INTRODUCTION. M. E. Abd El-Hack, M. Alagawany,,1 S. A. A. El-Sayed, and J. Fowler

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1 Influence of dietary inclusion of untreated or heat-treated Jatropha meal on productive and reproductive performances and biochemical blood parameters of laying Japanese quail M. E. Abd El-Hack, M. Alagawany,,1 S. A. A. El-Sayed, and J. Fowler Department of Poultry, Faculty of Agriculture, Zagazig University, Zagazig 44511, Egypt; Department of Nutrition and Clinical Nutrition, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44511, Egypt; and Department of Poultry Science, the University of Georgia, Athens, GA 30602, USA ABSTRACT Jatropha meal (JM) has been characterized egg mass compared with the control diet. There was as a potential animal feedstuff due to its high crude protein content and high levels of essential amino acids. However, it contains anti-nutritive and toxic compounds that may hinder its use. The present study was conducted to evaluate the effects of a dietary inclusion of raw Jatropha meal (RJM) and heat-treated Jatropha meal on the growth and production of laying Japanese quail using productive, reproductive, and biochemical blood parameters. A total number of 180 mature Japanese quail at 2 months of age (120 females and 60 males) was randomly divided into 4 treatment groups: control (0% JM); 3.5% RJM; 3.5% JM heated in an oven at 100 C for 24 h (JM 24 ); and 3.5% JM heated in oven at 100 C for 48 h (JM 48 ). After 8 wk on treatment diets, the inclusion of RJM to quail diets reduced (P < 0.05) feed intake, feed efficiency, egg number, and no difference between the heat-treated Jatropha (JM 24 and JM 48 ) treatments and the control. Fertility and hatchability percentages were improved (P < 0.001) with JM 24 and JM 48 compared with the RJM group. Feeding of 3.5% RJM or JM 24 in quail diets led to significant decrease (P < 0.05) in serum total protein, albumen, globulin, γ-glutamyl transpeptidase, total and HDL-cholesterol, immunoglobulin (IgG and IgM), and increased serum alanine transaminase (ALT) and creatinine along with the mortality rate when compared with birds fed JM 48. In conclusion, heat treatment of JM is essential to eliminate its hepatotoxic and nephrotoxic effects and, when properly heat-treated, can be used in laying Japanese quail diets without detrimental effects on production, reproductive performance, and health status. Key words: Jatropha meal, egg production, serum biochemistry, Japanese quail 2017 Poultry Science 96: INTRODUCTION The application of non-conventional feedstuffs in poultry diets in developing countries has received greater attention in recent years. Jatropha (Jatropha curcas) is a genus of flowering plants belonging to the family Euphorbiaceae, which grow in South and Central America, Southeast Asia, and Africa (Martínez- Herrera, 2006; Nesseim et al., 2015). By 2017, there will be around 330,000 km 2 of land cultivated worldwide producing 160 tonnes of seeds that will produce 53 tonnes of Jatropha seed meal (Devappa et al., 2010). The Jatropha plant and its products represent a potential supply of protein for livestock (Kumar et al., 2008; Barros et al., 2015), the need for which grows with increases in the world population. Jatropha seed meal contains a high crude protein level (56%), compared C 2017 Poultry Science Association Inc. Received December 5, Accepted March 17, Corresponding author: dr.mahmoud.alagwany@gmail.com with soybean meal (44 to 48%). However, through the oil extraction process, the shell is not completely removed. Therefore the by-product meal contains a higher crude fiber in its fractionations, such as lignin (Makkar and Becker 1999). Moreover, it contains anti-nutritional factors, such as trypsin inhibitor, lectin, phorbol esters, saponin, and phytate (Makkar and Becker, 1998). These compounds have deleterious effects on bird performance and feed utilization, as well as renal and hepatic functions (Johnson et al., 2013). To use Jatropha meal as a protein source in poultry diets, the anti-nutritional factors need to be removed by physical or chemical treatments (Aderibigbe et al., 1997), similar to the heattreatment required for highly digestible soybean meal. Therefore, the objective of this study was to evaluate the effects of the inclusion of raw Jatropha meal (RJM) and heat-treated Jatropha meal (JM) in the diet on productive and reproductive performance, egg quality, hepatic and renal function, lipid profile, and the immune responses of laying Japanese quails. 2761

