Effects of substituting amaranth silage for corn silage on intake, growth performance, diet digestibility, microbial protein, nitrogen retention and ruminal fermentation in fattening lambs

G Model

ARTICLE IN PRESS

ANIFEE-13050; No. of Pages 10

Animal Feed Science and Technology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci

Effects of substituting amaranth silage for corn silage on intake, growth performance, diet digestibility, microbial protein, nitrogen retention and ruminal fermentation in fattening lambs J. Rezaei a , Y. Rouzbehan a,∗ , H. Fazaeli b , M. Zahedifar b a b

Animal Science Department, Faculty of Agriculture, Tarbiat Modares University, P.O. Box 14115-336, Tehran, Iran Animal Science Research Institute, P.O. Box 1483, Karaj 315, Iran

a r t i c l e

i n f o

Article history: Received 21 May 2013 Received in revised form 18 January 2014 Accepted 9 March 2014 Available online xxx

Keywords: Amaranth silage Growth performance Intake Ruminal parameters Lamb

a b s t r a c t The effect of dietary substitution of corn silage (CS) with amaranth silage (AS) on feed intake, growth performance, digestibility, microbial protein, nitrogen (N) retention and ruminal fermentation was evaluated using 50 Moghani male lambs (initial body weight 28.5 ± 1.9 kg). Five iso-energetic and iso-nitrogenous diets were randomly assigned to the five groups of 10 lambs each in a completely randomized design for a period of 98 days. The diets were offered ad libitum at a forage to concentrate ratio of 40–60 in which CS was replaced by different levels (0, 75, 150, 225 or 300 g/kg of dietary DM) of AS. Diets were offered twice daily (at 08:00 and 17:00 h). Daily feed intake, average daily gain (ADG), in vivo digestibility, rumen fermentation parameters, microbial N supply (MNS) and N retention in the lambs were determined. Increasing dietary level of AS improved feed intake and ADG (P<0.051), but there was no detectable effect on feed efficiency (FE) and diet digestibility. Increasing the proportion of AS in diet increased N retention, MNS and ruminal butyrate (P<0.05) but decreased branched-chain fatty acids. Dietary treatment tended to increase rumen content of total volatile fatty acids (P=0.09). It is concluded that partial substitution of AS for CS, up to 300 g/kg DM, in diet of Moghani lambs had positive effects on feed intake, growth performance, N balance and microbial N, without any effect on FE. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Climatic conditions and shortage of water resources in the semi-arid and arid regions reduces availability of forage. Alternative forages, such as amaranth, however, can be useful in overcoming this problem under certain circumstances.

Abbreviations: ADFom, ash-free acid detergent fibre; ADG, average daily gain; AS, amaranth silage; BW, body weight; CP, crude protein; CS, corn silage; DM, dry matter; DMI, dry matter intake; DOMI, digestible organic matter intake; EE, ether extract; FE, feed efficiency; Lignin(sa), lignin measured by solubilization of cellulose with sulphuric acid; ME, metabolisable energy; MNS, microbial nitrogen supply; N, nitrogen; NFC, non-fibre carbohydrates; NDFom, ash-free neutral detergent fibre; OM, organic matter; OMI, organic matter intake; PD, purine derivatives; RDP, rumen degradable protein; RUP, rumen undegradable protein; TPD, total purine derivatives; VFA, volatile fatty acids. ∗ Corresponding author. Tel.: +98 21 48292336; fax: +98 21 48292200. E-mail addresses: rozbeh [email protected], [email protected] (Y. Rouzbehan). http://dx.doi.org/10.1016/j.anifeedsci.2014.03.005 0377-8401/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Rezaei, J., et al., Effects of substituting amaranth silage for corn silage on intake, growth performance, diet digestibility, microbial protein, nitrogen retention and ruminal fermentation in fattening lambs. Anim. Feed Sci. Tech. (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.03.005

G Model ANIFEE-13050; No. of Pages 10

ARTICLE IN PRESS J. Rezaei et al. / Animal Feed Science and Technology xxx (2014) xxx–xxx

2

Table 1 The chemical analyses (g/kg DM or as stated) of corn and amaranth. Chemical analyses

Dry matter (g/kg fresh matter) Organic matter Crude protein Rumen degradable protein Rumen undegradable protein NDFom ADFom Lignin(sa) Ether extract Non-fibre carbohydratesa Starch Calcium Phosphorous Metabolizable energyb (MJ/kg DM) pH Ammonia-N (g/kg total N) Lactate Acetate Propionate Butyrate

Corn

Amaranth

Fresh

Ensiled

Fresh

Ensiled

211 936 87.3 – – 509 300 45.4 33.9 306 – 2.2 2.1 10.8 – – – – – –

220 923 77.0 52.4 24.6 480 281 45.0 34.2 332 213 2.3 2.2 10.1 4.0 51.1 76.7 19.2 3.1 0.9

217 908 134 – – 449 284 35.1 24.7 299 – 17.9 2.7 9.5 – – – – – –

235 890 122 87.4 34.6 440 278 35 24.1 304 101 17.8 2.6 9.2 3.9 69.3 81.8 17.3 1.1 14.6

NDFom, ash-free neutral detergent fibre; ADFom, ash-free acid detergent fibre; Lignin(sa), lignin measured by solubilization of cellulose with sulphuric acid. a Calculated as: 1000 − (NDFom g/kg DM + crude protein g/kg DM + ether extract g/kg DM + ash g/kg DM). b The ME values were estimated using a gas production technique, as described by Menke and Steingass (1988).

