Abstract
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.
Highlights
Freeze-dried spermatozoa typically shows a reduction in fertility primarily due to the DNA damage resulting from the sublimation process
The type of DNA damage was quantified with the following parameters: (a) mean alpha-T; quantifies the degree of abnormal chromatin structure with an increased susceptibility to acid-induced denaturation and is expressed as the ratio of red to total fluorescence intensity [red/(red + green)]. (b) Standard Deviation of Alpha-T both in Single-Strand Breaks than Double-Strands Breaks populations, that shows the extent of abnormality in chromatin structure within a population. (c) Single-Strand Breaks Count (SSBs %), represents the percentage of Single-Strand Breaks on the total number of sperm cell acquired. (d) Double-Strand Breaks Count (DSBs %) represents the percentage of DSB on the total number of sperm cell acquired
A minor trend was found in the degree of abnormal chromatin structure in Double-Strand Breaks population in SF-group
Summary
Freeze-dried spermatozoa typically shows a reduction in fertility primarily due to the DNA damage resulting from the sublimation process. 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. The first significant result of this method was achieved by Wakayama and Yanagimachi[1], that demonstrated for the first time the birth of healthy offspring from epididymal freeze-dried mouse spermatozoa Since this pioneer work, dry spermatozoa successfully supported embryo development till the blastocyst stage in a wide range of experimental and farm animals: p ig2, bovine[3] and sheep[4]. Our previous work has reported a clear negative correlation between the extent of DNA damage in dry spermatozoa and embryo cleavage, with developmental arrest with double strand breaks (DSBs) exceeding 2%9
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