# Protective Effect of Raphanus sativus Seed Extract on Damage Induced by In Vitro Incubation and Cryopreservation of Human Spermatozoa

## TL;DR

This study shows that Raphanus sativus seed extract protects human sperm from damage during in vitro incubation and cryopreservation.

## Contribution

The study identifies sinapine and sinapic acid as active compounds in Raphanus sativus that mimic the protective effects of the whole extract.

## Key findings

- Raphanus sativus extract reduced sperm DNA fragmentation and reactive oxygen species during in vitro incubation.
- The extract improved sperm viability, motility, and DNA integrity after cryopreservation.
- Sinapine and sinapic acid replicated the protective effects of the whole extract.

## Abstract

In vitro manipulation of human spermatozoa during Assisted Reproductive Technology (ART) can induce several damages to sperm structure and functions. This study investigated the protective effects of Raphanus sativus seed extract and its active compounds on several sperm parameters during in vitro incubation and cryopreservation. Extracts from five seed-batches were characterized by HPLC-DAD-MS and 1H-NMR, identifying sinapine and sinipic glycosides as the main characteristic compounds. Sperm DNA fragmentation (sDF) was detected by the Sperm Chromatin Dispersion test and LiveTUNEL. Excessive reactive oxygen species (ROS) production was detected by MitoSOX Red in viable spermatozoa. Caspase activity was detected by FLICA. Cryopreservation was conducted with two alternative freezing media. In vitro incubation with the extract protected against the loss of motility and reduced the induction of sDF, sperm ROS production, and caspase activity. Similarly, during cryopreservation, it allowed much better recoveries of sperm viability, motility, and DNA integrity by decreasing sperm ROS production with both freezing media. Sinapine and sinapic acid completely mimicked the protective effects of the whole extract during both in vitro incubation and cryopreservation, suggesting that they are included among the active principles. These findings support Raphanus sativus seed extract and its active compounds as candidates for inclusion in handling and freezing media for human spermatozoa in ART.