Protocol for Cryopreservation of snowtrout semen
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-Mounibs 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 Mounibs extender with 5% DMSO as CPA, even after 375 days had hatching rate-35.91 賊0.15% and fry survival rate 21.28 賊0.41% while those from fresh semen it was 52.97賊0.26% and 30.43賊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.
Protocol for Cryopreservation of snowtrout semen
- 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 0-4尊C (ii) Quality analysis of semen/milt before cryopreservation (vi) Equilibration time on ice (0-4尊C) 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(-196尊C) 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 (20-25尊C). 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 Mounibs 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 賊0.07 7.34 賊 0.04 Sperm density 3.77 賊0.78x108 sp.-ml 8.67 賊0.50x108 sp.-ml Spermatocrit value 63.13 賊10.27% 58.24 賊 4.35 % Sperm motility rating 3.16 賊1.23 (>75%) 3.64 賊0.56 (>75%) Sperm motility duration 59.7 賊16.55 s 64.57 賊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 (尊C) 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 : Mounibs medium Semen-diluents ratio : 1:4 (7.19賊0.25x107 sperm/ml) Equilibration time : 45 min. for DMSO & 60 min. for Glycerol Freezing rate : 30属C/min Thawing temperature : 20-25属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 賊0.32 98.27 賊0.28 97.82 賊0.12 98.60 賊0.01 98.25 賊0.03 Hatching % 52.97 賊0.26 35.91 賊0.15 27.63 賊0.25 34.44 賊0.50 23.95 賊0.40 Swim-up fry survival % 30.43 賊0.50 21.28 賊0.41 16.11 賊0.24 19.01 賊0.38 12.33 賊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 Mounibs 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