Schizothorax richardsonii is an indigenous snow-trout in the Himalayan region of Uttarakhand. It contributes a lot to the fish food basket in the hilly area of the state. In past few years snowtrout capture fishery has registered continuously decreasing trend due to over-exploitation and habitat destruction. For reestablishing its status and propagation of the species, first successful attempt was made to cryopreserve the semen of S. richardsonii to make all time availability of viable sperm to increase hatchery production for river ranching and culture practices. To develop the cryopreservation protocol for S. richardsonii, initially semen quality was evaluated as per species trait and for sperm motility behaviour. Good quality semen samples (>75% motile sperm after activation) were pooled and frozen within 2 hours of collection. Four extenders and two cryoprotectants (CPA) were tested to cryopreserve the S. richardsonii semen. The extender-Mounib’s medium was found most successful. The DMSO proved better CPA than glycerol. Equilibration time was standardized as 45 min for DMSO and 60 min for glycerol. The semen is diluted with the different diluents (extender + cryoprotectant) before freezing. Two semen dilution ratios 1:4 and 1:10 were tested for better viability after cryopreservation. French medium straws of 0.5 ml capacity were used to store the semen in liquid nitrogen. Thawing temperature was standardized as 20-250C. The cryopreservation success was evaluated by post-thaw motility and fertilizing ability of cryopreserved semen. Post-thaw motility percentage and duration was significantly reduced. Fertilizing capacity of 375 days old cryopreserved semen at 1:4 dilution ratio was almost equal to that of fresh semen (>98%). Semen cryopreserved in Mounib’s extender with 5% DMSO as CPA, even after 375 days had hatching rate-35.91 ±0.15% and fry survival rate 21.28 ±0.41% while those from fresh semen it was 52.97±0.26% and 30.43±0.50% respectively. Though the hatching and survival rates were significantly superior with fresh semen (P<0.001) than the cryopreserved semen, but these results are still encouraging. This developed cryopreservation protocol for S. richardsonii semen has ensured year round availability of viable sperm for optimal utilization in hatchery production. It would open up new perspective such as establishing genetic material reserves, sperm / gene bank for selective breeding, cross breeding and ex-situ conservation of fish germplasm.

1. FISH SEMEN BANKING: A TOOL FOR PROPAGATION AND EX-SITU CONSERVATION OF SNOWTROUT
Prof. N. K. Agarwal
FISH REPRODUCTION AND CONSERVATION BIOLOGY RESEARCH LABORATORY, DEPARTMENT OF ZOOLOGY
HNB Garhwal University Campus Badshahithaul, -249199, Tehri Garhwal (Uttarakhand ) INDIA
e-mail : agarwalnareshk3@rediffmail.com
(i) Procurement of ripe male brooders and collection of semen sample
The Semen is stripped in 4.5 ml
cryovials and kept on ice at 0-4ºC
(ii) Quality analysis of semen/milt before cryopreservation
(vi) Equilibration time on ice (0-4ºC)
• For effective protection during cooling,
sufficient time must be allowed to
facilitate the penetration of
cryoprotectants into cells. This time is
termed as equilibration time.
Equilibration time has been standardized as-
 For Schizothorax richardsonii
45 min. for DMSO and 60 min. for Glycerol.
 For Schizothoraichthys progastus
50 min. for DMSO and 75 min. for Glycerol
(v) Filling of milt in straws
Milt is filled in 0.5 ml French
medium straws & sealed
manually by PVA powder
(viii) Plunging of cooled straws in LN2(-196ºC) for storage
Transferring the cooled straws in canisters for plunging in liquid
nitrogen (Cryocans) for long-term storage.
(ix) Thawing before artificial
fertilization
Thawing is done in water bath at room
temperature (20-25ºC).
After thawing, the cryopreserved straw
is being cut open to observe the post-
thaw motility under the microscope for
artificial fertilization of snowtrout eggs.
The snowtrout sperm Bank
• The snowtrout semen bank ensured all time availability of viable sperm
for snowtrout seed production in hatcheries .
• It reduces the cost of maintenance of live brooders in the hatchery.
• It makes possible ex-situ conservation of snowtrout gene pool.
