REGULAR ARTICLES

Nutrient intake, digestibility, and blood metabolites of goatsfed diets containing processed jatropha meal

Shrikant Katole & Subodh Kumar Saha & Asit Das &

Vadali Rama Bhadra Sastry & Munna Haridas Lade &

Bhukya Prakash

Accepted: 11 March 2013# Springer Science+Business Media Dordrecht 2013

Abstract This study was conducted to record the idealsource and level of alkali treatment to treat jatropha meal(JM) and to determine the effect of inclusion of variouslyprocessed JM (pJM) on nutrient intake, digestibility, bloodmetabolites and hormonal status in goats. The JM wastreated with 10 g/kg sodium chloride and 5 g/kg calciumhydroxide. The content of phorbol ester and hemagglutina-tion (HA) activity of JM and pJM were assessed. A feedingtrial for 90 days was conducted in short-haired multipurposegoats (n=15; five per group). The experimental animalswere offered oat (Avena sativa) straw ad libitum throughoutthe experimental period of 90 days. Each group wasassigned to one of the three diets, viz. R1—soybean meal,R2—sodium chloride (10 g/kg dry matter, DM), and R3—calcium hydroxide (5 g/kg DM), with pJM substituting250 g/kg DM of crude protein (CP) of control (R1). At theend of the feeding period, digestion trial of 7 days wasconducted. Blood samples were collected at the end of theexperimental period to assess the blood metabolites andhormonal status. The phorbol ester and HA activity werereduced considerably in pJM. The intake of DM, organicmatter, CP, and nitrogen-free extract were comparableamong all the groups. However, the intake of ether extractwas significantly higher in pJM-fed groups. The

hemoglobin, packed cell volume, serum urea, triiodothyro-nine and testosterone contents decreased in R2 and R3 ascompared to R1. Concentration of glucose and activity ofserum glutamic pyruvic transaminase and lactate dehydro-genase increased (P<0.01) in goats fed pJM. It was con-cluded that phorbol ester content and HA activity markedlydecreased by processing JM with sodium chloride and cal-cium hydroxide. However, they were not reduced to thelevels of safe feeding, as reflected in unusual values ofblood metabolites among the experimental animals.

Keywords Jatropha (Jatropha curcas) meal . Processing .

Nutrient intake . Blood metabolites . Goats

AbbreviationsCP Crude proteinDM Dry matterHA HemagglutinationJM Jatropha mealLDH Lactate dehydrogenaseOM Organic matterPCV Packed cell volumepJM Processed JMSGOT Serum glutamic oxaloacetic transaminaseSGPT Serum glutamic pyruvic transaminase

Introduction

Lack of feedstuffs for livestock in terms of concentrates androughages is a major problem throughout the globe, whichcauses inadequate supply of nutrients to the animals,resulting in suboptimal livestock production and makinganimal rearing an extravagant activity in the tropical

S. Katole : S. K. Saha :A. Das :V. R. B. Sastry :M. H. LadeCentre of Advanced Studies in Animal Nutrition, Indian VeterinaryResearch Institute, Izatnagar 243 122 Uttar Pradesh, India

B. Prakash (*)Project Directorate on Poultry, Indian Council of AgriculturalResearch, Rajendranagar,Hyderabad 500030 Andhra Pradesh, Indiae-mail: drbhukyaprakash@gmail.com

Trop Anim Health ProdDOI 10.1007/s11250-013-0400-9

countries. Therefore, animal nutritionists are in constantsearch of alternate feeds for feeding the animals.

