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i
NUTRITIONAL EVALUATION OF AZOLLA
(Azolla pinnata) AND ITS SUPPLEMENTARY EFFECT ON
IN VITRO DIGESTIBILITY OF CROP RESIDUES AND
TOTAL MIXED RATION
KAVYA, K.
DEPARTMENT OF ANIMAL NUTRITION
VETERINARY COLLEGE, BANGALORE
KARNATAKA VETERINARY, ANIMAL AND FISHERIES
SCIENCES UNIVERSITY, NANDINAGAR, BIDAR
JUNE, 2014
ii
NUTRITIONAL EVALUATION OF AZOLLA
(Azolla pinnata) AND ITS SUPPLEMENTARY EFFECT ON
IN VITRO DIGESTIBILITY OF CROP RESIDUES AND
TOTAL MIXED RATION
Thesis submitted to the
KARNATAKA VETERINARY, ANIMAL AND FISHERIES
SCIENCES UNIVERSITY, BIDAR
In partial fulfillment of the requirements
for the award of the degree of
MASTER OF VETERINARY SCIENCE
in
ANIMAL NUTRITION
By
KAVYA, K.
DEPARTMENT OF ANIMAL NUTRITION
VETERINARY COLLEGE, BANGALORE
KARNATAKA VETERINARY, ANIMAL AND FISHERIES
SCIENCES UNIVERSITY, NANDINAGAR, BIDAR
JUNE, 2014
iii
KARNATAKA VETERINARY, ANIMAL AND FISHERIES
SCIENCES UNIVERSITY, BIDAR
DEPARTMENT OF ANIMAL NUTRITION
VETERINARY COLLEGE, HEBBAL, BANGALORE
CERTIFICATE
This is to certify that the thesis entitled “NUTRITIONAL EVALUATION OF
AZOLLA (Azolla pinnata) AND ITS SUPPLEMENTARY EFFECT ON IN VITRO
DIGESTIBILITY OF CROP RESIDUES AND TOTAL MIXED RATION’’
submitted by Ms. KAVYA, K., ID No. MVHK 1204 in partial fulfillment of the
requirements for the award of MASTER OF VETERINARY SCIENCE in ANIMAL
NUTRITION of the Karnataka Veterinary, Animal and Fisheries Sciences University,
Bidar is a record of bonafide research work carried out by her during the period of her
study in this University under my guidance and supervision and the thesis has not
previously formed the basis for the award of any degree, diploma, associateship,
fellowship or other similar titles.
Place: Bangalore (Dr. T. M. PRABHU)
Date: June, 2014 Associate Professor
Major advisor
Approved by:
Chairman : ______________________________
(Dr. T.M. PRABHU)
Members : 1. ______________________________
(Dr. R. GIDEON GLORIDOSS)
2. ______________________________
(Dr. K. CHANDRAPAL SINGH)
3. ______________________________
(Dr. Y. B. RAJESHWARI)
4. ______________________________
(Dr. SIDDARAMANNA)
iv
Affectionately Dedicated To
My Beloved Parents
v
ACKNOWLEDGEMENT
I wish to express my deep sense of gratitude to Dr. T. M. Prabhu, Associate
Professor, Department of Animal Nutrition, Veterinary College, Bangalore and the
Chairman of my Advisory Committee. My sincere thanks are due to his constant
supervision, valuable suggestions, continuous encouragement and his efforts for
providing me the atmosphere and facilities that were needed for completing this work
successfully.
I wish to express my profound gratitude and sincere respect to
Dr. R. Gideon Gloridoss, Professor and Head, Department of Animal Nutrition,
Veterinary College, Bangalore and member of my Advisory Committee for his inspiring
guidance, valuable suggestions, constant encouragement throughout my study.
I express my gratitude to my Advisory Committee members
Dr. K. Chandrapal Singh, Head, Division of Animal and Poultry Science, KVAFSU,
Bidar, Dr. Y. B. Rajeshwari, Professor, Department of Livestock Production and
Mangement and Dr. Siddaramanna, Senior Technical Officer, NDRI, Adugodi,
Bangalore for their kind assistance, co-operation and suggestions
I am extremely thankful to Dr. U. Krishnamoorthy, Professor and Head,
Department of Livestock Production and Management, Veterinary College, Bangalore for
providing me the laboratory facilities for in vitro studies.
My sincere thanks to Dr. N. K. Shivakumar Gowda, Principal Scientist (Animal
Nurtrition), NIANP, Adugodi, Bangalore for providing me the Laboratory facilities to
analyse mineral profile of samples.
My sincere thanks to Mr. Hemanth Pandey, Research Scientist, Alltech
Ruminant Nutrition Laboratory, KVAFSU, Bangalore for providing me the Laboratory
facilities for in vitro studies.
vi
My sincere thanks to Dr. Kiran Doranalli, Regional Technical Manager (Asia
South), Health and Nutrition –Feed Additives, Evonik (SEA) Private Limited, Singapore
for his help to analyze the amino acid composition of Azolla at Evonik laboratory,
Singapore.
I express my heartfelt thanks and gratitude to my seniors Dr. Shivasharanappa
Biradar, Dr. Vikram, Dr. Venkatesh B. S, Dr. Anjaneya and Dr. Basvanth Kumar
for their constant help, encouragement and support.
It is my privilege to extend sincere thanks to my friends Dr. Thahir Ahamed,
Dr. Kavya. P. S, Dr. Sharadha and Dr. Pradeep for their cordial help and cooperation
during my research work.
My special thanks to the supporting staff such as Sri Rangaswami and
Smt. Shakuntala, Department of Animal Nutrition for their constant help during the
period of study.
It is family members without whose support and encouragement, I would not be
in this position today. At this explicable moment, I wish to express my gratitude to my
father Sri. R. Kalidas, mother Smt. G. Lakshmi and sister Soumya. K for their moral
support and encouragement which enabled me to achieve this today.
June, 2014
Bangalore (KAVYA, K.)
vii
CONTENTS
CHAPTER TITLE PAGE No.
I INTRODUCTION 1-4
II REVIEW OF LITERATURE 5-25
III MATERIALS AND METHODS 26-37
IV RESULTS 38-66
V DISCUSSION 67-76
VI SUMMARY 77-80
VII BIBLIOGRAPHY 81-89
VIII ABSTRACT 90
IX APPENDICES 91-106
viii
LIST OF TABLES
Table
No. Title of Table
Page
No.
2.1 Per cent chemical composition (DMB) of aquatic plants and alfalfa. 6
2.4.1 Per cent chemical composition of different species of Azolla (Dry matter
basis). 14
2.4.2 Chemical composition (on per cent dry matter basis) of Azolla pinnata. 16
2.4.3 Chemical composition of Azolla varieties 18
2.4.4 Amino acid composition of azolla. 20
2.5.1 Chemical composition of varieties of paddy straw. 22
2.5.2 Digestibility of OM and ME content of different varieties of paddy straw. 22
2.5.3 Chemical composition of rice straw in early, mid and late growing period. 23
4.1.1 Proximate principles (on per cent dry matter basis) of Azolla pinnata,
Crop residues and CFM. 39
4.1.2 Fiber fractions, total silica, biogenic silica and sand silica (on per cent dry
matter basis) of Azolla pinnata, crop residues and CFM. 41
4.1.3 Mineral profile of Azolla pinnata. 43
4.1.4 Amino acid profile of Azolla pinnata 44
4.2.1
Chemical composition (Percentage on DMB) of various diets comprising
paddy straw supplemented with different levels of azolla from 0 to 9 per
cent and the TMR comprising paddy straw and CFM supplemented with
different levels of azolla from 0 to 9 per cent.
45
4.2.2
Chemical composition (Percentage on DMB) of various diets comprising
ragi straw supplemented with different levels of azolla from 0 to 9 per
cent and the TMR comprising ragi straw and CFM supplemented with
different levels of azolla from 0 to 9 per cent.
46
4.2.3
Chemical composition (Percentage on DMB) of various diets containing
maize stover supplemented with different levels of azolla from 0 to 9 per
cent and the TMR comprising maize stover and CFM supplemented with
different levels of azolla from 0 to 9 per cent.
47
ix
Table
No. Title of Table
Page
No.
4.2.4
Chemical composition (Percentage on DMB) of various diets containing
maize husk supplemented with different levels of azolla from 0 to 9 per
cent and the TMR comprising maize husk and CFM supplemented with
different levels of azolla from 0 to 9 per cent.
49
4.2.5
Chemical composition (Percentage on DMB) of various diets containing
sorghum stover supplemented with different levels of azolla from 0 to 9
per cent and the TMR comprising sorghum stover and CFM supplemented
with different levels of azolla from 0 to 9 per cent.
50
4.2.6
Chemical composition (Percentage on DMB) of various diets comprising
Bengal gram husk supplemented with different levels of azolla from 0 to 9
per cent.
51
4.3
Rumen in vitro net gas production at 24 hours (IVGP-24h) and predicted
metabolizable energy (ME) content of Azolla pinnata, Crop residues and
CFM.
52
4.4.1 Rumen in vitro net gas production of diets comprising roughage replaced
with different levels of azolla from 0 to 9 per cent (Type I diet). 54
4.4.2
Rumen in vitro net gas production of diets (TMR) comprising roughage
(60 per cent kept constant) and CFM (31 to 40 per cent) replaced with
different levels of azolla from 0 to 9 per cent (Type II diet).
55
4.4.3
Rumen in vitro net gas production of diets (TMR) comprising CFM (40
per cent kept constant) and roughage (51 to 60 per cent) replaced with
different levels of azolla from 0 to 9 per cent (Type III diet).
56
4.5.1 Metabolizable energy (ME) content of diets comprising roughage
replaced with different levels of azolla from 0 to 9 per cent (Type I diet). 57
4.5.2 Metabolizable energy (ME) content of diets comprising roughage
replaced with different levels of azolla from 0 to 9 per cent (Type II diet). 59
4.5.3 Metabolizable energy (ME) content of diets comprising roughage
replaced with different levels of azolla from 0 to 9 per cent (Type III diet). 61
4.6.1
Apparent digestible dry matter (ADDM %) and True digestible dry matter
(TDDM %) of Azolla pinnata and the diets comprising roughage replaced
with azolla at 0 per cent and 9 per cent (Type diet).
63
4.6.2
Apparent digestible dry matter (ADDM %) and Total digestible dry
matter (TDDM %) of the diets (TMR) comprising CFM (40 per cent kept
constant) and roughage (51 to 60 per cent) replaced with azolla at 0 per
cent and 9 per cent (Type III diet).
65
x
LIST OF PLATES
Plate No. Title of Plate Page No.
3.1 Cultivation of Azolla (Azolla pinnata) in cement tanks 30
xi
LIST OF APPENDICES
Appendix No. Title Page
No.
Appendix I Gas production data of Hay standard and Concentrate standard
of 4 trials at different time intervals over 24 hours 91-92
Appendix II Gas production data of Azolla at different time interval over 24
hours 93
Appendix III Gas production data of Crop residues at different time interval
over 24 hours 94
Appendix IV
Gas production data of various diets comprising paddy straw
supplemented with different levels of azolla from 0 to 9 per cent
and the TMR comprising paddy straw and CFM supplemented
with different levels of azolla from 0 to 9 per cent.
95-97
Appendix V
Gas production data of various diets comprising Ragi straw
supplemented with different levels of azolla from 0 to 9 per cent
and the TMR comprising paddy straw and CFM supplemented
with different levels of azolla from 0 to 9 per cent.
98-99
Appendix VI
Gas production data of various diets comprising Maize stover
supplemented with different levels of azolla from 0 to 9 per cent
and the TMR comprising paddy straw and CFM supplemented
with different levels of azolla from 0 to 9 per cent.
100-101
Appendix VII
Gas production data of various diets comprising Maize husk
supplemented with different levels of azolla from 0 to 9 per cent
and the TMR comprising paddy straw and CFM supplemented
with different levels of azolla from 0 to 9 per cent.
102-103
Appendix VIII
Gas production data of various diets comprising Sorghum stover
supplemented with different levels of azolla from 0 to 9 per cent
and the TMR comprising paddy straw and CFM supplemented
with different levels of azolla from 0 to 9 per cent.
104-105
Appendix IX
Gas production data of various diets comprising Bengal gram
husk supplemented with different levels of azolla from 0 to 9
per cent
106
xii
LIST OF ABBREVIATIONS
ADDM -
Apparent digestible dry matter
ADF - Acid detergent fiber
ADL -
Acid detergent lignin
ANOVA -
Analysis of Variance
AOAC -
Association of Official Analytical Chemists
BS - Biogenic silica
CF - Crude fiber
CFM -
Compounded feed mixture
CP - Crude protein
DM -
Dry matter
DMB - Dry matter basis
DMD
Dry matter digestibility
EE - Ether extract
g - Gram
GP -
Gas production
ICP-AES -
Inductively coupled plasma-atomic emission spectrophotometer
Kg - Kilogram
ME - Metabolizable energy
NDF -
Neutral detergent fiber
NFE -
Nitrogen free extractives
OM -
Organic matter
RIVGP -
Rumen in vitro gas production
SS -
Sand silica
TA -
Total ash
TDDM -
True digestible dry matter
TMR -
Total mixed ration
IInnttrroodduuccttiioonn
I. INTRODUCTION
India has the largest livestock population in the world. The livestock population is
expected to grow at the rate of 0.55 per cent in the coming years, and the population is
likely to be around 781 million by 2050. Though India is among the leading producers of
milk, meat and eggs; productivity of our animals is 20-60 per cent lower than the global
average due to improper nutrition, inadequate health-care and management. Half of the
total losses in livestock productivity are contributed by the inadequacy in supply of feed
and fodder (ICAR, 2012).
The demand for milk and meat has been increasing and animal husbandry as a
profitable occupation, is expanding. However, there is a substantial decline in fodder
production, owing to the decreasing area under forest and grasslands. The fodder
availability from various crops has also decreased largely due to the introduction of high
yielding dwarf varieties. The shortage of fodder is therefore, being compensated with
commercial feed, resulting in increased cost of production of meat and milk. (Pillai et al.,
2002).
A major constraint to livestock production in developing countries is the scarcity
and fluctuating quantity and quality of the year-round feed supply. Providing adequate
good quality feed to livestock to raise and maintain their productivity is a major challenge
to agricultural scientists all over the world. The increase in population and rapid growth
in world economies will lead to an enormous increase in demand for animal products, a
large part of which will be from developing countries. Future hopes of feeding the
2
2
millions and safeguarding their food security will depend on the enhanced and efficient
utilization of alternative feed resources (FAO report, 2006).
Forage-based economical feeding strategies are required to reduce production cost
of quality livestock products; as feed alone constitutes 60-70% of production cost. At
present, the country faces a net deficit of 35.6 per cent of green fodder, 26 per cent of
dry-crop residues and 41 per cent of concentrate feed ingredients(ICAR, 2012).
In India, Karnataka is second state next to Rajasthan in terms of total geographical
area which is drought prone. Out of the 29 districts of Karnataka, 6 districts belonged to
adequate dry matter (DM) available category with the mean availability of 87.51 %. Five
districts belonged to moderately adequate (60 -79%) and 10 districts belonged to
deficient DM available categories (40-59%). However, one in every four districts
belonged to severely DM deficient category (below 40%). The mean DM availability for
the state was 56.46 %. Total contribution of crop residues to DM in the state was 72.59
%, with coarse straw contribution being one third of it. The contribution of green fodder
to the total DM availability in the state was 23.6 % and concentrates was 3.81%
(Nagaratna and Vinod, 2013).
The estimates by different group of workers have consistently pointed out the
deficit of the feed resources for livestock in terms of dry roughages, greens and
concentrates. Conventional sources of feeds are not enough to mitigate the shortage of
feeds and fodder and to make animal production viable and profitable. The gap between
the demand and supply is also increasing. In order to bridge this gap and to ensure
optimum production of livestock throughout the year use of non-conventional feed
3
3
resources as supplement or replacement of conventional feed without compromising the
quality is the area of focus in recent years. (Chatterjee et al, 2013).
There are varieties of unconventional feedstuffs available which find use as
protein and energy source for livestock. The supplementary resources in India include
aquatic macrophytes which have rich nutrient and mineral profile. Throughout the world,
and particularly in Asia, farmers have harvested naturally produced aquatic plants for
number of purposes which includes animal feed, green manure and for their family food.
The best known of these include the free floating plants; water lettuce (Pistia), water
hyacinth (Eichhcornia), duckweed (Lemna) and azolla and some bottom growing plants.
In recent years commonly occurring aquatic plant, azollahas become prominent and has
attracted the attention of scientists because of its apparent high potential as a feed
resource for livestock. Therefore, some workers also called it as Green gold mine due to
its high nutritive value and Super plant due to its fast growth (Wagner, 1997). Azolla is
considered as the most economic and efficient feed substitute and a sustainable feed for
livestock. It is a potential source of nitrogen and thereby a potential feed ingredient for
livestock (Lumpkin, 1984; Pannaerker, 1988).
Azolla is a free floating aquatic fern belonging to family Azolaceae, order
Pteridophyta distributed throughout the tropical, subtropical and temperate fresh water
ecosystems. It is used as nitrogen source for paddy cropping systems. Azolla can supply
around 25-30 kg of nitrogen per hectare (Pillai et al., 2002). In India Azolla is found
floating on the water in the shallow ditches and in channels. Azolla can fix atmospheric
nitrogen with help of blue green algae, Anabaena azollae, found in cavities of dorsal part
4
4
of leaves. This fact makes the azolla tend to contain relatively high levels of nitrogen and
can be a protein source for animal feeding.
The farmers, particularly in South East Asia and probably elsewhere had
developed the use of azolla as a source of nutrients for livestock. There are some reports
on the use of azollaas feed supplement for poultry and livestock, in which normal feed
protein sources have been replaced by azolla on an iso-nitrogenous basis (Chatterjee,
2013). An attempt has been made to evaluate azolla as a nutrient supplement by invitro
studies to assess its utility as a ruminant feed.
The present study was conducted with the following objectives:
1. To evaluate azolla in terms of its chemical composition, digestibility and
metabolizability by in vitro techniques.
2. To study the supplementary effect of azolla on in vitro digestibility and
metabolizability of crop residues and total mixed ration.
RReevviieeww ooff LLiitteerraattuurree
II. REVIEW OF LITERATURE
Inadequate availability of good quality feed is regarded as a major constraint to
the prevalent livestock production system. The conventional feed ingredients, particularly
protein supplement are expensive and are not always available at affordable prices. Since
the cost of feeding is a significant factor dictating the economic viability of livestock
industry, it must be reduced by adopting new measures in the ration formulation. Hence
to make livestock production a profitable enterprise, there is a great need to use alternate
feedstuffs replacing the traditional sources. This leads to a search for cheap and easily
available protein sources. Aquatic plants have long been used in many developing
countries as feed source for livestock and poultry.
The aquatic plants such as water hyacinth and hydrilla species (Boyed, 1969);
Duck weed (Culley and Epps, 1973) are approximately equal to alfalfa in crude protein
and crude fiber contents (Spencer et al., 1969). The lower level of fiber content in some
of the aquatic species depends on location and stage of growth. However, the ash and
xanthophylls contents are fairly high, when compared to the conventional forages.
Although, high ash content is being one of the apparent limitation in nutritional value of
such aquatic plants, but high xanthophylls contents possibly offset the limitation and thus,
makes these aquatic plants superior to other plants.
2.1 Nutritive value of Aquatic plants
Mutzar et al. (1976) studied the nutritional value of five different species of dried
ground aquatic plants, Azolla pinnata, caladophora, pond weed, milofoil and vallisneria
duck weed. These species of aquatic plants revealed (Table 2.1) that, the water weeds had
6
6
extremely variable levels of ash (18.4-62.5 per cent) and crude protein (5.6-15.5 per cent)
but contained relatively less crude fiber and ether extract than dehydrated alfalfa meal.
The calcium content varied from 3.0 to 16.7 per cent as compared to 1.9 per cent in
dehydrated alfalfa meal. Lipstein and Hurwitz (1980) reported 574 g of total protein, 22 g
fiber, 89 g moisture, 11.7 g phosphorus and 2.0 g calcium per kg of algae meal.
Table 2.1: Per cent chemical composition (DMB) of aquatic plants and alfalfa
Plant species Crude protein Crude fiber Ether extract Ash Ca P
Azolla pinnata 21.4 12.7 2.7 16.2 1.16 1.29
Cladophora 12.93 14.5 1.22 59.83 4.93 0.28
Duck weed 15.53 16.64 3.14 18.43 3.03 0.39
Milofoil 5.61 17.04 0.94 62.53 16.72 0.10
Pond weed 10.54 20.0 2.90 40.44 11.90 0.19
Alfalfa meal 17.7 24.2 3.65 9.9 1.9 0.27
2.2 Azolla
Azolla (mosquito fern, duckweed fern, fairy mass and water fern) is a floating
fern in shallow water. It floats on the surface of water by means of numerous, small,
closely overlapping scale like leaves with their roots hanging in the water. They are
extremely reduced in form and specialized, looking nothing like conventional fern but
more resembling duckweed. It can readily colonize areas of fresh water and grows at
great speed doubling its biomass every two to three days (Van Hove and Lejeune, 2002).
7
7
2.2.1 Morphology
Azolla is an aquatic fern consisting of a short, branched, floating stem, bearing
roots which hang down in the water. The leaves are alternately arranged and each
consists of a thick aerial dorsal lobe containing green chlorophyll and a thin floating
ventral lobe of slightly larger size that is colorless. Under certain conditions, an
anthocyanin pigment gives the fern a reddish-brown color. Plant diameter ranges from 1-
2.5 cm for small species, such as Azolla pinnata, to 15 or more cm like Azolla nilotica.
Azolla plants are triangular or polygonal in shape, and float on the water surface
individually or in mats. They give the appearance of a dark green to reddish carpet,
except Azolla nilotica which does not produce the red anthocyanin pigment. The most
remarkable characteristic of azolla is its symbiotic relationship with the nitrogen-fixing
blue-green algae (cyanobacterium) Anabaena azollae. The fern provides nutrients and a
protective cavity in each leaf to Anabaena colonies in exchange for fixed atmospheric
nitrogen and possibly other growth-promoting substances (Lumpkin, 1984).
