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MATERIALS AND METHODS
In order to find out the optimum requirements of cryoprotectants,
extenders and the optimum requirement of dilution ratio of the best performed
cryodiluents, equilibration time, prefreezing and thawing temperatures on the
successful preservation of spermatozoa of Heteropneustus fossilis, Channa
striatus and Channapunctatus, the experiments were designed as follows.
1. To find out the optimum requirement of cryoprotectant concentration of
glycerol and DMSO for H.fossilis, C.striatus and C.punctatus sperm, 5
different concentrations of glycerol and DMSO namely 5, 10, 15, 20 and
30% were tested separately for post thaw sperm motility.
2. To find out optimum requirements of extender in the cryodiluents of
experimental fishes, 6 different types of extenders namely cortland
medium, modified cortland medium, extender 1, extender 2, V 2e extender
and NaCl + KC1 medium with 10 % glycerol and 10 % DMSO separatly
were tested for post-thaw sperm motility of H.fossilis,. C.striatus and
C.punctatus after one month of preservation.
3. To find out optimum dilution ratio of selected optimum cryodiluents of 10
% glycerol and 10 % DMSO along with cortland medium extender for
H.fossilis, C.striatus and C.punctatus sperm, 6 different dilution ratios of
1:1, 1:3, 1:5, 1:10, 1:20 and 1:50 were tested after one month of
preservation. -
4. To find out the optimum requirements of equilibration time, the diluted
milt samples of experimental fish with the best performed cryodiluents
63
were equilibriated at 5, 10, 20, 60, 120, 240, 480, 720, 960 and 1440 mm
and tested their motility performance at every equilibration period.
5. To find out the optimum freezing temperature for long-term preservation,
the diluted milt samples of H.fossilis, C.striatus and C.punctatus were
frozen at four different freezing temperatures of —70°C, —90°C, —154°C,
—200°C and studied their sperm motility after cryopreservation.
6. To find out the optimum requirement of thawing temperature to obtain the
best post-thaw motility and fertilization performance, the frozen sperm
samples of H.fossilis, C.striatus and C.punctatus were thawed at 6 different
thawing temperatures of 25 (air thawing) 35, 45, 55, 65 and 75°C in water.
7. To find out the influence of cryopreservation on biochemical constituents
on the spermatozoa of H.fossilis, C.striatus and C.punct at us during
preservation, the levels of protein, carbohydrate and lipid and ions of Ca
and K ions were tested before and after cryopreservation.
8. To find out the best performance of fertilization and hatching, the
cryopreserved spermatozoa of H.fossilis, C.striatus and C.punctatus were
fertilized with normal eggs of the same species at different long-term (1, 2,
3, 4, 5 & 6 months) and short-term (5, 10, 20, 40, 60 & 90 days) storage
periods.
54
3.1 DESCRIPTION OF EXPERIMENTAL FISH
MURRELS
The two species of snakehead murrels of the genus Channa found in
rivers, ponds and swamps were chosen for present study. Over 15 species have
been reported from Asia and Africa. Among these, Channa striatus and
Channa punctatus have a wide range of distribution in South Asian countries
and they are more popular priced fish in India. In peninsular India, murrels
form the mainstay of freshwater capture fisheries (Alikunhi, 1953). They are
stocked in village ponds, irrigation wells and shallow waters in Southern India
(Alikunhi, 1953; Anon, 1962). Murrels are estimated high for their keeping
quality, flavour and nutritive and recuperative properties in Punjab, Madhya
Pradesh, and the Peninsular states of India. They invariably fetch a much
higher price than other freshwater fishes.
Systematic position of Channa striatus
Phylum : Chordata
Sub Phylum Vertebrata
Grade : Pisces
Class Teleostomi or Osteichthyes
Order : Channiforms
Family : Channidae
Genus : Channa
Species striatus
55
- .------.- -I-V
---4..-
C
I __ b
C
Heteropneustus fossilis, b1 Channa striatus, c channa punctatus,Injection of ova prim to brood Channa striatus,Injection of ova prim to Heteropneustus fossilisid ft Genital papillae of Heterropneustus fossilis
This is the most widely distributed and economically the most important
member of the genus. It attains a length of 60 - 75 cm, and matures at a size of
about 30 cm when one year old. It breeds during monsoon months
(Sepetember, October and November).
Systematic position of Channa punctatus
Phylum : Chordata
Subphylum : Vertebrata
Grade Pisces
Class : Teleostomi or Osteichthys
Order Charmiforms
Family : Channidae
Genus : Channa
Species : punctatus
The species attains a length of 31 cm, and prefers stagnant water. They
are found in muddy streams. This species is a prolific breeder and peak
breeding season is before and during monsoon months and matures in first
year.
