Effect of harvest date and nitrogen fertilization rate on the nutritive value of amaranth forage (Amaranthus hypochondriacus)

Animal Feed Science and Technology 171 (2012) 6–13

Contents lists available at SciVerse ScienceDirect

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

Effect of harvest date and nitrogen fertilization rate on the nutritive value of amaranth forage (Amaranthus hypochondriacus) D. Abbasi, Y. Rouzbehan ∗ , J. Rezaei Animal Science Department, Faculty of Agriculture, Tarbiat Modares University, Tehran, P.O. Box 14115-336, Iran

a r t i c l e

i n f o

Article history: Received 23 April 2011 Received in revised form 22 September 2011 Accepted 23 September 2011

Keywords: Amaranth forage Nutritive value Harvest date Nitrogen fertilization rate

a b s t r a c t This study was conducted to assess effects of harvest date (i.e., 40 and 60 d after planting) and N fertilization rate (i.e., 120, 180, 240 kg N/ha) on the nutritive value of amaranth forage (Amaranthus hypochondriacus) using a factorial experiment with a randomized complete block design. The content of dry matter (DM), crude protein (CP), true protein (TP), ether extract (EE), water soluble carbohydrates (WSC), ash-free neutral detergent fiber (NDFom), ash-free acid detergent fiber (ADFom), lignin(sa), ash, Ca, P, Na, K, oxalic acid and nitrate were determined. Soluble CP (SP) and protein fractions non-protein N (A), true protein rapidly degraded in the rumen (B1 ), true protein degraded in the rumen at a moderate rate (B2 ), true protein associated with the cell wall and slowly degraded in the rumen (B3 ) and acid detergent insoluble CP (C) were measured according to the Cornell Net Carbohydrate and Protein System. In vitro gas production (IVGP), OM disappearance (OMD) and NDFom disappearance (NDFD) were determined using a gas production technique. Results showed that the later harvest date increased (P<0.05) DM, EE, WSC, NDFom, ADFom, lignin(sa), B3 and C; while CP, TP, ash, Ca, P, K, SP, A, B1 , B2 , nitrate, total and soluble oxalic acid, IVGP, b (i.e., gas production from the insoluble fermentable fractions at 120 h), c (i.e., rate of gas production during incubation), OMD and NDFD decreased (P<0.05). With increasing N fertilization rate, CP, TP, EE, P, nitrate, oxalic acid, SP, A, b, OMD and NDFD increased (P<0.05), however B2 declined (P<0.05). Increasing N fertilization increased yield, CP concentration and nutrient digestibility. At 40 d after planting use of amaranth forage as a ruminant feed is limited due to its high nitrate content. However, at 60 d, although a depression in digestibility and CP content occurred, this forage has the potential as a ruminant feed due to the much lower nitrate levels. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Amaranth, genus Amaranthus, belongs to the Amaranthaceae family and includes more than 60 species (Pisarikova et al., 2006). Selected genotypes of Amaranthus hypochondriacus are characterized by a low optimal temperature for germination, fast growth after germination, early establishment of canopy (Pisarikova et al., 2006), early maturity, low water requirement

Abbreviations: A, non-protein N; ADFom, acid detergent fiber; ADICP, acid detergent insoluble CP; b, gas production from slowly fermentable OM; B1 , true protein rapidly degraded in the rumen; B2 , true protein degraded in the rumen at a moderate rate; B3 , true protein associated with the cell wall and slowly degraded in the rumen; C, ADICP; c, rate of gas production; CP, crude protein; DAP, days after planting; DM, dry matter; EE, ether extract; F, effect of fertilization; H, effect of harvest date; IVGP, in vitro gas production; lignin(sa), lignin determined by solubilisation of cellulose with sulphuric acid; L, linear effect; ME, metabolisable energy; NDFom, neutral detergent fiber; NDFD, NDFom disappearance; NDICP, neutral detergent insoluble CP; NPN, non-protein N; OM, organic matter; OMD, in vitro OM digestibility; SP, soluble CP; TP, true protein; WSC, water soluble carbohydrates. ∗ Corresponding author. Tel.: +98 21 48292336; fax: +98 21 48292200. E-mail address: rozbeh [email protected] (Y. Rouzbehan). 0377-8401/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2011.09.014

