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1
MOLECULAR CHARACTERIZATION OF INDIAN BREED KADAKNATH CHICKENS
Thesis submitted in partial fulfillment for the award of Degree of Doctor of
Philosophy in
BIOTECHNOLOGY
By
DEEPAK PRAKASH SAXENA, M.Sc., M.PHIL,
Under the Guidance of
DR. R. STEPHAN, M.Sc., M.C.A., M.TECH., Ph.D.,
Assistant Professor, Department of Botany,
Government Arts College, Ariyalur-13
VINAYKA MISSION UNIVERSITY SALEM, TAMILNADU, INDIA
SEPTEMBER -2014
2
VINAYAKA MISSION UNIVERSITY
DECLARATION
I DEEPAK PRAKASH SAXENA declare that the thesis entitled
“MOLECULAR CHARACTERIZATION OF INDIAN BREED
KADAKNATH CHICKENS” submitted by me for the Degree of Doctor of
Philosophy in Biotechnology is the record of work carried out by me
during the period from October 2008-2014 under the guidance of
DR.R.STEPHAN,M.Sc.,M.C.A.,M.Tech.,PhD., and has not formed the
basis for the award of any Degree, Diploma, associate ship, fellowship,
titles in this or any other university or other similar institutions of higher
learning.
Place:
Date: Signature of the Student
3
VINAYAKA MISSION UNIVERSITY
CERTIFICATE BY THE GUIDE
I DR.R.STEPHAN, M.Sc., M.C.A., M.Tech., PhD., Certify that the thesis
entitled “MOLECULAR CHARACTERIZATION OF INDIAN BREED
KADAKNATH CHICKENS” submitted for the degree of Doctor of
Philosophy in Biotechnology by Mr. Deepak Prakash Saxena is the
record of research of work carried out by him during the period from
2008- 2014 under my guidance and supervision and that this work has
not formed the basis for the award of any degree, diploma
associateship,fellowship or other titles in this university or any other
university or institutions of higher learning.
Signature of the
Supervisor with Designation
Place:
Date:
4
ACKNOWLEDGEMENT
On the accomplishment of the present study it is with deep sense
of reverence and gratitude, I deem it a proud privilege and feel immense
pleasure to acknowledge all those who have helped me during the
pursuit of my present study.
At the outset, I sincerely thank authorities of the Vinayaka
Mission University, Salem. For kindly permitting me to register for PhD,
Degree and granting permission to complete my research studies.
I express my sincere thanks to Dr.A.Shanmuga Sundaram,
Chairman, Vinayaka Mission University, Salem who gave an opportunity
to do this dissertation work.
First and foremost I find great pleasure in expressing my deep
sense of gratitude heartfull thanks to my research advisor
Dr.R.Stephan,M.Sc.,M.C.A..,M.Tech.PhD.,Lecturar Department of
Botany ,Government Arts College ,Ariyalur.who supported encouraged
and guided to my work with at most interest ,enthusiasm and patience
,discussion with him guided me in developing new ideas and inferences
with ease.
I would like to express my sincere thanks to the Dr.K.Rajedran,
Dean of Research, Vinayaka Mission University, Salem for having
given me an opportunity to do this dissertation work.
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I wish to place on record person who never showed prosaic
attitude in helping me at the best of their skills, Dr. D.P. Singh, Principal
Scientist & I/c Desi Fowl Unit, CARI, Izatnagar , I am sincerely highly
thankful to him for his kind cooperation and support.
I feel great pleasure to express my deep respect for Dr. Deepak
Sharma, Dr. S.K. Mishra CARI for their constructive suggestions and
guidance to accomplish this assignment.
I feel a great sense of elation and fulfillment to submit this tiny drop
of research in the ocean of human knowledge. Though this was a short
episode in the long journey of life, a thousand hands were there to help,
thousand smiles to make it joyful and thousand caring hearts to make it
memorable.
I am highly thanks to my dear Sir Dr.Ajay Kumar, Scientist, IVRI,
without him help I am not able to do this biggest opportunity in my life.
I lack to say words of reverence, gratification and affection for my
venerated parents who brought me to this stage and because of whom I
was able to pursue my higher study.
Friends are forever no matter they are far or away and we owe
them something for shaping us. At this moment I have no words to
thanks to my dearest, closest and beloved friend Dr. Sanjeev Kumar
Shukla and Dr.Kuldeep Shivalaya whose love, affection, wish, blessing,
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courage and moral support was always with me and with god’s grace the
care and concern shown by her will remain forever.
I apologize for faux pas of the persons who have extended the
help to me in completion of this work in a way or other and deserved
such thanks.
Least but not last I records my thanks for giving me patience and
strength to overcome the difficulties and remain stoic throughout my stay
away from home.
Faith remains as the surest and strongest hope during darkest
days and the Almighty God Lord Krishna leads us to light. Faith in Him
has been the prime support that leads me to this position in life.
(DEEPAK PRAKASH SAXENA)
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CONTENTS
CHAPTER NO. TITLE PAGE
NO.
1. INTRODUCTION 8-11
2. REVIEW OF LITERATURE 12-31
3. AIM AND OBJECTIVES 32-33
4. MATERIALS AND METHODS 34-55
5. RESULTS 56-91
6. DISCUSSION 92-103
7. SUMMARY AND CONCLUSION 104-108
8. REFERENCES
109-128
9. LIST OF PUBLICATION -----
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1- INTRODUCTION
__________________________________________________
Increasing population of India, increased demand of nutritious food and
shrinkage of land has drawn the attention of people towards the
livestock production and poultry farming. Due to high nutritive value and
less input cost, the poultry farming has grown up nicely. Poultry is
considered to be the best experimental animal because of its small size,
shorter generation interval, low feed intake, easy handling and fast
metabolism. Poultry production has been increasing world over because
of affordable prices, health consciousness and successful use of
scientific knowledge in poultry enterprises. Eggs and poultry meat are
protective food, are the cheapest source of quality animal protein, and
have wide acceptability in India.
Consistent with the increase in production and productivity, the per
capita availability of eggs and poultry meat in India has also increased to
44 eggs and 1.76 kg poultry meat per annum, which is still lower than
the recommended levels of 180 eggs and 11 kg of poultry meat, as per
the Nutrition Advisory Committee to Government of India (Indian Poultry
Industry Year Book., 2004).
Poultry industry has made tremendous growth during last three
decades and it has expanded multifold. Commercial strains of poultry
9
have attained almost the plateau of production traits because of
maximum exploitation of available genetic variations; hence further
improvement in production performance through conventional breeding
seems to be impractical. Advanced genetic selection has improved the
economic performance parameters, but reduced the immune status
(Yegani et al., 2005).
The term poultry includes a number of avian species such as chicken,
ducks, turkeys, geese, guinea fowls and pea fowls which have been
domesticated, but is very often used as synonymous to chicken. Chicken
alone account for about 90% of the total poultry. We have a fairly good
knowledge and understanding with regards to its breeding and
husbandry practices.
The common country hen, the desi, is as a rule the best mother for
hatching. She is a good forager. Some of the Indian fowls resemble the
Leghorn in size and shape, but have poor laying qualities. They are
found in various colours. One variety found in India resembles the
Sussex or Plymouth Rock in shape, but is smaller. These birds lay fairly
well and are more common in the eastern parts of the country. The
Indian birds are mostly non-descript, and are of very little value as
layers. There are only 4 pure breeds of fowls indigenous to India. They
are the Chttagong, the Aseel, the Karaknath and the Busra.
10
In India, the Kadaknath breed of the Jhabua District of Madhya Pradesh
is known to be well adapted to the harsh environment, which is
characterized by extreme climatic conditions and poor management,
housing and feeding. Because, long ago, the Kadaknath breed was
reared by tribal people, and following many years of selection mainly for
plumage characteristics, a recent study of their growth potential
recorded a daily weight gain of 6.2 grams from 0 to 20 weeks of age –
based on growth in the breed’s usual production environment with very
little supplemental feed.
The colour of the day old chicks is bluish to black with irregular dark
stripes over the back. The adult plumage varies from silver to gold
spangled to blue black without any spangling. The skin, beak, shank,
toes and soles of feet of males and females are dark gray colour. Even
the comb, wattles and tongue also show a purplish hue. The shining
blue tinge of the ear-lobes adds to its unique features. The peculiarity of
this breed is that most of the internal organs show the characteristic
black pigmentation which is more pronounced in trachea, thoracic and
abdominal air sacs, gonads, elastic arteries, at the base of the heart and
mesentery. The black colour of muscles and tissues is due to the
deposition of melanin pigment, a genetic condition called
"Fibromelanosis".
11
The immune system is the natural means by which animals resist
infection, and immunological parameters may reflect the immuno-
competence of the immune system and, in turn, the ability of the animal
to resist infection. So far, only a few genes have been identified in
influencing disease resistance. Heritability of antibody response of
chickens to an inoculation of SRBC is moderate and the trait responds to
divergent selection (Siegel and Gross., 1980; van der Zijppand Leenstra,
1980; Martin et al., 1990; Pinard et al., 1992). The literature is
inconsistent concerning the role of nonadditive genetic variation in
antibody responses to SRBC (Siegel et al., 1982; Ubosi et al., 1985;
Pinard and van der Zijpp., 1993; Boa-Amponsem et al., 1997).
In a study, chickens from different ecotypes were immunized with
the non-pathogenic multi determinant SRBC and breeds from India
showed the highest humoral response to SRBC, suggesting higher
immunocompetence (Baelmans et al., 2005).
Owing to the recent development in molecular genetics and
biotechnology, the immune system is an ideal component for
multidisciplinary analysis and efforts to improve immunocompetence in
chicken, which would be helpful to economize poultry production.
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2-REVIEW OF LITERATURE
_________________________________________________________
2.1. IMMUNOCOMPETENCE
Several workers have studied different immunocompetence traits
in selection experiments based on immune response to a single antigen
such as Sheep red blood cells (SRBC) in chicken (Siegel and Gross.,
1980; Van der Zijpp., 1983; Dunnington et al., 1984; Van der Zijpp et al.,
1986; Scott et al., 1988; Boa-Amponsem et al., 1997, and 2000;
Shivakumar., 2003; Sivaraman., 2004; Singh et al., 2008; Kumar et al.,
2007; and Jaiswal et al., 2008). Nucleotide polymorphism in important
candidate genes viz., Natural resistance-associated macrophage protein
1(NRAMP1) and Prosaposin (PSAP), Annexin A1 (ANXA1), Osteoclast
Stimulating Factor (OSF), Thrombospondin-4 (THBS4), Programmed
Cell Death Proteins (PDCD), Follistatin (FST), Growth Hormone
Receptor (GHR), Interferon (IFN) alpha and beta (Georgina and Aggrey.,
2005), etc. results in varied response of individuals to variety of
pathogens/antigens. The divergently selected groups or lines have been
evaluated for DNA polymorphism for such genes having bearing on
immune response by DNA methods viz., PCR-RFLP. The available
literature on the proposed objectives has been reviewed.
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2.1.1. Evaluation of Immunocompetence status
The Immunocompetence status can be evaluated by assessing
important parameters related to various facets of immunity such as
antibody response to SRBC; cell mediated immune response to PHA-P,
serum Lysozyme activity and serum IgG level etc. Various genetic
groups/ varieties/breeds/species have also shown significant differences
in antibody titres against SRBC, serum lysozyme level, CMI response to
PHA-P and serum immunoglobulin (IgG) level (Toro et al., 1997;
Saxena., 1993; Saxena et al., 1997; Sarker et al., 1998; Santosh., 1999;
Shivakumar., 2003; Singh et al., 2003; Singh., 2005; Kumar., 2006 and
Jaiswal et al., 2008).
Genetic differences in immune response have been observed by
several workers (Balcarova et al., 1973; Van der Zijpp., 1983; Sivaraman
et al., 2005). The capacity of antibody formation is inherited as a
dominant trait (Van der Zijpp., 1983).
2.1.2. Antibody response to SRBC
Sheep red blood cell (SRBC) is a non-pathogenic, non-specific,
natural multideterminant antigen. It provides good indication of innate
humoral immune response status of an individual because of its broad
immune response characteristics, thus valid for multiple pathogens.
14
The use of antibody responses to SRBC in a multitrait selection
programme is a well-known concept in avian immunology that reveals
various aspects of immune responses and their genetic basis. (Kean et
al., 1994).Several workers have studied different immunocompetence
traits in selection experiments based on immune response to a single
antigen such as Sheep red blood cells (SRBC) in chicken (Gross et al.,
1980; Van der Zijpp., 1983; Dunnington et al., 1984; Van der Zijpp et al.,
1986; Scott et al., 1988; Pinard et al., 1992; Boa-Amponsem et al., 1997,
and 2000; Shivakumar 2005; and Sivaraman., 2004).
Bi-directional selection experiment based on antibody response to
Sheep RBC in Cornell random bred chicken and their crosses were
carried out. The response to selection was immediate as highly
significant differences in the titre values between the High and Low
SRBC lines were observed. The response in first generation was 1.27
for males and 1.21 for females in first generation and progressively
increased to 1.77 and 2.42 in second generation. They observed that
the mean 5 dpi antibody titres of the crosses intermediate of the parental
lines were significantly greater than the mid parent values and
suggested the influence of non-additive gene action on antibody titres.
