Effect of chelating and antioxidant agents on morphology and DNA methylation in freeze‐drying rabbit (Oryctolagus cuniculus) spermatozoa

Abstract

Freeze-drying (FD) has been exhaustively tried in several mammalian species as an alternative technique to sperm cryopreservation, but few studies have been done in rabbits (Oryctolagus cuniculus). The main objective of this study was to compare the protective effect of various antioxidants added to EDTA medium on structural and functional components of FD rabbit spermatozoa and on their status of global DNA methylation. FD media used were composed of basic FD medium (10 mM Tris-HCl buffer and 50 mM NaCl) supplemented with either 50 mM EDTA alone (EDTA) or added with 105 µM of rosmarinic acid (RA, EDTA-RA) or 10 µM of melatonin (MLT, EDTA-MLT). The effect of each medium on the preservation of FD spermatozoon structure was evaluated with light and scanning electron microscopy (SEM). Global DNA methylation was quantified in all FD sperm samples as well as in fresh spermatozoa. Morphologically, fracture points were evidenced in the neck, mid and principal piece of the spermatozoon tail. No differences in spermatozoon fracture pointswere evidenced among FD treatments: intact spermatozoa were the largest (p < .01) category, whereas the most frequent (p < .01) injury was the neck fracture, resulting in tailless heads. At SEM, the head of spermatozoa showed a well-conserved shape and intact membrane in all treatments. DNA methylation status was the same in all FD treatments. In conclusion, supplementation of EDTA, EDTA-RA and EDTA-MLT during FD preserved rabbit sperm morphological integrity and methylation status as well. Therefore, the difficulty of getting viable offspring using FD semen is likely unrelated to the impact of the lyophilization process on DNA methylation and morphology of lyophilized spermatozoa.

Long-term preservation of germ cells and gonadal tissues at ambient temperatures

“…The process includes freezing samples followed by primary drying through sublimation of ice at freezing temperature under vacuum and secondary drying through desorption by slowly elevating temperature under vacuum (Weng 2021). Protectants and supplements used in this procedures include trehalose (Martins et al 2007, Sánchez-Partida et al 2008, Ito et al 2019, Shahmoradi et al 2021, buffers like EGTA (Liu et al 2004, Martins et al 2007, Ringleb et al 2013 or EDTA (Mercati et al 2020), fetal bovine serum (Choi et al 2011), or media/ buffer only (Keskintepe et al 2002, Kwon et al 2004). Sperm cells from many species have been preserved using that method (Patrick et al 2017a, Saragusty et al 2020.…”

Dry storage of mammalian spermatozoa and cells: state-of-the-art and possible future directions

“…Membrane-permeable trehalose would, in theory, allow better control of its uptake (and removal) by changing concentrations and, even more, by longer exposure to trehalose gradients. Surprisingly, a recent report claimed that trehalose lacks any DNA protection activity (Ito et al 2019); given that this report comes from an authoritative group, alternative compounds with antioxidant activity, like rosmarinic acid or melatonin, should be explored, as suggested by Mercati et al (2020). However, in our opinion, natural xeroprotectants found in tardigrades, midges or other anhydrobiotic organisms should guide our choices.…”

Damages and stress responses in sperm cells and other germplasms during dehydration and storage at nonfreezing temperatures for fertility preservation

“…Assessments of epigenetic patterns and gene expression after dehydration have also been performed (Table 1 and Figure 1). Global DNA methylation (5‐methylcytosine) level appears not to be affected by lyophilization in rabbit sperm (Mercati et al, 2020). The proportion of cat germinal vesicles with tri‐methylation of histone H3K4me3 (detected by immunostaining) after microwave drying is lower as observed during cryopreservation (P. C. Lee & Comizzoli, 2019).…”

“…Supplementation of drying medium with chelators like EDTA/ EGTA ensures long-term stability of mouse or hamster sperm DNA integrity during drying and storage, as well as successful embryo development after sperm injection (Kaneko & Nakagata, 2006;Muneto & Horiuchi, 2011). During the lyophilization of rabbit sperm, adding antioxidants (rosmarinic acid or melatonin) to a medium already containing a chelating agent like EDTA does not improve morphology or DNA methylation level (Mercati et al, 2020). However, desferal (an iron chelator) provides a synergistic effect with intracellular sugar (trehalose or sucrose) to improve the membrane integrity of bovine sperm during air drying (Sitaula et al, 2009).…”