Optimisation of cryovials and milt dilution ratios to upscale carp sperm cryopreservation for use in hatchery seed production: Upscaling of Carp Sperm Cryopreservation

Santosh Kumar; Aditya Kumar; Rama Shankar Sah; Ajay Kumar Singh; Ajay Yadav; Arvind Verma; Deepti Negi; Hayin Tamut; Kuldeep Kumar Lal

doi:10.21077/ijf.2023.70.3.134401-12

Authors

Santosh Kumar
Aditya Kumar
Rama Shankar Sah
Ajay Kumar Singh
Ajay Yadav
Arvind Verma
Deepti Negi
Hayin Tamut
Kuldeep Kumar Lal

https://doi.org/10.21077/ijf.2023.70.3.134401-12

Keywords:

Cryobanking, Cryovials, Premix, Dilution

Abstract

This study reports the experimental results carried out to optimise containers and milt
dilution ratios for carp sperm cryopreservation, in order to make the process simple and
compatible with the needs of commercial carp seed production, which is a small-scale and
low technology sector in Asia. The procedure optimisation for up-scaling was assessed
by the ability of cryopreserved sperms to fertilise Cyprinsu carpio eggs and hatching
percentage, in three different experiments. In the first experiment, we tested the effect of
milt to different extender dilutions (1:4, 1:6 and 1:8); the second experiment tested the
effect of container capacities (0.5 ml French straw; 2 ml and 5 ml cryovials) with two dilution
ratios (1:3 and 1:6) and in the third experiment, the efficacy of pre-mix of extender salts and
individually pre-weighed salts stored over 5 months was compared with fresh preparation
of extender. Out of the three dilutions tested, two dilutions viz., 1:4 and 1:6, yielded hatching
above 50%. We also used the milt cryopreserved at dilution of 1:3 which was post-thaw
diluted with extender, to make final dilution of 1:6, to fertilise the eggs. This post-thaw
dilution in the ratio 1:6, yielded the results comparable to the milt cryopreserved in 1:6
dilution. This approach can help small-scale seed producers to store more sperm within
the limits of the cost of liquid nitrogen. The extender salt composition, pre-weighed and
pre-mixed, was also found to give comparable results to the fresh composition after storage
over 5 months. This can assist in developing ready to use, cost-effective, working kits
available to semi-technical hatchery seed producers. The parameters optimised in the study
has potential to be transformed as an easy strategy with the use of 5 ml vial, cryopreserved
at 1:3 dilution, with further post-thaw dilution at site, thus aiding in fertilisation of large
volume of eggs produced by high fecund carp species within the limited time available for
maintaining good gamete quality.

Keywords:
Cryobanking, Cryovials, Dilution, Salt premix

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