2.2.2 Taxonomy and biogeography
Azolla is the only genus in the family Azollaceae and has a worldwide
distribution from temperate to tropical climates. There are seven species of azolla, which
are widely distributed in different parts of the world as Azolla caroliniana (North
America and the Caribbean), Azolla filiculoides (South America), Azolla microphylla
(Tropical and subtropical America), Azolla mexicana (North and South America), Azolla
nilotica (upper reaches of the Nile to Sudan), Azolla rubra (Japan, Korea, Australia and
New Zealand) and Azolla Pinnata commonly found in India (Van Hove and Lejeune,
2002).
8
8
2.2.3 Symbiotic interaction with Anabaena azollae
Azolla hosts symbiotic blue green algae Anaebaena azollae, which is responsible
for the fixation and assimilation of atmospheric nitrogen. Azolla, in turn, provides the
carbon source and favorable environment for the growth and development of the algae. It
is the unique symbiotic relationship that makes azolla, a wonderful plant with high
protein content (Pillai et al., 2002).This symbiotic relationship gives azolla a competitive
advantage over other floating hydrophytes in environments such as rice fields, which are
relatively low in available nitrogen. The symbiosis is of interest to agriculturists, since
incorporation and decomposition of azolla crop in rice field soil can result in, increased
amount of available nitrogen to a companion or succeeding rice crop. Azolla can
accumulate more than 10 kg N / ha per day. Some strains of azolla can fix as much as 1-3
kg of nitrogen / ha / day and its annual yield is 730 tonnes / ha as a green azolla for
feeding animals (Lumpkin and Plucknett, 1982).
2.2.4 Azolla in agriculture
Azolla can be used as a food, mosquito inhibitor, green manure, herbicide, water
saver, water purifier, nitrogen fertilizer saver, drug and for reclaiming saline soils (Van
Hove and Lejeune, 2002). It has long been used by farmers, mainly in Asia, as feed for
the animals and as green manure. A number of laboratory and field studies have shown
an unquestionable beneficial effect of azolla as an organic nitrogen fertilizer, mainly in
terms of increasing rice grain yield. In addition, presence of an azolla mat on the surface
of the water body has been shown to significantly reduce weed development, reduce
volatilization of applied N fertilizer and purify water.
9
9
Azolla is used as a nitrogenous crop in Vietnam, China, Taiwan and other South
East Asian countries. Azolla is capable of assimilating atmospheric nitrogen efficiently,
due to presence of an algal symbiont in the leaves (Moore, 1969).
Fresh azolla is used in the preparation of compost. Since, the fern has an excellent
carbon nitrogen ratio, it decomposes rapidly and accelerates the decomposition of other
organic residues inside the compost pit and used as a biofertilizer in coffee plantations
(Anandand Geetha., 2007).
2.2.5 Yield
The plant multiplies rapidly and gives good dry matter yield in spite of its high
water content. Singh (1982) reported that under conditions in Cuttack regions of East
coast, an annual production of fresh azolla was 347 tonnes / ha in field and 321 tonnes /
ha in concrete tank.
Pillai et al. (2002) reported that 500-600g of fresh azolla can be harvested daily
from a pit of 2x 2 x 0.2 m size after 10-15 days of adding culture.
2.2.6 Azolla production and multiplication
The cultivation of azolla is a continuous process requiring a certain amount of
attention throughout the year (Lumpkin and Plucknett, 1980). Usually a certain area of
field within a production unit is set aside for such cultivation.
Azolla nurseries of small plot (50-100 m2) area are prepared to avoid wind action.
A standing water of 3 to 5 cm depth is essential. A pH of 5.0 to 7.0 is ideal and acidic
10
10
soils with pH 4-6 are not suitable unless lime is used to correct pH. Winter temperature in
the range of 14oC to 35
oC is preferable but the optimum range is 20
oC to 30
oC. The azolla
inoculums at 0.1 to 0.4 kg per hectare is most desirable for production of 8 to 10 tonnes
per hectare green manure in 20 days. Application of super phosphate at 75 kg per hectare
is essential for rapid growth of azolla. Use of carbofuran/ furadon at one to two kg per
hectare or 0.625 g per m2 prevents the rapid spread of insects, parasites and the
consequent destruction of azolla nurseries (Pillai et al., 2002). At the good growth period
the azolla mat should be separated once in a week. Azolla doubles its weight in 3-5 days.
From a start of 1 tonne / ha, it can reach a fresh weight of 15-20 tonnes / ha in about 20
days (Khan, 1983).
2.3 Azolla as a livestock feed
Azolla is rich in protein. On a dry weight basis, it contains 25 - 35 per cent
protein, 10 - 15 per cent minerals and 7 – 10 per cent of amino acids, bio-active
substances and bio-polymers. The carbohydrate and fat content of azolla is very low. Its
nutrient composition makes it a highly efficient and effective feed for livestock. Azolla
can be used in animal feed and it is a potential feed ingredient for broilers; it is an income
generating crop (Reddy, 2007). Azolla is one of the most nutritive aquatic plant, owing to
its high protein and carotenoid contents and of generally good amino acid profile. It can
be incorporated into the feed of ruminants (Nik Khah and Motaghi, 1992), small
ruminants (Tamang et al, 1993; Ali and Leeson, 1995), pigs (Bacerra et al., 1995), broiler
chickens (Basak et al., 2002) and rabbits (Sreemannarayana et al., 1993).
11
11
Feeding trials with azolla in dairy cows and buffaloes, growing buffaloes, sheep
and goats have been carried out in India. Since the 2000s, azolla is being promoted in
India for dairy production (Pillai et al., 2002). Azolla meal (dried azolla) can be included
upto 15 per cent of total concentrate requirement of growing Osmanabadi goats (Ghodake
et al., 2012).
Azolla is used as a feed ingredient for ruminants and non-ruminants (Singh and
Subudhi, 1978). Azolla has long been used as green manure and as a feed for poultry
(Basak et al., 2002). The use of azolla as a green feed in cattle, swine, poultry and fish
has been tested with favourable results (Alalade and Iyayi, 2006). Azolla can serve as a
potential alternative nutrient supplement for the crossbred cattle for the improvement of
productivity in terms of growth, milk, meat etc (Chatterjee et al., 2013).
Azolla can be fed to the livestock either in a fresh or dried form. It can be given
directly or mixed with concentrates to cattle, poultry, sheep, goat, pigs and rabbits.
(Giridhar et al., 2012). Nik Khah and Motaghi (1992) reported that, the azolla can be
incorporated in the concentrate mixture at the level of up to 35 per cent in lactating cows
without any deleterious effect.
Murrah buffalo bulls fed with sun dried azolla at 225 g / day replaced 25% of
protein in concentrate feed (Kumar et al., 2012). Tamang et al. (1993) indicated that the
sun dried azolla can be incorporated up to 20 per cent in the concentrate mixture without
any deleterious effects on the performance, digestibility of various nutrients, carcass
characteristics of Black Bengal goats. Duran (1994) reported that the aquatic azolla can
replace up to 20 per cent of the soya bean protein with no adverse effect on the
performance of growing and fattening pigs.
12
12
Replacement of groundnut cake nitrogen with azolla at 50 per cent level for 90
days period improved the digestibility of major nutrients and resulted weight gain in
buffalo calves (Indira et al., 2009).
Five feeds (mixture of gram straw and concentrate in 60:40 ratio) were evaluated
for methane emission and digestibility with goat rumen liquor as inoculums in an in vitro
gas production test. These five feeds were formulated by replacement of oil cake by
azolla meal at 0%, 25%, 50%, 75% and 100% in the concentrate mixture. There was
significant difference (P<0.05) in the net methane production per g DM among feeds.
With the replacement of oil cake with azolla meal, a decreasing trend of dry matter and
organic matter digestibility was reported. It was concluded that 50% replacement of oil
cake with azolla meal reduces the methane production in in vitro condition with no
satisfactory significant effect on digestibility (Kumar et al., 2013).
Utilization of Sun-dried azolla at 10 and 20% in the diets of Marwari and
Patanwadi weaner lambs for 4 months resulted in lower DM and protein digestibility but
higher DM and protein intake and no effect on carcass traits (Wadhwani et al., 2010).
Different diets were formulated and subjected for in vitro gas production kinetic
studies by using cow rumen liquor. The paddy straw and ragi straw were supplemented
with 0%, 2%, 4% and 8% azolla. There was no significant difference in gas production
and dry matter digestibility in different feeds. Addition of azolla at different levels in two
different straws has failed to improve the gas production and it was similar to
unsupplemented straw (Ramesh, 2008).
13
13
2.4 Nutritional assessment of azolla
2.4.1 Chemical composition
Chemical composition of different species of azolla are reported by various
workers (Table 2.4.1). As cited by various authors the crude protein content of azolla
varies from 15.4 to 27.93, crude fibre content between 9.07 and 22.25%, ether extract
value varies between 1.60 and 5.05 % while total ash was in the range of 10.15-36.10%
and NFE values were found to vary between 30.08 and 52.46%. Van Hove (1989) noted
that the crude protein content of azolla might vary from 13.0 to 34.5%. These variations
in the nutrient composition of azolla meal is due to differences in the response of azolla
strains to environmental conditions such as temperature, light intensity and soil nutrients
which consequently affect their growth morphology and chemical composition.
Furthermore, contamination with epiphytic algae could also be important to such a degree
as to affect the results of chemical composition (Sanginga and Van Hove, 1989). The cell
wall composition of azolla is highly variable depending upon the species and the season
of cultivation of azolla. NDF content of azolla was found to be in the range of 36.88-70%
(Chatterjee et al., 2013) while ADF was reported to be in range of 25.24-47.08%.
Cellulose and hemicelluloses content was found to be in the range of 6.8 to 36.7% and
10.09 to 17.8, respectively. Lignin was reported to vary between 9.27 and 28.24% and
silica content varies between 4.8 and 16%. Chemical composition indicated that it was a
fair source of plant protein (210.7-296.7 g kg−1 DM). The mean concentration (per cent)
of organic matter, crude protein, crude fibre, ether extract, total ash, NFE, NDF, ADF and
ADL in Azolla microphylla meal were 80.53±0.59, 24.06±0.35, 13.44±1.20, 3.27±0.18,
19.47±0.59, 37.71±1.83,45.52±1.93, 30.16±1.12 and 8.96±0.56, respectively.
14
Table 2.4.1: Per cent chemical composition of different species of Azolla (Dry matter basis)
Proximate principles Azolla filiculoides Azolla pinnata Azolla microphylla Azolla pinnata
Beckingham et al., 1978 Parnerker et al., 1986 Taklimi, 1990 Alalade and Iyayi, 2006
Dry matter - 90
Crude Protein 23-42 15.63 25.33 21.4
Ether Extract 5.05 2.84 3.01 2.7
Crude Fiber - 13.01 11.60 12.7
Total Ash 15.54 23.59 16.2
Nitrogen free extract - 52.70 16.91 47.0
Neutral detergent fiber 39.16 52.76 40.36 36.88
Acid detergent fiber 26.58 25.24 47.08
Calcium 0.937 1.64 1.70 1.16
Phosphorus 0.487 0.56 1.05 1.29
15
15
In general azolla was reported to be rich in mineral profile, the fern was found to
be a rich source of calcium, phosphorous, potassium, ferrous, copper, magnesium and
zinc. Calcium content of azolla varies from 0.8- 4.99 %, while phosphorus between 0.3
and 1.3%. Querubin et al. (1986) reported the following mineral composition, Ca-2.07%,
P-0.77%, Mn-0.27%, Fe-0.25%, Mg-0.17%, Na-0.49%, K-4.93%, Cu-17.6 ppm, Zinc-
71.8 ppm in A. pinnata and the carotene content ranged from 206 to 619 mg / kg on a dry
matter basis and differed significantly between strains. Carotene content was maximal
during the linear phase of growth and minimal during the stationary phase for the all
strains (Lejeunea et al., 2000).
Variations in the chemical composition of Azolla pinnata as been reported by
different authors Singh and Subudhi (1978), Tamang et al. (1993), Anand Titus and
Geetha Pereira (2007), Ali and Leeson (1995), Khatun et al., (1999), Parthasarathy et al.
(2001), Alalade and Iyayi (2006) and Balaji et al. (2009) is presented in the Table 2.4.2.
Singh and Subudhi (1978) reported that azolla contained 24 to 34 per cent crude
protein, 3.18 per cent ether extract, 9.1 per cent crude fiber and 10.5 per cent total ash on
dry matter basis. The calcium and phosphorus contents were found to be in the range of
0.4 to 1.0 per cent and 0.5 to 0.9 per cent, respectively. They also reported that azolla is
rich in vitamins, minerals, xanthophylls and carotenoids.
The freshly harvested azolla contains 85 to 99 per cent of moisture and 0.4 to 6.0
per cent nitrogen (Moore, 1969). The moisture content varied with the stage of maturity
of the aquatic plant harvested and the time allowed for the drain/ drip, post harvesting.
16
Table 2.4.2: Chemical Composition (on per cent dry matter basis) of Azolla pinnata
Source
Singh and
Subudhi
(1978)
Tamang
et al.,
(1993)
Ali and
Leeson
(1995)
Khatun
et al.
(1999)
Parthasarathy
et al. (2001)
Alalade
and Iyayi
(2006)
Anand Titus and
Geetha Pereira
(2007)
Balaji
et al.
(2009)
Proximate Principles
Dry Matter 90.12 92 90.59 89.70
Crude Protein 24.3 15.37 16.5 28.54 26.02 21.4 20-25 24.5
Crude fiber 9.1 14.13 12.5 12.38 13.6 12.7 - 14.9
Ether extract D3-3.6 2.73 1.6 - 2.37 2.7 3-3.5 3.7
Total ash 10.5 20.35 36.1 - 12.3 16.2 17
NFE - 47.42 33.2 - 45.71 47 - 39.9
Cell wall fractions
Neutral detergent fiber - 67.54 47.8 44.57 - 36.88 - -
Acid detergent fiber - 51.96 46.7 33.41 - 47.08 - -
Cellulose - 15.61 - - - 12.76 - -
Lignin - 17.48 - - - 28.24 - -
Mineral
Calcium 0.4-1.0 1.54 1.43 - 1.24 1.16 0.45-1.25 2.14
Phosphorus 0.5-0.9 0.35 0.31 - 0.72 1.29 0.15-11 0.44
Potassium 2-4.5 - - - - 1.25 0.25-5.5 -
Mananesium 0.5-0.65 - - - - 0.35 0.25-0.5 -
Sulphur - - - - - - 0.2-0.75 -
Sodium - - - - - 23.79 - -
Manganese,ppm - - - - - 174.42 60-2500 -
Zinc.ppm - - - - - 87.59 25-750 -
Copper, ppm - - - - - 16.74 2-250 -
Iron, ppm 60-260 - - - - 755.73 40-500 -
Silica, ppm - 15.98 - - - 3.82 0.15-3.5 -
17
17
Basak et al. (2002) reported that azolla meal contains 25.78 per cent crude
protein, 3.47 per cent ether extract, 15.76 per cent total ash and 30.08 per cent NFE. The
gross energy was found to be 4243 kcal per kg. The NDF and ADF values were 47.85
and 42.16 per cent respectively. Azolla microphylla found to contain 80.53% OM, 24.06
% CP, 13.44% CF, 3.27% EE, 19.47% TA, 37.71% NFE, 45.52% NDF, 30.16% ADF
and 8.96% ADL (Chatterjee et al., 2013).
Parnerkar et al. (1986) analyzed the proximate principles of azolla and reported
15.63 per cent CP, 13.01 per cent CF, 2.84 per cent EE, 1.624 per cent calcium, 0.56 per
cent phosphorus on dry matter basis. Bacerra et al. (1995) analysed the chemical
composition of azolla and reported 26.7 per cent CP, 15.1 per cent TA, 94.4 per cent
moisture, 4.6 per cent EE, 11.2 per cent CF and 0.4 per cent phosphorus and 0.8 per cent
calcium.
Ramesh, (2008) has reported that azolla contains 4.1 % DM, 28.52 % CP, 4.13%
EE, 14.43 % TA, 57.71 % NDF and 26.22 % ADL. The in vitro gas production was
found to be 29.3 ml / 200mg DM / 24 hour and ME of 7.1 MJ/kg DM. The chemical
composition of azolla varieties reported by Bolka (2011) is presented in Table 2.4.4
Pillai et al. (2002) reported that azolla is very rich in proteins, essential amino
acid, vitamins (vitamin A, vitamin B12 and Beta-carotene), growth promoter
intermediaries and minerals like calcium, phosphorus, potassium, iron, copper,
magnesium. Sujatha et al. (2013) reported that azolla contains 21.17 per cent CP, 14.6
per cent CF, 3.39 per cent EE, 19.91 per cent of TA, 1.05 per cent Calcium, 0.52 per cent
Phosphorus and 0.49 per cent Iron.
18
18
Table 2.4.3: Chemical composition of Azolla varieties (Bolka, 2011)
Constituent
Azolla
pinnata
(KVAFSU
sample)
Azolla
pinnata
(Raichur
sample)
Azolla
pinnatae
(Hessaraghatta
sample)
Azolla
pinnata
(TRRI
sample)
Azolla
microlophylla
(GKVK sample)
Organic matter 81.25 80.75 81.02 70.83 75.83
Total ash 18.75 19.25 18.98 29.17 24.17
Crude protein 25.82 24.15 24.55 24.12 24.56
Crude fiber 17.53 19.75 19.85 16.25 15.17
Ether extract 4.85 3.95 4.12 3.80 3.38
NFE 33.05 32.90 32.50 26.66 32.72
Calcium - - - 1.95 -
Phosphorus - - - 0.40 -
Buckingham et al. (1978) has reported that azolla contains 31.36% CP, 5.08 %
EE, 12.2 % CF, 13.3 % TA, in vitro gas production was found to be 13.6 ml / 200 mg
DM / 24 h, digestibility of organic matter was 49.9% and ME was around 5.91 MJ / kg
DM. Khan et al. (2002) reported that the rate of gas production was highest in azolla and
lowest in water-hyacinth.
Alalade and Iyayi (2006) reported the gross energy and ME content of Azolla
pinnata as 8.53 MJ/kg DM and 6.99 MJ/kg DM, respectively. Balaji et al. (2009)
recorded the gross energy of azolla as 1807 Kcal/kg DM. Parthasarathy et al. (2001)
reported that, the apparent and true metabolisable energy values of Azolla pinnata as
19
19
1529 and 1855 Kcal/kg DM, respectively. Khatun et al. (1999) has reported the ME
content of azolla as 7.59 MJ / kg DM.
Among different aquatic plants, the in-vitro dry matter digestibility (52.2%) was
found to be highest for Azolla (Becerra et al., 1995). It was reported that replacement of
50% of oil cake with azolla meal in concentrate portion of TMR (Total Mixed Ration)
reduced the methane production under in vitro condition (Kumar et al., 2013).
On the basis of in vitro analysis (Rumen Simulation Technique, RUSITEC)
Ahirwar et al. (2009) reported that the Azolla pinnata could be used as protein
supplement by replacing 30 per cent of the conventional nitrogen source in complete
ration of ruminants. Khan et al. (2002) reported that, the rate of gas production was
highest in azolla as compared to water-hyacinth. A similar trend was observed with in
situ DM degradability.
Parashuramulu and Nagalakshmi (2013) reported that, azolla is a good protein
supplement with 21.37% crude protein, crude fibre - 12.5%, ether extract - 2.3%. The
average in vitro dry matter digestibility was 79.5 per cent, organic matter digestibility
was 63.8 mg / 200 mg DM and ME of 1759 kcal ME / kg or 7.36 MJ / kg DM.
2.4.4 Amino acid composition
The amino acid profile of azolla reported by different authors is presented in the
Table 2.4.2. Sanginga and Van Hove (1989) compared the total nitrogen and amino acid
composition of seven azolla strains at four different growth phases. Azolla microphylla
strain was found to be the best source of amino acids and A. filiculoides strain found to be
20
20
the poorest under the cultural conditions used for green manure production. Data on the
amino acid analysis reported by Alalade and Lyayi (2006) indicated that lysine, arginine,
isoleucine, leucine, phenylalanine, glycine and valine were predominant. However, the
sulphur-containing amino acids did not meet the recommended value of 3.5g / 100g
protein. Mandal et al. (2012) also reported azolla as a rich source of protein (21.6%) with
all essential amino acids, including lysine, arginine and methionine.
Table 2.4.4 Amino acid composition of azolla
Amino acids Ali and Leeson (1995)
Alalade and Iyayi
(2006)
Buckingham
et al., (1978)
% DM g /100 g CP %DM g/100g CP g/100g CP
Lysine
Methionine
Cystine
Threonine
Tryptophan
Arginine
Isoleucine
Leucine
Phenylalanine
Tyrosine
Glysine
Serine
Valine
Alanine
Histidine
Proline
Aspartic acid
Glutamic acid
0.62
0.25
0.15
0.66
0.08
0.82
0.69
1.28
0.77
0.49
0.86
0.66
0.84
0.95
0.26
0.67
1.37
1.56
3.80
1.50
0.90
4.00
0.50
5.00
4.20
7.70
4.60
4.00
5.20
4.00
5.10
5.80
1.60
4.00
8.30
9.60
0.98
0.34
0.18
0.87
0.39
1.15
0.93
1.65
1.01
0.68
1.00
0.90
1.18
4.58
1.59
0.84
4.07
1.82
5.37
4.35
7.71
4.72
3.18
4.60
4.21
5.51
6.45
1.88
2.26
4.70
4.10
6.62
5.38
9.05
5.64
4.10
5.72
4.10
6.75
6.45
2.31
4.48
9.39
12.72
2.5 Chemical composition of different crop residues
Chemical composition and digestibility of organic matter and ME content of
different varieties of Paddy straw as reported by Rahaman et al. (2010) are presented in
21
21
Table 2.5.1 and 2.5.2, respectively. Chemical composition of different stages of rice
straw Table as reported by Sarnklong et al. (2010) is presented in Table 2.5.3.
Preston et al. (2000)reported that rice straw contains 90 per cent DM, 88.3 per
cent OM, 8.4 per cent CP, 62.5 per cent NDF, 36.6 per cent ADF, 11.7 per cent TA, in
vitro organic matter digestibility was found to be 27.7 per cent.
