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International Buffalo Information Centre
BUFFALO BULLETIN
ISSN : 0125-6726
(IBIC)
Aims
IBIC is a specialized information center on
water buffalo. Established in 1981 by Kasetsart
University (Thailand) with an initial financial
support from the International Development
Research Center (IDRC) of Canada. IBIC aims at
being the buffalo information center of buffalo
research community through out the world.
Buffalo Bulletin is published quarterly in March,
June, September and December. Contributions on
any aspect of research or development, progress
reports of projects and news on buffalo will be
considered for publication in the bulletin. Manuscripts must be written in English and follow the
instruction for authors which describe at inside of
the back cover.
Editor
Main Objectives
S. Sophon
1. To be world source on buffalo
information
2. To provide literature search and
photocopy services
3. To disseminate information in
newsletter
4. To publish occasional publications
such as an inventory of ongoing
research projects
Publisher
International Buffalo Information Centre,
Office of University Library,
Kasetsart University
Online availible:
http://ibic.lib.ku.ac.th/e-Bulletin
BUFFALO BULLEITN
IBIC, KASETSART UNIVERSITY, P.O. BOX 1084
BANGKOK 10903, THAILAND
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http://ibic.lib.ku.ac.th
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66-2-9428616 ext. 344
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Buffalo Bulletin (June 2013) Vol.32 No.2
CONTENTS
Page
Review Article
Infectious causes of infertility in buffalo bull (Bubalus bubalis)
P. Perumal, T. Kiran Kumar and S.K. Srivastava............................................................................71
Case Report
Dystocia due to fetal anasarca coupled with amelia of one foetus in twin pregnancy in a buffalo
V.S. Dabas, V.K. Sharma and C.F. Chaudhari.................................................................................83
Short Communication
Prevalence of different spontaneous liver lesions in buffaloes
Y. Verma, U.K. Vyas and Suneeta Rani............................................................................................85
Original Article
Evaluation of cystourethroplasty vis-a-vis caecourethroplasty technique
in buffaloes (Bubalus bubalis)
Md. Moin Ansari and S.P. Sharma...................................................................................................91
A study on buffalo management practices in Khammam district of Andhra Pradesh
K. Kishore, M. Mahender and Ch. Harikrishna...............................................................................97
Sub-clinical mastitis in murrah buffaloes with special reference to prevalence,
etiology and antibiogram
Pankaj, Anshu Sharma, Rajesh Chhabra and Neelesh Sindhu......................................................107
Effect of yeast culture (Saccharomyces cerevisiae) on ruminal microbial population
in buffalo bulls
D. Srinivas Kumar, Ch. Srinivasa Prasad and R.M.V. Prasad......................................................116
Buffalo Bulletin (June 2013) Vol.32 No.2
CONTENTS
Page
Original Article
Early pregnancy diagnosis by transrectal ultrasonography
in Mehsana buffaloes (Bubalus bubalis)
Mehrajuddin Naikoo, D.M. Patel and H.J. Derashri..................................................................120
A study of protease inhibitor gene polymorphism in Mehsana buffalo (Bubalus bubalis)
Manisha Deshpande, D.N. Rank, P.H. Vataliya and C.G. Joshi..................................................126
Sox-2 gene expression pattern in stem cells derived from different stages of
in vitro produced buffalo (Bubalus bubalis) embryos
Ch. Srinivasa Prasad, A. Palanisamy, S. Satheshkumar, V.S. Gomathy,
G. Dhinakar Raj and A. Thangavel.............................................................................................131
Sequence characterization and polymorphism detection in the bubaline CD14 gene
Aruna Pal, Arjava Sharma, T.K. Bhattacharya, A. Mitra and P.N. Chatterjee...........................138
Review Article
Buffalo Bulletin (June 2013) Vol.32 No.2
INFECTIOUS CAUSES OF INFERTILITY IN BUFFALO BULL (Bubalus bubalis)
P. Perumal1,*, T. Kiran Kumar1 and S.K. Srivastava2
Keywords: buffalo bull, Bubalus bubalis, infertility,
infectious diseases
ABSTRACT
The artiﬁcial insemination (AI) technique
plays an important role in improvement of
conception rate, prevention of sexually transmitted
diseases and transmission of genetic material to the
next generation. The breeding soundness evaluation
(BSE) plays an important role in selection of male
buffalo for the said purposes. This evaluation
is an effective, inexpensive and easy method for
selection of breeding buffalo bulls. In buffalo
breeding management programmes, seasonal
variation, nutrition, congenital defect, hormonal
changes and hereditary play critical roles in
determining the reproductive efﬁciency in buffalo
bulls. The information regarding the reproductive
health of buffalo bulls for breeding soundness
evaluation is meager. The venereally transmitted
infection causes early embryonic death, infection
in the female reproductive tract and infertility or
sterility in female animals and also infertility and
disease condition in the male animals. To avoid
such infective conditions, proper and careful
investigation of all parts of the reproductive
tract and evaluation of semen and treatment are
important. So, a male buffalo calf being purchased
for breeding purpose must be evaluated for breeding
soundness.
INTRODUCTION
The buffaloes are in the order of
Artiodactyla, the cloven-hooved mammals, genus
Bubalus and species bubalis. Two main species of
buffalo are found in the world: the Asiatic (water)
buffalo (Bubalus bubalis) and the African buffalo
(Syncerus caffer). The two buffalo types are have
different habitats and chromosome numbers. There
are about ~170 million buffaloes in the world
(Perera et al., 2005). Out of this 97 percent of them
are water buffaloes and are mainly found in the
Asian region. Riverine buffaloes are characterized
by black colour and have long curled horns (e.g.
Murrah Breed) and the Swamp buffaloes are dark
grey, but may also be black, black and white, or even
all white, have long, gently curved horns. Riverine
buffaloes (70 percent of the total world population)
are reared in high numbers in South Asia, especially
in India and Pakistan. The name ‘swamp’ has
probably arisen from their preference for wallowing
in stagnant water pools and mud holes (Subasinghe
et al., 1998). Swamp buffaloes are found mainly in
southern China Sri Lanka, and the South-East Asian
1
National Academy of Agricultural Research Management (NAARM), Indian Council of Agricultural
Research (ICAR), Rajendranagar, Hyderabad - 500 407, India, *E-mail: perumalponraj@gmail.com
2
Division of Animal Reproduction, Indian Veterinary Research Institute, Izatnagar - 243 122, India
71
Buffalo Bulletin (June 2013) Vol.32 No.2
countries of Thailand, the Philippines, Indonesia,
Vietnam, Burma (Myanmar), Laos, Cambodia
and Malaysia (Chantalakhana and Falvey, 1999).
Riverine buffaloes are predominantly used for
milk production and they are also used for meat,
fuel and fertilizer production, as well as for draught
power, whereas swamp buffaloes are traditionally
kept as draught animals and are also (to a lesser
extent) used for meat production. The river-type
in two ways: they directly affect the reproductive
system and the quality of semen, and they affect
other systems, and this interferes with semen
production, affects the libido and mating ability and
prolongs reaction time. Lagerlof (1934) established
a relationship between semen charactertics and
testicular pathology. The condition of the testis is
clearly reﬂected in the type of semen produced.
Semen charactertics and fertility have a close
relationship. Bacterial contaminants of the semen
lead to the production of number macrophages and
polymorphonuclear granulocytes as the ﬁrst line of
defence against bacteria in the semen, and this in
turn leads to the generation of ROS (Ochsendrof,
1998) and also the production of a greater number
of dead sperm, which will enhance the production
of ROS in semen (Aitken, 1995). Increased ROS
generation impairs sperm function and fertilizing
capacity (Aitken, 1995; Griveau et al., 1995).
Various types of microorganism cause conditions
like balanitis, posthitis, seminal vesiculits,
prostatitis, urethral inﬂammation, testicular
degeneration, orchitis, epididymitis, and ampulitis
which result in male infertility. Under practical
conditions, it is not possible to produce semen free
from microorganisms as contamination with few
nonpathogenic organisms is unavoidable (Binda et
al., 1994). Commensal microorganisms also infect
the reproductive tract and affect sperm motility and
viability when increasing stress to the animal; such
organisms include E. coli (Schirren and Zander,
1966; Huwe et al., 1998) and Streptococcus faecalis
(Bisson and Czyglick, 1974; Makler et al., 1981;
Huwe et al., 1998).
The organisms present in semen may be
bacteria, viruses, protozoa, chlamydia, rickettsia
and fungi and generally they are classiﬁed as
pathogenic, potentially pathogenic, and not
pathogenic (Gangadhar et al., 1986). Their
buffalo has a diploid number of 50 as compared
to 48 for the swamp-type buffaloes. The Murrah
has two extra acrocentric chromosomes, which
are presumably translocated onto the short arms
of the number 1 autosome of the swamp buffalo.
Differential staining revealed that Egyptian water
buffaloes, with a diploid number of 50, are closely
related to the Murrah buffalo (Cribiu and Obeidah,
1978). Hybridization between Murrah and swamp
buffaloes is possible and the hybrids are fertile.
Improvement of buffalo reproduction helps in
production and enhances signiﬁcantly the economy
and living standards of many rural communities
throughout the world. The common defects
affecting the fertility of the male are congenital
defects in the male reproductive tract, hormonal
imbalances between different glands, infectious
diseases of the reproductive system or any other
system, hereditary defects, imbalanced nutrition,
psychological defects and abnormal climatic
conditions. The purpose of this paper is to describe
the different types of infectious disturbance which
affect the reproductive performance of the buffalo
bull.
GENERAL VIEW OF INFECTIOUS
DISEASES IN BUFFALO BULLS
Infectious microorganisms affect fertility
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Buffalo Bulletin (June 2013) Vol.32 No.2
Staphylococcus sp., E.coli, Bacillus sp., Aeromonas
sp., and yeast (Ghanghadar et al., 1986; Ramasamy
et al., 2002). Sensitivity (chloramphenicol (100%),
ciproﬂoxacin (100%) gentamicin (100%), neomycin
(100%), streptomycin (86.62%), tobramycin (80%),
co-trimoxazole (73.33%), erythromycin (53.33%),
polymycin-B (13.33%), cephalexin (6.67%),
nitrofurantoin (6.67%), tetracycline (6.67%) and
resistant penicillin-G (100%), Oxytetracycline
(100%), carbenicillin (100%), amoxicillin (100%),
ampicillin (100%) to microorganisms has also been
reported for buffalo semen in fresh and frozenthawed semen (Singh et al., 1992; Ramasamy et
al., 2002). Microorganisms Balakrishnan et al.
(2006) and Prabhakar et al. (1993) isolated from
frozen buffalo semen were Staphylococcus sp.
(56%), Bacillus sp. (45%), Micrococcus sp. (5%),
E. coli (5%), Klebsiella sp. (3%), Proteus sp. (3%)
and Pseudomonas sp. (10%). Balakrishnan et al.
(2006) reported that the sensitivity test for buffalo
semen were perﬂoxacin (95.2%),ciproﬂoxacin
(94.4%), gentamicin (93.60%), enroﬂoxacin
(92.8%), penicillin (31.2%), streptomycin (16.8%),
Oxytetracycline (40.8%), ampicillin (34.8%),
amoxicillin (38.4%), chloramphenicol (36.8%) and
triple sulpha (27.2%). Sharma et al. (1994) reported
that addition of gentamicin sulphate (500 mcg per
ml), chloramphenicol sodium succinate (500 mcg
per ml) or ampicillin sodium (500 mcg per ml) in
Tris egg yolk glycerol proved to be more beneﬁcial
rate than combination of streptomycin sulphate (500
mcg per ml) and penicillin G sodium (500 IU per
ml) at every stage of cryopreservation. Gentamicin
sulphate was most effective in controlling bacteria
in semen at all stages of deep freezing. Gentamicin,
being poorly soluble in lipids, has limited ability
to cross cell barriers (Prescott and Baggot, 1988).
Consequently, its distribution in the extended semen
is primarily extra spermatozoal and the whole of
presence in the semen may be due to from the
bull’s systemic or local speciﬁc infections and
they may also come from the normal prepuce
ﬂora or from the contamination that follows the
semen collection because of inappropriate manual
procedures or contaminated equipment. Many of
these microorganisms may survive during storage
carried out at low temperatures and they may
represent a real danger for the spread of diseases
if we consider that the semen is inserted directly
into the uterus without exposure to the bactericidal
action of the vaginal and cervical secretions
produced during estrus.
The mean microbiological load was increased
by four fold in the second ejaculate (Gangadhar et
al., 1986), hence use same artiﬁcial vagina for two
successive ejaculates is not accepted under clean
semen production. The bacterial contamination
of semen is major concern for semen production
laboratories as the pathogens adversely affect
the semen quality (Diemer et al., 1996). Bacteria
contaminate approximately 50 percent of frozen
semen doses as reported by Wierzbowski et al.
(1984). Moreover it has been reported that the
frozen semen of the buffalo bulls has a higher
microbial load (Shukla, 2005 (4.860 ± 0.73 X 102
ml-1); Rathnamma et al. (1997) (5.05 - 171.4 X 103
ml-1 ); Jaisal et al. (2000) (1.0 - 50.0 X 102 ml-1)
and there is a highly signiﬁcant negative (P<0.01)
correlation of standard plate count with progressive
sperm motility (Shukla, 2005), live sperm (Shukla,
2005; Ahmad and Mohan, 2001) and HOS %
(Shukla, 2005) both in neat as well as cryopreserved
semen, whereas a positive correlation of sperm
abnormalities with standard plate count was also
recorded. The SPC and type of microorganism
vary among different bulls (Shukla, 2005). The
common microorganisms isolated from buffalo
bull semen are Pseudomonas sp., Streptococcus sp.,
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Buffalo Bulletin (June 2013) Vol.32 No.2
the isolations and the serologic test results lead us
to believe that this infection is particularly spread
among buffaloes (De Carlos et al., 2004). This
disease is transmitted through respiratory contact
and ocular and reproductive secretions, with the
latter route seeming to be the most important for
entry into herds (Afshar and Eaglesome, 1990). In
all probability, the viruses are eliminated through
the semen of bulls both during the acute phase of
the disease and during latent infections in clinically
normal animals, and bulls may shed virus in semen
during both clinical and subclinical infections (van
Oirscholt et al., 1993). Viral reactivation from
the latent state is generally thought to be stressinduced but can also be induced by the injection of
corticosteroids (Pastoret et al., 1982). The excretion
may occur even in lack of antibody response;
therefore, only sero-negative and viruses-free bulls
can be used for artiﬁcial insemination. There are
differences in susceptibility to the disease among
breeds. I It has been reported that the Murrah breed
(85.8%) is more susceptible (p = 0.0004) than the
Mediterranean type (67.7%). Moreover, age is also
a major factor for susceptibility to this disease: rates
of infection increase progressively with advancing
age (Ferreira et al., 2010). Diagnosis is carried out
through ELISA, ﬂuorescent antibody tests (FAT)
of tissues, serum neutralization test, viral isolation
or direct demonstration of the viral DNA through
PCR (Polymerase Chain Reaction).
the drug is available for killing bacteria and this
is might be the reason for its better bactericidal
effect. The better efﬁcacy of chloramphenicol
sodium succinate and ampicillin sodium than that
of streptomycin sulphate and penicillin sodium G
might be due to their higher sensitivity to bacterial
ﬂora of semen (Sharma et al., 1994).
INFECTIOUS BOVINE
RHINOTRACHEITIS (IBRT)
The disease is caused by Bubaline
Herpes Virus 1 (BuHV-1) and Bovine Herpes
Virus 1 (BoHV-1), which are members of
the Alphaherpesvirinae. BHV-1 (Gibbs and
Rweyemamu, 1977) and which infect the
respiratory and genital tracts of cattle and buffaloes,
causing various diseases, such as infectious bovine
rhinotracheitis, infectious pustular vulvovaginitis,
and infectious pustular balanoposthitis, abortion,
mastitis, infertility, tracheitis, conjunctivitiskeratoconjunctivitis, encephalitis and fatal disease
in newborn calves (Gibbs and Rweyemamu,
1977) and are pathogens responsible for causing
signiﬁcant losses in the livestock industry,
through the failure of reproduction and increased
mortality in cattle (Cortez et al., 2001; De Carlos
et al., 2004). Phylogenically, this virus is closest
to Bovine Herpes Virus-5 (BoHV-5), which causes
encephalitis in cattle. On the basis of current
knowledge, therefore, it cannot be claimed that
BuHV-1 is responsible for abortion in buffalo
cows. By contrast, BuHV-1 is suspected of being
responsible for cross reactions with the IBR virus
when Bovine Herpes Virus-1 (BoHV-1) or its parts
(glycoproteins) are used as an antigen in serological
tests (Galiero, 2007). But the pathogenic role of the
BuHV-1 and BoHV-1 has not been clariﬁed yet and
FOOT AND MOUTH DISEASES (FMD)
Foot and mouth disease virus could be
transmitted in bull semen before the infected bull
shows any signs of disease. The virus carrier stage
has been shown to persist in some cattle for many
months following recovery from the disease or
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Buffalo Bulletin (June 2013) Vol.32 No.2
that the same viruses isolated in cattle are not also
present in the buffalo, nor that they act through the
complex pathogenic mechanisms that have long
been known to operate in cattle (Galiero, 2007).
Analysis of the molecular characteristics of the two
strains made it possible to classify the isolates as
BVDV-1, sub-genotype 1b. Evidence suggests that
this virus is present within the buffalo population,
and is associated with abortion, thus bringing to
light a previously unknown health problem for
the buffalo (Galiero, 2007). There are too little
data to clarify the pathogenic role of BVDV in
buffalo. Further research could shed light on the
role of this virus and make it possible to determine
pathogenesis, clinical characteristics and lesions.
following prophylactic immunization. The disease
has been shown to be transmissible when susceptible
females were inseminated with semen from virus
shedding bulls. It is found that FMD virus can
survive in semen in freezing procedures in liquid
nitrogen. Foot and mouth disease causes severe
degeneration of germinal epithelium of the testes
of bulls. The semen picture was severely adversely
affected. The motility was poor and the total sperm
abnormalities range from 34-62%. Signiﬁcant FMD
virus type speciﬁc antibody titers (IgG1, IgG2 and
IgA) were detected in milk and serum of female
buffaloes and serum of male buffalo. FMD virus
type speciﬁc IgG1 was found to be the predominant
subclass as compared to IgG2 and IgA both in milk
and serum of vaccinated buffaloes. Milk and serum
IgG1, IgG2 and IgA antibody titres were positively
correlated with values of regression coefﬁcient (R)
as 0.506, 0.434 and 0.396, respectively (Yadav et
al., 2007).
BRUCELLOSIS
Buffaloes are susceptible to infection
with Brucella abortus and Brucella melitensis. B.
melitensis biovar 3 and B. abortus biovars 1 and
6 predominates in Italian buffalo herds. Brucella
infection in bulls may affect the testicles, the
epididymis, the seminal vesicle, and the ampulla.
The infected males eliminate the Brucella spp.,
with the semen, so they play an active role in
the spread of the disease (Eaglesome and Garcia,
1992). Brucellosis is a contagious infectious
disease that affects bovines of traditional domestic
and nondomestic species such as the buffalo. It is
characterized by the production of abortions in the
last third of gestation, retention of placenta, metritis,
infertility, stillbirth , mastitis, poor production and
quality of milk. Affections of the male are arthritis,
orchitis, and epididymitis Brucellosis is a zoonosis
of world importance (Radostits et al., 2000). In
this regard, artiﬁcial insemination represents a
good tool to control this pathology on condition
BOVINE VIRAL DIARRHOEA (BVD)
This disease is caused by Bovine Viral
Diarrhoea Virus (BVDV) and the virus belongs to
genus Pestivirus and family Togaviridae (Andrews
et al., 1978). Although cattle are the primary hosts,
BVDV can infect most even-toed ungulates. The
main mode of transmission is through the oral
route, but the disease may also be transmitted
by inhalation and artiﬁcial insemination using
semen of persistently infected bulls. Interspecies
spread of this virus has been demonstrated but the
epidemiological signiﬁcance of this is uncertain.
Seroepidemiological studies seem to indicate that
these viruses circulate to some extent within the
buffalo population in Italy (Galiero, 2007). To date,
however, there is no conclusive scientiﬁc proof
75
Buffalo Bulletin (June 2013) Vol.32 No.2
that the donors are carefully monitored. Since
infected bulls may give serologically negative
results, bacteriological tests are necessary. The
tests have to be applied to the semen and carried
out in successive stages. The seminal plasma has
to undergo the serum-agglutination test. Freezing
procedures for the preservation of embryos cause
a 64% decrease of the Brucella abortus vitality
(Martinez et al., 2007). The administration of the
2007).
CAMPYLOBACTERIOSIS
The infection of buffaloes with
Campylobacter foetus may be widespread.
Campylobacter fetus subsp. venerealis causes
a type of venereal disease in the cow that is
transmitted through natural service or artiﬁcial
insemination. The disease is characterized by
infertility, prolonged estrous, premature embryo
death and in some instances, untimely abortion
with placental retention (Das and Paranjape, 1987).
The use of communal bulls and the use of males
that have not been tested for Campylobacter foetus
at artiﬁcial insemination centers are important
factors in spreading infection. It has been reported
that there are seven strains of Campylobacter
sputorum subsp. Bubulus (Modulo et al., 1997) and
Campylobacter fetus, Campylobacter fetus subsp.
venerealis and Campylobacter fetus subsp. fetus
(Joshi et al., 2006) have isolated from the prepuce
of buffalo bulls. The disease has been recorded in
India, Malaysia and the former U.S.S.R. (Eaglesome
and Garcia, 1992). The animals used for artiﬁcial
insemination have to undergo quarantine and have
negative results in three consecutive cultural tests
carried out on preputial scraping. Afterwards their
sanitary conditions have to be checked every six
months.
appropriate antibiotics causes a 99% inactivation
of the microorganism and the national eradication
schemes are more beneﬁcial in eradicating this
organism when based on the detection and slaughter
of infected buffaloes.
ARCANOBACTERIOSIS
Arcanobacterium pyogenes is commonly
present on the nasopharyngeal mucosa of buffalo;
in bulls. The usual habitat is the preputial mucosa
(Radostis et al., 2003). Arcanobacterium pyogenes
is a common cause of suppurative lesions in
buffalo. This bacterium has been associated with
mastitis, metritis, pyometra and abortion in buffalo
cows. In bulls Arcanobacterium pyogenes is an
important cause of orchitis, epididymitis or seminal
vesiculitis, so the organism can be eliminated
with semen. Specimens suitable for diagnostic
laboratory procedures include exudates, aspirates,
tissue samples and semen. Arcabacterium pyogenes
is a Gram positive pleomorphic rod and produces
a characteristic haemolytic pin-point colony in
48 hours of incubation. The diagnosis is based
on bacteriological examination of the organs.
Arcanobacterium pyogenes develops in 24-48 h,
forming haemolytic colonies on agar supplemented
with 5% sheep erythrocytes. Biochemical tests
yield deﬁnitive microbial identiﬁcation (Galiero,
LEPTOSPIROSIS
The microorganisms belonging to this genus
are mobile, helical bacteria, the terminal part of
the bacterial body being hooked-shaped. Although
cytochemically Gram-negative, they do not stain
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Buffalo Bulletin (June 2013) Vol.32 No.2
bacteria. Their developmental cycle comprises
two forms: infecting elementary bodies and
non-infecting reticular bodies. The former are
small and metabolically inert, and penetrate the
host cell by means of endocytosis. The reticular
bodies are metabolically active and replicate
by means of binary ﬁssion inside an endosome.
Two genera are recognized on the basis of
ribosomal RNA analysis: Chlamydophila spp.
and Chlamydia spp. Some of the species that
cause chlamydiosis are zoonotic: Chlamydophila
abortus, Chlamydophila psittaci, Chlamydophila
felis and Chlamydophila pneumoniae, while others
are not: Chlamydophila caviae, Chlamydophila
pecorum, Chlamydia suis, Chlamydia muridarum
and Chlamydia trachomatis. Ruminants can be
infected by two species: Chlamydophila abortus
and Chlamydophila pecorum. Chlamydophila
abortus causes abortion in small ruminants.
This pathogen is also deemed to be responsible
for abortion in buffaloes. Abortion, which may
even become epidemic, occurs in the second
half of pregnancy. The Chlamydophila abortus
infection may cause abortion and hypofertility.