2 2762 ABD EL-HACK ET AL. Table 1. Proximate analysis of raw and heat treated Jatropha meal. Item RJM 1 JM 24 1 JM 48 1 Composition (%) 2 Crude protein Crude lipid Crude fiber Calcium Total phosphorus Ash ME, Kcal/Kg (calculated) RJM = raw Jatropha meal. JM 24 and JM 48 = heat treated Jatropha meal at 100 C for 24 and 48 h, respectively. 2 Determined according to AOAC (2006). 3 Calculated metabolizable energy according to Carpenter and Clegg (1956). MATERIALS AND METHODS Source and Preparation of the Jatropha Meal Jatropha curcas seeds were collected from trees in Upper Egypt (Luxor Governorate) and transported to the Department of Agricultural Engineering, Faculty of Agriculture, Zagazig University, for further processing. Shelled seeds were mechanically pressed using a screw press to extract the oil. The jatropha oil and the residual meal were separated and the JM was stored at 10 C until further analysis and use. The JM was divided into 3 portions. The first portion was dried at room temperature and considered as RJM, the second portion was heated at 100 C for 24 h (JM 24 ), and a third portion washeatedinanovenat100 C for 48 h (JM 48 ). The proximate composition of RJM, JM 24,andJM 48 were determined using the standard methods of the AOAC (2006). All samples were analyzed in triplicate for crude protein, ether extract, crude fiber, ash, calcium, and phosphorus (Table 1). Experimental Design and Animal Husbandry This study was carried out at the Zagazig University Research Farm in Egypt. All procedures were carried out according to the Local Experimental Animal Care Committee and approved by the Ethics of Institutional Committee of Department of Poultry, Faculty of Agriculture, Zagazig University. A total of 180 mature laying Japanese quails (Coturnix coturnix japonica), at 8 wk of age (120 females and 60 males; initial BW: ± 2.14 g), was randomly divided into 4 treatment groups. Every treatment had 5 replications with 6 females and 3 males in each replicate. From the age of 8 to 16 wk, birds were fed 4 treatment diets (Table 2): 1) a control diet without Jatropha meal, 2) a diet containing 3.5% RJM; 3) a diet containing 3.5% JM 24,and 4) a diet containing 3.5% JM 48. All experimental diets were formulated to meet the nutrient requirements of laying Japanese quail as recommended by NRC (1994) as shown in Table 2. All birds were housed during the Table 2. Ingredients and nutritional composition of the experimental diets (% of the as-fed diet). Jatropha meal (JM) levels 2 Items 0 RJM JM 24 JM 48 Ingredient, % Corn Soybean meal 44% CP Jatropha meal Corn gluten meal 62% CP Soybean oil Limestone Di-calcium phosphate Salt Vit-Min-premix L-Lysine Dl-Methionine Determined composition, % 3 ME, kcal/kg (calculated) Crude protein Calcium Total phosphorus Non-phytate P (calculated) Lysine (calculated) Methionine (calculated) Met+Cys (calculated) Crude fiber Crude fat Supplied per kg of diet: vitamin A IU; vitamin D IU; vitamin E 30 IU; vitamin K3 5 mg; vitamin B1, 3 mg; vitamin B2 12 mg; vitamin B6 4 mg; vitamin B mg; nicotine amid 55 mg; calcium-d-pantothenate 15 mg; folic acid 2 mg; D-biotin mg; choline chloride 600 mg; Mn 80 mg; Fe 40 mg; Zn 60 mg; Cu 5 mg; I 0.4 mg; Co 0.1 mg; Se 0.15 mg. 2 RJM = raw Jatropha meal. JM 24 and JM 48 = heat treated Jatropha meal at 100 C for 24 and 48 h, respectively. 3 Determined according to AOAC (2006). 4 Calculated according to NRC (1994). experimental period in conventional type cages ( cm 3 ; 300 cm 2 of floor space per bird) and kept under the same managerial and environmental conditions. Birds were exposed to a 17:7 L:D cycle throughout the trial. Feed was provided in mash form and provided ad libitum with water. Data Collection Data collection began 4 wk after feeding the experimental diets (12 wk old). Body weight was recorded individually at the start of the experiment (initial BW) and at the end of the experiment (final BW). Feed intake was recorded weekly and calculated as grams of feed consumed over 7 d divided by the number of birds in each replicated group, while feed efficiency (g egg/g feed) was calculated as the egg mass value divided by the amount of feed consumed. Egg weight and egg number were recorded daily to calculate the egg mass (egg number egg weight). Egg quality parameters were measured monthly using 3 eggs from each replicate. Exterior and interior egg quality parameters (percentages of yolk, albumen, shell, and egg shape index [ESI], shell thickness, unit surface shell weight [USSW], and Haugh unit) were determined according to Romanoff and Romanoff (1949).

