Effect of crude enzymes from rumen liquor and yeasts culture on performance of goats fed diets containing palm kernel cake
EFFECT OF CRUDE ENZYMES FROM RUMEN LIQUOR
AND YEASTS CULTURE ON PERFORMANCE
OF GOAT FED DIETS CONTAINING
PALM KERNEL CAKE
ROBERT PAULIS
THE GRADUATE SCHOOL
BOGOR ARGICULTURAL UNIVERSITY
BOGOR
2013
STATEMENT OF RESEARCH ORIGINALITY
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unpublished papers are stated clearly in text as well as in the references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
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.
Hasil penelitian menunjukkan bahawa konsumsi bahan kering dan
kecernaan bahan kering, bahan organik tidak menunjukkan perbedaan yang
nyata (p>0.05) pada semua kelompok perlakuan. Karakteristik cairan rumen
menunjukkan bahwa konsentrasi ammonia, plasma urea dan pH tidak berbeda
nyata di antara perlakuan. Konsentrasi total produksi VFA menunjukkan
perbedaan yang nyata (p35%
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.
The inclusion of exogenous enzymes and yeasts culture in animal
nutrition hopefully has a positive effect that improved NSPs degradation which
can increase the energy concentration and the release of NSPs-trapped nutrients
(Sheppy 2001). This may contribute to low cost productions in ruminant systems
using poor quality agriculture industries by products as major components
replacing the much expensive cereal grain and protein plant ingredient.
4
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 agroindustrial 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
This study focuses on addition of feed additives in basal diets for growing
male Feral x Local crossbreed goats. The experiment was done in the Pusat
5
Pembiakan Kambing (PPK) farm where Feral Local crossbred growing goats
were selected among the farm animals (Bongawan, Papar district, Sabah,
Malaysia) to carry out this study. The concentrate used in this study was supplied
to the farm from Feed Mill, Department of Veterinary Services and Animal
Industry, Sabah Malaysia. The local yeasts was purchased from Local Trader
(Sabah, Malaysia), it contains Saccharomyces cerevisiae 1.5 x 1010 cfu/g. The
rumen liquor was obtained from abattoir at the Sabah Meat Technology Centre
(SMTC) Sabah, Malaysia.
6
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
Rumen liquor were collected from slaughtered cattle from the abattoir
Sabah Meat Technology Centre (SMTC), Sabah Malaysia and transported to the
laboratory used flasks in ice container at 4°C. Samples were filtered in cold
condition using four layer of cheesecloth to separate the undigested matter. The
filtrate was centrifuged at 10,000 rpm (4°C) for10 minutes to separate
supernatant from microbial cells (Lee et al. 2002). The supernatant was filtered
used Wathman filter paper (No. 4) under suction to remove any micro particles.
Supernatant was then taken as a source of crude enzyme and stored at 4°C until
use.
7
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
The data obtained from the variable was statistically analyzed according
to descriptive analyses.
8
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
The in vitro procedure was modified from that reported by Tilley and
Terry (1963). Rumen liquor was taken from slaughtered cattle which were
obtained from abattoir at Sabah Meat Technology Centre (SMTC), Sabah,
Malaysia.
The fluid was strained through four layers of cheese cloth. The
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%
OM samples
10
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)
YC2
= B + 2.5 g/h/d Commercial Yeasts (5g/kg)
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.
Table 1 Ingredients and chemical compositions of the experimental diets
.
Ingredients
Proportion (%)
Palm cake kernel, PKC
50
Corn meal, CM
10
Soybean meal, SBM
5
Rice bran, RB
20
Pollard wheat
10
Salt
1
Mineral mix1
calcium phosphate
Molasses
1
1
2
1
Minerals and vitamins (each kg contains): Vitamin A: 10,000,000 IU;
Vitamin E:70,000 IU ; Vitamin D:1,600,000 IU; Fe: 50 g; Zn: 40 g;Mn: 40 g;
Co: 0.1 g; Cu: 10 g; Se: 0.1 g; I: 0.5
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
12
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
All the data were analysed as a Randomized Complete Block Design
(RCBD) using one way analyses of variance (ANOVA). . Treatment means were
statistically compared using Duncan‟s Multiple Range Test to identify
differences between means. Analysis of covariance (ANCOVA) was used to test
for differences in ADG weight change between the supplemented diets groups. In
each ANCOVA, the initial body weight of the animals was used as the covariate
to control for pre-existing differences. . A linear regression was used to correlate
individual ADG rates within each supplemented diets group. Homogeneity of
regression assumptions were tested and met in each analysis. Significance was
set at a p value of 0.05 for differences in means and correlation coefficients
13
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.
.
