Title of Invention

"A MANUALLY OPERATED LOW COST HANDY CRYOFREEZER FOR GAMETE CRYOPRESERVATION"

Abstract

The manually operated hand cryofreezer was developed to cryopreserve the milt samples of Indian major carps and other cultivable fish species. We found it suitable and more advantageous over the programmable cryofreezers in terms of cost of the equipment, liquid nitrogen use, and size of the unit, on-farm utilization, and easy operation utilizing locally available materials. The manually operated handy cryofreezer is able to freeze the semen samples of Indian major carps, L. calbasu, catla catla, Cirrhinus mrigala and Labeo rohita successfully in a desired rate of -15°C/ min. The manually operated handy cryofreezer use less amount of liquid nitrogen than a programmable cryofreezer. For freezing 5ml milt in visotube (5ml capacity) till -60°C, 3-4 liters of liquid nitrogen. We found no significant difference in the fertilization rates of eggs of Indian major carp's eggs produced by utilizing cryopreserved milt frozen by programmable cryofreezer and a manually operated handy cryofreezer. Thus, suggesting its applicability for cryopreservation of gametes suceddfully.

Full Text

a) Nature of the invention: To increase productivity in fisheries, we need quality fish seed. This can be achieved by either genetic selection or by biotechnological tools. But for sure genetic upgradation has played an important role in increasing productivity in other farm animals. In fisheries an immediate possibility to increase productivity could be to upgrade the stocks by introducing quality cryopreserved milt from good male fishes. Before developing a crypreservation protocol for any types of gametes, the basic requirements is a cryofreezer for freezing the samples to -196 °C. The programmable cryofreezer used is an expensive equipment which many farmers may not afford or like to buy. The programmable cryofreezers presently in use at different parts of the world is expensive and requires more liquid nitrogen for milt cryopreservation. In this context a manually operated low cost handy cryofreezer for gamete cryopreservation was developed for cryopreserving fish milt. In this very few less expensive materials are used such as hot water bath, Styrofoam box, Liquid nitrogen pump, galvanized iron box to fit into the styrofoam box and liquid nitrogen. Apart from this extenders and cryoprotectant chemicals generally used for the specific protocols for different types of fishes are used. This handy cryofreezer can be operated on-farm sites also. This cryofreezer is an innovative method developed due to the constraints faced on farms and also is an improvement of "cryopreservation by keeping samples over liquid nitrogen fumes". Here, we have used a pump, heater and thermometer to regulate the cooling rate. This handy cryofreezer has a great scope for utilization by farmers, researchers and also hatchery managers for cryopreservation of fish milt at different locations with a much lower cost than any other programmable cryofreezers being used. By using this handy manual cryofreezer, multilocational trial cum demonstration of utilization of cryopreserved milt was carried out at different states such as Madhya Pradesh, Tamilnadu, Orissa and Punjab states of India. b) Prior Art: Prior to this design, there was no such equipment which can be used to cryopreserve the fish milt at any remote and difficult location with changing cooling rates apart from the programmable cryofreezers which are very expensive, requires more liquid nitrogen, mostly it has to be imported from foreign countries, size is very big when compared to the present one and maintenance problems. In fisheries sector, the hatchery owners and fish farmers face the problem of inbreeding can get benefit of this. The conservation of many endangered fishes available at very difficult locations can be attempted by using this cryofreezer. So designing a cryofreezer which is less expensive, easy to use with different cooling rate manipulation was needed for to the researchers and other people in the field of cryobiology, aquaculture and other related field also. c) Details of the invention : While working with simple cryofreezing by holding samples of milt over liquid nitrogen and advanced programmable cryofreezer "a manually operated low cost handy cryofreezer for fish gamete cryopreservation" has been designed using very simple materials such as: (1) liquid nitrogen along with can, (2) styrofoam box, (3) inner galvanized iron box to hold liquid nitrogen, (4) hot water bath, (5) hand pump (Bicycle pump) and (5) tripod stand to place visotubes over it. This method has been proven suitable for carp sperm cryopreservation in bulk. Indian major carp milt samples after collection was equilibrated for 30-40 minutes at 4 °C including filling and sealing of French straws / visotubes. These straws were kept over liquid nitrogen for 5 minutes over tripod stand in a galvanized iron container which is placed in a Styrofoam chamber. The Styrofoam chamber was placed in a hot water bath. The heat generated in the water bath allowed better rate of cooling (-15 °C/min) inside the chamber which can be monitored by the thermometer placed over it. The liquid nitrogen vapour in the chamber provide a good enough freezing rate for freezing milt samples filled in 0.25-0.5 ml straws. Thus cryopreserved spermatozoa can be utilized in hatcheries across India. Such a method is being used at CIFA, Bhubaneswar and also being advocated for hatchery managers due to its convenience and cost effectiveness in cryopreserving carp spermatozoa. Detail procedure (step-wise): i) Collect milt samples in sterilized tubes with enough airspace (more than 50%). ii) Store at 4 °C and process it under the same temperature, iii) Mix the milt and diluent at 1:3 ratio and keep for 30-40 min. iv) Fill the straw in 5ml visotubes and seal by cotton and soft wax powder, v) Keep the sample filled visotube over the tripod stand in the galvanized iron box. vi) Prior to this fill 30 % liquid nitrogen of the total capacity of the galvanized box placed inside the Styrofoam box. vii) Place the Styrofoam box over the water bath in hot water (80-100 °C). viii) At this rate of heating the liquid nitrogen fumes will spread evenly inside the chamber and cooling rate of-15 °C/min can be achieved, ix) After 10-15 minutes the samples will attain -60 to -80 °C. x) Remove the samples and store in liquid nitrogen cans in canisters, xi) Thawing of the samples should be done in water at 37-39 °C. Schematic view of "the manually operated handy cryofreezer for fish gamete cryopreservation": (Figure Removed) We Claim: 1. A manually operable low cost handy cryofreezer, said cryofreezer comprises: a styrofoam box (3); a galvanized iron box (4); a tripod stand (5); a hot water bath (7); a liquid nitrogen can (8); and a hand pump (9) connected to said liquid nitrogen can, wherein said cryofreezer utilizes 3 liters to 4 liters of liquid nitrogen for freezing 5 ml milt sample in viscotubes (1) till -60°C. 2. The manually operable low cost handy cryofreezer as claimed in claim 1. wherein the milt sample is from L. calbasu, Catla catla. Cirrhinus mrigala or Lebeo rohita.

Documents:

1123-DEL-2005-Abstract-(25-05-2011).pdf

1123-del-2005-abstract.pdf

1123-DEL-2005-Claims-(25-05-2011).pdf

1123-del-2005-claims.pdf

1123-DEL-2005-Correspondence Others-(04-11-2011).pdf

1123-DEL-2005-Correspondence Others-(25-05-2011).pdf

1123-del-2005-correspondence-others.pdf

1123-del-2005-description (complete).pdf

1123-DEL-2005-Drawings-(25-05-2011).pdf

1123-del-2005-form-1.pdf

1123-del-2005-form-13.pdf

1123-del-2005-form-18.pdf

1123-del-2005-form-2.pdf

1123-del-2005-form-26.pdf