3 LAYING JAPANESE QUAIL AND JATROPHA MEAL 2763 Table 3. Effect of dietary Jatropha meal on productive performance traits of laying Japanese quail from 8 to 16 wk of age. Performance Jatropha meal (3.5%) 1 SEM P-value 2 Control RJM JM 24 JM 48 Live body weight (g) Initial Final Daily feed intake (g) 8 to 12 wk to 16 wk a c b a Overall(8to16wk) a b a a Feed efficiency (g egg/g feed) 8to12wk 0.31 a 0.07 c 0.22 b 0.16 b to 16 wk 0.20 a 0.04 b 0.19 a 0.19 a 0.02 <0.001 Overall (8 to 16 wk) 0.26 a 0.06 c 0.20 b 0.17 b 0.02 <0.001 Egg weight (g) 8 to 12 wk to 16 wk 8.55 a 7.27 b 7.71 a,b 8.45 a Overall (8 to 16 wk) 8.35 a 7.38 b 7.70 a,b 7.80 a,b Egg number (hen/month) 8to12wk a 7.00 c b b 1.89 < to 16 wk a 3.68 b a a 2.22 <0.001 Overall(8to16wk) a 5.34 c b b 2.00 <0.001 Egg mass (g) 8to12wk a c b b < to 16 wk a c b a,b <0.001 Overall (8 to 16 wk) a c b b <0.001 Mortality rate 8to12wk 0.00 c a 2.22 b 0.00 c to 16 wk 0.00 c a b 4.44 c Overall (8 to 16 wk) 0.00 c a b 4.44 c Different superscripts within rows are significantly different (P < 0.05). At the fourth wk of each mo, eggs from each treatment replicate were collected over 2 d and incubated. After hatching, chicks were counted and non-hatched eggs were broken to determine the percentages of fertility and hatchability. The hatchability was expressed as hatched chicks from fertile eggs and as hatched chicks from total eggs set. Fertility and hatchability percentages were calculated as follows: Fertility % = (number of hatched eggs plus eggs containing embryos/total eggs set) 100; hatchability % = (number of hatched chicks/total number of set eggs) 100. At the end of the experimental period, blood samples were collected from 2 females per replicate. Blood samples were obtained by venipuncture of the brachial vein. Blood samples were centrifuged (2100 g) for 15 min, and serum was separated and stored at 20 C until analysis. The total protein (g/dl), albumin (g/dl), creatinine (mg/dl), bilirubin (mg/dl), urea- N (g/dl), triglycerides (TG; mmol/l), total cholesterol (mmol/l), high-density lipoprotein (HDL) cholesterol (mmol/l), low-density lipoprotein (LDL) cholesterol (mmol/l), and immunoglobulin G (IgG; mg/dl) and IgM (mg/dl) levels as well as alanine transaminase (ALT; U/L), aspartate transaminase (AST; U/L) and alkaline-phosphatase (ALP; U/L), lactate dehydrogenase (LDH; U/L), and γ-glutamyl transpeptidase (GGT; U/L) activities were determined spectrophotometrically according to Akiba et al. (1982), using commercial diagnostic kits from Biodiagnostic Company (Giza, Egypt). Statistical Analysis The study was a completely randomized design with 4 treatments and 5 replications with 9 birds for each replicate (6 females and 3 males). Data were subjected to one-way ANOVA for a completely randomized design using the GLM procedure of SPSS (2008), version The differences among means were determined using the post-hoc Newman-Keuls test when statistical significance was P < RESULTS Egg Production, Quality, and Fertility Compared with the control diet, the body weight among treatments was not significantly influenced (P = 318; Table 3) by the JM treatments throughout the experimental period. However, the final BW of quail fed RJM did show the lowest weights among the other treatments. There were significant (P < 0.05) differences in feed intake, feed efficiency, egg weight, egg number, and egg mass due to JM treatments. The inclusion of RJM in quail diets resulted in a decrease in feed intake, feed efficiency, egg number, and egg mass