Jatropha is a drought-resistant plant and can be grown oninfertile land (Moore et al. 2011). This is extensively plantedin Central and South America, Africa, and Southeast Asia(Al-Masri 2003). Due to the importance of biodiesel pro-duction from the jatropha seeds, its cultivation is increasingday-by-day in many countries including India, resulting inavailability of oil cake in sufficient quantity (Katole et al.2011). Jatropha meal (JM) is rich in protein (244 g/kg drymatter, DM) and has many medicinal values (Li et al. 2005;Makkar et al. 2008; Abdel-Shafy et al. 2011). However, JMcontains anti-nutritional factors such as phorbol ester,curcin, and trypsin inhibitor (Makkar and Becker 1999),which are toxic to animals. The literature indicates thatfeeding of raw JM is fatal for livestock (Ahmed and Adam1979; Makkar and Becker 2009). The major toxic com-pounds present in JM are curcin and phorbol ester (Rakshitet al. 2008), which restrict its use as a protein-rich feedsubstitute for livestock feeding. The trypsin inhibitor andcurcin present in jatropha can be minimized by heat treat-ment (Aderibigbe et al. 1997). However, phorbol ester pres-ent in jatropha is heat-stable and cannot be minimized byheat treatment. Nevertheless, it is possible to reduce itsconcentration by certain chemical treatments (Makkar andBecker 1997). The curcin, a toxic lectin has proteolytic andhemagglutination (HA) activity on red blood cells (Bolleyand Holmes 1958). Therefore, HA test can be performed forscreening of curcin content in variously processed JM(pJM). Chemical treatment such as sodium hydroxide andsodium hypochlorite along with heat treatment decreasedthe anti-nutritional factors in JM (Haas and Mittelbach2000). More information regarding toxic factors present inJM is available (Makkar and Becker 1999). However, littleinformation is available on the effect of feeding pJM tolivestock. Hence, the present study was aimed to detectresidual toxin present in pJM by comparative qualitativetest and to observe the effect of inclusion of pJM on nutrient

intake, digestibility, blood metabolites, and hormonal statusin goats.

Materials and methods

Chemical trial, HA test, and phorbol ester estimation

Solvent extracted JM was procured from Ayurvet Ltd, NewDelhi, India, and subjected to various chemical processingmethods separately to reduce its toxin content. The JM wastreated with various levels of sodium chloride (5-, 10-, 20-,and 25 g/kg DM), sodium hydroxide (2, 5, and 10 g/kg DM),calcium hydroxide (2.5, 5, and 10 g/kg DM), and urea (10, 20,and 30 g/kg DM) to reduce its toxin content. All the chemicalsused for processing of JM were procured from Merck Speci-alities Pvt. Ltd, Mumbai. The JM was also subjected tovarious physical processes like soaking (12 h) and roasting(100 °C for 30 min). Furthermore, the chemical and physicalprocessing methods were separately employed and not incombination.

HA test (Akande and Odunsi 2012) was performed toscreen for toxins present in JM using the RBCs of chicken,goat, sheep, guinea pig, and rabbit. HA activity of theextract of JM and pJM was analyzed based on the matrixformation and was expressed as reciprocal of end point ofdilution.

Phorbol-12-myristate-13-acetate (Calbiochem Ltd.,KGaA, Darmstadt, Germany) was used as standard forestimation of phorbol ester in JM using the HPLC(Shimadzu 10 A; Kyoto, Japan) method (Makkar et al.1997). Stock solution (1 mg/mL) was prepared by dissolv-ing pure standard in tetrahydrofuran and was stored at 4 °C.Working solution was prepared for various concentrations(10–500 μg/mL) of phorbol-12-myristate-13-acetate. Thephorbol ester concentration in JM and pJM was calculated(in milligram per kilogram DM) by comparing peak area ofeach standard and sample as follows.

Phorbol ester mg kg=ð Þ ¼ Area of sample� concentration of standard� final volume

Area of standard� weight of sample

Feeding trial

Fifteen short-haired multipurpose goats of about 1 year ofage with mean body weight 19±4 kg were used for thisstudy. They were randomly divided into three equal groupsof five and were housed in a well-ventilated shed withprovision for individual feeding, watering, and collectionof feces and urine. Prior to the experiment, all the animalswere dewormed with a 7.5-mg/kg body weight of levamisol

hydrochloride (Lemasol-75; Ranbaxy Laboratories, NewDelhi, India) administered subcutaneously in order to con-trol internal parasites. To control external parasites, Butox(Intervet, India Ltd., Pune) 2 mL/L water was sprayed allover the body of all experimental animals.

