Assessing Optimal Cell Counts in Sperm Shape Abnormality Assays in Rodents

Abstract

Rodents have been the preferred models for the evaluation of the toxicity of pollutants and drugs and their genotoxic effects, including sperm shape abnormalities. The scientific literature is dominated by studies conducted with model animals in laboratory conditions, but a generally accepted and standardized protocol addressing the optimal number of sperm cells to count is still lacking. In this study, we reviewed the literature regarding the number of counted sperm cells in such assessments, published from 1969 to 2023. To infer the number of counts providing the best cost/benefit regarding the robustness of the assay results, a new dataset involving the analysis of two populations of wild rodents was produced. We evaluated the frequency of sperm shape abnormalities in a total of 50 wild brown rats (Rattus norvegicus) captured in two port cities, aiming to detect the impact of differential sperm cell counts in the obtained results. During necropsy, the fresh epididymis tail of adult male rats was excised, and sperm cells were fixated in slides. For each animal, a total of 300, 500, 1000, and 2000 cells were sequentially counted, and head abnormalities were registered. Counting 300 sperm cells failed to detect significant differences between groups and 500 counts resulted in marginally significant differences. Only when 1000 or 2000 sperm cells were counted, significant differences emerged between groups. We propose that studies addressing sperm shape abnormalities should standardize counts to an optimal value of 1000 cells per animal, warranting robust statistical results while providing the best compromise concerning labor time.

Keywords: genotoxicity; lab rodents; sperm abnormalities; standardization; wild rodents.

Figure 1

Flowchart illustrating the literature selection and review process.

Figure 2

Evolution of the number of cells counted in published papers, by decade, between 1969 and June 2023, to assess genotoxic effects in rodents.

Figure 3

World heat map showing the sum of publications originated in each country [1969–June 2023]. Bar plots illustrate the distribution of publications through the years in each continent. Note: Oceania is not included in this temporal analysis because a single publication originated from this continent.

Figure 4

Number of abnormal sperm cells detected per animal in 300, 500, 1000, and 2000 cell counts, in Rattus norvegicus from Lisbon and Ponta Delgada; Mann–Whitney U test: NS. non-significant, * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).