D. Abbasi et al. / Animal Feed Science and Technology 171 (2012) 6–13

7

(Kauffman and Weber, 1990) and a yield performance of up to 103 t fresh mass/ha and 15.7 t dry matter/ha (Mehrani and Fazaeli, 2008). Several studies (Pond and Lehmann, 1989; Stordahl et al., 1999; Sleugh et al., 2001; Rezaei et al., 2009a) have shown that the nutritive value of amaranth as a ruminant feed is equal to, or better than, commonly used forages such as alfalfa. Its favorable composition (i.e., high crude protein (CP) and low lignin(sa) ranging from 80 to 285 and from 17 to 73 g/kg DM, respectively), low nitrate and oxalic acid levels (i.e., below toxic levels) and its high in vitro DM disappearance of 590–790 g/kg DM suggest high potential as a ruminant feedstuff (Sleugh et al., 2001; Rezaei et al., 2009b). Forage quality depends on harvest date (Nordheim-Viken and Volden, 2009) and N fertilization rate (Almodares et al., 2009). Plant maturity was found to affect neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin(sa), CP and nitrate content of amaranth forage (Sleugh et al., 2001). Soil N was the primary limiting factor to amaranth production and research has shown positive effects of N fertilization on plant growth and development, plant height and inflorescence length, DM yield, CP content and in vivo digestibility (Peyraud and Astigarraga, 1998; Pospisil et al., 2006). However, research with amaranth as a forage has mainly focused on yield traits, such as green mass and DM and grain yield rather than its nutritional value as a forage (Pospisil et al., 2006, 2009). Hence, the aim of this study was to determine effects of harvest date and N fertilization rate on biomass yield, chemical composition, secondary compound levels, protein fractions and in vitro digestibility of whole crop amaranth (A. hypochondriacus L.). 2. Materials and methods 2.1. Amaranth forage This investigation was in an experimental field near Tehran (Iran) in 2009 at an altitude of 1215 m above sea level with a mean annual rainfall and temperature of 306 mm and 15 ◦ C, respectively. The soil at the experimental site is soft loam-sandy. The forage was planted on 27th July (2009). Three rates of N fertilizer as urea (i.e., 120, 180, 240 kg N/ha) and two harvest dates (i.e., 40, 60 d after planting; DAP) were assessed with a factorial experiment in a randomized complete block design with four replications. There were 24 plots and each measured 9.6 m2 , with 12 preselected for whole-plant harvest at 40 DAP (i.e., early flowering) and 12 at 60 DAP (i.e., milk stage of seed). Plots were harvested at early flowering and at milk stage by cutting the plants with knife 5 cm above ground level. Fresh biomass production from each plot (9.6 m2 ) was determined at harvest. Samples were dried to a constant weight for DM content determination. Each individual plot sample was analyzed chemically and by in vitro gas production in triplicate. 2.2. Chemical composition For chemical analyses, samples were milled to pass a 1 mm screen in a CyclotecTM 1093 Sample Mill (Foss Companies, Hillerød, Denmark). Samples were analyzed for DM (oven-dried at 60 ◦ C for 48 h), ash (# 924.05), N (# 984.13) and ether extract (EE) (# 954.02) of AOAC (1990). Ash-free neutral detergent fiber (NDFom) was determined without use of sodium sulphite or amylase according to Van Soest et al. (1991). Acid detergent fiber (ADFom) was determined (# 973.18; AOAC, 1990) and expressed exclusive of residual ash. Lignin(sa) was determined by solubilization of cellulose with 720 g/kg sulphuric acid (Robertson and Van Soest, 1981). Water soluble carbohydrates were measured using the anthrone method (MAFF, 1982). Calcium was determined by atomic absorption (Temminghoff and Houba, 2004), P by spectrophotometer (Chapman and Pratt, 1961) and Na and K by flame emission spectrometer (Temminghoff and Houba, 2004). Contents of total and soluble oxalic acid were determined by High Performance Liquid Chromatography (Savage et al., 2000). From one gram of sample ground by the CyclotecTM 1093 Sample Mill, total oxalate was extracted with 50 ml HCl (2 M) in a water bath at 80 ◦ C for 15 min. Soluble oxalate was extracted by the same method, but with 50 ml distilled water (Savage et al., 2000). Nitrate was determined by the colorimetric method of Singh (1988). The CP in the forage samples were partitioned as proposed by Cornell Net Carbohydrate and Protein System; CNCPS (Licitra et al., 1996). Soluble CP (SP; fraction A plus fraction B1 ) was determined using the borate–phosphate buffer method of Krishnamoorthy et al. (1982). Non-protein N (NPN; fraction A) was calculated as the difference between the total N and TP N precipitated with tungstic acid (Licitra et al., 1996). Neutral detergent insoluble CP (NDICP) was determined after NDF determination and acid detergent insoluble CP (ADICP; fraction C) was determined after ADF determination (Licitra et al., 1996). True protein rapidly degraded in the rumen (fraction B1 ) was calculated as SP minus A. True protein degraded in the rumen at a moderate rate (fraction B2 ) was estimated as insoluble CP minus NDICP, and TP associated with cell wall and slowly degraded in the rumen (fraction B3 ) was estimated as NDICP minus ADICP. All N analyses were conducted using the FOSS KjeltecTM 2300 Analyzer (Foss Companies, Hillerød, Denmark). 2.3. In vitro gas production parameters and NDF disappearance For samples (i.e., 24 samples in triplicate), gas production kinetics, estimated organic matter (OM) disappearance (OMD) and metabolisable energy (ME) were determined as described by Menke and Steingass (1988). Kinetic parameters were estimated using the exponential equation of Ørskov and McDonald (1979) as: Y = b(1 − e−ct )

8

D. Abbasi et al. / Animal Feed Science and Technology 171 (2012) 6–13

where ‘b’ is gas production from the fermentable fraction (ml/200 mg DM), ‘c’ is the gas production rate for ‘b’ (/h), ‘t’ is the incubation time (h) and Y is the gas produced at t. The ME and OMD of samples were calculated using equations of Menke et al. (1979) as: ME(MJ/kgDM) = 2.20 + (0.1357 × GP) + (0.0057 × CP) + (0.00002859 × (CP)2 )

