Effect of partial or complete replacement of cottonseed meal by jojoba meal on gas production, rumen fermentation and produced amylase and carboxymethyl cellulase activity, in vitro

M E A W Nasser

Department of Animal Production, Faculty of Agriculture, Alexandria University, Alexandria, Egypt
menassero@yahoo.com

Abstract

An in vitro gas production technique was used to study the effect of jojoba meal on gas production, rumen fermentation, true dry and organic matter digestibility, the activity of amylase and carboxymethyl cellulase (CMC_ase) and protozoa count. Jojoba meal was added to a concentrate diet at 6%, 9% and 18% levels, substituting for cottonseed meal. A gas production technique was performed using rumen fluid collected from three fistulated Santa Ines sheep. Cumulative gas production was recorded at 3, 6, 9, 12, 24, 48, 72 and 96 h of incubation time. Kinetics of gas production was fitted to an exponential model. Volatile fatty acids (VFA), Ammonia-N (NH₃-N) concentrations, true dry and organic matter degradability and enzyme activity (CMCase and amylase) were determined at 24 h of incubation time.

The cumulative volume of gas production was increased by adding jojoba meal. Total gas produced at 96 h of incubation time was higher for the first level of jojoba meal. The values of iso-butyrate, butyrate and iso-valerate were significantly decreased for control sample compared to the third level of jojoba meal. No significant effects of jojoba meal on acetate, propionate and valerate concentration were seen. Also, no significant effects were observed on CH₄, NH₃-N levels or true dry and organic matter degradability at 24 h of incubation time. pH value was significantly decreased at  the third level of jojoba. No significant effects of jojoba meal were observed on the specific activity of amylase in the supernatant of sonicated bottle contents, while the specific activity of CMCase was significantly decreased. The lowest protozoa count was at the third level of jojoba meal.

Key words: enzyme assay, gas production, In vitro, jojoba meal, protozoa and rumen fermentation

Introduction

Agro-industrial and agricultural by-products can play an important role in animal production in developing countries. Jojoba (Simmondsia chinensis) is a native oilseed shrub being grown in the deserts or new lands, is being advocated and developed as a potential cultivated crop for warm, arid regions of the world (Hogan 1979). It produces highly marketable oil which is a unique mixture of unsaturated liquid wax esters (Spencer and Plattner 1984). The liquid wax (about 50% by weight) has characteristics similar to sperm whale oil (Verbiscar and Banigan 1978) and also, has applications in cosmetics, pharmaceuticals, and numerous other products. The residue (meal) that remains after extraction of oil from the seeds contains from 26 to 33% crude protein (Verbiscar and Banigan 1978; Verbiscar et al 1980; Nasser et al 2007) and would increase the economic value of this crop if it could be used as a feed ingredient. Practically, the meal is underutilized because it contains 11% anti-nutritional factors (ANF), 5-demethylsimmondsin (DMS), 4,5-didemethylsimmondsin (DDMS), simmondsin (S), and simmondsin 2'-ferulate (SF), that have adverse effects on animals (Elliger et al 1973; Verbiscar and Banigan 1978). In monogastric animals the ANF decompose on ingestion and apparently cause death by cyanide poisoning on the basis of reports of CN- and its metabolites in mice fed simmondsin (Weber et al 1983). Ruminants are somewhat more tolerant of the ANF but do not use the protein efficiently or gain weight well on unmodified meal substituted at a 10% level in a normal diet (Manos et al 1986).

There were very few studies with diets supplemented with jojoba meal conducted with broiler chicks (Ngou Ngoupayou et al 1981), rabbits (Ngou Ngoupayou et al 1985) and lambs (Verbiscar et al 1980, 1981 and Nasser et al 2007). There is no published information on the response of rumen microbes to diets containing jojoba meal. Therefore, the objective of this study to study the effects of replacing cotton seed meal with jojoba meal on in vitro gas production, fermentation and amylase and carboxymethyle cellulase activity.

Material and methods 

The present study was carried out at the Laboratory of Animal Nutrition (LANA), Center of Nuclear Energy in Agriculture (CENA), University of Sao Paulo (USP), Brazil.

Feedstuffs

Tifton-85 (Cynodon sp.) (H) and concentrate mixtures (C) were ground in mills to pass a 1 mm sieve prior to chemical analysis and in vitro gas production measurement. The ingredient and chemical composition of concentrate and hay mixtures are presented in Tables 1 and 2.

