Nutritional Evaluation and Effectiveness of Detoxified Jatropha curcas Kernel Meal in Finishing Pig Diets on Biogas and Methane Production from Slurry

Corresponding Author: Bernardo Berenchtein Agronomy College, Federal University of South Frontier, 99700-970, Erechim, RS, Brazil Tel: +55 54 33217050 Email: bernardo.berenchtein@uffs.edu.br Abstract: The purpose of this work was to evaluate Phorbol Ester’s (PE), Total Fenols (TF), Total Tannins (TT), Condensed Tannins (CT), Saponins Content (SPN), amino acids profiles and apparent digestibility of detoxicated Jatropha curcas meal in finishing pigs diets, as well as biogas and methane production by the manure from cross-bred pigs fed this meal. Apparent digestibility coefficients of Dry Matter (DM), Crude Energy (CE), Crude Protein (CP), Crude Fiber (CF) and Ether Extract (EE) were evaluated. Twenty crossbreed pigs with 70±1.95 kg initial weight were allotted in individual metabolic cages, according to body weight in a Complete Randomized Block Design with ten replications per treatment. On the last day of the adaptation phase of the digestibility experiment, the manure were collected and put it in bottles with hydraulic retention time of seven days for further evaluation of biogas and methane produced in liters and liters % of total solids. The content of PE, TF, TT, CT, SPN, were, respectively, 0.06 mg/g, 26.08 and 10.43 equivalent gram of tannic acid, 0.05 equivalent gram of leucocyanidin and 0.005%. The results of apparent digestibility coefficients were 83.80% (DM), 3.500 kcal/kg (CE), 13.45% (CP), 1.5% (CF) and 2.3% (EE). Regarding the generation of biogas and methane of manure from pigs fed detoxified Jatropha curcas meal, biogas production (1201.4, 1089.3 mL/g Total Solids) and methane (246.7 and 218.2 mL/g Total Solids) from manure weren't significantly different (p>0.05) between the treatments. So, concludes that detoxicated Jatropha curcas meal is a promising byproduct due its nutritional composition and metabolic response which may be used in finishing pig diets without affecting the generation of renewable energy.


Introduction
The feed spend is about 70% of average costs of swine production for slaughter in Brazil (Girotto and Santos Filho, 2000). In this context, the financial success of any production is directly related to the cost of the feed ingredients. Considering that the diets are constituted basically of corn and soybean meal, which present constant price instability, it's clear the need to search for new alternatives that can replace economically these ingredients. Thus, the research involving the use of alternative feeds, especially the byproducts or waste resulting from the industrial processing of agricultural products and of modern practices of agricultural mechanization, are gaining attention (Gomes, 2006).
The growing global concern about the environment and the search for energy renewable sources put the biodiesel on the spotlight of interests. Several countries, including Brazil, are seeking the path of technological domain of biofuel, both in agronomic and industry levels, which should cause a strong impact on the Brazilian economy and on the social inclusion policies of this country (Abdalla et al., 2008).
Brazil has nearly two hundred species of oil plants and palmaceae for oil production and biodiesel (Beltrão, 2006). Jatropha curcas is considered promising for used in animal feed due its high oil content (25-40%), higher than the other oilseeds used in the biofuels market (Arruda et al., 2004).
The production of biodiesel generates some byproducts, which can be the focus of more detailed analysis. The use of Jatropha curcas meal in animal feed arouses the interest of many researchers and it could be used in animal feed, provided that appropriate treatments should be performed for the reduction or elimination of bioactive molecules, such as phorbol esters (Neiva Junior et al., 2007).
The chemical analysis and feeding tests are the first items to determine the potential and nutritional value of a food to be used on animal nutrition. Different animal species metabolize food differently; being this variation quantified upon the determination their digestibility coefficients. The digestibility of a feed is defined as the skill with which the animal digests and absorbs the nutrients and energy it contains. The digestibility determination of an ingredient is the first concern when it comes to evaluating their potential for inclusion in rations for animal (Pezzato et al., 2004). Thus, the purpose of this study was to determine the chemical composition, phorbol esters, total phenols, total tannins, condensed tannins, saponins content, amino acid profile, the apparent digestibility of detoxified Jatropha curcas meal (Jatropha curcas L.) for finishing pigs and to evaluate the biogas and methane generation from the manure obtained from pigs fed this product.

