Dr. Vikas Sachan
Department of Veterinary Gynecology and Obstetrics, DUVASU, Mathura, UP
Artificial insemination (AI) is first generation reproductive biotechnology which has revolutionized the animal production. AI can be done with the fresh semen, semen preserved at room temperature, refrigerated semen as well as with the cryopreserved semen. Artificial Insemination with approven quality standard frozen semen has been proved the best tool worldwide for genetic improvement through dissemination of superior germplasm. It also enhances the breeding value of a bull with achieved rapid genetic progress along with less risk of venereal disease transmission and harmful recessive traits. To achieve better conception rate, it is not only to focus the importance of genetic merit of bulls, quality of semen used, AI technique but also upkeepment and handling of the LN2 cryocans, AI equipments and semen etc. is equally important.
LN2 provider/supplier and receiver should maintain a log book for all LN2 distribution schedules at different AI centres as well as its consumption at AI centre. The AI centre should maintain at least a bigger container for LN2 for storage (May be 52 liter), a frozen semen straws storage cryocan preferably of 35 litre capacity, and a small portable LN2 container, preferably 3-5 litre capacity, to carry the semen doses to the place where the AI is to be carried out. Regular checking for the LN2 level should be carried out with help of dip stick.
Handling of semen as well as liquid nitrogen (LN2) cylinder management has its own importance. Frozen semen straws are always preserved in liquid nitrogen (BP -1960C and FP -2120C) in special container called as LN2 cylinder or cryocan. Cryocan is a double-walled metal/aluminium vacuum vessel with an efficient insulation. There is an inner chamber, an outer chamber and a bad heat conductor non-metallic neck. A high quality insulator material (No heat exchange) with maintained vacuum system is filled between inner and outer chamber. Failure of this system will lead to loss of the gas rapidly. Frost line at the top of container is sign of increased evaporation due to damaged container. Weight of LN2 in the container is supported by neck tube which is a delicate part and easily get damaged on mishandling. With proper knowledge about structure and function of cryocan, One get the maximum efficiency of cylinder. LN2 cylinder should be kept on rubber, jute or wooden carpet in order to avoid the direct contact with hard floor. Wet floor, hard cement or chemicals may cause corrosion to the cylinder with due course of time and may lead to loss of vacuum system. LN2 cylinder should be kept always in vertical position. It should never be tilted or rolled to avoid any damage or dent. Piling one cylinder above other is a bad practice. To shift the cryocan from one to another platform base trolley should be used. Cryocan should be placed in cool place with no direct sunlight exposure as in exposed light the LN2 holding capacity is reduced. There should be an arrangement of extra cryocan in case of appearance of frost line or condensed moisture on outer shell. Handling should be careful to avoid damage to vacuum knob of the cylinder to avoid vacuum loss. Handling of cryocan should be gentle as any jerk or shock/vibration during lifting, putting or carrying may cause damage to the neck tube and loss of vacuum system. Punching or drilling on the container wall is prohibited. Neck plug or lid of cryocan should be opened only during filling of gas, measuring of LN2 gas level or taking straw out. Neck should be kept dry with use of cloth or tissue paper time to time to avoid jamming of the neck plug. The lid should always be kept in vertical position to avoid damage. Lids or canisters are never be recommended to exchange, as they are specific for individual models of cryocan otherwise there may be damage due to tight fitting or frost formation with LN2 loss due to loose fitting. Frequent opening, closing or shifting of the cryocan should be avoided to keep evaporation rate minimum. For proper charging and filling of the container, the gas should be filled slowly and in non-splashing manner. Funnel should be used to transfer the gas from one to another cryocan and avoid overfilling of gas. LN2 cylinder can be secured with rubber band rings at body and and at the base during transportation as shock absorbent. One should always be careful while transporting filled cryocans in bus or train or in gathering, as sudden evaporation may cause any unwanted or panic situation among people. Regular checking for frost formation, leakage and also the LN2 level in the container should be practised and replace immediately, if there is increased evaporation rate or any damage diagnosed. Metal or wooden dipstick should be used to measure the LN2 level in cryocan regularly. Plastic or any hollow rod is not recommended for the purpose. On filling new container with LN2 gas, one should wait for 24 hr for charging and cooling of the can, then it should be refilled to its full capacity. It is found that first filling lead to excess evaporation due to charging so, one should not worry about any damage to container. Also there be is frost appearance on first filling but it vanishes by its own within one or two hours. First noting of the LN2 level should be done after 10 days of first filling, as it is the normal time taken for thermal balance by the new container. Use of protective clothing and eye glasses is recommended to avoid any direct contact of the LN2 to body surface. One should know that although LN2 is a non-toxic and non-inflammable gas but if there is any direct exposure of the gas to body tissue, it may cause tissue damage or frost bite. In this situation, water should be used immediately to wash off the affected part. In poorly ventilated room, it may cause suffocation and lead to unconsciousness and dizziness, so quick movement to fresh ventilated area is needed.