Preparation of inoculum

Rumen liquor was collected from three fistulated sheep fed on Coast cross hay and concentrate mixture. The rumen liquor was sampled just before feeding (0 h) and transported in insulated flasks under anaerobic conditions to the laboratory, combined, mixed and strained through four layers of surgical gauze and flushed with CO₂. The well mixed and CO₂ flushed rumen fluid was added to the buffered mineral solution (1:2 v/v), which was maintained in water bath at 39 ^oC and mixed.

In vitro gas production test

In vitro gas production technique was carried out using a pressure transducer and data logger for measuring the produced gas in 160 ml serum bottle incubated at 39 ^oC (Mauricio et al 1999). Ground samples (1 g DM) of mixture of H and C (70% H:30% C, w/w) were incubated with 75 ml of diluted rumen fluid (25 ml mixed rumen fluid + 50 ml of Menke’s buffered medium) into 160 ml serum bottle. The bottles were closed by rubber stoppers, shaken and placed in the incubator at 39 ^oC.  Four bottles with only buffered rumen fluid were incubated and considered as the blank. The gas headspace pressure was recorded before incubation (0) and 3, 6, 9, 12, 18, 24, 30, 36, 48, 72 and 96 h after incubation using a pressure transducer (Theodorou et al 1994). Total gas values were corrected for blank incubation and expressed as milliliter of gas produced per 200 mg of dry matter. Cumulative gas production data were fitted to the model of Ørskov and McDonald (1979). Four bottles containing 1 g samples and 75 ml Buffered rumen fluid were incubated for determination pH, ammonia nitrogen (NH₃-N), volatile fatty acids (VFA) concentrations, protozoa count, digested dry and organic matter and enzyme activity at 24h of incubation.

Estimation of methane production

After 12 and 24 h incubation, 5 ml gas was sampled from the headspace of bottle in a test tube for methane estimation. Methane estimation was carried out according to Patra et al (2006).

Enzyme assay

At 24 h of incubation time, the whole content of two bottles was transferred to a 100 ml beaker. The contents were carefully mixed then sonicated at 4 ^oC using a sonicator (Labsonic U model; B. Braun Biotech International). The sonicated samples were centrifuged at 24 000×g for 20 min at 4 ^oC and clear supernatant was used for the estimation of enzyme activities. The reaction mixture contained 0.5 ml phosphate buffer (0.1 M, pH 6.8), 0.250 ml carboxymethylcellulose (1.0 g/100 ml phosphate buffer) and 0.250 ml extracted supernatant for the estimation of carboxymethylcellulase (CMCase). For amylase activity, the reaction mixture contained 0.5 ml phosphate buffer, 0.250 ml corn starch (2 g/100 ml phosphate buffer) and 0.250 ml extracted supernatant. The reaction mixtures were incubated for 45 min (amylase) and 60 min (CMCase) at 39 ^oC. The reducing sugars thus released were estimated according to Somogyi method (1960) using glucose as standard.

Volatile fatty acid estimation

At the end of incubation (24 h) 1ml of the supernatant was collected in a microfuge tube containing 0.20 ml metaphosphoric acid (25 ml/100 ml). The mixture was allowed to stand for 2 h at room temperature and centrifuged at 5000×g for 10 min. The clear supernatant was collected and stored at −20 ^oC until analyzed. The VFA’s were measured by gas chromatography (ThermoQuest mod. 8000top, FUSED SILICA capillary column 30m×0.25mm×0.25mm film thickness) as described by Cottyn and Boucque (1968).

In vitro dry matter degradability

At the end of the incubation period (24 h), contents of each serum bottle were filtered through pre-weighed Gooch crucibles and residual dry matter was estimated.  The per cent loss in weight was determined and presented as IVDMD. The dried feed sample and residue left above was ashed at 550 ^oC for determination of IVOMD.

Protozoa counts

After termination of incubation, the contents of the bottle were mixed properly and 1 ml sample was mixed with 1 ml methyl green formalin saline solution (MFS). The stained sample was kept overnight and protozoa were counted microscopically following the procedure described by Dehority (2005).

Proximate analyses

The dry matter (DM), organic matter (OM), crude protein (CP, N×6.25), ether extract (EE), crude fiber (CF) and ash of substrates were determined by AOAC,995) procedures. Protein concentration of the crude enzymes (amylase and CMCase) was determined by the Bradford method (1976).