4 2764 ABD EL-HACK ET AL. Table 4. Effect of dietary Jatropha meal on reproductive performance traits of laying Japanese quail from 8 to 16 wk of age. Parameters Jatropha meal (3.5%) 1 SEM P-value 2 Control RJM JM 24 JM 48 Fertility (%) 8to12wk a b a a 6.91 < to 16 wk a b a a 6.97 <0.001 Overall(8to16wk) a b a a 6.94 <0.001 Hatchability (%) 8to12wk a b a a 5.61 < to 16 wk a b a a 5.66 <0.001 Overall(8to16wk) a b a a 5.64 < Different superscripts within a row are significantly different (P < 0.05). Table 5. Effect of dietary Jatropha meal on egg quality criteria of laying Japanese quail during 8 to 16 wk of age. Egg quality criteria Jatropha meal (3.5%) 1 SEM P-value 2 Control RJM JM 24 JM 48 Albumin % Yolk % Shell % Egg shape index Shell thickness Yolk index Yolk: albumin ratio Haugh unit score b b b a Different superscripts within a row are significantly different (P < 0.05). when compared to the control diet. In the heat-treated Jatropha meal treatments (JM 24 and JM 48 ), those parameters were not affected. Also, it was observed that the inclusion of RJM into the quails diet resulted in higher mortality (at the 8 to 12, 12 to 16, and 8 to 16 wk time points) in comparison with the other treatment groups (P = 0.001; Table 3). Regarding the effect of JM on reproductive performance of Japanese quail, fertility and hatchability percentages were significantly (P < 0.001; Table 4) decreased by feeding quail RJM while quails fed JM 24 and JM 48 had fertility and hatchability percentages similar to control groups (Table 4). There were no effects on the egg quality parameters throughout this trial, with the exception of Haugh units, which showed the highest score in birds fed 3.5% of JM 48 (P = 0.01; Table 5). Liver and Kidney Functions Data presented in Table 6 indicate that JM treatments caused significant (P < 0.05) changes in the measures of liver function, such as serum total protein, albumin, and globulin concentrations as well as ALT, LDH, GGT, and ALP activities. Feeding 3.5% RJM or JM 24 in quail diets led to significant reductions (P < 0.05) in total protein, albumen, globulin, and GGT when compared with birds fed the diet containing JM 48. Also, birds fed the diet containing 3.5% of RJM recorded the lowest values of ALP and highest value of ALT enzyme when compared to the other treatment groups. The level of creatinine in the blood, which is the sensitive indicator of kidney function, was significantly (P = 0.008) affected by JM treatments. Birds fed the diet containing 3.5% of RJM recorded the highest value of creatinine when compared to the other treatment groups. The activity of the AST enzyme, concentrations of blood urea-n (P = 0.07), and total bilirubin (P = 0.66; Table 5) were not affected by the JM inclusion in these layer quail diets. Lipid Profile and Immunoglobulin It was observed that total cholesterol and HDL cholesterol were significantly decreased (P < 0.05; Table 6) in birds fed any of the diets containing JM, when compared with birds fed the control diet while triglycerides and LDL cholesterol were only numerically decreased by the different JM treatments. Regarding immunological parameters, feeding of RJM or JM 24 led to a significant decrease in IgG and IgM concentrations when compared with quail fed the diet containing JM 48 or the control diet (P = 0.001; Table 6). Immunoglobulin levels were similar for groups fed the control diet and that which contained JM 48 (IgG was 7.85 and 7.20 mg/dl, respectively, and IgM was 8.76 and mg/dl, respectively).

