Boar Semen Cryopreservation Protocol

- 2007 -

Boar semen is collected using the hand glove technique and the gel fraction is removed. The semen is diluted 1:3 (v:v) with Androhep Plus (Minitube, Verona, WI), cooled to 23ºC on the laboratory bench and then to 15ºC over 1.5 hours. The samples are then shipped to the repository at 15ºC for cryopreservation.

Upon receipt, the samples are centrifuged for 10 minutes at 800 x g, at 15ºC. The supernatant is removed and the sperm pellets are consolidated by boar. The sperm concentration and motility are determined using spectrophotometry (Hammerstedt, 1975) and a Hamilton Thorne motility analyzer (Beverly, MA), respectively (at least 5 fields of analysis and 1000 cells). The sperm samples are diluted to a final concentration of 200 x 10⁶ sperm cells per ml in a two-step process. The samples are first diluted to two-thirds of the final volume with the BF5 cooling extender (CE; please see recipe section) at 15ºC.   The diluted samples are then cooled to 5ºC over 2 to 2.5 hours. The second dilution is performed using the freezing extender (FE; please see recipe section), which is one-third of the final volume, over 5 minutes, at 5ºC. The samples are loaded into 0.5ml straws and frozen using the Cryo Bio System Mini Digitcool UJ400 (IMV Corporation, Minneapolis, MN) with the following curve: 5ºC to -8ºC at 20ºC per minute, -8ºC to -120ºC at 69ºC per minute, -120ºC to -140ºC at 20ºC per minute.  The samples are then plunged in liquid nitrogen for storage.

Samples are thawed for 20 seconds in a 50ºC water bath and motility analysis is performed as described previously.

Recipes: Modified from Pursel and Johnson, 1975, J Anim Sci 40:1, 99-102.

Beltsville Freezing Extender 5 (BF5)

Cooling extender (CE)
52mM TES
16.5mM Tris
178mM D-glucose
20% Egg yolk (v:v)

The CE should be centrifuged at 10000 x g for 25 minutes to remove egg yolk particles

Freezing extender (FE)
6% Glycerol (v:v)
2.5% Equex paste (v:v)
91.5% CE (v:v)

Artificial insemination:

Deep intrauterine insemination of swine is performed according to Roca et al. (2003).  Ten straws of frozen semen (1 x 10⁹ sperm) are thawed, as described previously, and pooled.  The sample is loaded into a syringe and inseminated as described by Martinez et al. (2001).  After deposition of the sample into the uterine horns, an additional 2 mL of BTS is used to flush the insemination catheter and remove the remaining sperm sample. 

Standard intracervical insemination can also be performed using insemination doses of 3 to 6 x 10⁹ sperm (30 to 60 straws) diluted to a final volume of 80 mL in Beltsville Thawing Solution (Pursel and Johnson, 1975) and inseminated.

References:
Hammerstedt, RH. Tritium release from [2-3H] D-glucose as a monitor of glucose consumption of bovine sperm. Biol Reprod 1975; 12:545-551.

Martinez, E.A., Vazquez, J.M., Roca, J., Lucas, X., Gil, M.A., Parrilla, I., Vazquez, J.L., Day, B.N. Successful non-surgical deep intrauterine insemination with small numbers of spermatozoa in sows. Reproduction 122: 289-296.

Obtain information from Harvey Blackburn at: Harvey.Blackburn@ars.usda.gov