Extrusion of sorghum starch enhances ruminal and intestinal digestibility, rumen microbial yield and growth in lambs fed on high-concentrate diets

Animal Feed Science and Technology 189 (2014) 30–40

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

Extrusion of sorghum starch enhances ruminal and intestinal digestibility, rumen microbial yield and growth in lambs fed on high-concentrate diets M. Yahaghi a , J.B. Liang b,∗ , J. Balcells c , R. Valizadeh d , M.F. Jahromi b , R. Alimon e , Y.W. Ho f a

Northeast Animal Breeding Station, Mashhad, Iran Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia c Department of Animal Production, ETSEA, Lleida University, 25198, Spain d Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran e Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia f Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia b

a r t i c l e

i n f o

Article history: Received 1 June 2013 Received in revised form 20 December 2013 Accepted 21 December 2013

Keywords: Extrusion Ruminal Small intestine Starch digestibility

a b s t r a c t This study consisted of two experiments that investigated the effect of extrusion of sorghum starch on in situ ruminal and post-ruminal disappearance of dry matter (DM), starch and nitrogen (N) and the effects of substituting barley by the extruded sorghum on digestion characteristics, microbial N supply and growth in finishing lambs. Sorghum was extruded using the following four temperature and pressure combinations: T1 = 90 ◦ C/17 bars, T2 = 115 ◦ C/24 bars, T3 = 150 ◦ C/55 bars and, T4 = 175 ◦ C/95 bars. Ruminal, intestinal and total tract DM content, starch and N degradability of the extruded sorghum (T1, T2, T3, T4), unprocessed sorghum (T0; control) and barley grain (B) were determined using nylon bag and mobile bag techniques in a completely randomized block design (CRBD). Extrusion under T3 conditions increased in vitro ruminal digestibility, solubility (a), fractional disappearance rate (c) and effective degradability (ED) of DM and starch in sorghum to near similar to those of B. T3 also had higher (P<0.05) ruminal and intestinal digestion rates for DM, starch and N compared to T0, T1, T2 and T4 suggesting that extruding sorghum under T3 conditions could be used as replacement of barley in lamb diets. In the second experiment, eighteen male Iranian Baluchi lambs were randomly allocated into three dietary treatments in a complete randomized design (CRD). Dietary grain in the treatment-concentrates were barley (B) as control, barley and extruded sorghum (under T3 conditions) in equal proportion (BSE ) and extruded sorghum (SE ). Dry matter and N intakes were not affected by source of grain, but because of the higher starch content in sorghum, lambs on SE diet had greater starch intake (995 g/d) and higher (P<0.01) starch (291 g/d) and N (53.1 g/d) outflow to the small intestine. Of the total input of starch to the small intestine, higher (P<0.01) amount (253 g/d) disappeared in SE compared to B and BSE lambs which was reflected by the higher

Abbreviations: ADG, average daily gain; bun, blood urea nitrogen; DMF, dry matter flow; CP, crude protein; DM, dry matter; DOMI, digestibility of organic matter intake; ED, effective degradability; FCR, feed conversion ratio; GIT, gastro intestinal tract; MN, microbial nitrogen; N, nitrogen; NDF, neutral detergent fibre; OM, organic matter; PD, purine derivatives; TMR, total mixed rations; VFA, volatile fatty acid. ∗ Corresponding author. Tel.: +60 3 89471390; fax: +603 8938 1612. E-mail address: [email protected] (J.B. Liang). 0377-8401/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anifeedsci.2013.12.009

