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lubricant should be used but in reality, all lubricants are spermicidal to some extent. A recent study demonstrated the AI lubricant jelly, Clarity®, had the least impact on sperm quality when tested against other lubricants (Serafini et al., 2019 ). Finally, if more than two col- lection attempts are required with an AV to obtain an ejaculate, we recommend replacing the used AV with a clean, freshly prepared AV to reduce dirt and bacterial contamination. When evaluating semen, one should look at the colour of the
ejaculate and a raw sample under a microscope, to determine the presence of sperm or other material such as blood, white blood cells or urine crystals as these can not only impact the accuracy of the evaluation but also the longevity of the sperm. Concentration can be evaluated using a haemocytometer, sphectrophotometer (Equine Densimeter [Animal Reproduction Systems], Spermacue ® [Minitube International], etc) or fluorescent- based system (nucleo- counter ® [Chemometec A/S]). Each system has its advantages and disadvantages which can impact accuracy. The haemocytometer al- lows differentiation of cells (sperm vs. other) but is subject to human error either in dilutions, counting sperm or mathematical mistakes. A spectrophotometer- based system works well with a clean sample reading in the middle of the standard curve. This system will over- estimate sperm numbers if debris, red blood cells, urine, extenders or other material is present. Additionally, accuracy declines when samples fall too far to the left (dilute) or right (concentrated) on the standard curve. Fluorescent- based systems stain the DNA of the sperm, so non- cellular or red blood cell contamination should not interfere with its accuracy. However, in cases of pyospermia, this system will overestimate sperm numbers. In situations where the spectrophotometer and fluorescent- based systems are unavailable or lacking accuracy, we recommend evaluation with a haemocytom- eter as a quality control measure. More in- depth reviews on semen processing and evaluation can be found elsewhere (Jasko, 1992 a; Love, 2016 ).
SEMEN EXTENDER
Several commercial extenders are available for processing semen for both on- farm and cooled shipping. It is beyond the scope of this review to discuss the composition and function of extenders and several reviews are available (Aurich, 2008 ; Brinsko & Varner, 1992 ; Gibb & Aitken, 2016 ). Nevertheless, each stallion is an individual and evaluating semen in different extenders can be useful to optimise semen quality and longevity.
BREEDING DOSE
The industry standard is to use 500 × 10 6 progressively motile sperm for fresh AI and 1 × 10 9 progressively motile sperm for cooled- shipped semen AI (Brinsko, 2006 ; Douglas- Hamilton et al., 1984 ; Pickett & Voss, 1975 ). In reality, some stallions can breed with a lower number of progressively motile sperm and still achieve
CENTRIFUGATION FOR CONCENTRATING SPERM
Centrifugation can be performed either with or without a cush- ion medium. Cushion is an iodixanol solution that allows the use of higher centrifugation g- forces, which reduces sperm damage and improves sperm recovery rates. Briefly, to perform cushion centrifugation (Cushion Fluid™, Minitube International), after semen is collected and analysed, it is diluted 1:1 using a semen extender and transferred to 50- mL conical tubes. If cushion is not being used, the tubes are balanced and centrifuged at 400– 650 × g for 15– 20 min (Cochran et al., 1984 ). If cushion is being used, after the diluted semen is added to the 50- mL conical tubes, the cushion solution is layered underneath the diluted semen. The amount of cushion solution used can vary on user preference and stallion but is typically between 0.5 and 3 mL.
KELLEY
acceptable pregnancy rates (Brinsko, 2006 ). Determining the mini- mum number of progressively motile sperm in a breeding dose for a particular stallion can be done by keeping detailed breeding records and systemic analyses.
INSEMINATION VOLUME
An interaction between the total number of sperm inseminated, volume inseminated and sperm concentration has been suggested (Pickett & Shiner, 1994 ) and sperm concentration may be more im- portant than volume (Katila, 2005 ). When mares were bred with the same total number of sperm but at different concentrations, those bred with high volumes and low concentrations (<25 × 10 6 /mL) had lower pregnancy rates than those bred with lower volumes and higher concentrations (50 × 10 6 /mL; Bedford & Hinrichs, 1994 ; Jasko, Hathaway, et al., 1992b ; Jasko, Martin, & Squires, 1992c ; Pickett & Shiner, 1994 ; Rowley et al., 1990 ). Additionally, when the concentration of sperm was consistent (50 × 10 6 sperm/mL) there was no difference in pregnancy rate between 30 and 120 mL (Bed- ford & Hinrichs, 1994 ) of semen. Large insemination volumes (>50 mL) are not thought to be beneficial as most of the semen will be lost through the mare ' s dilated cervix post- insemination (Brinsko et al., 2011 ; Jones, 1995 ; Rowley et al., 1990 ). When considering the inflammation in the mare ' s uterus post-
breeding, the data is conflicting. Two studies have found an increase in uterine inflammation with increased sperm concentration (Fiala et al., 2007 ; Kotilainen et al., 1994 ). Another study found no effect of sperm concentration on uterine inflammation (Sinnemaa et al., 2005 ) while a second study found mares infused with 40 mL of extended semen with a concentration of 500 × 10 6 sperm/mL had significantly less uterine inflammation than those infused with 40 mL at a con- centration of 50 × 10 6 (Nikolakopoulos & Watson, 2000 ). Thus, it is this author ' s recommendation to minimise the insemination volume while having enough sperm to produce acceptable pregnancy rates.
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