Based on the results of HA test and phorbol ester content,JM processed with 10 g/kg DM sodium chloride and 5 g/kgDM calcium hydroxide was selected for experimental trial(90 days) in goats. Three different concentrate mixtures

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were computed, viz. control (R1) and two test diets (R2 andR3). The R1 contained soybean meal, R2 contained sodiumchloride (10 g/kg) treated pJM, and R3 contained calciumhydroxide (5 g/kg) treated pJM. The pJM in R2 and R3

replaced 250 g/kg protein of control (R1), with an adjust-ment of the other ingredients. The concentrate mixtureswere made iso-nitrogenous and offered to the respectivegroups to meet the crude protein (CP) requirement as perthe NRC (1981). All the animals were offered oat (Avenasativa) straw ad libitum throughout the experimental period.Green tree leaves were provided once a week to meetvitamin A requirement. Leftover feed was weighed individ-ually next day morning at 9.00 a.m. to record the daily DMintake. The animals were offered ad libitum water in themorning and evening.

Digestibility study

At the end of the 90-day feeding trial, a digestion trialof a 7-day collection period was conducted duringwhich all feed, feces, and urine samples were collecteddaily, composited, and stored for laboratory analysis.The composite dried feed samples, refusal, and fecalsamples were pooled for 7 days, ground to pass througha 1-mm screen, and preserved for chemical analysis.The samples of feed and feces were analyzed for DMafter drying at 100 °C for 24 h. The contents of CP,ether extract, and ash were analyzed according to themethods of AOAC (1990).

Blood metabolite and hormonal status

About 4 mL of blood was collected in a sterile test tube fromall the animals at the end of experimental feeding. Theserum was separated and stored at −20 °C until furtheranalysis. Equal volume of blood was also collected in vialscontaining anticoagulant for estimation of packed cell volume(PCV) by capillary tubes and Hb by cyanmethemoglobinmethod (Decie and Lewis 1968). Serum samples were assayedfor glucose, total proteins, albumin, globulin and urea, andenzyme activities, like serum glutamic pyruvic transaminase(SGPT), serum glutamic oxaloacetic transaminase (SGOT),and lactate dehydrogenase (LDH) using commercial di-agnostic kits (Span Diagnostics Pvt. Ltd. Bombay, India).Testosterone, triiodothyronine, and thyroxine were assayedusing a radioimmunoassay kit (Immunotech, Marseille,France).

All the statistical analyses were performed using SPSSsoftware package, version 10 (SPSS, Chicago, IL, USA).The mean values were calculated using the descriptive sta-tistics. The variations in different recorded parameters wereanalyzed using the multivariate general linear model proce-dure. The model included different dietary treatments as

source of variation. Treatment means were separated byTukey’s test.

Results and discussion

Chemical treatment, HA test, and phorbol esterconcentration

Ingredient and nutrient composition of the experimental ra-tions, oat straw and JM, is presented in Table 1. The nutrientcomposition of JM is comparable with the reported values ofAnandan et al. (2005). In the present study, HA activity waslower in RBC of sheep, and goat in comparison to RBC ofchicken, rabbit, and guinea pig (Table 2). Highest HA activitywas observed in rabbit RBC. Among the different methods ofprocessing tried, soaking and roasting of JM was found to bethe most ineffective in reducing the HA activity on RBC ofchicken and rabbit. Processing the JM with NaCl (10 and20 g/kg DM) and Ca(OH)2 (5 and 10 g/kg DM) showedsimilar HA activity on sheep and goat RBC. Furthermore,JM processed with NaOH and urea was also able to reduceHA activity, but the amount of chemicals required to achievethis response was higher as compared to those in JMprocessed with NaCl and Ca(OH)2. Therefore, JM processedwith 10 g/kg NaCl and 5 g/kg Ca(OH)2 was selected forfurther in vivo study in goats. The JM contains curcin, a lectinsimilar to the ricin of castor (Ricinus communis) bean alongwith other toxicants, which might be the reason for maximumHA activity in the raw JM (Bolley and Holmes 1958).