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glucose concentration in the portal vein of SE lambs. Lambs fed SE diet also had greater ruminal microbial N (MN) yield (24.1 g/d), although ruminal pH and total VFA did not show any significant difference among treatments. Consequently, SE lambs recorded greater averaged daily gain (ADG) and more efficient (P<0.01) feed conversion ratio (FCR) compared to B or BSE lambs. It can be concluded that extrusion under 150 ◦ C/55 bars improves digestibility of sorghum starch and provides an option to completely replace barley in lambs. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Use of cereal grains with different starch degradation rates allows for a more synchronized release of energy and nitrogen (N) that prevents a sudden drop in ruminal pH (Horadagoda et al., 2008; Xu et al., 2009). It also increases outflow of starch from the rumen for more efficient digestion in the small intestine (McLeod et al., 2001; Abramson et al., 2005). However, excessive outflow of ruminal starch may lead to energetically less efficient fermentation in the hind gut (Huntington et al., 2006; Harmon, 2009). Yahaghi et al. (2012) reported that partial substitution of barley with sorghum grains improved starch digestion, microbial N production and growth rate in growing lambs. However, when barley was fully replaced by sorghum, ruminal outflow rate of starch was increased but a large proportion of the escaped starch was undigested and fermented in the hind gut or excreted as waste in the faeces (Yahaghi et al., 2013). Starch granules in the sorghum grain are embedded in a dense protein matrix rich in disulphide bonds which make them highly resistant to microbial fermentation and enzymatic digestion in the small intestine (Ljøkjel et al., 2003a; Mahasukhonthachat et al., 2010). Thus, in order to increase the substitution rate of quickly fermentable grain such as barley with sorghum, the highly resistant structure of sorghum starch must be destroyed or reduced. Extrusion of grains has been shown to alter the physicochemical characteristics of its starch because starch gelatinizes when treated at adequate temperature and pressure (Dehghan-Banadaky et al., 2007). The objective of this study was to examine whether extrusion will increase the outflow and digestibility of sorghum starch in the small intestine to allow for a more efficient replacement of barley grain by sorghum in high concentrate diet in lambs. Prior to the in vivo trial, the optimal extrusion conditions for sorghum grain was tested based on in situ rumen and intestinal nutrient disappearance studies. 2. Materials and methods 2.1. In situ trial 2.1.1. Experimental feed preparation Sorghum grain (Sorghum bicolor L. Moench) obtained from Khorasan-Razavi Agricultural and Natural Resource Research Centre, Mashhad, Iran, was ground to 2 mm and extruded. Extrusion was carried out using a single-screw extruder (Yemmak Makina Sanayi A.S. Turkey), running at screw speeds of 160, 185, 210 and 235 rpm and 8 mm die-plate nozzle to generate a combination of temperature and pressure to produce the following treatment groups: T1 = 90 ◦ C/17 bars; T2 = 115 ◦ C/24 bars; T3 = 150 ◦ C/55 bars and T4 = 190 ◦ C/95 bars. The above extrusion conditions were selected based on previous studies which used temperatures ranging between 100 and 180 ◦ C to extrude sorghum (Al-Rabadi et al., 2011; Lankhorst et al., 2007). After extrusion, the grains were transferred into a container to cool, dry (8–9% humidity) and to equilibrate for 30 min. After that, the extruded sorghum was re-ground through 2 mm sieve and kept for later use. Untreated sorghum (T0) and barley (Hordeum spp.) (B) grains were used as negative and positive controls respectively. The chemical compositions of barley and various sorghum samples used for this study are shown in Table 1. 2.1.2. Management of rumen fluid donor animals Three rumen-cannulated and two duodenal-fistulated Holstein steers (316 ± 31 kg), fed a mixed ration (67:33; concentrate:roughage) near to maintenance level (circa 6 kg DM) were used, respectively to estimate in situ DM, N and starch disappearances from nylon bags and disappearances in the intestine using mobile bags. Animals were fed alfalfa hay (NDF: 430, CP: 160 g/kg) as roughage source. The diet was offered twice daily in equal meals at 8:30 and 16:30 h. 2.1.3. Experimental design In situ DM, N and starch disappearances from nylon bags were determined following the methods of Ørskov and McDonald (1979) in the rumen and that of Donnem et al. (2010) through the lower gut. For the former, four nylon bags (13 cm×7 cm and 45 ␮m pore size) contained approximately 5 g of ground samples (2 mm) of each experimental ingredient (6 ingredients×6 incubation durations×2 duplicates×2 replicates) were prepared and incubated for 2, 4, 8, 16, 24 and 48 h. At any one time, a total of 36 bags [6 treatments in duplicates (12)×3 incubation durations] were incubated in each animal [considered as the experimental unit (block), n = 3]. When bags were removed at their assigned incubation time, they were replaced by duplicate sets of the same ingredient for another incubation duration. Similar procedure was repeated for the second run to obtain the experimental replicates for each treatment. Prior to insertion into the rumen, bags were soaked (5 min) in

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Table 1 Chemical compositions of barley (B) and sorghum extruded under various conditions. Parameters (g/kg DM)

B

T0*

T1

T2

T3

T4

DM (g/kg fresh matter) Starch CP NDF Ash

890 581 125 218 38

880 657 112 187 32

910 667 107 184 33

920 652 117 192 31

920 684 113 176 32

910 643 102 184 33

*

Unprocessed sorghum (T0), and T1, T2, T3 and T4, sorghum extruded at 90 ◦ C/17 bars, 115 ◦ C/24 bars, 150 ◦ C/55 bars and 190 ◦ C/95 bars, respectively.

