Effect of crude enzymes from rumen liquor and yeasts culture on performance of goats fed diets containing palm kernel cake

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EFFECT OF CRUDE ENZYMES FROM RUMEN LIQUOR

AND YEASTS CULTURE ON PERFORMANCE

OF GOAT FED DIETS CONTAINING

PALM KERNEL CAKE

ROBERT PAULIS

BOGOR 2013

THE GRADU_{ATE SCHOOL}

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Hereby, I state that the thesis entitled Effect of Crude Enzyme from Rumen Liquor and Yeats Culture on Performance of Goats Fed Diet Containing Palm Kernal Cake is my own work, which has never previously been published in any university. All of incorporated originated from other published as well as unpublished papers are stated clearly in text as well as in the references

Bogor, March 2013 Robert Paulis NIM D152098021

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RINGKASAN

ROBERT PAULIS. Pengaruh Penambahan Enzim dari Cairan Rumen dan

Kultur Ragi Terhadap Performan Kambing diberi Pakan Berbasis Bungkil Inti Sawit. Dibimbing oleh IDAT GALIH PERMANA, NAHROWI, MUTARIN DAMSHIK.

Pemanfaatan bahan lokal sebagai pakan alternatif telah banyak dilakukan. Sumber bahan pakan alternatif yang mempunyai potensi untuk dimanfaatkan adalah bungkil inti sawit (BKS). Hasil analisa proksimat menunjukkan bahawa kndungan bungkil inti sawit setanding dengan jagung dan dedak padi. Namun, kerana kandungan serat kasar yang tinggi yaitu mencapai 18.33%-21.33% menyebabkan bahan baku tersebut perlu diolah lagi sebelum digunakan.

Upaya pengolahan yang sering digunakan dalam bidang penternakan dengan penambahan bahan additif dalam pakan adalah penggunan enzim dan ragi. Tujuan penelitian ini adalah untuk menetukan penggunaan penambahan enzim dan ragi dalam meninggikan nilai nutrisi dan kecernaan BKS pada kambing dan dengan demikian BKS akan dapat menjadi sumber pakan alternatif.

Penelitian ini dilakukan dalam dua tahapan. Tahap pertama adalah penelitian in vitro dilakukan yaitu menguji akitivitas enzim mannanase dimana didapati nilai akitivitas enzim adalah sekitar 0.0128 U/L. Manakala hasil ujian degradasi enzim cairan rumen sapi terhadap PKC pada tingkat 0, 5, 10, 15 dan 20 ml/kg terhadap kandungan gula terlarut menunjukkan terdapat kenaikan sebanyak 3.4 -5.7%. Dalam penelitian koefeisien kecernaan in vitro sebanyak 5 perlakuan pakan (3 per ulangan) terdiri (1) kontrol: (tanpa penambahan suplemen (2) YC1: penambahan suplemen ragi tape pada tingkat 1% (3) YC2: penambahan suplemen yis pada tingkat 0.5% (4) EZ1: penambahan suplemen enzim dari cairan rumen pada tingkat 1.5% dan (5) EZ2: penambahan suplemen enzim pada tingkat 0.02% dari pakan konsentrat. Hasil penelitian menunjukkan koefisien kecernaan bahan kering (KCBK) dan koefisien kecernaan bahan organik (KCBO) bungkil inti sawit tidak berbeda nyata (p>0.05) diantara perlakuan.

Tahap kedua menguji pemanfaatan nutrient pada pakan kambing berbasis bungkil inti sawit dengan cara suplementasi penambahan enzim cairan rumen dan kultur yis. Dua puluh ekor kambing jantan muda dengan bobot badan awal sekitar 15 ± 2.5kg digunakan dalam penelitian dengan 5 perlakuan pakan (4 ekor per ulangan) terdiri atas (1) kontrol: (tanpa penambahan suplemen (2) YC1: Ragi tape 5g/h/d (3) YC2: Yis 2.5g/h/d (4) EZ1: Enzim cairan rumen 1,5% dan (5) EZ2: Enzim 0,02% dari pakan konsentrat. Penelitian dilakukan selama 35 hari dengan masa adaptasi 14 hari dalam rancangan acak kelompok. Parameter yang diamati adalah perubahan konsumsi dan kecernaan bahan kering, bahan organik, konversi pakan, karakteristik cairan rumen dan populasi mikroba rumen.

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nyata di antara perlakuan. Konsentrasi total produksi VFA menunjukkan perbedaan yang nyata (p<0.05) dengan penambahan supplement. Konsentrasi asam butirat berbeda nyata (p<0.05) dimana suplementasi ragi dan enzim meningkatkan konsentrasi butirat dibandingkan dengan kontrol. Proporsi rasio molar asetat propionat berbeda nyata pada penambahan suplemen enzim lebih tinggi sedangkan penambahan suplemen yis rasio asetat:propionate lebih rendah dibandingkan kontrol. Populasi mikroba rumen meningkat menunjukkan perbedaan yang nyata (p<0.05) pada suplemen penambahan yis. Hasil penelitian in di simpulkan bahawa tiada perbeda yang nyata diantara dua jenis kapang and enzim digunakan dalam perlakuan ini. Suplemen kultur ragi and enzim dapat digunakan sebagai aditivif makanan dalam meningkatan nutrisi bungkil inti sawit. .

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SUMMARY

ROBERT PAULIS. Effect of Crude Enzymes from Rumen Liquor and Yeasts Culture on Performance of Goats Fed Diet Containing Palm Kernel Cake. Supervises by IDAT GALIH PERMANA, NAHROWI, MUTARIN DAMSHIK.

Global price of raw material for conventional feed has increased recently over the year and become the most important factor constrains the development of animal production. Palm kernel cake (PKC) is the by product from the extraction of palm oil kernel can have a major influence on reducing the production cost. The proximate analysis of PKC has showed that it can be classified as energy feed, with the chemical component, very similar to that of corn gluten or rice bran. The usage of palm kernel cake has its limitations due to high in crude fiber and very low palatability.

Under the feeding of low quality agricultural residues, supply animal’s

rumen microbes with the necessary nutrients needed allow the animal to maximize the usage of the low quality feed. Manipulating rumen digestion system through the addition of yeasts and enzyme in ruminant rations as to enhance cellulose digestion and improves the performance of the animal is the most interest in recent year.

The aim of present study was to determine the nutritive value and digestibility with and without addition of enzyme and yeasts culture to growing goats fed containing palm kernel cake (PKC). The study was conducted by 2 affiliated experiments.

Experiment 1: Preparation of crude enzymes from rumen liquor and enzymes isolation. An enzymatic assay analysis was done to determine the activities of manananase enzymes which estimated the mannanase activities 0.0128 IU/L. Determination the total reducing sugar was carry out by added 5, 10, 15 and 20ml/kg crude enzymes mixed in palm kernel cake. There were an increased in total reducing sugar from 3.4 to 5.7% in total soluble sugar respectively. Meanwhile in vitro digestibility experiment was done using 5 different treatments with PKC as basic ration supplemented with 1% local ragi, 0.5% yeast, 1.5% rumen liquor and 0.02% enzymes. This results indicated that there no significant increased (p>0.05) on IVDMD and IVOMD in supplemented diets compare with the control ration.