Table 2 Increment of total soluble sugar of degraded palm kernel cake with
rumen liquor enzymes
Rumen Liquor
enzymes
vol (ml/kg)
5
0h
4.149
24 hr
4.290
Increase of Total
Soluble Sugar
(%)
3.4
10
4.045
4.228
4.5
15
4.253
4.481
5.3
20
4.175
In Vitro Digestibility DM and OM
4.415
5.7
Incubation time (hr)
14
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.
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
IVDMD=In Vitro Dry Matter Digestibility, 2IVOMD=In Vitro Organic Matter Digestibility,
C=Control, YS1=Local Ragi, YS2=Commercial Yeasts, EZ1=Rumen Liquor ,
1
EZ2=Commercial Enzymes
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.
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).
Table 4 Chemical compositions concentrate, Brachiaria grass and Palm Kernel Cake
(DM)
Items
1
Unit
Concentrate
Brachiaria
Palm
grass
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
NDFDry Matter Intake
% and digestibility
37.1
67.7
DM OM
72.68
NDF = Neutral detergent fiber, 2ADF= Acid detergent fiber
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
16
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 (p0.05) 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
DMI, kg/d
Brachiaria grass,
kg/d
Concentrate,
kg/d
Total DMI, kg/d
DMI, %BW
DMI, g/kg W0.75
Apparent
digestibility %
DM
OM
1
Treatment
YS1
YS2
EZ1
EZ2
SE1
Pvalue
0.248
0.255
0.265
0.251
0.259
0.01
0.99
0.208
0.456
2.78a
58.89
0.240
0.495
3.01b
60.70
0.244
0.509
3.02b
60.81
0.233
0.484
2.98b
59.87
0.239
0.498
2.99b
60.07
0.01
0.02
0.03
0.57
0.84
0.96
0.05
0.34
69.29
68.76
74.41
73.34
73.51
72.42
71.17
70.30
72.40
71.36
1.25
1.26
0.44
0.52
C
SE: standard error of the means, C =Control, YS1=Local Ragi, YS2=Commercial
Yeasts, EZ1=Rumen Liquor, EZ2=Commercial Enzymes
17
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.
C
Initial weight
(kg)
Final weight
(kg)
ADG (g/h/d)
Total gain (kg)
Daily feed
intake (g)
Feed
conversion
ratio
Feed cost
(Rm/kg)
Sales price
(Rm/kg)
IOFC
Rm/day/hd)
YS1
17±1.41
17.5±1.73
YS2
16.8±1.26
19.25±1.71 20.25±1.72 20.00±1.63
64.29±14.29 78.57±14.29 92.80±14.29
2.25±0.50
2.75±0.50
3.25±0.82
EZ1
16.25±1.89
EZ2
16.25±0.96
19.00±1.41 19.25±1.50
78.57±14.29 85.21±23.33
2.75±0.50
3.00±0.82
456±90
498±80
509±110
484±80
498±50
7.09
6.34
5.48
6.16
5.84
1.10
1.20
1.35
1.20
1.25
15.00
15.00
15.00
15.00
15.00
0.46
0.58
0.70
0.60
0.66
C =Control, YS1=Local Ragi, YS2=Commercial Yeasts, EZ1=Rumen Liquor, EZ2=Commercial
Enzymes, ADG=Average Daily Gain, IOFC=Income Over Feed Cost
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
presented in (Table 9) indicate that addition of yeasts and enzymes in the
18
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.
The blood urea nitrogen (BUN) was 2.3, 2.8, 2.6, 2.0 and 2.3 mM/L at 0
hour post feeding, and 2.7, 2.9, 3.6, 3.1 and 3.7 mM/L at 4 hours post feeding
when goat was fed with supplementary diet of yeasts (YS1, YS2) and enzyme
(EZ1, EZ2), respectively (Table 9). Blood urea nitrogen concentration in this
study, show no significant differences (p>0.05) among the dietary supplemented
compared with control group.
19
Table 7 Effect of enzyme and yeasts culture addition on ruminal fermentation, pH,
NH3 and BUN concentration.
Treatment
P
C
YS1
YS2
EZ1
EZ2
SEM
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
39.4
39.4
39.5
39.4
39.3
0 h post feeding
6.65
6.78
6.74
6.63
6.64
0.02 0.23
4 h post feeding
6.22
6.31
6.23
6.18
6.18
0.04 0.47
6.44
6.55
6.48
6.41
6.41
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
48.16
45.15
44.77
46.19
45.77
0 h post feeding
2.3
2.8
2.6
2.0
2.3
0.23 0.26
4 h post feeding
2.7
2.9
3.6
3.1
3.7
0.25 0.26
2.5
2.8
3.1
2.6
3.0
0.27
Item
Rumen
value
temperature, °C
Mean
Ruminal pH
Mean
NH3-N (mmol/L)
Mean
BUN (mmol/L)
Mean
1
SE: standard error of the means, C =Control, YS1=Local Ragi, YS2=Commercial Yeasts, EZ1=Rumen
Liquor, EZ2=Commercial Enzymes
20
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
AND YEASTS CULTURE ON PERFORMANCE
OF GOAT FED DIETS CONTAINING
PALM KERNEL CAKE
ROBERT PAULIS
THE GRADUATE SCHOOL
BOGOR ARGICULTURAL UNIVERSITY
BOGOR
2013
STATEMENT OF RESEARCH ORIGINALITY
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
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.