Akinfemi and Ogunwole (2012) reported that rice straw contains 93 per cent DM,
CP 4.69 per cent, CF 32.89 per cent, EE 1.66 per cent, TA 11.95 per cent, NFE 48.81 per
cent, NDF 69.96 per cent, ADF 56.28 per cent, ADL 12.54 per cent, cellulose 43.74 per
cent, hemicellulose 13.68, invitro gas production found to be 30 ml/ 24 h, ME 6.49 MJ/kg
DM and OMD 51.17%.Kumar et al. (1999) reported that rice straw contains 88.9% DM,
83.3% OM, 3.33% CP, 36.5% CF, 1.25% EE, 42.45% NFE.
Bhatta et al. (2000) reported that finger millet straw contains 91.38% OM, 3.33%
CP, 0.51% EE, 36.68% CF, 8.62% TA, NDF 80.73%, ADF 51.41 %, ADL 5.38%, ME -
6.02 MJ/Kg DM. Ramachandra (1997) has reported that ragi straw contains 89.69 % DM,
91.55 % OM, 13.68 % CP, 2.32 % EE, 32.58 % CF, 8.45 %TA, 6.55 AIA, 52.31 %NFE,
66.69 %NDF, 46.83% ADF and 19.84% hemicellulose.
Madibela and Modiakgotla (2004) had reported that, finger millet straw contains
16.9 per cent TA, 7.45 per cent CP, 69.7 per cent NDF, 40.9%ADF, 4.76 per cent ADL,
40.9 per cent NFE, Ca of 0.88 per cent, P of 0.03 per cent and in vitro dry matter
digestibility found to be 50.1 per cent.
22
22
Finger millet straw found to contain 92.7% OM, 5% CP, 0.8% EE, 75.1% NDF,
46.2% ADF, 6% ADL, in vitro gas production of 27.3 ± 0.45 ml / 200 mg DM / 24 h and
ME of 6.5MJ/kg DM (Sreerangaraju et al., 2000).
Table 2.5.1 Chemical composition of varieties of paddy straw (Rahaman et al.,
2010)
Varieties DM OM CP NDF ADF ADL Ca P
BRRI 29 93.05 84.49 5.1 72.53 41.38 6.97 0.165 0.146
BINA 92.81 81.21 4.64 72.16 43.62 4 0.10 0.08
Pajam 92.84 82.56 4.39 74.2 42.83 4.34 0.2450 0.074
Kablabadam 92.84 85.08 3.49 74.95 44.22 5.94 0.14 0.06
Nijerhail 92.8 86.24 3.61 74.86 46.32 4.3 0.17 0.068
BR 11 92.2 84.91 4.52 477.57 43.64 4.3 0.245 0.046
Table 2.5.2 Digestibility of OM and ME content of different varieties of paddy
straw (Rahaman et al., 2010)
Different varieties of Paddy
straw
Digestibility of OM
(%)
ME content (MJ/kg DM) ±
SE
BRRI 29 44.58a ± 0.565 6.68
a ± 0.021
Nijershail 39.94b
± 0.047 5.99b
± 0.031
BINA 5 39.87b
± 0.243 5.97b
± 0.070
Pajam 38.30c ± 0.093 5.73
c ± 0.010
Kablabadam 37.67c ± 0.055 5.65
c ± 0.015
BR 11 37.53c ± 0.104 5.61
c ± 0.021
23
23
Table 2.5.3: Chemical composition of rice straw in early, mid and late growing
period (Sarnklong et al., 2011)
Cultivation season DM NDF ADF Hemicellulose Cellulose ADL
Early 96.40 72.53 43.52 29.01 35.81 4.90
Mid 96.20 70.03 41.09 29.08 32.80 4.66
Late 96.87 71.97 39.83 32.24 31.96 4.63
Akinfemi et al. (2009) had reported that, maize husk contains 88.85 per cent DM,
7.44 per cent CP, 1.27 per cent EE, 3.32 per cent TA, 30.45 per cent CF, 42.48 per cent
NFE, 49.15 per cent ADF, 71.14 per cent NDF, 14.87 per cent ADL, 34.25 per cent
cellulose, 21.99 per cent hemicellulose, in vitro gas production found to be 20.33 ml /
200mg DM / 24 h and ME of 5.45 MJ / kg DM and Organic matter digestibility (OMD)
found to be 38.28%.
Kiangi et al. (1981) reported the maize stover contains 93.4 per cent DM, 12.1 per
cent TA, 2.3 per cent CP, 45.3 per cent CF, 1.8 per cent of EE and 38.4 per cent NFE and
the in vitro DMD was found to be 51.7%, while Ojiet al. (1997) recorded 3.5 per cent CP,
39.4 per cent CF, 0.6 per cent EE, 48.4 per cent NFE, 80.7 per cent NDF and 56.7 % of in
vitro DMD.
Sreerangaraju et al.(2000) had reported that, Bengal gram husk contains 5.1 per
cent CP, 1.1 per cent EE, 76 per cent NDF, 65.2 per cent ADF, 6.1 per cent ADL, in vitro
gas production found to be 45±0.39 ml / 200 mg DM / 24 h, ME 8.6 MJ / kg DM.
24
24
Sorghum stover contains 91.23% DM, 2.54 CP, 31.65% CF, 6.19% EE, 6.28%
TA, 53.34% NFE, 70.23% NDF, 46.69% ADF, 15.21% ADL, 31.48% Cellulose, 23.54%
Hemicellulose, in vitro gas production was 26 ml/200 mg DM/24 h, ME of about
5.97MJ/kg DM and OMD was about 42.99% (Akinfemi and Doherty, 2010).
2.6 Fiber degradation in ruminants
In ruminants forages are necessary for normal rumen function. However, high
proportions of structural carbohydrate found in forages limit the voluntary intake and
digestion of feedstuffs by ruminants because they slow down the rates of microbial
fermentation. Abundance and diversity of rumen microorganisms, particularly of
cellulolytic bacteria and anaerobic fungi, are necessary to degrade the fibrous dietary
components (Wilson, 2008). Thus, a method for promoting the growth of fiber-degrading
microorganisms would be beneficial in increasing the degradation of cellulose and
hemicellulose in the rumen. Most ruminal cellulolytic microorganisms, such as
Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenes, and
Butyrivibio fibrisolvents, require branched-chain volatile fatty acids (BCVFA, i.e.,
isobutyric, isovaleric, valeric, and 2-methylbutyric acids) for growth. Ruminal branched-
chain volatile fatty acids primarily originates from dietary protein or recycling of
bacterial protein by ruminal oxidative deamination and decarboxylation of valine,
leucine, and isoleucine (Argyl and Baldwin, 1989). Studies have shown that branched
chain fatty acids can improve apparent dry matter digestibility and microbial growth, and
enhance microbial functions and enzyme activities in the rumen (Moharrery, 2004).
Dietary supplementation with branched-chain volatile fatty acids improves rumen
fermentation and enhances digestion in cattle (Liu et al., 2009). Branched-chain volatile
25
25
fatty acids are used to synthesize branched-chain amino acids (BCAA, i.e., valine,
leucine, and isoleucine) by ruminal microorganisms. Several researchers have
demonstrated that amino acids are often stimulatory for ruminal microorganisms, even
when ammonia and carbohydrates exceed the requirements. These results indicate that
ruminal microorganisms could benefit more by direct provision of branched chain amino
acids rather than the corresponding branched-chain volatile fatty acids (Yang, 2002).
Low concentration of valine, leucine or isoleucine supplementation resulted in
higher VFA production during in vitro rumen fermentation of wheat straw. So branched
chain amino acids could be used as additives in ruminants to improve forage utilization
(Zhang et al., 2013).
In general, it can be inferred that the azolla appears to be a valuable non
conventional feedstuff for livestock and it may improve the utilization of crop residues.
The positive and in some respect controversial data of relevant literature have prompted
us to study supplementary effect of azolla on utilization of crop residues and total mixed
ration.
MMaatteerriiaallss aanndd MMeetthhooddss
III. MATERIALS AND METHODS
A brief account of experimental procedures and analytical techniques adopted
during the course of study are presented in this chapter.
3.1 Location
The experiment was conducted at the Department of Animal Nutrition and
Livestock Production and Management, Veterinary College, KVAFSU, Bangalore and
Alltech Ruminant Nutrition Laboratory, Veterinary College, KVAFSU, Bangalore.
3.2 Feed samples used
Crop residues:
1. Finger millet (Eleusine coracana) straw
2. Paddy (Oryza sativa) straw
3. Maize (Zea mays) stover
4. Sorghum (Sorghum bicolor) stover
5. Maize (Zea mays) husk
6. Bengal (Cicer arietinum) gram husk
Crop residues commonly used for dairy cattle feeding in Karnataka were selected.
The crop residues were dried at 65oC for 2 days and ground to pass through a one mm
sieve and preserved in air tight bottles at room temperature while using for chemical
analysis and in vitro studies.
Azolla pinnata was harvested from azolla tanks maintained at College of
Agriculture, University of Agricultural and Horticultural Sciences, Shivamogga. Fresh
27
27
samples of azolla was dried in hot air oven at 50oC for 48 h and dried samples was
ground to pass through a one mm sieve and preserved in airtight bottles at room
temperature.
3.3 Dietary treatments
Three types of diets were formulated by using azolla, crop residues and
compound feed mixture. The first type of diet consisted only roughage which was
supplemented with different levels of azolla that is 0%, 3%, 6%, and 9%. The second
type of diet consisted Total mixed ration (TMR) prepared using roughage and
concentrate in the ratio of 60:40 and the portion of concentrate in the TMR was replaced
with 0%, 3%, 6% and 9% azolla. The third type of diet consisted TMR, prepared using
roughage and concentrate in the ratio of 60:40 and the portion of roughage in the TMR
was replaced with 0%, 3%, 6% and 9% azolla.
The compounded feed mixture (CFM) or concentrate feed was formulated with
18% CP and 70% TDN by using following ingredients
Ingredients Percent
Maize 43%
Wheat bran 31%
Groundnut cake 23%
Salt 1%
Mineral mixture 2%
28
28
The treatment design is as follows
Type I diet (Crop residue + Azolla)
Crop residues Levels of azolla supplementation
0% (T1-control) 3% (T2) 6% (T3) 9% (T4)
Paddy Straw (R1) T1R1 T2R1 T2R1 T4R1
Ragi straw (R2) T1R2 T2R2 T3R2 T4R2
Maize stover (R3) T1R3 T2R3 T3R3 T4R2
Maize husk (R4) T1R4 T2R4 T3R4 T4R4
Sorghum stover (R5) T1R5 T2R5 T3R5 T4R5
Bengal gram husk (R6) T1R6 T2R6 T3R6 T4R6
Where, T1=Treatment with 0% azolla (Control), T2= Treatment with 3% azolla, T3= Treatment
with 6% azolla, T4= Treatment with 9% azolla, R1=Paddy straw, R2=Ragi straw, R3=Maize
Stover, R4=Maize husk, R5=Sorghum Stover, R6=Bengal gram husk.
Type II diet (TMR)
TMR Levels of azolla supplementation
0% (T5- control) 3% (T6) 6% (T7) 9% (T8)
Paddy straw(60% kept constant) +
Concentrate feed+ Azolla T5R1 T6R1 T7R1 T8R1
Ragi straw(60% kept constant) +
Concentrate feed +Azolla T5R2 T6R2 T7R2 T8R2
Maize Stover(60% kept constant) +
Concentrate feed+Azolla T5R3 T6R3 T7R3 T8R3
Maize husk(60% kept constant) +
Concentrate feed +Azolla T5R4 T6R4 T7R4 T8R4
Sorghum stover(60% kept constant) +
Concentrate feed +Azolla T5R5 T6R5 T7R5 T8R5
Where, T5= Treatment with 0% Azolla (Control), T6= Treatment with 3% Azolla, T7= Treatment
with 6% Azolla, T8 = Treatment with 6% Azolla, R1=Paddy straw, R2=Ragi straw, R3=Maize
Stover, R4=Maize husk, R5=Sorghum Stover, R6=Bengal gram husk.
29
29
Here roughage and concentrate taken in the ratio of 60:40. The portion of
concentrate in the TMR was replaced with azolla at 0%, 3%, 6% and 9% and the
roughage portion was kept constant (60%).
Type III diet (TMR)
TMR Levels of azolla supplementation
0%(T9- control) 3%(T10) 6% (T11) 9% (T12)
Paddy straw + Concentrate feed (40
% kept constant) T9R1 T10R1 T11R1 T12R1
Ragi straw+ Concentrate feed (40 %
kept constant) +Azolla T9R2 T10R2 T11R2 T12R2
Maize Stover+Concentrate feed(40
% kept constant) +Azolla T9R3 T10R3 T11R3 T12R3
Maize husk + Concentrate feed(40
% kept constant) +Azolla T9R4 T10R4 T11R4 T12R4
Sorghum Stover + Concentrate
feed(40 % kept constant) +Azolla T9R5 T10R5 T11R5 T12R5
Where, T9 = Treatment with 0% azolla (Control), T10 = Treatment with 3% azolla, T11= Treatment
with 6% azolla, T12 = Treatment with 6% Azolla, R1 = Paddy straw, R2 = Ragi straw, R3 = Maize
stover, R4 = Maize husk, R5 = Sorghum atsover, R6=Bengal gram husk
Here roughage and concentrate taken in the ratio of 60:40. The portion of
roughage in the TMR was replaced with azolla at 0%, 3%, 6% and 9% and the
concentrate portion was kept constant (40%).
30
Plate 3.1: Cultivation of Azolla (Azolla pinnata) in cement tanks
31
31
3.4 Chemical Analysis
The DM content of feed samples were analyzed by drying to a constant weight in
a forced hot air oven at 105oC. The ash content in the samples was estimated as residue
after incineration of samples at 600oC for 3 hours. Crude protein (N X 6.25) was
analyzed using Gerhardt digestion and distillation unit (AOAC, 2005). The Ether extract
(EE) content in the sample was analyzed after extraction with petroleum ether using the
procedure of AOAC (2005). The fiber fractions were determined according to the
methods described by Van Soest et al. (1991).
3.5 Donor cow and collection of rumen fluid
A crossbred (Holstein Friesian x Bos indicus) lactating dairy cow, weighing 400
kgs, producing 9 kg milk per day, fitted with a flexible rumen cannula of large diameter
(Bar Diamond, Inc. USA), served as the donor of the rumen inoculum. It was fed with
basal diet consisting of finger millet straw at the rate of 6.5 to 7 kg per day and CFM
(Maize- 54%, Wheat bran - 41%, mineral mixture - 2%, salt -1%, urea – 2%) of 4.0
kg/day in two equal portions at the time of milking at 6.00 A.M and 1.30 P.M. The rumen
fluid was collected between 9 A.M and 9.30 A.M before offering finger millet straw.
3.6 Rumen fluid collection
The rumen inoculum collected from rumen‐cannulated cow was brought in a
closed plastic container (Thermos) pre‐warmed with hot water. Completely filled
container with rumen fluid content brought to the laboratory, filtered through four layers
of cheese cloth and then mixed with the medium prepared (Menke and Steingas, 1988).
32
32
The buffered rumen fluid was supplied with CO2 to minimize changes in microbial
populations and to avoid O2 contamination while handling at 39oC at all the time.
3.7 Preparation of Medium for Rumen in vitro gas production kinetic study
(Incubation of 102 in vitro glass syringes)
Micro mineral solution – 0.28 ml
Macro mineral solution – 542.5 ml
Buffer solution – 542.5 ml
Resazurin – 2.8 ml
All these solution were mixed with 1085 ml of distilled water to get 2 liters of
buffer solution.
3.8 Estimation of Metabolizable energy (ME) content (Menke and Steingass, 1988)
1. The medium was prepared on the previous day of incubation in a water bath set at
39oC and carbon dioxide was supplied to the medium for 15 to 20 minutes and the
medium was continuously stirred with a magnetic stirrer.
2. On the day of incubation rumen fluid was collected from rumen fistulated animal
and was filtered through four layers of cheese cloth.
3. Reducing solution was added to the medium prepared. The medium turns pink to
colorless when the CO2 was supplied to the medium.
4. About 1137.5 ml of rumen fluid was added to the medium and mixed. CO2
wassupplied continuously until the completion of filling 102 syringes with rumen
fluid to maintain the anaerobic environment.
33
33
5. Air equilibrated samples of crop residues and TMR (200 ± 5mg) were incubated
in 100 ml calibrated glass syringes supplemented with azolla at 0, 3, 6, 9 percent
and azolla as such in triplicates with 30 ml of buffered rumen fluid with three
blank incubations and the reference standards of roughage and concentrates.
6. The incubation was done in a water bath maintained at 39oC
7. The readings of displaced syringes were recorded at different time intervals over
24 hours that is at 2 hours, 5 hours and 24 hours. Whenever the syringe readings
exceed 90 ml, the readings were reset to 30 ml. The cumulative gas production
was calculated.
8. For determination of ME content, 24 hours net cumulative gas production was
corrected for reference standards.
9. Using chemical composition and net gas production (corrected for blank and
reference standard) at 24 hr of incubation, ME was calculated by using the
following equations.
Roughages:
ME = 2.2 + 0.1357 GP + 0.0057 CP + 0.0002859 EE2
For Azolla and compounded feed or TMR
ME= 1.06 + 0.1570 GP + 0.0084 CP + 0.022 EE - 0.0081 TA
Where, ME = Metabolizable energy (MJ / Kg DM), GP = gas production for 24 h in
ml/200mg DM, CP = Crude protein (g / kg DM), EE = ether extract (g / kg DM), TA=
total ash in g / kg DM.
34
34
3.9 Estimation of ADDM and TDDM by Modified in vitro two stage digestion
technique (Goering and Van Soest, 1970)
1. The medium was prepared on the previous day of incubation by dissolving 3.35
liters of buffer solution with 250 ml of reducing solution and the CO2 was
dispensed untill the medium turns blue to colourless and the medium was stored
at 39oC incubator overnight.
2. On the day of incubation rumen fluid was collected from rumen fistulated animal
and was filtered through four layers of cheese cloth.
3. Air equilibrated samples in duplicates of about 400 ± 5mg was weighed in pre-
labeled and weighed filter bags and sealed with thermo sealer and transferred into
respective Erlenmeyer flask and 80 ml of buffered rumen fluid was added to the
each flask
4. Incubation was done in the water bath at 39oC for 48 hours, blank bags was also
kept for incubation.
5. Anaerobic condition of the inoculums was maintained by supplying carbon
dioxide.
6. After incubation for 48 hours, the bags were taken out and gently squeezed to
remove excess liquid, washed with distilled water and dried at 55oC for minimum
of 5 hours.
7. After cooling weight of bag was recorded for calculation of ADDM (Apparent
digestible dry matter).
35
35
8. The dried bags were refluxed with neutral detergent solution (NDS) for 1 hour, 15
minutes and the bags washed with hot distilled water and dried at 105oC for 2 to 3
hours.
9. After cooling weight of bag was taken for calculation of TDDM (True digestible
dry matter).
Calculation
NDF residue = [W3 - (W1xC1)] / W2 x 100
Where, W 1= Empty bag weight, W2 = Sample weight, W3 = Dried weight of bag with
fiber after extraction process, C1 = Blank bag correction (final oven dried weight divided
by original blank bag weight)
TDDM % =100 - NDF residue
ADDM% =100 - weight of residue before NDS refluxing
3.10 Estimation of mineral profile of Azolla
Preparation of sample
1. About 2 g of azolla samples was taken into the silica crucible
2. The sample in the crucible was kept in the muffle furnace at 550oC for 3 hrs to
make it ash
3. Crucible was taken out from muffle furnace after cooling
4. The ash was transferred quantitatively to 250 ml beaker using little quantity of
distilled water.
36
36
5. To the beaker containing ash 25 ml of dilute HCl (1:2) was added and boiled for
10 minutes.
6. The cooled solution was filtered through whatman No. 1 filter paper into a
volumetric flask of around 250 ml capacity and the volume was made upto the
mark with distilled water and mixed thoroughly.
Analysis of azolla for minerals by Inductively coupled plasma-atomic emission
spectrophotometer (ICP-AES).
The following steps were performed while working with Atomic Emission Spectrometry
1. The computer, printer and main power of the ICP was turned on.
2. The valve on argon tank was opened and the tank was filled upto 500 lbs and the gas
regulator on the argon tank was set at 80 psi.
3. The inlet and outlet tubing on peristaltic pump was set and the pump was switched on
to the low setting.
4. Plasma was ignited and the instrument was allowed to warm up to 20 minutes to
stabilize the wavelength.
5. The method file from the operating software was verified for all the elements of
interest.
6. The right wavelength for each element and the concentrations of the calibration
standards were set.
7. Analyses was done in the order of blanks, standards and azolla sample.
37
37
8. After analyses, the azolla sample was run with 5% HNO3 through the ICP for 5
minutes, followed by distilled water for 10 minutes.
9. The peristaltic pump and the main power was turned off.
10. The valve of argon tank was closed.
11. The ICP analysis program on the computer was closed.
12. The concentration of the liquid digest sample was reported in milligrams/liter (mg/L).
Calculation
Concentration of mineral in sample solution (mg / L ) x Volume made (ml)
µg / g = --------------------------------------------------------------------------------------------------
Weight of sample (g)
3.10 Estimation of amino acid profile of Azolla
The amino acid composition of azolla was analyzed at Evonik (SEA) Laboratory,
Singapore using the fast and reliable Near Infrared Reflectance Spectroscopy (AMINO
NIR).
3.11 Statistical Analysis
The mean values of ME content of the various diets were subjected to statistical
analysis using the software Graph pad prism version 5.0. One way ANOVA was used to
test the hypothesis according to the procedures described by Snedecor and Cochran
(1994). Individual differences between the mean values of ME were tested using
Bonferoni ‘t’ test when the treatment effect was significant. Individual differences
between the mean values of ADDM and TDDM were tested using Unpaired ‘t’ test.
RReessuullttss
IV. RESULTS
The results of the present study are presented in the form of Tables at the end of
this chapter. Detailed experimental results and data are provided in the appendices.
