Effect of monensin on performance in growing ruminants reared under different environmental temperatures

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Animal Feed Science and Technology 147 (2008) 279–291

Effect of monensin on performance in growing ruminants reared under different environmental temperatures M´arcia Saladini Vieira Salles a,∗ , Marcus Antonio Zanetti b , Evaldo Antonio Lencioni Titto b , Renata Maria Consentino Conti c a

Agˆencia Paulista de Tecnologia dos Agroneg´ocios, Avenida Bandeirantes 2419, CEP: 14030-670 Ribeir˜ao 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 environmental 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: [email protected] (M.S.V. Salles). 0377-8401/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2008.01.008

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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 ruminant performance by enhancing feed efficiency (Goodrich et al., 1984; Rumsey, 1984; Schelling, 1984). Thus, unraveling monensin action on heat-stressed animals is important 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 propionate 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 production in rumen increases and methane production declines (McGuffey et al., 2001). Another important effect of monensin is the decreased protein and peptide degradation by rumen microorganisms (Wallace et al., 1990). The decreased microbial protein synthesis is compensated by increased dietary protein, which reaches the intestine without changing the total amount of absorbed amino acids (NRC, 1989). This ionophore action consists of decreasing the number of monensin-sensitive bacteria, which are ammonia 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 performance. Since performance is affected by monensin and environmental temperature, the question is whether environmental temperature modulates monensin action on performance. Thus, this study investigated the hypothesis that heat stressor affects monensin supplementation action on performance, rumen parameters and feed digestibility in Holstein calves.

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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 climatic 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 (%) 17:00

Mean ± S.E.M.

Inside the climate chamber 1st 28.6 34.7 2nd 29.5 36.4 3rd 30.3 36.5 4th 29.6 36.9

35.1 34.5 33.0 32.9

32.8 33.5 33.2 33.1

± ± ± ±

Outside the climate chamber 1st 14.8 22.6 2nd 16.6 24.3 3rd 17.6 29.1 4th 17.7 29.2

23.2 23.9 24.1 27.8

20.2 21.6 23.6 24.9

± ± ± ±

7:00

13:00

7:00

13:00

17:00

Mean ± S.E.M.

0.1 0.2 0.1 0.2

74.8 78.7 75.1 74.7

62.7 56.6 54.7 55.0

60.3 63.6 49.3 57.9

65.9 66.3 62.8 63.0

± ± ± ±

0.4 0.5 0.5 0.6

0.3 0.2 0.3 0.4

82.4 90.1 73.7 72.8

58.4 60.7 42.3 40.5

59.4 58.9 39.6 37.7

66.8 69.9 51.9 50.3

± ± ± ±

1.0 0.9 0.8 1.2

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Table 2 Ingredients ratio and chemical composition of experimental diet Ingredients (g/kg) Coast-cross haya Soy meal Corn meal Wheat meal Mineral supplementation Kaolin

300 200 320 125 15 40

Chemical composition (g/kg DM) Dry matter (g/kg) Crude protein Neutral detergent fiber Ash Ether extract Ca P Mg Na K Cu (mg/kg) Zn (mg/kg)

891 148 108 25 42 3.9 3.8 2.2 1.5 6.6 21 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.

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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 buckets 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 collection 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 according 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 probability of P<0.10 was accepted as significant. Variables associated to nitrogen balance include: intake, urine and feces concentration, absorbed and retained amount (all variables 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 nitrogen 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.

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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 S.E.M.