Experiment 2: Twenty growing crossbred (Feral x Local) goats, aged about 6 months, weighed 14.8±2.5 kg, were allocated to 5 treatments according to RCBD with 4 goats in each treatment. The supplemented diets treatments were the control, 5g/h/d of local ragi, 2.5g/h/d yeasts, 1.5% rumen liquor and 0.02% enzymes in basal diets containing PKC basic ingredient.

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total VFA in ruminal fluid, molar proportions of propionate and acetate.

Depressed ruminal protozoal number and heightened ruminal total viable bacterial number were entailed by additional yeasts culture. There were no significant differences between the types of supplemented preparation of yeasts culture and enzymes effect on improved rumen fermentation and digestibility of nutrients.

Based on the experiments conducted, it could be concluded that the supplemented with yeasts and enzymes could use as feed additives in improving the PKC nutrition value and digestibility.

Keywords: goat, palm kernel cake, rumen fermentation, rumen enzyme,

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© Hak Cipta Milik IPB, Tahun 2013

Hak Cipta Dilindungi Undang-Undang

Dilarang mengutip sebagian atau seluruh karya tulis ini tanpa mencantumkan atau menyebutkan sumbernya. Pengutipan hanya untuk kepentingan pendidikan, penelitian, penulisan karya ilmiah, penyusunan laporan, penulisan kritik, atau tinjauan suatu masalah; dan pengutipan tersebut tidak merugikan kepentingan IPB

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A Thesis

Submitted in partial fulfillment of the requirement for the Degree of Master Science in Nutrition and Feed Science

EFFECT OF CRUDE ENZYMES FROM RUMEN LIQUOR

AND YEASTS ON PERFORMANCE OF GOAT FED

DIETS CONTAINING PALM KERNEL CAKE

ROBERT PAULIS

BOGOR 2013

THE GRADU_{ATE SCHOOL}

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Thesis Title : Effect of Crude Enzymes from Rumen Liquor and Yeasts Culture on Performance of Goats Fed Diets Containing Palm Kernel Cake Name : Robert Paulis

NIM : D152098021

Approved by Advisory Committee

Prof Dr Ir Nahrowi, MSc

Member Dr Ir Mutarin Damshik, MS Member

Acknowledged by

Head of Study Program of Nutrition and Feed Science

Dr Ir Dwierra Evvyernie A, M MSc

Dean of the Graduate School

Dr Ir Dahrul Syah, MScAgr

Date of Examination:

4 February 2013 Date of Graduation:

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ACKNOWLEDGEMENT

First of all, I would like to acknowledge the State Government of Sabah Malaysia for providing financial support during this course. I would also thank to the Director of Veterinary Services and Animal, for allowing me to undertake this study. I am also grateful for the help of my colleagues at the Veterinary Public Health Laboratory for their technical and logistical support.

I would like to express my greatest gratitude to my supervisors: Dr Ir Idat Galih Permana for their advice, encouragement, time and support.and Prof Dr Ir Nahrowi for sharing his special and inspirational thought at the Faculty of Animal Science, Bogor Agriculture University, Indonesia. I am also grateful for their critical and constructive comments on this study suggestion to Dr Ir Mutarin Damshik at the Department of Veterinary Services and Animal Industry, Sabah Malaysia.

My grateful appreciation to Dr Anuraga Jayanegara, my closed exam examiners and Dr Dwierra Evvyernie A our head of study program for your critical questions have helped me improve and make my work more in depth.

My appreciation and thank to all my lectures, staff and colleagues at the Bogor Agriculture University, Indonesia for their knowledge, friendship and cooperation.

I would like to dedicate this thesis to my beloved family, which includes my wife Bernaddette Mujah, Son Idy Detther Zedd, my mother, brother, sister and their family. I would like to thank them for supporting me spiritually, emotionally and otherwise.

Bogor, March 2013

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LIST OF TABLES

1. Ingredients and chemical composition of experimental diets 11 2. Total reducing sugar of degraded palm kernel cake

with rumen liquor enzymes 13

3. In vitro DM digestibility of concentrate with supplementary

with yeasts culture and enzymes during time of incubation 14 4. Chemical compositions of experimental diet (dry matter basis). 15 5. Effect of supplemented diets on dry matter intake,

average weight gain and digestibility. 16

6. Growth characters and performance of goats following

treatment with supplemented diets 17

10 Effect of supplemented diets on ruminal temperature,

ruminal pH, ruminal ammonia and blood urea nitrogen. 19 11 Effect of supplemented on volatile fatty acid 21 12 Effect of supplemented diets on ruminal microb 23

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LIST OF FIGURES

1 Diurnal variation of average of blood urea 33

2 A proposed scheme for mode of action of Saccharomyces

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1 INTRODUCTION

Background

Feed cost is a major cost burden in develop the livestock industry especially in developing countries because most of the diets are based on cereal grains and oil meals. This phenomenon encouraged and motivates nutritionists to explore for cheaper alternative high-energy feed ingredient from agro-industrial by-products (AIPD) and non competitive with human. These unutilised resources are plentiful and generated predominantly by the highly developed and organised palm oil industry. Indonesia and Malaysia both produces about eighty one percent (81%) of world oil palms production (MPOB 2008). Palm kernel cake (PKC) is the by product from the extraction of palm oil kernel (expeller/solvent) can have a major influence on reducing the production cost. Zahari and Alimon (2006) concluded that PKC as a high energy and cost effective feed ingredient that can be utilized in formulations ration for livestock feeding

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Under the feeding of low quality agricultural residues, supply animal‟s rumen microbes with the necessary nutrients needed allow the animal to maximize the usage of the low quality feed (Fadel et al. 2004). By manipulating rumen digestion system through the addition of direct feed microbial (DFM) and a fibrolytic enzyme in ruminant rations which could enhance cellulose digestion and improves the performance of the animal is gain interest in recent years (Salama et al. 2002, Haddad et al. 2005). Feeding yeast culture to dairy cows on concentrates diet could increased dry matter intake (DMI) and neutral detergent fiber (NDF) digestion (Fadel et al. 2007) decreased lactic acid production and milk yield (Fortina et al. 2011). Several work done by Erasmus et al (2005) suggested that feeding yeast products may be most beneficial to dairy cows during late gestation and early lactation when these effects of yeast cultures might be most valuable. Saccharomyces cerevisiae has caused beneficial changes inactivity and numbers of rumen microbes.

The approach usage of fibrolytic enzymes in the treatment of feedstuffs is to enhance their quality and digestibility. Feeding of exogenous enzymes in ruminants was previously an unacceptable practice, because it was assumed that the proteolytic activity in the rumen ecosystem would rapidly i_nactivate unprotected enzyme feed additives (Kung 1996). Improving the performance of enzyme supplements requires a thorough understanding of the structure and composition of the feedstuffs and of the digestive activities required for optimal nutrient utilization.

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Problems Statement

The low digestibility of PKC is attributed to the high level of non starch polysaccharides (NSPs) found in the cell wall materials. These NSP impair the digestibility and utilization of nutrients either by direct encapsulation of the nutrients or by increasing the viscosity of the intestinal content thereby reducing the rate of hydrolysis and absorption of nutrients in the diet. The NSP composition of PKC consisted mainly of mannans (major NSP), cellulose and xylans. Recently works concluded that inclusion level of PKC in diets (>35% PKC) in the rations decreased the digestibility of DM, protein and fibrous fractions and also decreased the protozoal populations (Chanjula et al 2010).

