Preparation
This review will not go into depth on collection and evaluation of semen; however, it is important to recognized that semen collection and evaluation can greatly impact the quality of the semen after processing. Two important factors in collecting semen which could impact the semen quality are proper washing of the penis (Bowen et al., 1982; Dascanio, 2014) and lubrication of the artificial vaginal. Proper washing reduces smegma in the ejaculate, which, even in small qualities, can be greatly increased if centrifugation is required. Water is recommended for washing, since detergents and disinfectants can increase the risk of pathogenic bacteria colonizing the penis (Bowen et al., 1982). Lubrication of the AV is important, but caution should be taken to minimize the amount of lubricant to reduce contamination of the ejaculate. A non-spermicidal lubrication should be used, but all lubricants are spermicidal to some extent. Clarity brand lubrication has been shown to have the least impact on sperm quality (Serafini et al., 2019). Finally, if more than 2 collection attempts are required with an artificial vagina (AV) to obtain an ejaculate, we recommend a replacing the used AV with a clean, freshly prepared AV to reduce dirt and bacterial contamination.
When evaluating semen, one should look at the color of the ejaculate and a raw sample 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 hemocytometer, sphectrophotometer (Equine Densimeter, Spermacue®, etc) or fluorescent based system (nucleocounter®). Each system to measure concentration has its advantages and disadvantages which should be recognized as this can impact accuracy. The hemocytometer allows 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 will in situations with little contamination of the ejaculate that is reading on the middle of the standard curve. This system will overestimate sperm numbers if debris, red blood cells, urine or other material is present such as extenders. Additionally, this system is not accurate in cases of low sperm concentration or high sperm concentration. Fluorescent-based systems stain the DNA of the sperm. Thus, non-cellular or red blood cell contamination will not interfere with the accuracy. In cases of pyospermia, this system will overestimate sperm numbers. In situations where the spectrophotometer and fluorescent-based systems are inaccurate, we recommend evaluation with a hemocytometer to try to gain a more accurate sperm concentration. For more in-depth reviews on semen processing and evaluation please see (Jasko, 1992; Love, 2016).