In vitro developmental ability of ovine oocytes following intracytoplasmic injection with freeze-dried spermatozoa

Abstract

Freeze-drying (FD) is a new and alternative method to preserve spermatozoa in refrigeration or at room temperature. Suitable protection is required to maintain the sperm DNA integrity during the whole process and storage. The aim of this study was to examine the effect of rosmarinic acid and storage temperature on the DNA integrity of freeze-dried ram sperm. In addition, we evaluated the in vitro developmental ability to the blastocyst stage of oocytes injected with freeze-dried sperm. Ram sperm was freeze-dried in basic medium and in this medium supplemented with 105 µM rosmarinic acid. The vials were stored for 1 year at 4 °C and at room temperature. Frozen sperm was used as control. After rehydration, sperm DNA damage was evaluated, observing that the percentage of spermatozoa with DNA damage decreased significantly in the presence of rosmarinic acid, without differences between the two storage temperatures. Moreover, no differences were observed between the freeze-dried group and the frozen-thawed group in terms of blastocyst formation rate. We proved for the first time that ovine spermatozoa can be lyophilized effectively, stored at room temperature for long term, reconstituted and further injected into oocytes with initial embryo development.

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

“…When EDTA FD medium is used, the balance between the amount of ROS and detoxification is compensated by the chelating agent and RA may interfere in its action. It should be noted that in the study of Olaciregui et al [18] in freeze-dried ovine spermatozoa, the percentage of fragmented DNA in EGTA (4.4%) and EGTA-RA (2.9%) was lower than our results, concluding that RA is more efficient when samples are diluted with EGTA and that DNA of rabbit spermatozoa diluted with EGTA is more sensitive to FD process than the ovine equivalent.…”

“…Zhang et al [26] reported that the antioxidants could minimize the detrimental effect of ROS and improve the quality of spermatozoa. Besides, Luño et al [16] showed that RA antioxidant provides a protection against oxidative stress during boar sperm cryopreservation and likewise, Olaciregui et al [18] observed that the addition of RA to EGTA FD medium decreases significantly (p = .003) the percentage of DNA damage in freeze-dried ovine spermatozoa. Lastly, Kodama et al [12] demonstrated that antioxidant treatment in humans with high level of oxidative sperm DNA damage may reduce the extensive oxidative DNA damage.…”

“…Therefore, freeze-drying is a method that can allow simple gene resources preservation among 4°C to 25°C and transportation without the need for liquid nitrogen or dry ice. Unlike other animals [17][18][19], EDTA chelating agent is the most suitable media for FD rabbit sperm under the conditions employed here and the addition of RA can protect the DNA against the oxidative stress caused during FD procedure.…”

“…Rosmarinic acid (RA) is a natural antioxidant [20] that has shown therapeutic effects against oxidative stress in in vitro cell studies [12]. It has been demonstrated that the incorporation of RA in freezing [16,26] and FD [18] medium provides protection on spermatozoa against oxidative stress and thus improves the quality of sperm preservation.…”

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

“…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.…”