clean water to remove the air and immediately introduced into the rumen. After retrieval from the rumen, nylon bags were immediately rinsed under tap-water and washed in washing machine with a 1 min agitation and 2 min spin cycle for four times to minimize microbial-N contamination and dried at 60 ◦ C for 48 h to determine DM disappearance, later pooled and ground through a 1-mm screen to determine nutrients content and disappearance. The same experimental design (CRBD) was applied to measure DM, N and starch disappearances in the intestine, Samples were first incubated in the rumen (for 16 h) and the residues (500 mg approx.) were then transferred into mobile bag (4 cm2 , 45 ␮m). The mobile bags were heat-sealed and pre-treated with pepsin–HCl (1 g pepsin in 1 L 0.01 N-HCl) solution at 38.5 ◦ C for 1 h before they were introduced into the duodenum at the rate of 1 bag per 20 min to each of the two duodenal-fistulated Holstein steers [considered as the experimental unit (block), n = 2], Two bags (duplicates) per experimental ingredient were applied, and the same procedure was continued for the second run (considered as experimental replicates). Bags were recovered from faeces the next day from 0830 to 1600 h and those recovered after the specified time were discarded. Bags were hand-rinsed, washed and dried at 60 ◦ C for 48 h. 2.2. In vivo trial 2.2.1. Animals and experimental diets The experiment was conducted in Abbas Abad Baluchi Breeding Station, northeast of Mashhad, Iran. Eighteen weaned male Baluchi lambs (65 days age and 32 ± 1.1 kg body weight) were randomly allocated into three dietary treatment groups (6 lambs/treatment) in a complete randomized design (CRD). Three iso-caloric (2.45 Mcal/kg DM) and iso-nitrogenous (CP: 140 g/kg) diets consisted of 32.5:67.5 roughage (alfalfa) to concentrate were used. Extruded sorghum grain (T3: 150 ◦ C/55 bars) was used in the sorghum concentrates. Compositions of the concentrates are shown in Table 2. The required amount of alfalfa hay was long/chopped, added daily to the respective concentrates and offered as total mixed rations (TMR). The animals were cared according to the experimental protocols approved for animal research by the Ethical Committee of University Ferdowsi of Mashhad, Iran (Code 1829). 2.2.2. Feeding and digestibility trials The experiment consisted of 10 weeks feeding trial followed by 10 days digestibility trial and finally 2 days of gut absorption kinetics study. During the feeding trial, lambs were housed in individual pens (1.5 m×2 m), offered the assigned experimental diets in equal portions twice daily (0800 and 1600 h) and their weekly weights recorded. During the digestibility trial, lambs were kept in individual metabolism cages (1.5 m×0.75 m) under the same feeding regime except that feed were Table 2 Ingredient and chemical compositions of concentrate diets for in vivo study. Dietary treatments* B

BSE

SE

Ingredients (g/kg DM) Barley grain Extruded sorghum grain Wheat bran Cotton seed meal Limestone Mineral and vitamin mix†

840 0 110 30 15 5

420 420 80 60 15 5

0 840 70 70 15 5

Chemical composition (g/kg DM) DM (g/kg fresh matter) Starch Crude protein Organic matter ME (MJ/kg DM)‡

910 597 141 940 12.02

910 657 140 945 11.93

910 721 139 947 11.84

* †

Dietary treatments, with barley (B), barley/extruded sorghum (BSE , 50:50) and extruded sorghum (S) as source of starch in the concentrates. Composition (mg/kg feed): 4.9 mg of Zn, 4.05 mg of Mn, 0.45 mg of Cu, 0.075 mg of I, 0.1 mg of Se, 2500 IU vitamin A, 400 mg of vitamin D, 2.5 IU vitamin

‡

Metabolizable energy, estimated from NRC (2007).

E.