Applied biotechnology and feed industries currently offer exogenous enzymes as feed additives for enhancing the nutritive value of animal diets. Studies about adding enzymes preparation to diets for ruminant still limited. Much of the variability can be attributed to factor such as production techniques, enzyme activity, mode of enzyme action and application techniques and portion of the diet (forage:concentrate) to which the enzyme is applied and differences in the physiological status of the test animals (Beauchemin et al. 2000).

The manipulation the microbial ecosystem of the rumen in order to improve the production efficiency by addition of direct feed microbial which could enhance feed digestion, to improve the performance of animals to boosts the health status of animals. Addition of yeasts in ration could enhance fiber digestion and producing nutrients that stimulate growth of rumen cellulolytic bacteria (Dawson et al. 1990) which are responsible for the bulk of fiber digestion.

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Objective

The objectives of this research were to determine the nutritive value and digestibility of diets containing palm kernel cake (PKC) with and without enzyme and yeasts culture addition in goat. This information could provide a better understanding the use of enzyme in enrichment of PKC nutritive value and influence of yeasts in dietary supplementary on performance of goats.

Experimental Benefits

The outcome from this experimental was to explore the advantage of addition feed additives such enzymes and yeasts in utilized low quality agro-industrial by product (PKC) by improvement the nutrition value which could be use to replaced more expensive ingredient.

Hypothesis

The use of enzymes and yeasts culture could increase the nutritive value and enhance feed digestibility, feed intake, rumen fermentation and weight gain of goat fed diet containing PKC.

Scope and limitation of the study

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MATERIALS AND METHODS

Place and Time

This experiment was carried out at the Laboratory of Veterinary Public Health (MKAV, Kepayan) Department of Veterinary Service and Animal Industry, Sabah Malaysia from Mei until June 2012.

Material

The materials used in this experiment were rumen liquor of cattle which was obtained from the abattoir (SMTC, Sabah), ice thermos for keeping the rumen liquor, cheesecloth, refrigerator, and centrifuge machine. The material used for determination enzyme activities was centrifuge, thermometer, water shaker water bath, pH meter, incubator, spectrometer, crude filtrate rumen enzyme, Gas Chromatography, Biochemical analyser and chemical reagent for measuring enzymes activities.

Methods

Sample preparation of rumen liquor

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Enzyme isolation

Supernatant containing enzymes was then reacted with ammonium sulphate (60% w/v) and stirred by magnetic stirrer for 1 hour and kept one night at 4°C. Supernatants were then centrifuged again at 10 000 rpm (4°C) for 15 minutes. The filtrate was taken and added with phosphate buffer pH 7 at the ratio of 10:1 (100 ml supernatant of rumen liquor is dissolved with 10 ml of phosphate buffer pH 7). The precipitates (enzyme source) in phosphate buffer are dialyzed before keeping in freezer for enzyme assay.

Enzymatic Activity Assay

Mannanase activity assayed using 0.5% (w/v) locust bean gum (LBG) in 50 mM sodium citrate buffer, pH 4.0 as substrate (Ghose 1987). The enzyme preparation (200 μL) is added into 1800 μL of substrate. The reaction mixtures are incubated at 40 °C in a water bath for 5 min and 30 min for mannanase. The quantity of reducing sugar released is measure using dinitrosalicylic acid (DNS) method (Miller 1959). One unit of enzyme activity is defined as the amount of enzyme producing 1 mmol of mannose per sec under the assay condition.

Experimental and data analyses

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In Vitro_Experimental

Method

This experiment was conducted to measures the enzyme capabilities to hydrolysis of carbohydrates by rumen fluid through in vitro test. This experiment carried out using method according by Boisen and Egum (1991) and Aslamyah (2006). A total of 25g of feed ingredients were weighed and placed in a plastic container with a lid. The feed was added with rumen filtrate enzyme and stir evenly. Enzyme doses used were 0, 0.5, 1.0, 1.5 and 2.0% (v/w). Solution volume for each treatment with the addition of enzyme equated with distilled water prior to incubation for 24 h at room temperature.

Determination of total soluble sugar.

Measurement of feed carbohydrate hydrolysis by the enzyme is performed by measuring total soluble sugars by the method of Dinitrosalycyclic (DNS) Acid Reagent Methods after incubation. A total of 1g sample rations that have been incubated with rumen fluid enzymes were weighed and put into test tubes. Add 5 ml of distilled water and then vortex for about 1 minute. Centrifuged the mixed at 3000 rpm for 15 min, supernatant is used to measure levels of total soluble sugar rations of feed.

Determination of reducing sugar using Dinitrosalycyclic Acid Reagent is based method tests for the presence of free carbonyl group, the so-called reducing sugars. The amount of carbohydrate present is determined by comparison with a calibration curve using a spectrophotometer.

In vitro _{digestibility test}

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9 McDougal buffer was prepared, then it was added mixed with rumen liquor at the ratio of (4 buffers: 1 rumen liquor). This mixture was saturated with CO2 and warmed at 39°C in a water bath.

This experiment was conducted according to Complete Randomized Design (CRD) with 5 treatments and each treatment with 3 replicate. The exogenous commercial enzymes contain of manananase (91 927.0 U/g), cellulases (526.0 unit/g), xylanases (3 869.0 U/g), alpha amylase (24 354.0 U/g), protease (345.4 U/g) in a powder form respectively and rumen liquor that used in this experiment consist of mannanase activities (0.0128 IU/L). Meanwhile the commercial yeasts and local yeasts content Sacchromyeces cerevisae about 4 x 1010 cfu g-1, 1.5 x 1010 cfu g-1 respectively.

About 500 mg experimental sample Basal ration (BS), BS + 1.5% filtrate enzymes (15ml/kg), BS+0.02% commercial enzyme (25mg/kg), BS+ 1% Local yeasts (10g/kg) , BS+ 0.5% commercial yeasts (5g/kg), then was mixed with McDougall buffer in a ratio 1:4. After gasifying with CO2, tubes were incubated at 39°C. After 48 h the fermentation, 6 ml of HCl solution (20 %) and 5 ml pepsin solution were added and the incubated for 48 h simulating post-ruminal degradation.

Samples were filtered using a dry weighed Whatman filter paper (No. 41) and the residue was washed with boiled water; the filter paper and residue was dried at 100°C overnight and weighed after cooling in desiccators. The content of the crucibles were then incinerated at 475-500°C until a constant weight is achieved. A blank correction without matter was included.

This was calculated by the following Equation:

% IVDMD = DM samples – (DM residue-DM blank) x 100% DM samples.

The organic matter digestibility (OMD) was calculated by the formula %IVOMD = OM samples-(Om residue- OM blank) x 100%

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In Vivo_Experimental

Material and Methods Location and Date

This experiment was conducted at the experimental facilities at the Pusat Pembiakan Kambing, Bongawan, Sabah Malaysia. The chemical feed was analysed at the Food Safety unit of the Veterinary Public Health Laboratory, Kepayan, Sabah, Malaysia and Laboratory of Food Technology, University Malaysia Sabah. This experiment was conducted from June until August 2012.