Chemical Analyses
This work was development in the premises of the Animal Nutrition Laboratory of the Nuclear Energy in Agriculture Center (CENA) of University of São Paulo, São Paulo, Brazil. The Detoxified Jatropha curcas Meal (DJCM) used was collected in a biodiesel industry producer, being later industrially detoxified.
The chemical composition analysis were performed according to AOAC (2005) and the amino acid profile obtained by HPLC process. The concentrations of phorbol esters were analyzed following the method by Makkar et al. (1997), the contents of total phenols and total tannins analyzed by Folin-Ciocalteu, condensed tannins (proanthocyanidins) by the butanol-HCl method (Makkar, 2000) and the concentration of saponins analyzed according to Makkar et al. (2007).

Apparent Digestibility and Experimental Set Up
Ten crossbred pigs, barrows, with average weight of 70±1,95 kg were used, distributed in a randomized block design in function of body weight in two treatments: The Reference Diet (RD) and Test Diet (TD), with five replicates (blocks), with an animal for metabolic cage (experimental unit).
Before the experimental period, feed intake tests to determine the maximum level of inclusion of DJCM in reference diet were performed, so that feed intake was not prejudice in the excreta collection period. Thus, the TD was composed of 92.3% of the RD and 7.7% of DJCM. The reference diet was formulated in accordance with the requirements proposed by Rostagno et al. (2005) as shown in Table 1.
The partial excreta collection method was used and chromic oxide (Cr 2 O 3 ) employed as a marker added to the experimental diets at a concentration of 0.05%. The animals were weighed before and after the trial, which lasted thirteen days with seven days of adaptation and measurement of voluntary feed intake, three days for adaptation and maintenance of flow of marker and three days of excreta collection. The quantities of diets supplied were calculated according to the metabolic weight (PV 0.75 ) and a proportion of 1:1 water and diet was supplied to the animals twice a day, at 08:00 and 16:00.
In adaptation period, the animals were encouraged to consume the maximum of feed aiming at avoiding leftovers and in the collection period the quantities of diets in each treatment were based on lower consumption among the animals of the block, obtained in the adaptation period.
From the average feed intake in the adaptation period, was established the Amount Restricted Feed (ARF) provided during the days of excreta collection, according to the following calculations: The index (I) was calculated for all animals, however, was adopted the lowest value of I, to calculate the ARF. Although the I value standardization, ARF were evaluated individually, also calculated being in function on the weight of each animal. Water was provided ad libitum throughout the experimental period.
Excreta samples from the rectum were collected twice daily (08:00 and 16:00) being these immediately placed in plastic bags, identified and frozen. After, these samples were thawed and homogenized in a single composite sample. These were dried in trays at appropriate ovens of forced air circulation at 65°C for 72 h, milled in mill through sieve riddled of 1 mm. The excreta and feed samples were analyzed for dry matter, crude energy, crude protein, crude fiber, ether extract and chromium oxide content.
Chromium oxide percentage in the samples was done in Instrumentation Nuclear Laboratory of Nuclear Energy in the Agriculture Center (CENA) of University of São Paulo by technique of X-ray fluorescence for dispersive energy (Zucchi and Nascimento Filho, 1995;Nascimento Filho et al., 1997). The apparent digestibility coefficients of RD and TD were calculated according to Serrano (1989) and from this, determined the apparent digestibility coefficients of dry matter, crude energy, crude protein, crude fiber and ether extract of DJCM using the equation proposed by Matterson et al. (1965).

