Ejaculate Volume Is Seriously Underestimated When Semen Is Pipetted or Decanted Into Cylinders From the Collection Vessel

Abstract

The total sperm count (number of spermatozoa per ejaculate) rather than sperm concentration (number of spermatozoa per unit volume of semen) is the more important semen variable related to fertility. It reflects testicular volume (Handelsman et al, 1984; Andersen et al, 2000; Behre et al, 2000), and thus is a measure of total testicular sperm output (MacLeod and Wang, 1979), which is directly related to the chances of pregnancy after coitus. The concentration of spermatozoa in the ejaculate, however, depends on the extent of dilution of epididymal spermatozoa by secretions of the prostate and seminal vesicles occurring at ejaculation and is therefore influenced by the secretory capacity of the accessory sex glands. This is an important distinction, for when comparing semen quality from older and younger men, sperm concentrations do not differ, yet semen volume is reduced in the older men, and so the total number of spermatozoa per ejaculate is lower in the older men (Ng et al, 2004; Nieschlag et al, 1982). The total number of spermatozoa per ejaculate is obtained by multiplying the concentration of spermatozoa by the semen volume. The latter is best measured by weighing (Eliasson, 2003), assuming a density of 1.0 g/mL (Auger et al, 1995; Jorgensen et al, 1997, 2001; Brazil et al, 2004), but alternative methods, such as collection into graduated cylinders (Behre et al, 2000), pipetting from the collection vessel (Mortimer 1994; Jorgensen et al, 1997), and pouring from the collection vessel into a graduated tube (Jorgensen et al, 1997), are in current practice.

Two recent studies have found that pipetting semen from the collection vessel leads to an underestimation of about 0.5 mL (range 0.3–0.8 mL; Brazil et al, 2004; Iwamoto et al, 2006) compared with weighing, but no data are available about losses incurred when pouring semen into graduated cylinders. Because the area of contact with the sides of the collection vessel while decanting semen into a graduated cylinder is likely to be far larger than that during pipetting, retention within the vessel could be much larger, leading to a larger underestimation of volume with this method. In this study, new data are obtained on the loss of semen volume during decanting to a cylinder and previously published results on losses because of pipetting, and the density of semen is reanalyzed together with additional data.

This study has shown that a consistent and significant reduction in the volume of semen is obtained when a pipette or a graduated cylinder is used to measure liquefied semen transferred from its collection vessel. These losses cannot be accounted for by evaporation because samples were capped during liquefaction at room temperature and pipetted or decanted immediately after weighing. It could be that with particularly viscous samples, transfer would result in even lower volumes because more would be retained on the side of the decanting vessel and some might adhere to the sides of the cylinder. The difference in estimates of semen volume by weighing and pipetting has been reported before (Brazil et al, 2004; Iwamoto et al, 2006) but only mean values were given. In this study, the loss of semen was similar (∼0.5 mL) and represented a mean of 14% loss of volume. The new data on loss of semen associated with pouring into a graduated cylinder revealed a similar underestimation of semen volume (∼0.4 mL) that represented a similar percent loss of semen (13%).

Iwamoto et al (2006) used their measured mean difference (0.49 mL) to correct semen volumes to compare results with other studies in which weighing was used to estimate semen volume. Jorgensen et al (1997) reported laboratories that assumed 0.1 mL of semen was left after decanting into a graduated tube and added this value to the volume measured. The results of this study suggest such a correction procedure would introduce even greater errors because the range of loss varied considerably, perhaps related to the inherent viscosity of the sample or the handling of the sample after collection and, thus, argues against this practice.

The density of human semen has been published before (Huggins et al, 1942; Brazil et al, 2004), but again, only mean values were reported. Reanalysis of the data from Brazil et al (2004) and analysis of additional samples provided values that are somewhat lower than the mean reported by Huggins et al (1942), for which no details of the methodology were given. The density of water established by exactly the same method was close to that reported for water at 20°C (Lentner 1981), confirming the accuracy of the value. A factor of 1.00 is thus sufficient for purposes of estimating semen volume from its weight.

Semen volume is best measured by weighing the sample in the collection vessel (and assuming a density of 1 g/mL, which is very close to the measured value of 1.014 g/mL) rather than pipetting or decanting the semen into a graduated cylinder because this subsequent transfer to measuring devices brings underestimates of volume that will compromise accuracy of total spermatozoan counts or other cells in the ejaculate.