Animal and diets

The exogenous commercial enzymes contain of manananase (91 927 U/g), cellulases (526.0 unit/g), xylanases (3 869.0 U/g), alpha amylase (24 354 U/g), protease (34.4 U/g) in a powder form respectively and rumen liquor that used in this experiment consist of mannanase (0.0128 IU/L). Meanwhile the commercial yeasts and local yeasts content Sacchromyeces cerevisae about 4 x 1010 cfu g-1, 1.5 x 1010 cfu g-1 respectively.

Twenty male growing crossbred goats (Feral x Local) averaging 15±2.5kg (initial mean BW±SD). The animals were allocated to 5 dietary treatments in which each treatment contain 4 replicate according to Randomized Complete Block Design (RCBD). The block was made according to their respective body weight. Dietary treatments are formulated to meet NRC nutrient requirements (NRC, 2007) which iso-protein (14%) and iso-energy (ME 2550 Kcal/kg) (Table 1).

The treatments are as follows:

C = Basal ration B (Control)

EZ1 = B + 1.5% Filtrate Enzyme (15ml/kg)

EZ2 = B + 0.02% Commercial Enzymes (25mg/kg) YC1 = B + 5 g/h/d Local Yeasts (10g/kg)

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11 The experiment was conducted for thirty five (35) days consisted of a 14 day preliminary period, in which animals were adapted to be experimental diet and 21 day trial period.

The basal ration composed of 40% concentrate feed mixture: 60% roughage (signal grass). The concentrate diets were fed in limited amounts (2 % of BW) once daily at 0800 h to minimize refusals and differences in intake among the dietary treatments. The refusals of signal grass were collected daily. Fresh water and mineral blocks were available ad libitum throughout the experiment. All goats were given injection for internal worms (Ivermectin) and vitamin A, D3, and E prior to commencing this experiment. Goats were monitored for any health problems which were treated accordingly. Records were kept of all the health problems. Body weights were measured at the beginning and at the end of experiment.

Data Collection and Laboratory Analysis

Feed offered and feed refused was recorded daily. Feeds and faecal samples were collected from the total collection of each individual goat on each treatment for the last 7 days at morning and afternoon feeding. Combined

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samples were dried at 60ºC and ground (1-mm screen) and then analysed for dry matter (DM), organic material (OM), crude protein (CP) content (AOAC 1997) and crude fibre ( Van Soest et al. 1994).

Rumen fluid was collected using stomach tube connected with a vacuum pump at 0 and 4h post feeding. The pH and temperature of the rumen fluid were immediately measured by means of a portable pH and temperature meter. Rumen fluid samples were then filtered through two layers of cheese cloth and divided into two portions.

The first portion of rumen fluid was used for analysis of volatile fatty acids (VFA) and Ammonia (NH3-N). 1M H2SO4 solution (5 mL) was added to 45 mL of rumen fluid. The mixture was centrifuged at 10,000 rpm for 15 minutes and the supernatant was stored at -20°C prior to VFA analysis by Gas Chromatography (GC). Ammonia concentration was determined by phenol-hypochlorite method of Broderick and Kang (1980). The second portion was use for a total direct count of bacteria and protozoa with a haemocytometer by the methods of Galyean (1989).

A blood sample (about 10 ml) was drawn from the jugular vein at the same time as rumen fluid sampling (at 0 and 4 h post-feeding) and centrifuged at 5,000 rpm for 10 minutes. The supernatants were stored at -20ºC until analysis of blood urea nitrogen (BUN) using Biochemical analyser (Microlab 300).

Experimental Design and Data analysis

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RESULT

In Vitro Experimental

Enzymes precipitation by ammonium sulphate

The concentrations of ammonium sulphate (NH4)2SO4 used to precipitation enzymes in this study indicated that the most active enzyme protein preparation could be obtained at the (NH4)2SO4 level of 60 %. Since (NH4)2SO4 fractionation process where inter protein molecules are aggregated and precipitated, this resulted an increased in mannanase enzymes specific activities to 0.0128 x 106 IU/L of rumen liquor enzymes.

Total soluble sugars tests

The data of reducing sugar analyses were summarized in Table 5. Different concentrations levels of crude enzyme from rumen liquor were mixed with PKC to determine the degradable of total reducing sugar production. The results presented showing an increase from 3.4 -5.7% of total reducing sugar content were recorded with increasing dietary enzyme inclusion level of 5 to 10%. The data clearly suggested that the enzyme effectively hydrolyzed the lignocelluloses in PKC to simple sugars which resulted in an increase in the total reducing sugar contents.

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In Vitro_{Digestibility DM and OM}

Table 2 Increment of total soluble sugar of degraded palm kernel cake with rumen liquor enzymes

Rumen Liquor enzymes vol (ml/kg)

Incubation time (hr) Increase of Total _{Soluble Sugar}

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In Vitro_{Digestibility DM and OM}

The effects of supplemented diet with yeasts (YS1, YS2) and enzymes (EZ1, EZ2) on rumen pH and in vitro dry matter (IVDMD) and organic matter digestibility (IVOMD) are given in the Table 6. There were no significant different in rumen pH which was found within the range of 6.62 to 6.86 in the control and treatments groups.

Data presented in Table 6 indicated that there were no significant increased (p>0.05) in vitro digestibility of dry matter (IVDMD) which value ranges from 77.09 to 77.77% among the treatment group and the control (76.00%). A similar result also observed in current study for in vitro organic matter (IVOMD) digestibility which the value ranges from 74.90% to 76.78%, respectively.

Table 3 In vitro DM and OM digestibility of concentrate added with yeasts culture and enzymes during time of incubation

In vitro digestibility (%)

Item pH IVDMD1 IVOMD2

C 6.67 76.00±0.51 74.90±0.64

YS1 6.62 77.09±0.78 76.31±0.91

YS2 6.72 77.25±0.85 76.40±0.79

EZ1 6.86 77.60±0.69 76.50±0.63

EZ2 6.79 77.77±0.63 76.78±0.72

1_{IVDMD=In Vitro Dry Matter Digestibility,}2_{IVOMD=In Vitro Organic Matter Digestibility,}

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15 In Vivo_Experimental

Chemical composition

All animals received the same Total mixed ration (TMR) contained a forage: concentrate ratio of 60:40. The ingredient and chemical compositions of roughage and basal experimental diets were summarized in Table 7. The five experimental basal diets supplemented with yeasts culture and enzymes contained similar concentrations of DM, ash, OM and CP. As to the concentrate, it contained DM 88.37%, CP 14.20%, and NDF 37.1%, whereas the roughage contained DM 26.7%, CP 7.7 %, and NDF 67.7% (DM basis).