They also observed the response to selection for persistence and non-
persistence of antibodies at 5 dpi and 21 dpi in the same population is
highly significant between the lines (Siegel and Gross., 1980).
15
(Van der Zijpp and Leenstra., 1980) observed highest mean total
Ab titre on 7 dpi as 5.2 using 2% SRBC in HA. Pullets generally showed
higher titres than the cockerels and from day 3 onward these differences
were highly significant.
(Van der Zijpp and Leenstra., 1980) observed highest mean total
titre (5.2; log2 values) on 7 dpi and females showed significantly higher
response than male chicken. The h2 estimates (Full sibs) for total
antibody titre, MES and MER antibody titre ranged from 0-0.5. The
genetic correlation between 2-β-ME resistant and susceptible antibodies
to SRBC ranged between -0.3 and -0.9.
(Yamamoto and Glick., 1982) could not observe any significant
difference between sexes within line during primary Ab response against
SRBC in the Hampshire birds having different sizes of bursa. (Van der
Zijpp., 1983) evaluated humoral immune response to SRBC in White
Plymouth Rock and White Leghorn (WL) chickens, with emphasis on
dose, genetic origin, interaction and correlation between primary and
secondary immune responses and also between humoral and cell-
mediated immune responses. They demonstrated significant differences
in total antibody titres among genetic groups.
16
(Ubosi et al., 1985) analyzed the age dependency of antibody
response to SRBC in divergently selected lines for this trait. There were
differences among populations for frequency of responders at 7 days of
age. The serological maturity was reached at 14 days of age. After the
attainment of serological maturity, the high SRBC line had significantly
higher antibody titre than low line and the reciprocal crosses were
intermediate to their parental lines. The weight of bursa and spleen was
more in the high line and the thymus weight was less than those in low
line.
(Gross, 1986) compared the doses required to get optimum
response in the divergently selected lines for SRBC response and
reported that the high line responded with low dosages of SRBC,
whereas low line could not respond to that dose. (Van der Zijpp and
Nieuwland, 1986) reported that in ISA Warren fowl line, selection for
high and low anti-SRBC titre did not affect the cell mediated immunity.
(Kim et al., 1987) estimated the correlation between the
immunological and production traits in S1 White Leghorn line. The
correlations (0.21-0.31) among response to SRBC and body weights in
all the ages were significant.
17
In HA line chickens; the heterozygous for MHC had higher titres
than either homozygous subclass. These findings suggested that the
response to SRBCs was dependant on MHC haplotypes as well as on
the background genotype. (Kreukniet et al., 1990) studied the effect of
SRBC dosage on the humoral responses of chicken lines selected for
high (H) and low (L) antibody production against SRBC and observed
that the H line had higher and early peak titre response than L line after
immunization. Further, the response was higher for pullets than
cockerels in H line.
(Martin et al., 1990) studied the correlation between antibody titre,
growth and reproduction traits in lines selected for high and low
response to SRBC. The genetic correlations were moderate among
most of the traits. They observed that the females of low lines for SRBC
response were heavier and had high egg number as compared to those
of high line. (Pinard et al., 1990) estimated the h2 for SRBC response in
fowl selected divergently for 8 generations. The estimates of the realized
heritability for response to SRBC in high line were 0.15 and 0.16 when
estimated by regression and animal model, respectively. The
corresponding values for low line were 0.22 and 0.26.
18
(Chao and Lee, 1991) reported higher immune response to SRBC
in country chicken than White Leghorn in Taiwan. Significantly higher
(P<0.01) Ab titre in females than did males in high, controls and low
response lines of SRBC response was reported by Pinard et al., (1992).
The Ab titres for females and males in 9th generation were 11.12, 10.11;
6.60, 5.87 and 2.35, 1.53 in high, control and Low lines, respectively.
(Miller et al., 1984) reported that additive genetic variation was
important in inheritance of both primary and secondary response to
antigen. Reciprocal differences and heterosis also influenced the
secondary response.
(Boa-Amponsen et al., 1997) measured SRBC antibody titre in
embryos and chicks of high and low SRBC lines. Maternal antibody was
detected earlier in high line as compared to low line chicks.
(Kundu ,1997) studied SRBC response in Aseel, Kadaknath,
Naked neck, Frizzle, Dahlem Red, White Leghorn, SDL broilers and
Naked Neck broilers and revealed that all genetic groups showed
highest titre on 5dpi except broilers which revealed peak titre on 12 days
post immunization (dpi). (Saxena et al., 1997) estimated the response to
SRBC in Guinea fowl, Kadaknath and broilers as 1.520±0.487,
1.525±0.068, and 1.386±0.119, respectively. Significant differences
among varieties and sire families were observed in guinea fowl.
19
(Parmentier et al., 1998) administered SRBC, LPS and BSA
intramuscularly in the fowl divergently selected for antibody response to
SRBC. Levels of antibody responses to SRBC and BSA were higher in
the High line than in the control and low line.
In different genetic groups of broiler chickens, (Nath, 1999) did not
observe any significant effect of sex on response to SRBC. Whereas,
(Kundu et al., 1999) reported that females had lower antibody titres in
Indian native and white leghorn chickens. The apparent differences
between sexes were not significant but interaction of breed and sex
were significant at 5 and 19 days post immunization.
(Santosh, 1999) evaluated the response to SRBC in White
Leghorn, Aseel, Kadaknath, and Dahlem Red (DR) and their crosses,
and reported significant genotype differences for SRBC response. The
DR birds showed the maximum titre. (Boa-Amponsem et al., 2000)
studied the primary and secondary antibody responses to different
dosages of SRBC, at different days of inoculation, in divergently
selected lines for SRBC response. They observed different patterns of
antibody response to SRBC dosages in different lines, which revealed
interactions between line x dosage x days. Antibody responses to the
booster inoculations differed between lines with a dosage effect for low
line chicks but not for high line chicks. They also observed greater
anamnesis response to SRBC in low lines than in high SRBC lines.
20
The females had higher antibody titres to SRBC response at 5 dpi
in divergent SRBC lines than males. The total antibody titre for females
was 10.1±0.5 and males had 5.8±0.7 (Boa-Amponsem et al., 2001).In
broiler chicken, (Ahmed, 2001) developed divergent lines based on
antibody response to SRBC at Central Avian Research Institute,
Izatnagar (India). Significant differences in the antibody titre even after
only one generation of selection were observed between lines.
(Shivakumar, 2003) estimated humoral immune response in
divergently selected (high & low antibody response) IWG & IWJ lines of
WLH chickens and observed that the SRBC response was higher in high
line than in low line. The respective least squares means in S1
generation were 8.89±0.29 and 8.63±0.19. (Sivaraman et al., 2003)
observed non-significant sex difference in the antibody response to
SRBC on 5 dpi in broiler chicken.( Sivaraman et al., 2004) observed
wide range of variability in the base population of SDL broiler chicken for
response to SRBC with their least squares mean as 6.289±0.246.
(Singh, 2005) observed non-significant effect of hatch and sex on
HA titre in IWG-WLH chicken. (Kumar, 2006) observed significant effect
(P<0.05) of age and sex on HA titre in Aseel native chicken. Jaiswal et
al. (2008) observed effect non-significant effect (P>0.05) of sire and sex
on HA titre in Kadaknath native chicken.
21
2.1.3. Serum Immunoglobulin-G (IgG) level
Humoral immunity is one of the most important body’s defense
mechanisms. Humoral immunity is the primary function/effect through
body fluid molecules, particularly the heterogeneous group of
immunoglobulins (IgG and IgM) and many other immunologically
relevant effecter substances. IgG is the most abundant antibody and it
constitutes approximately 80% of the total serum immunoglobulins
(Kuby, 1997) and is traceable in all body fluids.
In most cases, correlations between anti-SRBC response and
serum IgG level has been found to be positive but of low magnitude
(Chao and Lee, 2001). By moderating the humoral and cell mediated
immune responses, this correlation magnitude can be enhanced many
folds (Saxena et al., 1997). Similarly the phenotypic correlation (rp)
between HA titre and serum IgG level also been found to be positive but
lower in magnitude (Sivaraman et al., 2003).(Rees and Nordskog, 1981)
analyzed the serum IgG level of ten different inbred chicken lines with a
mean ranging from 6.6±0.33 to 13.5±0.68 mg/ml. The heavy breed line
W showed the lowest (6.6±0.33 mg/ml) and Leghorn HN showed the
highest (13.5±0.68 mg/ml) level.
(Sato et al., 1986) reported that IgG levels in high and low lines for
IgG in Plymouth Rock chicken, were 1200-1600 mg/ml and 500-600
mg/ml. The significant differences observed between high and low lines
22
were ascribed to the genetic factor or factors included in selection.
(Ahrestani et al., 1987) estimated the serum IgG levels in different
breeds of chicken by Single Radial Immunodiffusion (SRID) method.
Aseel (20.51±0.22 mg/ml) had significantly higher level than White
Leghorn (7.53±0.61 to 15.99±2.2 mg/ml).
(Scott et al., 1988) analyzed serum IgG levels in two lines i.e. large
bursal line (LBL) and small bursal line (SBL) of chicken and observed
that LBL line had higher serum IgG level (105+50.7 to 834.4+201.6mg)
than SBL line (3.8+7.9 and 576.3+122.7 mg).
(Saxena, 1993) studied serum Ig-G levels in Guinea fowl,
Kadaknath and broilers and estimated overall mean of serum Ig-G was
12.19+0.10, 10.01+0.4 and 8.1+0.4 mg/ml, respectively. The high Ig-G
levels were observed during first two weeks of age (15.7+0.49 and
14.3+0.32 mg/ml), which declined rapidly till fourth week of age and
finally attained a static level.
(Toro et al., 1997)) Studied the levels of lacrimal IgA and serum
IgG response to IBV vaccine by different doses, and routes of
immunization in different groups of chicken. Higher IgG levels were
detected throughout the experimental period after a vaccination by
ocular route as compared with vaccination via drinking water.
23
(Sarker et al., 1998) studied the effect of chicken MHC (B
complex) on IgM and IgG production, cell mediated immune response
and immunity against different disease agents in two pairs of B congenic
chicken lines. The MHC type was shown to have significant effect on
serum IgM and IgG production. Heterozygotic advantage of B haplotype
on IgM and IgG production was also observed.
(Sarker et al., 1999) studied the direct and correlated response to
divergent selection for serum IgM and IgG level in chicken and found
that the total antibody titres to SRBC were significantly (P<0.01) higher
in low IgG line than in the high IgG line at both 7 and 14 dpi. From the
results, it was suggested that selection of chicken on the basis of serum
immunoglobin isotypes might change antibody producing cells as well as
other immunocompetent cells that modulate the immune response of
selected lines.
(Chao-ChingHsein et al., 2000) studied the reproductive and
immunological traits in two Taiwan country chicken lines (H and L lines)
that had been divergently selected for high and low serum gamma-
globulin percentage at 34 weeks of age. The H lines had significantly
higher serum gamma-globulin percentage than that of L line. The h² of
serum gamma globulin estimated from sire variance component and
parent offspring regression was in the range of 0.218 - 0.529. Estimate
of realized h² for serum gamma globulin was 0.330.
24
(Sivaraman et al., 2001) recorded mean serum IgG level of
7.81±0.17 mg/ml in synthetic dam line of broiler birds. (Shivakumar,
2003) estimated mean serum IgG concentration in IWG and IWJ
genotypes of WLH chickens as 10.66±0.22 mg/ml and 10.52±0.16
mg/ml, respectively. High SRBC response line had significantly higher
titres than low SRBC line in both the genotypes.
(Singh, 2005) observed non-significant effect (P>0.05) of hatch
and sex on IgG level in IWG-WLH chicken. (Kumar, 2006) observed no
significant effect (P>0.05) of age and sex on IgG level in Aseel native
chicken. (Jaiswal et al., 2008) observed non-significant effect (P>0.05) of
sire and sex on IgG level in Kadaknath native chicken.
2.2. GENERAL GROWTH PERFORMANCE OF KADAKNATH
CHICKEN
The domestic chicken (Gallus gallus, 2n = 78) is believed to have
descended from the wild Indian and Southeast Asian red jungle fowl.
The evolutionary history of the domestic fowl can be divided into three
phases. The first phase started with the evolution of the genus Gallus,
followed by the emergence of the domestic fowl from its progenitors and
lastly the appearance of the large number of the current breeds,
varieties, strains and lines. The conservation and systemic study of this
breed using modern technologies is essential for assessment of its
25
productive and reproductive potential along with other traits as a pure
breed.
The KN breed reveals appreciable degree of resistance to
diseases compared with other exotic breeds of fowl in its natural habitat
in free range. These birds are also resistant to extreme climatic
conditions like summer heat and cold winter stress and thrive very well
under adverse environment like poor housing, poor management and
poor feeding (Thakur et al., 2006). However there is very little
information available regarding description, native breeding areas, and
geographical, demographical, morphological and productive traits of the
KN breed of poultry.
