Resource Manual - International Embryo Transfer Society

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10 min, the cryostraws were exposed to -20ºC for 15 min in the freezer, and afterwards cooled by exposure to liquid nitrogen gas vapor (10 cm above the liquid nitrogen level of the tank) for 10 min; the cryostraws were finally immersed into liquid nitrogen (-196ºC), and stored. In order to avoid excessive temperature fluctuations, fast transfer was performed at all steps. The same cryopreservation media can be used to store spermatozoa at 4 C for up to 72 hs with very good sperm function recovery rates. Thawing procedure For thawing, straws are rapidly immersed in a water bath (37ºC) for 60 sec and wiped dry. Depending on its use, samples can be processed without removing the cryoprotectant media (the content of the straws was released directly into 500 l of Tyrode´s buffered medium) or, the cryoprotectant can be removed by flushing the sperm suspension into an Eppendorf tube with 500 l Dulbecco´s phosphatebuffer saline-2% (w/v) polyvinylpyrrolidone-40 (DPBS-PVP, pH 7.4, Sigma Chemical Co.) per straw. The sperm suspension is washed twice by centrifugation (100 g, 10 min). The precipitate is then resuspended in Tyrode´s medium and adjusted to a final sperm concentration as needed. 136
Cortisol Enzyme Immunoassay Protocol Astrid Bellem 1 , Coralie Munro 2 and Nadja Wielebnowski 1 1 Brookfield Zoo, Chicago and 2 U.C. Davis, California March 2004 Protocol prepared by: Astrid Bellem Contact information for further details: asbellem@brookfieldzoo.org nawieleb@brookfieldzoo.org Publications using this protocol: 1) Shepherdson DJ, Carlstead KC, Wielebnowski N. 2004. Cross-Institutional assessment of stress responses in zoo animals using longitudinal monitoring of faecal corticoids and behaviour. Animal Welfare 13:S105-113. Reagents Antibody (R4866) and horseradish peroxidase (HRP) enzyme label can be purchased from Coralie Munro at the Clinical Endocrinology Laboratory at UC Davis. Standards were prepared from ICN Cortisol (Cat. #194569) by dissolving 1.000g in 10.00mL 100% EtOH for the primary stock and diluting downward into assay buffer. The high control was prepared by spiking assay buffer with standard stock to read about 30% on the %binding scale on the standard curve. 1. COATING BUFFER 1.59 g Na 2 CO 3 and 2.93 g NaHCO 3 per liter dH 2 O, pH to 9.6 2. ASSAY BUFFER 195 mL Stock A (0.2M NaH 2 PO 4 ), 305 mL Stock B (0.2M Na 2 HPO 4 ), 8.7 g NaCl and 1.0 g BSA/L dH 2 O, pH to 7.0 3. WASH SOLUTION CONCENTRATE (10X) 87.7 g NaCl and 5.0 mL Tween 20/L dH 2 O dilute 10-fold for working wash solution 4. SUBSTRATE BUFFER 9.61 g citric acid (anhydrous)/L dH 2 O, pH to 4.0 5. ABTS STOCK (40 mM) 0.55 g ABTS (azino-bis-ethylbenzthiazoline sulfonic acid)/25mL dH 2 O 6. PEROXIDE (0.5 M) 500 uL H2O2 (30% solution)/8mL dH 2 O EIA PROTOCOL 1. Plate Coating 1.1 Use Nunc Maxisorb plates only 137
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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
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ELISA Protocol: Measuring Anti-Gona
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Assay Notes: Blocking - If desired,
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~ 40 cm length ~ 5 cm ~ 6 cm ~ 4 cm
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Worksheet: Semen Collection by Rect
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6000iu. It is generally injected in
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Figure 1. Transcervical embryo coll
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Criteria for selection of recipient
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Contact Information for further det
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7. Clean up the microscope and aspi
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In Vitro Culture 1. The presumptive
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STOCK EDTA (edta) 100X 1 month expi
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Wild & Domestic Cattle, Buffalo and
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8. To thaw, individual straws shoul
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Cryodiluents: generally, a Tris-cit
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(semen:water) is made by adding 10
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A total of 200 sperm should be coun
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Epididymal Sperm Collection and Pro
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more aviculturists to attempt AI wh
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emaining semen is sufficient for mi
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Semen Collection in Snakes Submitte
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Artificial Insemination of Snakes S
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1996). Oocytes with >10 sperm penet
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Gorilla Sperm Cryopreservation Prot
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Gorilla Sperm Cryopreservation Prot
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Great Ape Semen Cryopreservation: E
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4. After a minimum of two hours in
Page 85 and 86: 8. Plunge the pellets directly into
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
Page 135: 1000 x magnification in an epifluor
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Resource Manual - International Embryo Transfer Society

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