Amaranth, genus Amaranthus, is an annual crop adapted to poor soils and areas with limited rainfall and high temperature (Kauffman and Weber, 1990; Myers, 1996). Good yield (a fresh and dry matter (DM) yields performance of up to 86.4, and 13.2 t/ha, respectively; Mehrani et al., 2012), high crude protein (CP; up to 285 g/kg DM) and DM digestibility (590–790 g/kg) and low nitrate and oxalic acid concentrations (below toxic levels) of the amaranth makes it a great potential forage for ruminants (Sleugh et al., 2001; Rezaei et al., 2009; Abbasi et al., 2012). Amaranth can be a suitable alternative for alfalfa in growing lambs at levels up to 500 g/kg of dietary DM (Pond and Lehmann, 1989). Presence of the main stem axis (Stallknecht and Schulz-Schaeffer, 1993) and high moisture content (Rezaei et al., 2009; Abbasi et al., 2012), however, make the rapid drying of a large quantity of grain type amaranth difficult, and this limits its use as dried forage. Ensiling has been more successfully used to preserve green amaranth (Rezaei et al., 2009; Rabbani, 2012). Because of a low water requirement (i.e., less than corn; Kauffman and Weber, 1990), amaranth silage (AS) may be a suitable choice as ruminant feed in regions where corn silage (CS) yields are low due to water limitation (Myers and Putnam, 1988). To our knowledge, the information on effect of AS on growth performance in ruminants is limited. Therefore, the aim of this study was to determine the effect of feeding different substitution rate of AS for CS on feed intake, growth performance, digestibility, microbial nitrogen (N) supply, N retention and ruminal fermentation in fattening lambs. 2. Materials and methods 2.1. Forage and silage preparation The forages (i.e., amaranth and corn) were planted on 17th July in a field (2 ha) located at the Animal Science Research Institute (Karaj city, Iran). The corn field was fertilized with 175 kg N (as urea) and 60 kg phosphorous (as triple super phosphate)/ha. The amaranth field was fertilized with 120 kg N (as urea)/ha, and, according to soil test, phosphorus and potassium were not applied. Weeds were controlled through row cultivation during the first several weeks after amaranth planting. In the early autumn, both forages were direct harvested at mid-milk stage of seeds by a corn harvester 10 cm above ground level. The chopped plants were ensiled into permanent horizontal silos (one 20 t silo per forage), with airtight lids to ensure good fermentation, for 5 months. The compaction density of the AS was approximately 850 kg wet matter/m3 . The representative samples were taken from the fresh silages and frozen (−20 ◦ C) for further analysis (Table 1). 2.2. Animals and treatments Fifty autumn born male Moghani lambs with average body weight (BW) of 28.5 ± 1.9 kg and 4.5 months of age were used for this study. Five iso-energetic and iso-nitrogenous diets were formulated according to NRC (1985), in which CS replaced by different levels (0, 75, 150, 225 or 300 g/kg of dietary DM) of AS (Table 2). The experimental diets were formulated to meet the requirements of growth rate of 290 g/d for fattening male lambs at 4–7 months of age. Please cite this article in press as: Rezaei, J., et al., Effects of substituting amaranth silage for corn silage on intake, growth performance, diet digestibility, microbial protein, nitrogen retention and ruminal fermentation in fattening lambs. Anim. Feed Sci. Tech. (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.03.005

G Model ANIFEE-13050; No. of Pages 10

ARTICLE IN PRESS J. Rezaei et al. / Animal Feed Science and Technology xxx (2014) xxx–xxx

3

Table 2 Ingredients and chemical composition (g/kg DM) of the experimental diets. Item

Ingredient Corn silage Amaranth silage Alfalfa Barley grain Corn grain, ground, dry Sugar beet pulp Soybean meal Cottonseed meal Di-calcium phosphate Limestone Vitamin-mineral premixa Salt Chemical compositionb Dry matter (g/kg as-fed basis) Organic matter Crude protein RDP RUP RDP:RUP NDFom ADFom Lignin(sa) Ether extract Non-fibre carbohydratesc Starch Calcium Phosphorous Calcium:Phosphorous Metabolizable energy (MJ/kg DM)

Level of AS in diet (g/kg DM) 0

75

150

225

300

300 0 100 340 61.4 7.0 135 27.7 7.0 7.0 10 5

225 75 100 345 70.3 7.0 135 16.9 6.0 4.8 10 5

150 150 100 350 79.0 7.0 131.3 10.0 3.6 4.1 10 5

75 225 100 355 85.0 9.5 120.8 10.0 1.4 3.3 10 5

0 300 100 360 85.0 18.4 110 10.0 0.4 1.2 10 5

469 924 147 86.9 60.1 1.4 309 173 28.1 32.5 435 280 9.2 3.9 2.4 11.2

474 923 147 88.0 59.0 1.5 305 171 27.5 31.4 440 279 9.2 3.9 2.4 11.2

478 922 147 89.0 58.0 1.5 301 171 27.0 30.4 444 278 9.2 3.9 2.4 11.2

483 920 147 90.1 56.9 1.6 299 171 26.9 29.4 445 275 9.2 3.9 2.4 11.2

488 918 147 91.0 56.0 1.6 300 172 26.8 28.3 443 269 9.2 3.9 2.4 11.2

RDP, rumen degradable protein; RUP, rumen undegradable protein; NDFom, ash-free neutral detergent fibre; ADFom, ash-free acid detergent fibre; Lignin(sa), lignin measured by solubilization of cellulose with sulphuric acid. a Premix contained (per kg): Ca, 120 g; P, 30 g; Na, 55 g; Mg, 20 g; Zn, 3 g; Fe, 3 g; Mn, 2 g; Cu, 280 mg; Co, 100 mg; Se, 1 mg; K, 215 mg; I, 100 mg; vitamin E, 100 mg; vitamin A, 500,000 IU; vitamin D3 , 100,000 IU; antioxidant, 400 mg; carrier, up to 1000 g. b Calculated from each feed composition, except for DM, and NDFom, which were analyzed. c Calculated as: 1000–(NDFom g/kg + crude protein g/kg + ether extract g/kg + ash g/kg).

