PROCESSING STALLION SEMEN


The layering of cushion is done by first drawing the cushion so- lution into syringes at the appropriate volume. Next, a Tom- cat catheter, pipette or blunted spinal needle can be used to deposit the cushion under the diluted semen. Once this is done the tubes are weighed and balanced and centrifuged at 1000 × g for 20 min. It is important for the centrifuge to be well balanced as any vibra- tion can disrupt the sperm pellet and reduce the recovery rate of sperm. After centrifugation, the supernatant is removed. This can be done with a pipette or a vacuum aspirator. It is important to remove the supernatant as soon as possible after centrifuga- tion to maximise the sperm recovery rate. Next, the samples are combined and resuspended into one tube and a concentration and volume measured. When using centrifugation to process semen, the reconstituted volume can be much lower and sperm concentration much higher than using simple dilution without re- ducing sperm longevity, since no difference in sperm longevity has been found after cooled storage between sperm stored at a concentration of 25 × 10 6 sperm/mL or 250 × 10 6 sperm/mL (Bliss et al., 2012 ). There is an excellent review on processing semen from subfertile stallions (Varner, 2016 ) which covers cushioned centrifugation techniques in more detail.


SIMPLE DILUTION— ON- FARM USE


For fresh semen, the industry standard is to use 500 × 10 6 progres- sively motile sperm with a semen extender added in a 1:1 (semen: extender) ratio and maintain a volume of less than 60 mL. This should then be inseminated into the mare after processing. If there is a short delay from processing to insemination (<12 h) then the dose should be stored in a dark location at room temperature until use (Love et al., 2002 ; Varner et al., 1989 ). Light and incubator tempera- tures will adversely affect sperm longevity. If there is a delay longer than 12 h then the semen should be processed for cooling and held cooled (~4°C) until use.


SIMPLE DILUTION— COOLED- SHIPPED SEMEN


The general guidelines for extending semen for cooled shipping are to have between 5% and 20% seminal plasma (Jasko, Hathaway, et al., 1992b ; Jasko, Martin, & Squires, 1992c ), a concentration of 25 × 10 6 – 50 × 10 6 sperm/mL (Varner et al., 1987 ), a volume less than 50 mL (Loomis, 1993 ) and 1 × 10 9 progressively motile sperm in the final breeding dose (Brinsko, 2006 ). Semen extended with 5– 20% seminal plasma had better motility


at 24, 48 and 72 h of cooled storage (Jasko, Hathaway, et al., 1992b ; Jasko, Martin, & Squires, 1992c ). This equates to a dilution ratio (semen: extender) from 1:4 to 1:19. Thus, the minimum recom- mended dilution ratio is 1:4 for cooled- shipped semen. The final ex- tended concentration of sperm should be between 25 and 50 × 10 6 sperm/mL. Dilution of semen to 25 × 10 6 sperm/mL resulted in


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the highest total and progressive motility after 24 h compared to 50 × 10 6 , 100 × 10 6 and no dilution, with 50 × 10 6 having the second highest motility values (Varner et al., 1987 ). Lastly, and as was pre- viously mentioned, the standard recommendation is to ship ≥1 × 10 9 progressively motile sperm in the insemination dose (Brinsko, 2006 ) and minimise the insemination volume (keep it ≤50 mL). There are situations when these guidelines cannot be met,


such as when a stallion has poorly concentrated semen or poor progressive motility. In these situations, the semen should be concentrated using a technique, such as cushion centrifugation, to reduce the total volume while maintaining an acceptable num- ber of progressively motile sperm in the breeding dose. A sim- plified table (Table 1 ) was developed to assist in determining the minimum dilution ratio (semen: extender) for cooled- shipped semen based on the raw sperm concentration of semen and the progressive motility cut- off values for centrifugation by sperm concentration in the ejaculate.


OTHER SEMEN PROCESSING TECHNIQUES


Continuous density gradient centrifugation (also known as single- layer colloid) can be a useful technique for some stallions to improve the sperm morphological profile, motility and DNA integrity post- processing (Edmond, 2010 ). It is important to note that this tech- nique does not improve the post- processing sperm quality in all stallions, and that a significant number of sperm are lost. Common recovery rates with density gradient centrifugation are from 30% to 40% but can be much lower (Edmond et al., 2012 ). Thus, it is impor- tant to test the effectiveness of this technique on any given stallion prior to shipping semen to mares, by not only looking at the recovery rate but also the morphological profile of sperm after the use of den- sity gradient centrifugation. Briefly, for this technique semen is extended 1:1 (v/v) with a pre-


warmed extender. Using either 15- or 50- mL conical tubes, the den- sity gradient media (EquiPure™, Nidacon International AB) is added to the tube first. The volume of EquiPure™ used is typically equal to the amount of extended semen to be added. Next, the extended semen is then added to form a layer on top, being careful not to mix the extended semen with the colloid. The tubes are then centrifuged at 400 × g for 20 min, and afterwards, the supernatant is removed and the pellet resuspended. For more details on the procedure, the reader is referred to Bradecamp ( 2014a ). Another method to concentrate sperm without the use of cen-


trifugation is through filtration. The recovery rate of sperm using this method was originally reported at 92% and 95%, depending on the size of the pores in the filter (Alvarenga et al., 2010 ). How- ever, in studies comparing filtration versus cushion centrifugation, the sperm recovery rate was lower for the filter relative to when centrifugation was used and there were no differences in motilities between the two techniques (Roach et al., 2016 ). Nevertheless, the authors did infer that filtration may be a useful technique when cen- trifugation is not available.


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