610 Method of differential breast tumour diagnostics

Abstract

The present study intended for displaying the effect of different freeze-drying media and temperature of storage on the ultrastructure and DNA of freeze-dried buffalo bull spermatozoa. The semen samples undergone freeze-drying were raw semen and frozen-thawed semen extended in Tris-Fructose-Egg yolk-Glycerol.Semen samples were processed in two portions: First portion was cryopreserved with Tris-Fructose-Egg yolk-Glycerol extender to be freeze-dried with the different media used in this study. Second portion was freeze-dried immediately with the different media used in this study. Semen samples were centrifuged in a percoll gradient (45–90%) for 20 min at 700 × g to remove seminal plasma. Subsequently, sperms were washed twice in Tyrode's albumen lactate pyruvate (TALP) to remove percoll remains, and allocated into the four freeze-drying media (media 1, 2, 3 and 4) respectively. The media tested were: medium 1 (EGTA solution), medium 2 (EDTA solution), medium 3 (TCM199 with Hanks salts and 10% FCS) and medium 4 (TCM199 with Hanks salts and 10% FCS and trehalose). For all the media used, samples were diluted, placed in tubes of 1.5 ml and kept at room temperature for 30 min. Then sperm cell suspensions were cooled in liquid nitrogen vapor (approximately −80 °C for 1 h), by keeping the tubes at a distance of 5 cm from liquid nitrogen surface before plunged into it. Frozen samples were immediately inserted into the freeze-drying machine, previously stabilized at (−40 °C) and 350 × 10−3 Mbar pressure. After 12–16 h of freeze-drying, the tubes containing the samples were covered with aluminum foil and stored for 3 months at different temperatures; 4 °C, −20 °C and −80 °C. Freeze-dried sperm samples were re-hydrated by adding 100 μL of milli-Q water at room temperature. To evaluate sperm ultrastructure, transmission electron microscopy was done. For detection of DNA fragmentation, commet assay was performed.Electron microscopy showed that the sperm cell component most affected by freeze-drying was the plasma membrane, which was destroyed in all media either in raw or frozen thawed sperm. Microtubules organization was also disorganized in the majority of the sperm from freeze-drying medium 2 and 3, diverging from freeze-drying media 1 and 4, in which microtubules were intact. Conversely, the acrosome and mitochondria were well protected in all media. However, the storage temperature has no effect. The freeze-drying medium with EDTA solution exhibited and the lowest percent of DNA damage (9.3) while the freeze-drying medium (TCM with trehalose) exhibited the highest percent of DNA damage (13.6) at temperature of storage (4 °C). In contrast, the freeze-drying medium with EGTA solution exhibited the lowest percent of DNA damage (15.3) while the freeze-drying medium (TCM with trehalose) exhibited the highest percent of DNA damage (18) at temperature of storage (−20 °C). On the other hand, the freeze-drying medium with EDTA solution exhibited the lowest percent of DNA damage (11.3) while the freeze-drying medium (TCM with trehalose) exhibited the highest percent of DNA damage (20) at temperature of storage (−80 °C). Consequently, the freeze-drying medium (TCM without trehalose) exhibited the lowest percent of DNA damage (6.5) while the freeze-drying medium with EDTA solution exhibited the highest percent of DNA damage (13.9) at temperature of storage (4 °C). On the contrary, the freeze-drying medium with EGTA solution exhibited the lowest percent of DNA damage (13.2) while the freeze-drying medium (TCM without trehalose) exhibited the highest percent of DNA damage (20.8) at temperature of storage (−20 °C). Conversely, the freeze-drying medium with EDTA solution exhibited the lowest percent of DNA damage (6) while the freeze-drying medium (TCM with trehalose) exhibited the highest percent of DNA damage (12) at temperature of storage (−80 °C).From the present study, we demonstrated that the freeze-drying medium containing EGTA and EDTA solution were more efficient in avoiding damage to components of buffalo bull sperm, especially the nuclei. Therefore, the medium used for freeze-drying process directly affected sperm nuclear integrity. Also, the storage temperature of freeze-dried sperm affects sperm nuclear integrity.