M

T

M×T

6 2.01

0.15

ns

<0.001

ns

0.74 0.22

0.13 0.06

0.06 0.02

0.036 0.040

<0.001 <0.001

ns ns

39.53

38.82

39.42

0.11

ns

<0.001

ns

26.16

31.69

26.49

31.19

0.29

ns

<0.001

ns

0.46 154.15

0.59 128.68

0.53 152.75

0.81 120.93

0.05 10.98

ns ns

0.040 0.017

ns ns

0 mg

n Feed ingestion (kg DM/day) Weight gain (kg/day) Feed efficiency (kg WG/kg DM) Rectal temperature (◦ C) Body’s radiant heat (◦ C) Cortisol (mg/dL) T3 (ng/dL)

85 mg

24.3 ◦ C

33.2 ◦ C

24.3 ◦ C

33.2 ◦ C

6 2.99

6 2.14

6 3.35

0.58 0.19

0.00 −0.01

38.71

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 proportion 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

n pH Ammonia-N (mg%) Total VFA (mM) VFA (mol/100 mol) Acetate Propionate Butyrate Acetate to propionate ratio

S.E.M.

M

T

M×T

6 6.52 26.34 65.00

0.07 1.54 2.75

ns ns 0.003

0.020 <0.001 0.071

ns ns ns

58.25 32.34 9.40 1.84

1.24 1.65 0.85 0.14

ns ns 0.034 ns

ns ns ns ns

ns ns ns ns

85 mg

24.3 ◦ C

33.2 ◦ C

24.3 ◦ C

33.2 ◦ C

6 6.68 17.04 60.71

6 6.58 23.58 55.02

6 6.77 18.94 70.19

58.51 29.13 12.37 2.08

57.93 31.42 10.66 1.86

57.63 32.80 9.57 1.78

n: number of calves; ns: non-significant.

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Table 5 Nitrogen balance in growing ruminants supplemented with monensin (M) at different environmental temperatures (T) Monensin

P

0 mg

n Ingested N (g/day) N in feces (g/day) N in urine (g/day) Absorbed N (g/day) N absorption Retained N (g/day) N retention Retained N:absorbed N

85 mg

24.3 ◦ C

33.2 ◦ C

24.3 ◦ C

33.2 ◦ C

6 44.23 10.00 12.12 34.23 0.77 22.12 0.47 0.61

6 32.45 10.37 22.40 22.09 0.73 −0.31 −0.01 −0.11

6 53.19 13.06 17.23 40.13 0.75 22.89 0.42 0.56

6 33.67 8.16 16.22 25.50 0.76 9.29 0.28 0.36

S.E.M.

M

T

M×T

4.00 1.60 1.64 3.10 0.02 3.54 0.06 0.09

ns ns ns ns ns ns 0.083 0.041

0.001 ns 0.011 <0.001 ns <0.001 <0.001 <0.001

ns ns 0.003 ns ns ns 0.018 0.012

n: number of calves; ns: non-significant.

The amount of urine N (P=0.003) changed in accordance with both environmental temperature 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 environmental 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 concentration (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

n DMD CPD ADFD EED CED DEI

85 mg

24.3 ◦ C

33.2 ◦ C

24.3 ◦ C

33.2 ◦ C

6 0.81 0.80 0.75 0.71 0.80 29.68

6 0.73 0.70 0.67 0.60 0.70 18.09

6 0.79 0.80 0.74 0.73 0.77 34.71

6 0.79 0.78 0.73 0.68 0.77 21.31

S.E.M.

M

T

M×T

0.03 0.03 0.03 0.03 0.03 2.80

ns 0.094 ns ns ns ns

ns 0.038 ns 0.030 ns <0.001

ns ns ns ns ns 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.