5 LAYING JAPANESE QUAIL AND JATROPHA MEAL 2765 Table 6. Effect of dietary Jatropha meal on some biochemical blood parameters of laying Japanese quail at 16 wk of age. Parameters 3 Jatropha meal (3.5%) 1 Control RJM JM 24 JM 48 SEM P-value 2 Liver and kidney functions Total protein (g/dl) 3.33 b 2.74 c 2.67 c 4.22 a 0.19 <0.001 Albumin (g/dl) 1.99 b 1.88 b,c 1.76 c 2.21 a Globulin (g/dl) 1.34 b 0.86 c 0.91 c 2.00 a 0.14 <0.001 AST (U/L) ALT (U/L) b a a a 2.58 <0.001 ALP (U/L) a c a,b b LDH (U/L) a b b b 5.90 <0.001 GGT (U/L) a b b a Urea-N (g/dl) Creatinine (mg/dl) 0.45 b 0.57 a 0.43 b 0.47 b Total bilirubin (mg/dl) Lipid parameters Triglycerides (mmol/l) Total cholesterol (mmol/l) 6.72 a 4.00 c 5.10 b 5.85 a,b HDL-cholesterol (mmol/l) 4.30 a 2.21 b 3.27 a,b 3.60 a,b LDL- cholesterol (mmol/l) Immunoglobulin IgG (mg/dl) 7.85 a 2.80 c 4.96 b 7.20 a IgM (mg/dl) 8.76 a 4.62 b 5.67 b a Different superscripts within a row are significantly different (P < 0.05). 3 AST = aspartate aminotransferase, ALT = alanine aminotransferase, ALP = Alkaline phosphatase, LDH = Lactate dehydrogenase, GGT = γ-glutamyl transpeptidase. DISCUSSION Egg Production, Quality, and Fertility This study found that the inclusion of either the RJM or heat-treated JM had no adverse effect on live body weight of laying Japanese quail throughout the experiment. These results were in disagreement with Pasaribu et al. (2010) who found that the inclusion of 4% raw Jatropha meal in broiler chickens caused a negative effect on live weight gain, while giving 4% physically and chemically treated Jatropha meal showed an increase in live weight. We saw that the inclusion of RJM in quail diets resulted in a significant decrease in feed intake, feed efficiency, egg number, and egg mass. In agreement with these results, inclusion of RJM in diets has been reported to decrease live weight gain (El Badwi et al., 1992; Samia et al., 1992). Also, our results are supported by Sumiati, Mutia and Darmansyah (2012) who observed that inclusion of Jatropha meal to diets decreased feed consumption, hen day egg, and egg mass in laying hens. Feeding RJM in this study decreased feed consumption, which thus lowered overall nutrient consumption. A lower nutrient consumption inevitably results in lower egg production from the hens. The altered productivity of birds in this study could have been a result of decreased nutrient intake, especially protein, which is mainly used for egg production, maintenance of body protein, and continual growth of birds. These negative impacts can be explained by the low nutritive value of JM when fed in its raw form. The low nutritive value of RJM could be attributed to the high content of both anti-nutritive and toxic compounds such as anti-trypsin, lectin, curcin, phytate, and phorbol esters (Makkar and Becker, 1999; Barahona et al., 2010). According to Leeson and Summers (2001), protein consumed from diets was utilized for production of an egg, maintenance of body protein for one d, growth per d, and feather growth per d. A decrease in feed consumption of chickens fed JM has previously been attributed to liver damage resulting from detoxification of compounds such as phorbol ester (Sumiati et al., 2011). The positive effect of heat-treating JM is in agreement with Pasaribu et al. (2010) who found enhancement in the productive performance of birds. Regarding feed efficiency, heat treatment of JM improves feed efficiency compared with RJM. However, feed efficiency values were not significantly different between JM 24 and JM 48. The negative effects of raw JM were previously reported by El Badwi et al. (1992). They used un-extracted Jatropha seed as a feed ingredient. In that case, the oil contains high toxic compounds so that the inclusion of 0.5% Jatropha seed in the diet caused a negative effect on live weight gain. Anti-nutritive compounds in Jatropha seed or JM may cause irritation in the digestive tract, which decreases feed intake and growth in the bird (Burrows and Tyrl, 2001). This study also saw that fertility and hatchability percentages were improved with JM 24 and JM 48, when compared with the RJM group. The deleterious effects on reproductive traits may be attributed to the toxic substances in the remaining oil of untreated Jatropha meal (Samia et al., 1992). The inclusion of RJM in quail diets also increased the mortality rate, which was decreased by heating the meal

