Materials and Methods - Information and Library Network …shodhganga.inflibnet.ac.in/bitstream/10603/6817/7/07... · · 2015-12-04MATERIALS AND METHODS ... multiplied and harvested

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III. MATERIALS AND METHODS

The materials and methods adopted for the present study are described under the

following headings:

3.1 Cultivation of Azolla

3.2 Chemical Evaluation of Azolla

3.3 Metabolism Assay in Broilers

3.3.1 Experimental Diets, Birds and Management

3.3.2 Collection of Samples and Analytical Procedure

3.3.3 Determination of Metabolizability of Energy and other Nutrients of Azolla

3.3.4 Ileal Nitrogen Absorbability

3.4 Metabolism Assay in Layers


3.4.2 Sample Collection and Analytical Procedure


3.5 Growth Performance in Broilers


3.5.2 Growth Performance Parameters

3.5.3 Carcass Characteristics and Organometry

3.5.4 Intestinal Tract Measurements

3.5.5 Production Performance Indices

3.5.6 Cost effectiveness of diets

3.6 Production Performance in Layers


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3.6.2 Egg Production Parameters

3.6.3 Egg Characteristics

3.6.4 Efficiency of Utilization of Protein and Energy


3.7 Statistical Analysis

3.1 Cultivation of Azolla

For initial screening, Azolla was cultivated, multiplied and harvested in cement

tanks near KVAFSU Library Bangalore (Plate-1 and 2). The cultivation was done in

28x3x6ft cement tank covered with plastic sheets. The culture Azolla pinnata for

multiplication was received from State Semen Collection Center, Hesseragatta and about

0.5kg/m2 of fresh Azolla seeds were inoculated into each tank. Each tank was dosed with

50 g of superphosphate at 15 days interval. First crop was harvested on 15th day of post-

inoculum and the subsequent harvest was at two days interval. The azolla immediately

after harvesting was washed with fresh water and dried in sun for 3 to 5 days and was

stored in plastic containers for further chemical evaluation (Plate-3).

However, the cultivated sample was not sufficient enough for carrying out

biological evaluation and hence the dried Azolla meal (AZM) (Plate-4) was procured

from Tamil Nadu Rice Research Institute, Aduthurai, Tanjore Dist. in bulk quantum. The

procured Azolla pinnata material was dried in sunlight for one day and dried material was

incorporated in the experimental rations.

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Plate 1 : Azolla pinnata grown in a Plate 2 : Fresh Azolla pinnata

cement tank

Plate 3 : Sun dried Azolla pinnata Plate 4 : Azolla meal (AZM)

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3.2 Chemical Evaluation of Azolla

The samples of fresh and dried Azolla pinnata which were cultivated at

Department of Animal Nutrition and the representative cultivated sample from farmers of

Raichur district and also sample obtained from Central Seed Procuring Institute,

Hessaraghatta and the sample from Tamil Nadu Rice Research Institute, Aduthurai,

Tanjore Dist (TNRRI), were ground through 1mm sieve and then subjected for analysis

of various nutrients.

The dried Azolla meal (AZM) samples were analyzed in triplicate for proximate

principles viz., dry matter (DM), crude protein (CP), ether extract (EE), total ash (TA),

crude fibre (CF) and nitrogen free extractives (NFE) as per the methods described by

AOAC (2005). The estimation of calcium (Ca) and phosphorus (P) was done by titration

method as per the procedures of Talapatra et al. (1975).

The gross energy content of dried AZM samples was determined using adiabatic

bomb calorimeter (AOAC, 2005) using benzoic acid as standard at the Dept. of Animal

Nutrition, Veterinary Research and Training Institute, TANUVAS, Namakkal, Tamil

Nadu.

3.3 Metabolism Assay in Broilers

The relative usefulness of a feed ingredient is measured by its metabolizable

energy (ME) content. Since no data is currently available as regards the energetic worth

of dried Azolla in poultry, an earnest effort was made to determine the ME value of dried

Azolla for broilers and layers separately. The procedures adopted to determine

metabolizabilty of energy in dried Azolla are explained hereunder:

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The experimental diets were prepared based on conventional system of replacing

varying level of basal diet by test ingredient as described by Sibbald and Slinger (1963).

A practical starter type of diet comprising of maize, soybean meal and vegetable oil was

prepared to serve as a basal mixture. The test diets were prepared by replacing 10 per

cent, 20 per cent and 30 per cent of the basal ration by the sun-dried AZM i.e., basal

mixture and test ingredient (dried Azolla) in the ratio of 90:10, 80:20 and 70:30,

respectively.

Further, the basal and test diets were supplemented with Fibre degrading enzyme

preparation @ 0.4g /kg to result in a set of 8 diets. The tailor made enzyme preparations

employed were received from M/s Kay Pee Yes Biotech Pvt. Ltd., Hebbal Industrial

Area, Mysore. The supplier reported that the fibre degrading enzyme preparation was

derived from Asperigillus species and claimed to contain fibre degrading enzymes viz.,

xylanase-2500, beta-glucanase–1000 and cellulase–500.

