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TABLE 1 Useful calculations for stallion semen processing. Parameter
Calculation
Testicular volume (based on ultrasonographic measurements)
Predicted daily sperm output
Expected sperm recovery post- centrifugation
Length (cm) × width (cm) × height (cm) × 0.523 Testicular volume (cc) × 0.024 − 0.076
Volume (mL) × concentration (sperm/mL) = total sperm number Total sperm number × 0.80 = conservative sperm recovery number Sperm recovery number/desired freezing concentration (millions of sperm/mL) = total expected volume (mL)
Total expected volume/2 = conservative estimate of number of 0.5 mL straws required to freeze
Comments
Calculated in cc (cubic cm) Estimated as volume of an ellipsoid
Calculated in billions (Love et al., 1991 )
This calculation assumes a conservative post- centrifuge sperm recovery rate of 80%.
PASCH
for cryopreservation, and by improving the efficiency of ejaculation one may also reduce the volume of gel (Sieme et al., 2004 ). Prior to freezing, 0.5 mL straws are printed and obtained; straws are labelled with the stallion ' s name, date of birth, breed, registration number, and other information the owner or breed registry requires. In the past, 5 mL ‘macro’ straws have been used to freeze equine semen but this is increasingly rare. An overview of the semen collection, processing, and cryopreservation process follows.
SEMEN COLLECTION AND CONCENTRATION
Prior to semen collection, the stallion ' s penis is washed with warm water to remove gross debris. Routine semen collection should be performed using any model of artificial vagina, based on stallion and operator preference. A nylon micromesh filter (Animal Reproduction Systems) should be in place to separate the gel fraction from the semen and reduce debris and dirt contamination of semen. After semen collection, the gel fraction is disposed of and the gel- free ejaculate is subject to analysis: volume, concentration, motility, and total sperm number. Volume can be determined with a graduated cylinder, measured in mL or a scale, measured in grams (g); 1 g of semen is equal to 1 mL of semen and may give the most accurate volume assessment. The concentration of raw semen is measured in millions of sperm per mL, using a densimeter (Animal Reproduction Systems) or nucleocounter (ChemoMetec), and motility is deter- mined using light microscopy or Computer Assisted Sperm Analysis (CASA) (Hamilton- Thorn, others). The raw semen is extended in a 1:1 volume ratio using an isosmotic extender, such as the commercially prepared INRA96 or BotuSemen. As always, care should be taken to ensure the semen and extender are temperature- matched prior to combining. The extender is designed to support sperm metabolism, buffer pH and osmolality, provide antimicrobial support, and offer protection against cold shock and oxidative damage. The concentration of stallion semen is necessary for cryopres-
ervation due to the large volume of the stallion ejaculate. Before centrifuging, the extended semen should be placed in one or more 50 mL conical tubes and a sperm cushion underlaid to reduce sperm damage during spinning at high speed. The sperm cushion is an
iodixanol preparation (OptiPrep, Eqcellsire, Cushion Fluid)
that
improves sperm recovery rate by protecting the sperm from me- chanical trauma during centrifugation. Semen centrifugation with a cushion can be performed at speeds as high as 1000 RCF for 20 min with no detrimental effect on sperm fertility (Ecot et al., 2005 ; Waite et al., 2008 ). Immediate removal of the supernatant must be per- formed after concentrating the sperm, as sperm will begin to swim up from the concentrated pellet. Several millilitres of supernatant should be left so as to reduce the risk of disturbing and inadver- tently aspirating the sperm pellet. Using a pipette, the sperm pellet is gently resuspended within the conical tube. A positive displacement pipette is preferred but, if using an air displacement pipette, care must be taken to avoid sperm loss secondary to aspiration into the bulb of the pipette.
SEMEN DILUTION
Following collection and concentration of sperm, the resuspended sperm pellet must be diluted with an extender containing cryopro- tectant agents. Cryoprotectants offer sperm varying degrees of pro- tection against the stressful process of cryopreservation, which will be addressed in more detail. Extender should be added slowly, with frequent gentle mixing, until the desired volume is reached. Equine sperm is typically frozen at a concentration of 200 million cells per mL, with a range of 100–400 million/mL (Sieme, 2011a ). In order to determine the necessary volume to reach this concentration, sev- eral calculations are made. When centrifuging with a cushion and using the optimal technique, one can theoretically expect a sperm recovery rate that approaches 90%–100% (Ecot et al., 2005 ; Waite et al., 2008 ). A conservative estimate of the number of 0.5 mL straws required per freezing session is determined by multiplying the total sperm number (calculated prior to extending then concentrating the semen) by 80%, then dividing this estimated total sperm number by 200 million sperm per mL (desired concentration) to obtain the total volume needed (Table 1 ). The semen will be loaded into and stored in 0.5 mL straws, therefore the total volume can be multiplied by two to determine the number of straws needed. This calculation provides an estimate, and actual sperm number should be determined via Nucleocounter (ChemoMetec) or haemocytometer following initial
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