Chlamydophila pecorum has long been recognized
as the etiological agent of encephalomyelitis in
buffalo calves. Recent studies conducted by means
of molecular biology techniques on positive foetal
tissues from archives have enabled the species
involved to be typed as pecorum. It can therefore
now be claimed that Chlamydophila pecorum is
the main agent responsible for abortion in buffalo
cows, as well as for encephalomyelitis (Galiero,
2007). The microorganism is eliminated through
the semen of sick bulls that appear clinically
normal, even if, sometimes, their semen has a large
number of leukocytes and a low concentration of
sperm with poor motility and high percentage of
sperm cell abnormalities. To isolate or demonstrate
well with the conventional bacterial stains, and are
normally observed under a dark-ﬁeld microscope.
In the past, leptospires were subdivided on the
basis of serological reactions into two species: L.
interrogans and L. biflexa. In nature, leptospires
survive in ponds, puddles and wet earth. They
can be hosted by animals and humans, causing
diseases of the urinary and genital apparatus or
serious systemic diseases. In the animal reservoir,
the micro-organism is hosted in the renal tubules
or genital tract. The pathogenic role of Leptospira
hardjo has long been known in cattle, in which it
causes abortion, stillbirth and agalactia. Serological
studies and the sporadic isolations described in the
literature seem to suggest that various serotypes of
Leptospira spp. are present in many buffalo herds.
Many serologic researches demonstrate that the
buffalo population has antibodies against several
Leptospira spp. (Eaglesome and Garcia, 1992).
Since these microorganisms cause hypofertility
and abortion and survive in the frozen semen,
particular attention has to be paid to bulls involved
in the artiﬁcial insemination. The seminal vesicles
of the bull are considered to be a major site for
the localization of Leptospira interrogans serovar
hardjo. The L. pomona, L. canicola and L. hardjo
serotypes have also been found in several foetal
buffalo kidneys (Galiero, 2007). The isolation
of Leptospira spp. from the semen is not easy;
therefore, the micro-agglutination test has to be
used even if it does not allow distinguishing the
vaccinated animals from the infected ones. Because
of these problems bulls should not be vaccinated.
CHLAMYDOPHILOSIS
Chlamydiae are members of the family
Chlamydiaceae, a group of obligate intracellular
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Buffalo Bulletin (June 2013) Vol.32 No.2
Chlamydophila abortus from the semen, preputial
or urethral swabs, ELISA, PCR, embryonated eggs
or culture tissue are the techniques generally used.
MYCOPLASMOSIS
Various workers have reported the recovery
of mycoplasma from bovine semen. It is established
fact that mycoplasma is common in the bull and
buffalo bull semen and survives during semen
processing, freezing and storage. The survival
of mycoplasma in frozen semen at -196oC for 18
MYCOBACTERIOSIS
Mycobacterium bovis can be responsible
of orchitis in the buffalo male. Therefore, the donor
buffalos’ semen has to be tested before using it and
then once a year. The tests applied are the single
intradermal test and, if necessary, the comparative
intradermal test. Blood based assays which have
been developed for use in conjunction with the
tuberculin test include the gamma interferon test,
ELISA and PCR.
months has been reported. Mycoplasma cause
granular vulvo-vaginitis and damage the inner lining
of the oviduct in females while impairing fertility in
males. Mycoplasma especially the bovigenitalicum
and agalactiae species are frequently associated
with seminal vesiculitis in the male and metritis
and salpingitis in the female.
FUNGI (MOULD AND YEAST)
INFECTIONS
TRICHOMONIASIS
Trichomoniasis is caused by Trichomonas
foetus, a pyriform protozoan that causes premature
abortions, pyometra and infertility. This ﬂagellate is
transmitted to the female buffalo during her mating
with an infected bull and vice versa. Trichomonas
foetus infection of the genital tract of buffaloes was
recorded only in India and Egypt (Eaglesome and
Garcia, 1992). This suggests that the buffalo is an
unusual host for this parasite and is not generally
susceptible to infection. The infected animals may
be carriers for their whole life. The parasite persists
in diluted semen and is resistant to freezing;
therefore, donors have to be tested frequently either
through a microscopic examination or through a
cultural test in order to exclude any infection.
Several genera of fungi have been
cultured from raw and extended semen and
preputial washings. These fungi may contribute to
reproductive failure under certain conditions. Their
source may be semen or anatomical loci within the
male or female reproductive tract or contaminated
semen collection equipments. Candida trapicalis,
Candida stellatoidea, C. albicans, Torulopsis
femata and Aspergillus fumigatus have been
isolated primarily by culture isolation and
Aspergillus sp., Fuzerium sp., Penicillum sp.,
Mucor sp., by staining examination from buffalo
semen and found to be associated with reduced
fertility (Kodagali, 1979). Management practices
leading to lowered resistance, feed fortiﬁed with
antibiotics, and nutritional disorders are supposed
to make animals liable for mycotic infection.
78
Buffalo Bulletin (June 2013) Vol.32 No.2
Escherichia coli INFECTIONS
OTHER MISCELLANEOUS MICRO
ORGANISMS
E. coli is a gram-negative, non-endospore
producing, facultative aerobic bacillus. Its antigenic
structure comprises the lipopolysaccharides of the
cell wall (O antigen), the polysaccharides of the
capsule (K antigen) and the ﬂagellar and ﬁbrillar
proteins (H and F antigens, respectively). Although
about 50,000 serotypes have been identiﬁed,
only a limited number of strains are able to cause
disease. The pathogenic action is linked to the
ability of the clone to produce so-called virulence
factors, which may be either structural (ﬂagellae,
capsule, lipopolysaccharides, adhesins or secreted
(cytotoxic and cytotonic toxins, haemolysins).
A wide variety of different serotypes of E. coli
can be found in buffalo herds. Many of these are
pathogenic in newborns, such as enterotoxaemic
and enterohaemorrhagic E. coli, which produce
heat-stable toxins, verocytotoxins and necrotising
cytotoxic factor. The buffalo is an important
reservoir of verocytotoxic E. coli serotypes,
especially O157. E. coli may also cause abortion,
albeit sporadically. To date, it is not certain
whether abortion is caused by the bacterium and
its structural antigens or by the cytolytic action of
its toxins. The diagnosis is based on the serological
examination of the foetal organs. E. coli develops in
MacConkey agar medium, fermenting lactose and
producing reddish-pink colonies. Any haemolytic
activity can be evaluated by means of blood agar.
PCR is a useful tool in detecting, from isolated
strains, the gene sequences responsible for coding
virulence factors or toxins (Galiero, 2007).
The other micro organisms that are
involved in infectious infertility in buffaloes are
Usteria monocytogenes, Pseudomonas aeruginosa,
Bacillus licheniformis, Streptococcus spp.,
Staphilococcus spp., Proteus spp., Aeromonas spp.,
yeast and moulds. Fungal infections may develop
during the processing of semen for preservation.
Fungal infections of the prostate and other
accessory sex glands may result in the presence of
fungal infections in semen, which reduces quality
of semen.
The principal part of contamination of
semen consists of saprophytic or opportunistic
organisms from the prepuce and upper parts of the
genital organs. Since the greatest amount of the
bacteria present in semen comes from the prepuce,
from the skin and from the equipment used, semen
collecting procedures have to be carried out
using sterilized tools and materials and the bull’s
preputial cavities must be cleaned constantly. In
spite of these preventive measures, sometimes the
ejaculate presents a non-speciﬁc microbial ﬂora
(5 x 106 CFU/ml). These concentrations may be
reduced by administering antibiotics and diluting
the semen, whihc lower the number of viruses
and bacteria about 100 times, and freezing until
the following count is obtained: 8 x 102 CFU/
ml (considered normal) (Visintin et al., 1997). A
permissible count of 500 bacteria per dose of semen
straw is the international standard. Accordingly, it
is recommended that preputial washings (maximum
50,000 count) and neat semen (maximum 1000 to
5000 count) should carry lowest minimum bacterial
load with no count for AI equipment and dilutors
as they are to be used only after strict sterilization
(Visintin et al., 1997). Liquid nitrogen used for
79
Buffalo Bulletin (June 2013) Vol.32 No.2
storage of frozen semen doses may act as vehicle for
contaminant pathogens with variety of organisms
and at various degrees. The same thus serves as
source of infection to cows and buffalos during AI.
Even fresh liquid nitrogen carries Staphylococcus
aureus. Certain bacterial contaminants acquire a
level of resistance to antibiotics and they are able
to survive at -190oC in liquid nitrogen (Ranold and
free semen service for the whole nation need to be
addressed.
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FUTURE PROSPECTS
The future prospects to improve the
reproductive traits and eliminate the reproductive
disorders in buffalo bulls are mentioned below.
There is a need to study to a greater extent
the sexual development, attainment of puberty,
sexual maturity, sex libido, semen production,
reproductive performance of buffalo bulls.
Study of molecular marker (DNA/gene) assisted
selection and cytogenetic studies linked to genes
of interest with major effects on reproduction need
to be strengthened for selection of breedable males
in bubaline species at an early age. A cytogenic
marker would help to cull/eliminate a particular
bull from the herd to prevent the spread of a genetic/
congenital defective gene from that male to future
offspring and to prevent reproductive disorders.
Breeding soundness evaluation technique
would help to improve the breeding efﬁciency of
buffalo bulls.
Realistic remedial measures for reducing
infertility and enhancing fertility need to be
emphasized for the effective control of various
reproductive disorders.
Biosafety measures for production diseasefree germplasm and registration of all A.I. bulls by
a national society to initiate a certiﬁed disease-
London, England.
Balakrishnan, G., K. Saravanabava and N.
Dorirajan. 2006. Microbial quality and
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Binda, D.S., G.R. Pangaonkar and J.S. Matharoo.
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Bisson, J.P. and F. Czyglick. 1974. Retentissement
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Chantalakhana, C. and L. Falvey. 1999.
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Amaral, P.S. Baruselli, T. Fujji and L.J.
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Buffalo Bulletin (June 2013) Vol.32 No.2
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Galiero, G. 2007. Causes of infectious abortion in
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Gangadhar, K.S., A. Ramamohana Rao, S.
Krishnaswami and S. Umamaheswara Rao.
1986. Bacterial and fungal types and their
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Gibbs, E.P.J. and M.M. Rweyemamu. 1977. Bovine
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Huwe, P., T. Diemer, M. Ludwig, J. Liu, H.G.
Schiefer and W. Weidner. 1998. Inﬂuence of
different uropathogenic microorganisms on
human sperm motility parameters in an in
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Jaisal, S., R.C. Katoch, D. Chachara and A.
Mahajan. 2000. Evaluation of bacterial load
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semen. Indian Vet. J., 56: 807-809.
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De Carlos, E., G.N. Re, R. Letteriello, V. Del
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M. Fabbi, C. Bazzocchi, C. Bandi and G.
Galiero. 2004. Molecular characterisation
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isolated from buffaloes (Bubalus bubalis)
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Diemer, T., W. Weidner, H.W. Michelmann, H.G.
Sciefer, E. Rovan and F. Mayer. 1996.
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Eaglesome, M. and M. Garcia. 1992. Microbial
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*Continued on page 96
82
Case Report
Buffalo Bulletin (June 2013) Vol.32 No.2
DYSTOCIA DUE TO FETAL ANASARCA COUPLED WITH AMELIA OF ONE FOETUS IN
TWIN PREGNANCY IN A BUFFALO
V.S. Dabas1, V.K. Sharma2 and C.F. Chaudhari3
ABSTRACT
CASE HISTORY AND OBSERVATIONS
This communication reports a case of
dystocia due to foetal anasarca coupled with amelia
of one foetus in a twin pregnancy and its successful
per-vaginal management in a Mehsana buffalo.
A five-year-old full term pregnant Mehsana
buffalo was presented with the history of dystocia.
Further, the water bags had ruptured three hours
before and there was no progression in foetal
delivery. The animal was straining repeatedly with
expulsive efforts and exhibiting all the external
signs of approaching parturition. Per-vaginally, the
cervix was completely relaxed and jam-packed with
a large, soft and smooth oedematous mass leading
difficulty in advancing the palpation. Accordingly,
the case was diagnosed to be the dystocia of foetal
origin and it was decided to manage per-vaginum.
Keywords: dystocia, fetal anasarca, amelia, twin
pregnancy, Mehsana buffalo
INTRODUCTION
Dropsical conditions viz. fetal ascitis, fetal
anasarca, edema of the allantochorion, hydrops of
the amnion or allantois or both etc. are reported
to be causes of dystocia (Roberts, 1971). Twin
pregnancy with foetal anasarca of one foetus
has also been observed as an occasional cause
of dystocia in bovine. Therefore, a rare case of
dystocia due to foetal anasarca coupled with amelia
of one foetus in twin pregnancy (Figure1) and its
successful per-vaginal management in a Mehsana
buffalo is placed on record.
TREATMENT AND DISCUSSION
Following cleaning of the perineum
and achieving caudal epidural analgesia using
injection lignocain HCL 10 ml between the last
sacral and first coccygeal vertebra; the lubricated
hand was introduced into the uterus through
vagina by pushing back the jam-packed foetal
parts. Eventually, it became possible to palpate
the atrophied jaws of the foetus, while the limbs
1
College of Veterinary Science and Animal Husbandry, Navsari Agricultural University (NAU), Navsari,
Gujarat - 396 450, India, E-mail: vsdabas@yahoo.co.in
2
Department of Veterinary Gynaecology and Obstetrics, College of Veterinary Science and Animal Husbandry,
Sardarkrushinagar Dantiwada Agricultural University (SDAU), Deesa, Gujarat, India
3
Department of Veterinary Gynaecology and Obstetrics, College of Veterinary Science and Animal Husbandry,
Navsari Agricultural University (NAU), Navsari, Gujarat, India
83
Buffalo Bulletin (June 2013) Vol.32 No.2
could not be traced. Subsequently, two obstetrical
hooks were separately placed between the
mandibles and in the oedematous mass. While
applying progressive gentle traction to each of
the hooks, one hand was kept inside the genital
tract for giving requisite force and the directions
to the foetal parts. Ultimately, a dead oedematous
foetal monster was delivered. While the genital
tract was examined to rule out the possibilities of
were given for three consecutive days. Soon, the
animal recovered uneventfully. The majority of
monstrosities are described as single with deviated
morphology of typical monster (Arthur et al., 2001).
However, two defects viz. anasarca and amelia all
together were observed in the present case.
REFERENCES
injury and removal of the afterbirths, one more
normal sized dead foetus was palpated inside the
uterus and could be easily removed. Subsequently,
both the easily detachable placentas were removed
manually and four Oriprim-U boluses were put in
each uterine horn. Injection calcium-borogluconate
450 ml intravenously was given once and injections
oxytetracycline 40 ml, chlorphenermine meleate
10 ml and ketoprofen 15 ml, intramuscularly
Arthur, G.H., D.E. Noakes, T.J. Parkinson
and G.C.W. England. 2001. Veterinary
Reproduction and Obstetrics, 8th ed. WB
Saunders Company Ltd., London, England.
Roberts, S.J. 1971. Veterinary Obstetrics and
Genital Diseases, 2nd ed. CBS Publishers,
New Delhi, India.
Figure 1. Fetal anasarca coupled with amelia of one foetus in twin pregnancy.
84
Short Communication
Buffalo Bulletin (June 2013) Vol.32 No.2
PREVALENCE OF DIFFERENT SPONTANEOUS LIVER LESIONS IN BUFFALOES
Y. Verma1, U.K. Vyas2 and Suneeta Rani2
ABSTRACT
immediate needs of the rural masses in several
communities. Of all domestic animals, the Asian
buffalo holds the greatest promise and potential for
production (Cockrill, 1994). India has world’s best
buffalo dairy breeds and provides superior buffalo
germplasm to several countries of the world
(Kaikini, 1992). The liver is one of the vital organs
of the body, susceptible to various affections which
influence the total health status of the animal.
Further, considerable liver damage may be present
before clinical signs are apparent largely due to
the high degree of reserve functional capacity. The
present investigation was carried out on the livers
of buffaloes to investigate the various spontaneous
lesions of the liver.
The present investigation was carried
out to study the various pathological conditions
occurring spontaneously in the livers of buffaloes.
A total number of 2,119 buffalo liver samples was
collected irrespective of sex, breed and age of
the animal from slaughterhouses located in and
around Bikaner, Rajasthan. Among the various
pathological conditions of the livers, fatty change
was found to be the highest followed by cirrhosis,
congestion, cell swelling, abscess, RES response,
haemorrahge, pigmentation, necrosis, apoptosis
and telangiectasis.
Keywords: buffalo, fatty changes, liver, prevalence,
spontaneous lesions
MATERIALS AND METHODS
INTRODUCTION
The materials for the present study
comprised livers obtained from buffaloes
slaughtered at slaughterhouses located in and
around Bikaner, Rajasthan. A total of 2,119 livers of
slaughtered buffaloes were examined irrespective
of age, breed and sex for spontaneous liver lesions.
Gross study was performed during collection
of samples from the slaughterhouses primarily
and then during trimming of the samples for
histopathology. Out of these, 476 livers exhibiting
The buffalo is one of the key animals in the
agricultural economy, contributing substantially
to the gross national income (GNP) by way of
good quality milk, meat, export quality leather
and physical power. The buffalo is a part of the
property, possession and profession of rural
farmers. Not only that, it is an easily ‘convertible
currency’ and a reliable ‘living bank’ to serve the
1
Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, Jabalpur,
Madhya Pradesh, India, E-mail: dryaminiverma@rediffmail.com
2
Department of Veterinary Pathology, College of Veterinary Science, Bikaner, Rajasthan, India
85
Buffalo Bulletin (June 2013) Vol.32 No.2
gross alterations were cleaned with normal neutral
saline and the changes were recorded. The lesion
containing tissue samples were collected and
preserved in 10% neutral formalin solution for
histopathological study. After 48-72 h, these tissue
pieces were washed overnight in running tap water,
dehydrated in ascending grades of alcohol, cleared
in benzene and embedded in paraffin wax (60-62oC
highest (6.09%), akin to the observation of Gupta
(1983). However, Kulkarni (1992) and Dhote et
al. (1992) reported much lower incidences, being
2.45% and 1.5%, respectively. While Purushotam
and Rajan (1985) noticed the condition in a much
higher number of cases (21.7%). Grossly these
livers appeared pale, soft, and greasy with rounded
edges. Histopathologically, the hepatic cells showed
presence of fat droplets as clear round spaces and
granular cytoplasm. Large clear cavities were
seen indicating that the scattered cells with fatty
changes coalescence (Figure 1). Sinusoides were
often reduced by swollen fatty cell cords (Figure
2). Focal, diffuse, pericentral and periportal types
of fatty changes noticed presently were comparable
to those described by Jubb et al. (1993) and Dhote
et al. (1992). Ketosis may be one of the possible
causes of fatty changes.
A total of 5.25% cases showed partial or
complete cirrhosis. The liver was constricted in
many cases. Thickened capsule, finally granular
surface, hard to cut and numerous newly formed bile
ducts in cirrhotic liver were observed in the present
melting point). Sections of 4-5 micron thickness
were cut and stained with haematoxylin and eosin
as per the standard procedure recommended by
Lille (1954).
RESULTS AND DISCUSSION
The various spontaneous liver lesions
observed during the present study were as shown in
Table 1. Examination of the livers of 476 buffaloes
revealed various types of lesions in 180 animals,
amounting to 37.82%.
Among the various pathological lesions
of the livers, fatty changes were found to be the
Table 1. Spontaneous liver lesions in slaughtered buffaloes (n = 476).
S.
No.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Spontaneous lesions
Fatty change
Cirrhosis
Congestion
Cell swelling
Abscess
RES response
Haemorrhage
Pigmentation
Necrosis
Apoptosis
Telangiectasis
86
No. of
Cases
(180)
Percentage of
affected animals
(37.82)
29
25
24
19
18
18
14
13
9
7
4
6.09%
5.25%
5.04 %
4.00 %
3.78%
3.78%
2.94%
2.73%
1.90%
1.47%
0.84%
Buffalo Bulletin (June 2013) Vol.32 No.2
study as described by previous researchers (Gupta,
1983; Moorty et al., 1984; Dhote et al., 1992; Vegad
and Katiyar, 1998). Microscopically considerable
proliferation of fibrous connective tissue was
marked mainly in the portal areas replacing hepatic
cells.. There was little lymphocytic infiltration of
fibrous strands. Portal veins and sinusoids were
dilated and irregular in shape. Cirrhosis was
frequently noticed concomitant with parasitic
condition; however; in many cases contributory
factors could not be incriminated.
Congested liver 24 (5.04%) showed
the character of “nutmeg” liver and mottled
appearance. Microscopically, sinusoids were
dilated and central veins were engorged with
erythrocytes. Slight periductal infiltration of round
cells was noticed. Hepatic cells around the central
veins were degenerated and atrophied Moderate
proliferation of fibrous tissue was also seen in
portal spaces, corroborating the descriptions of
Cohrs (1967) and Gupta (1983).
Cell swelling were found in 19 (4.00%)
of the cases. Grossly, the affected livers were
enlarged and of lighter color with rounded edges.
Microscopically, the enlarged rounded hepatocytes
containing eosinophilic granular cytoplasm which
obliterated sinusoidal space.
Abscess was found in only 2.94% cases.
Grossly, whitish foci on the surface of the liver
were found (Figure 3). Their size varied from 0.5 to
1.5 cm in diameter. Polymorphonuclear leukocytes
at the center surrounded by a thin fibrous capsule
were seen microscopically (Figure 4). Similar
histopathological changes have been reported
in earlier studies (Gupta, 1983 and Dhote et al.,
1992).
RES response was observed in 3.78%
cases. Grossly and microscopically the livers with
RES response showed variable extent of diffusely
distributed small foci in the form of Kuffer’s
cell hypertrophy and hyperplasia, degeneration,
necrosis and granulomas being comparable to the
descriptions of Chors (1967). These apparently
were consequent to the body defense against
underlying subtle infections.
In the 14 cases of haemorrhage, the liver
showed a dark brown petechial and/or ecchymotic
nature and superficial or deeper location
microscopically variable extents of degeneration,
necrosis and neutrophilic and lymphocytic
infiltrations in the involved area in conformity with
Dhote et al. (1992).
Pigmentation was seen in 13 (2.73%)
cases. The darker colored liver showing yellowish
brown granules of haemosiderin in the Kuffer’s
cells macrophages and free in the sinusoids in H&E
stain (Figure 5) being blue in Perls´ (Figure 6) were
similar to the reports of Gupta (1983) and Tripathi
and Chattopadhyay (1989).
In hepatic necrosis (1.90%), pale patches
were found on the surface of liver presenting a
mosaic appearance. White, opaque necrotic foci
were uniformly distributed throughout the liver.
Microscopically, the hepatic cells were very much
swollen and granular with pyknotic or lytic nuclei,
infilteration with neutrophils and lymphocytes,
engorged veins, fibroplasias and Kuffer’s cell
proliferation were observed. This presumably may
be due to involvement of some toxins in feed (Jubb
et al., 1993) possibility fungal, bacterial or plant
toxins or chemicals playing some role in causing
the condition.
Apoptosis was seen in seven (1.47%)
cases. Occurrence of apoptic cells and apoptic
bodies in the hepatocytes were similar to Kumar
et al. (1992) and Bhel and Tripathi (1999). These
apparently pointed to be the result of the mechanism
regulating the number of cells since these were
87
Buffalo Bulletin (June 2013) Vol.32 No.2
Figure 1. Photomicrograph of liver section showed presence of fat droplets as clear round space and granular
cytoplasm of hepatic cells. H&E X 200.
Figure 2. Photomicrograph of liver section showing fatty changes with coalescence forming large sized
cavities, sinusoidal congestion and edema is also seen. H&E X 400.
Figure 3. Photomicrograph of centrilobular hemosiderosis showing golden brown pigment in the Kuffer cells
and in the sinusoids. H&E X 200.
88
Buffalo Bulletin (June 2013) Vol.32 No.2
Figure 4. Photomicrograph showing blue haemosiderin pigment in the Kuffer cells. Pearls’ prussian blue
staining X 200.
Figure 5. Surface and cut surfaces of liver pieces showing abscesses (a,b) and congestion (d).
Figure 6. Photomicrograph showing within outside area of calcification, detritus and pyogenenic membrane.
H&E X 200.
89
Buffalo Bulletin (June 2013) Vol.32 No.2
neither frequent and nor abundant.
Telangiectasis was observed in 0.84%
cases. This was comparable to Gupta (1983) and
Hassib et al. (1995), who reported 1% and 0.58%.
respectively. The gross and microscopic character
of liver showed as dark reddish depressed single
or multiple areas of variable size and dilatation of
group of sinusoids and /or larger cavernous spaces
lined by endothelium and containing erythrocytes
with or without connections were in line with the
descriptions of Runnels (1976).
16: 42-44.
Gupta, P.P. 1983. Studies on pathology of buffaloes
livers. Indian J. Vet. Pathol., 7: 91.
Hassieb, M.M., E.M. El-Manakhly and O.R.
