The document describes the development of a novel, cost-effective variable freezing rate apparatus for cryopreserving fish semen for field use. The apparatus consists of an aluminum rack stand and straw racks that can hold 42 straws each. The racks can freeze samples simultaneously at the same or different rates by adjusting the height in liquid nitrogen. This allows for cryopreservation of endangered mahseer fish species' sperm in field conditions. The apparatus was successfully tested for cryopreserving two threatened mahseer species and provides a portable, inexpensive option for freezing fish genetic material in remote areas.

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DEVELOPMENT OF A NOVEL, COST EFFECTIVE,
VARIABLE FREEZING RATE APPARATUS FOR
CRYOPRESERVATION OF FISH SEMEN FOR FIELD USE
RAVINDRAGOUDA PATIL1*
, WAZIR SINGH LAKRA2
,
SHRINIVAS JAHAGEERDAR2
, GOPAL KRISHNA2
AND
ASIM KUMAR PAL2
1
CURRENT AFFILIATION: FISHERIES RESEARCH AND
INFORMATION CENTER, KVAFSU, HESSARAGHATTA,
BENGALURU, KARNATAKA STATE, INDIA.
2
FISH GENETICS AND BIOTECHNOLOGY DIVISION,
CENTRAL INSTITUTE OF FISHERIES EDUCATION,
VERSOVA, MUMBAI- 400 061, MAHARASHTRA STATE,
INDIA.
Corresponding author’s e-mail: : ravi.patil30@gmail.com
ABSTARCT:
In the present study, a variable freezing rate apparatus with a novel design
was fabricated using cost effective materials and tools for the
cryopreservation of the spermatozoa of threatened mahseer fish species
belonging to the genus Tor. The apparatus consists of a rack stand and
straw racks made of aluminium flat strips. The straw rack has a provision
to hold about 42 straws. The main advantage of the apparatus is that, the
three racks can be simultaneously frozen at the same or at three different
freezing rates. The rack stand with straw-loaded straw racks can be easily
kept into a styrofoam box with liquid nitrogen and frozen at desired
freezing rates. This apparatus was successfully used for the
cryopreservation of the endangered cold water fish species of the Deccan
mahseer, Tor khudree and the Golden mahseer, T. putitora.
KEY WORDS: Cost effective, Freezing apparatus, Straw rack, Rack stand,
Cryopreservation, Mahseer.
Universal Impact
Factor0.9285:2012;
1.2210:2013
Index Copernicus
ICV 2011: 5.09
ICV 2012: 6.42
ICV 2013: 15.8
ICV 2014:89.16
NAAS Rating
2012 : 1.3;
2013-2014-
2015:2.69
Received on:
5th May 2016
Revised on:
20th June 2016
Accepted on:
20th June 2016
Published on:
1st July 2016
Volume No.
Online & Print
77 (2016)
Page No.
113 to 117
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INTRODUCTION:
Various anthropogenic factors like dynamite fishing, use of piscicides, siltation due to deforestation,
construction of barrages, dams across streams and rivers have led to many fish species becoming
endangered/threatened at an alarming rate. Cryopreservation of gametes and embryos is one of the
very important methods of ex situ conservation of germ plasm of the endangered and /or threatened
plant as well as animal species on our planet. Mahseers, belonging to the family Cyprinidae, form one
of the most important food and game fishes of India. They once formed a major fishery of the rivers
and streams of the Shivalik Himalayas and the lower reaches of uplands of the deccan plateau
(Mahanta et al. 1988). But various anthropogenic factors like large scale killing of brooders and
juveniles of mahseer has led to the drastic decline in their population (Ogale, 1994; Basavaraja and
Keshavanath, 2000) and they have been enlisted as threatened species as per IUCN red list
(Dahanukar et al., 2004; NBFGR, 2000). FAO has endorsed cryopreservation as one of the major
strategies for conservation of fish resources (Khoshoo, 2002) and can be efficiently employed for the
conservation of mahseer. Often, it is very difficult to locate the endangered fish resources in the wild
and quite often, the fish resources will be in inaccessible, remote areas. It is always advisable to have
a portable cryopreservation set-up nearby. Programmable freezers are very expensive. With CPU-
control, they become very heavy and it is very difficult to mobilize them to the field area. Hence, in
the present study, efforts were made to design a cost-effective, variable freezing rate apparatus for the
cryopreservation of mahseer semen in the field conditions.
MATERIALS AND METHODS:
For cryopreservation of fish semen, a variable freezing rate apparatus with a novel design was
fabricated using cost effective materials and tools. The straw racks and the rack stands were designed
and fabricated in the workshop. Flat aluminium strips of 2 x 0.3 cm dimension were used in the study.
Aluminium rivettes of 4 mm diameter and 6 mm length were used.
Fabrication of Rack Stand: The rack- stand had a dimension of 52 x 29 x 20.5 cm with three sets
(two pairs per set) of vertical bars (Fig 1). Each set of vertical bars had one pair on either side to hold
one straw rack.
Fabrication of Straw Rack: The straw rack was rectangular with a dimension of 28 x 8.5 x 2 cm and
grooves 3 mm wide and 3 mm deep were made continuously along the top edge of the straw rack (Fig
1). Each straw rack had a provision to hold about 42 straws at a time, with a pair of holes 3 cm apart,
on either side. The straw racks could be put in place in between the vertical bars on either side, within
the rack stand, using two aluminium rods of 2 mm diameter and 30 cm length (Fig 2).

