USDA ARS National Animal Germplasm Program

Boar Semen Collection, Transportation, Processing and Cryopreservation Protocol Semen collection:

Prepare the semen extender (e.g. for Androhep Plus or Androstar mix 1 packet of powdered medium with 1 L of distilled/deionized water, Minitube, Verona, WI) and warm to 37 °C.

Collect the sperm-rich fraction of a boar semen sample using the hand-glove technique or a phantom mount and remove the gel fraction with sterile gauze or a semen filter.

Determine the sperm-rich ejaculate volume, sperm concentration, and the volume of the ejaculate that is needed:

sperm-rich ejaculate volume X sperm concentration = sperm count;
sperm count ÷ 45 billion sperm = sample volume needed by USDA.

NOTE: To meet repository targets, 45 billion cells are needed per boar collection for commercial boars and the entire ejaculate for minor/heritage breed boars; this will be determined by NAGP staff and communicated to the cooperator prior to the collection of the samples. Once determined, dilute the sample as follows:

Dilute the required sperm-rich fraction 1:1 (v:v) with 37 °C semen extender in a 37 °C beaker.

Aliquot the sample into 50 mL centrifuge tubes that are labeled with the name and identification number of the boar.

Cool the sample to 23 °C over 1 hour by placing it on the laboratory bench. The sample must be shielded from light during this time.

Further cool the sample over 1.5 hours by placing it in a 15 °C refrigerator and maintain it at this temperature prior to packaging for delivery. The samples are then at the appropriate temperature for overnight transportation to the repository.

Transportation:

Please see the Impact Shipper Protocol on the Animal GRIN webpage noting the specific temperatures for each species and type of tissue:

https://www.ars.usda.gov/plains-area/fort-collins-co/center-for-agricultural-resources- research/paagrpru/docs/animal/animal-protocols/

Semen processing:

Upon receipt, centrifuged the samples for 10 minutes at 800 x g at 15 °C. Remove the supernatant and combine the pelleted sperm by boar.

Determine the sperm concentration and motility using spectrophotometry and a Hamilton Thorne motility analyzer (Beverly, MA), respectively (at least 5 fields of analysis and 500 sperm) or microscopy and a hemocytometer.

Dilute the samples to 600 x 106 sperm/mL with 15 °C Beltsville Freezing Extender 5 (BF5) cooling extender (CE; see recipe below) and cool to 5 °C over 2 to 2.5 hours.

After cooling to 5 °C, dilute the samples drop-wise using 5 °C freezing extender (FE; see recipe below) over 5 minutes to 400 x 106 sperm/mL.

Load the samples into 0.5 mL CBS or wick and powder (aka French) straws and freeze them using:

a programmable freezer (e.g. Cryo Bio System Mini Digitcool UJ400, IMV Corporation, Minneapolis, MN) and the following curve: 5 ºC to -8 ºC at -20 ºC per minute, -8 ºC to - 120 ºC at -69 ºC per minute, -120 ºC to -140 ºC at -20 ºC per minute; OR
by placing the straws on a rack in liquid nitrogen vapor (4.5 cm above the liquid) for 10 min.

Plunge the samples into liquid nitrogen for storage.

Thawing:

Thaw samples for 20 seconds in a 50ºC water bath and evaluate motility as described previously.

Artificial insemination:

Perform standard intracervical insemination using 2 inseminations per sow or gilt and 1 x 109 motile sperm, per insemination. The semen sample should be diluted to a final volume of 80 mL in Beltsville Thawing Solution (Pursel and Johnson, 1975) per insemination dose.

Perform deep intrauterine insemination (1 insemination per sow or gilt; Martinez et al., 2001, Roca et al., 2003) using a dose containing 1 x 109 motile sperm diluted in Beltsville Thawing Solution as described previously. After insemination into the uterine horns, use an additional 2 mL of BTS to flush the insemination catheter of the residual insemination dose.

BF5 Recipes:

From Pursel and Johnson, 1975, J Anim Sci 40:1, 99-102

Cooling extender (CE)

52 mM TES

16.5 mM Tris

178 mM D-glucose

20% Egg yolk, by volume

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

Freezing extender (FE)

The ingredients are by volume:

91.5% CE

6% Glycerol

2.5% Equex paste

References:

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.

Pursel, V.G., Johnson, L. A. 1975. Freezing of Boar Spermatozoa: Fertilizing Capacity with Concentrated Semen and a New Thawing Procedure. J. Anim. Sci. 40: 99–102. https://doi.org/10.2527/jas1975.40199x

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.

Version updates: October 2019, April 2020