Freezing, Vitrification, and Freeze-Drying of Equine Spermatozoa: Impact on Mitochondrial Membrane Potential, Lipid Peroxidation, and DNA Integrity

Betancur, Giovanni Restrepo; Varela, Elizabeth; Duque, J.; Gómez, Jorge Enrique; Rojas, Mauricio

doi:10.1016/j.jevs.2018.10.006

Cited by 32 publications

(18 citation statements)

References 56 publications

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“…Different capital and small letters on the same row indicate statistically significant differences (P < 0.01 and P < 0.05, respectively) MMP was significantly correlated with sperm kinematic parameters, such as VCL, VSL and VAP, whereas it was not significantly correlated with total and progressive sperm motility (Boni et al, 2017). Hence, MMP does not seem to provide solid indications of sperm motility, as also demonstrated in freeze-dried spermatozoa that, even if immotile, showed high MMP values (Restrepo et al, 2019). However, it provides additional information on sperm quality that was not obtainable by other assays.…”

Section: Discussionmentioning

confidence: 91%

Kinematic, bioenergetic and oxidative evaluations of donkey sperm preserved at +4°C

Palma

Cecchini

Macchia

et al. 2020

Zygote

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Summary Information on donkey sperm bioenergetics, kinetics and oxidative status is scarce even though crucial for development of reproductive technologies and germplasm conservation. For these reasons, it is interesting to monitor sperm kinetics, bioenergetics, and oxidative status during sperm storage at +4°C and with several sperm extenders and concentrations. Donkey semen was collected from three jackasses, three times each. It was diluted with four extenders (Kenney, Equiplus, INRA96 or Hippex), set at three sperm concentrations (30, 50 or 70 × 106 spermatozoa/ml) and evaluated for its functionality after 0, 3, 24, 48 and 72 h storage at +4°C. Sperm kinetics was analyzed by Sperm Computer Analysis; sperm bioenergetics was assessed by mitochondrial membrane potential (MMP); sperm oxidative status was evaluated by lipid peroxidation (LPO), anti-LPO potential and nitroblue tetrazolium (NBT) assays. Incubation produced a progressive (P < 0.01) decline in sperm kinetics and MMP, whereas parameters related to oxidative status either increased (LPO, NBT) or decreased (anti-LPO). The anti-LPO potential was the index better related to sperm motility and kinetics. Extenders proved to be differently (P < 0.01) effective in preserving sperm kinetics, MMP, and oxidative status. The concentration of 30 × 106 spermatozoa/ml provided an optimum preservation of sperm functions. Significant correlations emerged between most parameters examined. This study identified reference criteria for storing donkey spermatozoa at +4°C. A low sperm concentration together with a proper extender are crucial requirements for optimum sperm cryopreservation efficiency. Field trials are, however, required to validate these findings, making them operational in practice.

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Section: Discussionmentioning

confidence: 91%

Kinematic, bioenergetic and oxidative evaluations of donkey sperm preserved at +4°C

Palma

Cecchini

Macchia

et al. 2020

Zygote

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“…Therefore, ultra-sensitive assays should be used to investigate DNA damage in dry spermatozoa. Generally, DNA status in lyophilized semen was performed using Sperm Chromatin Dispersion (SCD), Single cell gel electrophoresis (SCGE) or comet assay and SCSA 9 , 25 , 26 . However, the DNA parameters derived from SCD and SCGE analysis exhibited ample variation in each replicate, making an infeasible correlation with the fertilizing capacity of dry spermatozoa 25 .…”

Section: Discussionmentioning

confidence: 99%

Whole genome integrity and enhanced developmental potential in ram freeze-dried spermatozoa at mild sub-zero temperature

Palazzese

Anzalone

Turri

et al. 2020

Sci Rep

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Freeze-dried spermatozoa typically shows a reduction in fertility primarily due to the DNA damage resulting from the sublimation process. In order to minimize the physical/mechanical damage resulting from lyophilization, here we focused on the freezing phase, comparing two cooling protocols: (i) rapid-freezing, where ram sperm sample is directly plunged into liquid nitrogen (LN-group), as currently done; (ii) slow-freezing, where the sample is progressively cooled to − 50 °C (SF-group). The spermatozoa dried in both conditions were analysed to assess residual water content by Thermal Gravimetric Analysis (TGA) and DNA integrity using Sperm Chromatin Structure Assay (SCSA). TGA revealed more than 90% of water subtraction in both groups. A minor DNA damage, Double-Strand Break (DSB) in particular, characterized by a lower degree of abnormal chromatin structure (Alpha-T), was detected in the SF-group, comparing to the LN-one. In accordance with the structural and DNA integrity data, spermatozoa from SF-group had the best embryonic development rates, comparing to LN-group: cleaved embryos [42/100 (42%) versus 19/75 (25.3%), P < 0.05, SL and LN respectively] and blastocyst formation [7/100 (7%) versus 2/75 (2.7%), P < 0.05, SF and LN respectively]. This data represents a significant technological advancement for the development of lyophilization as a valuable and cheaper alternative to deep-freezing in LN for ram semen.

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“…These cryoprotectants remain liquid, and toxic, at room temperature and are therefore unsuitable for use in dry storage. Consequently, scientists started investigating solutions to this problem using milder cold temperatures to solidify water prior to sublimation (Restrepo et al 2019;Wakayama et al 2019;Palazzese et al 2020) with better results. After all, the aforementioned anhydrobiotes do not resort to freezing before dehydration; simply put, once a reduction in environmental water is sensed, they activate a timely, regulated gene expression leading to the accumulation of xeroprotectants in their cells.…”

Section: Somatic Cells: Current Situationmentioning

confidence: 99%

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

Loi

Anzalone

Palazzese

et al. 2021

Reprod. Fertil. Dev.

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This review provides a snapshot of the current state-of-the-art of drying cells and spermatozoa. The major successes and pitfalls of the most relevant literature are described separately for spermatozoa and cells. Overall, the data published so far indicate that we are closer to success in spermatozoa, whereas the situation is far more complex with cells. Critical for success is the presence of xeroprotectants inside the spermatozoa and, even more so, inside cells to protect subcellular compartments, primarily DNA. We highlight workable strategies to endow gametes and cells with the right combination of xeroprotectants, mostly sugars, and late embryogenesis abundant (LEA) or similar ‘intrinsically disordered’ proteins to help them withstand reversible desiccation. We focus on the biological aspects of water stress, and in particular cellular and DNA damage, but also touch on other still unexplored issues, such as the choice of both dehydration and rehydration methods or approaches, because, in our view, they play a primary role in reducing desiccation damage. We conclude by highlighting the need to exhaustively explore desiccation strategies other than lyophilisation, such as air drying, spin drying or spray drying, ideally with new prototypes, other than the food and pharmaceutical drying strategies currently used, tailored for the unique needs of cells and spermatozoa.

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Freezing, Vitrification, and Freeze-Drying of Equine Spermatozoa: Impact on Mitochondrial Membrane Potential, Lipid Peroxidation, and DNA Integrity

Cited by 32 publications

References 56 publications

Kinematic, bioenergetic and oxidative evaluations of donkey sperm preserved at +4°C

Kinematic, bioenergetic and oxidative evaluations of donkey sperm preserved at +4°C

Whole genome integrity and enhanced developmental potential in ram freeze-dried spermatozoa at mild sub-zero temperature

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

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