Dry Matter Intake and digestibility DM OM

Dry matter intake and apparent digestibility

The effects of dietary addition with yeasts (YS1, YS2) and enzymes (EZ1, EZ2) on daily feed intake and apparent digestibility of goats are presented in Table 8. Overall means for daily feed intakes for five diets in terms of roughage, concentrate were no significant different (p>0.05) for all dietary treatments. The data indicate that inclusion of supplementary had no effect on feed intake for Table 4 Chemical compositions concentrate, Brachiaria grass and Palm Kernel Cake

(DM)

Items Unit Concentrate Brachiaria

grass

Palm kennel cake

Dry matter % 88.37 26.7 95

Ash % 6.51 8.6 4.5

Crude protein % 14.20 7.7 15.9

Crude fibre % 8.21 32.4

Ether extracts % 3.62 2.2 9.4

2_ADF _% _25.60 _38.0 _47.3

1_NDF _% _37.1 _67.7 _72.68

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goats. All diets were accepted well by the goats, as evidenced by similar in total DMI between 456g/d to 509 g/d among goats receiving diets inclusion of supplementary with yeasts and enzymes. However in this study shows that DMI has a significant increase (p<0.05) in the supplemented diets ranged between 2.98 to 3.02% compare with control diets 2.78%, respectively. In contrast to DMI (g/kg 0.75) metabolic weight results indicate there were no significant different (p>0.05) seen among the treatment group.

The apparent digestibility of DM, OM, goats fed supplementary diet with yeasts and enzymes were slightly greater than the control (Table 8). Although in study shows that there were no significant differences (p>0.05) in digestibility of DM, however there were tend to be higher 73.31 to 74.41% with addition of yeasts culture (YS1, YS2) and 71.17 to 72.40% with addition of enzymes (EZ1, EZ2) compare to the control 69.29 % respectively. The OM digestibility also show no significant (p>0.05) differences were found between the control group (68.76%) compare with addition of yeasts culture 72.42 to 73.34 % (YS1,YS2) and 70.30 to 71.36% with addition of enzymes (EZ1, EZ2) respectively . Although the value of the control group was slightly lower that the other groups.

Table 5 Effect of diets on dry matter intake, average weight gain and digestibility. Item

1_{SE: standard error of the means, C =Control, YS1=Local Ragi, YS2=Commercial}

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Average weight gain and IOFC

The result was obtained with the ANCOVA with initial body weight as the covariable followed by adjusted means comparisons (Table 6). Average weight gain (ADG) was no significantly differences (p>0.05) for supplemented yeasts and enzymes compare to control diets. The use of ANCOVA and adjusted means comparisons further revealed that the highest values of ADG were recorded with addition of yeasts in supplemented diets (92.80g/day) and the lowest values were recorded in control ration (64.29g/day). This result could be interpreted by the improvement in the feed conversion ratio in feed consumption. Supplemented diets group had the best feed conversion ratio ranging from 5.48 to 6.34 in yeast and 5.84 to 6.16 in enzymes compare with control diets (7.09) respectively.

Table 6 Performance of goats following treatment of diet treated with yeasts culture and enzymes.

Analysis of income over feed cost (IOFC) was designed to evaluate the cost and net return of applying these rations in goat during the experiment. Data ADG (g/h/d) 64.29±14.29 78.57±14.29 92.80±14.29 78.57±14.29 85.21±23.33 Total gain (kg) 2.25±0.50 2.75±0.50 3.25±0.82 2.75±0.50 3.00±0.82

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supplemented diets, gave the highest net return, representing by Income Over Feed Cost (IOFC) for as much as Rm 0.58 to 0.70 and Rm 0.60 to 0.66/day/head compare to control diets (Rm0.46/day/head). This high net return was supported by the efficiency of using the ration with addition of yeasts as indicated by the feed conversion (5.46 to 6.58)

Ruminal Fluid Temperature, pH Ammonia and BUN

Rumen temperatures (Table 9) were similar among treatments and the mean values were quite stable at 39.3-39.5°C, which were within the optimal normal

range for microbial digestion (Van Soest, 1994). Rumen fluid pH at 0 post feeding and overall means were unchanged by dietary treatments (Table 9). Meanwhile at 4 h after the onset feeding, rumen pH of goats declined as active

fermentation of the newly ingested feed occurred. At this time, the pH values decrease ranged from 6.18-6.31. However, all treatment means were within the normal range and the values were quite stable at 6.41-6.55.

In this study shows there were no significant differences among supplements diets (p>0.05) on concentration of ammonia (NH3-N) in the rumen fluid (Table 9). At 0 a post feeding, ruminal NH3-N were ranged from 44.72 to 48.16 mM/L. Meanwhile at 4h post feeding, the ammonia, the ammonia concentration in the rumen tends to increased as a result degradation of protein by the rumen microbial. However, there were no significant differences (p>0.05) among the supplemented diets which the values ranges from 60.88 mM/L in control, 57.29 to 57.63 m/L in enzymes diets and 56.92 to 57.37 mM/L in yeasts culture diets respectively.

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19

Table 7 Effect of enzyme and yeasts culture addition on ruminal fermentation, pH, NH3 andBUN concentration.

Item

Treatment

SEM P value

C YS1 YS2 EZ1 EZ2

Rumen

temperature, °C

0 h post feeding 39.2 39.2 39.4 39.3 39.2 0.13 0.16 4 h post feeding 39.6 39.5 39.6 39.5 39.4 0.03 0.29

Mean 39.4 39.4 39.5 39.4 39.3

Ruminal pH

Mean 6.44 6.55 6.48 6.41 6.41

NH3-N (mmol/L)

0 h post feeding 35.43 34.00 32.48 34.76 34.24 0.10 0.65 4 h post feeding _60.88 _56.29 _57.07 _57.63 _{57.29 2.13 2.13}

Mean _48.16 _45.15 _44.77 _46.19 _45.77

BUN (mmol/L)

Mean 2.5 2.8 3.1 2.6 3.0 0.27

1 _{SE: standard error of the means, C =Control, YS1=Local Ragi, YS2=Commercial Yeasts, EZ1=Rumen}

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VFAs concentration

Data of the effect of supplemented diets on VFA concentration were summarized in Table 10. Addition of the supplemented diet for treatment group shown significantly increase (p<0.05) of total VFA content in the rumen fluid as well as other component of acids such as acetic, propionic and butyric. The total VFA concentration tend to increase after 4h post feeding ranged from 94.51 to 109.05 mM/ L compare at 0h post feeding ranged from 65.48 to 70.09 mM/ L.

In this study shows that total VFA significantly (p<0.05) increase between 107.91 to 102.81 mM//L with addition of yeasts in supplemented diets. There also a significant increase (p<0.05) in production of propionate acid and acetate with supplemented yeasts diets, meanwhile other diets treatment didn‟t show any significant differences (p>0.05). However, supplementation enzyme in the diets have higher concentration values ranges from 69.91 to 70.86 mmol L-1 compare to control diets value 63.99 mmolL-1 respectively. Regardless of the time when samples of ruminal fluid were collected (0 h and 4 h post feeding); the content of propionic and acetic acids was the highest in yeasts group, intermediate in enzymes diets and the was lowest the control diets. Meanwhile, control diets also have the lowest contents of butyric acid compare to other supplemented diets.

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21

Ruminal microbe population

Table 8 Effect of enzymes and yeasts culture addition in concentrate on ruminal fermentation, VFA proportion.