The dietary description is:

Enzyme supplementation Basal Mix 10% AZM 20% AZM 30% AZM

- T1 T3 T5 T7

+ T2 T4 T6 T8

The mineral sources and other additives were added over and above 100 per cent

to all the diets, so that a uniform supply of these was ensured after substitution of AZM in

the basal mixture. The chromic oxide (Cr2O3) as an inert indicator was added to all the

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diets at 0.5 per cent level to observe the ileal nitrogen absorbability. The detailed

ingredient composition of each diet is given in Table 3.1.

One hundred and sixty, one-day-old straight-run commercial broiler chicks

(selected crossbred of Hubbard strain) were distributed in to sixteen groups of 10 chicks

each (Plate-5). The birds were reared on raised wire floor battery brooders (plate-5) kept

in a well-ventilated and hygienic house. Ground maize was offered on first day while a

corn-soy type starter diet was prepared and offered to all the chicks uniformly for the

next 6 days. From 8th day onwards, the experimental diets were assigned randomly to two

duplicate of such groups. The diets were offered ad libitum to the corresponding birds in

colony feeders and potable water in PVC fountains for initial two weeks and in a

continuous channel from next week (3rd week) was provided. During the course of the

experiment, standard vaccination and managemental practices including brooding were

uniformly followed as per the commercial practice.

3.3.2 Collection of Samples and Analytical Procedure

On 19th, 20th and 21st day of the experiment, feed consumed by each group was

accurately measured and the excreta voided by such group was collected before the

beginning of the subsequent day at 09.00 a.m. for three consecutive days (Plate-6).

Utmost care was exercised to avoid contaminations from feathers, scales and debris. The

collected excreta samples were immediately weighed and dried for 24 h at 1000C in hot

air oven. The three-day dried excreta samples from each duplicate group were weighed

and pooled duplicate wise. Then the samples were allowed to equilibrate to atmospheric

conditions, ground to pass through 1mm sieve and well-homogenized samples were

stored separately in airtight polythene bags for further analysis.

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Table 3.1 : Ingredient and calculated nutrient composition of experimental diets

used in broilers' metabolism trial

Ingredients, Kg T1 T2 T3 T4 T5 T6 T7 T8

Basal Mix1 100 100 90 90 80 80 70 70

AZM - - 10 10 20 20 30 30Total 100 100 100 100 100 100 100 100

Di-calcium phosphate 1.93 1.93 1.93 1.93 1.93 1.93 1.93 1.93

Calcite powder 1.70 1.70 1.70 1.70 1.70 1.70 1.70 1.70

Salt 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35

Trace Minerals premix2 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10

Vitamin &additive mix3 1.04 1.04 1.04 1.04 1.04 1.04 1.04 1.04

Chromic oxide 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50

Fibredegradingenzyme4 - 0.40 - 0.40 - 0.40 - 0.40

Calculated composition

ME. Kcal/kg 3000 2700 2400 2100

CP, % 22.65 22.88 23.12 23.35

EE, % 3.41 3.34 3.27 3.2

CF, % 4.12 4.97 5.83 6.69

Ca, % 1.21 1.304 1.404 1.503

TP, % 0.808 0.889 0.97 1.051

Pav, % 0.501 0.526 0.552 0.578

1. Basal mix consisted of 61% maize, 38% soybean meal and 1% sunflower oil

2. Trace mineral premix contained Fe-90000, I-2000, Cu-15000, Mn-90000, Zn-80000 and Se-

300ppm

3. Vitamin and additive mix contained vitamin premix 0.05kg (each 1000g contained vit A-

25MIU, vit D3 –5.6MIU, vit E-60g, vit K -4g, vit B1- 4g, vit B2-10g, vit B6-6g, vit B12-0.03g,

Niacin-80g, Cal-d-pantothenate-30g, Folic acid-2g, Biotin-0.16g, Organic nutritive carrier-

qs), Hepatocare 0.05kg, Tylosin phosphate 0.05kg, Curotox 0.05kg, Choline chloride 0.05kg,

DL-methionine 0.195 kg and L-lysine 0.094kg.

4. Fibre degrading enzymes contained Xylanase 2500, β-glucanase1000 and cellulase 500 units

per gram

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Plate 5 : Metabolism trial in broiler birds

Plate 6 : Collection of excreta in broiler trial

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The dietary feed samples were also collected at the beginning and at the end of

the metabolism trial and dried in hot air oven to arrive at the dry matter per cent of such

samples. Further, the samples were ground to pass through 1mm sieve and stored in

plastic containers for further analysis.

The proximate analysis of experimental diets and excreta samples were performed

according to standard procedures (AOAC, 2005). Gross energy content of such samples

was determined by adiabatic bomb calorimeter (AOAC, 2005). Appropriate corrections

were made for differences in moisture content.