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(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 increasing 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 improving 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; Boucqu´e 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 administered in soft gels and feed palatability was not affected. The role of monensin in dairy and beef cattle weight gain has been established by many studies, which reported weight gain increases of up to 10%, often 5–8% above those obtained in control treatments (Lean et al., 1996). The supplemented animals at 33.2 ◦ C increased daily weight gain by 0.130 kg and feed efficiency by 0.07, whereas the supplemented animals at 24.3 ◦ C increased weight gain by 0.160 kg and feed efficiency by 0.03. Weight gain was higher in supplemented animals at 24.3 ◦ C, but feed efficiency was better in the animals at 33.2 ◦ C. Monensin improved performance of the animals under heat stress even though these individuals decreased feed ingestion by 1.1 kg DM/day. Ruminant performance is decreased by heat stress because maintenance requirements are higher while appetite is lower (Stokka et al., 1996). The negative impact caused by heat stress on animal performance can be more pronounced in tropical and subtropical areas (Beede and Collier, 1986). As a result, management practices, such as nutritional management, are proposed to relieve heat stress intensity. Monensin addition promoted better feed efficiency, which is in accordance with Tyler et al. (1992), who stated that the main benefit of ionophore use is increased feed efficiency in cattle. Monensin addition decreased the rumen molar proportion of butyrate from 2.80 mol/100 mol in 24.3 ◦ C and 1.26 mol/100 mol at 33.2 ◦ C, and increased VFA levels by 9.78 mM at 24.3 ◦ C and 9.98 mM at 33.2 ◦ C, which is a consequence of the increased molar proportion of propionic acid in the rumen liquid. Data of the supplemented animals

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at the two temperatures indicate that heat stress decreased the aforementioned variables as compared to the control animals (24.3 ◦ C), likely because the higher temperature decreased feed ingestion. Increased propionate and decreased acetate and butyrate in the rumen liquid of animals fed monensin is widely reported in the literature (Davis and Erhart, 1976; Dinius et al., 1976; Lemenager et al., 1978; Holzer et al., 1979; Chalupa et al., 1980; Shell et al., 1983; Oscar et al., 1987; Kalachnyuk et al., 1989; Clary et al., 1993; Zinn and Borques, 1993; Duff et al., 1994; Mbanzamihigo et al., 1996; Callaway and Martin, 1997; Garci´a et al., 2000; Oliveira et al., 2005), because ionophore promotes selectivity of rumen bacteria and growth of organisms that produce propionate over those that produce acetate and butyrate (Henderson et al., 1981). The intermediate metabolites used in methane production decreased while propionate production increased. Methane production is a consequence of the inefficient use of food energy. In the present study, the ratio between acetic and propionic acids decreased, although not at statistical levels. Brown and Hogue (1985) supplemented goats with 18 ppm monensin in a diet composed of 60% alfafa hay and detected an increase in acetic acid and propionic acid levels in the rumen liquid. They found, however, a significant decrease in the acetic acid/propionic acid ratio. In the present experiment, in which the animals received 70% concentrated ration, only a 0.2-unit decrease (1.97–1.81) in the ratio occurred when the animals were supplemented with ionophore. Lana and Russell (2001) found that after monensin supplementation the acetate/propionate ratio decreased 1.4 units (from 3.8 to 2.4) in bacteria cultures of foragefed animals and decreased 0.5 units (from 1.75 to 1.25) in bacteria cultures of animals fed 90% concentrated ration. This is because the bacteria of animals fed forage are more sensitive to monensin than those of animals receiving diets with a 90% concentrated ration. Given that the animals were fed 70% concentrated ration in this study, ionophore was not effective in changing acetic acid concentration, as was expected in a high-roughage diet. In the present study, different from literature reports, monensin did not decrease ammoniacal nitrogen levels. Salles and Lucci (2000) studied a similar diet and found a decrease in ammoniacal nitrogen along with an increase in monensin levels. Darden et al. (1985) studied concentrated diets and did not find any differences in N-NH3 values after monensin addition. Russell and Strobel (1989) reported that a greater monensin benefit is the decrease in rumen ammonia, an effect that increases when the diet is rich in soluble protein and limited in energy (forage-fed animals). Here, a more concentrated diet was used (soybean meal as the main protein source), which may explain why monensin did not affect N-NH3 . Lana et al. (2000) evaluated ruminant diets with different protein sources supplemented with monensin and found that soybean meal promoted higher ammonia production than other protein sources did because of its high protein content and high degradability. The effect of monensin on nitrogen retention depended of environmental temperature. Supplementation with monensin did not increase nitrogen absorption and retention in animals at 24.3 ◦ C. Thornton and Owens (1981) found similar results, in which N retention was not statistically changed, although there was a tendency toward a significance of addition to increase N retention. However, in the present study, monensin increased nitrogen retention by 29% in animals under heat stress. A 6.5% improvement in the efficiency of using protein from diet (expressed as a percentage of N retained from N ingested) was also observed