Khater. 1995. Incidence and pathology of
some hepatic lesions in buffalo. Egyptian J.
Comp. Path. Clinical Path., 8: 1-8.
Jubb, K.V. F., P.C. Kennedy and N. Plamer. 1993.
Pathology of Domestic Animals, Volume
2, 4th ed. Academic Press, Inc. San Diego,
USA.
Kaikini, A.S. 1992. Dimensions of infertility or
sterility in cattle and buffaloes. Indian J.
Anim. Reprod., 13: 10.
Kulkarni, V.G. 1982. Study on pathology of liver
in cattle and Indian buffaloes. Indian J. Vet.
Path., 7: 89-90.
Kumar, T.V., C.E. Sarraf and M.R. Alison. 1992.
The biology and pathology of programmed
cell death (Apoptosis), Vet. Hum. Toxicol.,
34(3): 251-254.
Lillie, R.D. 1965. Histopathologic Technique and
Practical Histochemistry. McGrew Hill
Book Co, New York and London.
Purushothaman, A.V. and A. Rajan. 1985. Incidence
and pathology of hepatic disorders in cattle.
Kerala J. Vet. Sci., 16(1): 85-94.
Runnels, R.A., W.S. Monlux and A.W. Monlux.
1976. Principles of Veterinary Pathology,
th
7 ed. Scientific Book Agency, Calcutta,
India.
Tripathi, B.N. and S.K. Chattopadhyay. 1989.
Study on spontaneous hepatic lesions in
Indian buffaloes. Indian J. Vet. Pathol., 13:
91-93.
Vegad, J.L. and A.K. Katiyar. 1998. A Text Book
of Veterinary Systemic Pathology. Vikas
Publishing House Pvt. Ltd. New Delhi,
India.
ACKNOWLEDGEMENT
The authors would like to acknowledge the
Indian Council of Agricultural Research, Delhi,
India for providing Junior Research Fellowships
during the course of study.
REFERENCES
Behl, D. and K.K. Tripathi. 1999. Apoptosis
programmed cell death. Post- Graduate
Medicine, 13: 13-25.
Cockrill, W.R. 1994. Present and future of buffalo
production in the world. In Proceedings of
the 5th World Buffalo Congress, Caserta,
Italy.
Cohrs, P. 1967. Special Pathological Anatomy of
Domestic Animals. Pergamon Press, Oxford,
England.
Culling, C.F.A. 1974. Histopathological and
Histochemical Techniques. Butterworths,
London, Butterworth.
Dhote, S.W., A.D. Ingle, A.G. Bhandarkar, M.V.
Joshi and S.S. Bhagwat. 1992. Hepatic
lesions in buffaloes. Indian J. Vet. Pathol.,
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Buffalo Bulletin (June 2013) Vol.32 No.2
Original Article
EVALUATION OF CYSTOURETHROPLASTY VIS-A-VIS CAECOURETHROPLASTY
TECHNIQUE IN BUFFALOES (Bubalus bubalis)
Md. Moin Ansari1 and S.P. Sharma2
free facial grafts, polyvinyl silicone patch,
peritoneum, omentum, gelatine sponge, OMS
membrane, Teﬂon felt, free autologous preputial
tissue, autogenous skin grafts, pedicle, autogenic,
allogenic, xenogenic, synthetic and processed
grafts, seromuscular graft, lyophilized human dura
and formalin preserved buffalo duramater, urinary
bladder grafts and intact caecum have been used for
surgical repair of bladder and urethral defects both
experimentally and clinically in man and animals
ABSTRACT
The present experimental urothroplasty
was undertaken in sixteen male buffalo calf models
apparently in good health status using urinary and
caecal grafts. The surgical technique was evaluated
on the basis of clinical observation, determination
of blood urea nitrogen, histopathological and postoperative complication for a period of 60 days
post-operatively. Urethroplasty using formalin
preserved urinary bladder and caecal grafts acted
as a scaffold around which there was gradual
regeneration of urethral tissue and resolution
of grafted material by 60th post-operative day.
However, both these techniques were worthy and
feasible in buffalo calves, yet caecourethroplasty
was preferred, because it was easier and safer than
the cystourethroplasty.
(Kelami, 1971; Kudale and Hattangady, 1971;
Prasad et al., 1973, Sharma and Khan, 1978, 1980;
Prasad et al., 1980; Sharma and Agrawal, 1997 and
Sengupta et al., 1998).
It is obvious from the available literature
that the clinical and experimental replacement of
the urethra and ureter is not a new concept for
human beings and canine but it has not been studied
further in large animals. The paucity of literature
on large animals as well as the high incidence of
urethral calculi leading to rupture of the urethra
have focused attention upon urethral substitution,
which can be achieved with a suitable substitute
if needed in many of the conditions: to repair the
wall of the urethra when it is excessively damaged
or badly replaced due to urethral calculi, violence
or non-speciﬁc reasons; concurrent papilloma and /
or carcinoma of the urethra; any anatomical defects
Keywords:
buffalo,
cystourethroplasty,
caecourethroplasy, urinary bladder, caecal graft
INTRODUCTION
Urethroplasty is an open surgical procedure
for the repair of an injury or a defect in the walls of
the urethra. Different grafts such as gut segments,
1
Division of Surgery and Radiology, Faculty of Veterinary Sciences and Animal Husbandry, Sher-E-Kashmir
University of Agricultural Sciences and Technology of Kashmir (SKUAST-K), Shuhama, Alesteng Srinagar
- 190 006, India
2
Department of Surgery and Radiology, Bihar Veterinary College, Patna - 800 014, Bihar, India
91
Buffalo Bulletin (June 2013) Vol.32 No.2
formalin solution for 15 to 30 days. The formalin
preserved urinary bladder/caecal grafts were kept
in running tap water for 24 h prior to grafting to
remove formalin from them. Then the graft was
kept in Povidine Iodine*** (Win-Medicare Ltd.
N. Delhi.) solution for 2 h prior to grafting. The
margin of the graft material was trimmed; mucosa
and sub-mucosa were scrapped to prepare a graft of
3.5 X 1.5 cm. It was stored in normal saline before
urethroplasty.
Xylazine* (Astra IDL Ltd.Bangalore)
0.05 to 0.2 mg/kg body weight was given
intramuscularly. Ten milliliters of 2% xylocaine**
(Astra IDL Ltd. Bangalore) was inﬁltrated on the
line of incision. Post-scrotal urethroplasty was done
in routine manner giving about 10 cm incision. The
ventral wall of the urethra was excised for about
3.5 cm length and about 1.5 cm breadth. Bleeding
vessels were ligated. A polythene tube about 2.5
mm diameter was passed inside the urethra towards
the ischial arch into the urethra and the lower end
of the polythene tube was passed through the
urethra in anterior direction, to take out through
the pre-preputial opening. The formalin-preserved
seromuscular urinary bladder /caecal graft was
placed over the excised urethra in such a position
that the serous layer faced outside. Then four
stay sutures were applied at the corners. Simple
interrupted sutures were applied using chromic
catgut no. 2/0 and Ethicon black braided silk no.
3/0 in both the groups. The protruding polythene
tube was anchored with the silk thread at the
preputial area. The cutaneous incision was closed
giving Halsted suture using black braided silk no.
2.The cutaneous stitches were removed on the 8th
post-operative day while the polythene catheter
was allowed to remain in the urethra for 2 weeks.
The operated buffalo calves were sacriﬁced on day
15, 30, 45 and 60 and materials were collected for
of the urethral wall, like irreparable urethral ﬁstula;
urethral stenosis, stricture or ill development,
congenital anomalies like hypospadia, episadia,
urethral diverticulum, urethrocele. Success attained
with vesiculoplasty in humans, canine and bovine
initiated this study on urethroplasty using urinary
bladder and caecal grafts. Based on a research and
experienced gained in urological surgery of buffalo
calves either experimentally or clinically, the study
was designed (Sharma and Khan, 1978; Sharma,
1995; Sharma and Agrawal, 1997, Ansari, 2002).
The damaged, diseased or congenital
urethral defects have been corrected and substituted
by preputial tissue. Various workers like Vyas et
al., 1987; Wessells and Mc Anich, 1996; Kocvara
and Dvoracek, 1997 and Sengupta et al., 1998
have replaced urethra partially or completely
with preputial graft. Uroepithelium is known for
its regenerating property (Bohne et al., 1955).
Therefore, the primary objective of urethroplasty
is to provide an environment for regeneration of
normal urethral tissue for urinary passage.
MATERIALS AND METHODS
Cystourethroplasty and caecourethroplasy
were conducted in 16 healthy male buffalo calf
models weighing about 80-115 kg. The animals
were divided into two groups consisting of eight
animals each. Formalin-preserved urinary bladder
and caecal allografts were used as urethral
prosthesis in Groups 1 and 2, respectively. Pre- and
post-operative temperatures, pulse, respiration and
blood urea nitrogen (Levine, et al., 1961) using a
photo-calorimeter were recorded in all the buffalo
calves.
Urinary bladder and caecum obtained
from buffalo calf cadavers were preserved in 10%
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Buffalo Bulletin (June 2013) Vol.32 No.2
lining on the 15th post-operative day (Figure
1) but it was evident on the 30th post-operative
day (Figure 2) and the epithelium of the urethra
was creeping towards the graft side. On the 45th
post-operative day (Figure 3), lymphocytic cells
were moderate in number and there was gradual
regeneration of uroepithelium and underlined
connective tissues at the operated area. On the 60th
post-operative day (Figure 4), all the inﬂammatory
changes were minimal and there was complete
regeneration of the uroepithelium over grafted
tissue from the host side. This observation was
in agreement with the results of Sharma (1995),
who tried formalin preserved urinary bladder and
terylene lined hemispherical hollow plastic balls
as bladderprosthesis in experimental buffalo.
In caecourethroplasty, all the operated
animals survived after the reconstruction. An
initial rise of temperature, pulse and respiration
for the ﬁrst few days were noticed in almost all
the animals, but these all became normal in due
course. All the operated animals were taking feed
normally after second post-operative day. Gross
observation in few cases showed echymotic
haemorrhage in the urethral mucosa and peelable
necrotic foci on the caecal graft even up to the
45th post-operative day. This type of haemorrhage
gross and histopathological studies (H&E stain).
RESULTS AND DISCUSSION
In cystourethroplasty, seven out of
the eight buffalo calves survived subjected to
urethroplasty. An initial rise of temperature,
pulse and respiration was marked in almost all
the animals after surgery, which might be due to
tissue reaction. The same was observed by earlier
studies in goats using caecal graft (Mukherjee,
1988) and PTFE, caecal and bladder grafts
Shivaprakash (1990) and in dogs using peritoneal
graft (Nair et al., 1988) following urinary bladder
reconstruction.
Estimation of blood urea nitrogen on
days 1, 2, 3, 7, 14, 21 and 28 after reconstruction
showed slight elevation from the pre-operative
level. During the post-operative period clinical
observation and the pattern of blood urea nitrogen
level proved that there was no obstruction to the
normal passage of urine. These ﬁndings on the
blood urea nitrogen levels in operated buffalo
calves were in agreement with the observation
made in human beings, canine and bovine
(Shoemaker and Marucci, 1955; Prasad et al.,
1973; Prasad et al., 1979; Sharma and Khan,
1978, 1980; Nair et al., 1988; Sharma, 1995)
after partial reconstruction of the urinary tract
experimentally and clinically. Gross observations,
which were made during the study, could not
detect untoward pathological changes in the wall
of grafted urethral tissue except in one buffalo
calf which died showing tympany. Histological
examination of the tissue taken from the operated
area of the urethra revealed gradual regeneration
of the urethral tissues and resolution of the grafted
material. There was lack of distinct epithelium
could be harmless as none of the animals showed
any complications up to the 60th post-operative
day. Necrotic foci which were present on caecal
graft up to the 45th day were also observed by
Prasad et al. (1973) and Sharma and Khan (1978,
1980) during intestinocystoplasty in canine and
bovine. Histological examination on the 15th postoperative day (Figure 5), showed epithelial lining
was not clearly discernible at the junctional zone.
At the end of the 30th post-operative day (Figure
6), epithelium of the urethra was creeping towards
the graft side and trying to overlap the grafted
93
Buffalo Bulletin (June 2013) Vol.32 No.2
Figure 1. Photomicrograph from the grafted urethral Figure 2. Photomicrograph from the grafted urethral
junction using urinary bladder on 15 POD (H junction using graft on 30 POD (H&E-150), showing
&E-150), showing not clearly discernible epithelium the epithelium creeping towards the graft side.
on of the urethral either side of the zone of junction
and marked inﬁltration of lymphocyte.
Figure 3. Photomicrograph from the grafted urethral Figure 4. Photomicrograph from the grafted urethral
junction using urinary bladder graft on 45 POD junction using graft on 60 POD (H&E-150), shows
(H&E-150), showing gradual regeneration of the completely uroepithelium and surrounding tissue.
regenerated uroepithelium and underlined connective
tissues.
Figure 5. Photomicrograph from the grafted urethral Figure 6. Photomicrograph from the grafted urethral
junction using caecal graft on 15 POD using caecal junction 30 POD (H&E-150), showing the gradual
graft on (H&E-150), showed epithelial lining was urethral tissues.
not clearly discernible at the regeneration of the
junctional zone.
Figure 7. Photomicrograph from the grafted urethral Figure 8. Photomicrograph from the grafted urethral
junction using caecal graft on 45 POD using caecal junction 60 POD (H&E-150) showed well developed
graft on (H&E-150), showed gradual regeneration of transitional uroepithelium.
transitiona uroepithelium.
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Buffalo Bulletin (June 2013) Vol.32 No.2
tissue at the junctional area. On the 45th postoperative day (Figure 7), gradual regeneration
of transitional epithelium was observed. The
lymphocytes and the blood vessels were fewer in
number. On the 60th post-operative day (Figure
8), the transitional epithelium of the urethra
had completely developed and lymphocytes
and blood vessels were scare in number. Thus,
the result of the study was agreement with the
work of Sharma and Khan (1980) who described
complete regeneration of uroepithelium over the
caecal graft in buffalo on the 75th post-operative
day during caecocystoplasty. Although both the
techniques were successful in buffalo calves,
caecourethroplasy was preferred because it was
easier and safer to perform.
Vet. J., 48: 1065-1069.
Levine, J.M., R. Leon and F. Steigmann. 1961. A
rapid method for the determination of urea
in blood and urine. Clin. Chem., 7: 488-493.
In Oser, B.L. 1965. Hawk’s Physiological
Chemistry, 14th ed. The Blankiston Division
Mc Graw Hill Book Co., London, England.
Mukherjee, C. 1988. Caecocytoplasty in goats.
M.V.Sc. Thesis, Indian Veterinary Research
Institute (IVRI), Izatnagar, India.
Nair, N.R., M.R. Patel and R.P. Awadhiya. 1988.
Cystoplasty with peritoneal grafts in dogs.
Indian J. Vet. Surg., 9: 82-85.
Prasad, B., R.V. Kumar and R.P.S. Tyagi. 1980.
Experimental evaluation of urinary diversion
technique in bovine calves. Indian Vet. J.,
57: 846-848.
Prasad, B., J. Singh, R.P.S. Tyagi and G.R. Singh.
1979. Subtotal cystoplasty using caecal
pedicle graft. Indian Med. Gazette., 17: 7377.
Prasad, B., R.P.S. Tyagi and R.V. Kumar. 1973.
Caecocystoplasty in buffalo calves. Indian
Vet. J., 50: 1046-1051.
Sengupta, S.K., D.K. De and D.B. Mukherjee. 1998.
Study of urethroplasty with free-autogenous
preputial tissue in repair of urethral ﬁstula
in dogs. Indian J. Anim. Health, 37: 25-28.
Sharma, S.P. 1995. Cystoplasty using formalin
urinary bladder grafts in buffalo calves.
Indian J. Vet. Surg., 16: 44-46.
Sharma, S.P. 1997. Urinary bladder reconstruction
using the intact caecum: experimental
studies. Indian Vet. J., 74: 51-53.
Sharma, S.P. and K.B.P. Agrawal. 1997. Cystoplasty
using the intact caecum in a male calf-clincal
case report. Indian Vet. J., 74: 95-96.
Sharma, S.P. and A.A. Khan. 1978. Studies on catgut
and silk as suture material for cystoplasty in
REFERENCES
Ansari, M.M. 2002. Studies on urethroplasty using
formalin preserved urinary bladder and
caecal grafts in buffalo calves (Bubalus
bubalis). M.V.Sc. Thesis, Rajendra
Agriculture University, Pusa, India.
Bohne, A.W., R.W. Osbrne and P.J. Hettle. 1955.
Regeneration of the urinary bladder in
the dog following total cystoplasty. Surg.
Gynecol. Obstet., 100: 159-264.
Kelami, A. 1971. Lyophilized human dura as a
bladder wall substitute: experimental and
clinical results. J. Urology, 105: 518-522.
Kocvara, R. and J. Dvoracek. 1997. Inlay-onley
ﬂap urethroplasy for hypospadias and
urethral stricture repair. J. Urology, 158:
2142-2145.
Kudale, M.L. and S.R. Hattangady. 1971. An
intestinal pedicle for correcting defects of
the wall of urinary bladder in dogs. Indian
95
Buffalo Bulletin (June 2013) Vol.32 No.2
buffalo calves. Haryana Agric. Uni. J. Res.,
8: 101-106.
Sharma, S.P. and A.A. Khan. 1980. Studies
on caecocystoplasty with “sheet Graft”
technique in buffalo calves. Indian J. Anim.
Health, 19: 35-36.
Shivaprakash, B.V. 1990. Experimental studies
on urinary bladder reconstruction using
PTFE, Caecal pedicle, fresh autogenous
Visintin, J.A., M.E.O.A. Assumpcao, P.S. Baruselli,
M.R.B. Mello and L.M.T. Tavares. 1997.
Use of biotechnologies in buffalo bred in
Brazil. In Proceedings of Third Course
on Biotechnologies of Reproduction in
Buffaloes, Caserta, Italy. Bubalus Bubalis,
4(Supp.): 189-204.
Wierzbowski, S., W. Nowakowski, E. Wayda and
S. Kuzniak. 1984. Antibiotic level and
and preserved allogenic bladder grafts in
goats. M.V.Sc. Thesis, Indian Veterinary
Research Institute (IVRI), Izatnagar, India.
Shoemaker, W.C. and H.D. Marucci. 1955. The
experimental use of seromuscular grafts in
bladder reconstruction: Preliminary report.
J. Urology, 73: 314-321.
Vyas, P.R., D.R. Roth and A.D. Permutter. 1987.
Experience with free grafts in urethral
reconstruction. J. Urology, 137: 471-474.
Wessells, H. and J.W. Mc. Aninch. 1996. Use of free
grafts in urethral stricture reconstruction. J.
Urology, 155: 1921-1923.
bacterial contamination of frozen bull’s
semen (streptomycin, penicillin). MedycynyWesterynaryjna (Poland), 40: 284-287.
Yadav, V., A. Sharma and R. Sharma. 2007.
Detection of FMD virus type speciﬁc IgG1,
IgG2 and IgA antibodies in milk and serum
of buffaloes vaccinated with oil adjuvant
polyvalent FMD vaccine. Ital. J. Anim. Sci.,
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*Continued from page 82
spermatozoa in relation to other semen
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Subasinghe, D.H.A., N.U. Horadogoda, H.
Abeygunawardena and J.A.D.S. Siriwardene.
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Van Oirschot, J.T., P.J. Straver, J.A.H. Van Lieshout,
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Exsel. 1993. A subclinical infection of bulls
with bovine herpesvirus type 1 at an artiﬁcial
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96
Buffalo Bulletin (June 2013) Vol.32 No.2
Original Article
A STUDY ON BUFFALO MANAGEMENT PRACTICES IN KHAMMAM DISTRICT OF
ANDHRA PRADESH
K. Kishore1, M. Mahender2 and Ch. Harikrishna3
buffaloes were predominant in this region and the
majority of the farmers were poor in certain aspects
of scientiﬁc feeding, breeding, housing, milking
and health care practices and needed to be educated.
Artiﬁcial insemination should be made available to
all the farmers, which in turn would result in the
ABSTRACT
A study was carried out to ﬁnd out various
buffalo management practices adopted by the
farmers in Khammam district of Andhra Pradesh.
More than half of the farmers under survey (58%)
were able to provide insemination or natural
service to their animals in heat at the right time,
while 42% of farmers were unable to inseminate
their animals in time due to various reasons. Only
16.66% farmers utilized the facility of artiﬁcial
insemination, while 37.51% farmers opted for
natural service. About 85.85% farmers fed green
and dry roughage in combination and provided
clean drinking water to their animals, but none of
the farmer practiced silage making or other special
treatments like chafﬁng, soaking or urea treating
paddy straw. Most of the farmers collected fodder
from the ﬁelds. Only 3% farmers fed additional
concentrates to pregnant animals; the rest did not
do this. As a part of summer management, 51%
farmers allowed their buffaloes to wallow in the
village tanks during the hotter parts of the day and
49.16% farmers washed their animals by splashing
water manually. Washing of the animals and of
their udders before milking were practiced by 15.68
and 98.40% farmers, respectively. Non-descript
upgrading of local buffaloes and improving their
performance.
Keywords: buffaloes, breeding, feeding, housing,
management, milking
INTRODUCTION
Buffaloes are the backbone of rural
economy in many developing countries of the
Asian region including India. Buffaloes occupy
a prominent place in the social, economic and
cultural life of Indian rural communities and are
useful as a triple purpose animal for milk, meat
and draft power. Dairying with buffaloes in India
is a closely interwoven integral part of agriculture.
India possess 283 million dairy bovines and stands
ﬁrst in milk production with more than 100 million
metric tonnes but the productivity of dairy animals
is very much less than in the developed countries.
1
Veterinary Dispensary, Khammam District, Andhra Pradesh, India
2
Department of Livestock Production and Management, College of Veterinary Science, Rajendra Nagar,
Hyderabad, Andhra Pradesh, India, E-mail: mallammahender@gmail.com
3
Livestock Research Institute (LRI), College of Veterinary Science, Rajendra Nagar, Hyderabad, Andhra
Pradesh, India
97
Buffalo Bulletin (June 2013) Vol.32 No.2
The performance and productivity of buffaloes in
the region appears to be at low level and the factors
responsible need greater attention. Productivity of
an animal is primarily the product of interaction of
its genetic makeup and the environment in which
it develops. Therefore, a study was undertaken
to assess various management practices such as
breeding, feeding, housing, milking practice etc.,
in Khammam district of Andra Pradesh.
RESULTS AND DISCUSSION
Results of buffalo management practices
adopted by the farmers in the villages under study
are appended in Tables 1, 2, 3, 4 and 5.
General observations
Occupation: It was observed (Table 1) that
agriculture was the main occupation among 84.5%
of the farmers and dairying was the secondary
occupation of 15.5% of farmers, whereas dairying
was the main occupation of 15.5% and agriculture
was the secondary occupation of 84.5%. Prasad
et al. (2001) reported that dairying was the main
occupation among 64% of the milk producers and
a subsidiary occupation for 36% in urban areas of
Andhra Pradesh. Similarly 75% farmers had dairying
as secondary occupation in the North east zone of
Tamil Nadu (Balusami, 2004). The present study
indicated that the majority of the farmers depended
on agriculture as the main source of livelihood and
also maintained livestock, especially buffaloes, as
these enterprises are interdependent on each other.
They maintain buffaloes for secondary source of
income i.e. through sale of milk or utilizing milk
for their home needs. These farmers utilized their
agricultural byproducts like paddy, jowar and
maize straws and green grasses, either cultivated or
natural fodders for their buffaloes, which converted
these poor roughages into valuable milk.
Land holding: In the present investigation,
it was found that 43.3% farmers possessed between
one to three acres of land, 47.5% possessed above
three acres of land and 9.2% were landless. Singh
and Thomas (1992) and Yadav and Yadav (1995)
observed that the land holding was positively
associated with the level of adoption of dairy
innovations among the respondents. It was
observed that, all the farmers irrespective of size
MATERIALS AND METHODS
Two revenue divisions in Khammam district
of Andra Pradesh were selected purposively, having
highest buffalo population. From each revenue
division two mandals and from each mandal ﬁve
villages were selected. Thus, 20 villages from four
mandals of two revenue divisions were selected.
From each village six farmers who were rearing
buffaloes were selected randomly, giving a sample
size of 120 farmers for the study. While conducting
survey, the assistance of the local Veterinary
Assistant Surgeon was sought.
The selected farmers were interviewed
by contacting them at their doorstep utilizing a
pre-tested interview schedule developed for the
purpose. While collecting data sufﬁcient time
was given to the farmer to arrive at values by the
memory recall method. The family members of the
farmers were also involved in collection of the data
so as to get accurate information as far as possible.