OMD(g/100 gOM) = 14.88 + (0.8893 × GP) + (0.0448 × CP) + (0.0651 × Ash) where GP is 24 h net gas production (ml/200 mg DM), CP is in g/kg DM and ash is in g/kg DM. The NDFom disappearance (NDFD) was determined by the in vitro incubation method of Menke and Steingass (1988) by collecting the NDF residues at the end of the incubation at 48 h. 2.4. Statistical analysis Obtained data were analyzed in a 2 (harvest date) × 3 (fertilization) factorial experiment in randomized complete block design with four plots replicates. Data were subjected to analysis using the GLM procedure of SAS (2001), using the statistical model: Yijk =  + Rk + Ai + Bj + ABij + eijk where Yijk is the measured value,  the general mean, Rk the block effect, Ai the effect of harvest date, Bj the effect of N fertilization rate, ABij the interaction of harvest date and N fertilization and eijk the error term. A polynomial linear contrast was used to test effects of N fertilization rate on parameters of forage quality. 3. Results 3.1. Yield and chemical composition Harvest date had positive effect on fresh and DM yields (P<0.001) (Table 1). Increased N fertilization increased fresh (L: P=0.007) and DM (L: P<0.001) yields. With increased plant maturity, DM content increased (P<0.001) and CP content decreased (P<0.001), which was accompanied by a reduction in true protein (TP). As expected, as N fertilization was raised, there was an enhancement (L: P<0.001) in the CP content of the forage. At 60 DAP, EE content was higher than at 40 DAP (P<0.001). Nitrogen fertilization increased EE content (L: P=0.014). The concentration of WSC was highest (P=0.036) at the second harvest date. The NDFom, ADFom and lignin(sa) contents increased (P<0.001) with advancing maturity. As the plant matured, ash content and the amounts of Ca, P and K declined (P<0.05). Increased N fertilization linearly increased P content (L: P=0.006).

Table 1 Yield and chemical composition (g/kg DM) of amaranth forage as affected by harvest date and N fertilization rate. Harvest date (DAP)

P

40 N level (kg/ha) Fresh yield (kg/ha) Dry matter yield (kg/ha) Dry matter (g/kg fresh wt.) Crude protein True protein Ether extract Water soluble carbohydrates NDFom ADFom Lignin(sa) Ash Ca P Naa K

120 46,253 6938 150 243 140 46.0 87 351 207 25.3 178 26.1 6.2 7.8 45.6

60 180 49,700 7554 152 253 145 47.9 92 348 210 25.5 175 26.0 6.5 8.1 46.5

240 53,375 8380 157 265 149 48.4 93 350 215 25.8 173 25.5 6.8 7.4 46.7

120 82,176 14,874 181 148 93 51.2 95 464 288 44.6 139 17.8 5.8 7.4 39.7

H 180 83,032 15,195 183 162 100 53.0 102 466 289 45.4 136 18.3 5.9 7.7 39.2

240 84,974 16,655 196 170 104 53.9 105 461 289 45.6 135 17.6 6.0 8.0 38.4

SEM

(Linear)

11.7 8.8 6.6 6.5 5.6 0.72 2.5 10.6 9.5 2.39 4.8 0.97 0.08 0.27 0.94

<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.036 <0.001 <0.001 <0.001 <0.001 <0.001 0.033 0.78 <0.001

F

H×F

0.007 <0.001 0.28 <0.001 <0.011 0.014 0.059 0.077 0.073 0.061 0.089 0.38 0.006 0.68 0.91

0.11 0.29 0.87 0.72 0.83 0.95 0.054 0.097 0.051 0.59 0.96 0.77 0.38 0.035 0.42

DAP, days after planting; NDFom, ash-free neutral detergent fiber; ADFom, ash-free acid detergent fiber; H, effect of harvest dates; F, effect of N fertilization. a The probability levels of the linear effect of N level within each harvest date (i.e., 40 and 60 DAP) are P=0.23 and P=0.028, respectively.

D. Abbasi et al. / Animal Feed Science and Technology 171 (2012) 6–13

9

Table 2 Secondary compounds (g/kg DM) of amaranth forage as affected by harvest date and N fertilization rate. Harvest date (DAP)

P

40

60

H

N level (kg/ha)

120

180

240

120

180

240

SEM

(Linear)

Nitrate NO3 -N (g/kg N) Total oxalic acid Soluble oxalic acid Insoluble oxalic acid

26.4 153 18.9 15.3 3.6

29.1 162 19.8 15.4 4.4

37.2 198 21.8 17.1 4.7

10.4 99.0 10.2 6.7 3.5

11.6 101 11.3 7.0 4.3

14.0 116 11.8 8.0 3.8

2.46 4.3 0.86 0.62 0.26

<0.001 <0.001 <0.001 <0.001 0.29

F

H×F

<0.001 0.007 0.037 0.041 0.12

0.099 0.22 0.69 0.89 0.52

DAP, days after planting; H, effect of harvest dates; F, effect of N fertilization.