4.1 Chemical composition of Azolla, Crop residues and CFM
4.1.1 Proximate priciples of Azolla, Crop residues and CFM
The proximate principles of Azolla pinnata, Paddy straw, Finger millet straw,
Maize stover, Maize husk, Sorghum stover, Bengal gram husk and Compound feed
mixture (CFM) are presented in Table 4.1.1. The moisture content of fresh azolla was
found to be 95.63 per cent (4.37 per cent DM). The Dry matter content of dried sample of
azollawas found to be 96.52 per cent. The DM content was found to be 95.63 per cent in
paddy straw, 84.64 per cent in ragi straw, 94.46 per cent in maize stover, 89.72 per cent
in maize husk, 87.61 per cent in sorghum stover, 87.44 per cent in bengal gram husk and
86.74 per cent in compound feed mixture. The organic matter content (DMB) was found
to be 82.16 per cent in azolla, 84.21 per cent in paddy straw, 89.25 per cent in ragi straw,
92.80 per cent in maize stover, 97.45 per cent in maize husk, 93.48 per cent in sorghum
stover, 96.38 per cent in bengal gram husk and 94.00 per cent in compound feed mixture
(CFM). The crude protein (DMB) content was found highest in azolla (21.66 per cent)
followed by 17.89 per cent in compound feed mixture , 4.27 per cent in bengal gram
husk, 3.55 per cent in ragi straw, 3.25 per cent in sorghum stover, 2.92 in maize stover,
2.51 per cent in paddy straw and 1.84 per cent in maize husk. The ether extract (DMB)
content was found to be 4.41 per cent in azolla, 0.84 per cent in paddy straw, 0.98 per
cent in ragi straw, 0.87 per cent in maize stover, 0.61 per cent in maize husk, 1.08 per
39
Table 4.1.1: Proximate principles1 (on per cent dry matter basis) of Azolla pinnata, Crop residues and CFM
Samples Proximate principles
DM % OM CP EE CF TA NFE
Azolla pinnata 4.371 82.16 21.66 4.41 15.15 17.84 40.94
Paddy straw 95.63 84.21 2.513 0.841 35.31 15.79 45.55
Ragi straw 84.64 89.25 3.558 0.983 32.49 10.75 52.23
Maize stover 94.46 92.80 2.921 0.873 30.51 7.217 58.49
Maize husk 89.72 97.45 1.844 0.611 31.83 2.551 63.17
Sorghum stover 87.61 93.48 3.253 1.084 30.64 6.522 58.51
Bengal gram husk 87.44 96.38 4.272 0.793 46.72 3.624 44.60
Compound feed mixture 86.74 94.00 17.89 4.081 8.351 6.00 63.68
1Mean of two replicates. Variations in duplicate measurements were within ± 3 % of the mean
40
40
cent in sorghum stover, 0.79 per cent in bengal gram husk and 4.08 per cent in compound
feed mixture. The Total ash (DMB) content was found highest in azolla (17.84 per cent)
followed by 15.79 per cent in paddy straw, 10.75 per cent in ragi straw, 7.2 per cent in
maize stover, 6.52 per cent in sorghum stover, 6.0 per cent in compound feed mixture,
3.62 per cent in bengal gram husk and 2.55 per cent in maize husk. The crude fiber
(DMB) content was found to be 15.15 per cent in azolla, 35.31 per cent in paddy straw,
32.49 per cent in ragi straw, 30.51 per cent in maize stover, 31.83 per cent in maize husk,
30.64 per cent in sorghum stover, 46.72 per cent in bengal gram husk and 8.35 per cent in
CFM.
4.1.2 Fiber fractions of Azolla, Crop residues and CFM
The fiber fractions of Azolla, paddy straw, ragi straw, maize stover, maize husk,
sorghum stover, bengal gram husk and compound feed mixture are presented in Table
4.1.2. The NDF (DMB) content was found to be 54.86 per cent in azolla, 71.50 per cent
in paddy straw, 66.26 per cent in ragi straw, 70.71 per cent in maize stover, 75.71 per
cent in maize husk, 66.72 per cent in sorghum stover, 71.09 per cent in bengal gram husk
and 19.80 per cent in CFM. The ADF (DMB) content was found to be 36.57 per cent in
azolla, 50.0 per cent in paddy straw, 37.33 per cent in ragi straw, 49.71 per cent in maize
stover, 42.18 per cent in maize husk, 38.21 per cent in sorghum stover, 58.2 per cent in
bengal gram husk and 10.11 per cent in compound feed mixture. The ADL (DMB)
content was found to be highest in azolla (24.03 per cent) followed by, 6.26 per cent in
maize stover, 5.14 per cent in ragi straw, 4.93 per cent in sorghum stover, 4.76 per cent in
bengal gram husk, 4.78 per cent in paddy straw, 2.16 per cent in maize husk and 1.13 per
cent in CFM.
41
Table 4.1.2: Fiber fractions1, total silica, biogenic silica and sand silica (on per cent dry matter basis) of Azolla pinnata, Crop
residues and CFM
Samples Azolla
pinnata
Paddy
straw Ragi straw Maize stover Maize husk Sorghum stover
Bengal
gram husk CFM
NDF 54.86 71.50 66.26 70.71 75.71 66.72 71.09 19.89
ADF 36.57 50.00 37.33 49.71 42.18 38.21 58.20 10.113
ADL 24.03 4.78 5.141 6.262 2.166 4.931 4.764 1.132
Hemicellulose 18.29 21.50 28.93 21.00 33.53 28.51 12.89 9.692
Cellulose 12.54 45.22 32.19 43.45 40.02 33.28 51.44 8.981
Total silica(ADF ash) 5.61 10.96 5.381 7.968 3.744 3.662 2.892 2.776
Biogenic silica(ADF
ash-NDF ash) 3.341 6.754 3.138 4.289 2.081 2.481 1.294 1.984
Sand silica
(NDF ash) 2.272 4.217 2.251 3.684 1.664 1.188 1.613 0.791
1Mean of two replicates. Variations in duplicate measurements were within ± 3 % of the mean
42
42
4.1.3 Mineral profile of Azolla
The mineral profile of Azolla pinnata is presented in Table 4.1.3. Azolla pinnata
was found to contain 1.64 per cent Calcium, 0.34 per cent magnesium, 2.71 per cent
potassium, 9.1 ppm of copper, 325 ppm of Zinc, 1569 ppm of Iron, 8.11 ppm of cobalt,
5.06 ppm of chromium, 2418 ppm of manganese, 31 ppm of boron, 5.33 ppm nickel, 8.1
ppm of lead, 1.2 ppm of cadmium.
4.1.4 Amino acid profile of Azolla
The amino acid profile of azolla is presented in Table 4.1.4. Azolla found to be
rich in all essential amino acids including branched chain amino acids like valine, leucine
and isoleucine.
4.2 Chemical composition of various diets
The chemical composition of various diets comprising paddy straw supplemented
with different levels of azolla from 0 to 9 per cent and the TMR comprising paddy straw
and CFM supplemented with different levels of azolla from 0 to 9 per cent are presented
in Table 4.2.1. The chemical composition of various diets comprising ragi straw
supplemented with different levels of azolla from 0 to 9 per cent and the TMR
comprising ragi straw and CFM supplemented with different levels of azolla from 0 to 9
per cent are presented in Table 4.2.2. The chemical composition of various diets
comprising maize stover supplemented with different levels of azolla from 0 to 9 per cent
and the TMR comprising maize stover and CFM supplemented with different levels of
azolla from 0 to 9 per cent are presented in Table 4.2.3. The chemical composition of
43
43
Table 4.1.3: Mineral profile of Azolla pinnata (on per cent DMB)
Minerals Percentage ppm
Calcium 1.64 -
Magnesium 0.34 -
Pottassium 2.71 -
Copper - 9.1
Manganese - 2418
Zinc - 325
Iron - 1569
Cobalt - 8.11
Chromium - 5.06
Boron - 31
Nickel - 5.33
Lead - 8.1
Cadmium - 1.2
44
44
Table 4.1.4: Amino acid profile of Azolla pinnata
Amino acids (AA) AA % DMB AA% in CP
Lysine 1.231 4.940
Methionine 0.413 1.657
Cystine 0.194 0.778
Threonine 1.164 4.671
Arginine 1.414 5.674
Isoleucine 1.160 4.655
Leucine 2.072 8.315
Phenylalanine 1.377 5.526
Glycine 1.341 5.381
Serine 1.124 4.510
Valine 1.445 5.799
Alanine 1.539 6.176
Histidine 0.488 1.956
Proline 1.032 4.141
Aspartic acid 2.303 9.242
Glutamic acid 2.740 10.995
Total (without NH3) 21.037 84.418
Ammonia 0.509 2.043
Total 21.546 86.461
45
Table 4.2.1: Chemical composition1 (Percentage on DMB) of various diets comprising Paddy straw supplemented with
different levels of azolla (0 to 9 per cent) and the TMR comprising paddy straw and CFM supplemented with
different levels of azolla (0 to 9 per cent).
Samples Chemical composition (% DMB)
DM % CP EE TA CF NFE NDF ADF ADL
T1R1(Control)* 0.982 2.5 0.84 15.5 35.3 44.6 71.5 50 4.7
T2R1 0.9815 3.08 0.95 15.6 34.7 44.4 71.0 49.5 5.28
T3R1 0.9809 3.64 1.05 15.6 34.1 44.3 70.5 49.1 5.87
T4R1 0.9805 4.22 1.16 15.7 33.4 44.2 70.0 48.7 6.46
T5R1(Control)** 0.973 7.72 2.14 11.7 24.5 53.1 50.8 34.0 3.40
T6R1 0.9732 7.90 2.15 12.0 24.7 52.4 51.8 34.8 4.09
T7R1 0.9733 8.9 2.16 12.4 24.9 51.6 52.9 35.6 4.7
T8R1 0.9735 8.27 2.17 12.7 25.13 50.89 53.97 36.42 5.46
T9R1 (Control)*** 0.973 7.72 2.14 11.71 24.5 53.1 50.8 34.04 3.4
T10R1 0.9725 8.29 2.24 11.93 23.9 53.0 50.3 33.6 3.9
T11R1 0.972 8.87 2.35 11.99 23.3 52.9 49.8 33.2 4.5
T12R1 0.9754 9.44 2.46 12.05 22.7 52.8 49.3 32.8 5.1
1Mean of two replicates. Variations in duplicate measurements were within ± 3 % of the mean,
* Type I diets
** Type II diet where CFM portion of TMR is replaced with azolla (0 to 9%)
*** Type III diet where roughage portion of TMR is replaced with azolla (0 to 9%)
46
Table 4.2.2: Chemical composition1 (Percentage on DMB) of various diets comprising Ragi straw supplemented with different
levels of azolla (0 to 9 per cent) and the TMR comprising ragi straw and CFM supplemented with different levels
of azolla (0 to 9 per cent).
Samples Chemical composition (% DMB)
DM% CP EE TA CF NFE NDF ADF ADL
T1R2(Control)* 0.9647 3.55 0.98 10.7 32.4 52.0 66.36 37.33 5.1
T2R2 0.9647 4.09 1.08 10.9 31.9 51.6 66.0 37.30 5.67
T3R2 0.9642 4.63 1.19 11.1 31.4 51.3 65.67 37.28 6.25
T4R2 0.964 5.17 1.29 11.3 30.9 50.9 65.3 37.26 6.82
T5R2(Control)** 0.9626 8.35 2.22 8.84 22.8 57.6 47.7 26.4 3.64
T6R2 0.9626 8.89 2.23 9.2 23.0 56.8 48.7 27.23 4.33
T7R2 0.9627 9.44 2.24 9.5 23.2 56.0 49.83 28.26 5.01
T8R2 0.9627 9.98 2.25 9.61 23.44 55.33 50.89 28.82 5.70
T9R1 (Control)*** 0.9626 8.5 2.22 8.84 22.8 57.6 47.7 26.4 3.64
T10R2 0.9626 8.89 2.32 9.06 22.3 57.2 47.39 26.41 4.22
T11R2 0.9627 9.44 2.43 9.27 21.7 56.9 47.04 26.39 4.8
T12R2 0.9633 9.98 2.53 9.49 21.2 56.5 46.7 26.36 5.37
1Mean of two replicates. Variations in duplicate measurements were within ± 3 % of the mean,
* Type I diet
** Type II diet where CFM portion of TMR is replaced with azolla (0 to 9%)
*** Type III diet where roughage portion of TMR is replaced with azolla (0 to 9%)
47
Table 4.2.3: Chemical composition1 (Percentage on DMB) of various diets containing Maize stover supplemented with
different levels of azolla (0 to 9 per cent) and the TMR comprising maize stover and CFM supplemented with
different levels of azolla (0 to 9 per cent).
Samples Chemical composition (% DMB)
DM% CP EE TA CF NFE NDF ADF ADL
T1R3(Control)* 0.9897 2.9 0.87 10.1 30.5 57.5 75.7 41.9 5.5
T2R3 0.9889 3.46 0.98 10.3 30.0 57.0 75.0 41.82 6.0
T3R3 0.9882 4.02 1.08 10.5 29 56.5 74.4 41.66 6.6
T4R3 0.9874 4.58 1.19 10.8 29.1 56.0 73.8 41.50 7.1
T5R3(Control)** 0.9776 7.96 2.15 8.4 21.6 60.9 50.3 33.8 4.3
T6R3 0.9778 8.14 2.16 8.8 21.8 60.1 51.3 34.66 4.9
T7R3 0.978 8.33 2.17 9.1 22.0 59.4 52.4 35.4 5.6
T8R3 0.9781 8.51 2.18 9.5 22.25 58.66 53.50 36.24 6.36
T9R3 (Control)*** 0.9776 7.96 2.15 8.4 21.6 60.9 50.3 33.8 4.3
T10R3 0.9769 8.52 2.26 8.7 21.1 60.4 49.8 33.4 4.8
T11R3 0.9762 9.09 2.37 8.8 20.7 59.9 49.3 33.0 5.3
T12R3 0.9808 9.1 2.47 8.9 20.2 594 48.9 32.6 5.93
1Mean of two replicates. Variations in duplicate measurements were within ± 3 % of the mean
* Type I diet
** Type II diet where CFM portion of TMR is replaced with azolla (0 to 9%)
*** Type III diet where roughage portion of TMR is replaced with azolla (0 to 9%)
48
48
various diets comprising maize husk supplemented with different levels of azolla from 0
to 9 per cent and the TMR comprising maize husk and CFM supplemented with different
levels of azolla from 0 to 9 per cent are presented in Table 4.2.4. The chemical
composition of various diets comprising sorghum stover supplemented with different
levels of azolla from 0 to 9 per cent and the TMR comprising sorghum stover and CFM
supplemented with different levels of azolla from 0 to 9 per cent are presented in Table
4.2.5 and the chemical composition of various diets comprising bengal gram husk
supplemented with different levels of azolla from 0 to 9 per cent are presented in Table
4.2.6.
4.3 Rumen in vitro net gas production and ME content of Azolla pinnata, Crop
residues and CFM
The rumen in vitro net gas production at 24 hours and predicted ME content of
Azolla pinnata, crop residues and CFM are presented in Table 4.3. The net gas
production (ml/200 mg DM) for 24 hours observed was highest in compound feed
mixture (48.73 ml) followed by 46.03 ml in maize husk, 45.44 ml in bengal gram husk,
41.51 ml in sorghum stover, 34.39 ml in ragi straw, 30.21 ml in maize stover, 22.37 ml in
paddy straw and 19.45 ml in Azolla pinnata. The ME (MJ / kg DM) content was found
highest for compound feed mixture (9.62 MJ / kg DM) followed by 8.57 MJ/kg DM in
bengal gram husk, 8.56 MJ / kg DM in maize husk, 8.13 MJ / kg DM in sorghum stover,
7.11 MJ / kg DM in ragi straw, 6.56 MJ / kg DM in maize stover, 5.47 MJ/kg DM in
paddy straw and 5.44 MJ/kg DM in Azolla pinnata. The gas production data of Azolla
and crop residues at different time intervals (2h, 5h) over 24 h period is presented in
Appendix II and III respectively.
49
Table 4.2.4: Chemical composition1 (Percentage on DMB) of various diets containing Maize husk supplemented with different
levels of azolla (0 to 9 per cent) and the TMR comprising maize husk and CFM supplemented with different
levels of azolla (0 to 9 per cent)
Samples Chemical composition (% DMB)
DM% CP EE TA CF NFE NDF ADF ADL
T1R4(Control)* 0.9904 1.8 0.61 2.55 31.8 63.5 75.7 41.9 5.5
T2R4 0.9896 2.39 0.72 3.01 31.3 62.5 75.0 41.8 6.06
T3R4 0.9888 2.99 0.83 3.47 30.8 61.8 74.4 41.6 6.63
T4R4 0.9881 3.58 0.95 3.93 30.3 61.1 73.8 41.5 7.19
T5R4(Control)** 0.978 7.30 1.9 3.93 22.4 64.3 50.3 33.8 4.31
T6R4 0.9782 7.48 2.00 4.29 22.6 63.5 51.4 34.6 4.99
T7R4 0.9784 7.67 2.01 4.64 22.8 62.8 52.4 35.4 5.68
T8R4 0.9786 7.85 2.02 5.0 22.25 62.0 53.5 36.2 6.36
T9R4 (Control)*** 0.9776 7.30 1.9 3.93 22.4 64.3 50.3 33.8 4.31
T10R4 0.9769 7.9 2.11 4.39 21.9 63.6 49.8 33.4 4.85
T11R4 0.9762 8.49 2.22 4.85 21.4 62.9 49.4 33.08 5.39
T12R4 0.9808 9.09 2.34 5.3 20.9 62 48.9 32.6 5.94
1Mean of two replicates. Variations in duplicate measurements were within ± 3 % of the mean
* Type I diet
** Type II diet where CFM portion of TMR is replaced with azolla (0 to 9%)
*** Type III diet where roughage portion of TMR is replaced with azolla (0 to 9%)
50
Table 4.2.5: Chemical composition1 (Percentage on DMB) of various diets containing Sorghum stover supplemented with
different levels of azolla (0 to 9 per cent) and the TMR comprising sorghum stover and CFM supplemented with
different levels of azolla (0 to 9 per cent).
Samples Chemical composition (% DMB)
DM% CP EE TA CF NFE NDF ADF ADL
T1R5(Control)* 0.9824 3.25 1.08 6.54 30.6 57.6 66.72 38.21 4.9
T2R5 0.9818 3.80 1.18 6.88 30.1 57.0 66.36 38.16 5.48
T3R5 0.9813 4.35 1.28 7.23 29.7 56 66.01 38.11 6.06
T4R5 0.9808 4.90 1.38 7.57 29.2 56.0 65.65 38.06 6.65
T5R5(Control)** 0.9819 8.17 2.28 6.32 21.7 60.9 47.95 26.97 3.53
T6R5 0.9814 8.35 2.29 6.68 21.9 60.2 49 27.76 4.21
T7R5 0.981 8.54 2.3 7.04 22.1 59.4 50.06 28.55 4.9
T8R5 0.9805 8.72 2.31 7.4 22.3 58.6 51.11 29.35 5.58
T9R4 (Control)*** 0.9819 8.17 2.28 6.32 21.7 60.9 47.95 26.97 3.53
T10R5 0.9814 8.72 2.38 6.67 21.2 60.4 47.6 26.92 4.11
T11R5 0.9809 92.7 2.48 7.01 20.7 59.9 47.24 26.87 4.69
T12R4 0.982 9.83 2.58 7.35 20.3 59.4 46.88 26.82 5.27
1Mean of two replicates. Variations in duplicate measurements were within ± 3 % of the mean
* Type I diet
** Type II diet where CFM portion of TMR is replaced with azolla (0 to 9%)
*** Type III diet where roughage portion of TMR is replaced with azolla (0 to 9%)
51
Table 4.2.6: Chemical composition1 (Percentage on DMB) of various diets comprising Bengal gram husk supplemented with
different levels of azolla (0 to 9 per cent).
Samples
Chemical composition (% DMB)
DM% CP EE TA CF NFE NDF ADF ADL
T1R6(Control)* 0.9857 4.27 0.79 3.63 46.7 44.6 71.09 58.2 4.76
T2R6 0.985 4.79 0.9 4.06 45.7 44.5 70.6 57.34 5.29
T3R6 0.9844 5.31 1.01 4.49 44.8 44.3 70.12 56.48 5.81
T4R6 0.9838 5.83 1.12 4.92 43.8 44.2 69.63 55.62 6.33
1Mean of two replicates. Variations in duplicate measurements were within ± 3 % of the mean.
* Type I diet
52
Table 4.3: Rumen in vitro net gas production1 at 24 hours (IVGP-24h) and predicted metabolizable energy
2 (ME) content of
Azolla pinnata, Crop residues and CFM.
Samples Rumen in vitro net gas production
(ml/ 200 mg/ 24h) ME(MJ/kg DM)
Azolla pinnata 19.45 5.44
Paddy straw 22.37 5.47
Ragi straw 34.39 7.11
Maize stover 30.21 6.56
Maize husk 46.03 8.56
Sorghum stover 41.51 8.13
Bengal gram husk 45.44 8.57
Compound feed mixture 48.73 9.62
1Mean of six replicates. Variations in six replicate measurements were within ± 3 % of the mean.
2Mean of six replicates. Variations in six replicate measurements were within ± 3 % of the mean.
53
53
4.4 Rumen in vitro gas production of various diets
The rumen in vitro gas production of type I diet, type II diet and type III diet are
presented in the Table 4.4.1., Table 4.4.2 and Table 4.4.3, respectively. The detailed gas
production data of various diets at different time intervals (2h, 5h) over 24 h period is
presented in appendix I to IX.