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offered 4 times (6 hourly intervals) a day. The digestibility trial consisted of 3 days of adaptation followed by 7 days of faeces and urine collection. Daily urine output was collected individually in an acidic solution, pH < 3 (100 ml of 1 M H2 SO4 ) to prevent bacterial contamination and then weighed, sampled (100 g/kg) and pooled on individual animal basis over 7 days and stored at −20 ◦ C for further analyses. Faecal samples were also collected in the same manner and pooled on individual animal basis over 7 days. 2.2.3. Nutrients disappearances through the intestine Ytterbium (Yb) acetate was used as a digesta flow marker and individual daily doses of 2 mg Yb/kg LW were diluted in distilled water (20 ml) and dosed orally 4 times a day (5 ml/time) after feeding during the 7 days digestibility trial. Immediately after the trial, lambs were randomly divided in two groups and euthanized the next two days to determine DM, starch and N disappearance through the intestinal tract following the procedure of Yahaghi et al. (2013). Briefly, two hours after feeding and with approximately 30-min interval, lambs were anesthetized (Xylazine, 0.3 mg/kg LW, Calier Laboratories, Barcelona, Spain) prior to intravenous euthanatization (Thiopental, 10 mg/kg LW Calier Laboratories, Barcelona, Spain). The digestive tract was immediately dissected and to prevent digesta mixing, intestinal segments were tied with cotton threads around the tissue before the intestine was released from the mesentery, sectioned to individual segments. Beside the reticulo-rumen and abomasum, the small intestine was sub-divided into 1 m-segments starting from the pylorus. Before slaughtering, blood samples were taken from portal and jugular veins using heparinzed vacuum tubes. Blood samples were then centrifuged at 2500×g for 20 min at 4 ◦ C and the plasma was stored at −20 ◦ C. The rumen was opened, pH of rumen content was immediately recorded, and total digesta content was taken, homogenized and strained through two layers of cheesecloth and two samples were taken from the filtrate for ammonia and volatile fatty acid (VFA) analysis. For the former, 4 ml rumen fluid were acidified with 2 ml of 0.2 N HCl, and frozen until NH3 -N analysis and for the latter, 3 ml were centrifuged (3500 rpm for15 min) and the supernatant mixed with 0.6 ml of a solution containing 2 g/l mercuric chloride, 20 ml/l orthophosphoric acid, and 0.5 ml of 20 mM 4-methylvaleric acid (internal standard) in distilled water, and frozen (−20 ◦ C) until subsequent analyses. For post-rumen digesta, samples from the abomasum, duodenum: proximal and distal, jejunum: proximal and distal also the distal ileum were taken following the procedure of Yahaghi et al. (2013). 2.3. Chemical analyses Dry matter was determined by drying samples to constant weight at 105 ◦ C and OM by combustion at 550 ◦ C for 8 h in a muffle furnace (AOAC, 2005; procedure no: 930.15). Neutral detergent fibre (aNDF) concentration was determined and expressed inclusive of residual ash (Van Soest et al., 1991). Total N in feed, refusals and digesta was determined following the Kjeldahl method (Kjeldahl unit Vapodest 30, C. Gerhardt, GmbH & Co., KG, Konigswinter, Germany, AOAC, 2005; index no: 977.02). NH3 -N in digesta was determined by Kjeldahl method (AOAC, 2005; index no: 977.02). Non-ammonia N (NAN) in abomasal digesta was assayed by the same method after removing the ammonia with 1 M NaOH. Volatile fatty acid concentration in deproteinized rumen fluid was determined by gas chromatography (Agilent 6890, Wilmington, DE, USA) following the protocol of Jouany (1982). Ytterbium in digesta samples was determined by spectrophotometry following the protocol of Hart and Polan (1984). Urinary purine derivative (PD; i.e. allantoin, uric acid, xanthine and hypoxanthine) were analyzed following the methods of Balcells et al. (1992). Starch was converted to soluble carbohydrates after ␣-amylase treatment followed by re-incubation with ␤-amyloglucosidase as described by Horadagoda et al. (2008). Concentrations of blood glucose and urea nitrogen (BUN) were determined using the method of Kaneko (1989). 2.4. Calculations and statistical analyses 2.4.1. In situ trial Hourly individual in situ DM, N and starch disappearances were calculated using the model (Ørskov and McDonald, 1979) p = a + b (1 − e−ct ), where p represents the ruminal disappearance at t time; a, the soluble fraction; b, the non-soluble but potentially degradable fraction and c is the fractional disappearance rate of b fraction (%/h). Adjustments were done using the Marquardt method of the PROC NLIN procedure of the SAS (2003) programme, with the limitation a + b ≤ 1. Ruminal effective degradability (ED) of DM, starch and CP were estimated using ED = a + b [bc/(c + kp )] at 0.05 and 0.08/h outflow rates (kp ). Intestinal digestibility of DM, starch and CP was calculated as: digested DM, starch or CP in small intestine/residue after 16 h rumen incubation. To examine the effect of different extrusion conditions on ruminal and post-ruminal starch and CP digestion, a CRBD were applied using GLM procedure (SAS, 2003) as follow: Yij =  + Ai + Tj + (AT )ij + eij where , mean value; Ai , block (animal effect); Tj , treatment effect; (A×T)ij , block/treatment interaction; and eij : the experimental error, respectively. Differences among all treatments were tested using the least significant difference (LSD) at P<0.05.

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Table 3 In situ dry matter (DM), starch and CP disappearance kinetic parameters in the rumen. Treatments*

SEM†

P<

B

T0

T1

T2

T3

T4

Dry matter a‡ b c ED5

0.45a 0.52d 0.21a 0.87a

0.35c 0.59a 0.10b 0.75b

0.38cd 0.56bc 0.12b 0.77b

0.40bc 0.54cd 0.13b 0.79a

0.43ab 0.56b 0.20a 0.87a

0.36de 0.58ab 0.12b 0.77b

0.012 0.009 0.0001 0.017

0.012 0.021 0.001 0.023

Starch a b c ED5

0.47a 0.52c 0.19a 0.88a

0.37d 0.60a 0.19c 0.79d

0.40cd 0.56b 0.13bc 0.81cd

0.43bc 0.56b 0.14b 0.84bc

0.46ab 0.53c 0.17ab 0.87ab

0.40cd 0.56b 0.13bc 0.80d

0.013 0.009 0.010 0.010

0.034 0.022 0.013 0.044

Crude protein a b c ED5

0.31a 0.61b 0.16a 0.78a

0.23b 0.67a 0.10b 0.70b

0.25b 0.69a 0.10b 0.65b

0.23b 0.71a 0.08bc 0.67b

0.24b 0.68a 0.09bc 0.68b

0.18c 0.60b 0.06c 0.51c

0.015 0.016 0.010 0.028

0.032 0.040 0.041 0.001

Means within a row with different letters differ (P<0.05). * Treatments, barley (B), unprocessed sorghum (T0), and T1, T2, T3 and T4: sorghum extruded at 90 ◦ C/17 bars, 115 ◦ C/24 bars, 150 ◦ C/55 bars and 190 ◦ C/95 bars respectively. † Standard error of the difference of the means. ‡ a, fraction solubilized at the initiation of incubation (time 0); b, not soluble but potentially degradable fraction; c, rate constant of disappearance of fraction potentially degradable; ED, effective ruminal degradability of DM. ED = a + b × c/(c + k) with k the passage rate ED represents DM effective degradability in kg/kg DM. ED5 = represent with k the passage rate from the rumen 0.05.