1_{SE: standard error of the means, C =Control, YS1=Local Ragi, YS2=Commercial Yeasts,}

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Ruminal microbial population

The ruminal microbial population count data were presented in Table 11. The protozoa count in ruminal fluid for treatment group were 2.10, 1.90, 1.80, 2.05 and 2.00 (104 cell/ml) at 0 hour post feeding, respectively. Meanwhile the populations of ruminal protozoa slightly increase 2.45, 2.25, 2.20, 2.40 and 2.30 (104 cell/ml) at 4 hours post feeding. There were no significantly different among the supplemented diets and control group (p>0.05). However, there were tendencies a decreased in protozoa population in supplemented diets with yeasts between 8.2 to 10.2% compare to control diets. As expected, even though the effectiveness of supplemented diet on protozoal population was no significant (p>0.05), an overt subtraction was found.

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DISCUSSION

Table 9 Effect on rumen microbial population with the addition enzymes and yeasts culture on rumen microbial of growing goats

Item Treatment P

C YS1 YS2 EZ1 EZ2 SE1 Value

Protozoal population

(x104)

Mean 2.30 2.05 1.98 2.15 2.50 0.07

Bacterial population

(x1010)

0 h post feeding .12a 1.28b 1.29b 1.13a 1.14a 0.13 0.09 4 h post feeding 1.32 1.61b 1.63b 1.39ab 1.35ab 0.16 0.07

Mean 1.22 1.44 1.46 1.26 1.25

1_{SE=standard error of the mean C =Control, YS1=Local Ragi, YS2=Commercial Yeasts,}

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DISCUSSION

In Vitro_Experiment

Enzymes precipitation

Extraction of enzymes cattle rumen fluid was done though enzymes precipitated process by added with high concentration of ammonium sulphate. Ammonium sulphate was used in enzymes precipitation because it has a high solubility, relatively inexpensive, non-toxic and can stabilize the enzyme (Chaplin and Bucke 1990). The selection for the optimum concentration of ammonium sulphate is based on the highest enzyme activity measurements. In this study the optimum concentration ammonium sulphate for deposition of bovine rumen fluid were at 60%. The enzymes activities obtain in this study 0.0128 x 106 IU/L mannose.

Precipitation with ammonium sulphate based on the nature of the polar equation of ammonium sulphate and water. The addition of salt to a solution of ammonium sulphate will damage the coat protein and attract water molecules from the surrounding surface of the protein molecule, the protein is no longer protected as a result of water molecules but rather aggregate with each other and then settles (Scopes 1987). Thus, the more the amount of protein in the solution will be more salt ammonium sulphate needed to attract water molecules and precipitate proteins.

Hydrolysis by Enzymes Feed Substance Rumen Fluid

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25 The hydrolysis of insoluble cellulose and hemicellulose into soluble sugars indicate an increase in usable energy in the enzyme treated PKC. Fiber-degrading enzyme activities are generally determined by measuring the rate of release of reducing sugars from pure substrates, with enzyme units expressed as the quantity of reducing sugars released per unit of time per unit of enzyme.

This study agreement with Lawal et al. (2010) who found that the fungal enzyme treated PKC increased the total soluble sugar content. It supported by Ng et al. (2002) which reported that enzyme treated and fungal fermented PKC increased reducing sugars content by about 69% (from 2.87 to 9.25 mg/g) and 65% (from 2.87 to 8.09 mg/g), respectively, which were much higher than values obtained in this study. The higher values of their soluble sugars obtained in the biodegraded PKC agree with the observed decrease in the contents crude fibre and NSPs. This correlation indicates that a NSPs in PKC which are high molecular weight substances are broken down by the fungal enzymes into soluble sugar, which are easily absorbed.

Although the enzymatic activities of crude enzyme from rumen liquor much lower compare with commercial enzyme properties, the concentration of the rumen liquor enzymes can be increased to level up the concentration to increase the effectiveness.

Effect of supplemented diets on In Vitro_{Digestibilities}

There were no significant increased (p>0.05) of IVDMD and IVOMD in current study. However there were slightly improved in DM digestibility observed by addition of enzymes 2.4% and yeasts 1.4% respectively. The slightly improvement of nutrient digestibility by addition of enzymes and yeasts probably due to the beneficial effects on fiber hydrolysis and rumen fermentation activity and this results were confirmed in vitro (Table 3).

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fill in the rumen over time thereby allowing higher voluntary feed intake (Dado and Allen 1995). This effect attributed to an increase in microbial colonization of feed particles and speculated that exogenous enzymes may act similarly to primary bacterial colonization Yang et al. (1999), and increase the dietary energy utilization and rumen fermentation. According to previous works reported by Yang et al. (1999) found that DM digestibility was increased significantly when alfalfa hay cubes were treated with enzymes and fed to lactating dairy cows. This statement supported by Feng et al. (1996) found that enzyme treatment of grass forage improved in vitro and in situ DM digestibility. Recently study by Colombatto et al. (2007) reported an improvement in vitro ruminal fermentation activity and cell wall digestibility of alfalfa stems by addition of exogenous fibrolytic enzyme in cow.

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27 In Vivo_Experiment

Diet composition

The diet was adequate to meet the requirements of crude protein (CP), net energy (NE) for growth and dry matter intakes of the goats under the condition of maintenance plus lower activity and 50 g/d weight gain Nutrients Requirements of Goats (NRC 2007). Average chemical composition of Brachiaria grass in these studies was quite similar values for Brachiaria grass have been previously reported by Nasrullah (2003). The CP and EE contents in PKC in this study were similar to those reported by O‟Mara et al. (1999); Carvalho et al. (2005) who found that PKC contained 14.10-16.42% CP and 7.83-9.40% EE. However the chemical composition of PKC is variable due to different processing methods and the degree of impurities such as shell content (Jalaludin et al. 1991). In general, the expeller samples had lower contents of CP, CF, NDF, and ADF than the extracted samples. These differences can be mainly attributed to the dilution effect of the oil in the expeller samples (O‟Mara et al, 1999).

Effect on DM intake and digestibility

In this experiment the DMI (%BW) in supplemented diets groups were similar with the requirement for goat in tropical regions, which reported to be 3.0-3.1% (Devendra and McLeroy 1982) and 2.83-2.97% (Burns et al. 2005). However, metabolic DMI was quite lower especially in control group (58.89 g/kgBW0.75) compare with the recommended level of DMI by AFRC (1998) for growing goats which about 66 g/kgBW0.75. This suggested by Kawas et al. (1999) works, reported that the low nutrient intake was the most important factor limiting animal performance. It is possible that low DMI could have been attributed to a high ADL with low fermentation rate and digestibility leading to a low rate of disappearance through digestion or passage and limited feed intake (Wanapat 2000).

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with supplemented enzymes diets compare to control diets. This studies supported by Dawson (1987) and Williams (1989) found that yeasts can increase DM intake, in response to more appropriate rumen conditions. Meanwhile Williams et al. (1991) found supplemented yeasts diets in total mixed diets for dairy cows, increasing DM intake, milk production and milk fat content. Response was greater with high levels of fermentable carbohydrates.