CAT FISH
The stinging catfish of the genus Heteropneustus are found in rivers,
ponds and shallow water bodies and it is confined to the Indian sub continent
and southeast Asia. H.fossilis and H.microps are the two known species of the
genus. Among these, H.fossilis has wide range of distribution and is very
common along the Peninsular India. H.microps is a Srilankan form, which has
a very restricted distribution in Utterpradesh and Bihar (Datta Munshi and
Srivastava, 1988). Recently this species has been reported from South India
(Arunachalam et al., 1999). H.fossilis is more popular in tank fishery and it is
highly priced for its low fat, high protein and high iron content. Because of its
air-breathing habits it is well adopted to the water bodies with low oxygen
content and it is to be sold as live fish in the market (Dehadry et al., 1985).
Systematic position of Heteropneustus fossilis
Phylum
Subphylum
Grade
Class
Order
Family
Genus
Species
Chordata
Vertebrata
Pisces
Teleostomi or Osteichthyes
Siluriforms
Heteropneustidae
Heteropneustus
fossilis
This species attains a length of 30 cm. It prefers stagnent water bodies in
muddy rivers. It matures in six months and breeds before and during monsoon
months (September, October and November).
57
3.2 COLLECTION, MAINTENANCE AND
ACCLIMATIZATION OF EXPERIMENTAL FISH
Matured males of Heteropneustus fossilis farmed in a private farm at
Tenkasi and the males of Channa striatus and Channa punctatus from
Palayankottai fish market were used for the present study. Male H.fossilis
aging about 8 - 10 months and weighing about 300 - 500 gms and male
Channa striatus of 1 - 2 years old weighing 1 - 1.5 Kg and male Channa
punctatus of 1 - 1.5 years old weighing 500 - 750 gms were transported from
Tenkasi and Palayankottai by train in aerated bags and maintained in cement
tanks of 1000 liter capacity separately. The experimental fishes were daily fed
with chicken intestine and beef liver at the rate of 3 % of their live body weight
per day during the study period. The females used for fertilization studies were
maintained in a separate tank. The water in the tank was exchanged once in two
days for clearing the waste products. The water temperature ranged from 28 to
29.50 Cduring the study period.
COLLECTION OF MILT
The milt was collected from the experimental fishes with hormonal
inducement. Channa striatus, Channa punctatus and Heteropneustus fossilis
were induced by administering the synthetic hormonal preparation, ovaprim at
0.1 ml I kg body weight through intramuscular injection. The milt was
collected from the fish after 12 hrs of hormonal administration by dissecting
the fish because the gametes of these species are difficult to obtain by stripping.
There are three fishes (replicates) were used for each species for milt
collection. The experimental fishes were killed and then testes were removed.
Adherent tissue was dissected away and testes were blotted dry and weighed.
The dissected testis from each fish species were homogenized in a loose fitted
58
glass-on-glass tissue grinder and the spermatozoa were suspended in 10 ml of
modified Hank's balanced salt solution (HBSS) (Table 1). (Wayman et.al.,
1996). The homogenized samples were filtered through nylon netting of 0.5
mm mesh.
59
Table!. Ingredients of a modified Hank's balanced salt solution used to
dilute fish sperms.
Ingredients Concentration (glut)
NaCl 8.00
KC1 0.40
CaC12 .2H20 0.16
M9SO4.7H20 0.20
Na2HPO4 .71120 0.12
KH2PO40.06
NaHCO3 0.35
C6H 1206 (Glucose) 1.00
The collected milt samples were kept on the crushed ice. The milt
collected from the individual experimental fish was kept separatively for
estimating the performance of milt (general appearance and motile capacity). A
drop of milt was placed on a clean glass slide and was activated by adding a
drop of distilled water. The percentage of motility of the spermatozoa was used
as a parameter to assess the viability of spermatozoa. Motility was evaluated by
estimating the number of sperm in a field of view (100 x magnification) that
exhibited a sustained directional movement after activation followed by Aas et
al. (1991). Cells that are uncontrollably spinning in place were considered as
non motile. Those milt samples showing more than 90 % motility alone were
selected for further processing for cryopreservation. The diluents extender,
cryoprotectant and glasswares used for this study were kept cool to avoid
temperature shock.
Colour of the milt was determined visually. The volume of milt was
measured by means of a micropipett. The number of sperm cells present in 1
ml of milt was counted by using an improved Neubauer haemocytometer.