3.2. Secondary compounds At 60 DAP, nitrate contents (Table 2) decreased (P<0.001). However with rising N fertilization, nitrate content increased linearly (L: P<0.001). With advancing maturity, concentrations of total and soluble oxalic acids decreased (P<0.001). Concentrations of total (L: P=0.037) and soluble oxalic acid (L: P=0.041) increased with increasing N fertilization. 3.3. Protein fractions Comparing harvest dates, amaranth at 40 DAP was higher (P<0.001) in SP (Table 3). Higher N application resulted in an increase in SP (L: P=0.037). As the plants advanced in maturity, the contents of fraction A and B1 decreased (P<0.001). With rising N fertilization, the content of fraction A increased (L: P=0.043). There was a tendency for fraction B2 to decrease (P=0.048) with maturity. With increased N fertilization, fraction B2 decreased (L: P=0.026). At 60 DAP, the contents of NDICP, fraction B3 and C were higher (P<0.001) than at 40 DAP. 3.4. In vitro gas production, estimated parameters and NDFD At 60 DAP, values of IVGP, b, c, OMD, ME and NDFD were less than those at 40 DAP (P<0.05). Apart from ‘c’, all in vitro parameters increased (L: P<0.05) with increasing rate of N application. The NDFD decreased (P<0.001) with plant maturity, while increasing N fertilization increased NDFD (L: P=0.018) (Table 4). 4. Discussion 4.1. Yield and chemical composition Similar to Peyraud and Astigarraga (1998), increased N fertilization increased fresh and DM yields. The increasing DM content with plant maturity is consistent with Johnson et al. (2001) and Mahmud et al. (2003). The reduction of CP content with increased maturity was related to a decline in the portion of leaves in the forage biomass, which has a higher CP concentration (Freer and Dove, 2002). Similar to our results, Sleugh et al. (2001) and Yu et al. (2004) observed that as forages advance in maturity the CP content decreases. As expected, as N fertilization increased, N uptake by the plants increased (Johnson et al., 2001) resulting in a linear enhancement in its CP content. However, in all samples, the CP content was more than 80 g/kg DM which, according to Norton (1998), should provide ruminal ammonia levels above the minimum required by rumen microorganism to support optimum growth. In our study, the EE content was high (at 50 g/kg DM) and at 60 DAP Table 3 Crude protein fractions (g/kg CP) of amaranth forage as affected by harvest date and N fertilization rate. Harvest date (DAP)

P

40

60

H

N level (kg/ha)

120

180

240

120

180

240

SEM

(Linear)

Soluble CP A B1 B2 NDICP B3 C

531 426 105 339 130 91.3 38.7

534 428 106 326 140 98.1 41.9

544 437 107 312 144 101 43.4

428 371 57.0 322 250 169 80.9

438 378 60.0 316 246 165 81.0

445 385 60.0 308 247 166 80.6

4.8 1.7 6.56 3.8 13.4 8.73 4.76

<0.001 <0.001 <0.001 0.048 <0.001 <0.001 <0.001

F

H×F

0.037 0.043 0.78 0.026 0.19 0.44 0.28

0.94 0.97 0.99 0.72 0.14 0.29 0.48

DAP, days after planting; A, non-protein N; B1 , true protein rapidly degraded in the rumen; B2 , true protein degraded in the rumen at a moderate rate; NDICP, neutral detergent insoluble CP; B3 , true protein associated with the cell wall and slowly degraded in the rumen; C, acid detergent insoluble CP; H, effect of harvest dates; F, effect of N fertilization.

10

D. Abbasi et al. / Animal Feed Science and Technology 171 (2012) 6–13

Table 4 In vitro fermentation of amaranth forage as affected by harvest date and N fertilization rate. Harvest date (DAP)

P

40

60

H

N level (kg/ha)

120

180

240

120

180

240

SEM

(Linear)

IVGP b c OMD ME NDFD

37.8 46.5 0.101 710 10.4 706

39.5 47.9 0.102 727 10.8 715

39.8 49.1 0.101 734 11.1 717

35.1 45.1 0.094 618 8.4 574

35.3 45.1 0.091 624 8.7 597

36.8 47.6 0.092 640 8.9 603

0.52 0.43 0.001 3.2 0.06 3.9

<0.001 0.029 0.042 0.010 0.011 <0.001

F

H×F

0.014 0.024 0.86 0.004 0.048 0.018

0.54 0.061 0.68 0.51 0.46 0.17

DAP, days after planting; IVGP, in vitro gas production at 24 h (ml/200 mg DM); b, the gas production from the insoluble but fermentable fractions at 120 h (ml/200 mg DM); c, rate of gas production during incubation (/h); OMD, estimated OM disappearance (g/kg OM); ME, metabolisable energy (MJ/kg DM); NDFD, NDFom disappearance (g/kg NDF); H, effect of harvest dates; F, effect of N fertilization.