In current study indicate that there were slightly increased in apparent 50±60% forage and 50±40% concentrate (Plata et al. 1994, Williams et al. 1991) and has been associated with changes in digestibility (Wallace, 1994). However in the other assays, dry matter intakes were not affected (AvendanÄo et al. 1997). The improvement of nutrient digestibility by addition of enzymes was probably due to the beneficial effects of enzymes on fiber hydrolysis and rumen fermentation activity (Gado et al. 2011). This effect may be due to the ability of enzymes to lose lignocellulotic bonds in fiber (Bassuny et al, 2003). Improved the nutrient digestibility in animals could be also refer to the improvement in ruminal fermentation activities, by addition of enzymes to diet (Gado et al. 2009 and 2011). Furthermore, Morgavi et al. (2000) reported a synergistic relationship between fibrolytic enzymes and ruminal enzymes in degrading soluble cellulose, xylans, and corn silage. The solubilised carbohydrate would also provide energy that would lead to rapid microbial growth, shortening the lag time for microbial colonization (Yang et al. 1999). Sutton et al. (2003) reported that enzyme treatments decreased the retention time of digesta in the rumen, which could be attributed in decreasing the lag time for bacteria to gain attachment to the feed. Increased rumen enzyme activity is another action by which exogenous enzyme treatment could increase feed digestibility.

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29 the roughage quality the greater is the response. However, at this moment responses to yeast culture are unpredictable. In some conditions, live yeasts have influenced growth and activity of fiber-degrading microorganisms in the rumen, although mostly in vitro. Germination of zoospores from a rumen fungal strain of Neocallimastix frontalis was stimulated in vitro by S. cerevisiae (Chaucheyras et al. 1995). It suggested that yeasts could enhance fungal colonisation of plant cell walls. In the same studies, cellulose filter paper degradation by N. frontalis was also stimulated in the presence of live yeast cells. Several modes of action were identified in this effect, one being the supply of thiamine, a vitamin required by rumen fungi for zoosporogenesis. The effectiveness of some yeast strains to stimulate growth and activities of fibrolytic bacteria has also been demonstrated.

In contrast with other experiment indicated that low digestibility could have been attributed to a high fibrous fraction (ADF and ADL) (Hart and Wanapat 1992). Digestibility appears to be negatively related to fibrous content (Van Soest 1994, O‟Mara et al. 1999), especially the large proportion of lignified cell walls with low fermentation rate and digestibility, leading to a low rate of disappearance through digestion or passage and limited feed intake. The poorly digested components are more concentrated in the ADF than the NDF, resulting in the lower digestibility.

Performance of average weight gain and IOFC

In current study, indicated that there were slightly increase (p>0.05) in ADG in addition of yeasts and enzymes in the supplemented diets compare with the control. The lack of significant difference for the majority of the variables, when including body weight as a covariable in the ANCOVA analyses supported that the differences between supplemented diets were really not related to initial body weight.

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experiment, an increased tendency (p>0.05) of DMI was found due to addition of yeast. More recently works done by Han et al. (2008) demonstrated that significant increases in DMI and ADG of growing goats resulted from additional probiotics that contained Saccharomyces cerevisia and Lactobacillus acidophilus. The effectiveness of additional yeasts on DMI, ADG, and feed conversion in goats were in the same case as in cattle.

Previous statement by Haddad and Goussous (2005) demonstrated that 3g/d of yeast supplementation to finishing Awassi lambs fed high energy rations improves weight gain, ADG and feed: gain ratio. This supported by Fallon and Hart (1987) suggested that the improvement occurred in animal performance of lambs raised on rations supplemented with yeasts due to the increased palatability of supplemented feeds which lead to an increase in animals feed intake. Meanwhile, supplemented enzyme in rations of ruminants can enhance their dry matter and ﬁbers digesting capabilities over non-supplemented animals (Lewis et al., 1996). Fibrolytic enzymes could help in freeing these proteins and reducing the ﬁber content of the palm kernel cake.

The income over feed cost (IOFC) in current study shows with the addition of yeasts in the supplemented diets have the highest net return between Rm 0.53 to Rm 0.67/day/head compare with other treatment diets. This could be concluded that the usage of yeasts in the supplement diets have the advantage in improve the feed conversion ratio efficiency between 5.48 to 6.34 compare with the control diets 7.09 respectively. This could increase the profit margin of production of per animal.

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Effect of supplemented diets on rumen fermentation parameters and blood

urea nitrogen

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Data presented in (Table 9) shows there were slightly deceased (p>0.05) in ammonia concentration with the addition of yeasts and enzymes in the supplemented diets compare with the control. The NH3 N produced in the rumen is the rumen either utilized by the rumen microorganisms for maintenance and growth or converted to urea in the liver, when the supply is higher than the requirement and excreted in the urine. Yeast culture has been found to stimulate microbial activity and increase the incorporation of nitrogen into microbial protein, which confirmed the suggestion by Erasmus et al. 1992 that yeast culture may exert an effect on the flow of protein. This was related to changes in the number and activity of rumen micro organisms. The efficiency of feed nitrogen utilization in ruminants supplied with yeast culture involved not only the increase of ammonia incorporation into microbial protein and a higher flow and absorption of amino acids but also an altered endogenous nitrogen metabolism. The addition of yeasts to the control ration improved NH3-N values in different experimental rations. Alshaikh et al. (2002) reported that lower ammonia concentration was observed when yeast culture was supplemented to the high concentrate diet, which was explained by increased flow of microbial N and efficiency of microbial protein synthesis.

Gado et al. (2006) suggested that addition of enzyme may improve the stimulation of ruminal microorganism‟s activity by reducing the NH3-N concentration in the rumen liquor throughout incorporation of NH3-N into microbial protein. This effect attributed to an increase in microbial colonization of feed particles and speculated that exogenous enzymes may act similarly to primary bacterial colonization Yang et al. (1999) and increase the dietary energy utilization and rumen fermentation. Colombatto et al. (2007) reported an improvement in vitro ruminal fermentation activity and cell wall digestibility of alfalfa stems by addition of exogenous fibrolytic enzyme in cow.

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33 the blood reached a maximum 3 h after feeding and then start decline at the optimal within 8.87 ± 0.37 mmol -1 (Khaled et al. 1999). Meanwhile, Gustafsson and Palmquist (1993) reported that the peak BUN concentration occurred 1.5-2.0 hours after the ruminal ammonia N peak. Blood metabolite urea is an indicator of the protein status of ruminants (Sykes 1978), and the plasma urea-N concentration is related to the level of ammonia absorption from the rumen and the deamination of amino acids not deposited in the tissue (Deaville and Galbraith 1992). The differences in NH3-N and BUN concentrations among treatment may have been related directly to crude protein level concentrate. Observation done by Hwang et al. (2001) indicated that the mean BUN value of dry cows in the 5 treatment groups increased after feeding in the morning and reached peak at 4 h after feeding and declined after ward (figure 1). This diurnal pattern agreed with the trend by Gustafsson and Palmquist (1993) that BUN reached peak level 2 to 4 h postprandial under feeding once or twice daily.

Excessive ruminal ammonia N is absorbed through the ruminal wall and converted to urea N in the liver and enters the circulatory system. The amount of ruminal ammonia N absorption is mainly determined by the concentrations of ruminal ammonia N and the ruminal pH (Ali et al. 2008). When energy is limited,

Time of day (h)

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the surplus ammonia is taken up by the liver and transformed to urea. Abo El-Nor and Kholif (1998) reported higher BUN values in response to probiotics supplementation. This higher BUN concentration may be due to incapacity of ruminal microflora to detain the ammonia optimally (Butler, 1998). Antunovic et al. (2006) also examined lower concentration of urea (5.51:7.97 mmol/l) in the blood serum of lambs fed diets with probiotics as compared to the control diets.

