VGO 511: Veterinary Andrology and Reproductive Techniques (1+1)

FREEZING OF SEMEN

(Freezing the straws in liquid nitrogen vapour (manual freezing)and Freezing in bio freezer)

Freezing the straws in liquid nitrogen vapour (manual freezing)

• Freezing is carried out in a wide mouthed container (LR 320, LR 250) especially designed for freezing of straws.
• The actual freezing is done by holding the equilibrated straws at 4-5 cm from the level of liquid nitrogen thereby exposing to the vapour of liquid nitrogen.
• When the temperature of semen reaches – 130°C to – 150°C they are transferred to goblets and submerged in liquid nitrogen for storage at – 196°C.
• The main advantage of horizontal freezing is the efficiency and simplicity of the method.
• The racks hold the straws at a constant level and the straws are frozen at even rate all along the length of the straw.
• Ensure that the level of liquids nitrogen is 1-2 mm above the safety freezing net.
• After that the vapour temperature is adjusted by wafting and the racks with straws are kept on the freezing net so that the straws are about 4-5 cm form the level of liquid nitrogen.
• The recommended temperature for freezing semen is LR 320 unit is
  • Medium straws: - 150 ° C to - 160 ° C for 7 minutes.
  • Mini straws : - 120 ° C to - 130 ° C for 9 minutes
• These containers are used for freezing as well as storage of semen.
• The maximum storage capacity of LR 250 will be about 70, 000 French medium straws can be stored in 3 layers.
• With LR 320 one lakh French medium straws stored in 4 layer.
• The height of canisters to be used for LR 250 and LR 320 containers should be 25 cm and 40 cm respectively.
• Normally semen banks should plan and store a minimum of 4 months of its supply to the field in advance.

• Manual freezing is done in wide mouth container (LR 320 or LR 250).
• Place the freezing net in the container and fill liquid nitrogen in such a way that the level should be 2 cm above the freezing net.
• Equilibrated semen straws arranged on freezing rack is placed on the freezing net.
• The distance between semen straw and freezing net should be within 4-5 cm.
• Straws are kept for 9 minutes, packed in goblet and submerged in liquid nitrogen.

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Freezing in bio freezer

(Click here for video demonstration)

• Where large scale freezing is carried out the freezing is done by using bio freezer. Here the freezing programme is already fed in a computer.
• It can be altered whenever required. The bio freezer is connected with liquid nitrogen tank and the chamber temperature is brought to +5⁰C.
• Transfer of freezing rack from cold handling cabinet to freezing unit should be done is the shortest possible time to avoid any increase of semen temperature above +5°C.
• To achieve this cold handling cabinet and freezing unit is kept in the same lab.
• The freezing racks are placed inside the bio freezer and it is allowed for 5 minutes to get stabilized at +5⁰C.
• Then freezing is started. The entire process will take 9 minutes where the temperature will reach from +5⁰C to -140⁰C.

• Bio freezer - used for large scale freezing.
• In bio freezer the temperature of the semen straws will reach to -140^oC from +5^oC in 9 minutes.
• Then the straws are packed in goblet and submerged in liquid nitrogen.

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