Biogas and Methane Production
On the last day of adaptation phase to the cage in digestibility experiment, the pigs manure were collected and added in proportion of 250 mL of excreta's, 250 mL of urine and 615 mL of water in PET bottles previously identified with total volume of 3,125 mL with rubber stoppers adapted to screw caps, sealed with silicone glue to prevent the gas produced was lost, placed in oven of forced ventilation at 40°C without agitation and with Retention Hydraulic Time (RHT) of seven days. These conditions were observed by Souza et al. (2005) as being ideal for biogas production by pigs manure, simulating the conditions of a batch bio digester, without continuous flow of effluents, for biogas and methane (CH 4 ) quantification. Two treatments were used, RD and TD (7.7% inclusion of DJCM), through ten bottles (replicates) for each treatment.
Each bottle was dimensioned for storing two liters of biogas and permit the biogas produced quantitation, through the use of a pressure gauge (transducer-model PDL800-LANA-CENA/USP) adapted of Theodorou et al. (1994) and graduated syringes, where two first days was held instrument calibration and preparation of the response curve. During the seven days, the pressure exerted by gases was measured for subsequent determination of the biogas volume of each bio digester (bottle). After each time of reading of gases production, a gas aliquot was stored in test tubes previously identified and subjected to vacuum where the determination of the methane levels (CH 4 ) were done using a gas chromatograph model CG-2014 GAS CHROMATOGRAPHA (Shimadzu). The equipment calibration was performed with standard gas supplied by White Martins. In addition, total solids levels (AOAC, 2005) of the waste incubated in each bio digester were evaluated. Considering the effective bio digester volume of 2,000 mL, the biogas and methane volumetric total production were determined, as well as daily thereof production in mL/g of total solids. The design was randomized blocks, with two treatments and ten replicates per treatment. vit. E -9.9 IU; vit. K3-0.72 mg; thiamine-404 mcg; riboflavin-1.98 mg; pyridoxine-404 mcg; vit. B12-8.1 mcg; folicacid-225.2 mcg; Pantothenic Acid-6.3 mg; Niacin-12.6 mg; growth promoter-10 mg; Se-0.24 mg; 2 Amountsper kg diet: Cu -9 mg; Fe -81 mg; I -0.9 mg; Mn-54 mg; Zn-135 mg

Statistical Data Analysis
The statistical analysis of the data was performed using SAS ® v.9.2 (Statistical Analysis). SAS LAB procedure was used to verify data adequacy to the linear model and then the variables obtained were submitted to analysis of variance using the PROC GLM.