2.4.2. In vivo trial Rumen microbial N production was calculated from urinary excretion of PD following the model proposed by Chen and Gomes (1995). The post-ruminal flow of DM (DMf) in different parts (i) of the intestinal tract was calculated using DM and Yb concentration in post rumen samples: DMFi =

DM (i, g) × Yb (i, mg) Daily Yb doses (mg/d)

Apparent and true nutrients disappearance from the different segments of the gut tract was calculated assuming no Yb absorption through the intestinal tract. Apparent digestibility of DM and other nutrients (starch and N) in the different intestinal tract parts (i) was calculated as follow: Apparent digestibility(i part) =

g DM or nutrient (i − 1) − g DM or nutrient (i) ; g DM or nutrient (i − 1)

Statistical analysis was conducted in CRD using GLM procedure (SAS, 2003) following the model: Yi =  + Ti + Aj + εij where  is the mean value, Ti is the treatment effect (fixed), A was the individual (random) variation nested within each treatment and εij, the error term. Differences in LSD were declared significant at P<0.05 3. Results 3.1. In situ ruminal and post ruminal DM, starch and N disappearance As expected, barley grain showed a higher rumen DM content, starch and N degradability than sorghum, (P<0.05) in terms of solubility (a), fractional rate of disappearance (c) and effective degradability (ED). Intensity of extrusion improved starch degradability up T3 (150 ◦ C/55 bars) to achieve similar ED as that of barley grain. Kinetic of rumen DM content disappearance showed similar trend as that of starch, however, the effect of extrusion on N disappearance was less pronounced (Table 3). Rumen DM content, starch and N disappearances from nylon bag at 16 h incubation time (Table 4) showed similar trends as those previously described (Table 3). Results of the DM content, starch and N disappearance through the lower gut are presented in Table 4. The untreated sorghum (T0) had lower intestinal digestibility for DM, starch and N than barley and digestibility of the above parameters increased with intensity of extrusion with T3 achieving the highest value. Higher extrusion intensity suppressed digestibility.

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Table 4 In situ rumen and lower gut disappearance of DM, starch and CP (g/g DM). Treatment†

SEM‡

P<

B

T0

T1

T2

T3

T4

Rumen degradability* Dry matter Starch Crude protein

0.87a 0.94a 0.74a

0.73d 0.81d 0.59c

0.79b 0.86c 0.61d

0.78bc 0.89b 0.62c

0.84a 0.91b 0.69b

0.75dc 0.78c 0.56f

0.013 0.001 0.001

0.012 0.014 0.001

Lower gut digestibility Dry matter Starch Crude protein

0.93a 0.99a 0.86a

0.86c 0.84d 0.75bc

0.87c 0.88cd 0.75bc

0.89bc 0.91bc 0.80ab

0.92a 0.95ab 0.85a

0.84c 0.86cd 0.70c

0.010 0.014 0.022

0.011 0.023 0.032

Total tract digestibility Dry matter Starch Crude protein

0.95a 0.99a 0.79a

0.77c 0.74d 0.65cd

0.82bc 0.78cd 0.63cd

0.87ab 0.86bc 0.68bc

0.94a 0.97ab 0.74ab

0.74c 0.80cd 0.58d

0.030 0.037 0.025

0.001 0.012 0.012

Means in a row with different letters differ (P<0.05). * Rumen degradability was measured after 16 h ruminal incubation. † Treatments, barley (B), unprocessed sorghum (T0), and T1, T2, T3 and T4: sorghum extruded at 90 ◦ C/17 bars, 115 ◦ C/24 bars, 150 ◦ C/55 bars and 190 ◦ C/95 bars respectively. ‡ Standard error of the difference of the means.

3.2. In vivo trial 3.2.1. Dry matter content and starch digestibility through the gastro-intestinal tract Dry matter content and starch flow through the different segments of the gastro-intestinal tract (GIT), apparent disappearance and digestibility values are presented in Table 5. Dietary treatments did not alter DMI (g/d), rumen disappearance (g/d) or rate of degradability. Although DMI was similar among treatments (Table 5), because of the higher starch content in the sorghum (Table 1), starch intake (g/d) was higher for SE than BSE and B diets. However, B and BSE diets had higher apparent ruminal degradability compared to SE diet. Along the small intestinal, bulk of the starch was digested in the duodenum and proximal-jejunum, with the remaining in the lower segments of the small intestine (Fig. 1). Of the total starch entering the small intestine, a higher amount was digested with the SE compared to B and BSE diets (Fig. 2). 3.2.2. N balance and flow through the intestinal tract Nitrogen intake averaged 44.3 g/d did not differ among treatments. The diets formulated with extruded sorghum (SE ) showed a high rumen outflow of total N, microbial-N and dietary N (Non-Microbial N). Duodenal flow of N was equivalent (B and SBE ) or higher (SE ) than their respective N intake (Table 6). Along the small intestine, N was mostly digested at the duodenum and proximal-jejunum, with SE lambs showed the highest (P<0.01) N digestibility in the lower gut. Lambs fed Table 5 Flow, apparent disappearance and digestibility of dry matter and starch in different parts of the intestinal tract in Baluchi lambs fed diets with different starch sources. Diet component

