The way insoluble fiber incorporated in the diet changes its physiological response

The physiological effect of insoluble fibers may change with diet composition. The present study aimed to determine the difference in the physiological responses of broiler chickens fed diet incorporated with rice hulls in different ways. Two hundred and forty broiler chicks at two-days old were randomly placed in 30 cages and fed: control diet (based on corn-soybean) (CO), rice hull supplementation diet (CO + 4% of rice hulls) (RS), and rice hull inclusion diet (inclusion of 4% rice hulls in diet) (RI). The significant effects were mostly found in the starter phase. The RI increased ADG (P<0.05) but reduced empty ceca weight (P<0.05) in comparison to CO and RS. The RS increased the jejunal crypt depth (P<0.05) and reduced the small intestinal content (P<0.05). Birds fed RS and RI had higher villus height (P<0.05) and thicker jejunal mucosa (P<0.05) than those fed the CO. In the finisher phase, birds fed the RS had the narrower width of the upper and lower jejunal villi than the others. Supplemen-


INTRODUCTION
The benefits of insoluble dietary fiber in broiler feed have been widely reported by previous researchers (Kheravii et al., 2018;Jiménez-Moreno et al., 2013a;Sacranie et al., 2012;Svihus et al., 2010), but in reality, each insoluble food fiber has a different physiological responses (Sadeghi et al., 2015;Alabi et al., 2014;Sacranie et al., 2012;Svihus et al., 2010). The level of insoluble dietary fiber in diet that provides benefits to broilers is also inconsistent (Svihus, 2011). On the other hand, age, strain, and composition of different rations were suggested to modify the effect of insoluble dietary fiber (Jiménez-Moreno et al., 2016).
The direct impact of providing insoluble feed fiber is the changes that occur in the small intestine, among others; changes in physiological function (Jiménez-Moreno et al., 2016;Sacranie et al., 2012), microbiota composition (Capuano, 2017), gut weight, and length (Yokhana et al., 2016;Sacranie et al., 2012;Jiménez-Moreno et al., 2009), and gut morphology (Rezaei et al., 2018;Adibmoradi et al., 2016;Nkukwana et al., 2015;Sarikhan et al., 2010). The small intestine will continue to experience changes both morphologically, biochemically, and molecularly after hatching (Bohórquez et al., 2011). Changes in intestinal morphology such as villous length (Adibmoradi et al., 2016;Rezaei et al., 2014;Sarikhan et al., 2010), villous thickness, and crypt depth (Rezai et al., 2018;Adibmoradi et al., 2016) are reported in broilers or quails fed insoluble dietary fiber. Any morphological changes that occur in the small intestine would affect its function, which eventually affects the function of other organs (Toman et al., 2015). The majority of digestion and absorption of all nutrients occurs in the jejunum, so morphological changes in the jejunum will have an impact on broiler performance. Rice hulls, as an agricultural waste product from rice milling industries, contain a higher level of celluloses and lignin (Podolske et al., 2013). Although many researchers reported the rice hulls effects as a source of insoluble fibers in broiler chickens (Hartini et al., 2019;Sabour et al., 2019;Hartini and Purwaningsih, 2017;Abazari et al., 2016;Adibmoradi et al., 2016;Alabi et al., 2014), but there is a lack of information regarding the different physiological effect of rice hulls when supplemented or included in the diet. Therefore, this study was conducted to observe the different physiological responses i.e. performance, gastrointestinal traits, and jejunal morphologies of broiler chickens fed diet incorporated with rice hulls in different ways.

