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