The dietary treatments were randomly assigned to five groups of 10 lambs each in a completely randomized design. The animals received the diets ad libitum, as total mixed rations, twice daily (at 08:00 and 17:00 h) to ensure 5% orts and fresh water was offered ad libitum. Lambs were housed in individual concrete floor pens (1.5 m × 1.5 m) with wood chips bedding, allowed an adaptation period of 14 days followed by a data collection period of 84 days. During the adaptation period, all the animals were treated for external (Azantole, Bayer, Germany) and internal (Triclabendazole + Levamisole, Darou-Pakhsh Co., Iran) parasites, and vaccinated against enterotoxaemia. 2.3. Feed intake and growth performance During the experimental period, feed offered and corresponding ort were recorded daily to estimate voluntary feed intake and representative samples were kept frozen at −20 ◦ C for subsequent analyses. Samples of silages, feeds offered, orts and faeces were oven-dried at 60 ◦ C until a constant weight to determine DM content, then ground to pass through 1 mm sieve (Wiley mill, Swedesboro, USA) and stored until analyzed. Samples were analyzed for ash (number 924.05), N (number 984.13) and ether extract (EE; number 954.02) of AOAC (1990) procedures. Starch content of the fresh and ensiled forages was analyzed according to AOAC (method 996.11; 1990). Ash-free neutral detergent fibre (NDFom) was determined without sodium sulphite, and expressed exclusive of residual ash (Van Soest et al., 1991). The acid detergent fibre was determined (number 973.18; AOAC, 1990) and expressed exclusive of residual ash (ash-free ADF; ADFom). The lignin(sa) was determined by solubilization of cellulose with 720 g/kg sulphuric acid, according to the procedure described by Robertson and Van Soest (1981). Non-fibre carbohydrate (NFC) content was calculated as NFC = 1000 − (NDFom g/kg DM + CP g/kg DM + EE g/kg DM + ash g/kg DM). Calcium and phosphorus concentrations were determined by atomic absorption (Temminghoff and Houba, 2004) and vanadate/molybdate method (Chapman and Pratt, 1961), respectively. The BWs of the animals were individually recorded at the beginning and the end of the experimental period (on 2 consecutive days to provide baseline weights) and at 21-days intervals before the morning feeding, after 16 h fasting, to Please cite this article in press as: Rezaei, J., et al., Effects of substituting amaranth silage for corn silage on intake, growth performance, diet digestibility, microbial protein, nitrogen retention and ruminal fermentation in fattening lambs. Anim. Feed Sci. Tech. (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.03.005

G Model ANIFEE-13050; No. of Pages 10

ARTICLE IN PRESS J. Rezaei et al. / Animal Feed Science and Technology xxx (2014) xxx–xxx

4

calculate average daily gain (ADG) and feed efficiency (FE). The average gain on a daily basis was calculated for each lamb by linear regression analysis of BW vs. time.

2.4. In vivo digestibility, microbial N supply (MNS) and N balance Whole tract apparent digestibility coefficients of DM, organic matter (OM), CP, NDFom and EE were estimated using the total faecal collection method (Givens et al., 2000). On day 68 of the trial, 5 animals per treatment (i.e., 25 lambs total) were placed into individual metabolism crates equipped with faeces and urine collectors, in a complete randomized design (5 animals for each treatment). The digestibility trial lasted for 13 days, with 7 days for adaptation to metabolism crates and 6 days for the collection period. During the 6 days of collection period, amount of feed offered, orts and faeces from each lamb were recorded daily and then 10% representative samples were taken. At the end of the period, daily samples were pooled for each lamb within the treatment, thoroughly mixed and stored at −20 ◦ C for later analysis. At the same time, total urine produced daily was collected in plastic vessels containing 100 mL of sulphuric acid solution (10%, v/v), to keep the final pH below 3, placed below the urine outlet in the metabolism crates. The volume of urine collected every morning from an individual animal was measured. To prevent the precipitation (particularly of uric acid) of purine derivatives (PD) in urine during storage (Chen and Gomes, 1992), 10% of daily amount was sampled, diluted by five times with distilled water and then stored at −20 ◦ C for the estimation of N and PD. Urinary PD was estimated by spectrophotometric methods (Chen and Gomes, 1992). Allantoin was measured in urine by colorimetric method at 522 nm after its conversion to phenylhydrazone. Sum of xanthine and hypoxanthine was calculated by their conversion to uric acid with xanthine oxidase (Sigma; Catalog No. X-1875, 5 Units, Germany) with subsequent optical density at 293 nm. Uric acid was measured from the reduction in optical density at 293 nm after degradation of uric acid to allantoin with uricase (Sigma; Product No. U-9375, Germany). Finally, total PD excretion per day was calculated as the sum of all four compounds (allantoin, uric acid, xanthine plus hypoxanthine); the daily absorbed exogenous purines estimated and MNS predicted. The concentration of urinary N was estimated by the Kjeldahl method (AOAC, 1990). Nitrogen retention was calculated as daily N excretion (urinary N plus faecal N) subtracted from daily N intake.

2.5. Rumen fluid parameters Rumen fluid samples were collected at 0 (just before morning feeding), 2, 4 and 6 h after morning feeding on days 38 and 65 of the experimental period from 5 animals assigned to each treatment. Rumen fluid samples were obtained with a stomach tube and pH was determined immediately by a digital pH metre (Sartorius PT-10, Germany) and then samples were strained through two layers of cheesecloth. Samples of strained rumen fluid (25 mL) submitted for the evaluation of ammonia-N were immediately preserved with 5 mL of HCl 0.2 N and stored at −20 ◦ C. The ruminal fluid was analyzed for ammonia-N using a phenol-hypochlorite assay according to Broderick and Kang (1980). For analysis of ruminal volatile fatty acids (VFA), 2 mL of strained rumen fluid was preserved, at −20 ◦ C, with 0.5 mL of an acid solution containing 200 mL/L orthophosphoric acid and 20 mM 2-ethyl-butyric acid. After thawing, rumen fluid samples were centrifuged (15,000 × g for 15 min; 4 ◦ C) and the supernatant was used to quantify the VFAs by GC using ethyl-butyric acid as the internal standard.