2.2.1. Growth traits
Growth involves simultaneous deposition of bones, muscle and fat;
each exhibiting an individual’s pattern of development. when based on
the percentage increase over the weight at the end of the pre-laying
phase, the most rapid growth or weight gain are made when the chick is
young.
(Mohammed et al., 2005; Devi and Reddy, 2005; Chatterjee et al.,
2007) reported that Body weight is the direct reflection of growth and it
influences the production and reproduction traits of birds. The significant
effect of genetic group on bodyweights of chicken was reported by many
workers similar to the present study.
26
2.2.1.1. Body Weight (BW)
(Singh et al., 1996) also observed better growth performance in
Nana chickens compared to their full feathered counterparts in India.
Naked neck chickens perform well in productive adaptability at hot-
humid climate, because of their association with the reduction in feather
coverage, which can increase heat loss, and so indirectly increase feed
intake and productive adaptability (Merat, 1986; Islam and Nishibori,
2009).The diversity of the local chickens reported so far is mostly on
phenotypes including adult bodyweight, egg weight, reproduction
performance and immune responses to various diseases (Gueye, 1998;
Msoffe et al., 2001; 2004).
2.2.1.2. Confirmation Traits
Growth and production traits of a bird indicate its genetic
constitution and adaptation with respect to the specific environment
(Ahmed and Singh, 2007).
(Vasu et al., 2004) reported lower body weights of 1413.96±7.97 at
40 weeks and 1405.79±9.47 at 64 weeks in White Leghorn control
populations which were lower than the present estimates. The C1 cross,
being developed as a dual purpose bird for backyard farming is excelling
Vanaraja in body weights and egg production.
27
(Niranjan and Singh, 2005) observed higher body weights, 1860
and 2773 g at 20 and 40 week of age in Gramapriya birds respectively.
They agreed that Gompertz and Logistic functions were more
appropriate function for chickens in general. These authors, however,
used only body weight-age data. Using other characteristics such as
body length, shank length, breast width and breast circumference helps
increase accuracy and detail for defining growth. The shape of growth
curves change depending on the species, gender, investigated
characteristic, time (age) and the environment (Ersoy et al., 2007).
The genetic components of body weight are mainly additive in
nature. Significant and positive effect of heterosis on body weight in
broiler was reported in literature (Iraqi et al., 2005). (Yalcin et al., 2000)
observed negative heterosis for body weight in chicken while (Nestor et
al., 2004) reported negative and significant heterosis for body weight at
8, 16 and 20 week of age in male and female turkey for fitness traits like
live ability in crossbred Breeding of rural chicken is important for small
farmers to produce more income and also to conserve genetic variation
of native breeds.
(Kiani-Manesh, 2000) showed that age at sexual maturity, number
of eggs, egg weight and body weight at eight weeks of age are the most
important traits for improving the economic efficiency of rural
chickens.The average day old weight was highest in RIR, intermediate in
28
Desi and lowest in Fayoumi. Similar trend was observed by (Farooq et
al., 2001). (Mostageer et al., 1975) indicated that live weight at hatching
averaged 28.5 and 34.5 g for the Fayoumi and RIR, with non significant
sex difference for the two breeds.(Halima et al., 2006) reported that day-
old weight, final body weight, body weight gain and mortality rate in RIR
were 35.2, 1394, 1359 g and18.3%, respectively.
The age at sexual maturity of indigenous Irani chicken were
reported 157.190.8 days and for three genetic groups of indigenous
chickens such as Naked Neck, Marandy and Public, sexual maturity age
were 23, 25 and 22 weeks, respectively (Nasrollah, 2008).
2.2.1.3. Heritability
The heritability is ratio of additive genetic variance to phenotypic
variance and therefore estimates of haritability vary from time to time
with change in additive and phenotypic variance .The importance of
haritability in breeding experiments lies in its predictive role expressing
the reliability of phenotypic value as a guide of the breeding value.
2.3. DNA POLYMORPHISM IN CHB6, CASPASE-1, IAP-1, AND ZOV3
GENES
Candidate gene analysis is a powerful approach to detect
variations in the genes controlling traits of economic importance in farm
animals, such as immune response (Rothschild and Soller., 1997).
Candidate genes chosen for studying immune response traits may have
29
known physiological functions with immune response or be in regulatory
or biochemical pathways affecting immune response. An amplified 215-
bp fragment of ChB6 gene showed a C→A substitution at base 470,
which caused a predicted amino acid change from Gln (Leghorn line) to
Lys (Fayoumi line) (Zhou and Lamont., 2003).
For the Caspase-1 gene, a 1,070-bp amplified fragment showed a
T→C substitution at −368 bp between the Leghorn and Fayoumi lines
(Zhou and Lamont., 2003). For the IAP-1 gene, a394-bp fragment
showed a T→A substitution from the Leghorn to the Fayoumi lines, and
a PCR-RFLP assay was developed to identify a Bgl I SNP to
characterize the polymorphism at Ala157 (Zhou and Lamont, 2003). For
ZOV3, the amplified 320-bp product showed a T→G SNP, which caused
a predicted amino acid change from Cys157 to Phe157 (Zhou and
Lamont., 2003).
2.3.1. Association among Immunocompetence traits
(Van der Zijpp, 1983) evaluated humoral immune response of
White Plymouth Rock and White Leghorn (WL) chickens to SRBC, but
did not observe any correlation between antibody titres to SRBC, on 3,
7, 14 dpi and PHA-P response, neither overall nor within groups of
different genotypes. It was suggested that the selection for general
immune responsiveness should include parameters of antibody and cell
mediated immune responses.
30
(Cheng and Lamont, 1988) conducted a detailed study for the
phenotypic correlations among immunological traits. Wherein, the
correlation among antibody response, phagocytosis and T-cell mediated
immunity were mostly found to be non-significant (0.14-0.17) but the
difference in correlation coefficient in males and females were
significantly different. Their results indicated the relative independence
of genetic control of these components.
(Saxena et al., 1997) observed the phenotypic correlation of anti-
SRBC antibodies in Guinea fowl to be 0.22±0.27, 0.62±0.46 and
0.27±0.25 with phagocytic index (PI), IgG and wing index (WI),
respectively.
The serum IgG concentration had a low rp with anti- SRBC
response (-0.043) in Taiwan country chicken (Chao and Lee, 2001).
(Sivaraman et al., 2003) reported positive (0.06) but significant rp
between HA titre and serum lysozyme level but observed a negative
(-0.01) and non-significant correlation between HA titre and serum IgG
level. The rp between serum lysozyme and IgG was positive (0.04) and
non-significant.
(Singh, 2005) observed highly positive genetic correlations among three
IC-traits viz., haemagglutination (HA), lysozyme (Lyso) and
immunoglobin-G (IgG) in WLH chicken. The rP among them was also
31
positive but very low in magnitude. (Kumar, 2006) observed genetic
correlations among three IC-traits viz., haemagglutination (HA),
lysozyme (Lyso) and immunoglobin-G (IgG) in Aseel native chicken. The
rP among them was also positive and low to medium in magnitude.
32
3-AIM & OBJECTIVES
__________________________________________________
The genetic resistance in poultry is controlled by immune system that
plays an important role in maintaining the normal health. Breeding
chickens for higher immunocompetence (IC) and disease resistance
provides a viable approach for commercial poultry production.
Indigenous chicken (IC) are mainly raised in resource poor rural
households of developing countries. Various economic traits viz. juvenile
body weights and body measurement sat 5 week-intervals from day old
to 20weeks of age, 4 weekly egg production, egg weight and feed
efficiency from 20to 72 weeks of age were studied for 29genetic groups
comprising purebreds, with the other native breeds and their reciprocals.
Juvenile body weights and body measurements revealed significant
breed differences at different ages. Genetic parameters were estimated
using animal model fitted with common environmental effects for growth
traits and ignoring common environment for egg production traits. The
aim of this study was to estimate heritabilities and genetic and
phenotypic correlations for growth and egg production traits to
understand which traits should be included in breeding programs for
Kadaknath chickens.
33
To evaluate the Immunocompetence status of Kadaknath.
To Measure the phenotypic traits of kadaknath.
To study DNA polymorphism of Kadaknath using PCR-RFLP
analysis.
34
4-MATERIALS AND METHODS
_________________________________________________________
4.1. IMMUNOCOMPETANCE
In the present investigation, chicks belonging to the selected lines
of Kadaknath chickens were used for the study of immunological traits
viz., humoral response to sheep RBCs, and serum IgG concentrations.
Phenotypic traits and growth rate of Kadaknath of different stages and
DNA polymorphism at candidate genes viz., ChB6, Caspase-1, IAP-1,
and ZOV3 genes. The statistical analysis was performed by using SPSS
10 (newer version) and ANOVA software for evaluating various
immunological parameters. Details of the materials used and the
procedures employed including various statistical models are given
below-
4.1.1. Selection of Parents
The birds were ranked from highest to lowest based on
immunocompetence index. 115 Randomly chicken were selected as the
parents of the first generation of high SRBC line.
35
4.1.2. Experimental Animals - Kadaknath Chickens
4.1.3. Birds and their genetic background
The current study involved approximate 115 chickens of Kadaknath
breed (indigenous chickens), randomly selected male and female from
a flock of 300 birds being maintained at experimental farm of CARI,
Izatnagar, India under standard management conditions will be used for
studying the genetic and phenotypic parameters. There are three main
varieties of Kadaknath breed, like Jet Black, Pencilled and Golden
Kadaknath. In all the three varieties of Kadaknath breed, most of the
internal organs exhibit intense black coloration which is due to the
deposition of Melanin pigment in the connective tissue of organs and in
the dermis.
36
Fig. 1.Experimental Birds of Kadaknath Native Chicken
37
4.1.4. Sheep
Healthy Muzaffarnagari breed of sheep maintained at Sheep and
Goat farm of Livestock Production Research Centre, Indian Veterinary
Research Institute, Izatnagar were used for collection of blood from the
Jugular vein to prepare SRBC suspension. Which was used in humoral
immune response studies.
4.2. Assaying of Immunocompetence traits
The Immunocompetence status of the birds can be assessed by
analyzing various components of immune system. A few important
facets of immunity response were evaluated in this investigation. Total
IgG antibody titre and humoral immune response manifested by its
components like antibody titres against an antigen were estimated. Non-
specific immunity conferred by humoral immune response to sheep
RBC and IgG concentration through SRID method were evaluated in this
investigation.
4.2.1. Humoral Response to Sheep RBC
The immune response to SRBC was assessed through HA test as
per Van der Zijpp and Leenstra (1980) using following procedure:-
4.2.1.1. Preparation of Sheep RBC antigen
Approximately 10 ml of heparinized (20 IU/ml) blood was collected
from jugular vein of healthy sheep. It was centrifuged at 40C 120C on 3-5
38
thousand rpm for 10 minutes to settle down the RBCs. The RBCs were
then washed thrice with PBS/NSS by mixing and centrifuging it to
remove other serum components. Finally, 1% sheep RBC suspension
was prepared by mixing 1 ml of packed sheep RBCs and 99 ml of PBS
which was then used for injection in the birds as antigen.
4.2.1.2. Administration of antigen
0.1 ml of 1% sheep RBC suspension was injected into the jugular
vein of each bird with tuberculin syringe. Jugular vein was the choice of
injection as it led to minimum bleeding in comparison to other veins like
brachial vein etc.
4.2.1.3. Harvesting of immune sera from SRBC sensitized birds
Two ml of blood was collected in sterilized glass tubes at 5 days
post immunization (5 dpi) and allowed to clot for 2-3 hours at 370C. The
hyper immune sera oozed out of the clot or the clot was broken. Sera
were collected by centrifuging the tube at 1000 rpm for 3-4 min. and
stored at -200C till further analysis.
4.2.1.4. Estimation of antibody titer against sheep RBCs
The antibody response to SRBC was assessed using
haemagglutination test (Vander Zijpp and Leenstra, (1980) as mentioned
below-
39
The test was performed in round bottom (U shaped) micro titer
plates.50 l of phosphate buffered saline (PBS) was added in each
well. Then, 50 l of serum was added in first well of each row
except the last row where 50 l of PBS was added, that acted as
control.
After thorough mixing thoroughly, the sera were two fold serially
diluted by taking 50 l from each of the well and adding it to the
subsequent wells, mixing thoroughly and it was continued like this
till last column, which was discarded. Equal volume (50l) of 1%
SRBC suspension was added in all the wells and was thoroughly
mixed with sera samples.
The plates were then incubated at 370C for 1 hour. The highest
dilution that gives complete agglutination (button shaped clumping
of RBCs indicated haemagglutination reaction) and it was recorded
as titer and was expressed as log2n.
4.2.2. Estimation of serum IgG by Single Radial Immuno-diffusion
(SRID) Assay
Chicken serum IgG neutralizes the anti chicken IgG. Agarose gel was
used as a solidifying base to assay IgG concentrations through Single
40
Radial Immunodiffusion (SRID) assay (Mancini et al., 1965). The
procedure is given below.