On completion of AME bioassay (end of 21st day), 5 birds from each duplicate

group were slaughtered by cervical dislocation. The body cavity was cut opened and the

entire intestinal tract was removed. The digesta content in the terminal 1/5 th segment of

the ileum was gently squeezed into a 120 ml reagent bottle containing 20 ml of 5%

H2SO4. The ileal digesta samples of birds from each duplicate group were pooled and

stored at -200C for further analysis.

At the time of analysis, the contents of ileal digesta were thawed, homogenized

and were made up to 100 ml volume. Duplicate samples of the homogenate were run

each for nitrogen and chromium oxide as per the methods described by Hill and

Anderson (1958). The nitrogen content of ileal digesta was obtained as per the standard

procedure (AOAC, 2005) while the chromic oxide content was analyzed

spectrophotometrically following the procedure of Fenton and Fenton (1979).

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The metabolizability of various nutrients of assay diets including gross energy

were calculated and then the metabolizability of energy and other components of AZM

were arrived at by using simultaneous equations.

a) Dry matter and other nutrients: The group wise dry matter metabolisability

(DMM) of diets was determined using the following formula (Han et al., 1976).

DMM (%) =Weight of the dry feed

consumed (g) } - { Weight of the driedexcreta voided (g) X 100

Weight of dry feed consumed (g)

Similarly, the metabolisability of organic matter (OM) and other nutrients, crude

protein (CP), ether extract (EE), crude fibre (CF) and nitrogen free extractives (NFE)

were determined using the formula given below.

Metabolisabilitycoefficient of nutrient

(%)=

(Unit nutrient intake) – (Unit nutrient out go)X 100

Unit nutrient intake

b) Metabolizable energy: Based on the gross energy (GE) values of assay diets and the

excreta samples determined by bomb calorimetry, the apparent ME (AME) values of

the diets were calculated by subtracting the GE of the excreta from GE intake divided

by total dry matter intake using the following formula:

AMEdiet (kcal/kg) =(Feed intake x GEdiet) - (Excreta output x GEexcreta)

Feed intake

The nitrogen corrected AME (AMEn) values were calculated using a factor of 8.22

kcal per g of nitrogen retained in the body (Hill and Anderson, 1958).

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c) Metabolizabile Energy Content of AZM: The ME content of the test AZM were

arrived by using simultaneous equation methods i.e., the difference between

measured digestibility or the calorific value for basal and test diets. Similarly,

metabolizability / digestibility coefficient of other nutrients of AZM were also arrived

at.

ME of test feedingredient (kcal/g) =

ME of test diet (kcal/g) - ME of control diet (kcal/g) x 0.7

0.3

3.3.4 Ileal Nitrogen Absorbability

The ileal nitrogen digestibility (IND) of assay diets was calculated using Chromic

oxide as a marker and given as follows:

IND (%) =[(N/Cr)d – (N/Cr)i]

X 100(N/Cr)d

Where (N/Cr) d = the ratio of nitrogen to Cr in diet and

(N/Cr) i = the ratio of nitrogen to Cr in ileal digesta

3.4 Metabolism Assay in Layers

Since there are differences within type of birds with respect to ability to

metabolize energy or digest other nutrients, a metabolism trial was also conducted with

egg type chicken to arrive at ME worth of AZM in layers as well.


The dietary design employed here is very much similar to that of broilers

metabolism trial and described under Sec. 3.3.1. A conventional practical type layer diet

comprising maize, soybean meal, sun flower extraction and DORB was prepared to serve

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as basal mixture (T1). The test diets were prepared by incorporating sun dried AZM at 10

(T3), 20 (T5) and 30 % (T7) respectively replacing the corresponding proportion of basal

mixture. Further, a parallel set of another 4 diets were prepared by supplementing each

diet with fibre degrading enzyme preparation @ 0.5g /kg to result in T2, T4, T6 and T8

correspondingly. The enzyme preparations added in such diets were same as those

employed in broiler metabolism trial. The rest of the components in the diets namely

shell grit, mineral mixture, common salt and vitamin premix were maintained in all diets

at uniform level. The chromic oxide (Cr2O3) as an external indicator was added to all

diets at 0.5 per cent level. The detailed ingredient composition of each diet is given in

Table 3.2.

A total of one hundred twenty-eight BV-300 commercial layers of about 32 weeks

age and uniform body weight with proper history were selected and randomly divided

into 32 groups of 4 birds each in a commercial poultry farm (Sri Rama Poultry Farm,

Channapatna) (Plate-7). The four groups were housed alternatively in colony cages with

each cage unit measuring 15’’x15’’x18” size. Each of the 8 diets described above was

offered to four replicates of four hens each. Partitioning was made in feed trough to avoid

mixing up of test diets. The rest of the managemental practices were kept uniform.