2.6. Statistical analyses Data were analyzed as a mixed model using the PROC MIXED (SAS Institute, 2001). Data on intake, ADG, FE, in vivo digestibility, MNS and N balance were analyzed as a completely randomized design. The model included only the fixed effect of dietary treatment because the experimental design was completely randomized. Data on rumen fermentation parameters were analyzed as repeated measurement. The effects of dietary treatment, sampling day (discrete variable) and sampling hour (continuous variable), and their interactions were considered fixed. The compound symmetry was used as a covariance structure in the model. Additionally, a polynomial contrast was used to test the linear or quadratic effects of AS feeding on parameters measured.

3. Results 3.1. Fresh and ensiled forages As shown in Table 1, ensiling the forages decreased OM, CP, rumen undegradable protein (RUP), NDFom, ADFom and ME, and increased DM and NFC in both feed items. Compared to CS, AS contained greater contents of CP, rumen degradable protein (RDP), calcium and butyrate, but less OM, NDFom, lignin(sa), NFC, starch, acetate, propionate and ME (Table 1). With inclusion of the increasing dietary rates of AS, the dietary RDP to RUP ratio, DM and NFC contents increased, but OM, lignin(sa), EE and starch decreased. Please cite this article in press as: Rezaei, J., et al., Effects of substituting amaranth silage for corn silage on intake, growth performance, diet digestibility, microbial protein, nitrogen retention and ruminal fermentation in fattening lambs. Anim. Feed Sci. Tech. (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.03.005

G Model

ARTICLE IN PRESS

ANIFEE-13050; No. of Pages 10

J. Rezaei et al. / Animal Feed Science and Technology xxx (2014) xxx–xxx

5

Table 3 Effect of level of amaranth silage (AS) on feed intake, and growth performance of lambs. Item

Intake (g/d) Dry matter Organic matter Crude protein Initial BW (kg) Final BW (kg) ADG (g/d) FE

Level of AS in diet (g/kg DM)

P-value

0

75

150

225

300

SEM

Linear

Quadratic

1339 1231 201 28.8 48.6 231 0.17

1349 1240 202 28.5 48.4 235 0.17

1375 1263 214 28.6 48.8 243 0.18

1404 1288 214 28.2 49.2 247 0.18

1460 1347 222 28.4 50.4 261 0.18

43.41 38.23 6.61 1.07 1.19 6.14 0.01

0.04 0.03 0.05 0.87 0.27 0.04 0.59

0.80 0.69 0.84 0.89 0.63 0.49 0.99

BW, body weight; ADG, average daily gain; FE, feed efficiency (gain:feed); SEM, standard error of means.

Table 4 Effect of level of amaranth silage (AS) on apparent digestibility (g/kg) in lambs. Apparent digestibility

Dry matter Organic matter Crude protein NDFom Ether extract ME (MJ/kg DM)

Level of AS in diet (g/kg DM)

P-value

0

75

150

225

300

SEM

Linear

Quadratic

709 726 663 588 752 10.5

701 719 660 579 751 10.4

699 724 662 582 748 10.4

704 722 673 580 744 10.4

700 720 669 586 740 10.4

4.64 4.33 9.82 6.52 4.86 0.09

0.16 0.53 0.35 0.76 0.32 0.54

0.42 0.67 0.89 0.42 0.68 0.62

NDFom, ash-free neutral detergent fibre; ME, metabolizable energy; SEM, standard error of means.

3.2. Feed intake The daily intakes of DM, OM and CP were linearly increased (P<0.051) as the dietary inclusion rate of AS increased (Table 3). The quadratic effect of AS level on feed intake was insignificant.

3.3. Growth performance There was a linear growth response to the dietary treatment. The final BW increased numerically when greater proportion of AS was included in the diet (Table 3). The inclusion of AS in diet resulted in an increase (P=0.04) of ADG.

3.4. In vivo digestibility, MNS and N balance Replacing CS by AS had no detectable effect on apparent digestibility coefficients of DM, OM, CP, NDFom and EE (Table 4). During the faeces and urine collection period, OM intake (OMI) and digestible OMI (DOMI) increased (P<0.01) by the substitution of AS for CS in diet (Table 5). There was a linear response to increasing dietary AS for urinary uric acid (P<0.001). Partially substituting AS for CS increased (P=0.02) daily MNS (g/d). Although the amount of faecal and urinary N were unaffected by the dietary treatment, statistical analysis showed that adding AS in the diet had a linear positive effect on daily retained N (P=0.02). However, efficiency of N balance as proportion of N intake was not affected by the treatment.

3.5. Rumen fermentation parameters Increasing AS in the diet had no detectable effect on the ruminal pH (Table 6). The rumen ammonia-N concentration was numerically decreased with increasing dietary AS. The partial inclusion of AS in the diet resulted in an elevated molar proportion of butyrate (P=0.03), whereas iso-valerate decreased (P=0.006) with partial inclusion of AS in the diet. The effects of sampling days and sampling hours on rumen fermentation parameters are presented in Table 6 and Figs. 1 and 2. On day 65, the ruminal concentration of total VFA, acetate and acetate to propionate ratio were greater (P<0.01), but ammonia-N, butyrate and valerate were less than those recorded on day 38 (P<0.05). There was a tendency for propionate to decline on day 65 compared to day 38. Rumen fluid sampling hour had a significant effect on rumen parameters measured (P<0.01), except butyrate (Table 6). Please cite this article in press as: Rezaei, J., et al., Effects of substituting amaranth silage for corn silage on intake, growth performance, diet digestibility, microbial protein, nitrogen retention and ruminal fermentation in fattening lambs. Anim. Feed Sci. Tech. (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.03.005

G Model

ARTICLE IN PRESS

ANIFEE-13050; No. of Pages 10

J. Rezaei et al. / Animal Feed Science and Technology xxx (2014) xxx–xxx

6

Table 5 Effect of level of amaranth silage (AS) on microbial N supply (MNS), and N balance of lambs. Item

Level of AS in diet (g/kg DM)

BW DMI (g/d) OMI (g/d) DOMI (g/d) Urinary excretion (mmol/d) Allantoin Uric acid Xanthine plus hypoxanthine TPD absorbed (mmol/d) Microbial N (g/d) Microbial N (g N/kg DOMI) N balance (g/d) N Intake Faecal N Urinary N Retention Retention (g/kg N intake)