The information regarding management practices
such as breeding, feeding, housing, milking, health
care including calf management were collected
and data were subjected to appropriate statistical
analysis (Snedecor and Cochran, 1994).
98
Buffalo Bulletin (June 2013) Vol.32 No.2
of land holding maintained buffaloes as source of
income. The majority of the dairy farmers held land
as major source of income.
Fodders cultivation: Among the selected
farmers, 45.0% grew fodders and 55% did not
cultivate fodder but depended on natural grasses
from their own ﬁelds, common lands, bunds and
other sources. Among the fodder growing farmers,
18.3% grew fodders on an area of about 0 to 0.5
acre and 26.6% on an area of about 0.5 acres
and above. These ﬁndings are in agreement with
the results of Garg et al. (2005) who reported,
about 48.43% farmers grew fodders. The results
indicated that almost 55% farmers in this study did
not grow any fodders and were dependent on either
sole grazing or cut natural grasses for feeding of
buffaloes. Most of the farmers grew sorghum as
a source of fodder, which may have been due to
utilisation of agricultural land for growing regular
crops during kharif season and sorghum as fodder
crop during rabi season because of suitable agroclimatic conditions.
simultaneously had their animals inseminated and
also undertook natural service with the expectation
that it would increase the conception rate. Breeding
bulls or scrub bulls were accessible to all farmers
in their villages for natural service. Sawarkar et al
(2001) reported that, most of the farmers preferred
natural service only due to various reasons. It was
also noticed that as many as 75.83% were unable
to detect pregnancy diagnosis at 3 months age.
Low adoption of artiﬁcial insemination by farmers
may have been due to the presence of scrub bulls,
the distance to artiﬁcial insemination centers, lack
of faith in artiﬁcial insemination, ignorance of
farmers, being busy with agricultural operations
and non availability of veterinary staff etc.
Feeding Practices
From the present investigation it was
observed that only 11.67% farmers fed balanced
rations to their animals; the rest of the farmers
(88.33%) had not adopted this practice (Table 3).
It was found that on an average 9.17, 5 and 85.83%
farmers fed only green fodder, dry roughages and
both in combination, respectively, and 36.67%
farmers fed 1 - 10 kg dry roughages per day while
63.33% farmers fed more than 10 kg. Out of the total
selected farmers, the majority (71.67%) fed 1 kg of
concentrate mixture as feed supplement, whereas
28.33% of them were feeding 2 kg. Kamboj and
Tomar (2000) and Sahu (2001) reported that farmers
were not feeding or feeding minimum quantities of
concentrates to their buffaloes. About 85.83% of the
farmers were feeding green and roughage fodder
in combination and provided clean drinking water
to their animals, but none of the farmers practiced
silage making or any other special treatments
like chafﬁng, soaking or urea treating paddy
straw. Most of the farmers collected fodder from
the ﬁelds. Dwaipayan et al. (2005) reported that,
Breeding Management
The present study revealed that, 95%
farmers were not able to detect heat in animals
(Table 2). This observation was not in agreement
with Dwaipayan et al. (2005), who observed
inability of the 6.11% farmers to identify heat
symptoms; on the contrary Balusami (2004)
reported that 82% farmers were able to detect heat
in buffaloes in Tamil Nadu. More than half of the
farmers under survey (58%) were able to provide
insemination or natural service to their animals
in heat at the right time while 42% farmers were
unable to inseminate their animals in time due to
various reasons. Only 16.66% farmers utilized the
facility of artiﬁcial insemination, while 37.51%
farmers opted for natural service and 45.83%
99
Buffalo Bulletin (June 2013) Vol.32 No.2
59.72% farmers fed poor quality concentrates. The
present results clearly indicated that there is a lot of
scope for improvement of buffalo performance by
better feeding. Only 3.5% farmers fed additional
concentrates to pregnant animals; the rest did not.
Similarly, 11.25% farmers providing additional
concentrates to pregnant animals was reported by
Garg et al. (2005) in rural areas of Baran district
of Rajasthan. About 13.33% farmers provided a
supplementary mineral mixture to their animals
while 86.67% did not follow this practice. Garg
et al. (2005) also observed that 62.5% farmers did
not feed mineral mixture or feed supplements to
their dairy animals. The non-feeding of additional
supplements to the pregnant animals and of
mineral mixture to all animals clearly indicates
that productive animals were facing shortages of
nutrients which would inhibit their exhibiting their
performance. This might have been due to a lack
of scientiﬁc feeding knowledge among the buffalo
farmers, high costs and feed and mineral mixtures
or non-availability etc.
shed, and the ﬂoor space available to the animals
of almost all (98.24%) was adequate. Similarly,
94% of the farmers provided proper ventilation
for their animals. These ﬁndings were similar to
the results of Deoras et al. (2004) who reported,
100 percent of the farmers provided adequate ﬂoor
space and ventilation for their dairy animals in
rural areas. Srivastava et al. (2000) also reported
that 99.5% the farmers studied kept their animals
in well-ventilated houses. It was also found that
locally made mangers were used by 83% farmers,
and 17% farmers maintained cement mangers.
Improper or no mangers may lead to wastage of
feed and fodder, which is already in short supply.
The majority of farmers (> 94%) provided adequate
drainage systems for their animals. It was noticed
that application of disinfectants was occasional by
most (88.33%) of the farmers, and about 11.67%
of them applied disinfectants to their sheds rarely.
Similarly, low use of disinfectants was also reported
by Lal (1999). One hundred percent of the farmers
in the present study had manure pits nearer to their
dwellings or farms and dumped solid manure into
these pits, which was used by them for agricultural
purposes. The reasons for disinfectants were not
used might have been a lack of awareness among
farmers, a high disinfectant cost, and an additional
burden which did not give any immediate return
to the farmer. As a part of summer management,
50.84% of the farmers allowed their buffaloes to
wallow in village tanks during hotter parts of the
day and 49.16% of the farmers washed their animals
by splashing water on them manually. Tailor and
Pathodiya (2000) reported 4.67% of the farmers
they studied in Rajasthan allowed their buffaloes
to wallow. Hot and dry climatic conditions and
heat intolerance of buffaloes requires summer
management like wallowing, sprinkling and
splashing of water etc. to improve the performance
Housing Management
Among the farmers who provided housing,
60% provided thatched roof sheds, 36.66% provided
asbestos roof sheds and 3.34% provided no housing.
Among the sheds, 84.17% had kutcha type ﬂooring
and 15.83% had pucca type (cement concrete)
ﬂooring. The present study is in agreement with the
reports of Deoras et al. (2004), who reported similar
types of housing for the animals of the majority of
farmers. On the contrary, Sohi and Kherde (1980)
reported that a large number of dairy farmers had
sheds for their animals as they were commercial
dairy farmers. The results of the present study do
not agree with the ﬁndings of the said authors as this
study was conducted in rural areas. The majority of
the farmers (62.5%) maintained cleanliness in the
100
Buffalo Bulletin (June 2013) Vol.32 No.2
of buffaloes during the summer.
before milking and of the udder after milking was
practiced by 15.68 and 1.6% farmers, respectively,
whereas 76.66% farmers did not follow any sanitary
practices before and after milking, and the majority
(95%) did not follow any mastitis prevention
programme. The majority of the farmers did not
wash the entire body of their buffaloes before
milking even in the summer (Verma and Sastri,
1994). The practice of not washing the udder after
milking might have been due to allowing the calf
to suckle after milking, when the calf consumes all
the leftover milk and leaves a layer of saliva on the
teats, or it might have been a way to save labour.
The present study concludes that
management practices had a signiﬁcant role on
the performance of buffaloes in the divisions of
Khammam district under study. Non- descript
buffaloes were predominant in this region, and
majority of the farmers were poor in certain
aspects of scientiﬁc feeding, breeding, housing
and milking practices and need to be educated.
Artiﬁcial insemination should be made available to
all the farmers, and this would result in upgrading
of the local buffaloes and improvement in their
performance.
Milking Practices
In the present study, all the buffalo farmers
allowed their calves to suckle their mothers before
and after milking twice a day and also used the
calf to let down milk from the udder (Table 5). It
was observed that 100 percent of the farmers were
following a regular milking interval, which is in
agreement with the reports of Malik and Nagpaul
(1999) wherein 88.88% of the farmers followed a
similar practice in Murrah buffaloes in Haryana. In
the present study, 88.33, 4.17 and 7.5% of farmers
used steel utensils, iron buckets and plastic vessels,
respectively. Very few farmers (8%) followed the
full hand method of milking; the remainder (92%)
followed the knuckling method, which was not
a recommended practice. Khupse et al. (1980)
reported that 8.18% of the farmers studied had
adopted full hand milking, whereas Malik and
Nagpaul (1999) reported that 36.11% farmers
followed the knuckling method of milking in
Haryana. These results indicate lack of awareness
among farmers and the urgent need for education on
the correct method of milking. Washing of animals
101
Buffalo Bulletin (June 2013) Vol.32 No.2
Table 1. General information about the selected farmers.
Sl. No.
Number of
farmers
Observation
1
Main occupation
2
Subsidiary
occupation
3
Land holding
(acres)
4
Area under fodder
crops
Agriculture
Dairying
Agriculture
Dairying
Landless
Medium( 1-3)
Large (> 3)
No fodder crops
0- 0.5 acres
> 0.5 acres
101
19
19
101
11
52
57
66
22
32
Percentage
84.50
15.50
15.50
84.50
9.20
43.30
47.50
55.10
18.30
26.60
Table 2. Breeding management practices followed by the selected farmers.
Sl. No.
Breeding management practices
1
Proper heat detection
procedures and methods
2
Insemination of buffaloes
at right time
3
Method of Insemination
4
Availability of breeding
bull in village
6
Pregnancy diagnosis at
the age of 3 months
Adopted
Not adopted
Followed
Not followed
Artiﬁcial Insemination
Natural service
Both
Yes
No
Adopted
Not adopted
102
Number
of farmers
following the
practice
Percentage
6
114
62
58
20
45
55
120
0
29
91
5.00
95.00
58.00
42.00
16.66
37.51
45.83
100.00
0.00
24.16
75.83
Buffalo Bulletin (June 2013) Vol.32 No.2
Table 3. Feeding management practices followed by the selected farmers.
Sl.
No.
Farmers
following the
practice
Percentage
Yes
No
1-10 kgs
Above 10 kgs
Not feeding at all
Up to 1 kg
2 kg
Dry only
Green only
Both combined
Yes
No
Yes
No
Chafﬁng
Soaking
Urea treated paddy straw
14
106
44
76
0
86
34
6
11
103
0
120
115
5
0
0
0
11.67
88.33
36.67
63.33
0.00
71.67
28.33
5.00
9.17
85.83
0.00
100.00
95.83
4.16
0.00
0.00
0.00
Mixture of green+ dry
+concentrate
120
100.00
Home grown
Purchased
Collected from the ﬁelds
26
0
94
21.67
0.00
78.33
Feeding management practices
1
Feeding balanced ration
2
Feeding dry roughages
per day
3
Feeding concentrate
supplements
4
Combination of roughages
fed
5
Feeding of silage fodder
6
Clean drinking water
access
7
Special treatment given to
feed and fodder
8
Source of fodder
Yes
3
3.50
9
Additional allowance
of concentrates during
advanced pregnancy and
lactation
No
117
97.50
Feeding mineral
supplements
Yes
No
16
104
13.33
86.67
10
103
Buffalo Bulletin (June 2013) Vol.32 No.2
Table 4. Housing management practices followed by the selected farmers.
Sl. No.
Housing management practices
1
Type of housing
2
Type of ﬂooring
3
Cleanliness in the shed
4
Adequacy of ﬂoor space
5
Proper ventilation to the
animals
6
Type of manger
7
Drainage system
8
Manure disposal
9
Use of disinfectants
10
Summer management
11
Intervals of the cleaning
sheds
Thatched roof shed
Asbestos roof shed
Pucca shed
No shed
Kutcha
Pucca (Concrete)
Satisfactory
Not satisfactory
Adequate
Not adequate
Available
Not available
Cement trough
Local made trough
Proper
Not proper
Manure pit
Bio gas
Direct application to
the ﬁelds
Regular
Occasional
Rare
Wallowing
Splashing of water
Sprinkling of water
Once in a year
Occasional
104
No. of
farmers
72
44
0
4
101
19
75
45
118
2
112
8
20
100
113
7
0
0
Percentage
60.00
36.66
0.00
3.34
84.17
15.83
62.50
37.50
98.24
1.76
94.00
6.00
17.00
83.00
94.16
5.84
0.00
0.00
120
100.00
0
106
14
61
59
0
13
107
0.00
88.33
11.67
50.84
49.16
0.00
10.84
89.16
Buffalo Bulletin (June 2013) Vol.32 No.2
Table 5. Milking management practices followed by the selected farmers.
Sl.
No.
1
Milking management practices
Let down of milk
2
Number of times animals are
milked
3
Interval between each milking
4
Type of utensils used for
milking and storage
5
Full hand milking
6
Sanitary practices followed
before and after milking
7
Care against mastitis
8
Washing of animals before
milking
9
Washing of udder after milking
Number
of farmers
following the
practice
Percentage
Use of calf
Use of oxytocin
Use of a phantom
calf
120
0
100.00
0.00
0
0.00
Twice
120
100.00
Regular
Irregular
Stainless Steel
Iron buckets
Plastic vessels
Adopted
Not adopted
Yes
No
Followed
Not followed
Followed
Not followed
Followed
Not followed
120
0
106
5
9
10
110
28
92
6
114
19
101
2
118
100.00
0.00
88.33
4.17
7.50
8.00
92.00
23.34
76.66
5.00
95.00
15.68
84.32
1.60
98.40
105
Buffalo Bulletin (June 2013) Vol.32 No.2
buffaloes. Indian Vet. J., 78(3): 257-259.
Sahu, S.P. 2001. The buffalo management practices
followed by the farmers of Karnal and
Kurukshetra district of Haryana. M.V.Sc.
Thesis, Haryana Agricultural University,
Hisar, India.
Sawarkar, S.W., M.M. Borkar, S.V. Upadhye and
S.B. Jadhao. 2001. Chararacteristics of
dairy owners, their awareness, adoption
and constraints in adoption of artiﬁcial
insemination practices in Vidarbha region.
Indian J. Dairy Sci., 54: 194-202.
Singh, L. and C.K. Thomas. 1992. Knowledge and
adoption of technology in the dairy farming
and its constraints. Indian Dairyman, 44:
445-450.
Sohi, J.S. and R.L. Kherde. 1980. A study of dairy
adoption behavior of small and marginal
farmers in Punjab. Indian J. Ext. Edu., 16:
84-86.
Srivastava, P.N., S.N.S. Parmar, B.C. Sarkhel, S.S.
Tomar and P.V.A. Pillai. 2000. Random
Survey of farmers status and geographical
distribution of Malvi cattle in Madhya
Pradesh, p. 642-647. In Proceedings of the
International Conference Held in Thrissur,
Kerala, India.
Snedecor, G.W. and W.G. Cochran. 1994. Statistical
Methods, 8th ed. Iowa State University Press,
Ames, Iowa, USA.
Tailor, S.P. and A.P. Pathodiya. 2000. Genetic
composition, feeding and management
practices for buffaloes to establish base of
dairy industry in Southern Rajasthan - A
survey, p. 787-789. In Proceedings of the
International Conference Held in Thrissur,
Kerala, India.
REFERENCES
Balusami. 2004. Performance of buffaloes in North
East Zone of Tamil Nadu. Ph.D. Thesis,
Tamil Nadu University of Veterinary and
Animal Sciences, Tamil Nadu, India.
Deoras, R., K. Nema, S.P. Tiwari and Mohan Singh.
2004. Feeding and housing management
practices of dairy animals in Rajanandgaon
of Chhatisgarh plain. Indian J. Anim. Sci.,
74: 303-306.
Dwaipayan, B., R.S.L. Srivastava and Y.P.S. Dabas.
2005. A study on constraints perceived by
farmers in rearing dairy animals. Indian J.
Dairy Sci., 58: 3, 214-220.
Garg, M.K., L.S. Jain and J.L. Chaudhary. 2005.
Studies on housing, feeding and milking
management practices of dairy cattle in
Baran district of Rajastan. Indian J. Dairy
Sci., 58: 123-128.
Kamboj, M.L. and O.S. Tomar. 2000. Feeds and
feeding managemental practices of Nagori
cattle under ﬁeld conditions. Indian J. Anim
Prod. Manage., 16: 129-131.
Khupse, T.S., V.P. Dhumal and S.D. Nimbalker.
1980. Adoption of improved dairy
management practices. Livestock Advisor,
5: 11-14.
Lal, H., 1999. Animal husbandry practices in
Mewat area of Haryana under different
farming systems. Ph.D. Thesis, Haryana
Agricultural University, Hisar, India.
Malik, D.S. and P.K. Nagpaul. 1999. Studies
on milking and calf rearing management
practices of Murrah buffalo in its home tract
of Haryana. Indian J. Anim. Prod. Manage.,
15: 52-54.
Prasad, R.M.V., G.N. Rao and V. Jayaramakrishna.
2001. Analysis on milk production from
*Continued on page 119
106
Buffalo Bulletin (June 2013) Vol.32 No.2
Original Article
SUB-CLINICAL MASTITIS IN MURRAH BUFFALOES WITH SPECIAL REFERENCE
TO PREVALENCE, ETIOLOGY AND ANTIBIOGRAM
Pankaj1, Anshu Sharma2, Rajesh Chhabra3 and Neelesh Sindhu4
revealed mixed infections with Staphylococcus
spp. + Streptococcus spp. Among Staphylococci,
Staphylococcus aureus and Staphylococcus
haemolyticus were the main isolates followed
by Staphylococcus epidermidis, Staphylococcus
simulans, Staphylococcus hyicus, Staphylococcus
pasteuri, Staphylococcus saprophyticus subsp.
saprophyticus,
Staphylococcus arlettae and
Staphylococcus gallinarum. All the strains of
staphylococci and streptococci were found sensitive
to cloxacillin, ceftriaxone and cefoperazone.
Streptococci revealed 100 percent sensitivity
towards penicillin, enroﬂoxacin, ciproﬂoxacin,
lincomycin and cephalexin.
ABSTRACT
This study was carried out to determine the
prevalence of sub-clinical mastitis, its etiological
agents and their antibiogram in Murrah buffaloes
at an organized farm. A total of 326 quarter milk
samples were screened from 82 apparently healthy
buffaloes. The percent prevalence of sub-clinical
mastitis was found to be lower on the basis of SCC
(>5x105/ml) alone (23.17) as compared to cultural
examination (29.26). However, the quarter-wise
percent prevalence on the basis of SCC (11.04) was
similar to bacteriological examination (11.65). On
the basis of International Dairy Federation criteria,
7.05% of the quarters (SCC above 500,000/ml
of milk and culturally positive), 4.60% quarters
(SCC below 500,000/ml of milk but culturally
positive) and 3.98% (culturally negative and SCC
above 500,000/ml) were found to suffer from
sub-clinical, latent and non-speciﬁc mastitis,
respectively. Out of 38 culturally positive quarters,
a total of 44 organisms were recovered. Of these,
15.90% were coagulase positive staphylococci and
47.72% were coagulase negative staphylococci
followed by Streptococcus dysgalactiae 25%,
Streptococcus agalactiae 9.09% and Streptococcus
uberis 2.27%. and 13.63% of the quarters
Keywords: sub-clinical mastitis, Murrah buffalo,
prevalence, etiology, antibiogram
INTRODUCTION
In India, the buffalo population is
approximately 94 million head. Of the total
production of milk, about 53 percent comes from
buffaloes and 43 percent from cows. Haryana
has the world’s best dairy type buffalo, the
Murrah, capable of milk yields as high as 35 kg
1
Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana - 125 004, India
2
College Central Laboratory, College of Veterinary Sciences, Haryana, India, E-mail: anshusharma_dr@
yahoomail.com
3
College of Veterinary Sciences, Haryana, India
4
J.C.B. Station, Jagadhari, Haryana, India
107
Buffalo Bulletin (June 2013) Vol.32 No.2
15-20 ml milk sample of each quarter was collected
separately in a sterilized test tube. These test tubes
were marked as right fore (RF), right hind (RH),
left fore (LF) and left hind (LH) and the collection
was done ﬁrst from the near side and then from the
off side to avoid contamination of teat apices. Each
test tube was given the number possessed by the
animal.
Bacteriological
examination:
The
prevalence of sub-clinical mastitis was determined
following International Dairy Federation Criteria
based on bacteriological examination of milk and
somatic cell count (SCC).
For bacteriological examination, the milk
samples were shaken thoroughly and 0.01 ml of the
milk sample was streaked on 5% sheep blood agar
and MacConkey’s lactose agar plates. The plates
were incubated aerobically at 37oC for 24 to 48 h.
Sub-cultures of the resulting growth were made on
blood agar for puriﬁcation of isolates and identiﬁed
on the basis of Gram’s reaction, morphology
and colony characteristics. All the isolates were
characterized up to species level following standard
bacteriological procedures.
Somatic cell count: The SCC on milk
samples was performed as described by Schalm et
al. (1971) and the milk smears were stained with
Newman-Lampert stain (methylene blue-1.2 gm,
ethyl alcohol (95%) 54 ml, tetra chloro ethane 40
ml, glacial acetic acid 6 ml).
Antimicrobial
sensitivity
testing:
Different strains of various organisms isolated
from udder infections were subjected to in-vitro
drug sensitivity testing, using 20 antimicrobials
by a disc-diffusion method as suggested by Bauer
et al. (1966). The sensitivity was observed on the
basis of a zone size interpretation chart provided
by the manufacturer. The results were recorded as
sensitive, intermediate and resistant.
a day. In a review on present status of mastitis in
buffaloes at periurban dairy farms in India, Joshi
and Gokhale (2006) stated mastitis was one of the
most important factors in dairy development in
the tropics. Sub-clinical mastitis has been reported
to be more important (5-20% in buffaloes) than
clinical mastitis (1-10) because it is 15-40 times
more prevalent than the clinical form, it drastically
reduces milk yield, and it usually precedes the
clinical form and is usually the basis of herd
problems when mastitis outbreaks occur. In India,
Dua (2001) has reported annual losses due to
mastitis to the tune of Rs 60.5321 billion of which,
Rs. 43.6532 billion has been attributed to subclinical mastitis. Therefore, the present study was
planned to determine the prevalence of sub-clinical
mastitis in Murrah buffaloes at an organized farm,
to determine the type of organisms responsible for
its causation and to determine their antimicrobial
sensitivity towards antimicrobials in common use
and some of the newer antimicrobials.
MATERIALS AND METHODS
Source of milk samples: A total of 326
quarter buffalo milk samples were collected from
82 apparently healthy buffaloes of Murrah breed
located at an organized farm. Animals which had
calved recently (less than two weeks) or those in
late lactation (more than nine months) were not
included in the study.
Collection of milk samples: Milk was
collected under aseptic conditions. The udders
of cows were cleaned thoroughly with a cloth
containing dilute potassium permanganate solution.
Hands were properly washed with soap and water
and teat apices disinfected with 70 percent alcohol.
The ﬁrst few milk strippings were discarded and
108
Buffalo Bulletin (June 2013) Vol.32 No.2
(Schultze, 1985). The lower rate of prevalence in
the present investigation in comparison to previous
studies on the same farm might be attributed to
adoption of proper management, hygienic and
control measures at the farm.
Tuteja et al. (1999) reported high percent
prevalence (26.67) of latent mastitis in comparison
to our study. Serieys (1985) and Roder and Gedek
(1986) reported that the SCC could be inﬂuenced
by the type of infecting organisms and season.
Thus, a low cell count does not reﬂect the true
bacteriological status of the udder. The signiﬁcance
of latent mastitis cannot be undermined since some
of these cases are likely to convert into the subclinical form and subsequently into clinical mastitis,
particularly under unfavorable environmental
conditions. Moreover, latent infection also reﬂects
the possibility of teat canal infections serving as a
potential source of infection to the milk secretory
tissue. Even mammary parenchyma may be damaged
due to liberation of bacterial toxins in the infected
teat canal (Nickerson et al., 1986). In comparison
to our study, on the same farm Tuteja et al. (1999)
observed a high percentage (7.3) of quarters
suffering from non-speciﬁc mastitis whereas
Sindhu et al. (2009) reported a lower percentage
(2.19) of quarters having non-speciﬁc mastitis.
Failure to detect pathogens in such cases might
be due to intermittent excretion of the organisms
or their disappearance because of spontaneous
recovery. Salsberg et al. (1984) observed that
somatic cell counts increased more during summer
months from June to August in Holstein cows than
in cooler months. The possibility of mycoplasmal
mastitis cannot be ruled out in such cases, since
the organism cannot be cultivated on common
bacteriological media.