Motility of spermatozoa was evaluated according to the method given by Guest
et at. (1976). The percentage of sperm mortility was estimated by counting the
number of active sperm cells in a field of view divided by total number of
sperm X 100. There are five replicates were taken to evaluate the mean
percentage of sperm motility.
3.3 PREFREEZE STORAGE PERIOD
The general freezing protocol was followed as the methods of
Lahnsteiner et a! (1994, 1995). The viability of sperm was affected by
prefreeze storage period, that is, the time interval between collection of milt
and freezing (Stoss and Holtz, 1983; Schmidt Baulain and Holtz, 1989;
61
Ciereszko et al., 1999). Milt collected from three different male specimens of
Heteropneustus fossilis, Channa striatus and Channa punctatus were pooled
species wise and stored at - 60 ± 1°C from 1, 3, 5, 7, 9 and 24 hrs in freezer.
The viability of prefrozen (in freezer at —6°C) spermatozoa was assessed after
thawing based on the percentage of spermatozoa. The sperm motility
percentage was measured by the use of Neubauer counting chamber
(Haemocytometer). This experiment was carried out only in freezer. There are
five readings were taken to estimate the mean percentage of sperm motility.
62
ri
4
b
qi
11111d
itU I
•3 JQ• ,
d V •
' .
1
Squeezing of eggs from brood Heteropneustusfossiis b. Testis of Heteropneustusfossüis,
Testis of channa striatus, d. Testis of channa punctatus, e. Homogenizing of testis in tissue
homogenizer. f. Sperm of Heteropneustus fossil is.
Table 2. Chemical composition of various extenders used in the present study.
Extenders I Source
Ingredients (mg / 100 ml distilled water)
0- 0 m
0C) 0
U 4 0 c
U) u
C)z
Cortland -
mediumWolf (1963) 725 23 38 41 100 23 - 100
ModifiedTruscott et
Cortlandal. (1968)
188 23 720 41 100 23 - 100
medium
Kurokura etExtender 1
750 - 20 - 20al. (1984)
Kurokura etExtender 2
440 - 620
20 I- 122 I- I-al.(1984)
V2 e Bayrle(1982) 750 - 38 -
NaCl+KC1Nalliappan 449. 573.
- -(1992) 5 5
-- -DIJIiiIi]
3.4 DILUTION OF MILT
3.4 1 EXTENDER
Semen samples were collected from Heteropneustus fossilis, Channa
striatus and Channa punctatus and the squashed suspension was kept on ice
before dilution with diluents. A sample from each experimental male was
examined for motility under the light microscope (Terner, 1991) and those
samples showing more than 90 % motility were diluted with diluents. All
extenders were prepared in double distilled water, and six different extenders
(cortland medium, modified cortland medium, extenderl, extender2, V2e and
NaC1+KC1 medium) (table 2) along with 10 % glycerol and or 10 % DMSO as
cryoprotectants were used individually in this experiment. The diluted milt
samples were equilibrated to 20 mm, then immediately stored in liquid nitrogen
at —196°C / mm. The effects of extender composition on the viability of
cryopreserved spermatozoa were tested after one month of preservation, by
water thawing at 45°C. There are five replicates per extender was used.
3.42 CRYOPROTECTANT
Glycerol and Dimethyl Suiphoxide (DMSO) were used as
cryoprotectants for this investigation. Various concentrations of glycerol and
DMSO at 5, 10, 15, 20 and 30 % levels were mixed separately with their
respective best extender (cortland medium) at 1:10 dilution ratio were used for
the preservation of spermatozoa of Heteropneustus fossilis, Channa striatus
and Channa punctatus. The milt samples were equibrated for 20 mm, the
performance of different concentrations of cryoptotectants was assessed.
64
WO100
MCOM
P.^Wrs
e
-- II
L71________________••i f
ie Sperm of channa striatus, b. Sperm of channa punctatus, C. The enlarged view
f sperm of Heteropneustusfossths, d. Cryocans, e. Cryovessel and
sle
illing apparatus with O.5m1 straws.
3.43 DILUTION RATIO
The collected milt samples of Heteropneustus fossilis, Channa striatus
and Channa punctatus were diluted with their respective diluents (cortland
medium and 10% glycerol and or 10% DMSO) at different dilution ratios, 1:1,
1:3, 1:5, 1:10, 1:20 and 1:50. The diluted samples were equilibrated for 20 mm
and frozen at —196°C. The dilution of milt with diluents and filling of straws
were done in a cold handling chamber (5°C). The performance of different
dilution ratios on the frozen spermatozoa were evaluated after one month of
preservation and subsequent thawing of 45°C. There are five replicates were
used for each dilution ratio.