P-value

0

75

150

225

300

SEM

Linear

Quadratic

44.9 1376 1274 925

44.6 1420 1312 943

44.8 1415 1306 946

45.1 1475 1361 983

45.6 1528 1405 1012

1.32 30.35 27.91 20.24

0.84 <0.001 0.004 0.007

0.64 0.35 0.37 0.35

11.6 4.0 1.1 19.1 13.9 15.0

11.6 4.1 1.1 19.3 14.1 14.9

11.7 4.3 1.2 19.7 14.3 15.1

12.0 5.1 1.2 21.0 15.2 15.5

12.2 5.1 1.1 21.3 15.5 15.3

0.31 0.22 0.06 0.53 0.35 0.55

0.29 <0.001 0.50 0.02 0.02 0.88

0.79 0.34 0.39 0.57 0.57 0.92

33.8 11.4 12.5 9.9 292

34.7 11.8 12.9 10.0 288

34.9 11.8 12.8 10.3 295

35.8 11.7 13.1 11.0 307

36.9 12.2 13.5 11.2 304

0.69 0.36 0.39 0.36 8.93

0.002 0.18 0.15 0.02 0.24

0.68 0.69 0.74 0.73 0.75

BW, body weight; DMI, dry matter intake; OMI, organic matter intake; DOMI, digestible OM intake; TPD, total purine derivatives; N, nitrogen; SEM, standard error of means.

Table 6 Effect of level of amaranth silage (AS) on ruminal fermentation parameters of lambs. Itemb

P-valuea

Level of AS in diet (g/kg DM)

pH Ammonia-N (mg/dL) Total VFA (mmol/L) VFA (mmol/100 mmol) Acetate Propionate Iso-butyrate Butyrate Iso-valerate Valerate Acetate:propionate

D

H

T×D

T×H

D×H

T×D×H

0.97 0.41 0.54

0.19 0.05 <0.001

<0.001 <0.001 <0.001

0.77 0.96 0.31

0.20 0.12 0.87

0.73 0.14 0.78

0.69 0.98 0.81

0.78 0.19 0.18 0.18 0.57 0.77 0.45

<0.001 0.11 0.23 <0.001 0.44 0.01 0.01

0.02 <0.001 <0.001 0.81 <0.001 <0.001 <0.001

0.83 0.63 0.61 0.36 0.31 0.75 0.68

0.98 0.97 0.78 0.25 0.30 0.02 0.93

0.60 0.52 0.24 0.17 0.01 0.15 0.64

0.95 0.99 0.95 0.64 0.97 0.97 0.96

0

75

150

225

300

SEM

Linear

Quadratic

6.7 24.2 91.8

6.6 24.0 92.4

6.6 23.5 96.4

6.6 23.6 96.0

6.6 23.2 96.0

0.05 0.64 1.65

0.88 0.35 0.09

45.2 27.3 3.0 18.8 3.0 2.7 1.7

44.9 27.5 2.8 19.1 2.9 2.8 1.6

44.6 27.4 2.9 19.4 2.8 2.9 1.6

44.7 26.7 2.8 20.3 2.7 2.9 1.7

44.8 26.6 2.7 20.6 2.4 3.0 1.7

0.91 1.69 0.12 0.37 0.15 0.12 0.06

0.95 0.46 0.29 0.03 0.01 0.18 0.77

T

N, nitrogen; VFA, volatile fatty acid; SEM, standard error of means. a T, effect of dietary treatment; D, effect of sampling day; H, effect of sampling hour. b Values are averages of repeated sampling of rumen fluid collected from 5 animals assigned to each treatment on two days (38 and 65 days of experimental period), just before lambs were offered the morning feeding (0 h) and 2, 4, and 6 h after feeding.

Fig. 1. Effect of substituting amaranth silage for corn silage on the pH and ammonia-N concentration in the rumen of the lambs.

Please cite this article in press as: Rezaei, J., et al., Effects of substituting amaranth silage for corn silage on intake, growth performance, diet digestibility, microbial protein, nitrogen retention and ruminal fermentation in fattening lambs. Anim. Feed Sci. Tech. (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.03.005

G Model ANIFEE-13050; No. of Pages 10

ARTICLE IN PRESS J. Rezaei et al. / Animal Feed Science and Technology xxx (2014) xxx–xxx

7

Fig. 2. Effect of substituting amaranth silage for corn silage on the ruminal VFA concentration of the lambs.

4. Discussion 4.1. Fresh and ensiled forages The alterations in the chemical composition of the forages after ensiling were likely because of microbial fermentation and also effluents and DM losses during ensilage (McDonald et al., 1991; Rezaei et al., 2009). The lesser CP content of the amaranth plant compared to other reports (up to 285 g/kg DM) was probably related to differences in amaranth species tested, harvest date and N fertilization rate (Sleugh et al., 2001; Abbasi et al., 2012). The AS was well preserved as determined by the pH value (∼4.0) and low ammonia-N concentration (<50 to 70 g/kg) in the high moisture (<300 g DM/kg fresh matter) silage (Demarquilly, 1990). Butyric acid content of AS (127 g/kg of total fermentative fatty acids) was however, greater than those outlined for a good silage quality and is indicative of degraded sugars and lactic acid by saccharolytic clostridia (McDonald et al., 1991). The DM contents of the both silages were low Please cite this article in press as: Rezaei, J., et al., Effects of substituting amaranth silage for corn silage on intake, growth performance, diet digestibility, microbial protein, nitrogen retention and ruminal fermentation in fattening lambs. Anim. Feed Sci. Tech. (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.03.005