Results and Discussion
In relation to bioactive molecules analyzed in DJCM (Table 2), phorbol esters content (0.06 mg/g) is lower than that found for Makkar et al. (1997), who evaluated the content of these in nontoxic to the animals varieties (0.11 mg/g), by Rakshit et al. (2008) evaluating DJCM in diet for rats (0.2 mg/g). Chivandi et al. (2006), evaluating the effect of different methods of detoxification in the Jatropha curcas meal with 0.8 mg/g of phorbol esters, observed significant losses in pigs blood parameters. The phorbol esters mimic the Diacylglycerol (DAG) action, inactivating the protein kinase C, which regulates different pathways signal of translation and other metabolic activities by acting as co-carcinogen and causing cellular and biochemical effects in many different animal species (Goel et al., 2007) and studied extensively in different animal models like goats, sheep, mice, rats and fish when fed with phorbol estercontaining feeds (Adam and Magzoub, 1975;Makkar et al., 1998). The phorbol esters content depends on the oil residue present on pie or meal after the processing (Rakshit et al., 2008), which justifies the values obtained in this study, since this is a specific research and the oil was almost completely extracted to obtain the meal and this passed over by the detoxification process.
The total phenols and total tannins values are similar to those reported by Makkar et al. (1998), but the content of condensed tannins is different from those observed by the same authors, that evaluating toxic and nontoxic varieties of Jatropha curcas not found condensed tannins in both varieties. It is important noting that there are factors that affect the tannin concentration in plants, among which stand out climate, plant mineral nutrition and growth stage (Waterman and Mole, 1994).
Tannins can affect negatively the feed intake, digestibility and the performance of animals. These effects depend greatly on the amount, the tannin type ingested and the animal tolerance, being more prominent in non-ruminants. Levels above 1% of condensed tannins in the diet, well above the values obtained in this study, affect mainly the intake, digestibility of protein and of the essential amino acids (McDonald et al., 1995).
The saponins content observed (0.005%) is similar to those reported by Makkar et al. (1998) assessed the nutritional content of seeds and meal of detoxified Jatropha curcas from non-toxic varieties in Mexico and even to those observed by Belewu and Sam (2010) who evaluated the content of different bioactive molecules of the DJCM by different microorganism's strains. The saponins are glycosides present in most part of the diets ingredients for animals. Is characterized by a bitter taste, capacity to form foam in aqueous solution, cause hemolysis and, also, of complexing in steroid. Moreover, their antinutritional effects also are related to changes in the permeability of the intestinal mucosa, inhibiting the transport of some nutrients, facilitating the absorption of other compounds (Francis et al., 2001).
The CE of DJCM determined in this study was 5,900 kcal/kg and the apparent digestibility coefficient of energy was 59.6%, from the value of digestible apparent energy of 3,500 kcal/kg (Table 4). Thus, DJCM can be considered an excellent source of energy, since the digestible energy value (3,500 kcal/kg) is similar to corn (Rostagno et al., 2005) and other cereals considered energy sources, such as the low tannin sorghum, triticale and the wheat meal, which have average digestible energy values of 3,794 (Ferreira et al., 1997), 3,236 (Furlan et al., 1999;Radecki and Miller, 1990) and 3,300 kcal/kg (Lima and Viola, 2001), respectively.
Regarding the CP content and their digestibility, value of 51.1% were observed, result considered excellent for protein sources. However, the apparent digestibility coefficient was only 26.3% with digestible protein value of 13.5%. These values are inferior when compared with digestible protein values of other oilseed meals considered as protein sources and oil source for biodiesel production.
In Turkey, Boguhn et al. (2010) evaluated the digestibility of DJCM amino acids and concluded that for young turkeys is higher compared with other ingredients to animals of the same species, therefore, can use it as an ingredient in diet.
In addition to soybean meal, worldwide used as ingredient protein in the animal diets, some foods considered by-products, such as cotton meal, peanut meal and canola meal, have respectively 25, 44 and 26% of digestible protein for pigs (Rostagno et al., 2005).    Thus, it's evident that DJCM, while possessing high level of crude protein in its composition, this nutrient is hardly been tapped by the pigs. Probably, the presence of bioactive molecules, such as protease inhibitors, saponins, tannins and phytates impaired the nutrient digestibility, which shows that the meal used in this study wasn't fully detoxified. Another possible explanation for the low crude protein digestibility coefficient observed in this study, may be due to low inclusion of the DJCM in the diet due low acceptance by the animals.
Regarding the crude fiber content, the value of 4.3% was observed with apparent digestibility coefficient of 34.5% with the digestible fiber value of 1.1%. It's verified that the DJCM has lower content of crude fiber and digestibility higher when compared to the values obtained in cotton meal (25%) (Rostagno et al., 2005). In relation to the ether extract content, the value of 21.9% were observed with the digestibility apparent coefficient of 10.2%, providing the digestible ether extract value of 2.2%. The nutritional value of some foods depends mainly on the content, nutrients biological availability and further the presence and levels of toxic substances or bioactive molecules, which can alter this composition or become unavailable some of these nutrients (Pezzato, 1995).
The mean values of volumetric cumulative production per gram of biogas and methane in total solids are shown in Table 5. The production curves of biogas and methane (mL/g of total solids) by the pigs manure, well as the accumulated production of biogas and methane (mL) are shown in the graphs 1 and 2, respectively.
In studies of Miranda (2005) average potentials of biogas production of 0.489; 0.566 and 0.768 m 3 /kg of Total Solids (TS) were verified in biodigesters supplied with excretas of pigs, subjected to temperature 35°C and Retention Hydraulic Time (RHT) of 30 days, these results are lower than those found in this study.
The biogas (1201.4; 1089.3 mL/g of total solids) and methane (246.7 and 218.2 mL/g of total solids) production from the finishing pigs manure fed with reference diet and diet containing DJCM are compatible with those observed by Souza et al. (2005), evaluated different retention hydraulic times and incubation temperatures of pigs manure. The methane proportion in relative to the biogas generated was of 20.5 and 20.35% for the treatments RD and RD8%, values well below of 60 to 70%, theorized by Miranda (2009) as normal. This happened, probably, due to the low retention hydraulic time (7 days) exercised in this study, corroborating Lucas Junior (1994) that assessing the effects of retention hydraulic times of 50, 30, 20 and 15 days, in the case of bio digestion anaerobic of pigs manure, found that the highest efficiency is obtained at RHT of 30 days, with worst use of the manure in the RHT of 15 days.

Conclusion
Detoxified Jatropha curcas Meal (DJCM) can be used in finishing pig diets without affecting the generation of renewable energy by its manure. Although the DCJM has shown low apparent digestibility of the nutrients for pigs, the biogas and methane generation wasn't compromised.