Clean and sterilized glass plate was placed on leveled horizontal
surface. About 50 ml of 0.1 M Tris-HCl was divided equally into
two halves. In first half, of 3% Agarose concentration was added
@ 3% (w/v) and boiled. In second half, 1.750 ml of anti-chicken
IgG (Sigma, USA) was added and after thorough mixing, it was
kept at 50ºC in a water bath.
The temperature of first half (boiled and cooled) was brought down
to about 50ºC and second half was mixed. The standards of IgG
(Sigma, USA) viz., 25g/ml, 12.5g/ml, 6.25 g/ml, 3.125 g/ml
and 1.5625 g/ml, prepared by serial dilution of stock solution were
loaded in the wells to plot standard curve.5 l of unknown sera
were diluted to 4 times with 0.1 M Tris and then loaded the wells.
The plate was incubated at 37ºC for 24 h in humid chamber. The
diameters of the ring around standard as well as unknown samples
were measured with the help of Digital Vernier Calipers.
The serum IgG concentrations in unknown samples were
determined with the help of regression equation obtained by
41
plotting log2 concentrations of IgG standards against diameter of
the precipitation ring.
4.2.2.1. Statistical analysis
The data generated on immunological traits were analyzed by LS
ANOVA using following Statistical model: -
Y= µ + Li + Li:Sij + Hk + Sxl + eijklm
Where, Y = value of a trait measured on ijklmth individual
µ = Overall mean
Li = Effect of line (i = 1, 2)
Li:Sj = Random effect of jth sire in ith line
Hk = effect of kth hatch
Sxl = Effect of lth sex (l = 1, 2)
eijklm = random error associated with mean ‘0’ and variance
σ2
42
4.3. BODY WEIGHT
The data utilized in this study were obtained from Desi Breeds for
present investigations were collected from the records of the indigenous
chicken maintained at Central Avian Research Institute, Izatnagar.
4.3.1. Management of Flock and Traits Measured
Chicks each of the breeds were obtained in more than one hatch
with seven to ten dams’ difference between the hatches. The selected
and controlled populations were maintained under uniform conditions of
feeding, management and disease control as far as possible over year’s
.following trait were measured in each of the generation in both the lines.
4.3.1.1. Pedigree Mating by Artificial Insemination
After identification of the parents of divergent lines, four females
were allotted to each male within each line. The semen from the cock
was collected by gentle massaging at the back and groin region of the
bird. The collected semen was diluted with normal saline and each of the
four females allotted to a particular male were inseminated with 0.2 ml of
the diluted semen.
4.3.1.2. Incubation and Hatching of eggs
Pedigreed eggs were marked for their sire as well as dam. They
were collected twice a day for 30 days, at 10 days interval and stored in
a cold chamber before setting. Cracked and grossly abnormal eggs were
43
discarded. Fumigation was done as per the standard procedure prior to
setting. The fertility was checked by candling of eggs on day 18 and the
fertile eggs were transferred to hatcher on 18th day of incubation by
placing the eggs in pedigree boxes transferred to hatching trays. On
22nd day, chicks were taken out from the hatcher after proper drying
and wing banded.
4.3.1.3. Fertility
Fertility of the birds was calculated as the percent age of eggs that
were found to be fertile after candling.
No. of Fertile eggs
Fertility (%) = X 100
Total number of egg set
4.3.1.4. Hatchability
Hatchability percentage were calculated on the basis of total eggs
set (TES) as well as fertile eggs set (FES) as given below-
Hatchability (TES) % = No. of chicks hatched/ Total no. of eggs set X
100
Hatchability (FES) % = No. of chicks hatched/ Total no. of fertile eggs
set X 100
All chicks of the S1 generation divergent lines were hatched in two
hatches.
44
4.3.1.5. Body Weight: Body weight at 4, 6, 8, 10 and 12 weeks of age
were recorded for individual birds. Gains in weight between 4 – 6
weeks, 6 – 8 weeks, 8 – 10 weeks, and 10 – 12 weeks of age were
recorded.
4.3.1.6. Age at Sexual Maturity: The age in days at the time of laying
its first egg was taken as measured of age at sexual maturity.
4.3.1.7. Egg Weight: The egg weight was recorded to nearest of 1g
accuracy. The average weight of three consecutive eggs at 12 weeks of
age was taken as average egg weight of egg for individual pullet.
4.3.1.8. Part Period Egg Production: it was measured as the number
of egg laid up to 280 days of age for each bird.
4.3.1.9. Statically Data Analysis
The collected data were analyzed using the general linear model
of SPSS 11.0 with sex and plumage colour as fixed factors. Significant
means were separated by the Duncan’s multiple range tests. Correlation
between measurements was determined by the Pearson’s Correlation
Coefficient.
Separate models (Linear and Multiple) for body measurements
singly and combined were enumerated. The regression model adopted
was as follows:
45
Y = a + b1 X
1 + b
2 X
2 + b
3 X
3
Where Y = body weight (kg)
X1 to X
3 = body measurements
a = Intercept
b (1-3) = regression coefficients of Y on X (i = 1, 2, 3).
4.4. DNA Polymorphism in ChB6, Caspase-1, IAP-1, and ZOV3
genes by PCR-RFLP
Nucleotide polymorphism at ChB6, Caspase-1, IAP-1, and ZOV3
genes were studied with PCR-RFLP.
4.4.1. DNA Sampling
Twenty four genomic DNA samples were isolated, twelve from
each breed and six from high IC group and six from low IC group in both
the breeds, using Phenol extraction method (Kagami et al., 1990).
Brief methodology is presented below-
0.1 ml of blood was collected from jugular vein in heparinized tube.
Centrifugation at 3000 rpm for 5 min. with PBS (for compositions
refer to the annexure) in refrigerated centrifuge for washing of
cells.
The supernatant was discarded and lysis buffer (composition is
given in annexure) was added. RBCs were suspended in the lysis
46
buffer and kept at room temperature for 15-20 min. Then
Proteinase K (200µg/ml) and SDS (0.5%) was added and it was
kept overnight for incubation.
In this mixture equal volume of Tris-saturated Phenol was added
and mixed gently for 10 minutes. Two phases were separated by
centrifuging at 5000 rpm for 5 min and the upper aqueous phase
was transferred to a new microfuge tube and this step was
repeated twice.
The aqueous phase was then extracted twice with equal volume of
Phenol: Chloroform: Isoamyl Alcohol (25:24:1).Lastly, the aqueous
phase was extracted twice with equal volume of Chloroform:
Isoamyl Alcohol (24:1).
DNA was precipitated from the aqueous phase by adding 2-2.5 ml
volume of chilled absolute ethanol and gentle mixing. The
precipitated DNA was centrifuged at 10000 rpm for 10 min and the
DNA pellet was washed twice with 70% ethanol. It was then air
dried and dissolved in sufficient volume of autoclaved distilled
water.
47
4.4.2. Checking of concentration, purity and quality of DNA
The concentration of DNA was calculated spectrophotometrically
by taking optical density (OD) at 260 nm using the following formula:
DNA concentration (µg/ml) = OD 260 x Dilution factor x 50
The purity of DNA was assessed by calculating ratio of optical
densities at 260 and 280 nm. The samples with OD ratio (260nm/
280nm) ranging from 1.7 to 1.9 were used in subsequent experiments.
Quality of DNA was assessed through 0.7% horizontal submarine
agarose gel electrophoresis as below-
The gel casting plate was sealed with adhesive tape and placed on
a leveling table. Agarose (0.7% w/v) was boiled in 1 X TBE (Refer
annexure for composition) buffer then cooled to 55°C, then
ethidium bromide (0.5 µg/ml) was added. The gel was poured into
the casting tray.
After solidification, the comb and adhesive tape were removed.
The gel casting tray was submerged in electrophoresis tank having
1 X TBE buffer.
48
DNA samples were prepared by mixing 3µl of genomic DNA, 7µl of
D.W. and 2µl of Bromo Phenol Blue dye (Refer Annexure for
composition). It was carefully loaded in the well. Electrophoresis was
performed at 2-3 volts/cm for one hour and then gel was visualized
under UV transilluminator.
4.4.3. PCR optimization
The reaction mixture and PCR programs for ChB6, Caspase-1, IAP-1,
and ZOV3 genes were optimized as per Zhou and Lamont (2003) with
slight modifications.
4.4.4. Optimized PCR reaction mixture
For ChB6, Caspase-1, IAP-1, and ZOV3 genes, the optimum
combination of various reaction components for each 25 µl PCR reaction
mix was 25 ng genomic DNA, 0.8 mM of each primer, 0.2 mM each
dNTPs and 1 U Taq DNA polymerase.
4.4.5. Optimization of PCR programme
To optimize the PCR conditions different annealing temperatures were
used. In case of all the four genes i.e., ChB6, Caspase-1, IAP-1, and
ZOV3 genes in Kadaknath breed of native chickens, initial denaturation
at 94˚C for 5 min, 35 cycles of (a) Denaturation at 96˚C for 1 min. in
case of ChB6, Caspase-1, and ZOV3 whereas in case of IAP-1, it was
49
94˚C, (b) Annealing at 53˚C, 65˚C, 62˚C, and 52˚C for ChB6, Caspase-
1, IAP-1, and ZOV3, respectively for 1 min., (c) Extension at 72˚C for 1
min. and final extension at 72˚C for 10 min. for ChB6, Caspase-1, and
ZOV3 whereas in case of IAP-1, it was 72˚C for 15 min. produced
distinct and robust amplified product.
The genomic DNA samples having good quality (Intact without
smearing) were used for further analysis.
4.4.6. PCR-RFLP Analysis
PCR-RFLP is a simple and reliable technique for studying
nucleotide polymorphism in nucleotide sequences in genes. It involves
designing of a set of primers for the locus of interest which are used for
PCR amplification of that segment of DNA, followed by restriction
enzyme digestion of the PCR product and visualization of restriction
fragments in gel.
4.5. Primer preparation
Following primer sequences were got synthesized utilizing the
published information and used in this study. The sequences for forward
and reverse primer for ChB6, Caspase-1, IAP-1, and ZOV3 genes were
obtained from Zhou and Lamont (2003) and presented below in table
no.1.
50
The upstream and downstream primers used to amplify ChB6,
Caspase-1, IAP-1, and ZOV3 were expected to yield a 215, 1070, 394,
and 320 bp product, respectively (Zhou and Lamont, 2003).
Table no. 1. Upstream and Downstream primer and their Sequences.
Name of
genes
Name of Primer Sequence
ChB6
Upstream primer
Downstream primer
5’-GCTTCCCCAATGGAACTG-3’
5’-GAGCACAATGGGCCTAGTC-3’
Caspase-1
Upstream primer
Downstream primer
5’-CCATGCTTGGGCTCTCAGTG-3’
5’-GGTCCCGCAGATCCCAGTG-3’
IAP-1
Upstream primer
Downstream primer
5’-TCACCATCTCTACGTTCCAT-3’
5’-CATTGAAACTTGGTTGGTCT-3’
ZOV3
Upstream primer
Downstream primer
5’-GCTTGGACCTGGTATATGAC-3’
5’-GGCTAAAGTAGGTCAAGTGAC-3’
4.5.1. PCR reaction mixture
The PCR was carried out in a total reaction volume of 25l. The
components and annealing temperature were optimized. The optimized
reaction that was finally used for amplification was as below in table.2.
51
Table. 2. PCR reaction mixture for ChB6, Caspase-1, IAP-1, and ZOV3
4.5.2. PCR amplification Program
PCR amplification was carried out in a thermal cycler (PTC-200,
MJ Research, USA) as per Zhou and Lamont (2003) using following
cyclic conditions.
S. No. Reaction component Volume Final conc.
1. Template (Genomic DNA) 1.0 l 25 ng
2. Up stream primer 1 l 0.8µM
3. Down stream primer 1 l 0.8µM
4. 10 X PCR buffer (without MgCl2) 2.5 l 1.5 mM
5. MgCl2 1.5 l 1.5 mM
6. dNTP mix 2 l 0.2 mM
7.. Autoclaved triple glass distilled water 15.8 l
8. Taq DNA polymerase 5U/l 0.2 l 1 U
Total 25 l
52
Table. 3. PCR Amplification Program.
ChB6 Caspase-1 IAP-1 ZOV3 1. Initial
denaturation at
94ºC for 5 min. 2. 35 cycles of a). Denaturation
at 96ºC for 1
min. b). Annealing at
53ºC for 1 min. c). Extension at
72ºC for 1 min. 3. Final extension
at 72ºC for 10
min.
1. Initial
denaturation at
94ºC for 5 min. 2. 35 cycles of a). Denaturation at
96ºC for 1 min. b). Annealing at
65ºC for 1 min. c). Extension at
72ºC for 1 min. 3. Final extension
at 72ºC for 10 min.