G Model

ARTICLE IN PRESS

ANIFEE-13050; No. of Pages 10

J. Rezaei et al. / Animal Feed Science and Technology xxx (2014) xxx–xxx

8

(<250 g/kg fresh weight), because in Iran, corn and amaranth grown for ensilage are planted as second crops in summer behind wheat and barley, and usually harvested at autumn, at which time adequate heat and sunlight for best maturity may not be available. Thus, most of the crops are ensiled with <250 g DM/kg fresh weight (Nikkhah et al., 2011). The effect of adding AS on dietary contents of DM and lignin(sa) were likely related to the greater DM and lower lignin(sa) contents in AS compared to CS (Table 1). 4.2. Feed intake The linear increase of feed intake with inclusion of AS in diet could, in part, be associated with increasing DM and decreasing lignin(sa) contents of diets (Table 2) and their effects on gut-fill and retention time (NRC, 2001; McDonald et al., 2002). Furthermore, different physical characteristics of the experimental feeds such as texture and resistance to fracture (Baumont, 1996) may have had an effect. Among the nutrients intake, the minimum significance of quadratic effect of AS level was P=0.69 for OMI (Table 3). Evidently, the absence of a quadratic effect of different dietary rates of AS on nutrients intake in the present study indicates that feeding incremental rates of this silage up to 300 g/kg dietary DM had no adverse effects on diet palatability and its consumption by the lambs. Pond and Lehmann (1989) have shown a tendency for daily feed intake of lambs to increase when diets had amaranth forage replacing alfalfa. The linear increase of OM and CP intakes with increased dietary levels of AS was a consequence of the increase in DMI. 4.3. Growth performance The linear increase in ADG by the inclusion of AS in diet (Table 3) could be related to the greater nutrients intake (Haddad and Hussein, 2004; Olfaz et al., 2005) and the increased ruminal VFA concentration (Galina et al., 2004). Another reason may stem from an improved N utilization and greater MNS (Ben Salem et al., 2002; Galina et al., 2004), as shown in Table 5. The lack of a quadratic effect of AS on growth performance was expected, and may have resulted from the linear effect observed on feed intake (Table 3), MNS (Table 5), OM fermented in the rumen per day and ruminal VFA (Table 6). This also could be reflecting absence of toxic compounds in the amaranth as also reported by Pond and Lehmann (1989), although no direct measurement was performed regarding toxic compounds in the present study. Lack of a significant effect on FE was likely related to the parallel linear increases occurred in both DMI and ADG (Table 3) as dietary inclusion rate of ensiled amaranth was increased. In the other work, Pond and Lehmann (1989) reported similar FE in lambs offered alfalfa or amaranth forage. Obviously the prediction equations used to estimate lamb performance (i.e., NRC, 1985) were not obtained in Moghani breed sheep, which may explain the lower growth rate and FE in this breed in comparison to those predicted in NRC tables. 4.4. In vivo digestibility, MNS and N balance The lack of significant linear and quadratic differences of digestibility (Table 4) among treatments could be related to relatively similar chemical composition of the diets and similar ruminal pH, which affects the activity of ruminal microbes (Russell and Dombrowski, 1980; Petit and Castonguay, 1994). In contrast, Pond and Lehmann (1989) reported that digestibility was greater for lambs fed amaranth as the forage component of the diet than for those fed alfalfa. The linear increase in urinary uric acid excretion by the inclusion of AS in diet was probably because of increased dietary ratio of RDP to RUP (Table 2) (Khandaker and Tareque, 1997; Zuo, 2011), as well as increased CP intake (Table 3). The significance of quadratic effects of AS feeding was at least P=0.34 for PD and MNS (Table 5). The MNS, which did not show a significant quadratic response, reached greatest value at the highest level of AS feeding. The linear increase in MNS (g/d) with the reduction of CS in diet could be due to increasing intakes of digestible OM and N (Table 5; Ben Salem et al., 2002), and similar to that results reported by Kaur et al. (2010) was independent of rumen pH. In addition, the greater MNS (g/d) also may have been resulted from a better synchrony of ME and N in the rumen (Kaur et al., 2010). The similar efficiency of MNS (as proportion of DOMI), despite a linear increase in MNS (g/d), was probably due to the greater intake of DOM (Table 5) as the dietary inclusion of AS increased. The linear positive effect of the treatment on daily retained N in the present study was similar to that reported by Ben Salem et al. (2002). Lack of a significant quadratic effect of feeding AS on the retained N seemed consistent with the linear changes occurred in feed intake. Actually, the linear improvement in N retention could be explained by the linear increase in DMI as a result of the AS level in diets, which also may explain the linear increase in N intake (Table 5), because the diets were iso-nitrogenous. In the present study, increasing N retention in the lambs received AS in the diet was accompanied by increasing ADG, which might indicate that increased N consumption was likely utilized for body tissue accretion rather than the energy cycle and reflected protein deposition. 4.5. Rumen fermentation parameters The ruminal pH values were varied within a normal range from 6.1 to 6.8 as reported by Van Soest (1994). Please cite this article in press as: Rezaei, J., et al., Effects of substituting amaranth silage for corn silage on intake, growth performance, diet digestibility, microbial protein, nitrogen retention and ruminal fermentation in fattening lambs. Anim. Feed Sci. Tech. (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.03.005

G Model ANIFEE-13050; No. of Pages 10

ARTICLE IN PRESS J. Rezaei et al. / Animal Feed Science and Technology xxx (2014) xxx–xxx

