Standard Operating Procedures for Bull Management (1088.47 KB)

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e. Using a fully automatic filling and sealing machine the diluted semen is filled in French mini straws under a LAFU using sterilized nozzles and fresh rubber tubing’s.(room temperature ) f. When the straw filling is done at 4°C, spacious cold handling cabinet is used in order to maintain semen equilibration temperature while filling and sealing the straws g. The specially designed rubber tubing for filling on machine should not be reused h. Sufficient number of sets (3 nozzles with rubber tubing and straws) are prepared in advance based on the number of bulls under collection and sterilized in ULTRA VOILET overnight for use. i. The diluted semen is gently mixed before filling to get uniform number of sperms in all the straws. j. Air space of 1 cm and the quality of sealing of straws at laboratory end should be checked at random k. After filling and sealing of straws the unit is cleaned with 70% alcohol l. All glass wares are immersed in neutral detergent in a basin for washing and sterilization. Racking of filled straws and equilibration of semen a. Once filling and sealing of straws is done the straws are immediately racked on racks by use of the ramp and racks and then placed in a cold handling cabinet (CHC) placed in the freezing room. b. The straws are maintained at 4°C temperature for a period of 4 hours c. This pre freeze storage period is termed as equilibration period. During this period the sperm cells become permeated with glycerol and an ionic and osmotic equilibrium is established with the media. This period also helps the spermatozoa to gain resistance prior to freezing stress. Freezing of straws a. The filled and sealed straws can be frozen by two methods, namely: 1. Static vapour freezing 2. Forced vapour freezing Static vapour freezing a. This is carried out in wide mouthed liquid nitrogen containers (LR 320 or LR 250) b. The straws on racks are placed on the S.S grill (Standard Type) which is kept 32 cms away from the brim (mouth) of the container c. Level of Liquid nitrogen (LN) is maintained just above the surface of the grill
d. A ply wood/acrylic sheet is placed on the mouth of the container for settling of the LN vapours e. Three racks of 175 straws each is placed on the grill after settling of vapours for a period of 10 minutes f. The height of the freezing racks is 4 cm from the grill where the LN vapour has a temperature of minus 180 degree C g. After freezing the straws are collected and plunged into pre-cooled goblets for storage. h. Care needs to be taken during freezing since the nitrogen gas has a low heat capacity per unit volume (1 / 1500 of alcohol) whereby a small amount of heat will warm the gas a great deal and thus disturb the static vapour equilibrium drastically. Forced vapour freezing a. This is carried out using a programmable bio freezer b. The rate of freezing is highly precise and the program is fed in the computerized freezer c. The rate of fall of temperature is as follows: 1. The machine is put on at 20 degree C (room temperature) and brought down at 10 degree C per minute till 4 degree C. 2. Here it is paused automatically for transfer of straws from the Cold handling unit. 3. Each batch can freeze 30 racks of 175 straws or 5250 straws. (digitcool model of IMV) 4. A straw is cut and placed on the thermocouple to record graphically the fall of temperature within the straw. 5. The machine is then closed, once the temperature is stabilized at 4 degree C it is started again. 6. The rate of fall of temperature is @ 5 degree C from 4 degree to minus 10 degree C. 7. From minus 10 degree C the rate of fall has been programmed at @ 40 degree C per minute up to minus 100 degree C. 8. Finally from minus 100 degree to minus 140 degree C at @ 20 degree C per minute. 9. After 140 degree the freezer is opened and the straws are removed from the racks and placed into pre cooled goblets and then plunged into LN at minus 196 degree C. d. Samples as per requirement are drawn after freezing to assess post thaw motility and other quality control tests as per MSP.
Page 1 and 2: TABLE OF CONTENTS Sl.No. C h a p t
Page 3 and 4: Standard Operating Procedures for B
Page 5 and 6: Except for very tender and succulen
Page 7 and 8: In places where the individual bull
Page 9 and 10: Preparation of bulls for collection
Page 11 and 12: Hygiene and Precautions required by
Page 13 and 14: g. Hold the AV before and after eac
Page 15 and 16: . Standard Operating Procedures (SO
Page 17 and 18: d. No staff from the semen collecti
Page 19: ONE STEP DILUTION Match extender te
Page 23 and 24: Chapter - 4 Standard Operating Proc
Page 25 and 26: Definition Description 0- None No m
Page 27 and 28: B. Wet technique From the thawed fr
Page 29 and 30: -B- 0.25 ml of frozen thawed semen
Page 31 and 32: Stock solution B 1. Nacl -9.01 gm A
Page 33 and 34: Volume) inactivated and sterilized
Page 35 and 36: 2. Water bath capable of being main
Page 37 and 38: SELECTING A SOLUTION FOR DILUTION:
Page 39 and 40: Area of 80 small squares = 1/400X80
Page 41 and 42: -Prepare semen smear and allow it t
Page 43 and 44: 6-cleaning and drying After equilib
Page 45 and 46: Sl. No Date of completion of Quaran
Page 47 and 48: Annexure-5 Q.C. test conducted duri

Standard Operating Procedures for Bull Management (1088.47 KB)

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