0.036 <0.001 ns Feed efficiency (kg WG/kg DM) 0.19 −0.01 0.22 0.06 0.02 0.040 <0.001 ns Rectal temperature (◦C) 38.71 39.53 38.82 39.42 0.11 ns <0.001 ns Body’s radiant heat (◦C) 26.16 31.69 26.49 31.19 0.29 ns <0.001 ns Cortisol (mg/dL) 0.46 0.59 0.53 0.81 0.05 ns 0.040 ns T3 (ng/dL) 154.15 128.68 152.75 120.93 10.98 ns 0.017 ns n: number of calves; ns: non-significant. 3. Results Monensin increased weight gain (P=0.036) and improved feed efficiency (P=0.040) (Table 3), increased rumen VFA concentration (P=0.003) and decreased the molar pro- portion of butyrate (P=0.034); all of these effects were irrespective of environmental temperatures (Table 4). With respect to N balance (Table 5), the monensin effect on N retention (P=0.018) and N retained:N absorbed (P=0.012) depended on environmental temperature. At 33.2 ◦C, the animals fed monensin retained higher N amounts than those of the non-supplemented ones. Table 4 Rumen parameters in growing ruminants supplemented with monensin (M) at different environmental temperatures (T) Monensin P 0 mg 85 mg S.E.M. M T M×T 24.3 ◦C 33.2 ◦C 24.3 ◦C 33.2 ◦C n 6 6 6 6 pH 6.68 6.58 6.77 6.52 0.07 ns 0.020 ns Ammonia-N (mg%) 17.04 23.58 18.94 26.34 1.54 ns <0.001 ns Total VFA (mM) 60.71 55.02 70.19 65.00 2.75 0.003 0.071 ns VFA (mol/100 mol) Acetate 58.51 57.93 57.63 58.25 1.24 ns ns ns Propionate 29.13 31.42 32.80 32.34 1.65 ns ns ns Butyrate 12.37 10.66 9.57 9.40 0.85 0.034 ns ns Acetate to propionate ratio 2.08 1.86 1.78 1.84 0.14 ns ns ns n: number of calves; ns: non-significant. M.S.V. Salles et al. / Animal Feed Science and Technology 147 (2008) 279–291 285 Table 5 Nitrogen balance in growing ruminants supplemented with monensin (M) at different environmental temperatures (T) Monensin P 0 mg 85 mg S.E.M. M T M×T 24.3 ◦C 33.2 ◦C 24.3 ◦C 33.2 ◦C n 6 6 6 6 Ingested N (g/day) 44.23 32.45 53.19 33.67 4.00 ns 0.001 ns N in feces (g/day) 10.00 10.37 13.06 8.16 1.60 ns ns ns N in urine (g/day) 12.12 22.40 17.23 16.22 1.64 ns 0.011 0.003 Absorbed N (g/day) 34.23 22.09 40.13 25.50 3.10 ns <0.001 ns N absorption 0.77 0.73 0.75 0.76 0.02 ns ns ns Retained N (g/day) 22.12 −0.31 22.89 9.29 3.54 ns <0.001 ns N retention 0.47 −0.01 0.42 0.28 0.06 0.083 <0.001 0.018 Retained N:absorbed N 0.61 −0.11 0.56 0.36 0.09 0.041 <0.001 0.012 n: number of calves; ns: non-significant. The amount of urine N (P=0.003) changed in accordance with both environmental temper- ature and monensin supplementation. The N excretion level in animals fed monensin was similar at both environmental temperatures, and in non-supplemented animals N excretion increased at 33.2 ◦C. Monensin increased the digestibility of crude protein (P=0.094), irrespective of environ- mental temperatures (Table 6). Thermal stressor decreased feed intake (P<0.001), weight gain (P<0.001), feed efficiency (P<0.001) and T3 blood levels (P=0.017), and increased rectal temperature (P<0.001), radiant body heat (P<0.001) and blood cortisol concen- tration (P=0.040) (Table 3). The animals at 33.2 ◦C had higher levels of ammoniacal nitrogen (P<0.001), lower pH (P=0.020) and lower rumen VFA concentration (P=0.071) (Table 4), ingested lower amounts of N (P=0.001) and had lower values of apparent absorbed Table 6 Apparent digestibility of nutrients and digestible energy intake (MJ/kg) in growing ruminants supplemented with monensin (M) at different environmental temperatures (T) Monensin P 0 mg 85 mg S.E.M. M T M×T 24.3 ◦C 33.2 ◦C 24.3 ◦C 33.2 ◦C n 6 6 6 6 DMD 0.81 0.73 0.79 0.79 0.03 ns ns ns CPD 0.80 0.70 0.80 0.78 0.03 0.094 0.038 ns ADFD 0.75 0.67 0.74 0.73 0.03 ns ns ns EED 0.71 0.60 0.73 0.68 0.03 ns 0.030 ns CED 0.80 0.70 0.77 0.77 0.03 ns ns ns DEI 29.68 18.09 34.71 21.31 2.80 ns <0.001 ns n: number of calves; ns: non-significant; DMD: dry matter digestibility; CPD: crude protein digestibility; ADFD: acid detergent fiber digestibility; EED: ether extract digestibility; CED: crude energy digestibility; DEI: digestible energy intake. 286 M.S.V. Salles et al. / Animal Feed Science and Technology 147 (2008) 279–291 (P<0.001) and apparent retained (P<0.001) nitrogen (Table 5), CP digestibility (P=0.038), EE (P=0.030) and DEI (P<0.001) (Table 6). 4. Discussion Male Holstein calves subjects to a heat stressor respond by increasing rectal temperature and radiant body heat, decreasing food ingestion and, as a consequence, obtaining lower weight gain and worse feed efficiency. T3 levels decreased while serum cortisol levels increased at 33.2 ◦C (Table 3). All of these results were expected and corroborate other studies (Dukes and Swenson, 1984; Beede and Collier, 1986; Guyton, 1986; Phillips and Piggins, 1992), that investigated these effects, confirming that animals at 33.2 ◦C were under heat stress. Monensin increased weight gain because feed efficiency improved, possibly by increas- ing total VFA levels and crude protein digestibility in the supplemented calves at both environmental temperatures. The effects of ionophore monensin on animal performance have been studied over the last decades in various studies, most of them describing similar effects to those reported here, indicating that ionophore increases performance by improv- ing feed efficiency (Gill et al., 1976; Potter et al., 1976; Raun et al., 1976; Boling et al., 1977; Haddad and Lourenc¸o, 1977; Bartley et al., 1979; Hanson and Klopfenstein, 1979; Turner et al., 1980; Boucque´ et al., 1982; Boin et al., 1984; Beacom et al., 1988; Grings and Males, 1988; Sprott et al., 1988; Stock et al., 1990, 1995; Lana et al., 1997). In the present study, the daily weight gain increased by 0.140 kg and feed efficiency by 0.05 in monensin-fed animals compared to the non-supplemented ones. Feed ingestion was not affected by monensin because ionophore was