The relative frequency of various microorganisms from the apparently healthy milk quarters
RESULTS AND DISCUSSION
Results of cultural examination and somatic
cell count (SCC) on 326 quarters of 82 buffaloes
are presented in Table 1.
Figures in parentheses indicate percentage
The percent prevalence of sub-clinical
mastitis was found to be lower on the basis of SCC
(>5x105/ml) alone (23.17) as compared to cultural
examination (29.26). However, the quarter-wise
percent prevalence on the basis of SCC (11.04) was
similar to bacteriological examination (11.65).
On the basis of IDF criteria 7.05% of
the quarters (SCC above 500,000/ml of milk
and culturally positive), 4.60% quarters (SCC
below 500,000/ml of milk but culturally positive)
and 3.98% (culturally negative and SCC above
500,000/ml) were found to suffer sub-clinical,
latent and non-speciﬁc mastitis, respectively.
Similar prevalence of SCM has been reported
by Bansal et al., 1995; In contrast to our study,
several workers (Kalorey et al., 1983; Rahman et
al., 1983; Tuteja et al., 1999; Maiti et al., 2003
and Chavan et al., 2007) reported high animalwise and quarter-wise prevalence of SCM. These
differences in the prevalence rates of SCM as
reported by different workers are perhaps due to
difference in managemental and hygienic practices
adopted in different dairy herds. The incidence of
mastitis varied among farms and the risk increased
with increasing parity (Sargeant et al., 1998).
Thirunavukarasu and Prabaharan (1998) reported
that the incidence of mastitis was signiﬁcantly
associated with animal factors such as breed (Dego
and Tareke, 2003), milk yield, stage of lactation
(Sharma et al., 2007) and udder morphology,
besides farm practices and sanitation. The climatic
conditions also affect the prevalence of mastitis
109
Buffalo Bulletin (June 2013) Vol.32 No.2
been reported by several workers in India (Kalorey
et al., 1983; Javed and Siddique, 1999; Tijare et
al., 1999; Tuteja, 1999; Kaya et al., 2000; Sharma
et al., 2007) and abroad (Hawari and Dabas, 2008;
Tenhagen et al., 2009 and Nickerson and Stephen,
2009). The prevalence of a pathogen is inﬂuenced
by parity, type of sample and season (Sharma et
al., 2007; Hagnestan et al., 2009). Distribution
of pathogens changes over time; therefore,
bacteriological examination at the herd level must
be taken regularly to monitor udder health.
Similar to our ﬁndings, other workers
from India have also reported staphylococci and
streptococci to be the main etiological agents of
mastitis in different parts of the country (Chavan
et al., 2007; Sharma and Sindhu, 2007; Behera et
al., 2008; Palanivel et al., 2008; Roychoudhary and
Dutta, 2009; Sindhu et al., 2010). Among
staphylococci, Staphylococcus aureus and
Staphylococcus haemolyticus were found to be
including 11 quarters harboring mixed infection is
given in Table 2.
Out of 38 culturally positive quarters, a total
44 organisms were recovered. Of these, 15.90 %
were coagulase positive staphylococci and 47.72%
were coagulase negative staphylococci followed
by Streptococcus dysgalactiae 25% Streptococcus
agalactiae 9.09%, and Streptococcus uberis 2.27%
and 13.63% quarters revealed mixed infections
with Staphylococcus spp. + Streptococcus spp.
All the CPS and CNS were further
characterized to species level. Details are given in
Table 3. Staphylococcus aureus and Staphylococcus
haemolyticus were the main isolates followed
by Staphylococcus epidermidis, Staphylococcus
simulans, Staphylococcus hyicus, Staphylococcus
pasteuri, Staphylococcus saprophyticus subsp.
saprophyticus, Staphylococcus arlettae and
Staphylococcus gallinarum. Results indicated
substantial differences in the prevalence of
pathogens among different herds. In our study
contagious bacteria like staphylococci and
Streptococcus agalactiae caused most of the
infections. Such infections are usually spread from
animal to animal at the time of milking. The mastitis
the most prevalent followed by Staphylococcus
epidermidis. Many workers have found
Staphylococcus aureus to be more prevalent
than Staphylococcus epidermidis (Char et al.,
1983; Saini et al., 1994; Armenteros et al., 2006;
Unnerstad et al., 2009). Contrary to this, several
workers (Chavan et al., 2007; Ferguson et al.,
2008; Petzer et al., 2009; Tenhagen et al., 2009;
Sampimon et al., 2009) reported high prevalence
of coagulase negative staphylococci (CNS).
These ﬁndings show the increasing importance of
CNS, which were formally described as a minor
pathogen in the case of mastitis. Since, in the
veterinary ﬁeld, our attention has been mainly
directed towards coagulase-positive staphylococci,
the potential of pathogenicity of CNS remains unelucidated. Moreover, Staphylococcus epidermidis
is supposed to be normal ﬂora of teat skin, its
higher prevalence, as observed in this study, might
situation can be improved by improving milking
practices and hygiene. Our ﬁndings are in close
agreement with those of Bansal et al. (1995) and
Petzer et al. (2009) who reported isolation of CNS,
Streptococcus agalactiae and Streptococcus uberis.
Rani et al., 2008 reported that the prevalence of
mastitis varies with breed, age, lactation and season.
Amongst various mastitogenic bacteria isolated,
staphylococci were the most prevalent, accounting
for 63.62 percent of the infections, followed
by streptococci (36.36 percent), respectively.
Similar ﬁndings were reported in India by Babu
et al. (1983), Sharma and Kapur (2000) and Bulla
(2002). The high prevalence of staphylococci has
110
Buffalo Bulletin (June 2013) Vol.32 No.2
Table 1. Prevalence of sub-clinical mastitis in 326 quarters of 82 buffaloes at an organized farm.
Quarters showing
Buffaloes
culturally
positive
24
(29.26)
Buffaloes
SCC >
SCC <
Quarters
showing
SCC >
5 lac/
5 lac/
culturally
SCC > 5
5 lac/
ml and
ml and
positive
lac/ml
ml
culturally culturally
positive
positive
19
(23.17)
38
(11.65)
36
(11.04)
23
(7.05)
SCC > 5
lac/ml and
culturally
negative
15
(4.60)
13
(3.98)
Table 2. Frequency of isolation of different organisms.
Organisms
Coagulase positive staphylococci
Coagulase negative staphylococci
Streptococcus dysgalactiae
Streptococcus agalactiae
Streptococcus uberis
Number (percent)
7 (15.90)
21 (47.72)
11 (25.00)
4 (9.09)
1 (2.27)
Table 3. Characterization of staphylococci isolated from buffalo milk.
Coagulase test
Coagulase
positive
staphylococci
Coagulase
negative
staphylococci
Sr. No.
Organisms
Number
1.
Staphylococcus aureus
5
2.
Staphylococcus hyicus
2
1.
2.
Staphylococcus hyicus
Staphylococcus epidermidis
2
4
3.
Staphylococcus hominis subsp.
hominis
1
4.
5.
6.
Staphylococcus pasteuri
Staphylococcus arlettae
Staphylococcus haemolyticus
2
1
5
7.
Staphylococcus saprophyticus
subsp. saprophyticus
2
Staphylococcus gallinarum
Staphylococcus simulans
TOTAL
1
3
28
8.
9.
111
Buffalo Bulletin (June 2013) Vol.32 No.2
be a consequence of unhygienic milking practice,
due to which the organisms gained access into
mammary gland through milkers’ hands, causing
an increase in SCC and inﬂicting pathogenicity in
the alveolar tissue. Further studies are required on
role of Staphylococcus haemolyticus in causing
mastitis.
In the current study, amongst streptococcal
isolates, Streptococcus dysgalactiae were the
predominating organisms 25 percent) followed
by Streptococcus agalactiae (9.09 percent) and
Streptococcus uberis (2.27 percent). The higher
prevalence of Streptococcus dysgalactiae than
Streptococcus agalactiae was reported by Kalra
and Dhanda (1964) and Tuteja (1999). In contrast to
our study Hameed et al., 2007; Chavan et al., 2007;
Getahun et al., 2008; Ferguson et al., 2008, reported
higher prevalence of Streptococcus agalactiae than
Streptococcus dysgalactiae, whereas other workers
(Javed and Siddique, 1999; Sampimon et al., 2009)
found higher prevalence of Streptococcus uberis
than that recorded in this study. Our ﬁndings are
in close agreement with Petzer et al. (2009) who
reported isolation of CNS, Streptococcus agalactiae
and Streptococcus uberis.
A good amount of literature is available
on the antibiogram of different mastitogens. It
is not possible to compare our results with their
Table 4. Antibiogram of different organisms isolated.
Antimicrobials
Erythromycin
Penicillin
Streptomycin
Tetracycline
Chloramphenicol
Ampicillin
Neomycin
Cloxacillin
Enroﬂoxacin
Gentamicin
Amikacin
Amoxycillin
Ceftrioxone
Cefoperazone
Ciproﬂoxacin
Colistin
Co-Triamoxazole
Nitrofurantion
Lincomycin
Cephalexin
Percent sensitivity
Staphylococci (28)
Streptococci (16)
72.2
90.9
88.8
100
50
72.7
100
72.7
83.3
81.6
88.8
36.6
66.6
63.6
100
100
94.4
100
83.3
100
50
81.8
94.4
81.6
100
100
100
100
83.3
100
44.4
27.2
94.4
63.6
94.4
90.9
94.4
100
94.4
100
112
Buffalo Bulletin (June 2013) Vol.32 No.2
ﬁndings. While considering overall sensitivity, all
the strains of staphylococci and streptococci were
found sensitive to cloxacillin, ceftriaxone and
cefoperazone. Streptococci revealed 100 percent
sensitivity towards penicillin, enroﬂoxacin,
ciproﬂoxacin, lincomycin and cephalexin. Similar
to our study, Ranjan et al. 2010 also found high
sensitivity towards Enroﬂoxacin (91.67%)
whereas they observed lower sensitivity towards
Ceftriaxone (84.10%). It was interesting to note
that staphylococci isolates revealed 100 percent
sensitivity towards tetracycline. Studies conducted
by several workers (Sharma et al., 2007; Chavan
et al., 2007; Roychoudhury and Dutta, 2009;
Sharma et al., 2010) have showed increased
resistance towards different traditional and newly
introduced antibiotics. In contrast to these studies,
the antibiogram obtained in the current study
indicated high sensitivity towards newer and older
antibiotics, showing rational use of these antibiotics
at farms under study. Antibiotic resistance patterns
vary among different farms, regions, states and
countries depending upon the type of organisms
and use of antibiotics in a particular area; therefore,
antimicrobial sensitivity is suggested before
institution of treatment. The information obtained
by this study will also be of useful to the dairy
industry and individual farmers. It will be helpful
in prioritizing mastitis control efforts.
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115
Original Article
Buffalo Bulletin (June 2013) Vol.32 No.2
EFFECT OF YEAST CULTURE (Saccharomyces cerevisiae) ON RUMINAL MICROBIAL
POPULATION IN BUFFALO BULLS
D. Srinivas Kumar1, Ch. Srinivasa Prasad2 and R.M.V. Prasad3
ABSTRACT
INTRODUCTION
This experiment was carried out to study
the effect of yeast culture in the diet on rumen
microbial population in buffalo bulls. In a cross
over design, six graded Murrah buffalo bulls
(254.4±7.98 kg) were randomly divided into two
groups of three animals each. The animals in
both the groups were offered 1.5 kg concentrate
mixture and had access to Hybrid Napier fodder
ad libitum. In the treatment group, the concentrate
mixture was supplemented with yeast culture
(Saccharomyces cerevisiae CNCM I-1077 strain)
at the rate of 0.5 g/animal/day. The results indicated
that supplementation of the diet with yeast culture
increased signiﬁcantly the mean protozoal count
(P<0.05) and the total bacterial count (P<0.01) in
SRL of graded Murrah buffalo bulls as compared
with the control group.
For many years, ruminant nutritionists and
microbiologists have been interested in manipulating
the microbial ecosystem of the rumen to improve
production efﬁciency of domestic ruminants. Yeast
culture has displayed positive impact on the growth
and viability of rumen microﬂora and the fermenting
process in the rumen. But, the results have been
inconsistent due to confounding effects of the ration
composition, and variations in the strain of the yeast
culture and their administration protocol. Addition
of Saccharomyces cerevisiae live yeast cultures
to ruminant diets has improved ﬁbre digestibility
and stimulated cellulolytic bacteria (Dawson et
al., 1990), increased protozoal count (Singh et al.,
2008) while in other studies no changes in ruminal
protozoa (Corona et al., 1999) were observed by
the addition of yeast culture to the diet. Therefore,
the present experiment was carried out to study the
effect of yeast culture (Saccharomyces cerevisiae
CNCM I-1077 strain) on the rumen microbial
population in buffalo bulls.
Keywords: yeast culture, protozoal count, bacterial
count, buffalo bulls
1
Department of Animal Nutrition, Sri Venkateswara Veterinary University, NTR College of Veterinary
Science, Gannavaram, India, E-mail: kumardhulipalla@rediffmail.com
2
Department of Veterinary Physiology, College of Veterinary Science, Proddatur - 516 360, Dr YSR Kadapa
District, Andhra Pradesh, India
3
Department of Instructional Livestock Farm Complex, College of Veterinary Science, Proddatur - 516 360,
Dr YSR Kadapa District, Andhra Pradesh, India
116
Buffalo Bulletin (June 2013) Vol.32 No.2
out as per the procedures suggested by Snedecor
and Cochran (1976).
MATERIALS AND METHODS
In a crossover design, six graded Murrah
buffalo bulls (254.4±7.98 kg), each ﬁtted with a
permanant rumen cannula, were randomly divided
into two groups (Control and Treatment) of three
animals each. Animals in both the groups were
offered 1.5 kg concentrate mixture and had access
to Hybrid Napier (CO-1 variety) fodder ad libitum
to meet the nutrient requirements (ICAR, 1998). In
the treatment group, the concentrate mixture was
supplemented with yeast culture (Levucell SC 20;
Saccharomyces cerevisiae CNCM I-1077) at the
rate of 0.5 g/animal/day. The experiment lasted for
60 days and the crossover was made after 30 days of
feeding. Rumen liquor was collected from the bulls
at 0, 2, 4 and 6 h post feeding and strained through
four layers of muslin cloth and was referred to as
strained rumen liquor (SRL). The protozoal count
in SRL was determined as per Kamra et al. (1991),
while the total bacterial count was determined as
per Gall et al. (1949).
Statistical analysis of the data was carried
RESULTS AND DISCUSSION
The DMI expressed as kg / 100 kg b.wt
was 3.29 kg and 3.44 kg in control and treatment
groups, respectively. Supplementation of the diet
with yeast culture increased the DMI (kg / 100 kg b.
wt.) but the difference is not signiﬁcant (P>0.05).
Protozoal Count: The mean total
protozoal count (x104 / ml of SRL) in the yeastculture supplemented (YS) group (21.50±1.48) was
higher (P<0.05) than in the control (15.42±1.36).
Many studies have been conducted to study the
effect of yeast cultures on protozoa populations.
In some cases (Newbold et al., 1995; Yoon and
Stern, 1996) the yeast culture did not modify total
protozoa count while in other studies (Plata et al.,
1994; Kumar et al., 1994; Singh et al., 2008), the
total protozoal count increased.
Time of sampling had signiﬁcant (P<0.01)
Table 1. Effect of dietary supplementation of yeast culture on rumen microbial population in buffalo bulls.
Parameter/
Time of rumen liquor sampling (h)
0
2
4
Treatment
4
*
Protozoal Count ( x10 / ml of SRL)
Control
10.67±1.20
17.00±1.46
22.33±1.71
YS
16.33±1.54
23.33±1.89
29.33±1.14
a
b
Overall*
13.50±1.26
20.17±1.49
25.83±1.44c
Total Bacterial Count ( x109 / ml of SRL)**
Control
5.77±0.28
7.96±0.24
11.23±0.31
YS
9.13±0.29
14.07±0.42
18.33±0.49
a
b
Overall*
7.45±0.54
11.02±0.95
14.78±1.11c
a, b, c
6
11.67±1.14
17.00±1.46
14.34±1.20a
15.42±1.36a
21.50±1.48b
9.72±0.28
16.29±0.62
13.01±1.04bc
8.67±0.27a
14.46±0.44b
Means with different superscripts in a row and column differ signiﬁcantly.
*
(P<0.05) **(P<0.01)
117
Treatment
average
Buffalo Bulletin (June 2013) Vol.32 No.2
These results corroborated with those of Singh et
al. (2008) in buffalo calves. The probable reason
for increased rumen microbial numbers after yeast
culture supplementation could be assigned to
the capacity of yeast to remove oxygen from the
rumen.
Thus, it can be concluded that
supplementation of yeast culture (Levucell SC
20) 0.5 g/animal/day in the diet of graded Murrah
buffalo bulls increased the microbial population in
the rumen.
effect on the total protozoal count. The total
protozoal count was the highest (25.83±1.44) at
4 h post-feeding. It increased (P<0.01) from 0 h
to 4 h post-feeding, and then declined (Table 1).
Feed offer was associated with an abrupt increase
in the total protozoal population within the ﬁrst
4 h of post-feeding, which could be due to migration
of protozoa from the reticulo-ruminal wall where
they sequester from the rumen medium in response
to chemical stimuli originating from the diet.
The migration of protozoa into rumen liquor was
due to chemotactic movement towards the feed
entering the rumen. After the feed was utilized, the
protozoa gradually migrated back to the reticuloruminal wall after 6 h post-feeding resulting in
the observed drop in their numbers in the rumen
liquor in both groups. Further, Iqbal et al. (1993)
reported that irrespective of diets, the protozoal
number increased after feeding and reached a peak
at 4 h post feeding, thereafter, the concentration
decreased gradually. This increase in protozoal
count after feeding may be attributed to availability
of substrate for protozoal growth. These results
corroborated with those of Singh et al. (2008) in
buffalo calves.
Bacterial Count: The mean total
bacterial count ( x109/ml of SRL) in the YS group
(14.46±0.44) was higher (P<0.01) than the control
(8.67±0.27). The increase in the total bacterial
count in the YS group may be attributed to the
positive effect of the yeast culture. These results
were in agreement with the ﬁndings of Rita Sharma
et al. (1988), Dolezal et al. (2005) and Kowalik et
al. (2008).
Time of sampling had a signiﬁcant (P<0.01)
effect on the total bacterial count. The total bacterial
count concentration was the highest (14.78±1.11)
at 4 h post-feeding. It increased (P<0.01) from 0
h to 4 h post-feeding, and then declined (Table 1).
REFERENCES
Corona, L., G.D. Mendoza, F.A. Castrejon, M.M.
Crosby and M.A. Cobos. 1999. Evaluation
of two yeast cultures (Saccharomyces
cerevisiae) on ruminal fermentation and
digestion in sheep fed a corn stover diet.
Small Ruminant Res., 31(3): 209-214.
Dawson, K.A., K.E. Newman and J.A. Boling.
1990. Effects of microbial supplements
containing yeast and lactobacilli on
roughage fed ruminal microbial activities.
J. Anim. Sci., 68: 3392-3398.
Dolezal, P., J. Dolezal and J. Trinacty. 2005. The
effect of Saccharomyces cerevisiae on
ruminal fermentation in dairy cows. Czech
J. Anim. Sci., 50(11): 503-510.
Gall, L.S., W. Burroughs, P. Gerlaugh and B.H.
Edgington. 1949. Special methods for
rumen bacterial studies in the ﬁeld. J. Anim.
Sci., 8: 433-440.
ICAR. 1998. Nutrient Requirements of Livestock
and Poultry. Indian Council of Agricultural
Research, New Delhi, India.
Iqbal, S., R.V. Singh and M.S. Setia. 1993. Inﬂuence
of diets on the population of rumen bacteria
118
Buffalo Bulletin (June 2013) Vol.32 No.2
buffaloes. Indian J. Anim. Sci., 78(2): 172174.
Snedecor, G.W. and W.C. Cochran. 1976. Statistical
Methods, 7th ed. Oxford and IBH Publishing
Company, Kolkata, Imdia.
Yoon, I.K. and M.D. Stern. 1996. Effects of
Saccharomyces cerevisiae and Aspergillus
oryzae cultures on ruminal fermentation in
dairy cows. J. Dairy Sci., 79: 411-417.
and protozoa in buffalo calves (Bubalus
bubalis). Buffalo J., 1: 87-91.
Kamra, D.N., R.K. Sawal., N.N. Pathak., N.
Kewalramani and N. Agarwal. 1991.
Diurnal variation in ciliate protozoa in the
rumen of black buck (Antilope cervicapra)
fed green forage. Lett. Appl. Microbiol., 13:
165-167.
Kowalik, B., T. Michalowski., J.J. Pajat., M.
Taciak and J. Rawa. 2008. The effect
of supplementing cows with live yeast,
Saccharomyces cerevisiae, on ciliate and
ruminal fermentation. J. Anim. Feed Sci.,
17(2): 157-165.
Kumar, U., V.K. Sarean and S. Singh. 1994. Effect
of S. cerevisiae yeast culture supplement on
ruminal metabolism in buffalo calves given
a high concentrate diet. Anim. Prod., 59:
209-215.
Newbold, C.J., R.J. Wallace, X.B. Chen and
F.M. Mcintosh. 1995. Different strains of
Saccharomyces cerevisiae differ in their
effects on ruminal bacterial numbers in vitro
and in sheep. J. Anim. Sci., 73: 1811-1818.
Plata, P.F., M.G.D. Mendoza, J.R. Barcena-Gama
and M.S. Gonzalez. 1994. Effect of a yeast
culture (Saccharomyces cerevisiae) on
neutral detergent ﬁber digestion in steer fed
oat straw based diets. Anim. Feed Sci. Tech.,
49: 203-210.
Rita, Sharma, O.P. Nangia and M. Gupta. 1998. Effect
of yeast culture (Saccharomyces cerevisiae)
plus growth medium supplementation on
rumen fermentation in buffalo calves fed
high roughage diet. Indian J. Anim. Sci., 13:
121-126.
Singh, G., S. Kulkarni and R. Singh. 2008. Effect
of Saccharomyces cerevisiae (Yea-sacc1026)
supplementation on rumen proﬁle in
*Continued from page 106
Verma, A.K. and N.S.R. Sastri. 1994. Comparison
of Buffalo housing practices prevalent in
rural Haryana with organized farm, p. 2123. In Proceedings of National Symposium
on Livestock Production and Management
Held at Anand, Gujarat, India.
Yadav, B.L. and M.C. Yadav. 1995. Adoption level
and index of buffalo husbandry practices in
the home tract of Murrah. Indian J. Anim.
Prod. Mgmt., 11: 4-13.
119
Original Article
Buffalo Bulletin (June 2013) Vol.32 No.2
EARLY PREGNANCY DIAGNOSIS BY TRANSRECTAL ULTRASONOGRAPHY IN
MEHSANA BUFFALOES (Bubalus bubalis)
Mehrajuddin Naikoo1, D.M. Patel2 and H.J. Derashri3
for early pregnancy was recorded as 94.44% and
100% on day 26 and day 40, respectively. Plasma
progesterone concentrations ranged from 3.006.02 ng/ml with an average of 4.42±0.52 ng/ml in
pregnant animals and from 0.97-1.91 ng/ml with
an average of 1.22±0.06 ng/ml in nonpregnant
ABSTRACT
The aim of this study was to detect early
pregnancy and early embryonic mortality using
transrectal ultrasonography in Mehsana buffaloes.
The study was carried out on 18 postpartum anestrus
Mehsana buffaloes of a dairy farm during breeding
season, using different estrus synchronization
protocols, followed by ﬁxed time AI. Ultrasound
examinations were performed using a real time
B-mode ultrasound scanner (ALOKA SSD - 500)
equipped with 5 MHz linear array transducer on
day 26 and 40 post AI. Ultrasound scanning on
day 26 post AI revealed visualization of embryonic
vesicle around the conceptus. The embryo proper
was observed in nine out of 13 animals (69.23%)
on day 26 and all 12 conceived animals (100%)
on day 40 post AI. The amniotic and allantoic
vesicles were observed as non-echogenic cavities
closely surrounding the embryo proper on day 40
post AI. Ovarian corpus luteum and foetal heart
beats were observed in conceived buffaloes on
both 26 and 40 days post AI. Early embryonic
death occurred in one buffalo each between days
26 and 40 and between day 40 and 60 post AI. The
overall diagnostic accuracy of ultrasonography
animals on day 20 post AI. It was concluded that
ultrasound scanning is a useful tool in detection of
early pregnancy, early non-pregnancy and early
embryonic mortality in buffaloes.