MATERIALS AND METHODS
Two hundred and forty two-day-old Lohmann chicks with an initial body weight of about 53.6 g (CV of 1.3%) were assigned to groups of eight in 30 battery cages. Birds were given ad libitum feed and water until 35 days of age. The light was set out for 24 hours per day throughout the experiment. Three experimental diets used in the study were: 1. corn and soybean meal base control diet (CO), 2. rice hull supplementation diet (CO + 4% of rice hulls) (RS), and 3. rice hull inclusion diet (inclusion of 4% rice hulls in diet) (RI). All diets were formulated to be iso-protein and were offered as mash. Besides iso -protein, the RS and RI were formulated to have a similar crude fiber level (around 5 to 5.5%). Starter diets were offered until 21 d of age, at which point finisher diets were offered until the end of the experiment (35 d of age). The experimental diets and calculated nutrient composition are shown in Table 1.
The birds were weighed at the beginning of the experiment, at the end of the starter period (21 d of age), and at the end of the finisher period (35 d of age). Feed consumption was recorded during the experiment. Average daily gain (ADG) (g/bird/d), feed consumption (FC) (g/bird/d), and efficiency of feed ratio (EFR) (g/g) were then calculated. Mortality was recorded when it hap-pened to allow correction to be made in calculating the FC and EFR.
At the end of starter and finisher periods, the birds were fasted for approximately seven hours. Two birds from each of the ten replicates were selected randomly, weighed, and slaughtered. The gastrointestinal tract (GIT), including the pancreas, was then removed. The aim of sampling was to weigh the gizzard, small intestine (from the end of the gizzard to the ileo-cecocolic junction), and ceca before and after removal of content. The pancreas was also weighed. The emptied weight of digestive organs was denoted as g/100 g BW (without digesta), whereas the content of gizzard, intestinal, and ceca were set as g/100 g BW.
To measure the jejunal morphology, two centimeters of the jejunum samples with content were cut adjacent to Meckel's diverticulum. Samples were stored in 10% buffered neutral formalin for fixation. Samples were manually sectioned with a microtome to obtain 4-5 µm thick sections, placed on glass slides, stained with hematoxylin-eosin, and then examined by light microscopy. The histology observations made were villus height (VH), upper villus width (UVW), lower villus width (LVW), crypt depth (CD), and mucosa thickness (MT). The calculation was made to get the VH:CD ratio.
Data were analyzed by one-way analysis of variance of completely randomized design (SPSS 25.0). When the F test was significant, mean differences among the group were inspected using the Duncan's multiple range test. A significant effect was accepted at level of 5%.

