The impact of percutaneous epididymal sperm aspiration on sperm quality in mice

Abstract

Abstract: In laboratory mice, sperm quality is usually assessed in spermatozoa collected from the cauda epididymidis of freshly sacrificed males. Percutaneous epididymal sperm aspiration (PESA) is a non-terminal alternative that would allow repeated sperm collection for sperm quality assessment in living males. To test whether PESA is a suitable method to assess sperm quality, we compared sperm traits between samples collected by PESA vs the commonly applied terminal cauda epididymidis dissection. The collected sperm samples were analyzed using computer-assisted sperm analysis and various parameters, including sperm motility, swimming velocity and morphology, were determined. We were able to retrieve motile sperm from all mice using PESA and the terminal cauda epididymidis dissection. Based on computer-assisted sperm analysis, however, sperm motility and swimming velocity were significantly lower after PESA compared to samples obtained by cauda epididymidis dissection. In addition, we found significantly more morphological abnormalities in PESA samples, probably induced as a side effect of the sampling technique. Although sperm samples collected by PESA are successfully used for in vitro fertilization, we cannot recommend PESA as a suitable method to assess sperm quality in mice, since the procedure seems to impair various sperm traits.

Lay summary: In mice, sperm quality is usually assessed in sperm collected from the epididymis (organ where ripe sperm is stored) of euthanized males. However, there is one non-terminal and minimal invasive alternative to collect sperm, called percutaneous epididymal sperm aspiration (PESA), which allows repeated sample collections from the same individual. Given that individual sperm quality is variable and can change according to various factors, PESA could allow to track sperm quality over time and would be highly appreciated in different research fields. Here, we tested the suitability of PESA to determine sperm quality by comparing sperm samples collected by PESA vs the commonly applied terminal epididymis dissection. We used computer-assisted sperm analysis to determine various sperm quality traits. Surprisingly, we found that sperm collected by PESA showed significantly reduced motility, swimming velocity and more morphological abnormalities compared to sperm samples collected by epididymis dissection. Thus, we cannot recommend PESA as a suitable method to determine sperm quality traits as the procedure itself seems to affect collected sperm cells.

Keywords: CASA; epididymal sperm; mouse; non-terminal; sperm collection; sperm quality assessment; sperm traits.

Figure 1

Picture of a fixated cauda epididymidis in a male RjOrl:SWISS mouse. For PESA, the testis was moved to the scrotum. The cauda epididymidis was then visually identified and fixated with curved forceps by applying gentle pressure. Sperm aspiration was subsequently performed by puncturing the cauda epididymidis with a 30G needle on an insulin syringe.

Figure 2

Pictures of morphological abnormalities observed in spermatozoa from RjOrl:SWISS mice collected by PESA and TCED. (A) Normal shaped sperm for comparison, (B) sperm with detached head, (C) sperm with broken tail, (D) sperm with bent midpiece, (E) sperm with abnormal head shape, (F) sperm with bent neck and tail, (G) sperm with bent neck, (H) sperm with bent tail, (I) sperm with bent midpiece, (J) sperm with coiled tail and (K) sperm with cytoplasmatic droplet.

Figure 3

Boxplot of sperm motility (%) in PESA and TCED samples collected from RjOrl:SWISS mice and assessed (A) after sperm collection (T0) and (B) after 2 h of incubation (T1). White bars show data from left epididymal samples and patterned bars from right epididymal samples. Circles (○) refer to mild outliers (Q3 + 1.5 × IQR).

Figure 4

Sperm swimming velocities in PESA (patterned bars) and TCED (white bars) samples collected from RjOrl:SWISS mice. Sperm curvilinear velocity (VCL, µm/s), straight-line velocity (VSL, µm/s) and average path velocity (VAP, µm/s) assessed (A) after sperm collection (T0) and (B) after 2 h of incubation (T1). Circles (○) refer to mild outliers (Q1 + 1.5 × IQR).

Figure 5

Boxplot of the observed abnormalities in RjOrl:SWISS sperm samples collected by PESA and TCED. White bars show results from left epididymal samples and patterned bars from right epididymal samples.

Figure 6

Classification and frequency distribution of observed morphological sperm abnormalities in RjOrl:SWISS mice. Type and prevalence (%) of observed morphological abnormalities in all sperm samples collected by PESA and TCED (n = 2000).