3.4.2 Sample Collection and Analytical Procedure

After conditioning the laying hens with their corresponding experimental diets for

11 days, a conventional 3-day metabolism trial was carried out, during which the feed

intake and excreta output (Plate-8) from each group of birds was measured quantitatively

to obtain the ME data.

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Table 3.2 : Ingredient and calculated nutrient composition of experimental diets

used in layer metabolism trial

Ingredients, Kg T1 T2 T3 T4 T5 T6 T7 T8

Basal mix 1 100 100 90 90 80 80 70 70

Azolla - - 10 10 20 20 30 30Total 100 100 100 100 100 100 100 100

Shell grit 8 8 8 8 8 8 8 8

Mineral mixture 2 3.50 3.50 3.50 3.50 3.50 3.50 3.50 3.50

Salt 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.39

Vitamin and additive mix3 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25

Chromic oxide 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50

Fibre degrading enzymes4 - 0.5 0.50 - 0.50 0.50

Calculated composition

ME. Kcal/kg 2922.5 2630.25 2338 2045.75

CP, % 19.64 17.676 15.712 13.748

EE, % 2.645 2.3805 2.116 1.8515

CF, % 6.06 5.454 4.848 4.242

1. Basal mix consisted of 55% maize, 14% soybean meal, 18% DORB and 13% sunflower

extractions

2. Mineral mixture contained : Calcium-32%, Phosphorus-6% and other trace minerals

3. Vitamin and additive mix contained AB2D3K- 0.1kg (each 1000g contained vit A-50MIU, vit

D3 –5.6MIU, vit E-60g, vit K -4g, B-complex-0.2kg, (vit B1-4g, vit B2-10g, vit B6-6g, vit B12-

0.03g, niacin-80g, Cal-d-pantothenate-30g, folic acid-2g, biotin-0.16g, organic nutritive

carrier-qs), Hepatocare 0.05kg,., Lysine - 0.013kg, DL-methionine 0.07kg

4. Fibre degrading enzymes contained : Xylanase 2500, β-glucanase1000 and cellulase 500

units per gram

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Plate 7 : Metabolism trail in layer birds.

Plate 8 : Collection of excreta in layers

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The procedure adopted for collection and processing and chemical analysis of

excreta and diet samples were similar to that of broilers’ metabolism trial, described

under section 3.3.2.


The metabolizable energy and other components of AZM in layers were arrived

by methods which were very much similar to those of broilers’ metabolism study

(Section 3.3.3).

3.5 Growth Performance in Broilers

A feeding trial was conducted to asses the effect of dietary inclusion of AZM at

different levels on performance of broilers such as body weight gain, feed consumption

and feed efficiency, carcass characteristics and organometry, nutrient utilization and cost

effectiveness of AZM inclusion. A brief account about the performance trial in broilers is

presented hereunder:


Five iso-nitrogenous and iso-caloric diets were prepared by incorporating sun

dried AZM at 0, 2.5, 5.0, 7.5 and 10 per cent levels to meet the nutrient requirements

specified by Bureau of Indian Standards (BIS, 1992). Further, another set of five diets

was prepared by supplementing each diet with fibre degrading enzyme preparation @

0.4g /kg. Such diets were prepared separately for each phase i.e., starter (0-14 days),

grower (15-28 days) and finisher (29 to 42 days) phases. The ingredients calculated

nutrient composition of different diets compounded for different phases are detailed in

Tables 3.3.

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Table 3.3 : Ingredient composition of experimental diets compounded for different phases during performance trial of

broilers

Ingredients, KgBroiler starter diet Broiler grower diet Broiler finisher diet

0%AZM

2.5%AZM

5%AZM

7.5%AZM

10%AZM

0%AZM

2.5%AZM

5%AZM

7.5%AZM

10%AZM

0%AZM

2.5%AZM

5%AZM

7.5%AZM

10%AZM

Maize 55.4 55.6 53.4 52.0 53.5 59.8 60.0 57.0 55.4 53.6 63.6 63.6 62.0 60.0 58.4Soybean meal 38.5 37.0 36.0 35.0 30.0 34.3 33.2 32.1 31.0 30.0 30.0 28.0 26.7 26.0 25.0Lecithin oil 2.5 1.5 2.1 2.2 3.3 2.3 1.9 2.4 2.8 3.1 2.8 2.4 2.6 3.1 3.2Di-calcium phosphate 1.9 1.9 1.8 1.7 1.6 1.9 1.9 1.8 1.7 1.6 1.9 2.0 2.0 1.8 1.7Calcite powder 1.4 1.2 1.4 1.3 1.3 1.4 1.2 1.4 1.3 1.4 1.4 1.2 1.4 1.3 1.4Salt 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3Azolla meal 0 2.5 5.0 7.5 10.0 0 2.5 5.0 7.5 10.0 0 2.5 5.0 7.5 10.0Additives1 + + + + + + + + + + + + + + +