9

The levels of ruminal ammonia-N were in the optimum range, 8.5 to over 30 mg/dL, described by McDonald et al. (2002). Numerically decreasing rumen ammonia-N concentration with enhancing dietary level of AS can be justified by the raise in the MNS (Ahmed and Abdalla, 2005; Azizi-Shotorkhoft et al., 2012). The greater daily OMI, which resulted in more fermented OM per day, could be responsible for the linear increased concentration of ruminal total VFA (Table 6) as dietary AS increased (McDonald et al., 2002). A possible reason for the relatively greater molar proportions of ruminal propionate in this study was the high concentrations of lactate in our silages, which are mainly converted to propionate in the rumen (Martin et al., 1994). Ramos et al. (2009) reported that decline in proportion of branched-chain VFA might be related to reduce protein degradation in rumen, because branched-chain VFA are produced as a result of branched-chain amino acids fermentation. In the present experiment however, linear decline in the proportion of branched-chain VFA by the increasing dietary AS was associated with linear increase in MNS (Table 5), which possibly points out towards a shift in the balance between deamination of branched-chain amino acids and their utilization for microbial growth (Shingfield et al., 2002). The linear increase in molar proportion of ruminal butyrate observed as the dietary level of AS increased might, in part, be related to the proportional reduction of branched-chain VFA (Table 5), as well the greater concentration of butyrate in the AS compared to CS (Table 1). Increased ruminal concentration of total VFA and molar proportion of acetate, and decreased ammonia-N and butyrate on day 65 compared to day 38 could be attributed to animal adaptation and changes in responses of rumen microorganism to diets over the experimental period (McDonald et al., 2002; Dehority, 2003). Effect of the sampling hour on the rumen fermentation parameters could be associated to breakdown pattern of diet ingredients after feeding and absorption of the fermentation products from the rumen into the blood (Van Soest, 1994; McDonald et al., 2002). As observed in this study, for all the measured traits the quadratic effect of the dietary treatment was not statistically significant which indicate none experimental level of AS had negative effect on the performance of lamb. Though, linear or quadratic responses of the fattening lambs should be investigated with higher inclusion of AS (more than 300 g/kg DM) in diet. 5. Conclusions Partial dietary substitution of amaranth silage for corn silage, up to 300 g/kg DM, in fattening Moghani lambs had positive effects on feed intake, growth performance, N balance and microbial N, but did not affect the feed efficiency. However, further researches are needed to confirm the usefulness of amaranth silage in the ruminant diets reported in this study. Acknowledgments The authors wish to gratefully acknowledge Mr. Ali Karimzadeh from the ‘Panje Talaee Cultivation and Industry’ (Tehran, Iran) for the kind support of the production of amaranth forage for this research. The authors also would like to thank Dr. Pedram Rezamand for his English language correction of the manuscript, and Dr. Ali Mokhtassi-Bidgoli for his assistance with statistical analysis of data. References Abbasi, D., Rouzbehan, Y., Rezaei, J., 2012. Effect of harvest date and nitrogen fertilization rate on the nutritive value of amaranth forage (Amaranthus hypochondriacus). Anim. Feed Sci. Technol. 171, 6–13. Ahmed, M.M.M., Abdalla, H.A., 2005. Use of different nitrogen sources in the fattening of yearling sheep. Small Rumin. Res. 56, 39–45. AOAC, 1990. Official Methods of Analysis, 15th ed. Association of Official Analytical Chemists, Arlington, VA, USA. Baumont, R., 1996. Palatability and feeding behaviour in ruminants: a review. Ann. Zootech. 45, 385–400. Ben Salem, H., Nefzaoui, A., Ben Salem, L., 2002. Supplementation of Acacia cyanophylla Lindl. foliage-based diets with barley or shrubs from arid areas (Opuntiaficus-indica f. inermis and Atriplex nummularia L.) on growth and digestibility in lambs. Anim. Feed Sci. Technol. 96, 15–30. Azizi-Shotorkhoft, A., Rouzbehan, Y., Fazaeli, H., 2012. The influence of the different carbohydrate sources on utilization efficiency of processed broiler litter in sheep. Livest. Sci. 148, 249–254. Broderick, G.A., Kang, J.H., 1980. Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media. J. Dairy Sci. 63, 64–75. Chapman, H.D., Pratt, P.F., 1961. Methods of analysis for soils. In: Plants and Waters. University of California, Division of Agricultural Sciences, Oakland, CA, USA, pp. 309 pp. Chen, X.B., Gomes, J.M., 1992. Estimation of Microbial Protein Supply to Sheep and Cattle Based on Urinary Excretion of Purine Derivatives: An Overview of the Technical Details. International Feed Resources Unit, Rowett Res. Inst., Bucksburn, Aberdeen, UK, pp. 21 pp (Occasional publication). Dehority, B.A., 2003. Rumen Microbiology, 1st ed. Nottingham University Press, UK, pp. 372 pp. Demarquilly, C., 1990. Utilisation des conservateurs: quand et pourquoi les utiliser. Resultats zootechniques obtenus avec des ensilages d’herbe prepares avec des conservateurs efficaces. In: Symposium International sur l’ensilage d’herbe, Conseil de Recherche en Agro-Alimentaire de l’Abitibi-Temiscamingue, Rouyn-Noranda, QC, pp. 93–104. Galina, M.A., Hummel, J.D., Sanchez, M., Haenlein, G.F.W., 2004. Fattening Rambouillet lambs with corn stubble or alfalfa, slow intake urea supplementation or balanced concentrate. Small Rumin. Res. 53, 89–98. Givens, D.I., Owen, E., Axford, R.F.E., Omed, H.M., 2000. Forage Evaluation in Ruminant Nutrition, 1th ed. CABI Publishing, Wallingford, UK, pp. 480 pp. Haddad, S.G., Hussein, M.Q., 2004. Effect of dietary energy density on growth performance and slaughter characteristics of fattening Awassi lambs. Livestock Prod. Sci. 87, 171–177. Kauffman, C.S., Weber, L.E., 1990. Grain amaranth. In: Janick, J., Simon, J.E. (Eds.), Advances in New Crops. Timber Press, Portland, OR, USA, pp. 127–139. Kaur, R., Garcia, S.C., Fulkerson, W.J., Barchia, I.M., 2010. Utilisation of forage rape (Brassica napus) and Persian clover (Trifolium resupinatum) diets by sheep: effects on whole tract digestibility and rumen parameters. Anim. Prod. Sci 50, 59–67.