4.5 Metabolizable energy content of various diets
4.5.1 Metabolizable energy content of Type I diet
The ME content of diets comprising crop residues supplemented with different
levels of azolla from 0 to 9 per cent are presented in Table 4.5.1. The ME content of
paddy straw (T1R1-control) was 5.47 MJ / kg DM. The ME content of the diet comprising
paddy straw supplemented with 3 % (T2R1), 6% (T3R1) and 9% (T4R1) azolla was 5.39MJ
/ kg DM, 5.28 MJ / kg DM and 5.43 MJ/ kg DM respectively. There was no significant
difference (P>0.05) in the ME content of the diets supplemented with different levels of
azolla compared with the control. The ME content of ragi straw (T1R2-control group) was
7.11 MJ/ kg DM. The ME (MJ/kg DM) content of the diet comprising ragi straw
supplemented with 3 % (T2R2), 6% (T3R2)and 9% (T4R2) azolla was 6.91, 6.28 and 6.35,
respectively. There was significant difference (P<0.05) in the ME content of the diets
supplemented with 3%, 6% and 9% of azolla compared with control. The ME content of
maize stover (T1R3- control group) was 6.56 MJ/ kg DM. The ME (MJ/kg DM) content of
the diet comprising maize stover supplemented with 3% (T2R3), 6% (T3R3) and 9%
(T4R3) azolla was 6.17, 6.13 and 6.12, respectively. There was significant difference
(P<0.05) in the ME content of the diets supplemented with different levels of azolla
54
Table 4.4.1: Rumen in vitro net gas production1 of diets comprising roughage replaced with different levels of azolla from 0 to
9 per cent (Type I diet).
Samples
Rumen in vitro net gas production(ml/200 mg DM/24 h)
0% Azolla (T1) 3% Azolla (T2) 6% Azolla (T3) 9% Azolla (T4)
Paddy straw (R1) 22.37 21.49 20.42 21.23
Ragi straw (R2) 34.39 34.13 28.11 27.99
Maize stover (R3) 30.21 27.08 26.42 26.09
Maize husk (R4) 46.03 45.03 43.96 40.69
Sorghum stover (R5) 41.51 38.97 39.59 38.56
Bengal gram husk (R6) 45.44 44.98 44.67 44.05
1Mean of six replicates. Variations in six replicate measurements were within ± 3 % of the mean.
T1=Treatment with 0% azolla (Control group), T2= Treatment with 3% azolla, T3= Treatment with 6% azolla, T4= Treatment with 9%
azolla, R1=Paddy straw, R2=Ragi straw, R3=Maize Stover, R4=Maize husk, R5=Sorghum Stover, R6=Bengal gram husk
55
Table 4.4.2: Rumen in vitro net gas production1 of diets (TMR) comprising roughage (60 per cent kept constant) and CFM (31
to 40 per cent) replaced with different levels of azolla from 0 to 9 per cent (Type II diet).
Samples
Rumen in vitro net gas production(ml/200 mg DM/24 h)
0% Azolla (T5) 3% Azolla (T6) 6% Azolla (T7) 9% Azolla (T8)
Paddy straw + CFM+ azolla (R1) 41.6 39.56 38.01 36.31
Ragi straw + CFM+ azolla (R2) 43.36 42.16 41.49 40.67
Maize stover +CFM + azolla (R3) 40.26 39.57 39.00 38.4
Maize husk+ CFM+ azolla (R3) 46.65 46.49 46.14 45.91
Sorghum stover +CFM + azolla (R4) 47.3 46.9 45.84 44.15
1Mean of six replicates. Variations in six replicate measurements were within ± 3 % of the mean.
T5= Treatment with 0% Azolla (Control), T6= Treatment with 3% Azolla, T7= Treatment with 6% Azolla, T8 = Treatment with 6%
Azolla, R1=Paddy straw, R2=Ragi straw, R3=Maize Stover, R4=Maize husk, R5=Sorghum Stover, R6=Bengal gram husk.
56
Table 4.4.3: Rumen in vitro net gas production1 of diets (TMR) comprising CFM (40 per cent kept constant) and roughage (51
to 60 per cent) replaced with different levels of azolla from 0 to 9 per cent (Type III diet)
Samples
Rumen in vitro net gas production(ml/200 mg DM/24 h)
0% Azolla (T9) 3% Azolla (T10) 6% Azolla (T11) 9% Azolla (T12)
Paddy straw + CFM + azolla (R1) 41.6 40.88 37.91 37
Ragi straw + CFM+ azolla (R2) 43.36 39.73 37.84 36.84
Maize stover +CFM + azolla (R3) 40.26 38.23 37.5 36.18
Maize husk+ CFM+ azolla (R4) 46.65 46.22 46.13 45.42
Sorghum stover CFM + azolla (R5) 47.3 46.72 46.1 45.6
1Mean of six replicates. Variations in six replicate measurements were within ± 3 % of the mean.
T9 = Treatment with 0% azolla (Control), T10 = Treatment with 3% azolla, T11= Treatment with 6% azolla, T12 = Treatment with 6%
Azolla, R1 = Paddy straw, R2 = Ragi straw, R3 = Maize stover, R4 = Maize husk, R5 = Sorghum atsover, R6=Bengal gram husk
57
Table 4.5.1: Metabolizable energy1 (ME) content of diets comprising roughage replaced with different levels of azolla from 0
to 9 per cent (Type I diet).
Samples
ME (MJ / kg DM)(Mean ± SE)
0% Azolla (T1) 3% Azolla (T2) 6% Azolla (T3) 9% Azolla (T4)
Paddy straw (R1) 5.47 ± 0.04 5.39 ± 0.12 5.28 ± 0.09 5.43 ± 0.04
Ragi straw (R2) 7.11a ± 0.05
6.91
b ± 0.10
6.28
b ± 0.05
6.19
b ± 0.04
Maize stover (R3) 6.56a ± 0.06
6.17
b ± 0.06
6.13
b ± 0.04
6.12
b ± 0.04
Maize husk (R4) 8.56a ± 0.07
8.46
b ± 0.09
8.16
b ± 0.03
7.88
b ± 0.10
Sorghum stover (R5) 8.13 ± 0.04 7.80 ± 0.04 7.95 ± 0.05 7.85 ± 0.14
Bengal gram husk (R6) 8.57 ± 0.06 8.46 ± 0.14 8.58 ± 0.07 8.53 ± 0.07
Means bearing different superscripts in a row differ significantly (P<0.05). 1Mean of six replicates. Variations in six replicate measurements were within ± 3 % of the mean.
T1=Treatment with 0% azolla (Control group), T2= Treatment with 3% azolla, T3= Treatment with 6% azolla, T4= Treatment with 9%
azolla, R1=Paddy straw, R2=Ragi straw, R3=Maize Stover, R4=Maize husk, R5=Sorghum Stover, R6=Bengal gram hus
58
58
compared with the control. There was decreasing trend of ME content as the percentage
of supplementation of azolla was increased. The ME content of maize husk (T1R4-
control group) was 8.56 MJ/ kg DM. The ME (MJ/kg DM) content of the diet comprising
maize husk supplemented with 3% (T2R4), 6% (T3R4) and 9% (T4R4) azolla was 8.46,
8.16 and 7.88, respectively. There was significant (P<0.05) difference between the ME
content of diet supplemented with different levels of azolla compared to the control.
There was decreased ME content in the supplemented diet compared to control. The ME
content of sorghum stover (T1R5-control group) was 8.13 MJ / kg DM. The ME (MJ / kg
DM) content of the diet comprising sorghum stover supplemented with 3 % (T2R5), 6%
(T3R5) and 9% (T4R5) azolla was 7.80, 7.95 and 7.85, respectively. There was no
significant (P>0.05) difference between three supplemented diets and control. The ME
content of bengal gram husk (T1R6- control group) was 8.57 MJ/ kg DM. The ME
(MJ/kg DM) content of the diet comprising bengal gram husk supplemented with 3%
(T2R6), 6% (T3R6) and 9% (T4R6) azolla was 8.46, 8.58 and 8.53, respectively. The
difference among the three different levels of supplementation was not statistically
significant (P>0.05) compared with the control.
4.5.2 Metabolizable energy (ME) content of Type II diet
The ME content of diets comprising crop residue (60 per cent kept constant) and
CFM which was replaced with different levels of azolla from 0 to 9 per cent are presented
in Table 4.5.2. The ME content of Paddy straw based TMR: (T5R1- control) was 7.57 MJ
/ kg DM. The ME (MJ / kg DM) content of the supplemented diets T6R1, T7R1 and T8R1
was 7.26, 7.03 and 6.78, respectively. There was significant difference (P<0.05) in the
ME content of the diets supplemented with different levels of azolla compared with the
59
Table 4.5.2: Metabolizable energy1 (ME) content of diets (TMR) comprising roughage (60 per cent kept constant) and CFM
(31 to 40 per cent) replaced with different levels of azolla from 0 to 9 per cent (Type II diet).
Samples
ME (MJ / kg DM) (Mean ± SE)
0% Azolla (T5) 3% Azolla (T6) 6% Azolla (T7) 9% Azolla (T8)
Paddy straw + CFM+ azolla (R1) 7.57a ± 0.03
7.26
b ± 0.10
7.03
b ± 0.09
6.78
b ± 0.02
Ragi straw + CFM+ azolla (R2) 8.05 ± 0.29 7.91 ± 0.26 7.86 ± 0.21 7.78 ± 0.13
Maize stover +CFM + azolla (R3) 7.65 ± 0.17 7.55 ± 0.09 7.48 ± 0.07 7.40 ± 0.10
Maize husk+ CFM+ azolla (R3) 8.80 ± 0.02 8.78 ± 0.08 8.74 ± 0.1 8.89 ± 0.13
Sorghum stover +CFM + azolla (R4) 8.96 ± 0.16 8.91 ± 0.12 8.76 ± 0.19 8.51 ± 0.12
Means bearing different superscripts in a row differ significantly(P<0.05).
1Mean of six replicates. Variations in six replicate measurements were within ± 3 % of the mean.
T5= Treatment with 0% Azolla (Control), T6= Treatment with 3% Azolla, T7= Treatment with 6% Azolla, T8 = Treatment with 6%
Azolla, R1=Paddy straw, R2=Ragi straw, R3=Maize Stover, R4=Maize husk, R5=Sorghum Stover, R6=Bengal gram husk.
60
60
control. There was decreased ME content in the supplemented compared to the control.
The ME (MJ / kg DM) content of ragi straw based TMR: T5R2- control group, T6R2, T7R2
and T8R2 (supplemented diets) was 8.05, 7.91, 7.86 and 7.78, respectively. There was no
significant difference (P>0.05) in the ME content of the three different levels of
supplementation compared to the control. The ME (MJ / kg DM) content of maize stover
based TMR: T5R3- control, T6R3, T7R3 and T8R3 (supplemented diets) was 7.65, 7.55,
7.48, and 7.40, respectively. There was no significant difference (P>0.05) in the ME
content of the three different levels of supplementation compared to the control. The ME
(MJ / kg DM) content of maize husk based TMR: T5R4- control group, T6R4, T7R4 and
T8R4 (supplemented) was 8.8, 8.78, 8.74 and 8.89, respectively. There was no significant
difference (P>0.05) in the ME content of azolla supplemented diet compared to the
control. The ME (MJ / kg DM) content of Sorghum stover based TMR: T5R5- control,
T654, T7R5 and T8R5 (supplemented) was 8.96, 8.91, 8.76 and 8.51 respectively. The
difference among the three supplemented diet was not statistically significant (P>0.05)
compared to the control.
4.5.3 Metabolizable energy (ME) content of Type III diet
The ME content of diets (TMR) comprising CFM (40 per cent kept constant) and
roughage which was replaced with different levels of azolla from 0 to 9 per cent are
presented in Table 4.5.3. The ME content of Paddy straw based TMR: T9R1 (control) was
7.57 MJ/ kg DM. The ME (MJ/ kg DM) content of the diets T10R1, T11R1 and T12R1
(supplemented) was 7.52, 7.45 and 7.36, respectively. There was significant difference
(P<0.05) in the ME content of the diets (TMR) supplemented with different levels of
azolla compared with the control group. However there was no significant difference
61
Table 4.5.3: Metabolizable energy1 (ME) content of diets (TMR) comprising CFM (40 per cent kept constant) and roughage
(51 to 60 per cent) replaced with different levels of azolla from 0 to 9 per cent (Type III diet).
Samples
ME (MJ / kg DM) (Mean ± SE)
0% Azolla (T9) 3% Azolla (T10) 6% Azolla (T11) 9% Azolla (T12)
Paddy straw + CFM+ azolla (R1) 7.57a ± 0.03
7.24
b ± 0.24
7.11
b ± 0.19
7.05
b ± 0.26
Ragi straw + CFM+ azolla (R2) 8.05a ± 0.29
7.53
b ± 0.08
7.28
b ± 0.21
7.18
b ± 0.12
Maize stover +CFM + azolla (R3) 7.65 ± 0.17 7.57 ± 0.15 7.39 ± 0.12 7.40 ± 0.07
Maize husk+ CFM+ azolla (R3) 8.80 ± 0.02 8.77 ± 0.08 8.8 ± 0.12 8.72 ± 0.07
Sorghum stover +CFM + azolla (R4) 8.96 ± 0.16 8.91 ± 0.08 8.85 ± 0.13 8.80 ± 0.06
Means bearing different superscripts in a row differ significantly (P<0.05).
1Mean of six replicates. Variations in six replicate measurements were within ± 3 % of the mean.
T9 = Treatment with 0% azolla (Control), T10 = Treatment with 3% azolla, T11= Treatment with 6% azolla, T12 = Treatment with 6%
Azolla, R1 = Paddy straw, R2 = Ragi straw, R3 = Maize stover, R4 = Maize husk, R5 = Sorghum atsover, R6=Bengal gram husk
62
62
among the supplemented diets. The ME (MJ/kg DM) content of the diets T9R2 (control),
T10R2, T11R2 and T12R2 (supplemented) was 8.05, 7.79, 7.80 and 7.69, respectively. There
was significant difference (P<0.05) in the ME content of the three supplemented diets
compared to the control but there was no significant difference among the diets
supplemented with different levels of azolla. The ME (MJ / kg DM) content of maize
stover based TMR: T9R3- control group, T10R3, T11R3 and T12R3 (supplemented) was
7.65, 7.57, 7.39 and 7.40, respectively. There was no significant difference (P>0.05) in
the ME content of the three groups of diet compared to the control. The ME (MJ / kg
DM) content of maize husk based TMR: T9R4- control group, T10R4, T11R4 and T12R4
(supplemented) was 8.8, 8.77, 8.8 and 8.72, respectively. There was no significant
(P>0.05) difference between supplemented and control diets. The ME (MJ / kg DM)
content of sorghum stover based TMR: T9R5- control group, T10R5, T11R5 and T12R5
(supplemented) was 8.96, 8.91, 8.85 and 8.80, respectively. The difference among the
three treatment diets was not statistically significant (P>0.05) compared with the control.
4.6 Apparent digestible dry matter (ADDM, per cent DMB) and True digestible
dry matter (TDDM, per cent DMB)
4.6.1 Apparent digestible dry matter (ADDM) and True digestible dry matter
(TDDM) of Type I diet
The Apparent digestible dry matter (ADDM) and True digestible dry matter
(TDDM) of Azolla pinnata and the diets comprising roughage supplemented with azolla
at 0 and 9 per cent is presented in Table 4.6.1. The ADDM and TDDM of Azolla was
found to be 29.59 % and 54.49 %, respectively. The ADDM of diet comprising paddy
straw supplemented with 0% (control) and 9% azolla was 30.27 % and 23.06 per cent,
63
Table 4.6.1: Apparent digestible dry matter (ADDM %) and True digestible dry matter (TDDM %) of Azolla pinnata and the
diets comprising roughage replaced with azolla at 0 per cent and 9 per cent (Type I diet).
Samples
ADDM %(Mean ± SE) TDDM %(Mean ± SE)
0% Azolla (T1) 9% Azolla (T4) 0% Azolla (T1) 9% Azolla (T4)
Azolla pinnata 29.59 ± 0.31 - 54.49 ± 0.43 -
Paddy straw with azolla (R1) 30.27a ± 1.34
23.07
b ± 2.08
51.18
c ± 1.24
46.29
d ± 0.94
Ragi straw with azolla (R2) 46.12a ± 0.25
35.49
b ± 0.73
62.19
c ± 0.49
53.96
d ± 0.26
Maize stover with azolla( R3) 31.01 ± 4.77 28.66 ± 0.22 49.50 ± 4.6 49.27 ± 0.22
Maize husk with azolla( R4) 32.89 ± 0.78 28.71 ± 0.81 50.14 ± 0.56 48.62 ± 0.52
Sorghum stover with azolla( R5) 30.16 ± 0.061 29.34 ± 0.40 50.70 ± 0.82 48.45 ± 0.35
Means bearing different superscripts in a row differ significantly (P<0.05).
T1= Treatment with 0% azolla (Control), T4 = Treatment with 9% azolla, R1 = Paddy straw, R2 = Ragi straw, R3 = Maize stover, R4 =
Maize husk, R5 = Sorghum atsover, R6=Bengal gram husk
64
64
respectively. The TDDM of diet comprising paddy straw supplemented with 0% (control)
and 9% azolla was 51.18 % and 46.28 per cent, respectively. There was significant
(P>0.05) difference in the ADDM and TDDM between the control and diet supplemented
with 9% azolla. There was decreased ADDM and TDDM % in 9% azolla supplemented
diet. The ADDM and TDDM of ragi straw based diets T1R2 (control) andT4R2 (9% azolla
treatment)were 46.12%, 35.49% and 62.19%, 53.96%, respectively. There was
significant (P<0.05) difference in ADDM and TDDM content in 9% treated diet
compared to control. There was decreased ADDM and TDDM % in 9% azolla
supplemented diet. The ADDM and TDDM of maize stover based diets T1R3 (control)
andT4R3 (9% azolla treatment)were 31.01%, 28.66% and 49.50, 49.27%, respectively.
There was no significant (P>0.05) difference between control and treatment diets. The
ADDM and TDDM content of maize husk based diets T1R4 (control) andT4R4 (9% azolla
treatment)were 32.89%, 28.71% and 50.14%, 48.62%, respectively. There was no
significant (P>0.05) difference between control and treatment diets. The ADDM and
TDDM of sorghum stover based diets T1R5 (control),T4R5 (9% azolla treatment)were
30.16%, 29.34% and 50.70%, 48.45% respectively. The difference among the control and
treatment diet was not statistically significant (P>0.05).
4.6.2 Apparent digestible dry matter (ADDM) and True digestible dry matter
(TDDM) content of Type III diet
The Apparent digestible dry matter (ADDM) and True digestible dry matter
(TDDM) of the mixed diets comprising CFM (40 per cent kept constant) and roughage
supplemented with azolla at 0 and 9 per cent is presented in Table 4.6.2. The ADDM
content of paddy straw based TMR: T9R1 and T12R1 were 48.18% and 40.81%,
65
Table 4.6.2: Apparent digestible dry matter (ADDM %) and Total digestible dry matter (TDDM %) of the diets (TMR)
comprising CFM (40 per cent kept constant) and roughage (51 to 60 per cent) replaced with azolla at 0 per cent
and 9 per cent (Type III diet).
Samples
ADDM %(Mean ± SE) TDDM %(Mean ± SE)
0 %Azolla (T1) 9% Azolla (T12) 0% Azolla (T1) 9% Azolla (T12)
Paddy straw + CFM + Azolla (R1) 48.18a ± 1.96
40.81
b ± 0.740
67.58
c ± 2.13
60.76
d ± 2.8
Ragi straw + CFM + Azolla (R2) 49.84a ± 1.35
44.62
b ± 2.6
67.35
c ± 2.74
63.89
d ± 2.2
Maize stover + CFM + Azolla (R3) 42.66 ± 0.52 41.29 ± 2.72 61.07 ± 0.31 60.70 ± 1.86
Maize husk + CFM + Azolla (R4) 43.56 ± 0.57 42.31 ± 0.34 60.09 ± 0.54 58.9 ± 0.95
Sorghum stover + CFM + Azolla (R5) 41.2 ± 0.64
38.3 ± 0.71
62.36 ± 0.35
58.64 ± 0.64
Means bearing different superscripts in a row differ significantly (P <0.05).
T1= Treatment with 0% azolla (Control), T12 = Treatment with 9% azolla, R1 = Paddy straw, R2 = Ragi straw, R3 = Maize stover, R4 =
Maize husk, R5 = Sorghum atsover, R6=Bengal gram husk
66
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respectively. The TDDM content of paddy sraw based TMR: T9R1 and T12R1 were
67.58% and 60.76%, respectively. There was significant (P>0.05) difference between
control and the diet supplemented with 9% azolla. There was decreased ADDM and
TDDM % in 9% azolla supplemented diet. The ADDM and TDDM of the ragi straw
based TMR: T9R2 (control) andT12R2 (9% azolla treatment)were 49.84%, 44.62 % and
67.35%, 63.89%, respectively. There was significant (P<0.05) difference between control
and supplemented diet. There was decreased ADDM and TDDM % in 9% azolla
supplemented diet. The ADDM and TDDM of the maize stover based TMR: T9R3
(control) and T12R3 (9% azolla treatment) were 42.66%, 41.29% and 61.07%, 60.70%,
respectively. There was no significant (P>0.05) difference between control and treatment
diets. The ADDM and TDDM of the maize husk based TMR: T9R4 (control) and T12R4
(9% azolla treatment) were 43.56%, 42.31% and 60.09%, 58.9%, respectively. There was
no significant (P>0.05) difference between control and treatment diets. The ADDM and
TDDM of the sorghum based TMR: T9R5 (control) and T12R5 (9% azolla treatment) were
41.2%, 38.3% and 62.36%, 58.64 %, respectively. The difference between the control
and treatment diet was not statistically significant (P>0.05).
DDiissccuussssiioonn
V. DISCUSSION
The present study was conducted to determine the supplementary effect of Azolla
pinnata on the metabolizable energy (ME) and digestibility of the crop residues and total
mixed ration (TMR) by rumen in vitro gas production technique and modified in vitro
two stage digestion technique, respectively. As azolla is a good source of protein, amino
acids, it could be a supplementary source in enhancing the ME content of the diet. Hence,
the present study was conducted by formulating the different diets comprising crop
residues which were supplemented with different levels of azolla ranging from 0 to 9 per
cent and the TMR which were supplemented with azolla at different levels ranging from
0 to 9 per cent by replacing either roughage portion or concentrate portion and subjected
to rumen in vitro gas production study (Hohenheim gas test) and modified in vitro two
stage digestion study to know the supplementary effect of azolla. As the dry matter
content of azolla is very less, it cannot be considered at high proportion, it can only be
tried at supplementary levels. Hence, the present study considered azolla supplemention
at a maximum level of 9 per cent of the diet.