Keywords: ultrasonography, early pregnancy
diagnosis, Mehsana buffaloes
INTRODUCTION
Livestock production in general and milk
production in particular play an important role in
our national economy and thus dairying is one
of the most important agricultural activities in
India. Buffaloes are the major contributor to milk
production in our country, especially in Gujarat
state. Conditions like early embryonic mortality,
early fetal death and unobserved abortions due
to the tropical environment lead to an increased
1
Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary Science and
Animal Husbandry, Anand Agricultural University, Anand - 388 001, India, E-mail: drmehraj@gmail.com
2
Teaching Veterinary Clinical Services Complex, Anand Agricultural University, Anand - 388 001, India
3
Department of Animal Reproduction, Gynaecology and Obstetrics, Navsari Agricultural University, Navsari,
Gujarat - 396 450, India
120
Buffalo Bulletin (June 2013) Vol.32 No.2
The ultrasound examinations were
performed using a transrectal B-mode ultrasound
scanner (ALOKA SSD - 500, SN - M10408. Japan)
equipped with a 5 MHz linear array transducer
designed for intra-rectal placement. The scanning
of uterine horns was carried out on their dorsal
and lateral surfaces. Ovaries were also scanned for
the presence of a corpus luteum and/or any other
palpable structure. The images displayed were
thoroughly examined and frozen on the screen
and the hard copies (sonograms) were taken using
a video graphic thermal printer (Sony, UP-895
MDW, Japan).
Blood samples were collected twice from
all the buffaloes for estimation of plasma levels
of progesterone, i.e. on the day of AI and on day
20 post AI. Plasma progesterone concentrations
were estimated by employing the standard radioimmuno-assay (RIA) technique of Kubasic et al.
(1984). Labeled antigen (I125), antibody coated tubes
and standards were procured from Immunotech SAS, Marseille Cedex, 9, France. The sensitivity
of assay was 0.1 ng/ml. Intra-assay coefﬁcient of
variation was 5.4 percent and inter-assay variation
was 9.1 percent. Cross reactivity of the antibody
with progesterone, 17Į-dihydroprogesterone and
20Į-hydroxyprogesterone was 100, 0.13 and 0.96
percent, respectively. The sensitivity, speciﬁcity,
positive predictive value, negative predictive value
and accuracy were calculated based on ﬁndings
of ultrasound scanning on different days (day 26
and 40 post AI) and those of plasma progesterone
concentrations on day 20 post AI in relation to
ﬁndings of pregnancy diagnosis by rectal palpation
on day 60 post AI.
anestrus period and thereby increase the costly
calving interval. To overcome such problems,
reproductive ultrasonography may serve as a better
tool.
The use of ultrasonography has been
increasing as an imaging modality in bovine
reproduction. Its use can provide solutions to
a number of unanswered questions in dealing
with the bovine reproductive cycle and its
concurrent disorders, including early pregnancy
diagnosis. Ultrasonographic observation of the
postpartum changes in female reproductive
tract, early pregnancy diagnosis by reproductive
ultrasonography and detection of early embryonic
mortality are some of the efﬁcient means of
improving domestic animal reproduction and
production and help in fulﬁlling the basic
necessities of man in the day-to-day world.
Ultrasound pregnancy diagnosis is a reliable
method of determining the presence of a conceptus
and viability of an embryo, which is essential to
increase the proﬁtability of the animal (Tiwari et al.,
2002). The present study was conducted to detect
early pregnancy and early embryonic mortality
by transrectal ultrasonography in oestrus induced
postpartum anestrus Mehsana buffaloes.
MATERIALS AND METHODS
A total of 18 postpartum anestrus Mehsana
buffaloes of a private dairy farm at Vadodara, Gujarat
were subjected to different estrus synchronization
protocols and ﬁxed time AI with good quality
frozen semen. The buffaloes not returning to estrus
were subjected to ultrasonographic scanning for
early pregnancy diagnosis on days 26 and 40 post
AI and ﬁnally pregnancy was conﬁrmed by rectal
examination on day 60 post AI.
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Buffalo Bulletin (June 2013) Vol.32 No.2
Developmental proﬁle of embryonic vesicle and
embryo
The scanning of recently bred buffaloes
through B-mode transrectal ultrasonography on
day 26 allowed the visualization of non-echogenic
embryonic vesicle around the conceptus (Figure 1).
The embryo proper was observed on day 26 post
AI, however, it was observed in only nine out of 13
(69.23%) buffaloes. Thereafter, the embryo proper
was observed in all 12 conceiving animals (100%)
on day 40 post AI (Figure 2). The amniotic and
allantoic vesicles were observed as non-echogenic
cavities closely surrounding the embryo proper on
RESULTS AND DISCUSSION
The results of ultrasound scanning and
progesterone assay were correlated with the
ﬁndings of palpation per rectum on day 60 post
AI with respect to accuracy and reliability of these
two tests. Although the sensitivity on days 26 and
40 and the speciﬁcity on day 40 of ultrasound
scanning was 100 percent, the speciﬁcity was lower
on day 26 being 80 percent. On day 26 one animal
was incorrectly diagnosed pregnant. However,
the progesterone assay revealed two animals
having been diagnosed incorrectly pregnant. The
sensitivity, speciﬁcity, positive predictive value,
negative predictive value and overall diagnostic
accuracy of progesterone assay were comparatively
lower as compared to ultrasound scanning results
on days 26 and 40 (Table 1).
day 40 post AI. These cavities were surrounded
by large anechoic area i.e. embryonic ﬂuid. The
scanning of ovaries revealed the presence of a
pregnancy corpus luteum in conceiving buffaloes
on both the days i.e. day 26 and 40 post AI. The
heartbeats were observed as a pulsatile ﬂickering
Table 1. Accuracy of ultrasonographic and P4 assay for early pregnancy diagnosis in Mehsana buffaloes on
different days after insemination.
Diagnostic results/Predictive values
Diagnosis pregnant correct (a)
Diagnosis pregnant incorrect (b)
Diagnosis nonpregnant correct (c)
Diagnosis nonpregnant incorrect (d)
Sensitivity (%) 100 x a /(a + d)
Speciﬁcity (%) 100 x c /(c + b)
Positive predictive value (%)
100 x a /(a + b)
Negative predictive value (%)
100 x c /(c + d)
Overall diagnostic accuracy (%)
100 x (a + c)/(a + b + c + d)
Days of ultrasound scanning
Plasma P4 assay
by RIA†
26
13
1
4
0
100%
80%
40
12
0
6
0
100%
100%
11
2
4
1
91.66 %
66.66 %
92.85%
100%
84.61 %
100%
100%
80.00%
94.44%
100%
83.33%
(†) RIA performed at day 20 post-breeding.
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Buffalo Bulletin (June 2013) Vol.32 No.2
Figure 1. Sonogram on day 26 post-insemination.
a) Conceptus and
b) Fluid ﬁlled embryonic vesicle
Figure 2. Sonogram on day 40 post-insemination.
a) Embryo proper and
b) Fluid ﬁlled embryonic vesicle
123
Buffalo Bulletin (June 2013) Vol.32 No.2
(2000); Rosiles et al. (2005). They observed 97.90
and 100 percent accuracy in detecting pregnancy
through ultrasonography, respectively. The possible
explanation for higher overall diagnostic accuracy
in the present study might be because ultrasound
scanning was carried out on days 26 and 40 post AI
with proper restraining of the animals.
at a faster rate in the centre of the embryo proper
within the embryonic vesicle on day 26 and 40 post
AI. However, heartbeats were observed in only 10
out of 13 animals on day 26 post AI and in all 12
animals on day 40 post AI. The mean heart rate
was 174.60±0.81 beats per minute on day 26 post
AI, which decreased to 165.28±1.4 on day 40 post
AI. These ﬁndings were in accordance with the
ﬁndings of many workers (Rosiles et al., 2005 and
Agarwal et al., 2007).
In the present study it was possible to clearly
visualize the embryonic vesicle in all animals and
embryo proper in nine out of 13 animals on day 26
post AI. Moreover, both the embryonic vesicle and
the embryo proper were detected in all pregnant
animals on day 40 post AI. Thus, ultrasonography
facilitates diagnosis of all nonpregnant animals at
an early stage (day 26 post AI) and is really more
advantageous than pregnancy diagnosis by rectal
palpation method, wherein 100 percent reliable
results cannot be obtained at such an early stage of
pregnancy. Agarwal et al. (2007); Ali and Fahmy
(2008) reported similar ﬁndings in buffaloes. The
results of the present study showed that transrectal
ultrasound scanning of buffaloes on day 26 post AI
Early embryonic mortality and/or early fetal
death
In one animal, ultrasound scanning revealed
the presence of a conceptus inside the ﬂuid ﬁlled
embryonic vesicle on day 26 post AI. However,
ultrasonography on day 40 post AI revealed the
same animal as nonpregnant as evident by the
absence of a conceptus and resorption of embryonic
ﬂuid; suggestive of early embryonic death between
days 26 and 40 post AI. In one buffalo, ultrasound
scanning revealed presence of a conceptus and an
embryonic vesicle on both the occasions, i.e. days
26 and 40 post AI. Later on, palpation per rectum at
day 60 post AI revealed the animal as nonpregnant,
suggestive of early embryonic or fetal death
between days 40 and 60 post AI. Similar ﬁndings
were recorded by Campanile et al. (2005); Vecchio
et al. (2007).
was less accurate than on day 40 post AI.
The sensitivity of ultrasound scanning
was 100 percent on days 26 and 40 post AI (Table
1). The speciﬁcity of ultrasound scanning was
80.00 and 100 percent on days 26 and 40 post AI,
respectively. The positive predictive value (PPV)
of the ultrasound scanning was 92.85 and 100
percent on day 26 and 40 post AI, respectively. The
negative predictive value (NPV) of the ultrasound
scanning was 100 percent on both the days i.e. day
26 and 40 post AI. The overall diagnostic accuracy
in the present study was recorded as 94.44 and 100
percent on days 26 and 40, respectively. Similar
ﬁndings were recorded by Bhosreker and Hangarge
Hormone proﬁle
In the present study, plasma progesterone
concentrations ranged from 3.00-6.02 ng/ml with
an average of 4.42±0.52 ng/ml in pregnant animals
on day 20 post AI. In nonpregnant animals plasma
progesterone concentrations ranged from 0.97-1.91
ng/ml with an average of 1.22±0.06 ng/ml on day
20 post AI. These ﬁndings were in close accordance
with the ﬁndings of Muhammad et al. (2000).
The ﬁndings of the present study indicate
that ultrasound scanning of freshly bred animals
is helpful in detection of early pregnancy, early
124
Buffalo Bulletin (June 2013) Vol.32 No.2
during the seasonal decline in reproductive
function. Theriogenology, 63: 2334-2340.
Kubasic, N.P., G.D. Hallauer and R.G. Brodows.
1984. Evaluation of direct solid phase RIA
for progesterone, useful for monitoring
luteal function. Clin. Chem., 30: 284-286.
Muhammad, F., A. Sarwar, C.S. Hayat and
M.I. Anwar. 2000. Peripheral plasma
progesterone concentration during early
pregnancy in Holstein Friesian cows. Pak.
Vet. J., 20: 166-168.
Rosiles, V.A., C.S. Galina, M. Maquivar, R. Molina
and S. Estrada. 2005. Ultrasonographic
screening of embryo development in cattle
(Bos indicus) between days 20 and 40 of
pregnancy. Anim. Reprod. Sci., 90: 31-37.
Tiwari, R.P., M.K. Awasthi and R.P. Gautam.
2002. Principles, methods and clinical use
of ultrasonography in bovine reproduction.
Intas Polivet, 3: 78-85.
Vecchio, D., R. Di Palo, L. Zicarelli, C. Grassi,
A. Cammarano, M.J. D’Occhio and G.
Campanile. 2007. Embryonic mortality in
buffaloes naturally mated. Italian J. Anim.
Sci., 6: 677-679.
nonpregnancy and early embryonic deaths (EED)
in buffaloes. The detection of early pregnancy in
buffaloes may be accomplished on or after day 26
post AI through ultrasound scanning with positive
predictive value and accuracy over 92 percent and
the resul ts are instantly available.
ACKNOWLEDGEMENTS
The authors would like to thank Mr. Ravi
Shetty, owner of Geeta Dairy Farm, Vadodara for
providing buffaloes for this research purpose and
for his kind cooperation.
REFERENCES
Agarwal, S.K., S.K. Singh, T. Sarath, R. Rajkumar,
S. Mehrotra, M. Hoque and Uma Shankar.
2007. Ovarian and uterine changes during
pregnany in buffalo using real time B mode
ultrasonography, p. 331. In Proceedings of
23rd Annual Convention of Indian Society
for the Study of Animal Reproduction
(ISSAR) and National Symposium, OUAT,
Bhubaneswar, India.
Ali, A. and S. Fahmy. 2008. Ultrasonographic
fetometry and determination of fetal sex in
buffaloes (Bubalus bubalis). Anim. Reprod.
Sci., 106: 90-99.
Bhosreker, M.R. and I.M. Hangarge. 2000.
Ultrasonography for early pregnancy
diagnosis in buffaloes. Indian J. Anim.
Reprod., 21: 143-144.
Campanile, G., G. Neglia, B. Gasparrini, G. Galiero,
A. Prandi, R. Di Palo, M.J. D’Occhio and
L. Zicarelli. 2005. Embryonic mortality in
buffaloes synchronized and mated by AI
125
Original Article
Buffalo Bulletin (June 2013) Vol.32 No.2
A STUDY OF PROTEASE INHIBITOR GENE POLYMORPHISM
IN MEHSANA BUFFALO (Bubalus bubalis)
Manisha Deshpande, D.N. Rank, P.H. Vataliya and C.G. Joshi
ABSTRACT
INTRODUCTION
Protease inhibitor (PI) genes belong
to the superfamily of serine protease inhibitors
that includes C1 esterase, antithrombin, and α1antichymotrypsin, among others. The primary
role of PI is to protect tissues against proteolytic
digestion by neutrophil elastase. PI is also found
to be associated with increased milk and fat yields,
increased productive life and decreased somatic
cell score. The present study was carried out to
reveal PCR-RFLP polymorphism of protease
inhibitor exon 2 fragment in Mehsana buffaloes.
The buffaloes were registered under the progeny
testing programme of the Dudhsagar Research and
Development Association, Mehsana, Gujarat state
(India). The fragment of 448 bp of PI gene (exon
2) locus was amplified by PCR and subsequently
digested with restriction enzyme SfaN1. It revealed
monomorphic patterns. Further representative
samples were cloned and sequenced. On comparison
with published cattle sequences, the nucleotide
sequence variation between cattle and buffalo was
present at ten nucleotide positions, i.e., 110, 141,
146, 170, 236, 257, 277, 306, 350, and 396.
The present study aimed at exploring
polymorphism in the PI gene by PCR-RFLP
sequencing and its association with production traits
in the Mehsana buffalo. Very scanty information
is available on PI gene polymorphism in Indian
buffaloes (Bubalus bubalis) .
It has been reported that the PI gene is a
candidate gene for production traits in dairy cattle
(Khatib et al., 2005). The candidate gene approach
consists of the study of different genes potentially
involved in physiological processes (e.g., milk
protein synthesis and milk fat synthesis) and the
identification for each gene of the allele responsible
for the desired phenotype.
The protease inhibitor (PI) genes belong
to the superfamily of serine protease inhibitors
that includes C1 esterase, antithrombin and α1antichymotrypsin among others (Barra et al., 1987;
Anderson and Kingston, 1983; Blank and Brantly,
1994). Khatib et al. (2005) reported five SNP in
coding regions of the protease inhibitor (PI) gene
by direct sequencing of reverse transcriptionpolymerase chain reaction products from a wide
range of cattle tissues. A total of six different
intragenic haplotypes were identified in the North
American Holstein population, and these were
examined for associations with milk production
traits in 24 half-sib families comprising 1007 sons
Keywords: Mehsana buffaloes, Bubalus bubalis,
PCR-RFLP, PI gene, protease inhibitors, gene
polymorphism
Department of Animal Genetics and Breeding, College of Veterinary Science and Animal Husbandry, Anand
Agricultural University, Anand, Gujarat 388001, India
126
Buffalo Bulletin (June 2013) Vol.32 No.2
total reaction and incubated in a water bath at 37°C
for 16 h. The digestion products were separated by
electrophoresis on a 2% agarose gel in 0.5 X TBE
buffer.
Cloning and sequencing
PCR products from representative
sample (448 bp) were puriﬁed and cloned in
pTZ57R/T vector. Ligated recombinant vector was
transformed in competent E. coli (DH5-Į) cells.
Recombinant plasmids were extracted and used for
cycle sequencing and were subjected to automated
DNA sequencing.
Sequence data obtained were analyzed
in silico by employing software tools, viz. NCBI
BLAST, SeqScape and ClustalW, to access the
genetic variation.
utilizing a granddaughter design. One common
haplotype was associated with increased milk and
fat yields, increased productive life and decreased
somatic cell score. Another common haplotype
was associated with decreased productive life and
increased somatic cell score. One rare haplotype
was associated with decreased milk, fat, and protein
yields and increased milk protein percentage;
another rare haplotype was associated with
decreased milk yield, increased protein percentage,
and decreased productive life.
MATERIALS AND METHODS
Buffalo population, sampling and DNA
extraction
To analyze PI/SfaNI polymorphism, blood
samples were collected randomly from 60 unrelated
Mehsana buffaloes registered under the progeny
testing programme of the Dudhsagar Research
and Development Association, Mehsana, Gujarat
state. DNA was extracted using a standard protocol
by phenol: chloroform extraction procedure
(Sambrook et al., 1989).
Molecular genotyping
A primer pair reported by Khatib et al.
(2005) F: ATG GCA CTC TCC ATC ACG CG,
R: CCA CTA GCT TTG CAC TCT CA was used
to amplify the 448 bp region of PI exon 2.
PCR was carried out in a ﬁnal reaction
volume of 25 ȝl. Ampliﬁcation cycling conditions
for PI involved initial denaturation at 94°C for 10
minutes, followed by 35 cycles at 94°C for 1 minute,
56°C for 45 seconds and 72°C for 1 minute with a
ﬁnal extension at 72°C for 10 minutes) (Figure 1).
For the PCR-RFLP analysis, 10 ȝl of
each PCR ampliﬁed product was digested with 5
units of the SfaNI 5’-G C A T C (N)5^-3’ in a 30 ȝl
RESULTS AND DISCUSSION
On screening the PI/SfaNIin the 60 Mahsana
buffalo individuals, all the samples showed an
identical restriction pattern with RE site at 242 bp
and 366 bp on both chromosomes resulting in the
appearance of three bands of 242 bp, 124 bp, 82 bp
with BB genotype (Figure 2). This result indicates
that there was no SfaNI polymorphism at the PI
locus in Mehsana buffalo. Allele B for PI is ﬁxed in
Mehsana buffaloes. As there is no polymorphism
association, studies for production traits were not
undertaken.
The RFLP results are not in accordance
with Khatib et al. (2005) as there was no SfaNI
polymorphism at PI locus in Mehsana buffalo.
Khatib et al. (2005) reported ﬁve SNP in coding
regions of the protease inhibitor (PI) gene.
Published reports for PI/ SfaNI polymorphism in
buffaloes were not available to compare with till
preparation of this manuscript.
127
Buffalo Bulletin (June 2013) Vol.32 No.2
Figure 1. PCR product of protease inhibitor exon 2 locus.
Lane 1, 2, 4, 5: 448 bp PI gene product.
Lane 3: 100 bp ladder.
Figure 2. PI exon 2 -SfaNI RFLP: 448 bp PCR fragment in Mehsani buffalo digested by SfaNI and
electrophoresed on 2% agarose in 0.5 X TBE at 80 V.
Lanes 1,2: PI /SfaNI digest with three fragments of 242 bp, 124 bp, 82 bp.
Lane 3: PCR product of 448 bp.
128
Buffalo Bulletin (June 2013) Vol.32 No.2
Plate 1. Clustal W results of PI exon 2.
129
Buffalo Bulletin (June 2013) Vol.32 No.2
of a genomic clone for bovine pancreatic
trypsin inhibitor by using a unique-sequence
synthetic DNA probe. Proc. Natl. Acad. Sci.
U.S.A., 80: 6838-6842.
Barra, D., S.M. Simmaco, S.F. Bossa, E.F.
Angelettill and A. Franca. 1987. Primary
structure of a protease isoinhibitor from
bovine spleen: a possible intermediate in
the processing of the primary gene product.
J. Biol. Chem., 262(29): 13916-13919.
Blank, C.A. and M. Brantly. 1994. Clinical
features and molecular characteristics of
Į1-antitrypsin deﬁciency. Ann. Allergy, 72:
105-120.
Khatib, H., E. Heifetz and J.C.M. Dekkers. 2005.
Association of the protease inhibitor gene
with production traits in Holstein dairy
cattle . J. Dairy Sci., 88(3): 1208-1213.
Sambrook, J. and D.W. Russell. 2001. Molecular
Cloning: A Laboratory Manual. Cold Spring
Harbour Laboratory, Cold Spring Harbour,
New York, U.S.A.
Representative samples from Mehsana
buffaloes for PI locus were sequenced and
forward and reverse sequences were assembled
along with reference sequences on Seq Scape
software. A consensus sequence of 426 bp was
obtained. Consensus sequence was then aligned
with published in GenBank for PI gene exon-2 in
cattle using NCBI BLAST, ClustalW programme
revealed strong homology of 94 % to 98 % (Table
1). There was no sequence available in Genbank
database for buffaloes until this manuscript was
prepared to compare with. The nucleotide sequence
variation between cattle and buffalo was present
at ten nucleotide positions, i.e., 110,141,146,170,
236, 257, 277, 306, 350, and 396 (Plate 1 ).
PI (exon2) nucleotide sequences were
submitted to NCBI Genbank database accession
number GQ385225.
REFERENCES
Anderson, S. and I.B. Kingston. 1983. Isolation
Table 1. Bubalus bubalis Protease Inhibitor - Blastn in GenBank + EMBL DDBJ.
Accession
Description
Location/
Source
Max
Indent
BT025459.1
Bos taurus serpin peptidase inhibitor, clade
A (alpha-1 antiproteinase, antitrypsin),
member 1 (SERPINA1), mRNA, complete
cds
1-426
(164-589)
97%
2
BC102730.1
Bos taurus serpin peptidase inhibitor,
clade
A
(alpha-1
antiproteinase,
antitrypsin), member 1, mRNA (cDNA
clone MGC:127781 IMAGE:30957372),
complete cds
1-426
(72-497)
97%
3
X 63129.1
Bos taurus mRNA for alpha-1-antitrypsin
1-426
(55-480)
97 %
Sr. No.
1
130
Buffalo Bulletin (June 2013) Vol.32 No.2
Original Article
SOX-2 GENE EXPRESSION PATTERN IN STEM CELLS DERIVED FROM DIFFERENT
STAGES OF IN VITRO PRODUCED BUFFALO (Bubalus bubalis) EMBRYOS
Ch. Srinivasa Prasad1, A. Palanisamy2, S. Satheshkumar2, V.S. Gomathy3,
G. Dhinakar Raj2 and A. Thangavel3
cause of the low levels of Sox-2 gene expression
after fertilization. Based on the results it was
believed that Sox-2 was co-expressed with Oct-4
in the ES cells and acts synergistically with Oct-4
to activate Oct-Sox enhancers, which regulate the
expression of pluripotent stem cell-speciﬁc genes,
ABSTRACT
This study aims to compare the Sox-2 gene
expression in stem cells derived from various stages
of in vitro produced buffalo embryos. Primers were
designed based on the Sox-2 sequence (NCBI
Ac. No: DQ487021.1) of Chinese swamp buffalo
available in Pubmed GenBank by a Web-based
primer3 designing programme to obtain a product
of 413bp. For zonalysis and subsequent isolation of
ES cells 0.5 percent pronase was used. The DNA
sequence of the RT-PCR product submitted to NCBI
Pubmed GenBank was given accession number:
EU661361. Strong Sox-2 expression was observed in
the inner cells obtained from 16-cell stage embryos,
morulae and inner cell masses of blastocyst. Out of
six trials, in two trials the blastomeres/ inner cells
of 2-cell, 4-cell and 8-cell stage embryos did not
express Sox-2 gene even though they were believed
to be totipotent. But in four trials a faint band was
observed. The Sox-2 gene expression pattern was
low and variable in stem cells derived from early
embryos but gradually became more regular, with
100 percent expressing Sox-2 from the 16-cell stage
onward. This might be related to the exhaustion of
maternally generated Sox-2 transcripts and then
its recovery via expression of zygotic transcripts,
which takes place in buffalo embryos at the 8-16
cell stage. Epigenetic mechanisms might be the
including Nanog, Oct-4 and Sox-2 itself.