1. Initial
denaturation at
94ºC for 5 min. 2. 35 cycles of a). Denaturation
at 94ºC for 1 min. b). Annealing at
62ºC for 1 min. c). Extension at
72ºC for 1 min. 3. Final extension
at 72ºC for 15min.
1.Initial
denaturation at
94ºC for 5 min. 2. 35 cycles of a). Denaturation
at 96ºC for 1 min. b). Annealing at
52ºC for 1min. c). Extension at
72ºC for 1 min. 3. Final extension
at 72ºC for10min.
4.5.3. Documentation of PCR products by Agarose Gel
electrophoresis
Approximately, 8 l of PCR product was added with 2 l triple
distilled water and 2 l Bromophenol blue dye for loading in a 1.4%
agarose gel containing ethidium bromide (0.5 µg/ml). The
electrophoresis was done at 2-5 V/cm. 100 bp and low range DNA
ladder (Bangalore Genei, India) was used as molecular size marker for
identification of the desired product. The amplified product was
examined under UV illumination and photographed for documentation.
53
4.5.4. Restriction enzyme digestion
One restriction enzyme was used for each amplified product to
study RFLP. The RE digestion was carried out in 20µl under the
manufacturer’s recommended conditions. RE Pvu II, Hsp 92 II, Bgl I and
SnaB I were employed for ChB6, Caspase-1, IAP-1, and ZOV3 genes,
respectively (Zhou and Lamont, 2003).
Table .4. Pvu II RE digestion of 215 bp of ChB6 gene
Reaction components Amount (1x)
Pvu II (10U/µl) 0.1µl
10X buffer B 2 µl
Autoclaved triple distilled water 7.9µl
PCR product 10 µl
Total 20 µl
The Pvu II digestion was carried out for overnight at 37ºC in a
water bath. After digestion, the digested products were kept in
refrigerator at 4ºC till further study.
Table.5.Hsp 92 II RE digestion of 1070 bp of Caspase-1 gene
Reaction components Amount (1x)
Hsp 92 II (1U/µl) 1µl
10X buffer R 2µl
Autoclaved triple distilled water 7.0µl
PCR product 10 µl
Total 20 µl
54
The Hsp 92 II digestion was carried out for overnight at 37ºC in a
water bath. After digestion, the digested products were kept in
refrigerator at 4ºC till further study.
Table.6.Bgl I RE digestion of 394 bp of IAP-1 gene
Reaction components Amount (1x)
Bgl I (10U/µl) 0.1µl
10 X buffer R 2µl
Autoclaved triple distilled water 7.9µl
PCR product 10 µl
Total 20 µl
The Bgl I digestion was carried out for overnight at 37ºC in a water
bath. After digestion, the digested products were kept in refrigerator at
4ºC till further study.
Table .7.SnaB I RE digestion of 320 bp of ZOV3 gene
Reaction components Amount (1x)
SnaB I (5U/µl) 0.2µl
10 X buffer R 2µl
Autoclaved triple distilled water 7.8µl
PCR product 10 µl
Total 20 µl
The SnaB I digestion was carried out for overnight at 37ºC in a
water bath. After digestion, the digested products were kept in
refrigerator at 4ºC till further study.
55
4.5.5. Determination of molecular sizes of digests and recording of
RFLP pattern
The molecular sizes of the digests were determined with the help
of Quantity One Software of BioRad provided with the Gel Doc System
(BioRad, USA).
4.5.6. Statistical analysis
On the basis of molecular sizes of digests, genotypes were
grouped according to the reports of Zhou and Lamont (2003). Standard
statistical method was followed to calculate gene and genotypic
frequencies.
56
5-RESULTS
___________________________________________________________________
5.1. EVALUATION OF IMMUNOLOGICAL TRAITS
The Immunocompetence status can be assessed by analyzing various
components of immune system viz., antibody titres against SRBC,
serum IgG level.
Accordingly two important traits viz., humoral response to sheep
RBCs, and serum IgG level were analyzed in Kadaknath breeds of
native chicken.
The least square analysis of variance for these traits of Kadaknath
is presented in table no. 8 and their factor-wise least squares mean are
presented in table 9.
5.1.1. Antibody response to Sheep RBC
The antibody titres against sheep RBCs were measured through
HA test on 5th dpi. HA titres ranged from 2-15 in Kadaknath .Average HA
titers were 8.32±0.13 in Kadaknath.
57
Fig .2. Humoral Immune response against SRBC titer in kadaknath chicken.
HA
12.011.010.09.08.07.06.0
40
30
20
10
0
Std. Dev = 1.45 Mean = 8.3
N = 114.00
58
Fig .3.Effect of IGG against SRBC in Kadaknath Chicken.
IGG
3.002.001.00
Cou
nt
120
100
80
60
40
20
0
The wide variations observed in the present study might be due to
the reason that no artificial selection has been applied on this breed of
chicken for immune response or production traits. Kadaknath breed of
native chicken demonstrated higher HA titre than most of the other
breeds.
59
5.1.2. Serum IgG concentrations
Serum IgG is the most abundant antibody and constitutes
approximately 80% of the total immunoglobulins. The bird’s ability to
mount antibody responses to other antigens is primarily revealed by
serum IgG concentration. The average serum IgG concentration was
10.07±0.20 mg/ml in Kadaknath breeds, respectively.
60
Fig .4 .Effect of Heamagglutination on Kadaknath chicken.
Fig .5.Effect of IgG serum Concentration on Kadaknath Chicken.
61
5.2. GENERAL PERFORMANCE OF KADAKNATH CHICKEN
The data utilized in this study were obtained from the Indian native breed
kadaknath .The average weekly body weight from 4th weeks to 12th of
age for both the sexes of these breeds along with their slandered
deviation. The observed results are listed in the following tables and
graphs by using SPSS10 software of data Analysis for evaluating
various statistical parameters of Kadaknath native Chickens. Trends for
breeding values and phenotypic performance were obtained by
regression of average breeding values and phenotypic least squares
means, respectively, on generation number.
5.2.1. Growth Traits
5.2.1.1. Body Weight
Body weights of the birds were controlled every week. The difference in
day old body weight of different genotypes might be due to difference in
weight of egg obtained from different female lines whose body weight
differ significantly due to the difference in breed characteristics. The
body weight of female followed similarly pattern to that of males. Males
were heavier then the females in all the breeds .No significant difference
however could be observed for day old weight due breeds and sex.The
hatch effects and breed-hatch interaction effects were significant.
62
5.2.1.2. Mean and Slandered Errors
The mean value for body weight at four weeks of age followed
similar trend to that of body weight at 2 weeks of age. Effect due to
breed, sex hatch and hatch-breed interaction were highly significant.
Mean along with the slandered errors and coefficient of variation of
the body weight and confirmation traits at various age are shown in
tables. Least-squares analysis of variance revealed significant difference
(p<0. 01) for general combining ability (GCA) in body weight at all age
groups. Significance of GCA indicates importance of additive genetic
effect for inheritance of juvenile body weight.
The average mean body weight at 4, 6, 8, 10, and 12, weeks of age
were 162.92, 175.71, 340.88, 391.75 and 500.04 gm. respectively. there
was a study increase in the body weight from 4th day to 12th weeks of
age .Growth was accelerated up to 12 week of age .The gain in the body
weight was highest during 8 to 12 weeks of age. The average body
weight week’s difference from 4th week to 12 weeks is 113. The average
slandered error of mean from the 4th, 6th, 8th, 10th and 12th weeks of age
is continuously increasing as 2.50, 3.43, 6.43, 7.68 and 9.88gm
respectively. The genetic components of body weight are mainly additive
in nature. Since the body weight is not directly related with fitness trait;
therefore in general it was observed that all crosses in a diallel
experiment did not express positive heterosis. It is concluded that using
63
Gompertz and linear growth models would be sufficient in modelling the
growth, based on the 7 traits determined. At four weeks of age the
kadaknath indigenous chickens had the lowest and highest mean body
weight gain of all the strains with average daily growth rate of 3.30 g and
4.20 g per bird per day in the starter growth phase, respectively.
The study of least square of analysis of slandered deviation of
kadaknath chicken from 4th week of body weight to 12th weeks of body
weight is increasing like 26.73, 36.64, 68.67, 82.00 and 105.56
respectively.
The mean body weight gain at the age of twelve weeks from the
on-farm experiment in management system is comparable with mean
body weight gain at eight weeks of age under favourable management
conditions.
The probable reason for such differences in the ranking of body
weight might have been due to genetics diversity of the stock used in
different places or genotype environment interaction.
The general trend of coefficient of variation indicated an increase
in the variation during growing period. The high coefficient of variation
appears to be the consequences of the expression of the differentials
rates of growth during this period .The sudden exposer of the keets to
the external environment in the post hatching periods i .e. brooder
house, feeding condition and other managemental factors combined with
64
the inherent genetics factors could be the cause for the general raise in
the coefficient of variation.
5.2.1.3. Phenotypic correlation among by body weight at various
stages
The Phenotypic correlation between body weights at various ages.
Day old body weight was positively correlated with body weight at later
ages. The genotypic correlation mostly varied from low to medium. The
magnitude of genetic correlation gradually increase up to 16 week of age
and then decrease .the phenotypic correlation between these traits were
small and positive and follow similar trend.
Body weight at 4 week of age was positively correlated with body
weight at 8, 10, and 12 week of age. The phenotypic correlation of Body
weight 4, 6, 8, 10, and 12 was 1.000, 0.843, 0.738, 0.729 and 0.563
respectively. The magnitude of such phenotypic correlation gradually
decrease with increase in age. The phenotypic correlation although
followed similar trend .12 week body weight showed high Pearson
correlation with 10 week of body weight .The phenotypic correlation
between 10 week and 12 week body weight were almost similar age of
body weight. Phenotypic correlation among body weights at different
ages ranged from low to high .The keets weight at hatching is not
supposed to bear ay genetic relationship with subsequent body weights.
Low phenotypic correlation between 0 day body weight and body weight
65
at later ages indicates that Body weight at 0 day of ages is not suitable
criterion for selection for improving market weight. Body weights to 16
weeks of age were used to characterize the growth of chicken in this
study. The phenotypic correlation among body weight at other ages
were high and possitive.As the time interval between two body weight
increased there was corresponding reduction in the magnitude of these
correlation .This may be because of the fact that the body weight of two
successful stage are more closely related and perhaps governed by
more common gene than the weight with large time interval.
66
Fig.6.Analysis of Standard Deviation & Mean of Body weight 4 of Kadaknath
Chicken.
BW4
230.0220.0
210.0200.0
190.0180.0
170.0160.0
150.0140.0
130.0120.0
110.0100.0
90.0
30
20
10
0
Std. Dev = 26.73 Mean = 162.9
N = 114.00
Fig .7. Analysis of Standard Deviation & Mean of Body weight 6 of KN Chicken.
BW6
300.0280.0
260.0240.0
220.0200.0
180.0160.0
140.0120.0
100.080.0
30
20
10
0
Std. Dev = 36.64 Mean = 175.7
N = 114.00
67
Fig .8. Analysis of Standard Deviation & Mean of Body weight 8 of KN Chicken.
BW8
580.0560.0
540.0520.0
500.0480.0
460.0440.0
420.0400.0
380.0360.0
340.0320.0
300.0280.0
260.0240.0
220.0200.0
20
10
0
Std. Dev = 68.67 Mean = 340.9
N = 114.00
Fig.9. Analysis of Standard Deviation & Mean of Body weight 10 of KN
Chicken.
BW10
650.0625.0
600.0575.0
550.0525.0
500.0475.0
450.0425.0
400.0375.0
350.0325.0
300.0275.0
250.0225.0
200.0
20
10
0
Std. Dev = 82.00 Mean = 391.8
N = 114.00
68
Fig.10. Analysis of Standard Deviation of Body weight 12 of KN Chicken.
BW12
800.0750.0
700.0650.0
600.0550.0
500.0450.0
400.0350.0
300.0250.0
14
12
10
8
6
4
2
0
Std. Dev = 105.57 Mean = 500.0
N = 114.00
69
5.3. DNA POLYMORPHISM IN CHB6, CASPASE-1, IAP-1, AND ZOV3
GENES
DNA polymorphism at ChB6, Caspase-1, IAP-1, and ZOV3 genes
were studied using PCR-RFLP.
DNA isolation and its evaluation for quality, purity and concentration
Genomic DNA were isolated from 24 blood samples (6 each at
high and low extremes of HA titers from both the breeds) using Phenol
extraction method.
5.3.1. Agarose Gel Electrophoresis
2% Horizontal Submarine Agarose gel was prepared for
documentation of RE digested products.
5.3.1.1. Procedure
20µl of the digestion mixture was kept in water bath for digestion at
37ºC for overnight.4µl Bromophenol blue dye was added in the
entire digested product. Uncut sample was made by mixing 10µl of
the PCR product and 2µl Bromophenol blue dye was added to it.
Marker was made by adding 7µl triple distilled water, 2 µl
Bromophenol blue dye, and 1µl ladder (100 bp in case of ChB6,
IAP-1, and ZOV3 gene whereas in case of Caspase-1, low range
70
DNA marker was used).The gel casting plate was sealed with
adhesive tape and placed on a leveled table surface.