Table 1 Ingredient and nutrient composition (in gram per kilogramDM) of the experimental rations, oat straw (OS) and jatropha meal(JM)

R1 R2 R3 OS JM

Ingredient compositiona

Maize 320 310 310 – –

Wheat bran 340 311 310 – –

Rice bran 110 100 100 – –

Soybean meal 200 150 150 – –

Jatropha meal 00 100 100 – –

Mineral mixture 20 20 20 – –

Salt 10 10 10 – –

Nutrient composition

Organic matter 900 907 903 902 931

Crude protein 200 195 196 26 237

Ether extract 27 32 31 8 106

Ash 100 93 97 868 69

DM dry mattera Vitablend (AD3) containing vitamins A (50,000 IU) and D3

(5,000 IU/g) was added at a 6-g/kg concentrate mixture

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However, pJM showed reduced HA activity, which might bedue to the decreased curcin concentration in NaCl- andCa(OH)2-treated JM (Anandan et al. 2005). Among the vari-ous chemical methods employed, processing JM with 10 g/kgDM sodium chloride and 5 g/kg DM calcium hydroxide wasfound promising and equally effective in reducing the toxinsin JM (Table 3). Phorbol ester concentration was decreased inpJM, which might be attributed to the alkaline condition thatresulted from the processing with sodium chloride and calci-um hydroxide (Makkar and Becker 1997).

Feeding and digestibility trial

The intake of DM, organic matter (OM,) and CP didnot differ among the experimental animals (Table 4).However, intake of these nutrients was numericallyhigher in groups fed R2 and R3 compared to R1. Theapparent digestibility of DM, OM, and CP did not differamong the groups. Intake of digestible CP, total digest-ible nutrient, and N were comparable among all thedietary groups. The marginal increase in nutrient intakein R2 group could be attributed to lowering of toxinconcentration as a result of NaCl treatment, whichmight have probably improved palatability. Furthermore,the taste buds in goats are reported to be ill developed,and goats are believed to enjoy a wide variety of bitterplants that are distasteful to other animals (Jindal 1984).So far, little information is available on feeding of JMto goats. However, feeding of pJM reduced feed intake

in sheep (Katole et al. 2011). The lower intake ofjatropha-supplemented diet might be due to the phorbol

Table 2 Variation in hemagglu-tination (HA) titres of RBC ofdifferent species

Different lowercase letters in acolumn differ significantly(P<0.05)

JM jatropha meal, Ca(OH)2 cal-cium hydroxide, NaCl sodiumchloride, NaOH sodiumhydroxideaValues represent assays of fivesamples

Samples HA unitsa

Chicken Rabbit Guinea pig Sheep Goat

Control 0 e 0 c 0 e 0 d 0 c

Raw JM 4,096 a 4,096 a 4,096 a 4,096 a 4,096 a

Soaked JM 1,024 b 1,024 b 128 b 128 b 64 b

Roasted JM 1,024 b 1,024 b 128 b 128 b 64 b

NaCl 10 g/kg JM 32 d 1,024 b 8 cd 2 d 4 c

NaCl 20 g/kg JM 16 de 4,096 a 4 e 2 d 4 c

NaCl 30 g/kg JM 16 de 4,096 a 8 cd 2 d 2 c

NaOH 2.5 g/kg JM 16 de 4,096 a 32 c 32 c 8 c

NaOH 5 g/kg JM 8 de 4,096 a 8 cd 16 cd 4 c

NaOH 10 g/kg JM 4 e 4,096 a 4 e 4 d 4 c

Ca(OH)2 2.5 g/kg JM 16 de 4,096 a 4 e 8 cd 8 c

Ca(OH)2 5 g/kg JM 8d e 4,096 a 4 e 4 d 4 c

Ca(OH)2 10 g/kg JM 4 e 4,096 a 4 e 4 d 2 c

Urea 10 g/kg JM 64 c 4,096 a 16 cd 8 cd 8 c

Urea 20 g/kg JM 32 d 4,096 a 8 cd 8 cd 8 c

Urea 30 g/kg JM 32 d 4,096 a 8 cd 8 cd 8 c

SEM 11.45 37.48 10.85 10.85 10.80

P value 0.01 0.01 0.01 0.01 0.01

Table 3 Variation in phorbol ester (in gram per kilogram dry matter)content in raw and processed jatropha meal (JM)