the EE content was higher than at 40 DAP, probably because of seed formation at that stage which contains oil (Betschart et al., 1981; Lorenz and Hwang, 1985). Nitrogen fertilization also increased EE content. Maturity effects on sugar levels of forage crops are inconsistent. Up to the full-bloom stage for legume crops, WSC concentrations tend to decline with maturity (Smith, 1973). In contrast, WSC concentrations in cool season grasses increase with advancing maturity (McDonald et al., 1991). In cool and warm season whole crop cereals, WSC concentrations increase until the milk stage of seed development and then decrease as the seeds develop (Barnes et al., 2007). Similarly, in our study, the concentration of WSC was highest at 60 DAP, probably because the forage had reached the milk stage (Barnes et al., 2007). The NDFom, ADFom and lignin(sa) contents increased with advancing maturity due to an expected decline of leaf:stem ratio (Freer and Dove, 2002). Similarly, Sleugh et al. (2001) and Yu et al. (2004) showed that as plants advance in maturity their cell wall content increases. However, the NDFom content remained unaffected by N fertilization, which is in agreement with Peyraud and Astigarraga (1998) and Abarza et al. (2001). Amaranth contains a high ash content due to its C4 metabolism and a very high carbon uptake per unit area (Edwards et al., 1983). This high ash is in agreement with Rezaei et al. (2009a), and the low P values are similar to A. hypochondriacus L. (Alfaro et al., 1987; Rezaei et al., 2009a). In our samples, the overall mean value for Na was relatively high at 7.8 g/kg DM. Our amaranth had a K content of about 42 g/kg DM which is lower than that reported by Rezaei et al. (2009a). As plants matures their ash content and levels of Ca, P and K tend to decline (Underwood and Suttle, 1999; Givens et al., 2000) due to dilution since, as the photosynthetic areas increase, DM production outstrips mineral uptake resulting in a decline in mineral concentration. Increasing N fertilization rate linearly increased the P content of our amaranth, although N fertilization effects on P levels of plants are inconsistent. For example, uptake of P by whole plant was generally increased with N fertilization (Coblentz et al., 2004), and the P concentration of sorghum forage increased (Spooner et al., 1971; Hunt, 1974). In contrast, P concentration in the whole plant, and in each component of E. purpurea, did not have a relationship with N level applied (Bonomelli et al., 2005). Factors such as soil, climate, stage of maturity, season and management contribute to variations in the concentration of minerals in forages (Whitehead, 2000; Klemencic and Kramberger, 2006). 4.2. Secondary compounds The nitrate levels at 40 DAP, of 26.4–37.2 g/kg DM, were higher than the toxic level for cattle (i.e., >17.6 g/kg DM) reported by Vough et al. (1991). However, at 60 DAP, nitrate contents decreased to the safe range for ruminant consumption. This decrease in nitrate level was accompanied with a CP decline, similar to Sleugh et al. (2001). With rising N fertilization, nitrate content increased linearly, which is in agreement with Follett and Wilkinson (1995) and Peyraud and Astigarraga (1998). The reduction of nitrates to ammonia N and its incorporation into protein can be considered as a single step limited by nitrate reductase, which mediates the first reaction. Thus levels of this enzyme may be a limiting step to nitrate reduction at high levels of fertilization, thereby leading to its accumulation in the plant (Peyraud and Astigarraga, 1998). In our study, total oxalic acid concentrations of 10.2–21.8 g/kg DM were lower than the acute toxic levels for cattle and horse (i.e., <70 g/kg DM) suggested by Radostits et al. (2000). Although a wide range of oxalic acid values for amaranth (i.e., 2–114 g/kg DM) was reported by Teutonico and Knorr (1985), Blaney et al. (1982) stated that only soluble oxalate is harmful to cattle due to formation of insoluble complexes with Ca. In our study, the soluble oxalate content was lower than 20 g/kg DM which, according to McKenzie et al. (1988), could cause deaths in sheep. With increasing plant maturity, concentrations of total and soluble oxalic acids decreased due to the rise in stem proportion as it contains lower levels of oxalates than leaves (Bressani, 1993). Concentrations of total and soluble oxalic acid increased with increasing N fertilization rate, probably due to the increase in leaf proportion (Middleton and Barry, 1978). 4.3. Protein fractions The 428–544 g/kg CP of forage as SP is higher than the SP content (i.e., 411 g/kg CP) reported by Rezaei et al. (2009b). Comparing harvests, amaranth at 40 DAP was higher in SP, indicating that amaranth at this growth stage contained more