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Freezing the straws: The rack stand could be slid into a styrofoam box with an inner dimension of 53
x 31.5 x 19.5 cm (Fig 3). The inside of styrofoam box was marked with a vertical scale, the markings
1 cm apart for knowing the level of the LN2.
RESULTS AND DISCUSSION:
A cost-effective, variable freezing rate apparatus with a novel design was fabricated which consists of
mainly two components, Rack Stand and Straw Racks. The rack stand had a dimension of 52 x 29 x
20.5 cm and the vertical bars on either side had holes at every 1 cm interval so as to hold the straw
rack at the desired height over the liquid nitrogen (LN2) surface, to achieve the desired rate of
freezing. The straw rack had a dimension of 28 x 8.5 x 2 cm with a provision to hold 42 straws at a
time. The main advantage of this variable freezing apparatus is that the three racks can be
simultaneously frozen at the same or at different freezing rates. This variable rate freezing apparatus
was successfully used for cryopreservation of spermatozoa of endangered, deccan mahseer, Tor
khudree and the golden mahseer, T. putitora in the field conditions (Patil and Lakra, 2005; Patil et al.,
2016) (Fig 4).
CONCLUSION:
This freezing apparatus is very cost effective, simple and very convenient to use and cryopreservation
of fish semen can be carried out with ease in the field conditions.
ACKNOWLEDGEMENTS:
The financial assistance received from Indian Council of Agricultural Research, New Delhi for the
present work is gratefully acknowledged. We thank The Director, Central Institute of Fisheries
Education, Mumbai and The Environmental Officer, Tata Power Company, Lonavala for providing
lab and farm facilities.
REFERENCES:
Basavaraja, N. and Keshavanath, P. 2000. Conservation and management of fish genetic resources in
Karnataka. In Ponniah, A. G. and Gopalakrishnan, A. (eds.), Endemic fish diversity of Western
Ghats, pp. 152-154. NBFGR-NATP Publication, Lucknow.
Dahanukar, N., Raut, R. and Bhat, A. 2004. Distribution, endemism and threat status of freshwater
fishes in the Western Ghats of India. J. Biogeogr., 31: 123-136.
Khoshoo, T. N., 2002. Conservation of biological diversity. In Dadhich, L. K. and Sharma, A. P.
(eds.), Biodiversity- strategies for conservation, pp. 15-64. APH Publishing Corporation, New
Delhi.

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Mahanta P.C., Kapoor, D., Pandey, A. K., Srivastava, S. M., Dayal, R., Patiyal, R. S., Joshi, K. D.,
Singh, A. K. and Paul, S. K. 1988. Conservation and rehabilitation of mahseer in India. In
Ponniah, A. G., Das, P. and Verma, S. R. (eds.), Fish Genetics and Biodiversity Conservation,
pp.93-105. Nature Conservators, Muzaffarnagar.
NBFGR, 2000. Threatened fishes of India. In NBFGR (ed.), Annual Reports, pp. 16-17. National
Bureau of Fish Genetic Resources, Lucknow.
Ogale, S. N. 1994. Endangered deccan mahseer, Tor khudree (Sykes)- A case study. In Dehadrai, P.
V., Das, P. and Verma, S. R. (eds.), Threatened fishes of India, pp. 213-218. Nature
Conservators, Muzaffarnagar.
Patil, R. and Lakra, W. S. 2005. Effect of different cryoprotectants, equilibration periods and freezing
rates during cryopreservation of spermatozoa of mahseer, Tor khudree (Sykes) and T. putitora
(Hamilton). Aquacul. Res., 36:1465 – 1472.
Patil, R., Lakra, W. S., Jahageerdar, S., Krishna, G. and Pal, A. K. 2016. Studies on fresh milt
parameters and cellular changes during cryopreservation of spermatozoa of Deccan mahseer Tor
khudree (Sykes, 1839). Indian J. Fish., 63(1) : 67-73.
Figure 1. Straw racks and rack-stand used for freezing the straws
Figure 2. Straw racks with straws mounted on rack-stand at different heights
(variable freezing rates)

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Figure 3. Rack-stand with loaded straw racks kept inside a Styrofoam container and ready
for freezing
Figure 4. Field set up of Cryopreservation materials with loaded rack stand in the covered
Styrofoam box