Resource Manual - International Embryo Transfer Society

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Semen Freezing Protocol (Pellet Method) Materials Needed: Refrigerator or cold room (4-5 o C) Dry ice (5-10 lb block) Cryogenic gloves Nail board (for making ~ 5 mm depression in the dry ice block) Sterile Pasteur or transfer pipettes Long handled plastic spoon Insulated container or bucket Long forceps Cryovials Fine permanent marker Canes for cryovials Sleeves for canes Liquid nitrogen Freezing Procedure for Pellets 1. Thaw one aliquot each of the non-glycerated (Primate Extender A) and glycerated (Primate Extender B) cryodiluents. Place Primate Extender B into the refrigerator, along with a Pasteur or transfer pipette, labeled straws (estimate four straws for every 0.25 ml of raw semen) and sealing powder. 2. At room temperature, add a volume of Primate Extender A equal to the semen volume (1:1 dilution ratio) directly to the raw ejaculate in a sterile test tube (large enough to contain at least four times the volume of the raw ejaculate. 3. Place the tube containing this first extension into a beaker of room temperature water, then place the beaker into a refrigerator (the water slows the cooling of the sperm to refrigeration temperature). Be sure that the refrigerator is left undisturbed during this equilibration period. 4. After a minimum of two hours in refrigeration, slowly (drop by drop) add the cold glycerated cryodiluent (Primate Extender B) using the cold pipette at a volume equal to that of the semen plus the first extension with Primate Extender A. During the second dilution, continuously and gently mix the suspension with the pipette or by a gentle swirling motion. 5. At this time, prepare for freezing by using the nail board to make ~ 5 mm depressions on a flat surface of a dry ice block and filling the insulated container with a small volume of liquid nitrogen. 6. Use a cold Pasteur or transfer pipette to first gently mix the second extension, then drop the extended semen directly into the dry ice depressions (one drop per depression). Count the total number of pellets. 7. Allow the pellets to remain on the dry ice for 10 min. 84
8. Plunge the pellets directly into liquid nitrogen by using cryogenic gloves to lift the dry ice block and tapping on the opposite side to dislodge the pellets. 9. Secure labeled cryovials using long forceps and lower it into the liquid nitrogen. Using the plastic spoon, scoop up the pellets and place them into the cryovial. 10. The cryovials containing pellets should be carefully capped and placed onto canes. The canes should be covered by protective sleeves then placed into a liquid nitrogen storage container. Thawing Procedure for Pellets 1. Transport one or more cryovials containing the frozen pellets in an insulated container (preferably not white) containing a small volume of liquid nitrogen. 2. Fill several 10 ml round bottom sterile tubes with 0.5 ml of medium (e.g., Hepes-TL Solution, BioWhittaker, Walkersville, MD, USA; Cat # 04-616F). Place the rack containing these tubes into a 37 o C water bath. Use one tube to thaw two to three pellets. 3. Using a flamed, 25 gauge needle, poke three holes into the body of a long handled plastic spoon. 4. Carefully uncap the cryovial and release the pellets. Using the modified plastic spoon, scoop 2-3 pellets at a time and lift them out of the liquid nitrogen. As soon as the liquid nitrogen is drained through the holes in the spoon, drop the pellets into a tube containing the pre-warmed medium and swirl the tube gently in the water bath until the pellets are thawed. 5. Thawed semen should be examined immediately and two hours post-thawing for overall progressive motility and acrosomal integrity. 85
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Research Subcommittee Resource Manu
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VI. Marsupials (Taxon Leader: Moniq
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Large Felid Semen Collection by Rec
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eturn to 0. Once initiated, this pr
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In Vitro Fertilization and Embryo T
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3. After overnight soaking in the d
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6. To prepare, 15 mM NaHCO3, 15 mM
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9. After checking for patency the b
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2. Three abdominal entry sites are
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6 Poliglecaprone 25 is the preferre
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IVF 1. The IVF dishes are prepared
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Notes 1. Each new BSA lot is tested
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11. Pope C. E., McRae, M. A., Plair
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Cryodiluents: generally, a TES-Tris
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5. To isolate the live and motile s
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( Andersen, 1975, Farstad, 1984, Fa
Page 33 and 34: ELISA Protocol: Measuring Anti-Gona
Page 35 and 36: Assay Notes: Blocking - If desired,
Page 37 and 38: ~ 40 cm length ~ 5 cm ~ 6 cm ~ 4 cm
Page 39 and 40: Worksheet: Semen Collection by Rect
Page 41 and 42: 6000iu. It is generally injected in
Page 43 and 44: Figure 1. Transcervical embryo coll
Page 45 and 46: Criteria for selection of recipient
Page 47 and 48: Contact Information for further det
Page 49 and 50: 7. Clean up the microscope and aspi
Page 51 and 52: In Vitro Culture 1. The presumptive
Page 53 and 54: STOCK EDTA (edta) 100X 1 month expi
Page 55 and 56: Wild & Domestic Cattle, Buffalo and
Page 57 and 58: 8. To thaw, individual straws shoul
Page 59 and 60: Cryodiluents: generally, a Tris-cit
Page 61 and 62: (semen:water) is made by adding 10
Page 63 and 64: A total of 200 sperm should be coun
Page 65 and 66: Epididymal Sperm Collection and Pro
Page 67 and 68: more aviculturists to attempt AI wh
Page 69 and 70: emaining semen is sufficient for mi
Page 71 and 72: Semen Collection in Snakes Submitte
Page 73 and 74: Artificial Insemination of Snakes S
Page 75 and 76: 1996). Oocytes with >10 sperm penet
Page 77 and 78: Gorilla Sperm Cryopreservation Prot
Page 79 and 80: Gorilla Sperm Cryopreservation Prot
Page 81 and 82: Great Ape Semen Cryopreservation: E
Page 83: 4. After a minimum of two hours in
Page 87 and 88: Semen/Spermatozoa Collection and Ev
Page 89 and 90: Epididymal flushing If flush is c
Page 91 and 92: Component B = 5 ml of 2.4 mM propid
Page 93 and 94: Superovulation of Female Brushtail
Page 95 and 96: Superovulation in Wombat Species (e
Page 97 and 98: Once prepared, the solutions can be
Page 99 and 100: day 6 mature oocytes are collected
Page 101 and 102: Table.1 Drugs administered to super
Page 103 and 104: Biosciences NZ Ltd.), supplemented
Page 105 and 106: Light microscopy - acetic lacmoid s
Page 107 and 108: Gauze Elastic band (Garrot) to visu
Page 109 and 110: Wombat Faecal Progesterone Metaboli
Page 111 and 112: Preparation of dilutions for the MT
Page 113 and 114: Stop reaction (should turn yellow)
Page 115 and 116: 10. Count 2-5ul from each fraction
Page 117 and 118: Contact Information for further det
Page 119 and 120: Summary: Media and Products a. For
Page 121 and 122: Steps in preparing specimens for cr
Page 123 and 124: Hybrimax, D-2650)) and 0.1 M sucros
Page 125 and 126: Acknowledgements: I thank Monash IV
Page 127 and 128: acrosome presence in barbel sturgeo
Page 129 and 130: Next step is express assessment of
Page 131 and 132: Measurement Techniques for Fish Lar
Page 133 and 134: Chinchilla lanigera Order: Rodentia
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1000 x magnification in an epifluor
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Cortisol Enzyme Immunoassay Protoco
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Resource Manual - International Embryo Transfer Society

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