2% Agarose (w/v) (Biogene, USA) was boiled in 1 X TBE (Refer
Annexure for composition) buffer. After boiling it was cooled to 55ºC
and then ethidium bromide (0.5 µg/ml) was added. The gel was
gently poured into the casting tray avoiding bubble formation and
was allowed to solidify at room temperature.
After solidification, the comb and adhesive tape were removed. The
gel casting tray was submerged in gel tank of electrophoresis unit
having 1 X TBE buffer.
Digested DNA samples, uncut sample, and marker sample were
carefully loaded in the wells. Electrophoresis was performed at 2-5
volts/cm for two to three hour and then gel was visualized and
photographed under UV transilluminator.
The quality of genomic DNA was assessed by 0.7% horizontal Agarose
gel electrophoresis and found to be good. All samples gave intact bands
without smearing on 0.7% Agarose. Gel was visualized under UV
transilluminator.
71
Purity and concentration of genomic DNA was determined by
optical density (OD) determination at 260 nm and 280 nm in a UV
Spectrophotometer. Ratio of OD260 to OD280 of most of the DNA samples
were lying in the range of 1.7 to 1.9 indicating satisfactory purity of DNA
samples.
Concentrations of DNA ranged from 258.8–321.7 ng/µl in
Kadaknath breed. DNA samples were diluted with triple glass distilled
water to obtain a final concentration of approximately 50 ng/µl. These
diluted DNA samples were used in PCR reactions.
The optimized PCR reaction mixture and PCR programmes gave
satisfactory amplification of all the genes in Kadaknath breed of native
chicken.
5.3.2. DNA Polymorphism at ChB6 gene
For ChB6 gene, a 215 bp amplicon was obtained in Kadaknath
native chickens on PCR amplification. The Pvu II RE digestion of PCR
product generated fragments of 215 bp in Kadaknath. Smaller fragments
could not be resolved properly. In present investigation revealed in the
case of Kadaknath, only two genotype i.e. AA and BB with fragment size
of 147 and 215 bp fragments, respectively could be observed. Genotypic
and gene frequencies for ChB6 are presented below-
72
Table. 34. Gene and Genotypic frequencies of PCR-RFLP alleles of ChB6 gene
in Kadaknath.
Genotype Gene
AA AB BB A B
Frequency 0.5 0.0 0.5 0.5 0.5
Thus, PCR-RFLP of 215 bp amplicon of ChB6 gene with Pvu II
restriction enzyme revealed polymorphic banding pattern in the
Kadaknath native chicken.
5.3.3. DNA Polymorphism at Caspase-1 gene
For Caspase-1 gene, 1070 bp amplicon was obtained on PCR
amplification in Kadaknath native chickens. The Hsp 92 II RE digestion
of PCR product generated fragments of 109, 122, 227, 244, 316, and
421 bp in Kadaknath. Smaller fragments could not be resolved properly.
On the basis of presence and absence of restriction fragments,
Kadaknath demonstrated only one genotype i.e. AB in all the birds.
Genotypic and gene frequencies for Caspase-1 are presented below-
73
Table.35.Gene and Genotypic frequencies of PCR-RFLP alleles of Caspase-1 gene in Kadaknath.
Genotype Gene
AA AB BB A B
Frequency 0.0 1.0 0.0 0.5 0.5
Thus, PCR-RFLP of 1070 bp product of Caspase-1 gene with Hsp
92 II restriction enzyme revealed polymorphic banding pattern in
Kadaknath native chicken.
5.3.4. DNA Polymorphism at IAP-1 gene
For IAP-1 gene, a 394 bp amplicon was obtained in Kadaknath
native chickens on PCR amplification. The Bgl I RE digestion of PCR
product generated fragments of 140, 254, and 394 bp in Kadaknath. In
Kadaknath breeds, present investigation revealed two genotypes i.e. AA
and AB, in all the birds analysed. In Kadaknath, AA genotype had the
254 and 140 bp fragments, whereas AB genotype had the fragments i.e.
140, 254, and 394 bp fragments.
Genotypic and gene frequencies for IAP-1 are presented below-
74
Table.36. Gene and Genotypic frequencies of PCR-RFLP alleles of IAP-1 gene
in Kadaknath
Genotype Gene
AA AB BB A B
Frequency 0.38 0.62 0.0 0.69 0.31
Thus, PCR-RFLP of 394 bp product of IAP-1 gene in Kadaknath
with Bgl I restriction enzyme revealed polymorphic banding patterns in
Kadaknath native chickens.
5.3.5. DNA Polymorphism at ZOV3 gene
For ZOV3 gene, 320 bp amplicon was obtained on PCR
amplification in Kadaknath. The SnaB I RE digestion of PCR product
generated fragments of 270 and 320 bp in Kadaknath breeds of native
chicken. Smaller fragments could not be resolved properly. On the basis
of presence and absence of restriction fragments, Kadaknath birds
resolved into two genotypes i.e. BB and AB. In Kadaknath, BB genotype
had the fragments viz., 320 bp whereas AB genotype had the fragments
viz., 270, and 320 bp.
75
Genotypic and gene frequencies for ZOV3 are presented below-
Table.37. Gene and Genotypic Frequencies of PCR-RFLP alleles of ZOV3 gene in Kadaknath.
Genotype Gene
AA AB BB A B
Frequency 0.0 0.6 0.4 0.3 0.7
Thus, PCR-RFLP of 320 bp product of ZOV3 gene with SnaB I
restriction enzyme revealed polymorphic banding pattern in Kadaknath
native chicken.
76
(A)
(B)
Fig .11. PCR-RFLP analysis of ChB6 gene in Kadaknath. (A) Amplified product. (B) Pvu II PCR-RFLP patterns in Kadaknath. bp1 and bp2 indicate molecular sizesofspecificamplicon/REdigestsandDNAmolecularsizemarkers,respectively. Various genotypes have been indicated above the lane numbers.L-IC= Low Immunocompetence birds; H-IC= High Immunocompetence birds.
L-IC H-IC
1 2 3 M 5 6 7 8 bp2
300
100
200
147
bp1
1000
215
AA BB UC BB BB AA AA
bp1
L-IC H-IC 1 2 3 M 5 6 7
300
100
200 215
bp2
1000
77
(A)
(B)
L-IC H-IC
Fig .12. PCR-RFLP analysis of Caspase-1 gene in Kadaknath native chickens.(A) Amplified product of 1070 bp (B) Hsp 92 II PCR-RFLP patterns in Kadaknath. bp1 and bp2 indicate molecular sizes of specific amplicon/ RE digests and DNA molecular size markers, respectively. Various genotypes have been indicated above the lane numbers. L-IC= Low Immunocompetence birds; H-IC= High Immunocompetence birds.
1 2 3 M 5 6 7 8
bp2
300
100
200
123
bp1 bp2
1000
112
600
232 249 316 421
AB AB UC AB AB AB AB
L-IC H-IC
1 2 3 M 5 6 7 8
bp2
300
100
200
bp1
1000 1070
600
78
(A)
(B)
Fig .13. PCR-RFLP analysis of IAP-1 gene in Kadaknath native chickens.(A) Amplified product of 394 bp. (B) Bgl I PCR-RFLP patterns indicates various genotypes in Kadaknath. bp1 and bp2 indicate molecular sizes of specific amplicon/ RE digests and DNA molecular size markers, respectively. Various genotypes have been indicated above the lane numbers.L-IC= Low Immunocompetence birds; H-IC= High Immunocompetence birds.
L-IC H-IC
bp1
1 2 3 4 5 M 5 6 7 8
bp2
300
100
200
1000
394 400
254
140
AB AB AA AA UC AB AA AB AB
bp2
L-IC H-IC
1 2 3 4 M 5 6 7
bp1
300
100
200
1000
394 400
79
(A)
(B)
Fig .14. PCR-RFLP analysis of ZOV3 gene in Kadaknath native chickens. (A) Amplified product of 320 bp (B) SnaB I PCR-RFLP indicates various genotypes. bp1 & bp2 indicate molecular sizes of DNA markers and specific amplicon (A) or RE digests (B), respectively. Various genotypes have been indicated above the lane numbers.
L-IC= Low Immunocompetence birds; H-IC= High Immunocompetence birds.
L-IC H-IC
1 2 3 M 5 6 7
8 bp2
300
100
200
bp1
1000
320
270
AB BB UC BB AB AB
BB
1 2 3 M 5 6 7 8
bp1
300
100
200
bp2
1000
L-IC H-IC
320
80
Table .8. Least Square means and Standard Error for Humoral immune
response against SRBC and IgG for Kadaknath native chicken.
S.N. SEX HA IgG
1- Male Mean 8.1596 1.9255
Std. Error of Mean .1713 0.098
2- Female Mean 7.0714 1.8000
Std. Error of Mean .2028 0.066
3- Total Mean 7.6951 1.8720
Std. Error of Mean .1371 0.063
Means in a column with different superscripts differ significantly (P<0.05) *P ≤ 0.05, **P≤0.01 (samples were taken in three replicates). Table.9.Least squares analysis of variance of important immunological traits in Kadaknath breed of native chicken for Descriptive Statistics.
N Mean Std. Deviation
Minimum Maximum
HA 114 8.3246 1.4482 6.00 12.00
IgG 114 2.0439 .3854 1.00 3.00
(Note - Samples has been taken three replicates.)
81
Table .10.Least square analysis of Chi-Square Test- Frequencies of Heam-agglutination against SRBC of Kadaknath Chicken.
Frequency Observed N Expected N Residual
6.00 13 16.3 -3.3 7.00 20 16.3 3.7 8.00 32 16.3 15.7 9.00 27 16.3 10.7
10.00 11 16.3 -5.3 11.00 10 16.3 -6.3 12.00 1 16.3 -15.3 Total 114
Table .11. Least square analysis of Chi-Square Test- Frequencies of IgG against SRBC of Kadaknath chicken.
Observed N Expected N Residual
1.00 6 38.0 -32.0
2.00 97 38.0 59.0
3.00 11 38.0 -27.0
Total 114
Table .12.Least Square analysis of Test Statistics of HA and IgG of Kdaknath Chicken.
HA IgG Chi-Square 42.211 137.737
df 6 2 Asymp. Sig. .000 .000
A. 0 cells (.0%) have expected frequencies less than 5. The minimum expected cell frequency is 16.3. B. 0 cells (.0%) have expected frequencies less than 5. The minimum expected cell frequency is 38.0.
82
Table. 13. Least Square Pearson Correlations of HA and IgG of Kadaknath Chicken.
HA IgG HA Pearson Correlation 1.000 .006
Sig. (2-tailed) . .950
N 114 114 IGG Pearson Correlation .006 1.000
Sig. (2-tailed) .950 .
N 114 114
Table .14.Least Square analyses of Frequencies of Statistic mean of HA and IgG of Kadaknath Chicken.
HA IgG
N Valid 114 114
Missing 0 0
Mean 8.3246 2.0439
Samples were taken three replicates.
83
Table 15. Least Square analysis of Frequency Table of Heamaglutination.
N
Frequency Percent Valid Percent Cumulative Percent
Valid 6.00 13 11.4 11.4 11.4
7.00 20 17.5 17.5 28.9
8.00 32 28.1 28.1 57.0
9.00 27 23.7 23.7 80.7
10.00 11 9.6 9.6 90.4
11.00 10 8.8 8.8 99.1
12.00 1 .9 .9 100.0
Total 114 100.0 100.0
Table .16. Least Square analysis of Frequency Table of IgG of kadaknath chicken.
N Frequency Percent Valid Percent Cumulative Percent
Valid 1.00 6 5.3 5.3 5.3
2.00 97 85.1 85.1 90.4
3.00 11 9.6 9.6 100.0
Total 114 100.0 100.0
84
Table .17. Least Square analysis of One way descriptive analysis of IgG of kadaknath Chicken.
Valid N Mean Std. Deviation Std. Error 95% Confidence Interval for Mean
Lower Bound
6.00 13 2.0769 .2774 7.692E-02 1.9093
7.00 20 2.0000 .3244 7.255E-02 1.8482
8.00 32 2.0313 .4004 7.077E-02 1.8869
9.00 27 2.0741 .5495 .1058 1.8567
10.00 11 2.0909 .3015 9.091E-02 1.8884
11.00 10 2.0000 .0000 .0000 2.0000
12.00 1 2.0000 . . .
Total 114 2.0439 .3854 3.609E-02 1.9724
(Note-samples have been taken four replicates.) Table .18. Least Square analysis of Case Processing Summary of Means of HA, IgG & POP. Of kadaknath chicken.
Cases
Included Excluded Total
N Percent N Percent N Percent HA * POP 114 100.0% 0 .0% 114 100.0% IgG * POP 114 100.0% 0 .0% 114 100.0%
85
Table .19.Least Square Mean and Std. Error report of HA and IgG of Kadaknath Chicken.