Dry matter

Diet*

B

BSE

SE

SEM†

P<

B

BSE

SE

SEM

P<

Intake (g/d)

2083

2036

2009

37.6

0.390

922b

963ab

995a

16.4

0.018

Flow (g/d) Abomasum Ileum Faeces

1138a 672a 593a

1128a 653a 584a

1070b 507b 459b

9.7 8 8.7

0.012 0.001 0.012

184b 34 19b

202b 39 25ab

291a 45a 32a

8.2 3.3 2.8

0.011 0.078 0.013

Apparent disappearance (g/d) Rumen Small intestine Large intestine Total

945 467b 79a 1490b

909 475b 69ab 1452c

938 563a 48b 1550a

35.6 8.5 6.01 23.5

0.752 0.001 0.001 0.022

730 158b 15 904b

753 171b 14 938ab

697 253a 13 963a

24.5 8.6 1.9 13.0

0.304 0.001 0.261 0.023

0.09 0.01 0.01 0.02

0.167 0.011 0.124 0.018

Apparent digestibility Rumen (g/g intake) Small intestine (g/g of abomasal flow) Large intestine (g/g of ideal flow) Total (g/g intake)

0.45 0.41b 0.12 0.72b

Starch

0.45 0.42b 0.11 0.71b

0.47 0.53a 0.09 0.77a

0.79a 0.82 0.44a 0.98

0.78a 0.81 0.36b 0.97

0.70b 0.85 0.29c 0.97

0.11 0.17 0.02 0.06

Means within a row with different letters differ (P<0.05). * Dietary treatments, with barley (B), barley/extruded sorghum (50:50 BSE ) and extruded sorghum (SE ) as source of starch in the concentrates. † Standard error of the difference of the means.

0.014 0.368 0.032 0.359

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Fig. 1. Post-ruminal starch flow in Baluchi lambs fed diets with barley (B), barley-extruded sorghum (BSE , 50:50) and extruded sorghum (SE ) as starch sources. a,b,c Means in a segment with different superscripts differed significantly (P<0.05).

SE diet also had higher apparent total tract N digestibility and N retention than those fed BSE or B diets. Microbial yield efficiency (g MN/kg DOMI) was also influenced by diet treatments with lambs on SE diet having higher efficiency compared to B or BSE lambs. 3.2.3. Rumen characteristics and blood parameters Although inclusion of sorghum apparently improved ruminal pH and ammonia-N concentration, the above parameters were not statistically significant (Table 7). Rumen VFA concentrations tend to decrease with inclusion of extruded sorghum and the proportion of the individual VFA and the A/P ratio remained unaltered. Complete substitution of barley grain by extruded sorghum (SE ) increased the blood glucose concentration in the portal vein (P<0.01) whereas the glucose concentration of the BSE lambs remained unchanged compared to the B lambs. The above observation was not seen in the jugular vein. No changes in BUN were recorded among treatment groups. 3.2.4. Productive parameters Intake and animal performance data are presented in Table 8. Substitution of barley grain by extruded sorghum significantly improved ADG and FCR. SE lambs recorded an additional 80 g/d weight gain, equivalent to 0.38 improvement compared to the B lambs.

Fig. 2. Proportion of starch digestion in different segments of the gastrointestinal tract of lambs fed experimental diets with barley, barley-extruded sorghum and extruded sorghum, as sources of starch.

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Table 6 N balance, digestibility and characteristics of duodenal flow in Baluchi lambs fed different starch sources. Dietary treatments* B

BSE

SE

SEM†

P<

N intake (g/d)

45.0

44.0

44.0

0.81

0.377

N excretion (g/d) Urine Faeces N balance

21.2 15.7 8.0b

20.2 15.1 8.7b

18.0 13.0 13.0a

1.40 1.13 1.41

0.402 0.716 0.021

0.04 0.02 0.02

0.001 0.028 0.031

1.13 1.04 0.67 1.19

0.012 0.001 0.023 0.027

N digestibility (g/g intake) Rumen Small intestine Apparent N digestibility N outflow (g/d) Total rumen N outflow Rumen MN outflow Rumen MN outflow MNE‡

1.05b 0.61b 0.65b

1.07b 0.67ab 0.66b

45.5b 19.6b 25.9b 21.5b

1.22a 0.71 0.72a

46.9b 18.9b 26.9b 21.6b

53.1a 24.1a 29.0a 26.1a

Means within a row with different letters differ (P<0.05). * Dietary treatments, with barley (B), barley/extruded sorghum (BSE , 50:50) and extruded sorghum (SE ) as source of starch in the concentrates. † Standard error of the difference of the means. ‡ Microbial N efficiency (g MN/kg DOMI).