frozen semen doses should be kept in a well-ventilated, all weather safe storage area. Semen straws should be kept in the goblets in canisters with proper identification marking like date, species and sire information, goblet no., canister no., no. of straws etc on locator strips tagged with canister. Maintenance of the proper inventory of stored semen must be done by preparing the “Inventory map of storage container” with above information is required. Frozen semen while transferring or handling should not be exposed above liquid nitrogen as it may cause death of sperms due to thermal shock. The Canister should be not exposed over and above the neck of the cylinder and it is recommended to use the forceps to take semen straw out. While transferring the semen straws from one to another container a thermocole box can be used and transferring procedure should be fast within 5 seconds exposure of straw in air. Liquid Nitrogen should be replenished in both storage and distribution containers at regular intervals to ensure proper level of liquid nitrogen. Details of semen dose should be updated after every supply or dispatch to AI centers with record of used and remaining. If LN2 level reduces below the canister i.e. cold chain is disturbed, whole of the semen stored in particular container will get spoiled because of the thermal shock so all the semen doses should be discarded before replenish the LN2. At the time of AI semen straw should not be exposed above LN2 level more than 10 seconds otherwise sperms will be thermally damaged. If any way it takes more than 10 sec for straw retrieval, lower back the canister to tank floor to dip in LN2 and then lift it again to complete the task.
Generally AI kit has Scissors, Thermometer/thaw monitor, Thawing Tray, Forceps, Sheaths with sheath container, AI Gun with container, Plastic gloves, Lubricant, Isopropyl alcohol/ surgical spirit, Tissue papers, Clean towel, Thermos-flask with hot water, apron, Tags, pins and tag applicator etc. Stainless steel AI guns from an agency whose AI guns are tested and approved by the testing stations identified by BIS shall be used for AI. AI sheath shall be from an agency whose AI sheaths are tested by BIS (Bureau of Indian Standard). AI accessories like forceps and scissors shall be made of good quality stainless steel. Thermos flask and thermometer shall be of good quality. The cleanliness and sterilization of these AI utensil is must to achieve better conception rate. All the disposables or non disposables should be sterilized properly. Clean AI gun, scissors and other accessories whenever they get soiled or at least once a week with hot water and air dry them. Sanitize the AI gun and the scissor with Isopropyl alcohol after drying. The AI Gun piston and the scissors should be wiped clean with water and Isopropyl alcohol after every insemination. Surgical spirit and soaps are lethal to semen, hence should not be used to clean equipments. Aprons, towel etc should be washed properly. Cold fumigation in formaldehyde chambers can be used time to time to sterilize AI utensils.
Scientific knowledge and handling of the AI equipments along with proper handling of semen at AI centres is not only necessary for improving the inseminator efficiency but it is recommended to add in maximizing the conception rate in dairy animals to maintain their reproductive efficiency at maximum level. Above mentioned scientific standards and protocols will definitely help in achieving the ultimate goal of farmers and dairy owners for the betterment of their economical status.
