|Boar Semen Protocol|
Boar Semen Collection, Processing and Cryopreservation Protocol
Boar semen is collected using the hand glove technique and the gel fraction is removed using sterile gauze. The semen is diluted 1:3 (v:v) with Androhep Plus (37ºC; Minitube,
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 and a Hamilton Thorne motility analyzer (Beverly, MA), respectively (at least 7 fields of analysis and 1000 cells). The samples are first diluted to 300 x 106 sperm 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 drop-wise using the freezing extender (FE; please see recipe section) over 5 minutes at 5ºC so that the samples are diluted to a final concentration of 200 x 106 sperm cells per ml. 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.
Beltsville Freezing Extender 5 (BF5)
Cooling extender (CE)
The CE should be centrifuged at 10000 x g for 25 minutes to remove egg yolk particles
Freezing extender (FE)
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/intrauterine 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.
Roca, J., Carvajal, G., Lucas, X., Vazquez, J.M., Martinez, E.A. 2003. Fertility of weaned sows after deep intrauterine insemination with a reduced number of frozen-thawed spermatozoa. Theriogenology 60:77-87.