Statistical analyses

Data were subjected to analysis of variance (ANOVA) using the General Linear Model. Significant differences between individual means were identified using least significance difference (LSD) multiple range test (SAS 1999).

Results and discussion 

Effect on gas and methane production

The results of gas production as affected by different levels of jojoba meal are presented in Figure 1 and Table 3.

The cumulative volume of gas production increased with increasing time of incubation. Gas produced at 96 h incubation ranged between 45.03 and 48.10 ml per 0.2 g of substrate. The jojoba meal included in the substrates had significant effect (P<0.05) on gas production at 24 and 96 h of incubation time which indicated that the levels of jojoba meal used in the experiment were not detrimental for rumen microbes or that rumen microorganisms can destroy the toxic compounds of jojoba meal. Lactobacillus acidophilus and Lactobacillus bulgaricus were found to grow well on jojoba seed meal and reduce the levels of simmondsin and other cyano toxicants (Verbiscar et al 1981). By 24 h of incubation the total volume of gas produced was different for the different substrates. The volume of gas production for control was significantly (P< 0.05) lower than those for both level 1 and 2 of jojoba meal at 24, 48, 72 and 96 h incubation; but there was no significant (P >0.05) difference between both control and level 3 of jojoba meal  at 72 and 96 h of incubation. Values for the estimated parameters obtained from the kinetic models of Ørskov and McDonald (1979) are given in Table 3. The values of the soluble fractions (a) were 0.54, 3.21, 1.8 and 0.87 ml for control, level 1, level 2 and level 3 of jojoba meal, respectively. The gas production of soluble fraction (a) was significantly (P<0.05) different between substrates. There was no significant (P<0.05) difference between all substrates of insoluble fraction (b) (Table 3). There were significant (P<0.05) differences between control and level1, level 2 and level 3 of jojoba meal of the gas production rate (c) (Table 3). The jojoba meal did not show significant effect (P>0.05) on methane emission (Table 4).

Also, there was no correlation in the amount of methane produced per unit of DMD (Table 4). The results appear to indicate that jojoba meal is not effective against methanogenesis.

Effect on protozoa

The effects of jojoba meal on protozoa counts in in vitro gas production test are presented in Table 4. High level of jojoba meal (L3) resulted in a significant reduction in protozoa count (P<0.05), while other levels (L₁ and L₂) had no significant effect on total protozoal counts (P > 0.05). Although, total protozoa counts reduced significantly (P<0.05) in the third level of jojoba meal the percentage of methane in total gas produced did not differ between all substrates at 24 h of incubation time (Table 4). The results indicated that methane emission is not essentially associated with protozoa activity. The results are in agreement with Kamra et al (2008) and Ranilla et al (2007). Methanogenesis is not essentially related to the density of protozoa population in the rumen (Patra et al 2006). According to Newbold et al (1997) and Hess et al (2003) only a small portion of total methane production is due to the presence of methanogens attached with the ciliate protozoa. Dohme et al (1999) also reported inhibition of in vitro methane emission both in defaunated and faunated rumen liquor with coconut oil. Machmuller et al (2003) demonstrated an increased number of methanogens in defaunated sheep, and suggested that association between protozoa and methanogens does not play an important role in methanogenesis in rumen. The presence of the other protozoal species different than Entodinium caudatum, Eudiplodinium maggii and Isotricha intestinalis did not increase production of methane – either absolute or relative to the amount of substrate degraded – in these fermentors with the faunated supernatant (Ranilla et al 2007).

Effect on pH, ammonia nitrogen and degradability of feed

The values of pH, ammonia-N concentration (NH₃-N) and true dry matter and organic matter degradability at 24 h of incubation time are presented in Table 4. The average values of pH ranged from 6.60 to 6.70 at 24 h of incubation time. pH value was decreased significantly (P<0.05) at  the third level of jojoba meal compared to that of control, level 1 and 2 of jojoba meal. Nasser et al (2007) suggested that there were no significant differences in the ruminal pH of lambs, at 1, 3 and 6 h after feeding, fed rations contained jojoba meal. Although, jojoba meal contains 11 % ANF that have adverse effects on animals the present results showed that there were no significant (P > 0.05) differences among all substrates in NH₃-N levels or true degradability of dry (TDMD) and organic matter (TOMD) at 24 h of incubation time (Table 4). The levels of jojoba meal used in the experiment may be not detrimental for rumen microbes or rumen microorganisms can destroy the toxic compounds of jojoba meal. The results of NH₃-N are in agreement with Nasser et al (2007) who found that there were no significant differences at zero time and 1 h after feeding.