POP HA IgG
1.00 Mean 8.3246 2.0439
Std. Error of Mean .1356 3.609E-02
Total Mean 8.3246 2.0439
Std. Error of Mean .1356 3.609E-02
Samples were taken four replicates. Means in a column with different superscripts differ significantly
(P<0.05)
Table .20.Least Square of T-Test of One-Sample Statistics of HA and IgG of kadaknath chicken.
N Mean Std. Deviation Std. Error Mean
HA 114 8.3246 1.4482 .1356 IgG 114 2.0439 .3854 3.609E-02
Samples were taken three replicates. Table .21.Least Square analysis of One-Sample Test of mean difference of HA and IgG of kadaknath chicken.
Test Value = 0 t Df Sig. (2-
tailed) Mean
Difference 95%
Confidence Interval of the
Difference
Lower Upper HA 61.374 113 .000 8.3246 8.0558 8.5933 IgG 56.629 113 .000 2.0439 1.9724 2.1154
Sample were taken four replicates
86
Table .22.Least Square mean and Standard errors of one way Descriptive analysis of HA of Kadaknath chicken.
N Mean Std. Deviation
Std. Error 95% Confidence Interval for Mean
Minimum Maximum
Lower Bound
Upper Bound
1.00 6 8.3333 .8165 .3333 7.4765 9.1902 7.00 9.00 2.00 97 8.3196 1.5176 .1541 8.0137 8.6254 6.00 12.00 3.00 11 8.3636 1.1201 .3377 7.6112 9.1161 6.00 10.00 Total 114 8.3246 1.4482 .1356 8.0558 8.5933 6.00 12.00
Samples were taken three replicates. Table .23. Least Square Analysis of ANOVA of Sum Square of mean of HA of Kadaknath chicken.
Sum of Squares
Df Mean Square F Sig.
Between Groups
1.966E-02 2 9.828E-03 .005 .995
Within Groups 236.972 111 2.135
Total 236.991 113
Table .24. Least Square Analysis of ANOVA of mean square of IgG of Kadaknath chicken.
Sum of Squares
Df Mean Square F Sig.
Between Groups
.128 6 2.132E-02 .137 .991
Within Groups 16.653 107 .156
Total 16.781 113
Samples were taken four replicates
87
Table .25. Least Square analysis of Descriptive Statistics of NPar Tests body weight of KN Chicken.
Samples were taken four replicates.
Table.26. Least Square Statically Analysis of Chi-Square Test of Body Weight of KN Chicken.
BW4 BW6 BW8 BW10 BW12
Chi-Square 40.947 28.807 24.965 21.842 20.105
df 63 73 88 88 90
Asymp. Sig. .986 1.000 1.000 1.000 1.000
a 64 cells (100.0%) have expected frequencies less than 5. The minimum expected cell frequency is 1.8. b 74 cells (100.0%) have expected frequencies less than 5. The minimum expected cell frequency is 1.5. c 89 cells (100.0%) have expected frequencies less than 5. The minimum expected cell frequency is 1.3. d 91 cells (100.0%) have expected frequencies less than 5. The minimum expected cell frequency is 1.3.
N Mean Std. Deviation Minimum Maximum
BW4 114 162.9298 26.7333 86.00 233.00
BW6 114 175.7193 36.6404 71.00 299.00
BW8 114 340.8860 68.6741 190.00 570.00
BW10 114 391.7544 82.0032 204.00 659.00
BW12 114 500.0439 105.5695 249.00 803.00
88
Table.27.Least Square Analysis of Correlations of Body Weight of KN Chicken.
BW4 BW6 BW8 BW10 BW12
BW4 Pearson Correlation
1.000 .843 .738 .729 .563
Sig. (2-tailed)
. .000 .000 .000 .000
N 114 114 114 114 114
BW6 Pearson Correlation
.843 1.000 .713 .714 .558
Sig. (2-tailed)
.000 . .000 .000 .000
N 114 114 114 114 114
BW8 Pearson Correlation
.738 .713 1.000 .765 .651
Sig. (2-tailed)
.000 .000 . .000 .000
N 114 114 114 114 114
BW10 Pearson Correlation
.729 .714 .765 1.000 .796
Sig. (2-tailed)
.000 .000 .000 . .000
N 114 114 114 114 114
BW12 Pearson Correlation
.563 .558 .651 .796 1.000
Sig. (2-tailed)
.000 .000 .000 .000 .
N 114 114 114 114 114
* Correlation is significant at the 0.01 level (2-tailed). Table. 28. Descriptive Statistics Analysis of mean and Std. Error of Body Weight of KN Chicken.
N Mean
Statistic Statistic Std. Error
BW4 114 162.9298 2.5038
BW6 114 175.7193 3.4317
BW8 114 340.8860 6.4319
BW10 114 391.7544 7.6803
BW12 114 500.0439 9.8875
Samples were taken four replicates.
89
Table .29. Least Square analysis of Case Processing Summary of Body Weight of Kadaknath Chicken.
Cases
Included Excluded Total
N Percent N Percent N Percent
BW4 * POP 114 100.0% 0 .0% 114 100.0%
BW6 * POP 114 100.0% 0 .0% 114 100.0%
BW8 * POP 114 100.0% 0 .0% 114 100.0%
BW10 * POP
114 100.0% 0 .0% 114 100.0%
BW12 * POP
114 100.0% 0 .0% 114 100.0%
Table.30.Least square Analysis of Mean and Standard Error Report of kadaknath chicken.
POP BW4 BW6 BW8 BW10 BW12
1.00 Mean 162.9298 175.7193 340.8860 391.7544 500.0439
Std. Error of Mean
2.5038 3.4317 6.4319 7.6803 9.8875
Total Mea n
162.9298 175.7193 340.8860 391.7544 500.0439
Std. Error of Mean
2.5038 3.4317 6.4319 7.6803 9.8875
Samples have been taken three replicates.
90
Table .31.Least Square analysis of One-Sample Statistics of Mean and Standard .Deviation of kadaknath chicken.
N Mean Std. Deviation Std. Error Mean
BW4 114 162.9298 26.7333 2.5038
BW6 114 175.7193 36.6404 3.4317
BW8 114 340.8860 68.6741 6.4319
BW10 114 391.7544 82.0032 7.6803
BW12 114 500.0439 105.5695 9.8875
Samples have been taken three replicates. Table.32.Least Square Analysis of One-Sample Test of body Weight of KN Chicken.
Test Value = 0
t df Sig. (2-tailed)
Mean Difference
95% Confidence Interval of the
Difference
Lower Upper BW4 65.073 113 .000 162.9298 157.9693 167.8903
BW6 51.205 113 .000 175.7193 168.9205 182.5181
BW8 52.999 113 .000 340.8860 328.1432 353.6288
BW10 51.008 113 .000 391.7544 376.5383 406.9705
BW12 50.573 113 .000 500.0439 480.4550 519.6328
Samples were taken three replicates.
91
Table.33.Least Square Analysis of ANOVA for Sum Square and Mean Square. Sum of Squares df Mean
Square F Sig.
BW4 Between Groups
245274.431 63 10036.100 2.569 .001
Within Groups
412300.450 50 6052.009
Total 657574.876 113 BW6 Between
Groups 136043.351 63 2159.418 6.894 .000
Within Groups
15661.667 50 313.233
Total 151705.018 113 BW8 Between
Groups 437405.034 63 6942.937 3.634 .000
Within Groups
95518.483 50 1910.370
Total 532923.518 113 BW10 Between
Groups 599554.706 63 9516.741 2.968 .000
Within Groups
160316.417 50 3206.328
Total 759871.123 113 BW 12 Between
Group 947274.331 63 15036.100 2.409 .001
Within Groups
312100.450 50 6242.009
Total 1259374.781 113 Samples were taken four replicates.
92
6-DISCUSSION
___________________________________________________________________________
6.1. IMMUNOLOGICAL TRAITS
Antibody titers against SRBC and serum IgG level act as indicators for
humoral immune response, bacteriolytic activity of serum lysozyme
acts as indicator for non-specific immune response and CMI response
to PHA-P injection is a thymus cell dependent cutaneous basophil
hypersensitivity (CBH) in chickens (Corrier and Deloach, 1990) .
6.1.1. Antibody Response to Sheep RBC
Singh (2003) reported a titre of 7.4520.279 and 6.8700.254 in
black and white varieties of Turkeys on 5dpi.
Shivakumar (2003) reported a titre of 8.890.29 in the base
population of IWG of WLH chicken.
Sivaraman et al. (2005) observed a mean HA titre of 6.2890.246 in
SDL broiler chickens.
Singh (2005) estimated an average HA titre of 8.23±0.60 in IWG
genotype and 12.00±1.48 and 4.46±0.68 in high and low line,
respectively in IWG-WLH chicken. It reveals that third generation of
divergent lines differs significantly for humoral response to sheep
erythrocytes.
93
Kumar (2006) estimated the least squares mean of HA titre as
12.38±0.60 in Aseel native chicken. In males, the average was
12.80±0.74 whereas in case of females, it was 12.350.62.
Jaiswal et al. (2008) estimated the least squares mean of HA titre as
7.46±0.23 in Kadaknath native chicken. In male, the average was
7.49±0.31 whereas in case of female, it was 7.440.36.
Hatch effect was significant (P<0.05) on all the traits. Chicks in the
first hatch generally demonstrated highest magnitude of the traits
(P<0.05).
It has been reported that advanced genetic selection for economic
performance parameters reduced the immune status (Yegani, et al.,
2005).
Besides, genetic factors (Gyles et al., 1986, Saxena et al., 1997,
Sivaraman et al., 2003) other factors have also been reported to
influence the response to sheep RBCs. Higher dose elicited higher
response (Ubosi et al., 1985, Van der Zijpp, 1983a & Boa-Amponsem et
al., 2000); intravenous route elicited higher responses than other routes
(Boa-Amponsem et al., 2001).
94
In Kadaknath also the least squares mean indicated significant
(P<0.05) differences between all the three hatches, in which the first
hatch birds were having highest antibody titres (8.44±0.05) whereas the
second hatch had the lowest titres (6.61±0.39).
Van der Zijpp and Leenstra (1980) reported significant hatch
differences on HA titres at various dpis and attributed it to the difference
in the climatic conditions during different hatches.
The dietary feed intake also influenced the antibody titres (Siegel
et al., 1982) and genetic environmental interactions influencing antibody
responses in chickens (Gross et al., 1980), which might also contributed
in the present study as these factors may not be precisely same in all
the hatches.
Shivakumar (2003) also observed significant effect of hatch on
antibody response to SRBC.
Contrary to the present finding, non-significant effect of hatch was
noticed for antibody titres at 5 dpi in broiler chickens (Sivaraman et al.,
2003).
Singh (2003) also observed non-significant effect of hatch on
antibody response to SRBC in Black variety of turkeys but not in White
variety.
95
Singh (2005) also observed non-significant effect of hatch on
antibody response to SRBC in IWG-WLH chicken (Singh, 2005).
6.1.2. Serum IgG concentrations
Previous reports on IgG concentrations revealed 7.53±0.61 to
15.99±2.2 mg/ml IgG concentration in White leghorn chicken and
20.51±0.22 mg/ml in indigenous Aseel (Ahrestani et al., 1987).
The serum IgG concentrations in broiler & indigenous birds were
reported to be 8.01±0.4 and 10.01±0.4 mg/ml, respectively (Saxena,
1993). Another finding revealed IgG concentration of 13.5±0.68 mg/ml in
WLH (Rees & Norskog, 1981).
Singh (2003) estimated serum IgG concentrations of 0.672±0.105
mg/ml and 0.930±0.096 mg/ml in black and white varieties of Turkeys,
respectively.
Shivakumar (2003) estimated serum IgG concentrations of 5.65 ±
0.17 mg/ml and 5.24 ± 0.15 mg/ml in IWG and IWJ genotype of WLH
chicken, respectively.
Sivaraman (2004) estimated serum IgG concentrations of
7.361±0.53 mg/ml and 7.137±0.53 mg/ml in the high and low index line,
respectively in broilers.
96
Singh (2005) estimated serum IgG concentration of 55.59±7.37
mg/ml and an average of 61.73 ±15.48 mg/ml and 49.44±6.07 mg/ml in
high and low line of IWG-WLH chicken, respectively i.e. it remain
unaffected through divergent selection.
Kumar (2006) estimated serum IgG concentration of 69.87±6.57
mg/ml in Aseel native chickens. In males, the average was 74.09±8.13
mg/ml whereas in case of females, it was 65.64±7.05 mg/ml.
Jaiswal et al. (2008) estimated an average of serum IgG
concentration of 12.62±0.58 mg/ml in Kadaknath native chicken. In male,
the average was 13.35±0.75 μg/ml whereas in case of female, it was
11.89±0.89 μg/ml.
In Kadaknath also the least squares mean indicated significant
differences (P<0.01) among all the three hatches, in which the first hatch
birds were having highest values i.e. 10.69±0.41mg/ml) whereas the
third hatch had the values (8.98±0.32 mg/ml). There was not much
difference between the values of first and second hatch but the influence
of hatch on IgG values were statistically significant.
