Decrease, Sperm count

Sperm is produced in the seminiferous tubules of the testis through spermatogenesis (Sharma & Agarwal, 2011). This process begins with Type A spermatogonia, which divide by mitosis to maintain the stem cell pool. A subset of these cells differentiate into Type B spermatogonia, which divide by mitosis and give rise to primary spermatocytes. Subsequently, primary spermatocytes undergo meiosis I to form secondary spermatocytes, which undergo meiosis II to produce haploid spermatids. Spermatids then differentiate into spermatozoa through spermiogenesis, a process marked by several morphological changes, including condensation and elongation of the nucleus, acrosome formation, and development of the flagellum (Nishimura & L’Hernault, 2017; Sharma & Agarwal, 2011). Spermatozoa are released into the seminiferous tubule lumen, exit the testis through the rete testis, and enter the epididymis, where sperm maturation occurs. Spermatozoa acquire motility and acrosomal function during transit through the three distinct regions of the epididymis: caput, corpus, and cauda (Sharma & Agarwal, 2011).

Sperm count refers to the number of spermatids present in the testis, or the number of spermatozoa present in semen or the cauda epididymis. Reduced sperm count describes a decrease in spermatids or spermatozoa with respect to a control or reference number. In humans, a total sperm number below 39 million per ejaculate and a sperm concentration below 16 million per ml represent the fifth percentile lower limits, based on a reference group of men whose partners conceived within 12 months (World Health Organization, 2021). Reduced sperm count can be temporary, prolonged, or permanent depending on the cause, including genetic or other intrinsic problems, or an exposure that occurred during development that impaired the stem cell pool.

The toxicological interpretation of reduced sperm count depends on the biological compartment assessed. A decrease in testicular spermatid number suggests impairment of one or more stages of spermatogenesis (Creasy & Chapin, 2013; M. L. Meistrich, 1989). However, a reduction in cauda epididymal sperm reserves may reflect impaired spermatogenesis or spermatid retention in the testis, disruption of epididymal processes such as sperm transit, maturation, and storage, or a combination of these effects (Blazak et al., 1985; Creasy & Chapin, 2013).

The timing and duration of exposure are important considerations when evaluating sperm count due to the length of spermatogenesis and the spermatogenic cycle. Exposure durations spanning multiple spermatogenic cycles may be necessary to produce detectable changes in sperm count, as toxicants that target earlier stages of spermatogenesis may only affect sperm count after the damaged cells have progressed through subsequent stages of development (Amann, 1986; Mangelsdorf et al., 2003). For epididymal sperm counts, sperm transit time through the epididymis should also be considered.

OECD Test Guideline 416: Two-Generation Reproduction Toxicity, and OECD Test Guideline 443: Extended One-Generation Reproductive Toxicity Study, recommend estimating sperm count by quantifying cauda epididymis sperm reserves and spermatids in the testis (OECD, 2001, 2025).

Sperm counts can be estimated from the testis, epididymis, or semen. Testicular sperm counts are generally estimated by quantifying homogenization-resistant spermatids (Amann, 1986). During spermiogenesis, spermatid nuclei become highly condensed and resistant to mechanical or biochemical breakdown. Homogenization destroys most testicular cells and nuclei except for the late-stage spermatid nuclei, which can then be quantified (Amann, 1986). Testicular sperm counts can also be used to estimate daily sperm production (DSP) by dividing the number of homogenization-resistant spermatid nuclei by the number of days they spend in the testis (Amann, 1981).

Epididymal sperm counts are most often estimated using the cauda epididymis, where sperm is stored (Seed et al., 1996). Sperm can be isolated from the cauda epididymis using various methods, including diffusion, aspiration, or homogenization (Chapin et al., 1992; Seed et al., 1996; Slott et al., 1991). In the diffusion method, small incisions are made in the cauda epididymis to allow sperm to swim out into the surrounding medium. The aspiration method collects sperm directly from incised tissue using a capillary tube. Homogenization methods mechanically disrupt epididymal tissue to release sperm (Amann, 1986; Seed et al., 1996).

In species where semen can be collected, such as humans, dogs, and rabbits, sperm count can be evaluated from ejaculated semen samples (Seed et al., 1996).

The resulting sperm suspension is counted manually or by automated methods. Manual counting using a hemacytometer and phase-contrast microscopy is a widely used and accepted method for determining sperm count (Amann, 1986; Seed et al., 1996; Strader et al., 1996). Sperm counting with a hemacytometer, specifically the improved Neubauer hemacytometer, is considered the gold standard and is extensively described in the WHO laboratory manual for the examination and processing of human semen (World Health Organization, 2021). Hemacytometer counts are used for calibrating other automated techniques (Kuster, 2005; Prathalingam et al., 2006).

Automated methods include Computer-Assisted Sperm Analysis (CASA), in which a video camera attached to a microscope captures images or videos that are analyzed by specialized software (Akal, 2023). The CASA system objectively estimates sperm concentration and related sperm parameters. CASA-derived sperm counts have demonstrated strong agreement with hemacytometer-based methods while improving analytical efficiency (Dearing et al., 2014; Lammers et al., 2014; Strader et al., 1996). However, CASA systems have been reported to overestimate sperm count at lower concentrations due to misclassification of debris as sperm (Dearing et al., 2014).

Flow cytometry-based approaches have also been developed for counting sperm and assessing sperm membrane integrity. Sperm from zebrafish testis were stained with SYBR-14, a membrane-permeable nucleic acid dye, and propidium iodide, a DNA dye that can only permeate damaged cell membranes. Fluorescence filters were used to detect stained cells, and forward scatter (FSC) and side scatter (SSC) were used to differentiate sperm from debris. Resulting sperm counts were comparable to those obtained from using a hemacytometer (Yang et al., 2016).

This KE is plausibly applicable to all male animals that produce sperm through spermatogenesis.

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