Effect on VFA

The TVFA concentration (mM) was significantly (P<0.05) increased when jojoba meal were added at L2 and L3 but not in L1 (P > 0.05) (Table 5).

The average acetate and propionate concentrations for all substrates which contain jojoba meal were higher than control although not statistically significant. Compared to the value in the control, concentration of butyrate and isovalerate were significantly increased at L2 and L3.  The value for iso-butyrate of L3 was significantly increased (P < 0:05) compared to control. A concentration (mM) of valerate was similar among the substrates tested. The reduced protozoa numbers is sometimes associated with increase in propionate (per cent) and decrease in A:P ratio (Hess et al 2003; Machmuller et al 2003). According to Jouany et al (1988) changes in the VFA pattern due to reduction in protozoa population is not always consistent because nature of diet also plays an important role in VFA pattern.

Effect on enzyme profile

The effects of jojoba meal on enzyme activities are presented in Table 6.

The specific activities of amylase were not affected by any level of jojoba meal, whereas specific activity of CMCase was reduced significantly (P<0.05) at L2 and L3. The numerically lower activities in the presence high levels of jojoba meal on CMCase might be due to its antiprotozoal activity, as it has been reported that about 38% of cellulase activity is associated with protozoa fraction of rumen liquor (Agarwal et al 1991).

Conclusions 

• The results of this experiment indicate that replacement of cotton seed meal by the cheap protein source (jojoba meal) does not adversely affect rumen fermentation and appears nontoxic for rumen microbes.

  
  

• Jojoba meal appears to have defaunating properties.

  
  

• However, further studies are needed to evaluate the nutritive value and biological value of jojoba meal as a protein source for different species of ruminants.

Acknowledgements 

The author would like to express his deepest thanks to Profs Dorinha M.S.S. Vitti and Adibe L. Abdalla, Laboratory of Animal Nutrition (LANA), Center of Nuclear Energy in Agriculture (CENA), University of Sao Paulo (USP), Brazil for their help and useful discussion.

References 

Agarwal N, Kewalramani N, Kamra D N, Agarwal D K and Nath K 1991 Hydrolytic enzymes of buffalo rumen: comparison of cell free fluid, bacterial and protozoal fractions. Buffalo Journal 7: 203–207

AOAC (Association of Official Analytical Chemists) 1995 Official Methods of Analysis, 16th edition; AOAC: Arlington, VA, USA.

Bradford M M 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254

Cottyn B G and Boucque C V 1968 Rapid method for the gas-chromatographic determination of volatile fatty acids in rumen fluid. Journal of Agriculture and Food Chemistry 16: 105–107

Dehority B A 2005 Ciliate protozoa. In Methods in Gut Microbial Ecology for Ruminants, edited by H P S Makker and C S Mc Sweeney, IAEA, pp 67-78  http://books.google.fr/books?id=cfsXuJCewf8C&pg=PA67&lpg=PA67&dq=Ciliate+protozoa.+In+Methods+in+Gut+Microbial+Ecology+for+Ruminants&source=bl&ots=F3J2Lo4eFa&sig=lWA5JPpASJclGW4Z6vEBSUQJRXc&hl=fr&ei=gTvrSdm6L5yZjAfDo5GfCg&sa=X&oi=book_result&ct=result&resnum=1#PPA66,M1

Dohme F, Machmuller A, Estermann B L, Pfister P, Wasserfallen A and Kreuzer M 1999 The role of the rumen ciliate protozoa for methane suppression caused by coconut oil. Letter in Applied Microbiology 29: 187–192

Elliger C A, Waise A C and Lundin R E 1973 Simmondsin, an unusual 2-cyanomethylenecyclohexyl glucoside from Simmondsia californica. Journal of Chemical Society, Perkin Transactions 1: 2209-2212

Hess H D, Kreuzer M, Diaz T E, Lascano C E, Carulla J E and Solvia C R 2003 Saponin rich tropical fruits affect  fermentation and methanogenesis in faunated and defaunated fluid. Animal Feed Science and Technology 109: 79–94

Hogan L 1979 Jojoba: A new crop for arid regions. In: G. A. Richie (Ed.) New Agricultural Crops, AAAS Selected Symposium 38. pp 177-205. Western Press, Boulder, CO.