Total 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100Chemical composition, %

CP 22.71 22.76 22.72 22.75 21.01 21.01 21.01 21.00 21.01 21.01 19.11 19.13 19.10 19.10 19.12EE 4.68 3.77 4.33 4.63 5.60 4.61 4.27 4.74 5.13 5.36 5.17 4.82 5.04 5.52 5.62CF 4.03 4.26 4.45 4.67 4.61 3.79 4.00 4.20 4.41 4.61 3.52 3.74 3.95 4.15 4.36Ca 1.08 1.05 1.12 1.09 1.10 1.05 1.04 1.09 1.08 1.09 1.03 1.02 1.09 1.07 1.11TP 0.83 0.82 0.84 0.85 0.85 0.80 0.81 0.81 0.84 0.84 0.78 0.80 0.80 0.82 0.84Pav 0.53 0.51 0.51 0.51 0.52 0.51 0.51 0.50 0.52 0.50 0.51 0.51 0.51 0.51 0.51ME, kcal/kg 2943 2944 2943 2943 2944 2944 2944 2944 2944 2945 3007 3007 3007 3008 3008Cost, Rs/kg 11.46 11.35 11.53 11.78 11.76 11.03 10.94 11.21 11.49 11.68 10.76 10.69 10.91 11.20 11.39

1 Additives contained Brovit plus- 0.5kg (each 500g contained vit A-12.5 MIU, vit D3-2.8 MIU, vit. E-30g, vit K-2g, vit B1-2g, vit B2-5g, vit B6-3g, vit B12-0.015g, niacin-40g, Cal-d-panthothenate-15g, folic acid-1g, biotin-0.08g, organic nutritive carrier.-q.s), Tylosine phosphate-0.05kg;Hepatocare-0.1kg; Choline chloride-0.05kg; Curodox-0.05kg., DL-Methionine-200g, L-Lysine-100g.

Note: Parallely, another set of 5 diets with AZM at 0, 2.5, 5, 7.5 and 10% were also prepared using fibre degrading enzyme@ 0.05kg per quintalfor all stages.

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The design was aimed to study the influence of inclusion of AZM at different

levels on broiler performance as well as for increasing the efficiency of AZM utilization

through biotechnological approaches and is described as follows:

Enzymesupplementation

0%AZM

2.5%AZM

5%AZM

7.5%AZM

10%AZM

- T1 T3 T5 T7 T9

+ T2 T4 T6 T8 T10

A total of 200 one-day-old straight-run commercial chicks (Hubbard) were

randomly divided into 20 groups of 10 chicks each. The birds were housed in raised wire

floor battery brooders of single tier system, which were kept in a well-ventilated hygienic

house. The brooding was done up to two weeks of age as per the standard practice and all

the birds were vaccinated against New Castle disease and Infectious Bursal disease on 8 th

and 14th day of age, respectively. Each of the 10 diets were offered randomly to duplicate

groups of 10 chicks each. The diets in colony feeders were provided to the corresponding

birds ad libitum and potable water was provided in PVC fountains (initial 2 weeks) or in

a continuous channel along with PVC fountains (3rd week onwards) was made available

round the clock. The rest of the managemental practices were accomplished uniformly

for all the birds during the 42-day experimental period.

3.5.2 Growth Performance Parameters

Several parameters were studied to observe the effect of inclusion of AZM in

broiler diets with biotechnological approaches and are described hereunder:

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a) Body Weight Gain: The weekly individual body weights of birds were recorded and

the body weight gains were arrived at for each week. The body weight gain was also

calculated for starter, grower and finisher phases and on a cumulative basis.

Accordingly, the body weight gains in different dietary groups were compared as per

treatment wise and main factors (AZM levels and Enzyme supplementation) wise.

b) Feed Consumption: The daily feed offered to all individual groups was accurately

recorded and at the end of each week, the residual feed, as well as spilled over feed

from that group was accounted. Care was also taken to subtract the amount of feed

consumed by the birds that died during a particular week by calculating the daily feed

consumption during that week. Thus the average feed consumption in different

groups was calculated during each week as well as during starter phase (0 to 14 days),

grower phase (15 to 28 days) and finisher phase (29 to 42 days) and cumulatively (0

to 42 days).

c) Feed Conversion Ratio: The feed conversion ratio (FCR) expressed as the ratio of

amount of feed consumed to the body weight gained under each group of birds

arrived at each week and each phase wise and also cumulatively.

d) Mortality: Mortality in respective groups was recorded as and when the birds died.

The dead birds were necropsied to identify the dietary cause, if any. The physical

consistency and the colour of fecal droppings of various groups were under

observation from time to time.

