THE TRANSPORT OF SPERM

This protocol is based on studies of the short-term unfrozen storage of anuran sperm without (Browne et al., 2001) or without subsequent cryopreservation (Browne et al. 2002 a,b), and successful recent and current studies of sperm and oocytes induction by hormones and in vitro fertilization techniques at the Memphis Zoo (Browne et al., 2004-5).

Basically, three conditions must be met to sample sperm from recently deceased anurans and for its unfrozen storage and transportation.

1) The testes (whether in situ or in vitro) or sperm must be kept as close to 0ºC as possible. The reason for an ice slurry rather than on ice is that a slurry avoids problems with freezing caused by untempered ice at temperatures well below 0°C. Refrigeration normally runs at 4ºC at which temperature things will only store about 10-50% of the time in ice slurry at just above 0ºC.

2) Blood and extraneous (to the testes) tissue must be removed from the testes surface and not included in testes macerates. The testes can be rolled between paper moistened with simplified amphibian ringers.

3) If testes are transported these must be kept on a paper that is moist, but not moist enough to form a film of water over the testes (it is easier and safer to store and transport macerates and storage of testes is not recommended) (Browne et al., 2002a).

Other factors to consider.

1) With small testes the sample of sperm suspension produced will be minute and may best be stored in the smallest container available. Probably the best storage containers for sperm suspensions or for spermic urine are 1.5 or 0.75ml Eppendorf (bullet) tubes. To maintain the lowest temperature above freezing these tubes could be stored in plastic jars filled with ice slurry. These jars could then be stored in a Styrofoam box.

2) The best method is to ship sperm from testes is as a macerate in a cryodilutents. With spermic urine as a vehicle the maintenance of motility and fertility for days is possible (Browne, unpublished). The preferred cryodilutents for short-term storage of testes macerates would be simplified amphibian ringers. If the sperm is to be subsequently cryopreserved use 20% (w/v) trehalose with 20% (v/v) dimethyl sulphoxide added at an equal weight to volume of testes and diluent to give a sperm suspension with a concentration of 10% (w/v) trehalose and 10% (v/v) dimethyl sulphoxide (Browne et al., 2001, 2002b). Recent studies (Sargent and Mohun, 2005) have shown success in cryopreservation of Xenopus laevis and X. tropicalis sperm using an alternative cryodilutent of 20% (w/v) sucrose with 20% (v/v) egg yolk mixed 1:1 (w/v) with sperm suspension or spermic urine. I have not tried this cryodilutent for the short term storage of amphibian sperm before cryopreservation, however, egg yolk can lead to excessive bacterial growth. The addition of antibiotics may avoid this problem. To avoid bacterial growth, oxidation, etc., all cryodiluents should be stored frozen before use as small aliquots, which then are used, with the remainder discarded.

THE SHORT-TERM STORAGE OF SPERM

1) As per. above the short-term storage of sperm is best when the sperm is stored in Eppendorf tubes in an ice slurry, (in a refrigerator at 4°C) as a testes macerate in simplified amphibian ringers, or as spermic urine.

References:

Correspondence regarding references: ifg.bioteck@gmail.com

Browne, R.K., Clulow, J., Mahony, M. 2001. Short-term storage of cane toad (Bufo marinus) gametes. Reproduction. 121(1):167-173.

Browne, R.K., Clulow, J., Manony, M., 2002a. The short-term storage and cryopreservation of spermatozoa from hylid and myobatrachid frogs. Cryo Letters 23(2): 129-136.

Browne, R.K., Davis, J., Pomering, M., Clulow, J. 2002b. Storage of cane toad (Bufo marinus) sperm for 6 days at 0ºC with subsequent cryopreservation. Reproduction, Fertility and Development. 14 (5): 267-273.

Hollinger, T. G. and Corton, G. L. (1980). Artificial fertilisation of gametes from the South African clawed frog, Xenopus laevis. Gamete Res. 3, 57-64.

Sargent, MG., Mohun, TJ. 2005. Cryopreservation of sperm of Xenopus laevis and Xenopus tropicalis. Genesis. 41:41-46.