D. Abbasi et al. / Animal Feed Science and Technology 171 (2012) 6–13

11

rapidly degradable CP. The reduction in SP as maturity advanced was anticipated, and is in agreement with Michaud et al. (2003) with alfalfa. More N fertilization resulted in a linear increase of SP, which is probably due to accumulation of nonprotein organic N (Peyraud and Astigarraga, 1998). The concentration of fraction A (i.e., NPN) in amaranth was 371 to 437 g/kg CP, similar to values of 395 g/kg CP reported by Rezaei et al. (2009b). Although rumen microbes use NPN, much of it will be converted to ammonia if the diet CP is higher than the requirement of ruminants. If so, ammonia will be absorbed from the rumen, transported in the blood to the liver, converted to urea and excreted in urine (Martin et al., 2004). With maturity, fraction A decreased, likely due to increased CP bonded to cell wall constituents (McDonald et al., 1995). With rising N fertilization, the content of fraction A tended to increase. N uptake by plants increases rapidly with increased N fertilization and this leads to accumulation of non-protein organic N thereby decreasing the proportion of protein N (Peyraud and Astigarraga, 1998). With increased plant maturity, fraction B1 decreased, probably due to an increase in cell wall bound N (McDonald et al., 1995). Similarly, Ehsani (2007) observed a negative relationship between NDF and fraction B1 N. However, N fertilization had no effect on fraction B1 . There was a tendency for fraction B2 to decrease with advancing maturity, similar to Ehsani (2007). With rising N fertilization, fraction B2 decreased. As the plant matured, NDICP content increased, probably due to a rise in N in cell wall (McDonald et al., 1995). At 60 DAP, fraction B3 was higher than that at first harvest, similar to Michaud et al. (2003) with alfalfa. Different changes in NDF concentration of plant parts with maturity may explain the differences in proportions of fraction B3 (Alzueta et al., 2001). At 60 DAP, fraction C increased due to increasing lignification (Schmidt et al., 1988). However, the content of fraction C was lower than that reported by Schmidt et al. (1988) (i.e., <100 g/kg N), and this fraction is unavailable to the animal (Fox et al., 2003). It is noteworthy that these results on CP fractions indicate that those with rapid and moderate rates of degradation were altered to a greater extent than the slowly degraded and indigestible CP fractions when N fertilization was higher. 4.4. In vitro gas production, estimated parameters and NDFD At 60 DAP, the IVGP, b, c, OMD, and ME were lower than those at 40 DAP, probably due to a reduction in the leaf:stem ratio (Akin et al., 1977) and increased lignin(sa) (Van Soest, 1994; McDonald et al., 1995). Similar findings were obtained by Rinne et al. (1997) and Yu et al. (2004) with grasses and alfalfa respectively. Apart from ‘c’, all in vitro parameters increased linearly with increasing rate of N application, which was probably due to an increased leaf:stem ratio (McDonald et al., 1995) and WSC level (Freer and Dove, 2002). The NDFD decrease with plant maturity is likely due to the increased content of lignin. Many workers have observed the potential of lignin to inhibit NDFD in forages (e.g., Van Soest, 1994; McDonald et al., 1995; Moore and Jung, 2001). However, increasing N fertilization improved NDFD, possibly due to the increase in WSC which is a source of energy for microorganisms in the rumen (Van Soest, 1994). 5. Conclusions Increasing N fertilization led to improved yield, CP concentration and nutrient digestibility of amaranth forage. At 40 DAP there is a limitation to using amaranth forage as a ruminant feed due to a high nitrate content. However at 60 DAP, although a depression in digestibility and CP content had occurred, this forage has the potential to be used as a ruminant feed due to the many lower nitrate levels. However more research, especially on in vivo animal responses, is needed to affirm the generally positive nutritional characteristics of amaranth reported in our study. Acknowledgement The financial support from Ali Karimzadeh at the “Panje Talaee Cultivation and Industry” is gratefully acknowledged. References Abarza, S., Brevedan, R., Labrode, H.,2001. Forage quality of mixtures of Bromus wildenowii and B. parodii influenced by nitrogen fertilization or alfalfa interseeding. In: Proc. XIX International Grassland Congr. Brazilian Society of Animal Husbandry, Sao Pedro, Sao Paulo, Brazil, pp. 735–737. Akin, D.E., Robinson, E.L., Barton, F.E., Himmelsbach, D.S., 1977. Change with maturity in anatomy, histochemistry, chemistry and tissue digestibility of bermudagrass plant parts. J. Agric. Food Chem. 25, 179–186. Alfaro, M.A., Martinez, A., Ramirez, R., Bressani, R., 1987. Yield and chemical composition of the vegetal parts of the amaranth (Amaranthus hypochondriacus, L.) at different physiology stages. Arch. Latinoam. Nutr. 37 (1), 108–121. Almodares, A., Jafarinia, M., Hadi, M.R., 2009. The effects of nitrogen fertilizer on chemical compositions in corn and sweet sorghum. American-Eurasian. J. Agric. Environ. Sci. 6 (4), 441–446. Alzueta, C., Caballero, R., Rebole, A., Trevino, J., Gil, A., 2001. Crude protein fractions in common vetch (Vicia sativa L.) fresh forage during pod filling. J. Anim. Sci. 79, 2449–2455. AOAC, 1990. Official Methods of Analysis, 15th ed. Assoc. Off. Anal. Chem., Washington, DC, USA. Barnes, B.F., Nelson, C.J., Moore, K.J., Collins, M., 2007. Forages (Volume II): The Science of Grassland Agriculture, 6th ed. Blackwell Publishing, Ames, IA, USA, 791 pp. Betschart, A.A., Irving, D.W., Shepherd, A.D., Saunders, R.M., 1981. Amaranthus cruentus: milling characteristics distribution of nutrient within seed components, and the effects of temperature on nutritional quality. J. Food Sci. 46, 1181–1187. Blaney, B.J., Gartner, R.J.W., Head, T.A., 1982. The effects of oxalate in tropical grasses on calcium, phosphorus and magnesium availability to cattle. J. Agric. Sci. Camb. 99, 533–539.