6 2766 ABD EL-HACK ET AL. for 24 h and restoring it to levels comparable to the control treatment by heating for 48 hours. The high mortality rate is best explained by the accumulation of toxic compounds in the body. As shown previously, 5% heattreated JM was considered the safest level for growing Japanese quail, but the mortality rate was increased with increasing levels of JM above 10% (Agboola and Adenuga 2015). Also, Gross et al. (1997) found that the inclusion of raw JM in a fish diet caused 100% mortality, and chemically treated JM was able to decrease mortality to 8%. Egg quality parameters in this study were not affected by JM inclusion, with the exception of Haugh unit score, which was improved in the 3.5% of JM 4 8. Haugh unit is an indicator of the protein quality and freshness of an egg. The blood profile demonstrated that quail fed the JM 48 treatment had higher total protein, albumin, and globulin in circulation. This could explain the obtained decrease in productive performance in the RJM group, where the lower quality diet decreased the growth and egg characteristics by providing low-quality substrates for synthesis. Liver and Kidney Function The total protein, albumen, and globulin were decreased with RJM or JM 24, but increased in the JM 48 group when compared to the control. The hypoproteinemia observed in the RJM and JM 24 groups may be due to the toxic impact of phorbol esters, the main toxic component of JM, which led to necrosis and degeneration of hepatocytes (Aregheore et al., 2003), the main site of protein synthesis. Reddy and Salunkhe (1982) also attribute reductions in total protein to an inhibition of protein utilization in the birds, resulting in inefficient digestibility. Our results revealed that RJM in laying quail diets results in a significant increase in liver enzymes such as ALT. The increased liver enzymes in the serum is an indicator of liver damage, suggesting the cytotoxic effects of RJM on the liver cells led to leakage of this enzyme from damaged hepatocytes into the bloodstream (Nabil et al., 2011). The ALT enzyme is the most sensitive marker for liver damage as compared to the other liver enzymes and can provide quantitative assessment of the degree of damage sustained by the liver (Aniagu et al., 2004). Our results are in agreement with Kaneko, (1989) who found that the ALT enzyme was highly affected by Jatropha. The effects of the phorbol esters in RJM have greatly hindered its use as an animal feed (Makkar et al., 1997). Our results recorded the lowest values of ALP and LDH in quails fed RJM. Rivetz et al. (1975) mentioned that the main reason for ALP reduction is damage to the intestine or other internal organs. Barros et al. (2015) found that the dietary inclusion of RJM had negative effects on feed digestibility and the development of the GI tract and internal organs in broiler chicks. Other researchers have also found associated damage to the digestive tract when feeding RJM (Makkar and Becker, 1998). In our study, the inclusion of JM to quail diets did not cause an increase in total bilirubin. In contrast, El Badwi et al. (1992) found that inclusion of Jatropha curcas in brown Hisex chicks was associated with an elevation of serum bilirubin. In this study, RJM resulted in a significantly higher level of creatinine, which is an indicator of renal dysfunction. If the filtering capacity of the kidney is deficient, creatinine levels rise in the blood (Nwanjo et al., 2005). So RJM has both hepatotoxic and nephrotoxic effects in laying quail. Lipid Profile and Immunoglobulin The dietary inclusion of 3.5% RJM in the laying quail diet resulted in significant decreases in total cholesterol and HDL cholesterol and numerical decreases in triglycerides and LDL cholesterol when compared to the control group. Our results disagree with Johnson et al. (2013) and Ojediran et al. (2014) who found that administration of Jatropha curcas at different levels from 4 to 12% in broiler diets for 4 wk caused an increase in serum cholesterol. This contradiction may be attributed to 70% of the plasma cholesterol being synthesized endogenously, hence the negative effect of the RJM diet on the GI tract may have had little impact on circulating levels of serum cholesterol. The immune system plays a crucial role in guarding body health against foreign substances and from infection by potential pathogens. We studied the effect of JM on the humoral component of the immune system and found that groups fed on RJM or JM 24 had lower concentrations of IgG and IgM. The decreased humoral immunity could be attributed to the toxic effect of JM on lymphoid organs (Barros et al., 2015). Similarly, Dai-Gang et al. (2012) found that dietary JM at 3 to 12% caused histological damage to immune organs and decreased the blood concentrations of immunoglobulin and T-lymphocytes. The authors also found that a 48-hour heat treatment was sufficient to remove this toxic effect on the immune system. Jatropha meal is expected to be a potential soybean meal alternative protein source for poultry diets in the future. Using it, however, requires that the antinutritional factors be dealt with. 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