Treatments Phorbol estera %Decrease

Raw JM 2.14

NaCl 5 g/kg JM 0.40 81.3

NaCl 10 g/kg JM 0.32 85.0

NaCl 20 g/kg JM 0.32 85.2

NaCl 25 g/kg JM 0.31 85.6

NaOH 2.5 g/kg JM 0.45 79.0

NaOH 5 g/kg JM 0.45 79.2

NaOH 10 g/kg JM 0.47 78.1

NaOH 20 g/kg JM 0.47 78.2

NaOH 25 g/kg JM 0.45 79.2

Ca (OH)2 2.5 g/kg JM 0.36 83.2

Ca (OH)2 5 g/kg JM 0.36 83.2

Ca (OH)2 10 g/kg JM 0.36 83.2

Ca (OH)2 20 g/kg JM 0.34 83.9

Ca (OH)2 25 g/kg JM 0.36 83.4

Urea 10 g/kg JM 0.37 82.6

Urea 20 g/kg JM 0.37 82.7

Urea 30 g/kg JM 0.36 83.1

Ca(OH)2 calcium hydroxide, NaCl sodium chloride, NaOH sodiumhydroxidea Values represent assays of two samples

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ester and/or curcin (Aregheore et al. 2003; Katole et al.2011). The N excretion was higher (P<0.01) in groupsfed pJM-supplemented diets (R2 and R3). Similar in-crease in N excretion was also observed in sheep fedpJM-supplemented diets (Katole et al. 2011). Lowerintake and higher excretion of N could be attributed tothe anti-nutritional factors and purgative properties ofJM (Mampane et al. 2006).

Blood metabolite and hormonal status

Serum concentration of total protein, albumin, and glob-ulin did not differ among the groups. Blood Hb andPCV and serum concentration of urea were lower (P<0.01) in animals fed pJM-supplemented diets as comparedto those fed the control diet (Table 5). Similar resultsare also reported in rat (Oluwole and Bolarinwa 1997),

Table 4 Nutrient intake(in gram per day), digestibility,and nutritive value (in gram perkilogram dry matter) and nitro-gen (in gram per day) utilizationin goats fed diets supplementedwith processed jatropha meal

Means in the same row withdifferent lowercase letters differ(P<0.05)

DCP digestible crude protein,TDN total digestible nutrients,DM dry matter

Particulars R1 R2 R3 SEM P value

Nutrient intake

Dry matter 405 452 388 17.0 0.25

Organic matter 377 423 339 17.2 0.13

Crude protein 40.9 48.3 49.3 1.93 0.16

Ether extract 6.46 b 8.36 a 8.67 a 0.37 0.02

Nitrogen-free extract 189 212 187 7.71 0.37

Digestibility

Dry matter 600 591 583 13.9 0.90

Organic matter 633 612 609 13.4 0.75

Crude protein 615 599 571 16.2 0.56

Ether extract 734 b 805 a 808 a 13.4 0.02

Nitrogen-free extract 683 a 620 ab 605 b 15.6 0.08

Nutritive value

DCP 69.5 64.8 67.5 2.45 0.76

TDN 604 592 562 14.4 0.51

Nitrogen intake 6.55 7.72 7.88 0.31 0.15

N in feces 1.53 a 1.66 b 2.43 a 0.10 0.01

N in urine 1.83 2.15 1.78 0.13 0.53

Retained 3.17 3.90 3.66 0.34 0.71

Table 5 Variation in blood me-tabolites in goats fed dietssupplemented with processedjatropha meal

Means in the same row withdifferent lowercase letters differ(P<0.05)

BUN blood urea nitrogen, Hbhemoglobin, LDH lactate dehy-drogenase, PCV packed cellvolume, SGOT serum glutamicoxaloacetic transaminase, SGPTserum glutamic pyruvic trans-aminase, TP total protein, T3 tri-iodothyronine, T4 Thyroxine