Van der Zijpp & Leenstra (1980) obtained significant hatch
differences for MES titres at 3,7,10 and 13 dpis.
Singh et al. (2003) also obtained non-significant hatch differences
in Black variety of Turkey but not in white variety of Turkey. Contrary to
97
the present findings, the IgG concentration of the broiler birds was not
significantly affected by hatches (Sivaraman et al. 2003).
Shivakumar (2003) observed non-significant effects of hatch on
MES antibody titres in both genotypes of WLH chicken.
Singh (2005) observed non-significant (P>0.05) hatch effect on
IgG concentrations in Aseel native chicken.
It was evident that IgG antibodies are significantly affected by non-
significantly factors like hatch, unlike IgM antibodies (Shivakumar, 2005).
6.2. General Performance of Kadaknath Chicken
A recent study of their growth potential recorded a daily weight gain of
6.2 grams from 0 to 20 weeks of age – based on growth in the breed’s
usual production environment, with very little supplemental feed. Similar
trend was observed by Farooq et al. (2001), who reported higher day-old
chick weight in RIR (35.3290.86 g), in comparison to Desi (33.8490.67
g) and Fayoumi chicken (30.7490.72 g.
6.2.1. Growth Traits
6.2.2. Body Weight
The hatch effects and breed-hatch interaction effects were significant.
(Williams et al., 2002). Yalcin et al. (2000)observed negative heterosis
for body weight in chicken while Nestor et al. (2004) reported negative
98
and significant heterosis for bodyweight at 8, 16 and 20 week of age in
male and female turkey.
6.2.3. Mean and Slandered Errors
Significant and positive effect of heterosis on body weight in broiler was
reported in literature (Iraqi et al., 2005). Analysis of slandered errors of
mean revealed highly significant differences for six body weight due to
breed, hatch hatch-breed interaction and sex. Similar results were
reported by Hoque et al. (1975). For instance, growth curves may enable
us to forecast the future growth of an animal and, thereby, select the
animals for breeding purposes when they are young (Akbag, 1995).
Also, with the help of growth curves, it is possible to revise growing
systems in effect at early stages (Ersoy et al,, 2006; 2007; Dincer et al.,
2007). It is concluded that using Gompertz and linear growth models
would be sufficient in modeling the growth, based on the 7 traits
determined. These findings are in agreement with the earlier literature
stating “growth curve models differ based on the trait under
investigation” (Ersoyet al., 2006; 2007; Dinver et al., 2007). At four
weeks of age the kadaknath indigenous chickens had the lowest and
highest mean body weight gain of all the strains with average daily
growth rate of 3.30 g and 4.20 g per bird per day in the starter growth
phase, respectively.
99
The result of this study is similar to the work reported by
Shanawany (1987) who stated that differences in hatching weight may
be attributed to differences in the age of the breeder flock, which have
been reported to affect the subsequent growth performance (North,
1984). It was also reported that at egg laying stage, the mean
bodyweight of Thai indigenous hens reared under intensive
management systems was 1.45 kg (Bansidhi et al., 1988;
Gongrattananun et al., 1992). These results also indicated the presence
of a substantial amount of variation in growth rate among and between
the indigenous chicken populations.
The mean body weights of Horro chicken were generally within the
ranges reported for unselected indigenous populations in north western
Ethiopia (Halima et al. 2007)and many other countries of Africa (Gueye
1998).
6.2.4. Phenotypic correlation among by body weight at various
stages
The mean body weights of Horro chicken were generally within the
ranges reported for unselected indigenous populations in north western
Ethiopia (Halima et al. 2007)and many other countries of Africa (Gueye
1998).
100
Phenotypic correlation among body weights at different ages ranged
from low to high .The keets weight at hatching is not supposed to bear
ay genetic relationship with subsequent body weights. Low phenotypic
correlation between 0 day body weight and body weight at later ages
indicates that Body weight at 0 day of ages is not suitable criterion for
selection for improving market weight. Body weights to 16 weeks of age
were used to characterize the growth of chicken in this study. Selection
for rapid early growth at a market age (40–50 days) has been the most
common approach in broiler chicken breeding programs (Emmerson
2003).
6.3. DNA Polymorphism in ChB6, Caspase-1, IAP-1, and ZOV3
genes.
6.3.1. PCR optimization
The reaction mixture and PCR programs for ChB6, Caspase-1,
IAP-1, and ZOV3 genes were optimized as per Zhou and Lamont (2003)
with slight modifications.
6.3.2. Optimized PCR reaction mixture
For ChB6, Caspase-1, IAP-1, and ZOV3 genes, the optimum
combination of various reaction components for each 25 µl PCR
reaction mix was 25 ng genomic DNA, 0.8 mM of each primer, 0.2 mM
each dNTPs and 1 U Taq DNA polymerase.
101
6.3.3. Optimization of PCR programme
To optimize the PCR conditions different annealing temperatures
were used. In case of all the four genes i.e., ChB6, Caspase-1, IAP-1,
and ZOV3 genes in Kadaknath breed of native chickens, initial
denaturation at 94˚C for 5 min, 35 cycles of (a) Denaturation at 96˚C for
1 min. in case of ChB6, Caspase-1, and ZOV3 whereas in case of IAP-
1, it was 94˚C, (b) Annealing at 53˚C, 65˚C, 62˚C, and 52˚C for ChB6,
Caspase-1, IAP-1, and ZOV3, respectively for 1 min., (c) Extension at
72˚C for 1 min. and final extension at 72˚C for 10 min. for ChB6,
Caspase-1, and ZOV3 whereas in case of IAP-1, it was 72˚C for 15 min.
produced distinct and robust amplified product.
The optimized PCR reaction mixture and PCR programmes gave
satisfactory amplification of all the genes in Kadaknath breed of native
chicken.
6.3.4. DNA Polymorphism at ChB6 gene
For ChB6 gene, a Previous reports revealed the presence of sites
in the 215 bp of ChB6 which generated the fragments of 147 and 68 bp
(LL Genotype) and 215 bp (FF Genotype) (Zhou and Lamont, 2003). In
present investigation revealed in the case of Kadaknath, only two
genotype i.e. AA and BB with fragment size of 147 and 215 bp
fragments, respectively could be observed.
102
A 215 bp fragment of ChB6 gene showed a C→A substitution at
base 470, which caused a predicted amino acid change from Gln
(Leghorn line) to Lys (Fayoumi line)(Zhou and Lamont, 2003).
6.3.5. DNA Polymorphism at Caspase-1 gene
For Caspase-1 gene, Previous reports revealed the presence of
Hsp 92 II RE sites in the 1070 bp of Caspase-1 which generated the
fragments of 421, 244, 227, 122, and 56 bp (LL Genotype) and 312,
244, 227, 122, 109, and 56 bp (FF Genotype) (Zhou and Lamont, 2003).
For the Caspase-1 gene, a 1,070-bp amplified fragment showed a
TC substitution at 368 bp between the Leghorn and Fayoumi lines. A
PCR-RFLP assay was developed with Hsp 92 II (Zhou and Lamont,
2003).
6.3.6 DNA Polymorphism at IAP-1 gene
For IAP-1 gene, Previous reports revealed the presence of Bgl I
RE sites in the 394 bp of IAP-1 gene which generated the fragments of
254 and 140 bp (LL Genotype) and 394 bp (FF Genotype) (Zhou and
Lamont, 2003).
A 394-bp fragment showed a TA substitution from the Leghorn
to the Fayoumi lines for the IAP-1 gene, and a PCR-RFLP assay was
103
developed to identify a Bgl I SNP to characterize the polymorphism at
Ala157 (Zhou and Lamont, 2003).
6.3.7. DNA Polymorphism at ZOV3 gene
For ZOV3 gene, Previous reports revealed the presence of SnaB I
RE sites in the 320 bp of ZOV3 which generated the fragments of 270
and 50 bp (LL Genotype) and 320 bp (FF Genotype) (Zhou and Lamont,
2003).
An aamplified 320-bp product of ZOV3 demonstrated a TG SNP,
which caused a predicted amino acid change from Cys157 in the
Leghorn line to Phe157 in the Fayoumi lines. The SnaB I RE produced
fragment sizes of 270 and 50 bp for the Leghorn lines, whereas the
Fayoumi lines had a 320-bp fragment. Because ZOV3 maps to the Z
chromosome, no heterozygous pattern was generated from the hens
(Zhou and Lamont, 2003).
104
7-SUMMERY & CONCLUSIONS
___________________________________________________________________________
Several elite layer line have been developed in our country which
have resulted into bringing the country 4th rank in the world. Still there
exists a gap between availability and requirements; hence further growth
in the egg production sector is very much needed. Besides feed, health
coverage takes the major chunk of the input cost. Owing to wide variety
of pathogens and the poultry rearing by diversified group across the
country, development of lines for general higher imuunocompetent
seems more feasible. Immune response to sheep erythrocytes (SRBC)
is one of such parameter which is used to develop general
immunocompetance lines. Periodical evaluation of these lines for
various immunological traits is an essential feature. Molecular analysis
of genes that have bearing on immune response would add the
knowledge of understanding the mechanism of disease resistance.
Indian breeds of chicken are well known for their resistance to diseases
and tropical adaptability.
In present investigation, was undertaken to estimate genetic and
phenotypic parameter of Kadaknath breed of native chicken were
evaluated for immunocompetence traits viz., antibody response to
SRBC, and serum IgG level, Body Weight and DNA polymorphism at
105
ChB6, Caspase-1, IAP-1, and ZOV3 genes along with associations
among immunocompetence traits. Growth is multi-factorial and
multidimensional phenomenon. Many genes and gene products play
key role in development and growth of an individual.
One hundred and fifteen birds of Kadaknath breed of native
chicken were used in the investigation for evaluation of
immunocompetence traits. Humoral immune response to SRBC was
measured through HA test. Immunization was done by injecting 1 ml of
1% SRBC intravenously. Sera samples were collected 5th day post
immunization. The antibodies were titrated against 1% SRBC
suspension. Single radial immunodiffusion assay was used to estimate
the total IgG in serum. Diameter of precipitation ring of samples was
compared with that of standard to determine the concentration of serum
IgG. The antibody titres against sheep RBCs were measured through
HA test on 5th dpi. HA titers ranged from 2-15 in Kadaknath .Average HA
titers were 7.69±0.13 in Kadaknath. Serum IgG is the most abundant
antibody and constitutes approximately 80% of the total
immunoglobulins. The bird’s ability to mount antibody responses to other
antigens is primarily revealed by serum IgG concentration. The average
serum IgG concentration was 10.07±0.20 mg/ml in Kadaknath breeds,
respectively.
106
Least-squares analysis of variance revealed significant difference
(p<0. 01) for general combining ability (GCA) in body weight at all age
groups. The gain in the body weight was highest during 8 to 12 weeks of
age.
On the basis of HA titer birds were grouped high and low titre
group. Polymorphism in these genes were analyzed by PCR-RFLP
technique using Pvu II, Hsp 92 II, Bgl I and SnaB I RE, for ChB6,
Caspase-1, IAP-1, and ZOV3 genes, respectively. On the basis of
presence and absence of restriction fragments, birds were screened for
their genotypes i.e. AA, BB, and AB. In Kadaknath, only two genotype
was present i.e. AA and BB. AA genotype had 147 bp fragments
whereas BB genotype had 215 bp fragments.
Overall, the present study demonstrated that divergent selection
based on response to SRBC gave direct response but did not influence
other immunological and early layer traits, hence HA titer may be
considered as selection criterion along with production traits for
improving production and protection status of Kadaknath native chicken.
Kadakanath demonstrated relatively higher humoral response to
Sheep erythrocytes than other chicken breeds. The differences
between HA titers in males and females were not significant. Older
bird’s higher response than younger group.
107
Serum lysozyme level was also relatively higher in Kadakanath. It
did not differ between sexes or among age groups.
Serum IgG level did not vary significantly (P>0.05) between sexes
or age groups; although older birds demonstrated higher values.
Overall, the present study demonstrated that Kadakanath breed of
native chicken have high immunocompetence status. Varied levels
of humoral immune response in Kadakanath can be exploited for
development of higher immune tolerant birds through selective
breeding.
The Kadaknath birds attain 1 kg body weight between 6 to 7
months of age and the birds reached around 1.5 kg by 1 year of
age.
The growth trends in both sexes showed linear increase in body
weights; however the rate of increase in body weights were higher
from 6 to 52 weeks of age in males as compared to females, thus
showing clear sex-dimorphism.
The differences in body weights of male and females were
significant (P>0.05).
BB genotype of ChB6 had high frequency than the heterozygotes
in Kadaknath. Heterozygotes were more in Kadaknath for
Caspase-1 and IAP-1 gene. Heterozygotes were equal to BB in
Kadaknath for ZOV3 gene.
108
The unique characteristics of native chicken of various countries
are well established by research findings. Hence it is essential to
conserve the precious genetic resources and every effort from the
government and the public needs to be taken to conserve them for
the present and future. On the other hand, newer genomic tools
could be applied to utilize the potential of native chickens for the
betterment of mankind.
109
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