RESULTS AND DISCUSSION
In this study, to get a similar level of protein and crude fiber of the rice hull inclusion diet with the rice hull supplementation diet, the level of wheat bran and the level of palm oil of the rice hull inclusion diet was increased about 2 and 10 times, respectively. Wheat bran is a concentrated source of insoluble fiber (Stevenson et al., 2012). The proportions of insoluble and soluble fiber in wheat bran are approximately 42% and 3%, respectively (Vitaglione et al., 2008). Consequently, in addition to fat level, the rice hull inclusion diet obtains a higher concentration of insoluble fiber along with 40g/kg rice hulls than the other diets. While the higher insoluble fiber was reported to increase the rate of digesta passage (Hetland et al., 2004), the higher dietary fat was reported to slow down the digesta passage of broilers (Latshaw, 2008).
The effect of treatment diets on broilers performance is presented in Table 2. The effect of treatment diets was significant during the starter period but not in the finisher period. The rice hull inclusion diet increased ADG about 5% and 8% higher than the control and the rice hull supplementation diets, respectively, but did not affect feed consumption and EFR. This result is in contrast with Jimenéz-Moreno et al. (2016), who found that the inclusion of rice hulls up to 5% in broiler diets had no effects on ADG, feed consumption, and feed conversion ratio at 21 d of age. On the other hand, Adibmoradi et al. (2016) reported that the inclusion of 1.5% rice hulls in broiler diets increased feed consumption at 24 d of age and increased feed consumption and ADG at 42 d of age. Moreover, Sadeghi et al. (2015) reported that 3% of rice hull inclusion in broiler diets had no effect on performance either at 24 d or 42 d of age. The findings of various authors and our data suggested that the physiological effect of insoluble fiber was not due to its level but it is more likely due to diet composition, age, and growth potential of broiler (Jimenéz-Moreno et al., 2016). According to Hartini et al. (2019), an interaction between insoluble fiber and other feed components in diet would modify the physicochemical properties of an individual fiber. After 15 d of age, the GIT of the birds starts to develop and obtain its maturity (Sklan, 2001), therefore the older the birds the higher their capability to digest fiber. This might explain the non-significant result found in the finisher period.
The effects of diets on the relative weight of digestive organs and content varied and were only significant during the starter period ( Table  2). The rice hull inclusion diet decreased the relative weight of ceca as compared with the rice hull supplementation or the control diets. There are suggestions that the reduction in the weight of ceca was due to little or no microorganism activities that happened in the ceca. Activities of microorganisms in the ceca will increase with increasing fermentative substrates in the ceca, resulting in an increase the weight of the empty ceca (Jozefiak et al., 2006). The rice hull diet contains a high level of insoluble fibers and monogastric species lack specific microorganism species to ferment insoluble fiber (Hetland et al., 2004, Jha et al., 2010. A less fermentable diet would not support the proliferation or the activities of microorganisms in the ceca. The result showed that the weight of the gizzard, small intestine, and pancreas was not affected by treatment diets. This result was similar to Hartini et al. (2013), who reported that the GIT weight including gizzard, pancreas, and part of the small intestine was not affected by the addition of rice hull up to 6% in the diet based on corn and soybean. Sadeghi et al. (2015) and Incharoen (2013) also reported no increase in gizzard weight by the inclusion of rice hulls at 3% and 10%, respectively. However, Gonzaleź-Alvarado et al. (2007) found there was an increase in gizzard weight by including 3% oat hulls in the diet. This result implies that besides levels and particle size (Svihus, 2011), the structure and the source of insoluble fibers contribute to their physicochemical properties (Mateos et al., 2012;Gonzaleź-Alvarado et al., 2008). Oat hulls were higher in hemicelluloses while rice hulls were higher in celluloses and lignin (Podolske et al., 2013). In addition, water absorption from cellulose extraction of oat hulls was higher than rice hulls (392.1% vs 141.6%) (de Oliveira et al., 2016). All of these differences will affect the characteristic of the diet which eventually affects the response of fiber inclusion on GIT weight (Mateos et al., 2012). The small intestine content significantly increased in birds fed the rice hull inclusion and control diets, the result that is not consistent with Hetland et al. (2004) who reported that high insoluble fiber diets would increase the rate of digesta passage, resulting in less digesta in the small intestine. Our result indicates that the magnitude of the response may be due to the higher fat level in the rice hull contained diet. Latshaw (2008) reported that the higher dietary fat slow down the digesta passage of broilers, thereby increasing nutrient utilization. Thus, the physiological function of the rice hull was interrupted by a higher fat level of diet, eventually slowing down the digesta passage and elevating nutrient utilization. This leads to an increase in the daily gain of young broilers as shown in our result. Mandey et al. (2017) also reported a good value of carcass percentage in broilers fed a diet containing 11% of crude fiber and about 11% of fat level.
The data on jejunal morphology traits of broilers are shown in Table 2. The jejunum villus height (VH) and mucosa thickness significantly increased by rice hull supplementation or inclusion diets than the control group. The finding is in agreement with Rezaei et al. (2014), who reported that the inclusion of 3% raw rice hull in the diet increased villous height in the jejunum of broilers. Moreover, Rezaei et al. (2018) observed an increase in jejunal villous height due to mi- However, some authors have demonstrated that the addition or inclusion of rice hulls decreased jejunal villus height in broilers (Sabour et al., 2019;Abazari et al., 2016;Adibmoradi et al., 2016;Incharoen, 2013). The varied effect of dietary fiber on epithelial morphology depends on the physico-chemical properties of the fibers, the incorporation level of fibers in the diet, the length of the feed given, the species of animal and age (Montagne et al., 2003), and the composition of the diet (Mateos et al., 2012).
The villus height and villus surface area (Rezaei et al., 2014;Teirlynck et al., 2009), mucosa thickness (Jiménez-Moreno et al., 2013b), and crypt depth (CD) (Markovicva et al., 2009) of the jejunum reflect its capacity for nutrient absorption. A higher villus height and wider villus surface area are associated with greater capacity of nutrient absorption (Rezaei et al., 2014), whereas a deeper jejunal crypt is referred to an increase in intestinal maintenance, thus consuming energy and protein that is used in muscle deposition (Markovicva et al., 2009). In the present study, the value of the VH:CD ratio in birds fed the rice hull inclusion diets was higher than the others, whereas birds fed the rice hull supplementation diet showed a significantly higher value in the crypt depth. This might explain the lower daily gain observed in birds fed the rice hull supplementation diet in the starter period.
At 35 d of age birds fed the rice hull supplementation diet reduced the width of upper and lower jejunal villi. However, the mucosa thickness in birds fed either rice hull supplementation or inclusion diets was higher than the control diet. Therefore, although the villus surface area in birds fed the rice hull supplementation diet was reduced, the function of the intestinal mucosa was not affected (Jiménez-Moreno et al., 2013b), as shown by no significant effect of daily gain among diets in the finisher period. The representation of histological of the jejunal villi of broiler chickens at 21 d of age and 35 d of age from all the groups are shown in Figures 1 and 2.

CONCLUSION
Rice hull supplementation diet caused an increase in villus height and mucosa thickness, but a deeper crypt, resulting in poor performance of younger broiler chickens. In contrast, the rice hull inclusion diet increased villus height, mucosa thickness, and showed a higher value of VH:CD ratio, resulting in greater body weight gain. The way insoluble fiber is incorporated into the diet will lead to different physiological responses due to changes in diet composition. The physiological response was pronounced in younger broiler chickens but not in older age.

ACKNOWLEDGMENTS
The present study could be carried out due to the financial assistance from the Ministry of Research and Technology of Higher Education of the Republic of Indonesia through contract No. 080/SP2H/L. T/DRPM/2018 from the National Competitive Fund, Competency-based Research. Extended thank was delivered to PT. Japfa Comfeed Tbk for help in providing the feedstuffs used in this study.