3.5 FILLING OF MILT
The semen of Heteropneustus fossilis, Channa striazus and Channa
punctatus were diluted with the cold diluents at 1:10 dilution ratio (Semen
Diluent). The diluted milt samples were sucked in to 0.5 ml straws and filled
straws were sealed with a sealing powder of Polyvinyl Alcohol (PVA). Straws
and PVA powder of different colours were used for the ready identification of
samples. The sealed ends were dipped in cold water (5°C) for proper sealing.
Filling and sealing of straws were carried out in the cold handling chamber
5°C.
3.6 EQUILIBRATION TIME
The spermatozoa of Heteropneustusfossilis diluted in the diluent, 10 %
glycerol and 10 % DMSO separately with cortland medium as a extender, and
the spermatozoa of Channa striatus and Channa punctatus were diluted with
only 10 % glycerol and cortland medium at 1:10 dilution ratio were
equilibrated at 0°C in refrigerator for various periods of 5 - 1440 mm (5, 10,
65
i'i 4I
-ii
a
A
.7
ling of diluted milt samples, b. Freezing of filled straws in liquid nitrogen vapour, c.
'sing of straws with frozen milt samples in liquid nitrogen, d. The canister containing straws
reserved spermatozoa, e. Addition of cryopreserved milt with fresh eggs of Heteropneustus
is f. Embryo of HeteropneustusfossUis (fertilized by cryopreserved sperm).
20, 40, 60, 120, 480, 720 and 1440). In addition, the spermatozoa of
Heteropneustus fossilis, Channa striatus and Channa punctatus were diluted
with 5, 10, 15, 20 and 30 % of glycerol and or DMSO with cortland medium as
extender individually and equilibrated upto 180 mm (5, 10, 20, 40, 60, 120 and
180) to test the combined effect of equilibration time and different
concentrations of cryoprotectant. The three dimensional statistical test was used
to determine the combined effects between equilibration time at different
cryoprotectant concentrations.
3.7 FREEZING TEMPERATURE
The filled straws with diluted milt samples (milt:cortland
medium+glycerol and or DMSO at 1:10) were frozen after exposing them to an
equilibration time of 20 mm. The sealed straws were arranged on a rack in an
insulation box in the vapour of liquid nitrogen at different freezing
temperatures. The freezing temperature were controlled by adjusting the
distance between straws and the surface of liquid nitrogen. The experimental
fish spermatozoa were frozen at distance of 1, 2 and 3 cm at the rate of-154°C
I mm, —91'C / min and —70°C / min above the surface of liquid nitrogen
(Kurokura et al., 1986). In addition to this, the diluted spermatozoa were
directly plunged into liquid nitrogen at —200°C I mm (Gwo et al., 1991). The
frozen straws with spermatozoa samples in liquid nitrogen vapour phase were
immediately transferred to liquid nitrogen phase kept in cryocan for long- term
preservation (1 to 6 months). There are four replicates were used to determine
the mean percentage of motility for each freezing temperature.
3.8 THAWING TEMPERATURE
For thawing the frozen straws with dilluted milt samples of
Heteropneustus fossilis, Channa striatus, Channa punctatus were allowed in
air thawing at 25°C (room temperature) and in water bath at 35, 45, 55, 65 and
75°C of thawing temperatures. Exactly after 30 sec the straws were removed
from the water bath, the plug of the straws was cut off and the thawed semen
was taken to evaluate the viability (Lahnsteiner et al., 1996). The preserved
samples of spermatozoa were analysed to find out the optimum temperature for
the best viability. There are four replicates were taken to estimate the mean
percentage of motility for each thawing temperature.
67
-1 IIIIIIIIIIIIIIIIe
A All *StVr A4
Rd - A
•
<1
\
A
6'-IVMVol -44
• Fertilized and unfertilized eggs of Heteropneustusfossxlis, b. Developmental stage,
Hatchlings of Heteropneustusfossilis, d. Fresh eggs of Channa striatus, e. Fertilized and
infertilized eggs of Channa striatus, f. Hatchlings of Channa striatus.