Keywords: embryonic stem cells, Sox-2, Gene
expression, buffalo, Bubalus bubalis
INTRODUCTION
The pluripotency of ES cells is thought to
be maintained by a few key transcription factors,
including Oct-4, Sox-2 etc. These transcription
factors in ES cells have been extensively
characterized in mouse and human, but for those of
domestic animals and particularly buffalo (Anand
et al., 2008; Kumar et al., 2008) little information
is available.
Sox-2 is a member of the Sry (Sex
determining region-Y) related transcription factor
family. Sox-2 function in ES cells was ﬁrst identiﬁed
in relation to Oct-4. Sox-2 and Oct-4 expressions
overlap during early embryogenesis, and both are
important for the maintenance of the pluripotent
state. Sox-2 can be regarded as one of the cofactors
1
Department of Veterinary Physiology, NTR College of Veterinary Science, Gannavaram - 521 102, India
Department of Animal Biotechnology, Madras Veterinary College, Chennai - 600 007, India
3
Department of Veterinary Physiology, Madras Veterinary College, Chennai - 600 007, India
2
131
Buffalo Bulletin (June 2013) Vol.32 No.2
72oC, and a ﬁnal extension for 10 minutes at 72oC.
These primers ampliﬁed a fragment of 413 bp.
The Sox-2 RT-PCR product was analyzed by gel
electrophoresis along with a standard 100 bp ladder
as marker. Then the gel was visualized under a UV
light gel documentation unit.
The cDNA sequence of the Sox-2 RT-PCR
product submitted to NCBI Pubmed GenBank was
given accession number: EU661361.The sequences
were analyzed for phylogenic conservation
and the sequence homology across the species
was established. Homologies of the Sox-2 gene
(mRNAs) were compared with reported sequences
of other species retrieved from annotated databases
such as the National Centre of the Biotechnology
information (www.ncbi.nih.gov). The BLASTn
search of highly similar sequence homology were
explored. The cDNA sequences of the Sox-2 gene
of Oct-4, since it activates the transcription of target
genes, such as Fgf-4, Utf-1, Fbx-15, and Lefty-1 in
cooperation with Oct-4. Moreover, Sox-2 expression
is regulated by Oct-4 and Sox-2, indicating that a
positive feedback mechanism may be involved in
the maintenance of ES cell self-renewal.
MATERIALS AND METHODS
Embryos were incubated in solution
containing 0.5 percent pronase until the zona was
removed. To isolate the inner cell mass (ICM),
blastocysts were incubated for 3-4 minutes in
solution containing calcium ionophore until the
zona and trophectoderm were lysed. Embryos
were observed constantly under a zoom stereo
microscope until the zona and trophectoderm were
lysed. Residuary embryos/ zonafree embryos were
washed with phosphate buffered saline containing
10 percent FBS. Zonafree embryos were incubated
in Ca++/ Mg++ free PBS for 10 to 15 minutes
at 37oC in CO2 incubator. Repeated pipetting
through Pasteur pipette disaggregates the zonafree
blastomeres/ inner cells.
Sox-2 was detected by reverse
transcription-polymerase chain reaction (RT-PCR).
Primers were designed based on the sequence
(NCBI Ac. No: DQ487021.1) of Chinese swamp
buffalo available in Pubmed GenBank by using
a Web-based primer 3 designing programme.
RT-PCR for Sox-2 was carried out with 100
ng of RNA by using the forward primer (5’
GCCGAGTGGAAACTTTTGTC 3’) and the reverse
primer (5’ TGCGAAGCTGTCATAGAGTTG 3’)
with the following cycling proﬁle: cDNA synthesis
for 15 minutes at 50oC, initial denaturation at 95oC
for 2 minutes followed by 36 cycles of 30 seconds
at 94oC, 30 seconds at 55oC and 45 seconds at
were phylogenetically analysed using Lasergene
version 4.1 (DNASTAR Package, USA). The Sox-2
gene sequences used for comparison are presented
in Table 1.
RESULTS AND DISCUSSION
I In two out of six trials the blastomeres/
inner cells of 2-cell, 4-cell and 8-cell stage embryos
(Pre ZGA) did not express the Sox-2 gene whereas
inner cells obtained from 16-cell stage embryos,
morulae and inner cell masses of blastocyst
consistently expressed the Sox-2 gene. Representative
photographs of gel for Sox-2 gene expression are
presented in Figure 1.
The percentage of identity among the
sequences of the Sox-2 gene from various isolates
in comparison with MVC, TANUVAS is shown in
Figure 2. The phylogenetic analysis of the Sox-2
gene from various species by clustralW method with
132
Buffalo Bulletin (June 2013) Vol.32 No.2
Table 1. Sox-2 gene sequences used for comparison of homology.
Species
Bubalus bubalis
(Chinese swamp buffalo)
Bos taurus
Ovis aries
Canis familiaris
Sus scrofa
Homo sapiens
Homo sapiens
Mus musculus
Rattus norvegicus
Gallus gallus
Accession No
Percent homology
DQ487021.1
99
NM 001105463.1
X96997.1
XM 545216.2
EU503117.1
BC013923.2
Z31560.1
NM 011443.3
NM 001109181.1
D50603.1
99
98
97
96
95
95
93
93
84
Figure 1. Sox-2 gene expression in stem cells derived from different stages of in-vitro produced buffalo
embryos.
Lane I
Lane II
Lane III
Lane IV
Lane M
Lane V
Lane VI
= Sox-2 amplicon in Two celled embryos
= Sox-2 amplicon in Four celled embryos
= Sox-2 amplicon in Eight celled embryos
= Sox-2 amplicon in Sixteen celled embryos
= 100 bp DNA ladder
= Negative Control
= Sox-2 amplicon in Morulae
133
Buffalo Bulletin (June 2013) Vol.32 No.2
bootstrap analysis, presented in Figure 2, revealed
two major clusters. The Sox-2 gene lineage of
Chinese swamp buffalo, cattle, pig, amur tiger, rhesus
monkey, Norway rat, dog and lizard were grouped to
the ﬁrst cluster. The lineage of this ﬁrst cluster again
sub-grouped into three clusters of which dog, mouse,
human (US, France, Yugoslavia), Norway rat, rhesus
monkey, sheep and pig (USA) were grouped to one
cluster, Indian water buffalo of TANUVAS, platypus
and xenopus were grouped to another cluster and
lizard, amur tiger, cattle (Italy), Chinese swamp
buffalo and pig (China) were grouped to another
cluster. The second cluster revealed a lineage
covering the human (Italy), zebra ﬁsh and chicken.
The bootstrap analysis is shown on every node from
common ancestor.
Amino acid analysis of the Sox-2 gene
revealed hypervaraible regions at the positions 23682373 and 2380. The variations were in relation with
species speciﬁcity.
The HMG domain containing Sox-2
and the POU domain-containing Oct-4 were
the transcription factors known to be essential
for normal pluripotent cell development and
maintenance (Avilion et al., 2003). Their function
regulatory network.
Sox-2 mRNA was detected in oocytes as
well as in embryos at the different developmental
stages analyzed, resembling the proﬁle of Oct-4
(Srinivasa Prasad, 2008). Sox-2 was present as both
maternal and embryonic transcript; in particular,
a signiﬁcant increase from the 16-cell stage,
concomitant with embryo genome activation, was
observed suggesting that Sox-2 expression might
be regulated by Oct-4. The results of the present
study are in agreement with the ﬁndings of Brevini
et al. (2008). The Sox-2 gene expression patterns
were variable in stem cells derived from early
embryos but gradually became more regular, with
100 percent expressing Sox-2 from the 16-cell stage
onward. This might be related to the maternalzygotic transition (activation of the embryonic
genome), which takes place in buffalo embryos
at the 8-16 cell stage. This might be related to
the exhaustion of maternally generated Sox-2
transcripts and then its recovery via expression
of zygotic transcripts. An epigenetic mechanism,
consisting of DNA methylation and chromatin
remodeling, might be the cause of the low levels of
Sox-2 gene expression after fertilization as reported
by Hattori et al., 2004.
Sox-2 expression in ES cells is regulated
by Sox-2 itself and Oct-4, suggesting the possibility
that Sox-2 isactivated in primitive cells by a positive
autoregulatory loop. Therefore, it is speculated that
the same positive feedback loop maintained the
expression of Sox-2 and Oct-4 together and that the
Sox-2 and Oct-4 are regulated coordinately (Boyer
et al., 2005).
The essential function of Sox-2 was to
stabilize ES cells in a pluripotent state by maintaining
the requisite level of Oct-4 expression (Masui et
al., 2007). A critical amount of Oct-4 has recently
been reported to be crucial for the maintenance
in pluripotent cells is via a synergistic interaction
between the two to drive transcription of target
genes. Sox-2 is co-expressed with Oct-4 in the ES
cells and acts synergistically with Oct-4 to activate
Oct-Sox enhancers, to regulate the expression of
pluripotent stem cell-speciﬁc genes, including
Nanog, Oct-4 and Sox-2 itself as well as Fgf4,
Utf1, and Fbx15. Each of these target genes has a
composite element containing an octamer and a Sox
binding site. Genetic links between the Sox2-Oct4
complex and Sox-2 and Pou5f1 expression, as well
as their in vivo binding to these genes in mouse and
human ESCs (Chew et al., 2005), suggests that this
complex is at the top of the pluripotent cell genetic
134
Table 2. Percentage of identity among the sequences of sox-2 gene from various isolates in comparison with Indian water buffalo of TANUVAS.
Buffalo Bulletin (June 2013) Vol.32 No.2
135
Buffalo Bulletin (June 2013) Vol.32 No.2
25.0
20.0
14.3
61.8
66.0
62.0
45.2
Canis lupus familiaris (dog).seq
Mus musculus (house mouse).seq
Homo sapiens (human)-FRANCE(2).seq
Rattus norvegicus (Norway rat).seq
Homo sapiens (human)-YOGOSLAVIA.seq
Homo sapiens (human)-.seq
Macaca mulatta (rhesus monkey).seq
99.4
Bos taurus (cattle).seq
NA
Ovis aries (sheep).seq
53.8 47.9 Homo sapiens (human)-US.seq
Homo sapiens (human)-FRANCE.seq
58.3
78.5 Sus scrofa (pig).seq
Sus scrofa (pig)-US.seq
36.1
BUBALUS BUBALIS ±MVC.TANUVAS.seq
9.1
Ornithorhynchus anatinus (platypus).seq
Xenopus tropicalis (Silurana tropicalis
100.0
Eremias brenchleyi (LIZARD).seq
50.6
97.2
Panthera tigris altaica (Amur tiger).se
93.1
Bos taurus (cattle) ITALY.seq
57.2
BUBALUS BUBALIS (CHINA).seq
61.3
Sus scrofa (pig)-china.seq
Oreochromis mossambicus (Mozambique til
80.3
Homo sapiens (human)-ITALY.seq
100.0
Danio rerio (zebrafish).seq
Gallus gallus (chicken).seq
142.8
140
120
100
80
60
40
Nucleotide Substitutions (x100)
Bootstrap Trials = 1000, seed = 111
20
0
Figure 2. Phylogenetic analysis of Sox-2 gene from various species by ClustralW method with bootstrap
analysis.
(Niwa et al., 2000; Lanza et al., 2006).
Based on the results it is speculated that
the observed lack or low expression of Sox-2 in
cells derived from early embryos (pre ZGA) might
be the reason for inadequate ability of those cells
to retain the property of stemness, to form primary
stem cell colonies and subsequently ES-cell lines
compared to inner cells derived from morulae and
blastocysts (Ito et al., 1996; Hatoya et al., 2006) as
Oct-4 is essential for antiapoptosis of stem cells in
response to stress effects that might be mediated
through the STAT3/Survivin pathway, Sox-2 being
responsible for maintaining the requisite level of
Oct-4 expression (Masui et al., 2007).
of ES cell self-renewal. A 50 percent decrease in
the endogenous Oct-4 levels relative to that of
undifferentiated ES cells results in the commitment
of ES cells to trophoectoderm lineages, containing
both proliferating and endoduplicating giant cells
based on the culture conditions. However, an
increase beyond the 50 percent threshold level
of Oct-4 leads to the concomitant differentiation
of ES cells into extra-embryonic endoderm and
mesoderm. Interestingly, LIF withdrawal leads to
the speciﬁcation of the same lineages. Less subtle
changes in Oct-4 level (both increase and decrease)
do not affect ES cell self-renewal. In conclusion, the
precise level of Oct-4 protein governs commitment
of embryonic cells along three distinct lineages
136
Buffalo Bulletin (June 2013) Vol.32 No.2
stem cells. Mol. Cell. Biol., 25(14): 60316046.
Hatoya, S., R. Torii, Y. Kondo, T. Okuno, K.
Kobayashi, V. Wijewardana, N. Kawate,
H. Tamada, T. Sawada, D. Kumagai, K.
Sugiura and T. Inaba. 2006. Isolation and
characterization of embryonic stem-like
cells from canine blastocysts. Mol. Reprod.
Dev., 73(3): 298-305.
Hattori, N., K. Nishino., Y.G. Ko, N. Hattori, J.
Ohgane, S. Tanaka and K. Shiota. 2004.
Epigenetic control of mouse Oct-4 gene
expression in embryonic stem cells and
ACKNOWLEDGEMENT
The work was carried out at the Centralized
Embryo Biotechnology Unit, Department of
Animal Biotechnology in the DBT scheme on
“Establishment of Buffalo ES-Cell Line”. Authors
are thankful to the funding agency.
REFERENCES
Anand, T., D. Kumar, M.K. Singh, M.S. Chauhan,
R.S. Manik and P. Palta. 2008. Expression
of pluripotency-determining factors in in
vitro-produced buffalo embryos. Reprod.
Fert. Develop., 20(1): 163
Avilion, A.A., S.K. Nicolis, L.H. Pevny, L. Perez,
N. Vivian and R. Lovell-Badge. 2003.
Multipotent cell lineages in early mouse
development depend on SOX2 function.
Gene. Dev., 17(1): 126-140.
Boyer, L.A., T.I. Lee, M.F. Cole, S.E. Johnstone,
S.S. Levine, J.P. Zucker, M.G. Guenther,
R.M. Kumar, H.L. Murray, R.G. Jenner,
D.K. Gifford, D.A. Melton, R. Jaenisch
and R.A. Young. 2005. Core transcriptional
regulatory circuitry in human embryonic
stem cells. Cell, 122: 947-956.
Brevini, T.A.L., S. Antonini, F. Cillo, G. Pennarossa,
S. Colleoni, G. Lazzari, C. Galli and F.
Gandolﬁ. 2008. Expression pattern of the
sox-2 gene in bovine oocytes and in vitroderived embryos. Reprod. Fert. Develop.,
20(1): 165.
Chew, J-L., Y-H Loh, W. Zhang, Xi Chen, W-L
Tam, L-S Yeap, P. Li, Y-S Ang, B. Lim,
P. Robson and H-H Ng. 2005. Reciprocal
transcriptional regulation of Pou5f1 and Sox2
via the Oct4/Sox2 complex in rmbryonic
trophoblast stem cells. J. Biol. Chem.,
279(17): 17063-17069.
Ito, T., M. Konishi., T. Takedomi., H.Itakura.,
S.Yazawa and Y. Aoyagi. 1996. Inﬂuence
of stage and derivation of bovine embryos
on formation of embryonic stem (ES) like
colonies. J. Reprod. Dev., 42(5): 29-33.
Kumar, D., T. Anand, K.P. Singh, M.S. Chauhan, P.
Palta and R.S. Manik. 2008 Expression of
Oct-4 in buffalo (Bubalus bubalis) embryos
generated through in vitro fertilization or
parthenogenetic activation. Reprod. Fert.
Develop., 20(1): 162-163.
Lanza, R., J. Gearhart, B. Hogan, D. Melton, R.
Pederson, J. Thomson, E.D. Thomas and M.
West. 2006. Essentials of Stem Cell Biology.
Elsevier Academic Press.
Masui, S., Y. Nakatake, Y. Toyooka, D. Shimosato,
R. Yagi, K. Takahashi, H. Okochi, A.
Okuda, R. Matoba, A.A. Sharov, M.S.H. Ko
and H. Niwa. 2007. Pluripotency governed
by Sox2 via regulation of Oct3/4 expression
in mouse embryonic stem cells. Nat. Cell
Biol., 9: 625-635.
*Continued on page 156
137
Original Article
Buffalo Bulletin (June 2013) Vol.32 No.2
SEQUENCE CHARACTERIZATION AND POLYMORPHISM DETECTION
IN THE BUBALINE CD14 GENE
Aruna Pal1, Arjava Sharma1, T.K. Bhattacharya2, A. Mitra1 and P.N. Chatterjee3
fragment of the CD14 gene ranging from the 3rd
ABSTRACT
nucleotide to the 261st nucleotide of the 2nd exon
of the CD14 gene revealed identical nucleotide
sequences, indicating monomorphism.
The present study was undertaken with
the objectives of sequence characterization
and identiﬁcation of polymorphisms in the
bubaline CD14 gene. Single strand conformation
polymorphism analysis revealed a total of eight
different variants viz. CD14-A, CD14-B, CD14-C,
CD14-D, CD14-E, CD14-F, CD14-G and CD14-H
in exon 2 of the CD14 gene for four breeds of buffalo.
Variant CD14-A was observed to be the wild type
as it exhibits highest frequencies, whereas CD14-D
was observed to be genetically distant from others.
Polymorphism or variability may be regarded to be
the highest in the Mehsana breed of buffalo, which
exhibited six genotypes out of eight. Thus 42 SNPs
were identiﬁed for the CD14 gene of buffalo. The
CD14 gene ranging from the 592nd to the 856th
Keywords: CD14
polymorphism, SSCP
gene,
buffalo,
exon,
INTRODUCTION
Mastitis is one of the most common diseases
affecting dairy cattle and buffaloes causing huge
economic losses to dairy farmers. In India, about
3.84% and 24.43% of buffaloes, respectively,
are affected with clinical and subclinical mastitis
every year (Singh and Singh, 1994). The annual
loss due to clinical and subclinical mastitis in
buffaloes was estimated to be Rs. 6,962,900,000
and Rs. 17,233,200,000, respectively (Dua, 2001).
Incidences of diseases of milch animals is critical
to dairy industry, wreaking economic havoc in
terms of veterinary and treatment costs, reduced
milk production in the subsequent lactation, milk
condemnation due to antibiotic residues, early
culling, extra labour involvement and deterioration
in milk quality. Antibiotic therapy and vaccination
have their own limitations. Thus, it seems
nucleotide of the second exon, which corresponds
to the 198th to the 285th codon of the coding
sequence was found to be highly polymorphic and
may be regarded as the ‘mutational hot spot’, or
the hyper variable site, leading to ligand diversity.
Comparison of nucleotide sequences of different
regions of the buffalo CD14 gene with that of
taurine cattle revealed a total of 22 point mutations,
of which eleven were non-synonymous codon
with eleven amino acid substitutions. The 258 bp
1
Animal Genetics Division, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India, E-mail:
arunachatterjee@gmail.com
2
Project Directorate on Poultry, Rajendra Nagar, Hyderabad, India
3
Animal Nutrition Division, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India
138
Buffalo Bulletin (June 2013) Vol.32 No.2
(Pal and Chatterjee, 2009a; Pal et al., 2008)
Detection of SNPs is useful for analysis of
the evolutionary history of species development,
assessment of biodiversity, associative studies
between polymorphisms and disease resistance.
The variability at the nucleotide level of the CD14
gene leads to the variability in the CD14 encoded
molecule, which in turn gives rise to the phenotypic
variability in host immune response. Thus, the
variants of the CD14 gene and their association
with the incidences of disease occurrence may
be used as a marker for disease resistance.
Association of polymorphic candidate genes with
economic traits will help the breeders to search
some genetic markers for economic traits. This
may be used as an aid to the selection of bulls
at an early age and can save huge economic loss
for rearing the bulls till maturity. In spite of its
tremendous potential to be exploited, very little
work has so far been done whereas such type of
marker trait association studies have also been
documented by a number of workers in various
ETLs (Pal et al., 2004; Pal et al., 2005) No reports
are available so far for genetic polymorphism
of the CD14 gene in any animal at the coding
region; however CD14 gene polymorphism study
at the promoter region is available in human. In
the last decade, many ﬁne mapping experiments
have resulted in identiﬁcation of several QTLs
in cattle affecting milk production and udder
health emphasizing their potential value in
marker assisted selection programs. However,
beyond ﬁne mapping, the ultimate target of QTL
analysis is the identiﬁcation of casual gene itself
which would facilitate identiﬁcation of resistance
genes and alleles (Reinard and Riollet, 2005).
Scanty reports are available so far for genetic
polymorphism of the CD14 gene in an animal
at the coding region; however CD14 gene
understanding and subsequent manipulation of
the host immune response is the most precise and
effective tool to lower the disease incidences and
to nullify the limitations associated with antibiotic
treatment or vaccination. Selective breeding of
dairy cattle and buffaloes for reduced susceptibility
to/increased resistance against mastitis is difﬁcult
as it is a polygenic trait with very low heritability.
However, indirect selection based on somatic cell
count and candidate gene markers can help to
increase the efﬁciency of such breeding programs.
A fair amount of genetic research related to udder
health has already been performed due to its
importance from the economic as well as from
the quality control points of view (Ogorevc et al.,
2009)
CD14 is an important molecule for
innate immunity. The CD molecule ranges from
1 to 166 with differential structure and functions
(Goldsby et al., 2000), of these CD14 is the most
important molecule known so far, playing a vital
role against several endotoxigenic bacteria. Its
pattern recognition receptor binds mainly with LPS
(lipopolysaccharide), lipotechoic acid, arachidonic
acid and thus releases various cytokines which
acts for the body’s defence. The body’s immunity
thus can act against a wide range of pathogens
including gram-negative bacteria and gram positive
as Mycobacterium sp., Pseudomonas sp. and
Staphylococcus aureus etc. CD14 functions both
as a cell membrane receptor and a soluble receptor
for bacterial LPS. It has been considered as an
important molecule for its role in various diseases,
like mastitis (Lee et al., 2003), treponemiasis
(Schroder et al., 2000) and glomerulonephritis
(Yoon et al., 2003). Soluble CD14 enriched bovine
colostrums and milk induces B cell growth and
differentiation (Fillip et al., 2001). The CD14 gene
has been cloned and sequenced in other ruminants
139
Buffalo Bulletin (June 2013) Vol.32 No.2
Livestock Farm, Etawah (U.P.) respectively .
Blood was collected from the jugular
vein into EDTA containing vacutainer tubes and
DNA extraction was performed from whole blood
following a standard phenol-chloroform extraction
method (Sambrook and Russell, 2001).
polymorphism study at the promoter region
is available in human (Hubacek et al., 1999;
Hartel et al., 2004; Guerra et al., 2004).
Buffaloes are economically important
animals with higher milk producing ability (54%)
with high SNF and fat percentage, better capacity
to utilize coarse fodders and their innate resistance
to a wide range of diseases (Annon, 2001). Most
Indian buffaloes are of the riverine type; there are
a few swamp buffaloes in the northeastern region.
So far no reports are available regarding molecular
characterization of the CD14 gene in buffaloes and
very scanty reports are available regarding SNP
detection in any farm animal.
Keeping the above facts in view, the
present investigation was planned to identify
the polymorphism of the CD14 gene of buffalo,
analyze the sequences of the identiﬁed variants and
compare them with those of other species.
Ampliﬁcation of the Bubaline CD14 gene
The 2nd exon of CD14 gene was studied
in two fragments. A 258 bp fragment (the 3rd
nucleotide to the 261st nucleotide of CD14 cDNA)
of the coding sequence of Bubaline CD14 (the
587th to the 854th nucleotide of second exon)
was ampliﬁed using the forward primer as
ATGGTGTGCGTGCCCTACCTG and the reverse
primer as TATGCTGACACAATCAAGGCT and
the fragment obtained was of 258 bp. The reaction
mixture used was PCR buffer 1.2X, MgCl2 1.5
mM, dNTP 0.5 mM, forward primer and reverse
primer 60 ng each, Taq DNA polymerase 1 unit.
The reaction condition used was initial denaturation
at 94oC for 5 minutes, denaturation at 95oC for
45 seconds, followed by annealing for 61oC for
30 seconds, extension for 72oC for 45 seconds,
repeated for 34 cycles followed by ﬁnal extension
for 72oC for 7 minutes.