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3.5.3 Carcass Characteristics and Organometry

At the end of the experiment (42nd day), two birds (1 male and 1 female) from

each replicate, were separated and starved for 12 hrs however with a provision of plenty

of water. Then immediately after recording their live body weights (pre-slaughter bird

weight), the birds were sacrificed by cervical dislocation and the carcasses were

subjected for the study of following parameters:

a) Dressing Percentage: The slaughtered birds were defeathered, denecked and

eviscerated along with two legs beneath the hock joint to observe the effect of

various experimental diets on the dressing percentage. The dressing percentage was

calculated as the percent of the carcass weight obtained after removing the feathers,

neck, legs and internal viscera, to its live body weight.

b) Organometry: From the sacrificed birds, different organs viz., heart, liver, gizzard,

proventriculus, spleen and bursa and the breast meat portion as well as abdominal fat

were carefully separated and weighed to observe the effect of different dietary

treatments on growth and development of certain organs. The procedures followed

are briefed here under:

1. Giblets: The weight of the giblet organs viz., heart without pericardium, liver with

out gall bladder, gizzard with out the food contents and internal lining membrane and

proventriculus from each sacrificed bird were recorded (Plate-9) and expressed as the

per cent of pre-slaughter bird weight as well as dressed weight as follows:

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Relative heartpercentage =

Heart weigth (g)x 100

Live weight (g)

Relative liverpercentage =

Liver weight (g)x 100

Live weight (g)

Relative gizzardpercentage =

Gizzard weight (g)x 100

Live weight (g)

Relativeproventriculus

percentage=

Proventriculus weight (g)x 100

Live weight (g)

2. Lymphoid organs: The weights of the lymphoid organs viz., bursa and spleen from

each sacrificed bird was recorded and expressed as the percent of pre-slaughter bird

weight (g/100g).

3. Abdominal fat weight: The weight of the fat in abdomen including the fat

surrounding gizzard, bursa, cloaca and adjacent muscles of each bird was recorded

and expressed as percent of concerned pre-slaughter bird weight.

4. Breast meat yield: The weight of the breast meat from each sacrificed bird was

recorded (Plate-10) and expressed as the percent of pre-slaughter bird weight

(g/100g).

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Plate 9 : Organometry of broiler performance trial.

Plate 10 : Breast meat of broiler performance trial

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3.5.4 Intestinal Tract Measurements

Along with other parameters of organometry study, the intact digestive tract of all

sacrificed birds were carefully removed and the length of different segments of intestine

viz., duodenum, jejunum and ileum were measured as follows:

Length of duodenum = the length of pancreatic loop

Length of jejunum = from pancreatic loop to Meckel’s diverticulum

Length of ileum = from Meckel’s diverticulum to ileocaecal junction

The length of each segment was expressed in terms of percentage of pre-slaughter

body weight (cm/100g).

3.5.5 Production Performance Indices

The cost of the starter, grower and finisher types of all the experimental diets was

arrived at by considering the prevailing prices of the constituent feed ingredients and feed

additives including that of enzymes. The relative cost effectiveness of each diet was thus

assessed. Further, by considering the prevailing sale price of the broiler bird, the

Performance Index Score (PIS) and the Economic Index Score (EIS) were arrived at i.e.,

1) PIS =Average final body weight (g) x % livability

FCR x No, of days reared x 10

2) EIS =PIS

Cost of diet (Rs/kg)


The cost of each diet prepared during broilers’ feeding trial was arrived at by

considering the prevailing prices of the constituent feed ingredients, mineral salts and

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other additives including that of enzymes. Further, by considering the sale price of broiler

chicken including the expenditure on chicks, labour, medicine etc., the net profit for each

treatment was calculated separately for broilers under different treatments. The relative

cost effectiveness of each diet was thus assessed.

3.6 Production Performance in Layers

By making use of the ME value of AZM obtained from layers’ metabolism trial,

AZM was included at 0, 2.5, 5.0, 7.5 and 10 per cent levels in layers diets and evaluated

in terms of egg production, feed consumption, feed conversion efficiency, egg

characteristics (egg weight, Haugh unit, shell thickness, yolk colour etc.), nutrient

utilization and efficiency of utilization of energy and protein. At the end, the cost

effectiveness of AZM was also being assessed. The 84-day layer production performance

trial was conveniently divided into 3 periods of 28 days duration each. A brief description

about the materials and methods adopted for feeding cum production performance trial in

layers are presented hereunder:


a) Experimental dietary design : The practical type layer diet (control) involving

maize and DORB as the main energy sources and soybean meal and sunflower

extraction as the protein sources with shell grits, calcite powder, di-calcium

phosphate and salts of pertinent trace minerals was prepared to conformed to the

nutrient specification of Bureau of Indian Standards (BIS, 1992). In the test diets, the

sun dried AZM was included at four different levels (2.5, 5.0, 7.5 and 10 per cent,

respectively). Further, these five iso-nitrogenous and iso-caloric diets were

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supplemented with fibre degrading enzyme preparation @ 0.5g/kg diet to result in a

set of five test diets, a design similar to that described under section 3.4.1., was aimed

to study the effect of inclusion of AZM at different levels as well as with the hope of

increasing the efficiency of AZM utilization through biotechnological approach. The

detailed ingredient and calculated nutrient composition of each diet is given in Table