Hasil penelitian menunjukkan bahawa konsumsi bahan kering dan
kecernaan bahan kering, bahan organik tidak menunjukkan perbedaan yang
nyata (p>0.05) pada semua kelompok perlakuan. Karakteristik cairan rumen
menunjukkan bahwa konsentrasi ammonia, plasma urea dan pH tidak berbeda
nyata di antara perlakuan. Konsentrasi total produksi VFA menunjukkan
perbedaan yang nyata (p35%
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.
The inclusion of exogenous enzymes and yeasts culture in animal
nutrition hopefully has a positive effect that improved NSPs degradation which
can increase the energy concentration and the release of NSPs-trapped nutrients
(Sheppy 2001). This may contribute to low cost productions in ruminant systems
using poor quality agriculture industries by products as major components
replacing the much expensive cereal grain and protein plant ingredient.
4
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 agroindustrial 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
This study focuses on addition of feed additives in basal diets for growing
male Feral x Local crossbreed goats. The experiment was done in the Pusat
5
Pembiakan Kambing (PPK) farm where Feral Local crossbred growing goats
were selected among the farm animals (Bongawan, Papar district, Sabah,
Malaysia) to carry out this study. The concentrate used in this study was supplied
to the farm from Feed Mill, Department of Veterinary Services and Animal
Industry, Sabah Malaysia. The local yeasts was purchased from Local Trader
(Sabah, Malaysia), it contains Saccharomyces cerevisiae 1.5 x 1010 cfu/g. The
rumen liquor was obtained from abattoir at the Sabah Meat Technology Centre
(SMTC) Sabah, Malaysia.
6
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
Rumen liquor were collected from slaughtered cattle from the abattoir
Sabah Meat Technology Centre (SMTC), Sabah Malaysia and transported to the
laboratory used flasks in ice container at 4°C. Samples were filtered in cold
condition using four layer of cheesecloth to separate the undigested matter. The
filtrate was centrifuged at 10,000 rpm (4°C) for10 minutes to separate
supernatant from microbial cells (Lee et al. 2002). The supernatant was filtered
used Wathman filter paper (No. 4) under suction to remove any micro particles.
Supernatant was then taken as a source of crude enzyme and stored at 4°C until
use.
7
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
The data obtained from the variable was statistically analyzed according
to descriptive analyses.
8
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
The in vitro procedure was modified from that reported by Tilley and
Terry (1963). Rumen liquor was taken from slaughtered cattle which were
obtained from abattoir at Sabah Meat Technology Centre (SMTC), Sabah,
Malaysia.
The fluid was strained through four layers of cheese cloth. The
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%
OM samples
10
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)
YC2
= B + 2.5 g/h/d Commercial Yeasts (5g/kg)
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.
Table 1 Ingredients and chemical compositions of the experimental diets
.
Ingredients
Proportion (%)
Palm cake kernel, PKC
50
Corn meal, CM
10
Soybean meal, SBM
5
Rice bran, RB
20
Pollard wheat
10
Salt
1
Mineral mix1
calcium phosphate
Molasses
1
1
2
1
Minerals and vitamins (each kg contains): Vitamin A: 10,000,000 IU;
Vitamin E:70,000 IU ; Vitamin D:1,600,000 IU; Fe: 50 g; Zn: 40 g;Mn: 40 g;
Co: 0.1 g; Cu: 10 g; Se: 0.1 g; I: 0.5
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
12
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
All the data were analysed as a Randomized Complete Block Design
(RCBD) using one way analyses of variance (ANOVA). . Treatment means were
statistically compared using Duncan‟s Multiple Range Test to identify
differences between means. Analysis of covariance (ANCOVA) was used to test
for differences in ADG weight change between the supplemented diets groups. In
each ANCOVA, the initial body weight of the animals was used as the covariate
to control for pre-existing differences. . A linear regression was used to correlate
individual ADG rates within each supplemented diets group. Homogeneity of
regression assumptions were tested and met in each analysis. Significance was
set at a p value of 0.05 for differences in means and correlation coefficients
13
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.
.
Table 2 Increment of total soluble sugar of degraded palm kernel cake with
rumen liquor enzymes
Rumen Liquor
enzymes
vol (ml/kg)
5
0h
4.149
24 hr
4.290
Increase of Total
Soluble Sugar
(%)
3.4
10
4.045
4.228
4.5
15
4.253
4.481
5.3
20
4.175
In Vitro Digestibility DM and OM
4.415
5.7
Incubation time (hr)
14
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.