5.1 Chemical composition of Azolla pinnata
The results of the proximate analyses of azolla obtained in the present study are in
close agreement with the values reported by Bolka (2011). The crude protein content of
azolla obtained in present study is similar to the values reported by Alalade and Iyayi
(2006) and Mutzar et al. (1976). The total ash content of azolla obtained in the present
study is lower than the values reported by Tamang et al. (1993) and Ali and Leeson
(1995) but higher than the value reported by Singh et al. (1978) and Parthasarathy et al.
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(2002). The ether extract content obtained in present study is higher than the values
reported by Parthasarathy et al. (2001), Alalade and Iyayi (2006), Tamang et al. (1993)
and Balaji et al. (2009). The crude fiber content of azolla obtained in present study is
slightly higher than the observations of Alalade and Iyayi (2006) and Singh and Subudhi
(1978). The NFE content of azolla is in close agreement with the results of Balaji et al.
(2009) but lower than the values reported by Alalade and Iyayi (2006) and Tamang et al.
(1993). The NDF content of azolla is in close agreement with the value reported by
Parnerkar et al. (1986) but higher than the values reported by Alalade and Iyayi (2006),
Buckingham et al. (1978), Ali and Leeson (1995) and Taklimi (1990). The ADF content
of azolla is almost similar to the value reported by Khatun et al. (1996). The ADL content
of azolla obtained in present study is almost similar to the value reported by Ramesh
(2008) but higher than the value reported by Tamang et al. (1993). The variations in the
nutrient composition of azolla may be due to differences in the response of azolla strains
to environmental conditions such as temperature, light intensity and soil nutrients which
consequently affect their growth morphology and chemical composition.
5.2 Mineral profile of Azolla pinnata
The mineral profile of azolla obtained in the present study is almost similar to the
values reported by Anand and Geetha (2007). Calcium content of azollais similar to the
reports of Tamang et al. (1993). Magnesium content of azollaobtained in the present
study is similar to the value reported by Alalade and Iyayi (2006). Higher level of heavy
metals like nickel, lead, cadmium was obtained in the sample of azolla used for the
present study indicating biooccumilation of heavy metals by azolla. Jain, et al.
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(2007)studied the absorption of iron, copper, cadmium, nickel, lead, zinc, manganese,
and cobalt by Azolla pinnata indicating bioaccumilation of heavy metals by azolla.
5.3 Amino acid profile of Azolla
The data on amino acid profile indicate that lysine, isoleucine, leucine,
phenylealanine, glycine, arginine and valine were predominant. However, the
concentration of sulphur containing amino acids like methionine and cystine is
comparatively less in Azolla pinnata. Amino acid values obtained in this study were in
corroboration with the findings of Alalade and Iyayi (2006). Beckingham et al. (1978)
reported higher concentration of essential amino acids in Azolla filiculoides.
5.4 Chemical composition of crop residues
The chemical composition of paddy straw obtained in the present study is in close
agreement with the reports of Rahaman et al. (2010). The DM content of paddy straw is
almost similar to the reports of Sarnklong et al. (2012) but little higher than the values
reported by Kumar et al. (1999). The crude protein content of paddy straw is lower than
the values reported by Preston et al. (2000) and Akinfemi and Ogunwole (2012). The
ether extract content of paddy straw is little higher than the values recorded by Kumar et
al. (1999). The CF, NDF and the total ash contents of paddy straw obtained in the present
study are little higher than the values recorded by Preston et al. (2000) and Akinfemi and
Ogunwole (2012).The lignin content of paddy straw obtained in the present study is
almost similar to the value recorded by Sarnklong et al. (2011) but lesser than the value
reported by Rahaman et al. (2009).
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The crude protein content of ragi straw obtained in the present study is similar to
the value reported by Bhatta et al. (2000) but lower than the values reported by Madibela
and Modiakgotla (2004) and Sreerangaraju et al. (2000). The ether extract content of ragi
straw is little higher than the values recorded by Bhatta et al. (2000) and Sreerangaraju et
al. (2000). The NDF, ADF and ADL content of of ragi straw is almost similar to the
values reported by Madibella et al. (2004).
The DM, TA, CF, NFE, ADF, NDF content of the maize husk obtained in the
present is in close agreement with the values reported by Akinfemi et al. (2009) but it
contained lower content of CP, EE and ADL.
The CP, CF and TA content of sorghum stover is in close agreement with the
values reported by Akinfemi et al. (2010) but it contained lower content of NDF, ADF
and ADL.
The CP, EE, NDF, ADF and ADL content of bengal gram husk obtained in the
present study is in close agreement with the values reported by Sreerangraju et al. (2000).
The limitation of crop residues includes high fibre, low protein and less of
fermentable carbohydrates.
5.5 Metabolizable energy content and in vitro dry matter digestibility of Azolla
pinnata
The in vitro net gas production and metabolizable energy density of azolla
obtained in the study is almost similar to the value reported by Buckingham et al. (1978)
but lower than the values reported by Alalade and Iyayi (2006), Parashuramulu and
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Nagalakshmi (2013), Khatun et al. (1999) and Ramesh (2008). The in vitro dry matter
digestibility of azolla obtained in the present study is almost similar to the value reported
by Becerra et al. (1995). In comparison with crop residues, azolla contains low level of
organic matter and higher levels of Acid detergent lignin and biogenic silica which limits
the digestibility and ME content to the lower extent.
5.6 Metabolizable energy content and in vitro dry matter digestibility of crop
residues
The ME content of paddy straw obtained in the present study is lesser than the
value reported by Akinfemi and Ogunwole (2012). The in vitro gas production and ME
content of ragi straw obtained in the present study is more than the values reported by
Sreerangaraju et al. (2000). The in vitro gas production and ME content of maize husk
obtained in the present study is higher than the values reported by Akinfemi et al. (2009).
The in vitro dry matter digestibility of maize stover obtained in the present study is
almost similar to the value reported by Kiangi et al. (1981) but lower than the value
reported by Oji et al. (1997). The in vitro gas production and ME content of sorghum
stover obtained in the present study is higher than the values reported by Akinfemi et al.
(2010). The in vitro gas production and ME content of bengal gram husk obtained in the
present study is almost similar to the value reported by Sreerangaraju et al. (2000).
Among the crop residues, the high crude fibre content and sand silica limits the
digestibility.
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5.7 Metabolizable energy content of Type I diet
In case of the diet comprising paddy straw supplemented with azolla at different
levels there was no significant difference between the control and supplemented diets.
Similar observation was made in case of sorghum stover and bengal gram husk
supplemented with azolla at different levels indicating no supplementary effect of azolla
on crop residues. The results are similar to those of Ramesh (2008) who has reported that,
there was no significant difference in gas production and dry matter digestibility of the
paddy straw supplemented with azolla at different levels and the addition of azolla has no
supplementary effect on paddy straw. In case of diet comprising ragi straw supplemented
with azolla at different levels, there was significant (P≤0.05) difference between the
control and the diet supplemented with 6% and 9% per cent azolla. In the diet comprising
maize stover supplemented with azolla at different levels there was significant (P ≤ 0.05)
difference between the control and the diet supplemented with 3%, 6% and 9% per cent
azolla. Similar observations were made in case of diet comprising maize husk
supplemented with azolla at different levels. There was decrease in ME content of all the
treatments compared to the control. The lower ME content of the treatment diet
supplemented with different levels of azolla may be due to the presence of high lignin
content (24.30%) and high total ash content (17.84 %) of the azolla sample. This
indicates that there is negative effect of azolla supplementation on utilization of crop
residues. There is a negative correlation between lignin content and digestibility of forage
materials in ruminant. Lignin is considered as an antinutritive component of forages as it
cannot be readily fermented by rumen microbes. It has a negative impact on
metabolisable energy (ME) values of forages (Van Soest, 1994). Lignin was identified as
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a dominant factor limiting the feed value in perennial grasses, maize stems and tropical
forages. Apart from high lignin content, higher level of biogenic silica and presence of
heavy metals in azolla would also have been the reason for lower ME content and
digestibility of the diets. It has been reported that elevated concentration of heavy metals
like nickel, cadmium reduces the in vitro dry matter digestibility of fiber (cellulose) upto
50 to 60 % (Martinez and Church, 1990). In general we cannot replace any of the crop
residues with azolla.
5.8 Metabolizable energy content of Type II diet
In the diet (TMR) comprising paddy straw and CFM which was replaced with
azolla at different levels, there was significant (P ≤ 0.05) difference between the control
diet and the diet supplemented with 3%, 6% and 9% azolla. There was decrease in ME
content of all the treatment diets compared to the control. The results were similar to
those of Kumar et al. (2013) who has reported that 50% replacement of oil cake with
azolla meal reduced the gas production in in vitro condition with no satisfactory
significant effect on digestibility. In the diet comprising ragi straw and CFM which was
replaced with azolla at different levels there was no significant difference between the
control and the treatment diets. Similar observations were made in case of all other diets
(TMR) supplemented with azolla at different levels. This indicates that there is no
supplementary effect of azolla on TMR, where concentrate portion was substituted with
azolla at different levels. Compared to CFM, azolla contains good amount of protein,
fibre, lignin and biogenic silica but it has low energy content because of low fermentable
organic matter. So replacement of CFM with azolla reduces ME content and digestibility.
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5.9 Metabolizable energy content of Type III diet
In the diet comprising CFM and paddy straw which was replaced with azolla at
different levels, there was significant (P ≤ 0.05) difference between the control and diet
supplemented with 3%, 6% and 9% azolla. There was decrease in ME content of all the
treatment diets compared to the basal control diet. The lower ME content of the diet
incorporated with different levels of azolla may be due higher lignin content (24.30%)
and higher total ash content (17.84 %) of azolla. This indicates that there is no
supplementary effect of azolla on TMR where roughage portion was substituted with
azolla at different levels ranging from 0 to 9%.
5.10 Apparent digestible dry matter (ADDM) and true digestible dry matter
(TDDM) content of Type I diet
In case of the diet comprising either paddy straw as such and the paddy straw
supplemented with azolla at 9% there was significant difference between the
supplemented and control diet. In case of the diet comprising ragi straw supplemented
with azolla at 9% there was significant (P ≤ 0.05) decrease in digestibility. The decreased
ADDM and TDDM in the diet supplemented with 9% azolla might be due to high lignin
content of azolla sample. In case of the diet comprising maize stover supplemented with
azolla at 9% there was no significant difference between the treatment and control diet.
Similar observations were made in case of diet comprising maize husk and sorghum
stover. This indicates that there is no satisfactory effect on the digestibility by
supplementing azolla to various crop residues.
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5.11 Apparent digestible dry matter (ADDM) and true digestible dry matter
(TDDM) content of type III diet
In the diet comprising CFM and paddy straw which was replaced with azolla at
different levels, there was significant difference between the control and diet
supplemented with 9% azolla. In the diet comprising CFM and ragi straw which was
replaced with azolla at different levels, there was significant (P≤0.05) difference between
the control and diet supplemented with 9% azolla. There was decrease in ADDM and
TDDM content of supplemented diet. The cause for the lower dry matter digestibility of
the diet incorporated with 9% azolla might be high lignin and total ash content of the
azolla sample, indicating no effect of azolla supplementation on digestibility of TMR as
well. In the diet comprising CFM and maize stover which was replaced with azolla at
different levels, there was no significant difference between the control and diet
supplemented with 9% azolla. Similar observations were made in all other diet
supplemented with azolla (0 to 9%) indicating there is is no satisfactory effect on the
digestibility of TMR by supplementing azolla at different levels (0 to 9%).
The greatest problem of the Indian livestock farmers facing today, is the shortage
of fodder. The arrival of high yielding varieties, shifted the emphasis from the bio-mass
oriented agriculture to grain yield oriented agriculture. This in turn highly reduced the
sources of fodder from agricultural crops. The rapid shrinkage of common lands of the
villages and expansion of urban areas also led to disappearance of grazing lands and
pastures. Traditional sources of cattle feed like oil cakes and coarse grains also declined
as other cash crops occupied their areas. Such varied factors have contributed in an inter-
linked way to aggravate the fodder crisis. So to substitute the cattle feeds with natural
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feeds that are rich in useful nutrients, azolla has gained importance and is being used as a
protein substitute in the diet of cattle due to its good content of proteins, essential amino
acids, vitamins (vitamin A, vitamin B12, Beta Carotene), growth promoter intermediaries
and minerals, its ability to proliferate without inorganic nitrogen fertilization and its high
rate of growth in water without the need to displace existing crops or natural ecological
systems. It has been used for many years throughout Asia to feed the livestock. One of
the advantages of growing azolla, is that it not only requires less water, but is also cost-
effective for the dairy farmers. With azolla, there is no need to clear the weeds and level
the soil. Even the water left after the second rinsing of the plant could be used. Besides,
the field claims states that it helps in increasing milk yield by 15 to 20 per cent and
reduction in feed cost by 20-30 per cent (Pillai et al., 2002).
The results of the present study however, confirm that azolla can only be
considered as a source of protein, amino acids, major and trace mineral elements, since it
did not improve the digestibility or metabolizability of crop residues or total mixed ration
even at higher level of supplementation.
As the biomass yield of azolla is extremely low, it is practically difficult to
include even 3% of azolla in the diet of ruminants, therefore it could only be a
supplementary source of protein, amino acids and mineral elements.
SSuummmmaarryy
VI. SUMMARY
The present study was conducted to determine the supplementary effect of Azolla
pinnata on the metabolizable energy (ME) and digestibility of the crop residues and Total
mixed ration (TMR) by rumen in vitro gas production study (Hohenheim gas test) and
modified two stage digestion technique, respectively. As azolla is a good source of
protein, amino acids and mineral elements, it could be a supplementary source in
enhancing the ME content of the diet. Hence, the present study was conducted by
formulating the diets comprising crop residues which were supplemented with different
levels of azolla from 0 to 9 per cent and the TMR which were supplemented with azolla
at different levels from 0 to 9 per cent by replacing either roughage portion or concentrate
portion in the mixed diets and subjected to rumen in vitro gas production and digestibility
studies to verify the supplementary effect of azolla on utilization of different types of
diet.
The study included chemical analyses, in vitro gas production and digestibility
studies. The study was conducted at the Department of Animal Nutrition, Livestock
Production and Management, Veterinary College, KVAFSU, Bangalore and Alltech
Ruminant Nutrition laboratory, Veterinary College, Bangalore.
Crop residues commonly used for dairy cattle feeding in Karnataka were selected
for evaluation. The crop residues were ground to pass through a one mm sieve and
preserved in airtight bottles at room temperature while using for chemical analysis and in
vitro studies. Azolla pinnata was harvested from azolla tanks maintained at University of
Agricultural and Horticultural Sciences, College of Agriculture, Shivamogga. Fresh
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samples of Azolla was dried in hot air oven at 50oC for 48 h and the dried samples was
ground to pass through a one mm sieve and preserved in airtight bottles at room
temperature.
Three types of diets were formulated by using azolla, crop residues and
compound feed mixture. The first type of diet comprised only roughage which was
supplemented with different levels of azolla that is 0%, 3%, 6%, and 9%. The second
type of diet comprised of Total mixed ration (TMR) with roughage and CFM taken in
the ratio of 60:40 and the CFM portion of TMR was replaced with 0%, 3%, 6% and 9%
azolla. The third type of diet also comprised of TMR, roughage and concentrate taken in
the ratio of 60:40 and the roughage portion of TMR was substituted with 0%, 3%, 6%
and 9% azolla.
The samples of azolla and different types of diets were subjected to rumen in vitro
gas production study to know the supplementary effect of azolla on ME content of
various diets.
A crossbred (Holstein Friesian x Bos indicus) lactating dairy cow fitted with a
flexible rumen cannula of large diameter served as the donor of the rumen inoculum. The
ME content of the various diet formulated was obtained by in vitro gas test (Menke and
Steingass, 1988). The per cent ADDM and TDDM contents of the diets was obtained by
modified in vitro two stage digestion technique (Goering and Van Soest, 1970).
The chemical analyses of the azolla revealed that it is a good source of protein,
amino acids and minerals. Estimation of ME content of azolla revealed that it is a low
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energy feed resource compared to all other crop residues and TMR. Estimation of mineral
profile of azolla revealed that, it is a good source of major and trace mineral elements.
Reduction in the ME content was observed in the diet comprising ragi straw
supplemented with azolla at different levels (0 to 9%) as compared to the control diet.
Similar observations were made in case of diets comprising maize husk and maize straw.
This indicates negative effect of azolla supplementation on the ME values of the crop
residues. In the diet comprising paddy straw supplemented with azolla at different levels,
there was no significant difference between the control and all other supplemented diets.
Similar observations were made in case of the diet comprising sorghum stover and bengal
gram husk indicating that there is no supplementary effect of azolla on ME content.
Similarly there was no significant difference in the in vitro dry matter digestibility
between the treatment diet and control diet indicating no beneficial effect of azolla
supplementation on digestibility of crop residues.
In the type II diet there was no significant difference in the ME content between
the control and all other supplemented diets except for the one comprising paddy straw
and CFM, where ME content was reduced in the azolla supplemented diet compared to
the control diet. This indicates that, there is no beneficial effect of azolla supplementation
on ME value of TMR in which concentrate portion is replaced with different levels of
azolla ranging from 3 to 9 %.As ME value of azolla is less, it caused reduction of ME
value of other diets which was supplemented with azolla.
Identical observations were made even with type III diet preparation in which the
roughage portion was substituted with different levels of azolla ranging from 0 to 9%.
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There was no significant difference either in ME density or digestibility of DM with
respect to most of the type III diet preparations, except the mixed diet comprising paddy
straw and CFM and ragi straw and CFM, where in the ME content was reduced with
azolla supplementation.
Based on the results of the present study, following conclusions can be drawn.
1. The chemical composition analyses indicated that, the azolla is a good source of
protein, amino acids and mineral elements.
2. Estimation of ME content in azolla revealed that, it is a low energy feed resource
compared to all other crop residues and TMR preparations.
3. By considering the ME content of various diets supplemented with azolla, it was
concluded that, there is no difference between the control diet and the one
supplemented with different levels of azolla ranging from 0 to 9 per cent.
4. By considering the in vitro dry matter digestibility percentage of various diets
supplemented with azolla, it was concluded that, there was decrease in the in vitro dry
matter digestibility of the diet supplemented with 9% azolla as compared to basal diet
indicating that there is negative effect of azolla on the digestibility of various diets.
BBiibblliiooggrraapphhyy
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AAbbssttrraacctt
90
90
VIII. ABSTRACT
The objective of the study was nutritional evaluation of Azolla pinnata and its
supplementary effect on the metabolizable energy (ME) and digestibility of the crop
residues and total mixed ration (TMR) by rumen in vitro gas production technique and
modified in vitro two stage digestion technique, respectively. Three different types of
diets were formulated using either crop residues alone or combination of crop residue and
Compounded feed mixture (CFM). The first type of diet comprised of only roughage
which was supplemented with 0%, 3%, 6%, and 9% azolla. The second type of diet
consisted of TMR, where roughage and CFM taken in the ratio of 60:40 and the CFM
portion was replaced with 0%, 3%, 6% and 9% azolla. The third type of diet consisted of
TMR, where roughage and CFM taken in the ratio of 60:40 and the roughage portion was
replaced with 0%, 3%, 6% and 9% azolla. The CP, CF, EE, TA, NFE (on %DMB)
contents of azolla were at a level of 21.66, 15.15, 4.41, 17.84 and 40.79, respectively.
The NDF, ADF, ADL and biogenic silica (on %DMB) content of azolla were found to be
54.86, 36.5, 24.03 and 3.34, respectively. Azolla was found to contain 1.64 % Ca, 0.34 %
Mg, 2.71 % K, 9.1 ppm Cu, 325 ppm of Zn, 1569 ppm of Fe, 8.11 ppm of Co, 5.06 ppm
of Cr, 2418 ppm of Mn, 31 ppm of Bo, 5.33 ppm Ni, 8.1 ppm of Pb, 1.2 ppm of Cd and
also rich in all essential amino acids. The in vitro dry matter digestibility (IVDMD) of
azolla was found to be 54.49 per cent and the ME content was found to be 5.44 MJ/kg
DM. There was no significant difference in the ME content and IVDMD in the diet
comprising crop residues (paddy straw, sorghum stover and bengal gram husk)
supplemented with azolla at different levels (0 to 9%) indicating no significant effect of
azolla supplementation on utilization of crop residues. There was significant decrease in
the ME content of the diet comprising crop residues (ragi straw, maize stover, maize
husk) supplemented with azolla at various levels (0 to 9%). There was decrease in the
IVDMD of the diet comprising ragi straw supplemented with azolla at 9% compared to
the control diet or TMR comprising CFM and ragi straw supplemented with azolla at 9%
compared to control diet indicating negative effect of supplementing azolla on
digestibility of crop residues and TMR. It is concluded that, azolla can only serve as a
source of protein, amino acids and minerals with limited or no supplementary effect on
digestibility or metabolizability of crop residues or total mixed ration.