The second fragment ampliﬁed was a 265
bp fragment of the coding sequence of Bubaline
CD14 (the 587th to the 854th nucleotide of the
second exon). PCR was carried out in a ﬁnal
volume of 25 l of reaction mixture containing
80-100 ng DNA, PCR buffer 1.2X, MgCl2 1.5 mM,
dNTP 0.5 mM, forward primer and reverse primer
60 ng each, Taq DNA polymerase 1 unit. The DNA
was subjected to ampliﬁcation with polymerase
chain reaction in a thermocycler (PTC-200, MJ
Research, USA) using the forward primer as
AGCGAACGACAAATTGAGAGACCTTAGTG
and the reverse primer as AAGGTCTCTCAATT
MATERIALS AND METHODS
Animals
Blood samples were collected from a
total of 246 unrelated animals belonging to four
different breeds of Indian riverine buffaloes
(Bubalus bubalis) with Murrah (103), Mehsana
(69), Surti (36) and Bhadawari (38). Buffaloes
were maintained at different government farms in
India as well as farmers’herds, gaushals and NGOs.
Samples from Murrah buffalo were collected from
farmers’ herds, gaushals and NGOs at Dubrajpur
Block, Birbhum district, West Bengal and the
Indian Veterinary Research Institute, Izatnagar
(U.P.). Samples from Mehsana and Surti buffaloes
were collected from Gujrat Agricultural University,
S.K. Nagar (Gujrat), Gujrat Agricultural University,
Navasari, Anand (Gujrat), and the Government
140
Buffalo Bulletin (June 2013) Vol.32 No.2
A phylogenetic tree was constructed for detecting
the genetic distance between different variants
and their evolutionary signiﬁcance. (MegAlign
Programme of Lasergene Software, By ClustalW
Method, DNASTAR).
The genotypes were detected directly
by observing the SSCP pattern of each sample in
the polyacrylamide gel. The gene and genotype
frequencies were estimated by a direct counting
method (Falconer and Mackay, 1998). PCR
products from different genotypes were selected
and sequenced using respective forward and
reverse primers to detect variations if any, at the
nucleotide level. Then sequences were aligned with
those of the reported CD14 sequences of different
species using MegAlign Programme of Lasergene
Software DNASTAR, Inc, Madison WI, USA).
Multiple sequence alignments were performed with
the Megalign. program of LASERGENE software.
The coding DNA sequences of different exonic
regions were conceptually translated to amino acid
sequences using the same software.
TGTCGTTCGCTGGGC and the fragment
obtained was of 265 bp. The reaction condition
used was initial denaturation at 94oC for 5
minutes, denaturation at 95oC for 45 seconds,
followed by annealing for 61oC for 30 seconds,
extension for 72oC for 45 seconds, repeated for 34
cycles followed by ﬁnal extension for 72oC for 7
minutes.
Genotyping
by
PCR-Single
stranded
conformation polymorphism
SNPs were detected by PCR-SSCP and
identiﬁed by subsequent sequencing. PCR-SSCP
was performed for all the samples. In 0.5 ml PCR
tubes, 3 l of ampliﬁed PCR-product was mixed
with 9 l of formamide dye.. This 12 l of PCRSSCP solution was denatured at 95oC for 5 minutes
followed by immediate chilling in ice for 15 minutes.
The products were run in 12% polyacrylamide
gel at 4oC for 11 h at 200 V in case of the 268 bp
fragment and 6 h for the 261 bp fragment. SSCP
fragments were visualized by silver staining and
documented in the Gel Documentation System.
Silver staining was carried out according to the
procedure described by Basam et al. (1991) with
some modiﬁcations.
The variants were detected directly by
observing the SSCP pattern of each sample in
the polyacrylamide gel. PCR products from
different variants were selected and sequenced
using respective forward and reverse primer to
detect variations if any, at the nucleotide level.
Then sequences were aligned with those of the
reported CD14 sequences of different species using
MegAlign Programme of Lasergene Software
(DNASTAR). Moreover, sequence variations were
also compared with the cDNA (Gene bank acc no.
DQ457089) and genomic DNA of buffalo (Gene
bank acc no. DQ444324), we have reported earlier.
RESULTS AND DISCUSSION
PCR-SSCP analysis of 258 bp fragment
The 258 bp fragment from the 3rd
nucleotide to the 261st nucleotide of the 2nd exon of
CD14 gene (Figure 1) revealed identical nucleotide
sequences (Gene bank acc no. EU370404),
indicating monomorphism of the gene (Figure 2).
This indicates the conserved nature of the CD14
gene of buffalo pertaining to this region. It reveals
the conserved nature of this gene for the ﬁrst 258
nucleotides, encoding for 86 amino acids. This
region in buffalo may probably be under strong
purifying selection which may explain the lack of
SNPs in this region. This may be the reason that 1
141
Buffalo Bulletin (June 2013) Vol.32 No.2
position (Hartel et al., 2004, Guerra et al., 2004),
(-1619), (-550) from the transcription start site of
the CD14 gene (Guerra et al., 2004).
CD14-E may be considered as the breed
speciﬁc marker for the Mehsana breed. whereas
CD14-G and CD14-H may be considered as breed
speciﬁc markers for the Mehsana and the Surti,
repectively. In the present study, the Mehsana
breed exhibited six genotypes out of eight. Thus
polymorphism or variability may be regarded
to be the highest in this breed. Simultaneously
heterozygosity may also be expected to be high in
this breed.
The frequencies of different variants
estimated in the population of four different
breeds are listed in Table 1 and Figure 12. The
most frequent variant (genotype) was identiﬁed
as CD14-A, with the overall genotypic frequency
being 0.47. The least frequent were CD14-G and
CD14-H, with very low frequencies (0.018) for
both the genotypes. The frequencies for other
genotypes were intermediate. Thus CD14-A may
be the original wild type allele for the gene. The
other variants may therefore be the result of recent
mutational events. Among the breeds, Bhadawari
had the highest frequency (0.818) for CD14-A
and Surti had the lowest frequency for this pattern
(0.071) which showed the highest homozygosity
in the Bhadawari population and least in the Surti
population.
CD14-D was observed to be genetically
distant from other variants as evidenced from Figure
13. Since the Surti breed of buffalo was observed
to have highest frequency for CD14-D (Figure 11),
it is expected that the Surti is genetically distant
from the other breeds.
to 60 nucleotide codes for signal peptide, when no
variation is expected. The present ﬁnding is similar
to CB cattle (Pal and Chatterjee, 2009b), where
monomorphism was reported for this nucleotide
region except for one nucleotide change.
PCR-SSCP analysis of 265 bp fragment
The ampliﬁed product of the 265 bp
fragment of the CD14 gene was observed by
agarose gel electrophoresis (Figure 3). Eight
variants designated as CD14-A, CD14-B, CD14-C,
CD14-D, CD14-E, CD14-F, CD14-G and CD14-H
were detected in 265 bp fragment of CD14 gene of
buffalo. The frequencies of the different variants
of bubaline CD14 gene are depicted in Table 1. In
the Bhadawari breed of buffalo, only three variants
were identiﬁed as CD14-A, CD14-B and CD14-F
(Figure 4) with the respective frequencies being
0.818, 0.045 and 0.136 (Figure 5). In the Mehsana
breed, all variants, except CD14-D and CD14-H
were identiﬁed (Figure 6). The highest frequency
was observed for the CD14-A genotype (0.465),
whereas CD14-B (0.056) and CD14-G (0.056) were
found to be the least frequent genotypes (Figure
7). The Murrah breed revealed only four patterns
(Figure 8) with CD14-A being the most frequent
(0.596), whereas CD14-B (0.105) and CD14-D
(0.105) were the least frequent genotypes (Figure
9). The Surti breed of buffalo showed a different
picture where the highest frequency was observed
for the CD14-D genotype, and the CD14-A (0.071)
and CD14-H (0.071) genotypes were the least
frequent (Figure 10, Figure 11). Since this is the ﬁrst
report of study for polymorphism of the CD14 gene
in farm animals and ﬁrst time in the coding region
in any animal so far, comparison was not possible.
However, polymorphism has been identiﬁed in
the promoter region of the CD14 gene in human
being at C (-260)ĺT (Hubacek et al., 1999), (-159)
142
Buffalo Bulletin (June 2013) Vol.32 No.2
Bos taurus, 95% with Bos indicus, 96% with Bos
grunniens, 91.5 with Capra hircus and 94.6 with
Ovis aries. The percent sequence similarity of the
bubaline 265 bp region was greater than 90% with
ruminants (Bos taurus, Capra hircus and Ovis aries)
while it was less than 90% with monogastric species
(Sus scrofa, Canis familiaris and Equus caballus).
Similarly, the percent homology of bubaline CD14
was more than 90% with ruminants while it was
84% with Equus caballus. Comparative analysis
of the buffalo CD14 peptide sequence with that of
cattle has been depicted in Table 4. This represents
the species speciﬁcity. Phylogenetic closeness of
buffalo with cattle as observed in the present study
has also been reported while studying other genes
like growth hormone gene (Pal and Chatterjee,
2010). Chicken has been found to be genetically
most distant to buffalo.
Sequence analysis of PCR-SSCP 265 bp
fragments
Six different SSCP variants CD14-A
(Gene bank acc no. EU370398), CD14-B (Gene
bank acc no. EU370399), CD14-C (Gene bank
acc no. EU370400), CD14-D (Gene bank
acc no. EU370401), CD14-E (Gene bank acc
no. EU370402), CD14-F (Gene bank acc no.
EU370403) were sequenced for determining the
variation at the nucleotide level. The combined
effects of the SNPs were estimated through PCRSSCP. Moreover, sequence variations were also
compared with the cDNA (Gene bank acc no.
DQ457089) and genomic DNA of buffalo (Gene
bank acc no. DQ444324), we have reported
earlier. Since this is the ﬁrst report of CD14 gene
polymorphism in coding region, comparison was
not possible. However, polymorphism studies
have been conducted at promoter region in human
(Hubacek et al., 1999; Guerra et al., 2004; Hartel
et al., 2004; Geaghan et al., 2010). The SNPs have
been depicted in Figure 14. A very high degree of
nucleotide sequence variability was observed for
CD14-B (81.3), CD14-C (97.0), CD14-D (86.0),
CD14-E (85.8) and CD14- F (85.8) as compared
to CD14-A (Table 2). A high degree of nucleotide
variation was observed between the alleles/patterns
identiﬁed within the coding sequence of CD14 gene
for the 265 bp fragment ranging from the 587th to
the 854th nucleotide the of second exon of buffalo,
which corresponds to the 197th to the 285th codon
of the coding sequence.
SNP detection of bubaline CD14 gene
Very high degrees of nucleotide variation
with 42 SNPs were identiﬁed with 39 nonsynonymous changes, leading to 23 amino acid
changes. Twenty-nine transversional mutations and
18 transitional mutations were detected (Table 5).
At some sites, both transitional and transversional
mutations were detected: at 702, 703, 764, 767, 770
nucleotide positions of CD14 gene of buffalo. Nonsynonymous substitutions exceeding synonymous
substitutions indicates the evolution of this protein
through positive selection among domestic animals.
SNPs for the CD14 gene of buffalo was reported
here for the ﬁrst time and moreover, this is the
ﬁrst report of SNPs in any farm animal. Combined
genotypes of SNPs were analyzed using a PCRSSCP method. Thus, the 268 bp fragment of the
coding sequence of Bubaline CD14 was found to
be highly polymorphic and may be regarded as
the ‘mutational hot spot’ or the hyper variable site,
Phylogenetic analysis of CD14 gene of riverine
buffalo
The percent homology of different regions
of the bubaline CD14 gene with that of various
species is presented in Table 3. The sequence
homology of the 258 bp region was 97.7% with
143
Buffalo Bulletin (June 2013) Vol.32 No.2
leading to ligand diversity. Similar higher degrees
of mutations have been observed in some other
genes as 25 SNPs in the leptin gene in Korean
cattle (Chung et al., 2008), 96 SNPs from TLRs and
their associated intracellular signaling molecules
(Dhiman et al., 2008) in human.
Most of the SNPs identiﬁed were within
LRR at amino acid positions 209, 210, 215, 217,
246, 249, 252, 255, 256, 257, 266, 267, 271, 273,
274, 275, 277, 280. It has been reported that LRR
in the extracellular domain is responsible for the
recognition of pathogens and participates in receptor
and ligand interactions (Gordon, 2002). Grooves
within the CD14 molecule were responsible for
receptor recognition and binding (Kim et al.,
2005). Since CD14 molecules can bind to a wide
range of substances including lipopolysaccharides
from gram-negative bacteria, lipo-arabinomannans
of mycobacteria, manuronic acid polymers
of Pseudomonas sp. and to peptidoglycans of
Staphylococcus aureus, it is expected that there
should be sufﬁcient variabilty in the pathogen
recognition and receptor binding site, which is
primarily comprised of LRR region of the CD14
molecule (Kim et al., 2005). Thus, the presence
of the LRR coding region may be the region for
maximum variability to enable the CD14 molecule
to bind with a wide range of substances. From the
sequence alignment studies, it was observed that
the particular leucine moeity was almost unaltered
within the leucine rich repeats and the variations
were observed for other amino acids. Thus the basic
function of leucine rich repeats remains unaltered.
However, in the absence of any available
report regarding the polymorphism study of this
particular region in livestock species, comparison
is not possible. Moreover, similar nucleotide
variations were also observed for this particular
region of the CD14 gene between different species,
which may give some insight.
Further research need be directed to
ﬁnding out the association of allelic variants with
traits related to disease incidences, to establish
markers. There is a need to ascertain the differential
biological potency of different allelic variations of
CD14. On the basis of such functional study, the
most potent variant can be expressed in vitro for
further therapeutic or immuno competence study.
Gene inserts containing the resistant variety of
CD14 gene of buffalo may be approached for
somatic gene therapy, particularly against mastitis.
Transgenic animal production with the buffalo
CD14 gene insert may provide scope for future
research to develop disease resistant stock.
REFERENCES
Anonymous. 2001. Dairy Year Book. Special
Millennium Issue-2001, 2nd ed. Sadana
Publishers and Distributors, Gaziabad, U.P.,
India.
Basam, B.J., G. Caetano-Anolles and P.M.
Gresshoff. 1991. Fast and sensitive silver
staining of DNA in polyacrylamide gels.
Anal. Biochem., 196: 80-83.
Chung, E.R., S.C. Shin, K.H. Shin and K.Y. Chung.
2008. SNP discovery in the leptin promoter
gene and association with meat quality and
carcass traits in Korean cattle. Asian Austral.
J. Anim., 21(12): 1689-1695.
Dhiman, N., I.G. Ovsyannikova, A. Robert, R.A.
Vierkant, E. Jenna, J.E. Ryan, V.S. Pankratz,
R.M. Jacobson, A. Gregory and G.A.
Poland. 2008. Associations between SNPs
in toll-like receptors and related intracellular
signaling molecules and immune responses
to measles vaccine: preliminary results.
144
Buffalo Bulletin (June 2013) Vol.32 No.2
Figure 1. Ampliﬁcation of the 258 bp fragment of the CD14 gene of buffalo.
Lane 1: Ampliﬁed product of 258 bp fragment.
Lane M: 100 bp DNA ladder.
Figure 2. PCR-SSCP pattern of the 258 bp fragment of the CD14 gene of buffalo.
145
Buffalo Bulletin (June 2013) Vol.32 No.2
Figure 3. Ampliﬁcation of the 265 bp fragment of the CD14 gene of buffalo.
Lane 1: Ampliﬁed product of 265 bp fragment.
Lane M: 100 bp DNA ladder.
Figure 4. PCR-SSCP pattern of the 265 bp fragment in the Bhadawari buffalo CD14 gene in 12% PAGE.
Lane 1-4 and 8: CD14-A.
Lane 9-12: CD14-F pattern.
Lane 5-7: CD14- B.
146
Buffalo Bulletin (June 2013) Vol.32 No.2
Bhadawari
0.9
0.8
0.7
0.6
0.5
Bhadawari
0.4
0.3
0.2
0.1
0
CD14- CD14- CD14- CD14- CD14- CD14- CD14- CD14A
B
C
D
E
F
G
H
Figure 5. Frequency distribution for different SSCP variants in the Bhadawari breed of buffalo.
Figure 6. PCR-SSCP pattern of the 268 bp fragment in the Mehsana buffalo.
Lane 1-3: CD14- F.
Lane 4: CD14- G.
Lane 5, 6, 10: CD14- B.
Lane 8, 9,11 : CD14- A.
Lane 12-13: CD14- C.
Lane 7: CD14- E.
147
Buffalo Bulletin (June 2013) Vol.32 No.2
Mehsana
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Mehsana
CD14- CD14- CD14- CD14- CD14- CD14- CD14- CD14A
B
C
D
E
F
G
H
Figure 7. Frequency distribution for different SSCP variants in the Mehsana breed of buffalo.
Figure 8. PCR-SSCP pattern of the 265bp fragment in the Murrah buffalo.
Lane 5-8: CD14-A.
Lane 1-4: CD14- B.
Lane 12-13: CD14- D.
Lane 9-11: CD14- F.
148
Buffalo Bulletin (June 2013) Vol.32 No.2
Murrah
0.7
0.6
0.5
0.4
Murrah
0.3
0.2
0.1
0
CD14-A CD14-B CD14-C CD14-D CD14-E CD14-F CD14- CD14-H
G
Figure 9. Frequency distribution for different SSCP variants in the Murrah breed of buffalo.
Figure 10. PCR-SSCP pattern of the 268 bp fragment in the Surti buffalo.
Lane 1-2: CD14- C.
Lane 7-8: CD14- D.
Lane 3-4: CD14- A.
Lane 11-12: CD14- H.
Lane 5-6, 9-10: CD14- B.
149
Buffalo Bulletin (June 2013) Vol.32 No.2
Surti
0.7
0.6
0.5
0.4
Surti
0.3
0.2
0.1
0
CD14-A CD14-B CD14-C CD14-D CD14-E CD14-F CD14-G CD14-H
Figure 11. Frequency distribution for different SSCP variants in the Surti breed of buffalo.
Buffalo
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Buffalo
CD14- CD14- CD14- CD14- CD14- CD14-F CD14- CD14A
B
C
D
E
G
H
Figure 12. Frequency distribution for different SSCP variants of buffalo.
150
Buffalo Bulletin (June 2013) Vol.32 No.2
CD14
CD14
CD14
CD14
CD14
CD14
11.5
10
8
6
4
Nucleotide Substitutions (x100)
2
0
Figure 13. Phylogenetic tree constructed for variants of CD14 gene in riverine buffalo.
1 C T A G A C C T G T C T G A C A A T C C C A G T C T C/A G G/C C
31 G/C A C/G A/G C C G/T G G/T C T G A T G G C A G C T C T C T G T C C/T G/A
61 A A C A A/G G T T C C C G G C C C T C C A A T A/G T C T A G C G/A
91 C/G T A/C C G C A/C A C/T G/C C C G G G/T A/C T G/A G /A A G/A/C A/G C/A G
CTG AG C
121 G G A G T G T G C G C G G C G C T G G C G/T G C A G/A C/G G A G/A G
151 G/C T G C A/C G/T C/G C C C A A/T A/C G C C T/G G G A/C C C T /A/C C A G/A C C A/G/C C
181 A/T A/G C/T T/C C/T G C T G C G/A C G T C A/T/C C/G C G/A C C C C G/C G G/C C G C/A T/G
211 A C C C G A/T T/A G/A T/G G T C/A T G/A G/C C/G C C/A A G T G C/T A/G C T A A G/T G
241 T C T C T C A/G A T T T G T/G C G T/C T C G C T G G G C
Figure 14 . SNPs for different sscp fragments (265 bp) of CD14 gene in buffalo.
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Buffalo Bulletin (June 2013) Vol.32 No.2
Table 1. Frequencies of the identiﬁed variants of the 265 bp fragment of the CD14 gene in buffalo.
Breed
Bhadawari
Mehsana
Murrah
Surti
Overall
CD14-A
0.818
0.465
0.596
0.071
0.47
CD14-B
0.045
0.056
0.105
0.16
0.09
CD14-C
0.183
0.089
0.08
CD14-D
0.105
0.607
0.18
CD14-E
0.070
0.022
CD14-F
0.136
0.169
0.193
0.13
CD14-G
0.056
0.018
CD14-H
0.071
0.018
Table 2. Genetic identity of different variants of CD14-A with respect to others.
SSCP variants
CD14-B
CD14-C
CD14-D
CD14-E
CD14-F
Gene bank Accession no.
EU370399
EU370400
EU370401
EU370402
EU370403
Percent identity
81.3
97.0
86.0
85.8
85.8
Table 3. Percent sequence homology of ampliﬁed region of bubaline CD14 gene with various species.
258 bp
3 nucleotide to 261st
nucleotide
Percent
Accesion
homology
NM_174008
97.7
DQ457090
91.5
AY289201
94.6
NM_000591
76.7
EF051626
80.6
M85233.1
77.1
NM_009841
62
NP 068512
66.7
AF200416
80.2
XP_848746
79.1
AM933591
15.9
XP_517975
76.0
rd
Species
Cattle
Goat
Sheep
Human
Pig
Rabbit
Mouse
Rat
Horse
Dog
Chicken
Monkey
152
265 bp
th
587 to 854th nucleotide
of second exon
Percent
Accesion
homology
NM_174008
98.1
DQ457090
90.6
AY289201
96.6
NM_000591
80.8
EF051626.
81.5
M85233.1
73.2
NM_009841
75.5
NP 068512
73.2
AF200416
84.5
XP_848746
77.7
AM933591
19.2
XP_517975
80.4
Buffalo Bulletin (June 2013) Vol.32 No.2
Table 4. Comparative analysis of the buffalo CD14 peptide sequence with that of cattle.
1
2
3
4
Amino acid sequence
position
14*
62*
209**
235-236**
5
277**
Sl. No.
Buffalo
Cattle
Proline
Glycine
Serine
Serine,
Lysine
Serine
Serine
Alanine
Threonine
Threonine,
Proline
Arginine
Nucleotide
substitution
C/T
G/C
C/G
T/A
T/C
C/G
* indicates aa changes resulting due to the 258 bp fragment (the 3rd - 261st bp of the 2nd exon of the CD14
gene of buffalo).
**indicates aa changes resulting due to the 265 bp fragment (the 587th to 854th nucleotide of the second exon
of the CD14 gene of buffalo).
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Buffalo Bulletin (June 2013) Vol.32 No.2
Table 5. Identiﬁed SNPs in the CD14 gene of buffalo include 6 variants.
CD14-
CD14-
CD14-
CD14-
CD14-
CD14-
SNPs
Nucleotide
Synm/ Non-
A
B
C
D
E
F
identified
Position
synm
C
C
G
T
C
G
A
A
G
C
G
G
C
G
A
G
A
A
G
T
C
G
A
G
G
C
T
G
T
C
C
C
A
G
T
C
A
G
G
A
G
T
A
C
T
T
C
G
A
A
C
C
A
A
C
T
G
C
A
A
G
T
C
G
A
G
G
C
T
G
T
C
C
C
A
G
T
C
A
G
G
A
T
T
C
G
G
T
C
G
A
G
G
C
C
A
A
G
G
C
A
A
G
T
C
G
A
G
G
C
T
G
T
C
C
C
A
G
T
C
A
G
G
A
T
T
C
G
G
C
T
A
A
A
G
G
G
A
C
G
G
C
C
C
G
A
C
A
G
C
G
C
T
G
C
C
G
A
C
C
C
C
A
T
G
G
G
C
C
G
G
T
C
G
G
A
G
C
G
A
C
G
G
C
A
A
G
T
C
G
A
G
G
C
T
G
T
C
C
C
A
G
T
C
A
G
G
A
T
T
C
C
G
T
C
G
A
A
G
C
G
A
C
G
G
C
A
A
C
C
G
A
C
C
C
A
G
A
C
A
C
C
A
G
T
T
G
G
G
A
T
T
C/A
C/G
G/T
C/T
C/T
G/A
A/G
A/G
G/C
C/G
G/A/C
A/G/T
C/A
G/T
G/A
C/G
A/C
A/C
G/C
T/A/C
C/G
G/A
A/G/C
G/C
G/C
C/A
T/G
G/A
T/C
C/A
C/G
C/A
A/C
G/C
T/C
C/T
A/G
G/T
G/C
A/G
T/G
T/C
618
627
630
644
650
651
656
674
679
693
702
703
704
732
736
737
746
754
755
764
765
767
770
797
799
800
801
811
812
813
817
819
820
821
822
824
825
830
831
838
844
847
Synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Synm
Non-synm
Non-synm
Non-synm
Synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Non-synm
Synm: Nucleotide substitution leading to synonymous codon change.
Non-synm : Nucleotide substitution leading to non-synonymous codon change.
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