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by Beede and Collier (1986) in growing calves fed monensin. Considering the relation between N retained and N absorbed (Table 5), monensin had a stronger action on animals under heat stress (0.36 versus −0.11) than it did on animals at room temperature (0.56 versus 0.61). This indicates that heat stress promoted additional apparent absorbed N, which was completely used. Corroborating data on apparent N retention, monensin increased protein digestibility, irrespective of environmental temperature, an effect more pronounced in the heat-stressed animals (0.78 versus 0.70, Table 6). Thus, despite the increase in nitrogen digestibility, absorption and retention of this nutrient was also increased by the action of monensin, especially in heat-stressed animals. Protein digestibility after monensin supplementation increases if: (i) proteolysis and/or deamination, followed by ammonia absorption improves, resulting in lower fecal N loss, or (ii) a higher proportion of diet protein escapes rumen degradation and is digested and absorbed after passing through the rumen (Beede and Collier, 1986). Probably, in the present study, could have improved rumen proteolysis preceded an increase in absorption. Considering daily nitrogen consumption (Table 5) and ADCP (Table 6), daily protein consumption was 218 and 147 g (non-supplemented animals) and 266.31 and 163.92 g (supplemented animals) at 24.3 and 33.2 ◦ C, respectively. As per NRC (2001) guidelines, calves in the present study had a minimal protein consumption of 75 g to maintain weaning calves at a live weight of 80 kg (0 kg daily weight gain). Ingestion values for calves at 24.3 ◦ C were similar to those of calves with the same weight, but they gained 600 g a day. This indicates that the animals had minimal protein ingestion to supply the minimal protein requirements of rumen microorganisms. Protein degradation in the rumen may not have been changed by monensin because ammoniacal nitrogen levels in the rumen liquid were unchanged, indicating that protein did not escape from the diet to be degraded in the intestine, although the mean values show a tendency to increase after ionophore supplementation. Ionophore enhances rumen fermentation with increased VFA in rumen liquid of supplemented animals but without any alteration in proteolytic microorganisms. According to Zinn et al. (1981), the protein available for digestion and intestinal absorption in ruminants depends on the amount of protein degradation in the rumen as well as microbial protein production. The addition of monensin improved crude protein digestibility in animals at both environmental temperatures. Several studies about the effect of monensin on food digestibility can be found. According to Medel et al. (1991), the monensin effect on feed digestibility depends on the type of feed and the nutrition content of the diet, including true protein content. Salles and Lucci (2000) used a similar diet and found that adding monensin improved feed digestibility and promoted higher amounts of digestible nutrients. A review by Goodrich et al. (1984) found six studies where monensin improved protein digestibility by 6.5%. In the present study, we found a mean increase of 5.8% in the crude protein digestibility in the animals supplemented with ionophore, with a 1.3% increase in animals at 24.3 ◦ C and 10.5% in animals at 33.2 ◦ C. This resulted in improved nutrient use and promoted better feed efficiency, especially in the animals under heat stress that had lower feed ingestion. Summarizing, monensin improved animal performance at both environmental temperatures, but this effect was more pronounced in the heat-stressed animals.

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5. Conclusion Monensin affected the metabolism of the heat-stressed animals by improving rumen fermentation of the feed, increasing digestibility and nitrogen retention in animal organisms, and promoting better feed use efficiency and increasing weight gain. The action of monensin on nitrogen balance depends on environmental temperature and increases nutrient retention in animals under heat stress.

Acknowledgements We thank Fundac¸a˜ o de Amparo a` Pesquisa do Estado de S˜ao Paulo (FAPESP) for sponsoring this study, and Faculdade de Zootecnia e Engenharia de Alimentos da Universidade de S˜ao Paulo for providing facilities.

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