3.4.

b) Experimental birds: After a gap of 15 days following a metabolism trial, the same

birds were used for production trial. One hundred sixty BV-300 commercial layers of

about 35 weeks age were redistributed randomly into 10x4x4 birds. The birds were

housed in three tier colony cages. Four birds were housed in each cage measuring

15’’x15’’x18” size to serve as a replication. Each of the 10 diets (Sec. 3.6.1) were

offered to four such replications of 4 birds each in colony cage units. The birds were

placed in cages in such a way that there was one empty cage between the two groups

to limit the availability of particular diet to another group of birds. Added to it, the

continuous feed trough was partitioned with a partition material to avoid mixing up of

test diets, if any. By and large, the distribution of replications of treatments was

essentially uniform.

The birds were maintained under standard managemental conditions including

periodical deworming, preventive or therapeutic disease control, lighting programme,

feeding frequency, watering methods and other routine bio-security aspects. The

experiment lasted for 84 days which was conveniently divided into three 28-day interval

periods.

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Table 3.4 : Ingredient and nutrient composition of experimental diets compounded

during performance trial of layers

Ingredients, kg 0 % AZM 2.5% AZM 5% AZM 7.5% AZM 10% AZM

Maize 43.5 43.6 43.5 43.6 43.6

De-oiled rice bran 17.0 16.0 14.9 14.4 13.5

Soybean meal 17.4 16.9 16.1 15.8 15.8

Sunflower extraction 10.6 9.6 9.2 7.6 6.0

Di-calcium phosphate 1.1 1.0 1.0 0.9 0.9

Calcite powder 3.1 3.1 3.0 3.0 2.9

Shell grit 7.0 7.0 7.0 7.0 7.0

Salt 0.3 0.3 0.3 0.3 0.3

Azolla meal 0 2.5 5.0 7.5 10.0Additives1 + + + + +

Total 100 100 100 100 100

Chemical composition, %

CP 17.61 17.57 17.56 17.50 17.51

EE 1.95 1.99 2.04 2.08 2.12CF 7.15 7.05 7.12 6.91 6.81

Ca 3.93 3.93 3.92 3.92 3.91

TP 1.83 0.76 1.83 0.76 1.82

Pav 0.32 0.31 0.32 0.31 0.31

ME, Kcal/kg 2439 2439 2439 2439 2439

Cost, Rs/kg 10.82 10.91 11.04 11.13 11.25

1 Additives contained AB2D3K- 100g (each 500g contained vit A-12.5 MIU, vit D3-2.8 MIU, vit.

E-30g, vit K-2g), B-complex-200g (vit B1-2g, vit B2-5g, vit B6-3g, vit B12-0.015g, niacin-40g,

Cal-d-panthothenate-15g, folic acid-1g, biotin-0.08g, organic nutritive carrier.-q.s), Tylosine

phosphate-0.05kg; Hepatocare-0.1kg; Choline chloride-0.05kg and toxin binder-0.075kg., DL-

Methionine-0.05kg, L-Lysine-0.02kg

Note: Parallely, another set of 5 diets with AZM at 0, 2.5, 5, 7.5 and 10% were also prepared

using fibre degrading enzyme @ 0.05kg per quintal.

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3.6.2 Egg Production Parameters

Various egg production performance parameters were considered to critically

evaluate the possibility of inclusion of AZM in layer diets in combination with

biotechnological approach for enhancing its nutritive value and are described hereunder:

a) Body weights: Individual body weights of birds were recorded at beginning of the

experiment as well as at every 28-day interval to monitor the pattern of body weight

changes, if any, due to dietary regimen. Group wise average weights under different

treatments were arrived and the period wise changes (loss / gain) in body weight were

arrived at. The weighing of the birds was done in the early hours of the day before

feeding and oviposition.

b) Egg production: Every day in late hours, the number of eggs produced in a particular

replicate group was recorded and the hen day egg production was arrived at on the

basis of three 28-day periods as per various treatments for further statistical analysis.

The rate of egg production was calculated both for the individual periods and on

cumulative basis as well.

c) Daily feed consumption: An amount of 120 g of feed per bird per day was

collectively offered to the four birds of replicate group during the experiment period.

Subsequently, the daily required amount of concerned diet was adjusted based on the

pattern of feed being consumed by the particular group of birds. The diets were

offered in divided doses of about 50 per cent in morning and the remaining 50 per

cent in the late afternoon hours. The feed residue, if any, was recorded in each group

at the end of 28-day period.