Parameters R1 R2 R3 SEM P value

Hb, g/dl 11.3 a 9.15 b 8.15 b 0.45 0.01

PCV, % 46.6 a 38.6 c 41.9 b 0.92 0.01

Serum TP, g/dl 6.56 6.97 6.24 0.13 0.07

Albumin, g/dl 3.20 3.03 3.04 0.07 0.56

Globulin, g/dl 3.36 3.95 3.20 0.15 0.11

Glucose 12.2 b 20.7 ab 38.5 a 3.82 0.01

BUN, g/dl 28.4 a 21.4 b 16.5 c 1.14 0.01

Urea, g/dl 60.6 a 45.7 b 35.3 c 3.01 0.01

Enzymes

SGPT, IU/L 225 b 237 b 252 a 3.43 0.01

SGOT, IU/L 81.1 b 84.4 ab 93.4 a 2.40 0.09

LDH, IU/L 621 c 4,131 b 4,596 a 474 0.01

Hormones

Testosterone, ng/mL 0.11 a 0.03 b 0.02 b 0.02 0.03

T3, nmol/L 2.05 a 1.88 a 0.94 b 0.17 0.01

T4, nmol/L 85.6 b 114 a 97.6 ab 4.67 0.02

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goats (Belewu and Ogunsola 2010), and sheep (Katoleet al. 2011). Feeding of pJM resulted in increased (P<0.01) serum concentration of SGPT, SGOT, and LDH.Increased activities of these enzymes as observed inthe present study might be due to the presence ofresidual toxin in pJM. Hepatotoxins present in jatropha(Widaryanto 2012) may cause rapid disorganization ofhepatic tissue and increased cytosolic leakage of hepaticenzymes resulting in increased concentrations of theseenzymes in circulation (Petrie 1987; Dawson 1998).Similar increase in concentration of serum SGPT dueto feeding of JM had earlier been reported in goats(Gadir et al. 2003) and sheep (Katole et al. 2011).

Serum concentration of triiodothyronine and thyroxinwas lower (P<0.01) in goats fed pJM diets as comparedto those fed the control diet. Different toxins present injatropha are known to be cytotoxic (Devappaa et al.2010). The residual toxin of pJM might have damagedthe cell membrane of the thyroid gland, resulting indecreased synthesis of thyroid hormones. Many toxinsare reported to interfere in absorption of iodine andtyrosine (Cinar and Selcuk 2005). As these two nutri-ents are essential for synthesis of thyroid hormones,residual toxins of pJM may have induced the interfer-ence in their absorption, resulting in decreased serumconcentration of triiodothyronine and thyroxine. Similardecrease in serum concentration of thyroid hormoneswas also observed in rats fed diet containing ricin(Sadani et al. 1997). Serum concentration of testoster-one was lower (P<0.01) in goats fed pJM-supplementeddiets than those fed the control diet. Many toxins pres-ent in jatropha, e.g., phorbol ester, may cause degener-ative changes in a wide range of organs including thetestes (Awasthy et al. 2010).

The results of the present study indicate that feeding ofpJM had an adverse effect on the hematological and serumbiochemical parameters. Hemoglobin content of blood,PCV, and serum concentration of urea in goats fed pJM-supplemented diet was not within the normal range reportedfor the species (Kaneko 1989). Similarly, Katole et al.(2011) also reported that feeding of pJM to sheep resultedin abnormal values of blood metabolites.

Conclusions

The results of the study showed that processing JM with10 g/kg sodium chloride or 5 g/kg calcium hydroxide mark-edly reduced phorbol esters and HA activity. However,inclusion of pJM in the diets of goats decreased dietarynutrient intake and digestibility. It can therefore be conclud-ed that though the HA activity and phorbol ester contentshowed considerable reduction in pJM, it did not reach up to

the safe level of feeding for the goats, as its feeding reducednutrient intake and resulted in atypical values of bloodmetabolites.

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