12

D. Abbasi et al. / Animal Feed Science and Technology 171 (2012) 6–13

Bonomelli, C., Cisterna, D., Reciné, C., 2005. Effect of nitrogen fertilization on Echinacea purpurea mineral composition. Cienc. Inv. Agr. 32 (2), 85–91. Bressani, R., 1993. Amaranth. In: McRae, R., Robinson, R.K., Sadler, M.J. (Eds.), Encyclopedia of Food Science, Food Technology and Nutrition, vol. 1. Academic Press, London/England/San Diego, CA, USA, pp. 135–140. 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, 309 pp. Coblentz, W.K., Daniels, M.B., Gunsaulis, J.L., Turner, J.E., Scarbrough, D.A., Humphrey, J.B., Coffey, K.P., Moore Jr., P.A., Teague, K.A., Speight, J.D., 2004. Effects of nitrogen fertilization on phosphorus uptake in bermudagrass forage grown on high soil-test phosphorus soils. Prof. Anim. Sci. 20, 146–154. Edwards, G.E., Walker, D.A., 1983. C3, C4: Mechanisms and Cellular and Environmental Regulation of Photosynthesis. Blackwell Scientific Publications, Oxford, UK, 552 pp. Ehsani, P., 2007. Evaluation and comparing of qualitative and quantitative yield of four amaranth varieties in different harvesting date. MSc Thesis. Islamic Azad University of Saveh, Saveh, Iran. Follett, R.F., Wilkinson, S.R., 1995. Nutrient management of forages. In: Barnes, R.F., Miller, D.A., Nelson, C.J. (Eds.), Forages: The Science of Grassland Agriculture. Iowa State Univ. Press, Ames, IA, USA, pp. 55–82. Fox, D.G., Tylotki, T.P., Tedeschi, L.O., Van Amburgh, M.E., Chase, L.E., Pell, A.N., Overton, T.R., Russell, J.B., 2003. The net carbohydrate and protein system for evaluating herd nutrition and nutrient excretion: CNCPS Version 5.0. Model documentation. In: Animal Science Mimeo 213. Department of Animal Science, Cornell Univ., Ithaca, NY. Freer, M., Dove, H., 2002. Sheep Nutrition, 1st ed. CABI Publishing, Wallingford, Oxon, UK, 385 pp. Givens, D.I., Owen, E., Axford, R.F.E., Omed, H.M., 2000. Forage Evaluation in Ruminant Nutrition. CABI Publishing, Wallingford, UK, 480 pp. Hunt, I.V., 1974. Studies in response to nitrogen. Part 7. Residual response as mineral uptake. J. Brit. Grassl. Soc. 29, 225–231. Johnson, C.R., Reiling, B.A., Mislevy, P., Hall, B., 2001. Effects of nitrogen fertilization and harvest date on yield, digestibility, fiber, and protein fractions of tropical grasses. J. Anim. Sci. 79, 2439–2448. 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. Klemencic, S., Kramberger, B., 2006. Effects of growth stage and the season on the mineral concentrations in Cerastium holosteoides. J. Cent. Eur. Agric. 7 (4), 767–772. Krishnamoorthy, U., Muscato, T.V., Sniffen, C.J., Van Soest, P.J., 1982. Nitrogen fractions in selected feedstuffs. J. Dairy Sci. 65, 217–225. Licitra, G., Hernandez, T.M., Van Soest, P.J., 1996. Standardization of procedures for nitrogen fractionation of ruminant feeds. Anim. Feed Sci. Technol. 57, 347–358. Lorenz, K., Hwang, Y.S., 1985. Lipids in amaranths. Nutr. Rep. Int. 31, 83–89. Mahmud, K., Ahmad, I., Ayub, M., 2003. Effect of nitrogen and phosphorus on the fodder yield and quality of two sorghum cultivars (Sorghum bicolor L.). Int. J. Agric. Biol. 5, 61–63. Martin, N.P., Mertens, D.R., Weimer, P.J., 2004. Alfalfa: hay, haylage, baleage, and other novel products. In: Proceedings of the Idaho Alfalfa and Forage Conference, February 24–25, Twin Falls, ID, USA, 2004. Idaho Hay and Forage Association Inc., University of Idaho Cooperative Extension, pp. 9–18. McDonald, P., Henderson, A.R., Herson, S.J.E., 1991. The Biochemistry of Silage, 2nd ed. Chalcombe Publication, Marlow, UK. McDonald, P., Edwards, R.A., Greenhalgh, J.F.D., Morgan, C.A., 1995. Animal nutrition. In: Longman Scientific and Technical, 5th ed. John Wiley and Sons, Inc., New York, NY, USA, 607 pp. McKenzie, R.A., Bell, A.M., Storie, G.J., Keenan, F.J., Cornack, K.M., Grant, S.G., 1988. Acute oxalate poisoning of sheep by buffel grass (Cenchrus ciliaris). Aust. Vet. J. 65, 1–26. Mehrani, A., Fazaeli, H., 2008. Evaluation and comparing of qualitative and quantitative yield of four amaranth varieties in different harvesting date. In: Final Report of Research. Seed and Plant Improvement Institute, Agricultural Research and Education Organization, Karaj, Alborz, Iran, 44 pp. Menke, K.H., Raab, L., Salewski, A., Steingass, H., Fritz, D., Schneider, W., 1979. The estimation of the digestibility and metabolisable energy content of ruminant feedingstuffs from the gas production when they are incubated with rumen liquor in vitro. J. Agric. Food Sci. 93, 217–222. 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. Michaud, R., Tremblay, G.F., Belanger, G., 2003. Crude protein degradation in leaves and stems of alfalfa (Medicago sativa). J. Dairy Sci. 80, 315–325. Middleton, C.H., Barry, G.A., 1978. A study of oxalate concentration in five grasses in the wet tropics of Queensland. Trop. Grassl. 12, 28–34. Ministry of Agriculture, Fishier and Food (MAFF), 1982. The Analysis of Agricultural Materials, 2nd ed. MAFF, London, UK. Moore, K.L., Jung, H.G., 2001. Lignin and fiber digestion. J. Range Manage. 54, 420–430. Nordheim-Viken, H., Volden, H., 2009. Effect of maturity stage, nitrogen fertilization and seasonal variation on ruminal degradation characteristics of neutral detergent fibre in timothy (Phleum pratense L.). Anim. Feed Sci. Technol. 149, 30–59. Norton, B.W., 1998. The nutritive value of tree legumes. In: Gutteridge, R.C., Shelton, H.M. (Eds.), Forage Tree Legumes in Tropical Agriculture. Tropical Grassland Society of Australia Inc., Queensland, Australia. Ørskov, E.R., McDonald, I., 1979. The estimation of protein degradability in the rumen from incubation measurements weighed according to rate of passage. J. Agric. Sci. 92, 499–503. Peyraud, J.L., Astigarraga, L., 1998. Review of the effect of nitrogen fertilization on the chemical composition, intake, digestion and nutritive value of fresh herbage: consequences on animal nutrition and N balance. Anim. Feed Sci. Technol. 72, 235–259. Pisarikova, B., Peterka, J., Trckova, M., Moudry, J., Zraly, Z., Herzig, I., 2006. Chemical composition of the above-ground biomass of Amaranthus cruentus and A. hypochondriacus. Acta Vet. Brno. 75, 133–138. Pond, W.G., Lehmann, J.W., 1989. Nutritive value of a vegetable amaranth cultivar for growing lambs. J. Anim. Sci. 67, 3036–3039. Pospisil, A., Pospisil, M., Macesic, D., Svecnjak, Z., 2009. Yield and quality of forage sorghum and different amaranth species (Amaranthus spp.) biomass. Agric. Cons. Sci. 74 (2), 85–89. Pospisil, A., Pospisil, M., Varga, B., Svecnjak, Z., 2006. Grain yield and protein concentration of two amaranth species as influenced by nitrogen fertilizer. Eur. J. Agron. 25, 250–253. Radostits, O.M., Gay, C.C., Blood, D.C., Hinchcliff, K.W., 2000. Veterinary Medicine, 9th ed. Saunders Company, London, UK. Rezaei, J., Rouzbehan, Y., Fazaeli, H., 2009a. Nutritive value of fresh and ensiled amaranth (Amaranthus hypochondriacus) treated with different levels of molasses. Anim. Feed Sci. Technol. 151, 153–160. Rezaei, J., Rouzbehan, Y., Fazaeli, H., 2009b. An assessment of digestibility and protein quality of the fresh and ensiled amaranth forage according to CNCPS. Iranian J. Anim. Sci. 40 (3), 31–38. Rinne, M., Huhtanen, P., Jaakkola, S., 1997. Grass maturity effects on cattle fed silage-based diets. 2. Cell wall digestibility, digestion and passage kinetics. Anim. Feed Sci. Technol. 67, 19–35. 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 Fibre in Food. Marcel Dekker, NY, USA, pp. 123–158 (Chapter 9). Savage, G.P., Vanhanen, L., Mason, S.M., Ross, A.B., 2000. Effect of cooking on the soluble and insoluble oxalic acid content of some New Zealand foods. J. Food Compos. Anal. 13, 201–206. Schmidt, G.H., Van Vleck, L.D., Hutjens, M.F., 1988. Principles of Dairy Science, 2nd ed. Prentice-Hall Inc., Englewood Cliffs, NJ, USA, 466 pp. Singh, J.P., 1988. A rapid method for determination of nitrate in soil and plant extract. Plant and Soil 110, 137–139. 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.

