Long-term preservation of freeze-dried rabbit sperm by adding rosmarinic acid and different chelating agents

Abstract

A B S T R A C TFreeze-drying (FD) technique has been applied as an alternative technology to preserve gene resources to allow simple sperm preservation and shipment at 4°C. Nevertheless, DNA sperm might be damaged by mechanical or oxidative stress throughout FD procedure. Therefore, suitable protection to maintain DNA integrity is required. The aim of this study was to determine the effect of rosmarinic acid (RA) as an antioxidant and two chelating agents (EGTA and EDTA) on the DNA integrity of freeze-dried rabbit sperm after storage of the samples at 4°C and room temperature for 8 months. Rabbit sperm were freeze-dried in basic medium (10 mM Tris-HCl buffer and 50 mM NaCl) supplemented with 50 mM EGTA (1), 50 mM EGTA plus 105 μM RA (2), 50 mM EDTA (3) or 50 mM EDTA plus 105 μM RA (4). Semen samples were kept at 4°C and room temperature during 8 months. After rehydration, DNA integrity was evaluated with Sperm Chromatin Dispersion test observing that DNA fragmentation was higher when semen samples were freeze-dried with EGTA (10.9%) than with EDTA (4.1%) (p < 0.01). Furthermore, RA acted better under adverse conditions and no significant differences were found in temperature storage. Summarizing, FD is a method that can allow simple gene resources preservation among 4°C to 25°C during 8 months and transportation without the need for liquid nitrogen or dry ice. EDTA chelating agent is the most suitable media for freeze-dried rabbit sperm and the addition of RA protects the DNA against the oxidative stress caused during FD procedure. (P. Domingo).Cryobiology xxx (xxxx) xxx-xxx 0011-2240/

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

“…Finally, intact spermatozoa were the largest category in all the three media evaluated, attesting that they adequately protected the cell structure. Moreover, very few spermatozoa with plasma membrane damage were detected, suggesting that chelating agents plus addition of antioxidant treatments preserve DNA integrity as previously reported (Domingo et al, ).…”

“…Despite this relevant achievement, only a viable puppy was born and hence the need to study the possible factors that compromised the success of the technique. A later study on lyophilized rabbit sperm demonstrated that EDTA preserved DNA integrity of spermatozoa better than EGTA as well as the positive effect of RA supplementation (Domingo et al, ). However, the effect of chelating molecules on the morphology of freeze‐dried sperm is still not well defined.…”

“…Rosemary ( Rosmarinus officinalis ) and its extracts, such as rosmarinic acid (RA), are natural antioxidants that show antiproliferative, antiradical and protective properties against oxidant‐induced stress effect (Ho, Wang, Wei, Huang, & Huang, ). In several mammals (Luño et al, ; Olaciregui, Luño, Domingo, González, & Gil, ; Olaciregui, Luño, González, et al, ; Yeni et al, ), including rabbits (Amidi, Pazhohan, Shabani Nashtaei, Khodarahmian, & Nekoonam, ; Domingo, Olaciregui, González, De Blas, & Gil, ), addition of RA in FD or post‐thawing medium (Motlagh et al, ) protected spermatozoa against oxidative stress and improved sperm preservation. In contrast, in a sperm in vitro study, Lv et al () found a decreased intracellular Ca 2+ concentration compromising sperm function after the addition of RA in a dose‐dependent manner.…”

Rosmarinic acid improves boar sperm quality, antioxidant capacity and energy metabolism at 17°C via AMPK activation

“…Yeni et al (2018) showed that supplementation with RA reduced oxidative stress in freeze-thawed bull sperm and increased sperm quality parameters. Domingo et al (2018) showed that the addition of RA and different chelating agents preserved freeze-dried rabbit sperm for a long time. However, when the concentration of RA was too high (more than 100 μmol/L), the sperm quality decreased.…”

Exploring dry storage as an alternative biobanking strategy inspired by Nature

“…In most freeze-drying studies, the spermatozoa were snap-frozen without any attempt to maintain their membrane integrity (so called 'viability') [e.g. 80,98,99] while in other studies an attempt to obtain viable cells was made by using proper cryopreservation techniques [e.g. 90,100].…”