Please cite this article in press as: Rezaei, J., et al., Effects of substituting amaranth silage for corn silage on intake, growth performance, diet digestibility, microbial protein, nitrogen retention and ruminal fermentation in fattening lambs. Anim. Feed Sci. Tech. (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.03.005

G Model ANIFEE-13050; No. of Pages 10

10

ARTICLE IN PRESS J. Rezaei et al. / Animal Feed Science and Technology xxx (2014) xxx–xxx

Khandaker, Z.H., Tareque, A.M.M., 1997. Effect of rumen degradable protein (RDP) in straw based ration on purine derivatives excretion and microbial nitrogen supply in cattle. AJAS 10, 364–370. Martin, P.A., Chamberlain, D.G., Robertson, S., Hirst, D., 1994. Rumen fermentation patterns in sheep receiving silages of different chemical composition supplemented with concentrates rich in starch or in digestible fibre. J. Agric. Sci. (Camb.) 122, 145–150. McDonald, P., Edwards, R.A., Greenhalgh, J.F.D., Morgan, C.A., 2002. Animal Nutrition, 6th ed. Pearson Education Inc., Harlow, UK, pp. 672 pp. McDonald, P., Henderson, A.R., Herson, S.J.E., 1991. The Biochemistry of Silage, 2nd ed. Chalcombe Publication, Marlow, UK, pp. 340 pp. Mehrani, A., Fazaeli, H., Asadi, H., 2012. Effect of harvest in different growth stages on quantity and quality of forage of amaranth (Amaranthus sp.) varieties and its economic assessment. Seed Plant Prod. J. 28, 173–185 (in Persian). Menke, K.H., Steingass, H., 1988. Estimation of the energetic feed value obtained from chemical analysis and gas production using rumen fluid. Anim. Res. Dev. 28, 7–55. Myers, R.L., 1996. Amaranth, new crop opportunity. In: Janick, J. (Ed.), Progress in New Crops. ASHS Press, Alexandria, VA, pp. 207–220. Myers, R.L., Putnam, D.H., 1988. Growing Grain Amaranth as a Specialty Crop. Center for Alternative Crops and Products, Minnesota Extension Service. University of Minnesota, St. Paul, MN, USA. Nikkhah, N., Ghaempour, A., Khorvash, M., Ghorbani, G.R., 2011. Inoculants for ensiling low-dry matter corn crop: a midlactation cow perspective. J. Anim. Physiol. Anim. Nutr. 95, 623–631. NRC, 1985. Nutrient Requirements of Sheep, 6th rev. ed. National Academy Press, Washington, DC, USA, pp. 99 pp. NRC, 2001. Nutrient Requirements for Dairy Cattle, 7th rev. ed. National Academy Press, Washington, DC, USA, pp. 408 pp. Olfaz, M., Ocak, N., Erener, G., Cam, M.A., Garipoglu, A.V., 2005. Growth, carcass and meat characteristics of Karayaka growing rams fed sugar beet pulp, partially substituting for grass hay as forage. Meat Sci. 70, 7–14. Petit, H.V., Castonguay, F., 1994. Growth and carcass quality of prolific crossbred lambs fed silage with fish meal or different amounts of concentrate. J. Anim. Sci. 72, 1849–1856. Pond, W.G., Lehmann, J.W., 1989. Nutritive value of a vegetable amaranth cultivar for growing lambs. J. Anim. Sci. 67, 3036–3039. Rabbani, H., (M.Sc. thesis in Animal Science) 2012. Study on Silage Characteristics of Two Varieties of Amaranth Forage (Amaranthus hypochondriacus) and its Comparison with Corn Silage. Department of Animal Science, Bu-Ali Sina University, Hamadan, Iran, pp. 81. Ramos, S., Tejido, M.L., Martínez, M.E., Ranilla, M.L., Carro, M.D., 2009. Microbial protein synthesis, ruminal digestion, microbial populations, and nitrogen balance in sheep fed diets varying in forage-to-concentrate ratio and type of forage. J. Anim. Sci. 87, 2924–2934. Rezaei, J., Rouzbehan, Y., Fazaeli, H., 2009. Nutritive value of fresh and ensiled amaranth (Amaranthus hypochondriacus) treated with different levels of molasses. Anim. Feed Sci. Technol. 151, 153–160. Robertson, J.B., Van Soest, P.J., 1981. The detergent system of analysis and its application to human foods. In: James, W.P.T., Theander, O. (Eds.), The Analysis of Dietary Fiber in Food. Marcel Dekker, NY, USA, pp. 123–158 (chapter 9). Russell, J.B., Dombrowski, D.B., 1980. Effect of pH on the efficiency of growth by pure cultures of rumen bacteria in continuous culture. Appl. Environ. Microbiol. 39, 604–610. SAS, 2001. Statistical Analysis System. User’s Guide: Statistics, Version 8.2. SAS Institute, Cary, NC, USA. Shingfield, K.J., Jaakkola, S., Huhtanen, P., 2002. Effect of forage conservation method, concentrate level and propylene glycol on diet digestibility, rumen fermentation, blood metabolite concentrations and nutrient utilization of dairy cows. Anim. Feed Sci. Technol. 97, 1–21. Sleugh, B.B., Moore, K.J., Brummer, E.C., Knapp, A.D., Russell, J., Gibson, L., 2001. Forage value of various amaranth species at different harvest dates. Crop Sci. 41, 466–472. Stallknecht, G.F., Schulz-Schaeffer, J.R., 1993. Amaranth rediscovered. In: Janick, J., Simon, J.E. (Eds.), New Crops. Willey, New York, NY, USA, pp. 211–218. Temminghoff, E.J.M., Houba, J.G.V., 2004. Plant Analysis Procedures, 2nd ed. Kluwer Academic Publishers, Dordrecht, Netherlands, pp. 179 pp. Van Soest, P.J., 1994. Nutritional Ecology of the Ruminant, 2nd ed. Cornell Univ. Press, Itacha, NY, USA, pp. 476 pp. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597. Zuo, Z., (Master’s Thesis in Animal Nutrition and Feed Science) 2011. Effects of Different Ratios of Ruminally Degradable Protein to Ruminally Undegradable Protein on Ruminal Fermentation and Nutrient Digestion and Metabolism in Luxi Cattle Fed Distillers Grains-based Diets. Sichuan Agricultural University, Ya’an, Sichuan, China.

Please cite this article in press as: Rezaei, J., et al., Effects of substituting amaranth silage for corn silage on intake, growth performance, diet digestibility, microbial protein, nitrogen retention and ruminal fermentation in fattening lambs. Anim. Feed Sci. Tech. (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.03.005