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d) Feed efficiency: The amount of feed consumed to produce one kg egg mass and/or

dozen eggs in each dietary replication was calculated. Such feed efficiency values

(kg feed/kg egg mass or kg feed/dozen eggs) were calculated both period-wise and

cumulatively.

e) Mortality: The health status of the birds of various groups was under constant

observation from time to time. The dead birds were subjected for post mortem

examination to identify the dietary causes, if any. The physical consistency of fecal

droppings of various groups was under periodical observation.

3.6.3 Egg Characteristics

On the terminal day of every 28-day interval, all the eggs produced from different

replicate groups were collected and were weighed individually during the experimental

period of 84 days. Further, on the immediate next day, each egg was broken and the

entire contents were carefully placed on a glass slab for measurement of albumen and

yolk indices. The values were then arranged according to treatments and main factors

wise under each 28-day period as well as cumulatively.

a) Egg weight: On every Tuesday and Friday of each week, egg weights were recorded

and in addition, egg weights were also taken at the end of 28-day periods. The

weights so obtained on eight occasions during a particular 28-day (4-week) period

were averaged and the data were arranged as per treatments and main factors.

b) Egg shape index: The breadth and length of each egg (unbroken) was measured by

using Vernier Calipers as described by Reddy (1979) and egg shape index (ESI) was

calculated as:

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ESI (%) =Horizontal diameter (breadth) in cm

X 100Vertical diameter (length) in cm

c) Albumen index : The height and diameter of egg albumen were obtained using Ames

Haugh Unit Spherometer and Vernier Caliper (Plate-11), respectively and the

albumen index (AI) was calculated as:

AI=Albumen height in mm

X 100Albumen diameter* in mm

*Average of albumen length (mm) and albumen width (mm)

d) Haugh unit score: After recording the height of albumen at two places (one near to

yolk and the other at the end of dense albumen) by using Ames Haugh unit meter and

the relationship between egg weight and albumen height for each egg was calculated

as Haugh unit score (Haugh, 1937) which was calculated as follows:

Haugh unit = 100 x log (H + 7.57 – 1.7 x W0.37)

Where H= albumen height (mm) and W= egg weight (g)

e) Egg yolk colour: The colour of yolk of every broken open egg was scored by

matching (contrast) technique using Roche yolk colour fan (Roche Company, 1969).

Its colour value denotes the colour intensity from 1 to 14 according to the degree of

yolk colour (Plate-12, Plate-13 and Plate-14).

f) Yolk index: The height (at centre place) and diameter of the yolk was measured

using Ames Haugh Unit Spherometer and Vernier Calipers (Plate-15) and the yolk

index (YI) was calculated as:

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Plate 11 : Albumen index score

Plate 12 : 28th day yolk colour Plate 13 : 84th day yolk colour

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Plate 14 : The yolk colour of eggs collected during the 84-days layer production trial as influenced by AZM

at different levels

0% AZM 2.5%AZM 5%AZM

7.5%AZM 10%AZM Magnified yolk colour at 10%AZM

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Plate 15 : Yolk index score.

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YI (%) =Yolk height in mm

X 100Yolk diameter in mm

g) Shell thickness: Following the breaking of egg, the shell pieces devoid of shell

membranes at broad end and narrow end and at middle were carefully selected and

their thicknesses were measured using a digital calipers as described by Ogunmodde

and Oguntella (1971). The average of these two pieces was represented as shell

thickness.

3.6.4 Efficiency of Utilization of Protein and Energy

The gross efficiency of converting dietary protein and metabolisable energy to

those of eggs under all treatments and during different periods was calculated based on

intake and transfer of the said nutrients as described below:

a) Protein utilization: Gross efficiency of protein utilization (EPU) was calculated as

follows:

EPU (%) =Total egg mass produced X 0.12

X 100Total dietary protein intake

Where, 0.12 represents the unit of protein present in one unit of egg.

b) Energy utilization: Gross efficiency of energy utilization (EEU) was calculated as

follows:

EEU (%) =Total egg mass produced X 1.6

X 100Total dietary ME intake

Where, 1.6 represents the amount of kcal per every gram of egg mass as

described by Reddy (1979).

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The cost of each diet prepared during layers’ feeding trial was arrived at by

considering the prevailing prices of the constituent feed ingredients, mineral salts and

other additives including that of enzymes.. Further, by considering the sale price of eggs

including the expenditure on labor, medicine etc., the net profit for each treatment was

calculated separately for layers under different treatment. The relative cost effectiveness

of each diet was thus assessed

3.7 Statistical Analysis

The data generated during the biological trials in broiler and layers involving

AZM at different levels and enzyme supplementation were analyzed manually using

Microsoft excel based one way ANOVA (CRD) for the treatments and two way ANOVA

(CRD) for the main effect of dietary regimen (AZM level and Enzyme supplementation)

according to the procedures described by Snedecor and Cochran (1989). All the

percentage values were transformed to arc sin values before analysis. The differences in

means were tested by using Duncan’s Multiple Range Test (Duncan, 1955) when the test

was significant.

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