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
IVDMD=In Vitro Dry Matter Digestibility, 2IVOMD=In Vitro Organic Matter Digestibility,
C=Control, YS1=Local Ragi, YS2=Commercial Yeasts, EZ1=Rumen Liquor ,
1
EZ2=Commercial Enzymes
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.
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).
Table 4 Chemical compositions concentrate, Brachiaria grass and Palm Kernel Cake
(DM)
Items
1
Unit
Concentrate
Brachiaria
Palm
grass
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
NDFDry Matter Intake
% and digestibility
37.1
67.7
DM OM
72.68
NDF = Neutral detergent fiber, 2ADF= Acid detergent fiber
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
16
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 (p0.05) 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
DMI, kg/d
Brachiaria grass,
kg/d
Concentrate,
kg/d
Total DMI, kg/d
DMI, %BW
DMI, g/kg W0.75
Apparent
digestibility %
DM
OM
1
Treatment
YS1
YS2
EZ1
EZ2
SE1
Pvalue
0.248
0.255
0.265
0.251
0.259
0.01
0.99
0.208
0.456
2.78a
58.89
0.240
0.495
3.01b
60.70
0.244
0.509
3.02b
60.81
0.233
0.484
2.98b
59.87
0.239
0.498
2.99b
60.07
0.01
0.02
0.03
0.57
0.84
0.96
0.05
0.34
69.29
68.76
74.41
73.34
73.51
72.42
71.17
70.30
72.40
71.36
1.25
1.26
0.44
0.52
C
SE: standard error of the means, C =Control, YS1=Local Ragi, YS2=Commercial
Yeasts, EZ1=Rumen Liquor, EZ2=Commercial Enzymes
17
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.
C
Initial weight
(kg)
Final weight
(kg)
ADG (g/h/d)
Total gain (kg)
Daily feed
intake (g)
Feed
conversion
ratio
Feed cost
(Rm/kg)
Sales price
(Rm/kg)
IOFC
Rm/day/hd)
YS1
17±1.41
17.5±1.73
YS2
16.8±1.26
19.25±1.71 20.25±1.72 20.00±1.63
64.29±14.29 78.57±14.29 92.80±14.29
2.25±0.50
2.75±0.50
3.25±0.82
EZ1
16.25±1.89
EZ2
16.25±0.96
19.00±1.41 19.25±1.50
78.57±14.29 85.21±23.33
2.75±0.50
3.00±0.82
456±90
498±80
509±110
484±80
498±50
7.09
6.34
5.48
6.16
5.84
1.10
1.20
1.35
1.20
1.25
15.00
15.00
15.00
15.00
15.00
0.46
0.58
0.70
0.60
0.66
C =Control, YS1=Local Ragi, YS2=Commercial Yeasts, EZ1=Rumen Liquor, EZ2=Commercial
Enzymes, ADG=Average Daily Gain, IOFC=Income Over Feed Cost
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
presented in (Table 9) indicate that addition of yeasts and enzymes in the
18
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.
The blood urea nitrogen (BUN) was 2.3, 2.8, 2.6, 2.0 and 2.3 mM/L at 0
hour post feeding, and 2.7, 2.9, 3.6, 3.1 and 3.7 mM/L at 4 hours post feeding
when goat was fed with supplementary diet of yeasts (YS1, YS2) and enzyme
(EZ1, EZ2), respectively (Table 9). Blood urea nitrogen concentration in this
study, show no significant differences (p>0.05) among the dietary supplemented
compared with control group.
19
Table 7 Effect of enzyme and yeasts culture addition on ruminal fermentation, pH,
NH3 and BUN concentration.
Treatment
P
C
YS1
YS2
EZ1
EZ2
SEM
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
39.4
39.4
39.5
39.4
39.3
0 h post feeding
6.65
6.78
6.74
6.63
6.64
0.02 0.23
4 h post feeding
6.22
6.31
6.23
6.18
6.18
0.04 0.47
6.44
6.55
6.48
6.41
6.41
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
48.16
45.15
44.77
46.19
45.77
0 h post feeding
2.3
2.8
2.6
2.0
2.3
0.23 0.26
4 h post feeding
2.7
2.9
3.6
3.1
3.7
0.25 0.26
2.5
2.8
3.1
2.6
3.0
0.27
Item
Rumen
value
temperature, °C
Mean
Ruminal pH
Mean
NH3-N (mmol/L)
Mean
BUN (mmol/L)
Mean
1
SE: standard error of the means, C =Control, YS1=Local Ragi, YS2=Commercial Yeasts, EZ1=Rumen
Liquor, EZ2=Commercial Enzymes
20
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