Effect of supplemented diets on volatile fatty acid profiles

Volatile fatty acids are produced in the rumen as the end products of microbial fermentation and they represent the major energy source absorbed by ruminants which accounting for 50 to 80% of total metabolisable energy supplied to ruminants. In this current study result (Table 10) were similar with previous works show that there significantly increase (p<0.05) in total volatile fatty acids (VFAs) concentration produce with the addition of yeast and enzymes diets groups.

According to Fadel Elseed et al. (2007) works indicate that Saccharomyces cerevisiae resulted in a numerical increase in total VFA concentration. Church (1991) suggested that under any feeding regime VFA products that predominate were acetate, propionate and butyrate. Fermentations with high in propionate are more energetically and efficient that those high in acetate (Hungate 1966). Propionate concentration were significantly (p<0.05) increased with the addition of yeasts in supplemented diets. This result were coincides with Bunn et al. (1968) works, who observed that yeasts in the diet maintained a high rumen pH, and that molar percent propionate was higher than that of acetate. The increased proportion of propionic acid in total VFA, especially in the group of goat supplemented with live yeast culture may indicate increased ruminal degradation of concentrates and the higher utilization of acetic acid by yeast cells for growth compared to other feeding groups (Chu et al. 1981). Another probably reason as a result of stimulation of bacteria which can convert ruminal lactate into propionate by dietary yeast.

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35 Enjalbert et al. (1999) where a decrease in acetate to propionate ratio associated with yeast culture supplementation was reported. The decrease in acetate to propionate ratio was caused by yeast culture increasing the molar proportion of propionate. Meanwhile previous works done by Russell (1998) reported an increase in propionate but there were no change in acetate concentrations in vitro experiment. Although acetate concentration in current study was reduced, its production level does not change considerably. Meanwhile, it could be possible as the propionate production increases at the same time with the increased in acetate absorption (Davis et al. 1964). An increased in concentration of propionic acid in the ruminal contents of goats receiving the yeast preparation also reported by Lynch and Martin (2002). On the other hand, the increase in acetate concentration in the rumen of goats receiving a diet supplemented with live yeast might result from the increase in the number of cellulolytic bacteria, as the enhanced acetate level was associated with a higher activity of fibrolytic enzymes.

In current works the addition of enzymes in supplemented diets shows a decrease in percentage of propionic acid in total VFA with paralleled by the increased C2:C3 ratio in the ruminal contents. This may indicate the increased degradation of roughage fibers. It similar results to ours when feeding exogenous fibrolytic enzymes to growing goats were also reported by Beauchemin et al. (2000) when feeding exogenous fibrolytic enzymes to early-lactation dairy cows. However, these relationships were not confirmed by Giraldo et al. (2008) who fed enzyme preparations to sheep and by Krause et al. (1998) who used enzyme preparations to fatten young bulls. In accordance with the suggestion of Sutton et al. (2003), it can be assumed that the differences in the results obtained in different studies could be due to environmental factors, in particular the type of feed and the mode of enzyme application.

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enzymes to early-lactation dairy cows which reported by Beauchemin et al. (2000).

The influence of yeast supplementation on rumen parameters is greater when using high-concentrate diets or feeding systems that reduced cellulolysis the most. Yeast effect on rumen VFA appeared to be greater in high-concentrate or high-NDF diets than in intermediate ones (Desnoyers et al. 2009). The increased effect in high-NDF diets is certainly caused by an increase in cellulolysis or the number of cellulolytic bacteria. Feeding of yeast cultures has been shown to beneficially modify rumen metabolism which might increase DM intake (figure 2). Fibrous components that are fermented and passed from the reticulo-rumen more slowly, have greater filling effect in the rumen over time. Increased digestibility of NDF might decrease rumen filling effect which can lead to an enhancement in feed intake (Fadel et al. 2004).

Figure 2A proposed scheme for mode of action of Saccharomyces cerevisiae CNCM I-1077 on fibre degrading communities \

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Effect of supplemented diets on rumen microbial populations

Ruminal protozoa counts were no significant different (p<0.05) among the supplemented yeasts and enzymes diets. Results of supplemented with Saccharomyces cerevisiae in the diets have been inconsistent in previous studies. According to Plata et al (1994) works shown that protozoa counts were elevated with S. cerevisiae, meanwhile others they were reduced (Angeles et al, 1995) or remained unchanged (Miranda et al. 1996). Stimulatory effects of the yeast are probably associated with nutritional factors in cultures, such as L-malic acid (Nisbet and Martin 1991), whereas negative effects might be associated with accumulation of ethanol in the medium (Bruning and Yokoyama 1988).

Meanwhile other works done by Galp (2006) observed that Saccharomyces cerevisiae treatment decreased Diplodinium spp. protozoa significantly but did not affect total protozoal counts. Similarly with Galip (2006) mentioned the supplementation of Saccharomyces cerevisiae decreased protozoal counts (424.33 vs.383.33) before feeding, but it was not different for the average. In presently studies by Tripathi et al. (2007) described that ciliate protozoa population did not change due to yeast supplementation

In current study indicated that there was a negative relationship between protozoa and bacterial population. Although, in this result show that protozoa population no significantly decreased (p>p0.05) among the treatment diets but there were tendency decreased in protozoa population in yeasts supplemented diets. Meanwhile there are significant (p<0.05) bacteria population increased supplemented yeasts diets from 1.28, 1.29 x 1010 cell/.ml at 0 hour post feeding and to 1.61, 1.63 x 1010 cell/.ml at 4 hours post feeding. Whereas other treatment group supplemented with enzymes and control shown no significant differences (p>0.05) in total bacterial population. It has been reported that yeast culture stimulates growth of cellulolytic bacteria and improve anaerobiosis in the rumen (Wallace 1994).

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are known to be a source of vitamin, enzymes and some unidentified activity in the rumen as well as the beneficial effects of yeast supplementation reported so far include better growth rate, feed conversation efficiency and milk yield (Dawson et al. 1990). In addition, yeasts are usually related to stimulation of cellulolytic and lactate-utilizing bacteria in the rumen, increase fiber digestion and increased flow of microbial protein from the rumen which may be beneficial for cattle fed high-grain diets (Guedes et al. 2008)

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CONCLUSION

The addition with yeasts culture and enzymes in supplemented diet containing PKC did not show any adverse effect on dry matter intake, digestibility, rumen pH, NH3-N and BUN. Yeasts culture supplementation diets increased Total Volatile Fatty Acids, propionate, acetate acid production and reduced proportion acetic propionic ratio. Meanwhile addition of enzyme in the diets has no significant affect on the concentration of total VFA in ruminal fluid, molar proportions of propionate and acetate. Depressed ruminal protozoal number and heightened ruminal total viable bacterial number were entailed by additional yeasts culture. There were no significant differences between the types of supplemented preparation of yeasts culture and enzymes effect on improved rumen fermentation and digestibility of nutrients.

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