• Snowtrout sperm bank facilitate in seed production & propagation of the
species
Ripe male brooders has white
prominent tubercles on the snout
(iii) Extenders and cryoprotectants used for preservation
An extender is the solution of balanced salts, which inhibits the activation
of sperm and thereby increases the life span of spermatozoa. For
snowtrouts, following extenders have been used-
1 Mounib’s Medium 2. KCl Medium
• Cryoprotectant is the chemical compound, which is used for minimizing
the cryo-injuries associated with cooling and thawing.
Following cryoprotective agents are in use -
1 DMSO (@ 5% and 10% conc.) 2 Glycerol (@ 5% and 10% conc.
(iv) Dilution Ratio
• Milt is diluted with diluent (extender +
cryoprotectant).
• The milt : diluent ratio for successful
fertilization is 1: 4
After sealing, Straws are kept on
ice pads (0-4oC) for equilibration
Equilibrated straws are kept on
freezing rack in liquid nitrogen
vapours from 00 c to 1200 c
Semen Characteristics Schizothorax
richardsonii
Schizothoraichthys
progastus
pH 7.31 ±0.07 7.34 ± 0.04
Sperm density 3.77 ±0.78x108 sp.-ml 8.67 ±0.50x108 sp.-ml
Spermatocrit value 63.13 ±10.27% 58.24 ± 4.35 %
Sperm motility rating 3.16 ±1.23 (>75%) 3.64 ±0.56 (>75%)
Sperm motility duration 59.7 ±16.55 s 64.57 ±11.44 s
Characteristics of good quality snowtrout semen
(vii) Cooling of straws on LN2 vapor
0
1
2
3
4
15 20 25 30 35 40
Motility
Rating*
Thawing Temperature (ºC)
Evaluation
of
cryopreserved
semen
samples
SEM image of the S. richardsonii
sperm prior to cryopreservation
SEM image of cryopreserved sperm
showing shrinkage in the membrane of
sperm head.
Evaluation of cryopreserved semen samples
Evaluation of cryopreserved semen samples
Details of cryopreserved semen samples used for fertility test in S. richardsonii
Pre-freeze motility rating : 4 (75-100% motile sperm)
Extender : Mounib’s medium
Semen-diluents ratio : 1:4 (7.19±0.25x107 sperm/ml)
Equilibration time : 45 min. for DMSO & 60 min. for Glycerol
Freezing rate : 30°C/min
Thawing temperature : 20-25°C for 10-15 sec.
Post-thaw sperm
motility rating
5% DMSO 10% DMSO 5% Glycerol 10% Glycerol
50-<75% 50-<75% 50-<75% 50-<75%
Storage period : 375 Days
cryopreserved semen used for insemination : 1 ml (2 straws) for 1200 eggs
Fertility/Viability test of cryopreserved Semen
Incubation of
fertilized eggs with
cryopreserved
semen in Flow-
through hatchery
Incubating
water temp.
14.5 to 18.0oC
Fertility results of 375 days old cryopreserved semen of S richardsonii
Fertility/
Viability
Control
(n=3)
DMSO
(% mean value,
n=3)
Glycerol
(% mean value
n=3)
5% 10% 5% 10%
Fertilization
%
98.72
±0.32
98.27
±0.28
97.82
±0.12
98.60
±0.01
98.25
±0.03
Hatching % 52.97
±0.26
35.91
±0.15
27.63
±0.25
34.44
±0.50
23.95
±0.40
Swim-up fry
survival %
30.43
±0.50
21.28
±0.41
16.11
±0.24
19.01
±0.38
12.33
±0.61
(Sperm-egg ratio in control and experimental groups ~ 1 : 27,000).
•Among all sets of cryopreserved semen, 5% DMSO is significantly
superior over 10% DMSO (P<0.001), 5% glycerol (P<0.01) and 10%
glycerol (P<0.001) in terms of hatching rate & swim up fry survival %
•Thus use of Mounib’s extender and 5% DMSO as CPA for
cryopreserving the S.richardsonii semen is included in the protocol.
Cite this article as : Agarwal, N.K. (2015): Fish
Semen Banking: A tool for Propagation and ex-
situ conservation of Snowtrout , Conference:
Science and Technology for Human Development
At: Department of Zoology, Jammu University,
Jammu India DOI: 10.13140/RG.2.2.28782.51526/1