Table 7 Rumen characteristics and blood metabolites concentration in Baluchi lambs fed diets of different starch sources. Item

Dietary treatments* B

BSE

Rumen parameters pH NH3 (mg/100 ml) VFA (mM) Acetate (mol/100 ml) Propionate (mol/100 ml) Butyrate (mol/100 ml) Acetate: Propionate ratio

5.95 20.8 104.7 62 18.5 14.7 1.9

6.03 21.0 101.6 56 17.2 13.6 2.0

Blood Profile (Portal vein) Glucose (mg/dL) BUN (mg/dL) VFA (mM) Acetate (mol/100 ml) Propionate (mol/100 ml) Acetate: propionate ratio

114.2b 15.5 7.0 62 18.5 3.7ab

Blood profile (Jugular vein) Glucose (mg/dL) BUN (mg/dL)

81.5 10.7

SE

SEM

P<

6.19 22.7 96.3 64 16.2 12.8 2.2

0.09 0.96 2.54 3.8 1.1 0.64 0.12

0.211 0.331 0.092 0.301 0.362 0.130 0.174

111.3b 16.0 6.5 56 17.2 3.0b

136.3a 16.3 6.6 64 16.2 3.9a

4.5 1.4 0.32 3.8 1.1 0.23

0.001 0.913 0.530 0.296 0.362 0.043

82.0 9.5

92.7 11.8

3.5 0.93

0.071 0.242

Means within a row with different letters differ (P<0.05). * Dietary treatment, with barley (B), barley/extruded sorghum (50:50 BSE ) and extruded sorghum (SE ) diets.

Table 8 Production parameters in Baluchi lambs fed diets with different starch sources. Dietary treatment*

Initial live weight (kg) Final live weight (kg) Average daily gain (kg/d) Feed conversion ratio (kg dry matter/kg gain)

B

BSE

SE

29.33 43.72b 0.21c 10.2a

28.98 45.04b 0.23b 8.9b

29.13 49.14a 0.29a 7.0c

Means within a row with different letters differ (P<0.05). * Dietary treatments, with barley (B), barley and extruded sorghum (BSE , 50:50) and extruded sorghum (SE ). † Standard error of the difference of the means.

SEM†

P<

0.501 0.58 0.006 0.38

0.923 0.001 0.001 0.012

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4. Discussion 4.1. Effects of extrusion on in situ degradation kinetics Resistance of starch granules to microbial degradation is attributed to its non-starch component and the amylose to amylopectin ratio (Mahasukhonthachat et al., 2010). It has been reported that processing of grains may alter the physicochemical characteristics of its starch because starch gelatinizes when treated at adequate temperature and pressure (DehghanBanadaky et al., 2007), resulting in improved digestibility in the ruminant GIT (Goelema et al., 1999; Mahasukhonthachat et al., 2010). Our results reaffirmed the above, and further suggesting that extruding sorghum with 150 ◦ C/55 bars (T3) effectively improves its DM and starch degradation in the rumen and digestibility in the small intestine (Table 4). The present result is consistent with published data suggesting that heat treatment enhances sorghum starch degradability (Dahlin and Lorenz, 1993; Ljøkjel et al., 2003a,b) but the processing temperature should not exceed 180 ◦ C (Lankhorst et al., 2007). This could explain why Al-Rabadi et al. (2011) did not show any improvement in starch digestibility when sorghum was treated using temperatures between 100 ◦ C and 140 ◦ C. Likewise, in our study, extrusion at higher than 180 ◦ C (190 ◦ C for T4) resulted in poorer ruminal and intestinal nutrient digestibility, presumably because excessive temperature denatured the nutrient content. The improvement of the total tract starch digestibility from 0.74 in the unprocessed sorghum (T0) to 0.97 in T3 in this study is in agreement with Gaebe et al. (1998), who reported a total tract starch digestibility of 0.96 for extruded sorghum in steers. 4.2. In vivo trial 4.2.1. Ruminal DM and starch degradation characteristics Complete or partial substitution of barley by extruded sorghum did not negatively affect DMI, suggesting that extrusion had no effects on palatability. The three iso-caloric, iso-nitrogenous diets were formulated with fixed substitution rates of barley with extruded sorghum grain, however, because of the inherent higher concentration of starch in the sorghum, starch content in the diets (Table 2), and subsequently starch intakes (Table 5) for the SE treatment were highest, followed by the BSE and B. However, of the total starch ingested by lambs fed on B diet, approximately 0.80 fermented in the rumen and 0.18 digested in the small intestine (Fig. 2). In contrast, lambs in the SE diet recorded lower (0.70) starch fermented in the rumen and higher (0.27) quantity digested in the small intestine and absorbed, presumably mostly as glucose. The different digestion kinetics between lambs in the above two diet groups indicated an eight percent-unit higher starch digestion in the small intestine in the SE compared to the B diet lambs (Fig. 2). The starch digestion kinetic for BSE lambs (0.78 fermented in the rumen and 0.19 digested in the small intestine) was near identical to that of lambs in the B diet. Different grain sources neither affected VFA concentration nor individual VFA proportions, however, the numerically higher pH and acetate/propionate ratio values in lambs fed on SE diet seemed to suggest higher fermentation of fibre in the lambs fed on SE diet. The present result agrees with the study of Opatpatanakit et al. (1994) who reported that rumen conditions fed with sorghum grain diets favoured cellulolytic activity on fibre and structural components of the sorghum grain. 4.2.2. Nitrogen degradation and microbial N synthesis Lambs in the three dietary treatments had similar N intake, but lambs fed on SE diet recorded higher microbial N synthesis and microbial N efficiency compared to those fed on B and BSE diets. The results suggest that extrusion enhanced and synchronized the release of dietary energy and N to provide an optimal condition for rumen microbial N synthesis in the diet in which barley was fully replaced by extruded sorghum (SE ). In contrast, for untreated sorghum, optimal microbial N synthesis was achieved when barley was partially (0.50) replaced by sorghum, because under a full substitution diet, the low fermentation of sorghum starch could not support an optimal microbial N synthesis in the rumen (Yahaghi et al., 2013). The outflow of N from the rumen to proximal-duodenum exceeded the sum of N intake and microbial N yield in the sorghum inclusion diets, particularly in the SE lambs (Table 6) indicating an additional source of N to the rumen. Similar observation was noted in the previous study and since the recovery of the marker in the faecal-Yb was near complete, it was suggested that the excess N was of endogenous sources, presumably urea recycling via saliva or the rumen wall (Yahaghi et al., 2013). No differences were registered in NH3 -N concentration among lambs in the different diet groups but all the values were higher than 5 mg/100 ml, indicating that microbial growth was not limited by N availability (Kim et al., 2010). 4.2.3. Post ruminal nutrient digestion Previous study (Yahaghi et al., 2013) showed that in diet which barley was fully replaced by untreated sorghum, out of the 0.38 of the ingested starch that escaped rumen fermentation, only 0.22 was digested in the small intestine. The remaining 0.16 had either undergo less efficient fermentation in the caecum or was excreted as waste. The primary objective of this follow-up study was to examine whether the digestibility of the sorghum could be improved by extrusion process to allow for higher replacement of barley with sorghum when needed. The high amount (g/d) digested (Table 5) and high digestion rate (Figure 2) of starch in the small intestine of SE diet lambs (0.25 of the ingested starch) indicated that starch of the extruded sorghum was more readily hydrolyzed in the small intestine compared to the untreated sorghum reported previously