AAppppeennddiicceess
91
APPENDIX I: Gas production data of Hay standard and Concentrate standard of 4 trials at different time intervals over 24 hours
Sample
Weight
of
Sample
DM Initial
reading
Final
reading
Gas
production
(2hour)
Gas
production
(5hour)
Gas
production
(24hour)
Blank Net gas in ml
(Total gas -Blank)
Net gas
(ml/200
mg DM)
Correction
factor
Blank
28 39 2 4 11 11.66
TRIAL I
29 41 2 4.5 12
29 41 3 5 12
Hay
Standard
0.2016 0.9116 29 82 7 15 53 11.66 41.34 44.98 1.07
0.201 0.9116 29.5 81 6.5 15.5 51.5 11.66 39.84 43.48
0.204 0.9116 29.5 79.5 6.5 15.5 50 11.66 38.34 41.23
0 0 0 0
43.23
Concentrate
Standard
0.2006 0.9014 29.5 91.5 11.5 35.5 62 11.66 50.34 55.67
0.2037 0.9014 29 90 11.5 38 61 11.66 49.34 53.74
0.2055 0.9014 29 92 12 36 63 11.66 51.34 55.43
54.95
1
Blank
29.5 42 3 5 12.5 15.83
TRIAL II
28.5 46 3 7.5 17.5
29 46.5 4 8 17.5
3
Hay
Standard
0.2021 0.9116 29.5 87 6 15.5 57.5 15.83 41.67 45.23 1.07
0.2048 0.9116 29.5 87 6.5 15 57.5 15.83 41.67 44.63
0.2028 0.9116 29.5 85.5 5 14.5 56 15.83 40.17 43.45
0 0 0
44.44
Concentrate
Standard
0.2016 0.9014 30 95 12 34 65 15.83 49.17 54.11
0.2016 0.9014 29 95 12 35 66 15.83 50.17 55.21
0.2045 0.9014 29.5 94 10.5 35 64.5 15.83 48.67 52.8
54.04
Contd…
92
Sample
Weight
of
Sample
DM Initial
reading
Final
reading
Gas
production
(2hour)
Gas
production
(5hour)
Gas
production
(24hour)
Blank
Net gas in ml
(Total gas –
Blank)
Net gas
(ml/200 mg
DM)
Correction
factor
Blank
30 48 3 5 18 17.83
TRIAL III
29 47 3 7.5 18
29.5 47 4 8 17.5
1
Hay
Standard
0.2011 0.9116 29 87 5.5 14 58 17.83 40.17 43.82 1.06
0.2008 0.9116 30 87.5 4.5 14 57.5 17.83 39.67 43.3
0.2006 0.9116 29.5 88 3.5 15.5 58.5 17.83 40.67 44.48
0 0 0
43.88
Concentrate
Standard
0.2003 0.9014 29.5 96 13.5 34.5 66.5 17.83 48.67 53.91
0.2007 0.9014 29.5 97.5 12.5 33.5 68 17.83 50.17 55.46
0.2005 0.9014 29.5 98 13.5 34 68.5 17.83 50.67 56.07
55.14
Blank
29 41 3.5 6 12 12.33
TRIAL IV
28.5 41 2 5.5 12.5
27.5 40 3 5 12.5
1
Hay
Standard
0.2004 0.9116 29.5 81 6 12 51.5 12.33 39.17 42.88 1.07
0.201 0.9116 30 81.5 5.5 11.5 51.5 12.33 39.17 42.75
0.201 0.9116 29.5 80.5 5 11 51 12.33 38.67 42.2
42.6
1
Concentrate
Standard
0.2009 0.9014 29 90.5 11 33.5 61.5 12.33 49.17 54.30
0.2003 0.9014 29.5 92 11.5 33.5 62.5 12.33 50.17 55.5
0.2002 0.9014 28.5 92 11 34 63.5 12.33 51.17 56.71
55.52
93
Appendix II: Gas production data of Azolla at different time interval over 24 hours
Sample
Weight
of
Sample
DM Initial
reading
Final
reading
Gas
Production
(2hour)
Gas
Production
(5hour)
Gas
Production
(24hour)
Blank
Net gas in ml
(Total gas -
Blank)
Net gas
(ml/200
mg DM)
Correction
factor
Corrected
net gas
AZOLLA 0.2005 0.9652 31 63 3.5 8 32 12.33 19.67 20.32 1.07 21.75
0.2008 0.9652 30 60.5 3 7 31.5 12.33 19.17 19.78 1.07 21.16
0.201 0.9652 30.5 60 3.5 8 29.5 12.33 17.17 17.70 1.07 18.93
0.2004 0.9652 30 62 3.5 8.5 32.5 12.33 20.17 20.85 1.07 22.31
0.2009 0.9652 31 59 2.5 8 30.5 12.33 18.17 18.74 1.07 20.05
0.2012 0.9652 31 60 3 8.5 30 12.33 17.67 18.19 1.07 19.47
0.2002 0.9652 30 58 3.5 7.5 28 11.66 16.34 16.91 1.07 18.09
0.2038 0.9652 30.5 57 3 7.5 26.5 11.66 14.84 15.08 1.07 16.14
0.2030 0.9652 31 58.5 3.5 7.5 27.5 11.66 15.84 16.16 1.07 17.30
19.45
Calculation of Corrected net gas production of Azolla
Sample weight = 0.2005
Gas production for 24 h (ml) = 32 ml
Blank =12.33
Net gas (ml) = Total gas –Blank
= 32-12.33
= 19.67ml
Net gas / 200 mg DM = (19.67x 0.2)/0.2005
= 19.62ml
DM = 0.9652
Net gas / 200 mg DM = 19.62 / 0.9652
= 20.32
Correction factor = 1.07
Corrected net gas (ml) = 20.32 x 1.07
= 21.75ml
94
APPENDIX III: Gas production data of Crop residues at different time interval over 24 hours
Samples Weight of
Sample DM
Initial reading
Final reading
GP*
(2hour) GP
*
(5hour) GP
*
(24hour) Blank
Net gas in ml (Total gas Blank)
Net gas (ml/200 mg DM)
Correction factor
Corrected net gas
Paddy Straw
0.207 0.982 33 67 5.5 10 34 11.66 22.34 21.98 1.07 23.52 0.2072 0.982 29 63 5 8.5 34 11.66 22.34 21.96 1.07 23.5
0.2024 0.982 29 61 5 9 32 11.66 20.34 20.47 1.07 21.9
0.2015 0.982 29.5 66 4.5 9.5 36.5 15.83 20.67 20.89 1.07 22.35
0.205 0.982 29 66 6 13 37 15.83 21.17 21.03 1.07 22.5
0.2039 0.982 29 65 5 11 36 15.83 20.17 20.15 1.06 21.36
0.2026 0.982 29 67 3.5 7.5 38 17.83 20.17 20.28 1.06 21.49
22.37
Ragi Straw
0.2064 0.9647 30 73 6.5 18.5 43 11.66 31.34 31.48 1.07 33.68 0.2067 0.9647 29.5 74 8.5 15.5 44.5 11.66 32.84 32.94 1.07 35.24
0.2015 0.9647 30 74 7 14 44 11.66 32.34 33.27 1.07 35.6
0.2023 0.9647 28 77 10 20 49 17.83 31.17 31.94 1.06 33.86
0.2007 0.9647 29.5 78 10.5 20.5 48.5 17.83 30.67 31.68 1.06 33.58
34.39
Maize 0.2042 0.9897 28 67 4 8.5 39 11.66 27.34 27.06 1.07 28.95 Stover 0.2039 0.9897 28 68 4 9 40 11.66 28.34 28.09 1.07 30.05
0.2007 0.9897 29.5 69 4.5 9.5 39.5 11.66 27.84 28.03 1.07 29.99
0.2019 0.9897 29 77 6 11 48 17.83 30.17 30.2 1.06 32.01
0.2006 0.9897 28.5 74.5 5.5 11.5 46 17.83 28.17 28.38 1.06 30.08
30.22
Maize Husk
0.2002 0.9904 29 86 3 10 57 15.83 41.17 41.52 1.07 44.43 0.2024 0.9904 29 89 4 13 60 15.83 44.17 44.07 1.07 47.15
0.203 0.9904 29 88 4.5 13.5 59 15.83 43.17 42.94 1.07 45.95 0.2011 0.9904 29 90 3 11 61 17.83 43.17 43.35 1.06 47.21 0.2003 0.9904 30 91 2.5 10 61 17.83 43.17 43.52 1.06 45.46 46.04
Sorghum Stover
0.2029 0.9824 29.5 85 9 19 55.5 15.83 39.67 39.8 1.07 42.59 0.2044 0.9824 30 85 9 19.5 55 15.83 39.17 39.01 1.07 41.74
0.2081 0.9824 29.5 84 10.5 22 54.5 15.83 38.67 37.83 1.07 40.48 0.2028 0.9824 28.5 85 10.5 21.5 56.5 17.83 38.67 38.82 1.06 41.15 0.2032 0.9824 29 86 10 21.5 57 17.83 39.17 39.24 1.06 41.6 41.51
Bengal Gram Husk
0.2009 0.9857 28.5 85 6.5 15 56.5 15.83 40.67 41.08 1.07 43.95 0.2004 0.9857 28.5 87 5.5 14 58.5 15.83 42.67 43.2 1.07 46.23 0.2005 0.9857 29 88 6 14 59 17.83 41.17 41.66 1.06 45.7
0.201 0.9857 29 89 5.5 13 60 17.83 42.17 42.57 1.06 45.91 45.45
*GP = Gas production
95
Appendix IV: Gas production data of various diets comprising paddy straw supplemented with different levels of azolla from 0 to 9 per
cent and the TMR comprising paddy straw and CFM supplemented with different levels of azolla from 0 to 9 per cent.
Samples
Weight
of
Sample
DM Initial
reading
Final
reading
Gas
production
(2hour)
Gas
production
(5hour)
Gas
production
(24hour)
Blank
Net gas in ml
(Total gas
Blank)
Net gas
(ml/200 mg
DM)
Correction
factor
Corrected
net gas
T1R1 0.207 0.982 33 67 5.5 10 34 11.66 22.34 21.98 1.07 23.5
0.2072 0.982 29 63 5 8.5 34 11.66 22.34 21.96 1.07 23.5
0.2024 0.982 29 61 5 9 32 11.66 20.34 20.47 1.07 21.9
0.2015 0.982 29.5 66 4.5 9.5 36.5 15.83 20.67 20.89 1.07 22.4
0.205 0.982 29 66 6 13 37 15.83 21.17 21.03 1.07 22.5
0.2039 0.982 29 65 5 11 36 15.83 20.17 20.15 1.06 21.4
0.2026 0.982 29 67 3.5 7.5 38 17.83 20.17 20.28 1.06 21.5
0 0
22.4
T2R1 0.2059 0.9814 24 56 3 6.5 32 11.66 20.34 20.13 1.07 21.5
0.2089 0.9814 29.5 61 4.5 8.5 31.5 11.66 19.84 19.35 1.07 20.7
0.2035 0.9814 29 60 5 10 31 11.66 19.34 19.37 1.07 20.7
0.2009 0.9814 29 61 5 13.5 32 15.83 16.17 16.4 1.07 17.6
0.204 0.9814 27 67 6 13.5 40 15.83 24.17 24.15 1.07 25.8
0.2038 0.9814 29.5 68 3 9.5 38.5 17.83 20.67 20.67 1.06 21.9
0.2013 0.9814 29.5 68 4.5 10 38.5 17.83 20.67 20.93 1.06 22.2
0 0
21.5
T3R1 0.2012 0.9809 30 61 5 9.5 31 11.66 19.34 19.6 1.07 21
0.2078 0.9809 29 62 5 9 30 11.66 18.34 18 1.07 19.3
0.2096 0.9809 29 60 5 9.5 31 11.66 19.34 18.81 1.07 20.1
0.2012 0.9809 29.5 67 6.5 14 35.5 17.83 17.67 17.91 1.06 19
0.201 0.9809 30 69 6 13.5 39 17.83 21.17 21.47 1.06 22.8
0 0
20.4
T4R1 0.201 0.9804 29.5 60 4.5 8.5 30.5 11.66 18.84 19.12 1.07 20.5
0.2052 0.9804 29 61 5.5 10 32 11.66 20.34 20.22 1.07 21.6
0.2066 0.9804 30 61 4.5 10 31 11.66 19.34 19.1 1.07 20.4
0.2033 0.9804 28 64 6 13 36 15.83 20.17 20.24 1.07 21.7
0.2004 0.9804 29 65 5 12 36 15.83 20.17 20.53 1.07 22
1.07 21.2
Contd….
96
Samples
Weight
of
Sample
DM Initial
reading
Final
reading
Gas
production
(2hour)
Gas
production
(5hour)
Gas
production
(24hour)
Blank
Net gas in ml
(Total gas
Blank)
Net gas
(ml/200 mg
DM)
Correction
factor
Corrected
net gas
T5R1 0.2078 0.9816 29 80 10 20 51 11.66 39.34 38.57 1.07 41.3
0.2037 0.9816 29 80 9 19.5 51 11.66 39.34 39.35 1.07 42.1
0.2035 0.9816 27.5 78.5 8.5 18 51 11.66 39.34 39.39 1.07 42.1
0.2016 0.9816 29 85 11 23.5 56 17.83 38.17 38.58 1.06 40.9
0.2008 0.9816 29.5 86 11 23 56.5 17.83 38.67 39.24 1.06 41.6
41.6
T6R1 0.2023 0.9812 29 78 10 20 49 11.66 37.34 37.62 1.07 40.3
0.2025 0.9812 29 75 8.5 18 46 11.66 34.34 34.57 1.07 37
0.2056 0.9812 29 79 9 18 50 11.66 38.34 38.01 1.07 40.7
0.2005 0.9812 29.5 84 11 23 54.5 17.83 36.67 37.28 1.06 39.5
0.2041 0.9812 29 85 10.5 23.5 56 17.83 38.17 38.12 1.06 40.4
39.6
T7R1 0.2037 0.9807 29.5 76 8.5 18 46.5 11.66 34.84 34.88 1.07 37.32
0.2063 0.9807 29 76 10 20 47 11.66 35.34 34.94 1.07 37.38
0.2028 0.9807 29 77 9.5 19 48 11.66 36.34 36.54 1.07 39.1
0.2048 0.9807 29.5 82 10.5 23 52.5 17.83 34.67 34.52 1.06 36.6
0.2052 0.9807 29.5 85 11 24 55.5 17.83 37.67 37.44 1.06 39.68
38.02
T8R1 0.2077 0.9802 28.5 74 8.5 18.5 45.5 11.66 33.84 33.24 1.07 35.57
0.2046 0.9802 29.5 74 9 18.5 44.5 11.66 32.84 32.75 1.07 35.04
0.2012 0.9802 29 76 8.5 17 47 11.66 35.34 35.84 1.07 38.35
0.2045 0.9802 29.5 82 10.5 22.5 52.5 17.83 34.67 34.59 1.06 36.67
0.2026 0.9802 29.5 81 10.5 22 51.5 17.83 33.67 33.91 1.06 35.94
36.31
T9R1 0.2078 0.9816 29 80 5 10 51 11.66 39.34 38.57 1.07 41.27
0.2037 0.9816 29 80 5 10.5 51 11.66 39.34 39.35 1.07 42.1
0.2035 0.9816 27.5 78.5 6 10.5 51 11.66 39.34 39.39 1.07 42.15
0.2016 0.9816 29 85 4 9 56 17.83 38.17 38.58 1.06 40.89
0.2008 0.9816 29.5 86 4 9 56.5 17.83 38.67 39.24 1.06 41.59
Contd….
97
Samples
Weight
of
Sample
DM Initial
reading
Final
reading
Gas
production
(2hour)
Gas
production
(5hour)
Gas
production
(24hour)
Blank
Net gas in ml
(Total gas
Blank)
Net gas
(ml/200 mg
DM)
Correction
factor
Corrected
net gas
T10R1 0.2005 0.982 29.5 79 7 16.5 49.5 12.33 37.17 37.76 1.07 40.4
0.2011 0.982 29.5 81.5 6.5 16 52 12.33 39.67 40.18 1.07 42.99
0.2008 0.982 29 77.5 7.5 17.5 48.5 12.33 36.17 36.69 1.07 39.25
40.88
T11R1 0.2011 0.982 29.5 77.5 6.5 15.5 48 12.33 35.67 36.13 1.07 38.65
0.201 0.982 29.5 76 7.5 16.5 46.5 12.33 34.17 34.62 1.07 37.05
0.2015 0.982 29.5 77 8 16 47.5 12.33 35.17 35.55 1.07 38.04
37.91
T12R1 0.2008 0.982 29.5 79 6 16 49.5 12.33 37.17 37.7 1.07 40.34
0.2008 0.982 29.5 78.5 8 18 49 12.33 36.67 37.19 1.07 39.8
0.2005 0.982 29.5 76 6.5 16 46.5 12.33 34.17 34.71 1.07 37.14
37.01
98
Appendix V: Gas production data of various diets comprising Ragi straw supplemented with different levels of azolla from 0 to 9 per cent
and the TMR comprising paddy straw and CFM supplemented with different levels of azolla from 0 to 9 per cent.
Samples
Weight
of
Sample
DM Initial
reading
Final
reading
Gas
production
(2hour)
Gas
production
(5hour)
Gas
production
(24hour)
Blank
Net gas in ml
(Total gas –
Blank)
Net gas
(ml/200
mg DM)
Correction
Factor
Corrected
net gas
T1R2 0.2064 0.9647 30 73 6.5 18.5 43 11.66 31.34 31.48 1.07 33.7
0.2067 0.9647 29.5 74 8.5 15.5 44.5 11.66 32.84 32.94 1.07 35.2
0.2015 0.9647 30 74 7 14 44 11.66 32.34 33.27 1.07 35.6
0.2023 0.9647 28 77 10 20 49 17.83 31.17 31.94 1.06 33.9
0.2007 0.9647 29.5 78 10.5 20.5 48.5 17.83 30.67 31.68 1.06 33.6
34.4
T2R2 0.2072 0.9647 29.5 72 8.5 17 42.5 11.66 30.84 30.86 1.07 33
0.2041 0.9647 28 72 10 18.5 44 11.66 32.34 32.85 1.07 35.1
0.2026 0.9647 28.5 73 10.5 20.5 44.5 11.66 32.84 33.6 1.07 36
0.2027 0.9647 30 80 8.5 18.5 50 17.83 32.17 32.9 1.06 34.9
0.2024 0.9647 29.5 76.5 9.5 19.5 47 17.83 29.17 29.88 1.06 31.7
34.1
T3R2 0.2004 0.9647 28 65 8 16 37 11.66 25.34 26.21 1.07 28
0.2026 0.9647 27.5 64 4.5 10.5 36.5 11.66 24.84 25.42 1.07 27.2
0.2095 0.9647 29 68 4 9 39 11.66 27.34 27.06 1.07 28.9
0.2018 0.9647 29 72 9.5 18.5 43 17.83 25.17 25.86 1.06 27.4
0.2022 0.9647 29.5 74 10.5 20.5 44.5 17.83 26.67 27.35 1.06 29
28.1
T4R2 0.2091 0.9647 28.5 66 6 0.5 37.5 11.66 25.84 25.62 1.07 27.4
0.2055 0.9647 28 66 6 11 38 11.66 26.34 26.57 1.07 28.4
0.2075 0.9647 30 75 5 12 45 17.83 27.17 27.15 1.06 28.8
0.2021 0.9647 30 73 9 19 43 17.83 25.17 25.82 1.06 27.4
28
T5R2 0.2044 0.9713 29.5 85 11 14.5 55.5 17.83 37.67 37.95 1.06 40.2
0.2042 0.9713 28.5 87 9.5 20.5 58.5 17.83 40.67 41.01 1.06 43.5
0.2089 0.9713 25 78 14 25 53 11.66 41.34 40.75 1.07 43.6
0.2025 0.9713 28 81 11 28 53 11.66 41.34 42.04 1.07 45
0.2044 0.9713 28.5 81.5 12 27.5 53 11.66 41.34 41.65 1.07 44.6
43.4
Contd….
99
Samples
Weight
of
Sample
DM Initial
reading
Final
reading
Gas
production
(2hour)
Gas
production
(5hour)
Gas
production
(24hour)
Blank
Net gas in ml
(Total gas –
Blank)
Net gas
(ml/200
mg DM)
Correction
Factor
Corrected
net gas
T6R2 0.205 0.9708 29.5 85 7.5 18.5 55.5 17.83 37.67 37.86 1.06 40.1
0.2059 0.9708 30 87 10 21.5 57 17.83 39.17 39.19 1.06 41.5
0.2023 0.9708 28 79 9 24 51 11.66 39.34 40.06 1.07 42.9
0.2066 0.9708 29.5 82.5 13 23.5 53 11.66 41.34 41.22 1.07 44.1
42.2
T7R2 0.2037 0.9703 29.5 88 8.5 19 58.5 17.83 40.67 41.15 1.06 43.6
0.2048 0.9703 29 86 5.5 17 57 17.83 39.17 39.42 1.06 41.8
0.2033 0.9703 28 77 10 20 49 11.66 37.34 37.86 1.07 40.5
0.2003 0.9703 25 73 17 31.5 48 11.66 36.34 37.4 1.07 40
0.2088 0.9703 28 79 13.5 28 51 11.66 39.34 38.84 1.07 41.6
41.5
T8R2 0.202 0.9698 29 82 8.5 20 53 17.83 35.17 35.91 1.06 38.1
0.2016 0.9698 28 82.5 10 21 54.5 17.83 36.67 37.51 1.06 39.8
0.2023 0.9698 28.5 79.5 10 19 51 11.66 39.34 40.1 1.07 42.9
0.2092 0.9698 28.5 80 11 23.5 51.5 11.66 39.84 39.27 1.07 42
0.2055 0.9698 28.5 78 9.5 21.5 49.5 11.66 37.84 37.97 1.07 40.6
40.7
T9R2 0.2044 0.9713 29.5 85 5.5 13 55.5 17.83 37.67 37.95 1.06 40.2
0.2042 0.9713 28.5 69 7.5 15 58.5 17.83 40.67 41.01 1.06 43.5
0.2089 0.9713 25 78 10 17.5 53 11.66 41.34 40.75 1.07 43.6
0.2025 0.9713 28 81 6.4 15.5 53 11.66 41.34 42.04 1.07 45
0.2044 0.9713 28.5 81.5 6 14.5 53 11.66 41.34 41.65 1.07 44.6
43.4
T10R2 0.2007 0.9708 29.5 76 8.5 21 46.5 12.33 34.17 35.08 1.07 37.5
0.2007 0.9708 28.5 78 9.5 21 49.5 12.33 37.17 38.15 1.07 40.8
0.2006 0.9708 29 78.5 8 19.5 49.5 12.33 37.17 38.17 1.07 40.8
39.7
T11R2 0.2006 0.9703 28.5 76 7 18.5 47.5 12.33 35.17 36.14 1.07 38.7
0.2006 0.9703 28.5 79.5 8.5 21 46 12.33 33.67 34.6 1.07 37
37.8
T12R2 0.2005 0.9698 28.5 77.5 8.5 20.5 47 12.33 34.67 35.66 1.07 38.2
0.2009 0.9698 28.5 76 7.5 19 44.5 12.33 32.17 33.02 1.07 35.3
0.2006 0.9698 29 79 7.5 19 46 12.33 33.67 34.61 1.07 37
0 0 36.8
100
Appendix VI: Gas production data of various diets comprising Maize stover supplemented with different levels of azolla from 0 to 9 per
cent and the TMR comprising Maize stover and CFM supplemented with different levels of azolla from 0 to 9 per cent.