D. Abbasi et al. / Animal Feed Science and Technology 171 (2012) 6–13

13

Smith, D., 1973. The nonstructural carbohydrates: 105–155. In: Butler, G.W., Bailey, R.W. (Eds.), Chemistry and Biochemistry of Herbage, vol. 1. Academic Press Inc., London, England. SAS, 2001. Statistical Analysis System, User’s Guide: Statistics, Version 8.2. SAS Institute, Cary, NC, USA. Spooner, A.E., Jeffery, R., Huneycutt, H.J., 1971. Effect of Management Practices on Johnson Grass for Hay Production. University of Arkansas, Fayetteville, AK, USA, Report series 99. Stordahl, J.L., Sheaffer, C.C., DiCostanzo, A., 1999. Variety and maturity affect amaranth forage yield and quality. J. Prod. Agric. 12, 249–253. Temminghoff, E.J.M., Houba, J.G.V., 2004. Plant Analysis Procedures, 2nd ed. Kluwer Academic Publishers, Dordrecht, Netherlands. Teutonico, R.A., Knorr, D., 1985. Amaranth: composition, properties, and applications of a rediscovered food crop. Food Technol. 39, 49–60. Underwood, E.J., Suttle, N.F., 1999. The Mineral Nutrition of Livestock, 3rd ed. CABI Publishing, Wallingford, UK. Van Soest, P.J., 1994. Nutritional Ecology of the Ruminant, 2nd ed. Cornell Univ. Press, Itacha, NY, USA, 476 pp. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597. Vough, L.R., Cassel, E.K., Barao, S.M., 1991. Nitrate poisoning in livestock. Animal Agriculture Update Newsletter, 6, 7–11. Cooperative Extension Service, University of Maryland, College Park, MD, USA. Whitehead, D.C., 2000. Nutrient Elements in Grassland. Soil–Plant–Animal Relationships. CABI Publishing, Wallingford, UK. Yu, P., Christensen, D.A., McKinnon, J.J., 2004. In situ rumen degradation kinetics of timothy and alfalfa as affected by cultivar and stage of maturity. Can. J. Anim. Sci. 84, 255–263.