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(Yahaghi et al., 2013). Reasons for the improved starch digestibility could be because extrusion alters the protein structure surrounding the starch granules allowing the starch to be more accessible to the intestinal amylases hydrolysis, and/or the high microbial N synthesis induced by the SE diets. This leads to the presence of additional true protein (amino acids) which stimulates secretion of pancreatic ␣-amylase enzyme necessary for starch digestion (Richards et al., 2002; Abramson et al., 2005). The higher digestion and absorption of starch in the SE diet lambs were supported by the significant increased blood glucose concentration in the portal vein of lambs in the treatment. The blood glucose concentrations recorded in this study seemed to be rather high and could be partly because the animals were slaughtered between 2 and 4 h after feeding when most of the concentrates portion (containing starch) of the diets had already reached the small intestine (Fig. 1). In addition, the blood glucose level was expressed as its concentration and not the absolute quantity of glucose that flowed through the portal vein. Since the lambs were under similar management protocols except for the source of starch in the diets, it is believed that the blood glucose concentrations could serve as an indicator to compare the rate of starch digestion and absorption for this study. 4.2.4. Animal performance Lambs fed on SE diet had 0.39 and 0.24 higher ADG than lambs on the B and BSE diets, respectively. Except for the differences in the starch sources and their contents, the experimental diets were iso-caloric and iso-nitrogenous. Therefore the higher digestion rate and absorption (evident by the higher blood glucose concentration) of the ingested starch in the small intestine together with the higher microbial N synthesis recorded in lambs fed on SE diet were the primary reasons for the higher growth rate of SE lambs compared with those on other diets. 5. Conclusion Results of this study showed that extrusion of sorghum grain at 150 ◦ C/55 bars enhanced the outflow of rumen unfermented starch which was effectively hydrolyzed in the small intestine and absorbed, primarily as glucose, and together with the increased microbial N yield, resulted in significant increased growth rate of lambs fed on diet in which barley was fully replaced by sorghum. This finding provides an option to allow barley to be replaced fully by sorghum in finishing-lamb diet. Whether to replace barley partially with untreated sorghum or fully with extruded sorghum should be determined by comparative cost advantage and availability of the feed ingredients. References Abramson, S., Bruckental, I., Lipshitz, L., Moalem, U., Zamwel, S., Arieli, A., 2005. Starch digestion site: influence of ruminal and abomasal starch infusion on starch digestion and utilization in dairy cows. J. Anim. Sci. 80, 201–207. 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