Jouany J P, Demeyer D I and Grain J 1988 Effect of defaunating the rumen. Animal Feed Science and Technology 21: 229–265

Kamra D N, Patra A K, Chatterjee P N, Kumar R B, Agarwal N A and Chaudhary L C 2008 Effect of plant extracts on methanogenesis and microbial profile of the rumen of buffalo: a brief overview. Australian Journal of Experimental Agriculture 48(2): 175–178

Machmuller A, Soliva C R and Kreuzer M 2003 Methane suppressing effect of myristic acid in sheep as affected by dietary calcium and forage proportion. British Journal Nutrition 90: 529–540 http://journals.cambridge.org/download.php?file=%2FBJN%2FBJN90_03%2FS0007114503001570a.pdf&code=780c207a2f2d2f6d1e3a1a9fe14560df

Manos C G, Schrynemeeckers P J, Hogue D E, Telford J N, Stoewsand G S, Beerman D H, Babish J G, Blue J T, Shane B S and Lisk D J 1986 Toxicologic studies with lambs fed jojoba meal supplemented rations. Journal of Agriculture and Food Chemistry 34: 801-805

Mauricio R M, Mould F L, Dhanoa M S, Owen E, Kulwant C S and Theodorou M K 1999 A semi-automated in vitro gas production technique for ruminant feedstuff evaluation. Animal Feed Science and Technology 79: 321– 330

Nasser M E A, El-Waziry A M, Sallam S M A and Mahmoud S A S 2007 Evaluation of jojoba meal as protein source for sheep. Agricultural Research Journal, Suez Canal University 7(3): 1-7

Newbold C J, Hassan S M E, Wang J, Ortega M E and Wallace R J 1997 Influence of foliage from African multipurpose trees on activity of rumen protozoa and bacteria. British Journal Nutrition 78: 237–249 http://journals.cambridge.org/download.php?file=%2FBJN%2FBJN78_02%2FS000711459700130Xa.pdf&code=89c2e3c46e4986385e74d9e8bd98cb77

Ngou Ngoupayou J D, Maiorino P M and Reid B L 1981 Jojoba meal in Poultry diets. Poultry Science 61:1692-1696

Ngou Ngoupayou J D, Maiorino P M, Schurg W A and Reid B L 1985 Jojoba meal in rabbit diets. Nutrition Reports International 31 (1): 11-19

Ørskov E R and McDonald I 1979 The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. Journal of Agricultural Science 92: 499

Patra A K, Kamra D N and Agarwal N 2006 Effect of plant extracts on in vitro methanogenesis, enzyme activities and fermentation of feed in rumen liquor of buffalo. Animal Feed Science and Technology 128: 276–291

Ranilla M J, Jouany J P and Morgavi D P 2007 Methane production and substrate degradation by rumen microbial communities containing single protozoal species in vitro. Letters in Applied Microbiology 45: 675–680

SAS 1999 SAS users guide statistical analysis systems institute. Cary, USA.

Somogyi M 1960 Modifications of two methods for the assay of amylase. Clinical Chemistry 6:  23-35

Spencer G F and Plattner R D 1984 Compositional analysis of natural wax ester mixtures by tandem mass spectroscopy. Journal of American Oil Chemistry SOC., 61: 90-94

Theodorou M K, Williams B A, Dhanoa M S, McAllan A B and France J 1994 A simple gas production method using a pressure transducer to determine the fermentation kinetics of ruminant feeds. Animal Feed Science and Technology 74: 3583–3597

Verbiscar A J and Banigan T F 1978 Composition of jojoba seeds and foliage. Journal of Agriculture Food Chemistry 26: 1456-1459

Verbiscar A J, Banigan T F, Weber C W, Reid B L, Swingle R S, Trei J E and Nelson E A 1981 Detoxification of jojoba meal by Lactobacilli. Journal of Agriculture Food Chemistry 29: 296-302

Verbiscar A J, Banigan T F, Weber C W, Reid B L, Trei J E, Nelson E A, Raffauf R F, and Kosersky D 1980 Detoxification of jojoba meal. Journal of Agriculture Food Chemistry 28: 571-578

Weber C W, Berry J W and Cook E M 1983 Influence of jojoba meal upon growth and reproduction in mice. In Jojoba and Its Uses Through 1982, Proceedings of the Fifth International Conference on Jojoba and its Uses; Elies-Cesnik A, Editor. Office of Arid Lands Studies, University of Arizona: Tucson, AZ.

Received 11 January 2009; Accepted 14 March 2009; Published 1 May 2009

Go to top