Felis margarita (spermatozoa / sperm)
The study from which this protocol is derived sought to evaluate cryopreservation protocols for sperm cryopreservation in the sand cat (Felis margarita) and the black-footed cat (Felis nigripes). The authors determined that “Motility and acrosomal integrity of fresh and frozen-thawed spermatozoa from BFCs and SCs were similar,” “cryopreservation in straws was superior to cryopreservation in pellets for both species,” and the BFC and SC spermatozoa fertilized 60.0%–79.4% of homologous and 37.7%–42.7% of heterologous oocytes”.
· Feline-optimized culture medium (FOCM; Sigma Chemical Co.)
· Hepes (Sigma Chemical Co.)
· Test Yolk Buffer (Irvine Scientific)
· 1.5-ml microcentrifuge tube
· Plastic container
· Refrigerator or cold room
· Liquid nitrogen
· Plastic straws (Agtech, Inc., Manhattan, KS)
· Plastic bag
· Water bath
· Metal test tube racks
· Styrofoam cooler
1. After collection, dilute the semen samples 1:1 with Hepes-buffered FOCM (FOCMH; 20.0 mM Hepes and 5.0 mM NaHCO3 in FOCM)
2. Centrifuge the semen at 300 x g for 10 min.
3. To ensure maximum sperm recovery, recentrifuge the supernatant at 1100 x g for 10 min.
4. Combine the bottom 20 µl from the bottom of the recentrifuged sample with the sperm pellet from the initial centrifugation.
5. Slowly dilute the concentrated sperm samples to 25-50 x 106 motile sperm/ml with room temperature (~22°C) Test Yolk Buffer containing 4% glycerol.
6. Aspirate aliquots (~30 µl) of the diluted sample into 0.25-ml plastic straws.
7. Heat seal the straws on both ends.
8. Place the straws in a plastic bag.
9. Place the bag in a plastic container filled with 250 ml of room temperature water and place the container in a refrigerator or cold room for 3.5 hours.
10. Partially fill a styrofoam container with liquid nitrogen. Place two metal test tube racks in the Styrofoam container such that the top of the first tube rack is 7.5 cm above the surface of the liquid nitrogen and the top of the second tube rack is 2.5 cm above the liquid nitrogen.
11. Hold the straws for 1 min on the upper rack then transfer to the lower rack for 1 minute.
12. Plunge the straws into liquid nitrogen. They are now ready for long-term storage in liquid nitrogen.
1. Thaw the cryopreserved samples in air for 10 sec.
2. Place directly into a ~38°C water bath (straws) and hold for 30 seconds.
3. Empty the contents of each straw into a 1.5-ml microcentrifuge tube and slowly dilute 1:3 with FOCMH.
4. Your samples are now ready for downstream use.
J.R. Herrick, M. Campbell, G. Levens, T. Moore, K. Benson, J. D’Agostino, G. West, D.M. Okeson, R.Coke, S.C. Portacio, K. Leiske, C. Kreider, P.J. Polumbo and W.F. Swanson. In vitro fertilization and sperm cryopreservation in the Black-Footed Cat (Felis nigripes) and Sand Cat (Felis margarita). Biology of reproduction. 2009 Nov;82:552-562
Added by: Carlton Hoyt on September 26, 2012
Please help improve Cryosearch!
No one has given their opinion on this protocol yet. If you have used this protocol in a lab, please mark whether or not it worked for you. If you had any difficulties, please discuss in the comments.