3.9 FERTILITY STUDIES
The sperm samples of Heteropneustusfossilis, Channa striatus, Channa
punctatus were cryopreserved for I to 6 months and the stored samples were
tested for their fertilising capacity. The female fishes of Heteropneustus
fossilis, Channa striatus, Channapunctatus were induced to spawn by injection
of 0.3 ml / kg body weight of ovaprim. Eggs were collected by abdominal
stripping 12 hrs after injection. About 200 to 300 ripe ova were stripped
directly into petri dishes and were fertilized with cryopreserved sperm from
single 0.5 ml straw. The frozen samples in the straws were taken out from the
cryocan and dipped in hot water at 45°C (water thawing) temperature, which
was found to be the optimum in this present study. After thawing both the ends
of the straw were cut and the milt was allowed to flow over the stripped eggs.
The cryopreserved milt and eggs were mixed well using a feather. Sperm and
eggs were activated by the addition of 20 - 25 ml aerated well water. After 1-2
minutes the eggs were rinsed and incubated at 25 - 26°C in 1.51 of
dechlorinated water with continuous aeration. The water was changed for every
30 minutes to oxygenate the eggs. The viability of cryopreserved sperm was
assessed by the percentage recovery of fertilized eggs with embryo. The control
fertilization test was carried out in the same fashion with fresh sperm. The
stored sperm samples were tested for their fertilizing capacity at various
storage periods of 1, 2, 4 and 6 months. The fertilization test was carried out at
1:10 dilution ratio (sperm:cryodiluent) of Heteropneustus fossilis, Channa
striatus, Channa punctatus. The fertilized eggs were inspected in a dissecting
microscope for development in to the neurula stage. Neurulation was employed
as the basis for identification of fertilization success because unfertilized eggs
begin to develop but become arrested at gastrulation (Withier, 1982). In
addition to this the hatching percentage of eggs fertilized with cryopreserved
spermatozoa was assessed after fertilization at different storage periods. In
68
fertility studies five replicates were also used to determine the mean
fertilization percentage.
3.10 BIOCHEMICAL CHANGES
Changes in the macro elements like protein, carbohydrate and lipid and
microelements like calcium and potassium of semenial plasma were estimated
prior and after freezing in order to study the fluctuations in the biochemical
composition of Heteropneustus fossilis, Channa striatus, Channa punctatus
spermatozoa before and after preservation. The seminal plasma was obtained
by subjecting the diluted milt samples of semen through centrifuge at 3000 rpm
for 10 min and the supernatants with seminal plasma was used to estimate
carbohydrate, protein, lipid and ions of Ca and K levels. Protein was
estimated by Lowry's method (Lowry et al., 1951), using bovine serum
albumin as the standard. The supernatant was precipitated with 10 % TCA
(Trichioro acetic acid). The protein in the precipitate was estimated and the
obsorbance was measured at 660 nm. The carbohydrate content of the seminal
plasma was estimated using the anthrone method (Roe, 1955). Glucose was
used as standard and the optical density was measured at 620 rim. Lipid level
was estimated by Suipho phosphovanillin method (Barnes and Black stock,
1974) in which the lipid was extracted using chloroform and methanol mixture.
Cholesterol was used as the standard and the colour developed was measured at
520 rim.
The calcium ion concentration was estimated by Webster's
spectrophotometric method (Webster, 1962). The precipitated calcium was
dissolved in tetra sodium Ethylene Diamine Tetra Acetate (EDTA), and
coloured content formed was measured at 490 nm. The potassium ion
concentration of seminal plasma was determined spectrophotometrically (Snell
and Snell, 1959). The colour intensity developed was read at 430 nm. There are
three replicates were used for each biochemical change (Protein, carbohydrate
and lipid).
3.11 SHORT TERM PRESERVATION
The samples of semen were collected from Heteropneustus fossilis,
Channa striatus and Channa punctatus by surgically removing the testis. There
are three fishes from each fish species was used in this experiments. The testis
was squeezed to release sperm. The collected milt samples were diluted with
their best diluents and equilibrated for 20 mm. were found in the present study
and preserved in freezer at —6°C for short-term preservation. The spermatozoa
of Heteropneusius fossilis, channa striatus and channa punctatus were diluted
with diluent of 10 % glycerol and or 10% DMSO individually with cortland
extender medium at 1:10 dilution ratio and immediately placed at —6°C in
refrigerator for different short-term storage periods, such as 5, 10, 20, 40, 60,
90 and 120 days. The viability performance of frozen thawed short-term stored
spermatozoa of Heteropneustus fossilis, Channa striatus, Channa pun claws
were analysed at various short-term storage periods (5, 10, 20, 40, 60, 90 and
120) until zero percentage of motility was obtained. There are five replicates
were used to determined the mean percentage of sperm motility for each short-
term storage period.
70