Dilute Semen for Artificial Insemination: New procedures

Since artificial insemination is currently practiced in most equine breeds (except Thoroughbreds), processing semen for cooling and transport is a common task in most equine reproductive centers. However, processing a dilute ejaculate--one with a relatively low concentration of sperm--requires modified procedures to get enough viable sperm in each breeding dose. At the 2008 American Association of Equine Practitioners convention, held Dec.6-10 in San Diego, Calif., procedures and calculations for this modified technique were presented by Dirk Vanderwall, DVM, PhD, Dipl. ACT, an academic faculty member at the University of Idaho.

Vanderwall reported that ejaculates with less than 100 million sperm per milliliter are considered dilute and require the modified technique. Normal ejaculates with 100 million or more sperm/mL are typically mixed with extender in a 3:1 ratio to achieve a final concentration of approximately 25-50 million sperm/mL. However, with a dilute ejaculate, this dilution ratio would give you too few sperm for successful breeding. He explained that you can't just use less extender for a dilute sample because seminal plasma (fluid in semen) needs to be diluted to 25% or less of its original concentration to maximize sperm viability during cooling/storage.

Processing a dilute sample requires mixing the semen with extender, centrifuging the sample at 300-400 Gs (300-400 times the force of gravity) for 10-12 minutes to concentrate the sperm into a soft "pellet" at the bottom of the tube, drawing off some of the liquid (half seminal plasma and half extender), and resuspending the sperm pellet with some additional extender. With this procedure, at least 75% of the sperm are kept, seminal plasma concentration is reduced to a viable concentration for cooling/shipping, and adequate breeding doses can be achieved.

"The objective is to produce a final concentration of spermatozoa between 25 and 50 million/mL in a mixture of extender and seminal plasma containing 5-20% seminal plasma," Vanderwall summarized. It is helpful, especially for very dilute ejaculates, to use the sperm concentration of the raw semen to calculate the amounts of extender needed to achieve this target.

An additional assist is a "cushion," or a dense liquid that prevents excessive compaction of the sperm during centrifugation (Vanderwall likened it to a trampoline). With a cushion, sperm can be safely centrifuged for longer periods at higher G forces to concentrate them further, allowing recovery of nearly 100% of sperm without excessive damage.

"If dilute semen is routinely encountered, and/or the total number of spermatozoa in an ejaculate are extremely limited, the use of a centrifugation cushion might be particularly beneficial to optimize recovery of the spermatozoa," he noted.