Sample
Weight
of
Sample
DM Initial
reading
Final
reading
Gas
production
(2hour)
Gas
production
(5hour)
Gas
production
(24hour)
Blank
Net gas in ml
(Total gas –
Blank)
Net gas
(ml/200
mg DM)
Correction
factor
Corrected
net gas
T1R3 0.2039 0.9897 28 68 4 9 40 11.66 28.34 28.1 1.07 30.05
0.2007 0.9897 29.5 69 4.5 9.5 39.5 11.66 27.84 28 1.07 29.99
0.2019 0.9897 29 77 6 11 48 17.83 30.17 30.2 1.06 32.01
0.2006 0.9897 28.5 74.5 5.5 11.5 46 17.83 28.17 28.4 1.06 30.08
30.22
T2R3 0.2062 0.9889 27 64 5 10 37 11.66 25.34 24.9 1.07 26.59
0.202 0.9889 28.5 65.5 3.5 8 37 11.66 25.34 25.4 1.07 27.15
0.2049 0.9889 28.5 64.5 2.5 7 36 11.66 24.34 24 1.07 25.71
0.2003 0.9889 29 72.5 3 8 43.5 17.83 25.67 25.9 1.06 27.47
0.2005 0.9889 28.5 73 4.5 9 44.5 17.83 26.67 26.9 1.06 28.52
27.09
T3R3 0.2038 0.9882 28.5 64 4.5 9 35.5 11.66 23.84 23.7 1.07 25.33
0.2078 0.9882 29.5 67 5 10 37.5 11.66 25.84 25.2 1.07 26.93
0.2023 0.9882 29 65.5 3 8 36.5 11.66 24.84 24.9 1.07 26.59
0.2009 0.9882 28.5 70.5 3.5 9.5 42 17.83 24.17 24.3 1.06 25.81
0.2005 0.9882 28 71.5 5 9 43.5 17.83 25.67 25.9 1.06 27.47
26.43
T4R3 0.2014 0.9874 29 64.5 5 11 35.5 11.66 23.84 24 1.07 25.65
0.2017 0.9874 29 65.5 3 8.5 36.5 11.66 24.84 24.9 1.07 26.69
0.2005 0.9874 29.5 65 4 8.5 35.5 11.66 23.84 24.1 1.07 25.77
0.2003 0.9874 29 70.5 5 11 41.5 17.83 23.67 23.9 1.06 25.37
0.2002 0.9874 29 72 5.5 11 43 17.83 25.17 25.5 1.06 26.99
26.1
T5R3 0.2079 0.9863 29 82 9.5 21 53 11.66 41.34 40.3 1.07 43.14
0.2048 0.9863 28.5 80.5 8.5 19.5 52 11.66 40.34 39.9 1.07 42.74
0.2015 0.9863 28 75 8 19 47 11.66 35.34 35.6 1.07 38.05
0.2045 0.9863 27.5 82.5 9.5 21 55 17.83 37.17 36.9 1.06 39.07
0.2056 0.9863 29.5 84 7.5 19 54.5 17.83 36.67 36.2 1.06 38.34
40.27
Contd….
101
Sample
Weight
of
Sample
DM Initial
reading
Final
reading
Gas
production
(2hour)
Gas
production
(5hour)
Gas
production
(24hour)
Blank
Net gas in ml
(Total gas –
Blank)
Net gas
(ml/200
mg DM)
Correction
factor
Corrected
net gas
T6R3 0.2038 0.9858 28.5 76 8.5 18.5 47.5 11.66 35.84 35.7 1.07 38.18 0.2026 0.9858 29.5 78.5 8.5 20.5 49 11.66 37.34 37.4 1.07 40.01 0.2033 0.9858 29 76.5 7.5 17.5 47.5 11.66 35.84 35.8 1.07 38.27 0.2 0.9858 28.5 84.5 10 19.5 56 17.83 38.17 38.7 1.06 41.04 0.2007 0.9858 28.5 84 9.5 20 55.5 17.83 37.67 38.1 1.06 40.36 39.57
T7R3 0.2009 0.9853 30 77 7 17.5 47 11.66 35.34 35.7 1.07 38.21 0.2036 0.9853 29.5 78 8.5 19.5 48.5 11.66 36.84 36.7 1.07 39.3 0.2005 0.9853 28.5 75.5 8.5 18.5 47 11.66 35.34 35.8 1.07 38.28 0.2001 0.9853 28.5 82 8.5 18 53.5 17.83 35.67 36.2 1.06 38.36 0.2009 0.9853 28.5 84.5 7.5 18 56 17.83 38.17 38.6 1.06 40.88 39
T8R3 0.2004 0.9848 30 75 7 16.5 45 11.66 33.34 33.8 1.07 36.15 0.2014 0.9848 29 76 8 18.5 47 11.66 35.34 35.6 1.07 38.13 0.2004 0.9848 30 78.5 7 16 48.5 11.66 36.84 37.3 1.07 39.95 0.2028 0.9848 28.5 82.5 7.5 16.5 54 17.83 36.17 36.2 1.06 38.39 0.203 0.9848 28.5 83.5 7.5 18 55 17.83 37.17 37.2 1.06 39.42 38.41
T9R3 0.2079 0.9863 29 82 4.5 17 53 11.66 41.34 40.3 1.07 43.14 0.2048 0.9863 28.5 80.5 7.5 18.5 52 11.66 40.34 39.9 1.07 42.74 0.2015 0.9863 28 75 9 20.5 47 11.66 35.34 35.6 1.07 38.05 0.2045 0.9863 27.5 82.5 9.5 21 55 17.83 37.17 36.9 1.06 39.07 0.2056 0.9863 29.5 84 6.5 18 54.5 17.83 36.67 36.2 1.06 38.34 40.27
T10R3 0.2005 0.9858 28.5 75 6 16 46.5 12.33 34.17 34.6 1.07 37 0.2005 0.9858 28 77 6.5 16.5 49 12.33 36.67 37.1 1.07 39.7 0.2009 0.9858 28.5 76 6 16.5 47.5 12.33 35.17 35.5 1.07 38 38.23
T11R3 0.2008 0.9853 28.5 75.5 7.5 18.5 45 12.33 32.67 33 1.07 35.34 0.2007 0.9853 29.5 76 7.5 19 47 12.33 34.67 35.1 1.07 37.52 0.2008 0.9853 28.5 77.5 7 17.5 49 12.33 36.67 37.1 1.07 39.66 37.51
T12R3 0.201 0.9848 28 76 7 18 48 12.33 35.67 36 1.07 38.56 0.2011 0.9848 29 74 6 15.5 45 12.33 32.67 33 1.07 35.3 0.2016 0.9848 28.5 76 7 18 44.5 12.33 32.17 32.4 1.07 34.68
0 0 36.18
102
Appendix VI1: Gas production data of various diets comprising Maize husk supplemented with different levels of azolla from 0 to 9 per
cent and the TMR comprising paddy straw and CFM supplemented with different levels of azolla from 0 to 9 per cent.
Sample
Weight
of
Sample
DM Initial
reading
Final
reading
Gas
production
(2hour)
Gas
production
(5hour)
Gas
production
(24hour)
Blank
Net gas in ml
(Total gas –
Blank)
Net gas
(ml/200
mg DM)
Correction
factor
Corrected
net gas
T1R4 0.2002 0.9904 29 86 3 10 57 15.83 41.17 41.52 1.07 44.43
0.2024 0.9904 29 89 4 13 60 15.83 44.17 44.07 1.07 47.15
0.203 0.9904 29 88 4.5 13.5 59 15.83 43.17 42.94 1.07 45.95
0.2011 0.9904 29 90 3 11 61 17.83 43.17 43.35 1.06 47.21
0.2003 0.9904 30 91 2.5 10 61 17.83 43.17 43.52 1.06 45.46
46.04
T2R4 0.2001 0.9896 28.5 89.5 4.5 13 61 17.83 43.17 43.6 1.06 46.22
0.2003 0.9896 29 89.5 3 11 60.5 17.83 42.67 43.05 1.06 45.64
0.2091 0.9896 29.5 90 2.5 10.5 60.5 15.83 44.67 43.17 1.07 46.2
0.207 0.9896 29 87.5 2 12 58.5 15.83 42.67 41.66 1.07 44.58
0.2016 0.9896 28.5 84 3 13 55.5 15.83 39.67 39.77 1.07 42.55
45.04
T3R4 0.2072 0.9888 28 86.5 4 11.5 58.5 15.83 42.67 41.65 1.07 44.57
0.2096 0.9888 29.5 87.5 3.5 10.5 58 15.83 42.17 40.69 1.07 43.54
0.2076 0.9888 29 87.5 3 11 58.5 15.83 42.67 41.57 1.06 44.07
0.2003 0.9888 28.5 86.5 2.5 12.5 58 17.83 40.17 40.56 1.06 43
0.2002 0.9888 28.5 88 3 12.5 59.5 17.83 41.67 42.1 1.06 44.63
43.96
T4R4 0.204 0.9881 28.5 85.5 4 10 57 17.83 39.17 38.86 1.06 41.2
0.2051 0.9881 28.5 87 3 9 58.5 17.83 40.67 40.14 1.06 42.54
0.2054 0.9881 29.5 82.5 3 10 53 15.83 37.17 36.63 1.07 39.19
0.2021 0.9881 29 82 2 9 53 15.83 37.17 37.23 1.07 39.83
T5R4 0.2073 0.9867 29 89.5 8 21.5 60.5 15.83 44.67 43.68 1.07 46.74
0.2008 0.9867 29 88 7.5 26 59 15.83 43.17 43.58 1.07 46.63
0.2027 0.9867 28.5 87.5 6.5 19 59 15.83 43.17 43.17 1.07 46.19
0.2062 0.9867 28.5 91.5 7.5 20.5 63 17.83 45.17 44.4 1.06 47.07
46.66
Contd….
103
Sample
Weight
of
Sample
DM Initial
reading
Final
reading
Gas
production
(2hour)
Gas
production
(5hour)
Gas
production
(24hour)
Blank
Net gas in ml
(Total gas –
Blank)
Net gas
(ml/200
mg DM)
Correction
factor
Corrected
net gas
T6R4 0.2002 0.9862 28 89.5 7 20.5 61.5 17.83 43.67 44.24 1.06 46.89
0.2009 0.9862 29 91.5 6 19.5 62.5 17.83 44.67 45.09 1.06 47.8
0.2057 0.9862 27 86.5 7 20.5 59.5 15.83 43.67 43.05 1.07 46.07
0.2048 0.9862 28.5 87 6 18.5 58.5 15.83 42.67 42.25 1.07 45.21
46.49
T7R4 0.2072 0.9857 29.5 91 9.5 24.5 61.5 15.83 45.67 44.72 1.07 47.85
0.2015 0.9857 29.5 88.5 11.5 27.5 59 15.83 43.17 43.47 1.07 46.51
0.2034 0.9857 29 89 8 20 60 17.83 42.17 42.07 1.06 44.59
0.2059 0.9857 29 90.5 10 28 61.5 17.83 43.67 43.03 1.06 45.62
1.06 46.14
T8R4 0.2038 0.9852 28 91 9 23.5 63 17.83 45.17 44.99 1.06 47.69
0.2026 0.9852 28.5 89.5 9 23.5 61 17.83 43.17 43.26 1.06 45.85
0.2073 0.9852 29.5 87.5 8.5 20.5 58 15.83 42.17 41.3 1.07 44.19
0.2041 0.9852 29.5 90.5 9 22 61 15.83 45.17 44.93 1.07 48.07
0.2019 0.9852 29 90.5 9 21.5 61.5 15.83 45.67 45.92 1.07 49.13
45.91
T9R4 0.2073 0.9867 29 89.5 8 21.5 60.5 15.83 44.67 43.68 1.07 46.74
0.2008 0.9867 29 88 7.5 26 59 15.83 43.17 43.58 1.07 46.63
0.2027 0.9867 28.5 87.5 6.5 19 59 15.83 43.17 43.17 1.07 46.19
0.2062 0.9867 28.5 91.5 7.5 20.5 63 17.83 45.17 44.4 1.06 47.07
46.66
T10R4 0.2006 0.9862 29 85 6 15 56 12.33 43.67 44.15 1.06 46.8
0.2009 0.9862 29 84 7 18 55 12.33 42.67 43.07 1.06 45.66
46.23
T11R4 0.2005 0.9857 28.5 83 6.5 18.5 54.5 12.33 42.17 42.68 1.06 45.24
0.2007 0.9857 29 86 5.5 16 57 12.33 44.67 45.16 1.06 47.87
0.2005 0.9857 29 85 6 18 56 12.33 43.67 44.19 1.06 46.84
46.13
T12R4 0.2005 0.9852 28.5 83 7.5 20 54.5 12.33 42.17 42.7 1.06 45.26
0.2006 0.9852 29 84.5 6.5 17 55.5 12.33 43.17 43.69 1.06 46.31
0.2006 0.9852 29 83 7 18.5 54 12.33 41.67 42.17 1.06 44.7
45.42
104
Appendix VIII: Gas production data of various diets comprising Sorghum stover supplemented with different levels of azolla
from 0 to 9 per cent and the TMR comprising Sorghum stover and CFM supplemented with different levels
of azolla from 0 to 9 per cent
Sample
Weight
of
Sample
DM Initial
reading
Final
reading
Gas
production
(2hour)
Gas
production
(5hour)
Gas
production
(24hour)
Blank
Net gas in ml
(Total gas –
Blank)
Net gas
(ml/200
mg DM)
Correction
factor
Corrected
net gas
T1R5 0.2029 0.9824 29.5 85 9 19 55.5 15.83 39.67 39.8 1.07 42.59
0.2044 0.9824 30 85 9 19.5 55 15.83 39.17 39.01 1.07 41.74
0.2081 0.9824 29.5 84 10.5 22 54.5 15.83 38.67 37.83 1.07 40.48
0.2028 0.9824 28.5 85 10.5 21.5 56.5 17.83 38.67 38.82 1.06 41.15
0.2032 0.9824 29 86 10 21.5 57 17.83 39.17 39.24 1.06 41.6
41.51
T2R5 0.2082 0.9818 29 82.5 9 19.5 53.5 15.83 37.67 36.86 1.07 39.44
0.2086 0.9818 29.5 82 8.5 18.5 52.5 15.83 36.67 35.81 1.07 38.32
0.2072 0.9818 30 83.5 8 18.5 53.5 15.83 37.67 37.04 1.07 39.63
0.2025 0.9818 29 82.5 8 18 53.5 17.83 35.67 35.88 1.06 38.04
0.2024 0.9818 29.5 83.5 8 17.5 54 17.83 36.17 36.4 1.06 38.59
1.06 38.8
T3R5 0.2005 0.9813 29.5 83.5 8.5 19.5 54 17.83 36.17 36.77 1.06 38.97
0.2033 0.9813 30 85 7.5 17.5 55 17.83 37.17 37.26 1.06 39.5
0.2064 0.9813 29.5 82 8.5 19 52.5 15.83 36.67 36.21 1.07 38.74
0.2021 0.9813 29 81.5 8 20 52.5 15.83 36.67 36.98 1.07 39.57
0.202 0.9813 29.5 83.5 7.5 19.5 54 15.83 38.17 38.51 1.07 41.21
1.07 39.6
T4R5 0.2002 0.9808 30 79.5 8 18.5 49.5 15.83 33.67 34.29 1.07 36.7
0.2012 0.9808 29.5 81.5 8 17.5 52 15.83 36.17 36.66 1.07 39.22
0.2005 0.9808 30 84.5 8 18 54.5 15.83 38.67 39.33 1.07 42.08
0.2031 0.9808 29.5 83.5 7.5 17.5 54 17.83 36.17 36.32 1.06 38.49
T5R5
(Control)
0.2022 0.9819 29.5 90.5 11 25 61 17.83 43.17 43.49 1.06 46.1
0.2038 0.9819 29 89.5 11.5 26 60.5 17.83 42.67 42.65 1.06 45.21
0.2008 0.9819 29.5 89.5 10 26.5 60 15.83 44.17 44.8 1.07 47.94
0.2079 0.9819 29 92.5 9 25 63.5 15.83 47.67 46.7 1.07 49.97
47.3
Contd….
105
Sample
Weight
of
Sample
DM Initial
reading
Final
reading
Gas
production
(2hour)
Gas
production
(5hour)
Gas
production
(24hour)
Blank
Net gas in ml
(Total gas –
Blank)
Net gas
(ml/200
mg DM)
Correction
factor
Corrected
net gas
T6R5 0.2003 0.9814 29.5 88.5 10 23.5 59 15.83 43.17 43.92 1.07 47
0.2058 0.9814 29.5 91.5 11 24.5 62 15.83 46.17 45.72 1.07 48.92
0.2033 0.9814 29.5 90.5 10 24.5 61 15.83 45.17 45.28 1.07 48.45
0.2027 0.9814 29.5 89.5 8.5 25 60 17.83 42.17 42.4 1.06 44.94
0.2015 0.9814 29.5 89.5 8.5 25 60 17.83 42.17 42.65 1.06 45.21
46.9
T7R5 0.2015 0.9809 28.5 88.5 10.5 25.5 60 17.83 42.17 42.67 1.06 45.23
0.2031 0.9809 28.5 87 10.5 24.5 58.5 17.83 40.67 40.83 1.06 43.28
0.2081 0.9809 28 89 11.5 24 61 15.83 45.17 44.26 1.07 47.36
0.2065 0.9809 30 93 10.5 21.5 63 15.83 47.17 46.57 1.07 49.84
0.2063 0.9809 29.5 86.5 10.5 22 57 15.83 41.17 40.69 1.07 43.54
0 0 45.85
T8R5 0.2045 0.9804 30 89 10 23.5 59 15.83 43.17 43.06 1.07 46.08
0.2003 0.9804 29.5 86.5 11 24.5 57 15.83 41.17 41.93 1.07 44.87
0.2035 0.9804 30 88 10 23 58 15.83 42.17 42.27 1.07 45.23
0.2002 0.9804 29.5 86.5 11.5 23.5 57 17.83 39.17 39.91 1.06 42.31
0.2002 0.9804 29 86 11 23 57 17.83 39.17 39.91 1.06 42.31
44.16
T9R5 0.2022 0.9819 29.5 90 11.5 22.5 60.5 17.83 42.67 42.98 1.06 45.56
0.2038 0.9819 29 89 12.5 24 60 17.83 42.17 42.15 1.06 44.68
0.2008 0.9819 29.5 90 12 23.5 60.5 15.83 44.67 45.31 1.07 48.48
0.2079 0.9819 29 92.5 -29 -29 63.5 15.83 47.67 46.7 1.07 49.97
92.5 0 0 47.17
T10R5 0.2023 0.9814 29.5 87.5 11.5 20.5 58 15.83 42.17 42.48 1.07 45.45
0.2032 0.9814 29.5 90 11 21 60.5 15.83 44.67 44.8 1.07 47.94
0.2012 0.9814 29.5 88.5 11 17.5 59 15.83 43.17 43.73 1.07 46.79
46.73
T11R5 0.2043 0.9809 28.5 91 11.5 22.5 62.5 17.83 44.67 44.58 1.06 47.26
0.2011 0.9809 28.5 88.5 11 21 60 17.83 42.17 42.76 1.06 45.32
0.2012 0.9809 28 86 10.5 21 58 15.83 42.17 42.73 1.07 45.73
46.1
T12R5 0.2031 0.9804 30 86.5 10 18.5 56.5 15.83 40.67 40.85 1.07 43.71
0.2002 0.9804 29.5 87.5 8.5 18.5 58 15.83 42.17 42.97 1.07 45.98
0.2023 0.9804 30 89.5 8 19 59.5 15.83 43.67 44.04 1.07 47.12
45.6
106
Appendix IX: Gas production data of various diets comprising Bengal gram husk supplemented with different levels of azolla
from 0 to 9 per cent.
Sample
Weight
of
Sample
DM Initial
reading
Final
reading
Gas
production
(2hour)
Gas
production
(5hour)
Gas
production
(24hour)
Blank
Net gas in ml
(Total gas –
Blank)
Net gas
(ml/200
mg DM)
Correction
factor
Corrected
net gas
T1R6
0.2009 0.9857 28.5 85 6.5 15 56.5 15.83 40.67 41.08 1.07 43.95
0.2004 0.9857 28.5 87 5.5 14 58.5 15.83 42.67 43.2 1.07 46.23
0.2005 0.9857 29 88 6 14 59 17.83 41.17 41.66 1.06 45.7
0.201 0.9857 29 89 5.5 13 60 17.83 42.17 42.57 1.06 45.91
45.45
T2R6 0.2004 0.985 29 86 6 13.5 57 17.83 39.17 39.69 1.06 42.07
0.2005 0.985 29 85 5 12.5 56 17.83 38.17 38.65 1.06 40.97
0.2012 0.985 29 87 3.5 11 58 15.83 42.17 42.56 1.07 45.54
0.2004 0.985 29 88 5 11.5 59 15.83 43.17 43.74 1.07 46.8
0.2007 0.985 28.5 85.5 5.5 12 57 15.83 41.17 41.65 1.07 44.57
44.99
T3R6 0.2012 0.9844 28.5 85 5.5 12 56.5 15.83 40.67 41.07 1.07 43.94
0.2029 0.9844 28 84 7 15.5 56 15.83 40.17 40.22 1.07 43.04
0.2025 0.9844 28.5 86.5 5.5 13.5 58 15.83 42.17 42.31 1.07 45.27
0.2007 0.9844 29.5 89.5 3.5 11.5 60 17.83 42.17 42.69 1.06 45.25
0.2003 0.9844 29.5 90 5.6 13 60.5 17.83 42.67 43.28 1.06 45.88
44.68
T4R6 0.2005 0.9838 28 85.5 7 15.5 57.5 17.83 39.67 40.22 1.06 42.64
0.2009 0.9838 28 87 5 13 59 17.83 41.17 41.66 1.06 44.16
0.2000 0.9838 29 84.5 7 15.5 55.5 15.83 39.67 40.32 1.07 43.15
0.2009 0.9838 27.5 85 6.5 15.5 57.5 15.83 41.67 42.17 1.07 45.12
0.2004 0.9838 29.5 87 5 14 57.5 15.83 41.67 42.27 1.07 45.23
44.06