Highly efficient vitrification for cryopreservation of human oocytes and embryos: the cryotop method

Preservation of female genetics is currently done primarily by means of oocyte and embryo cryopreservation. The field has seen much progress during its four-decade history, progress driven predominantly by research in humans, cows, and mice. Two basic cryopreservation techniques rule the field--controlled-rate freezing, the first to be developed, and vitrification, which, in recent years, has gained a foothold. While much progress has been achieved in human medicine, the cattle industry, and in laboratory animals, this is far from being the case for most other mammals and even less so for other vertebrates. The major strides and obstacles in human and other vertebrate oocyte and embryo cryopreservation will be reviewed here.

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Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification

The predictable answer to the provocative question of whether programmable freezers are still needed in the embryo laboratory is an even more provocative 'no'. However, such a radical statement needs strong support. Based on the extensive literature of the past 5 years, the authors collected arguments either supporting or contradicting their opinion. After an overview of the causes of cryoinjuries and strategies to eliminate them, the evolution of vitrification methods is discussed. Special attention is paid to the biosafety issues. The authors did not find any circumstance in oocyte or embryo cryopreservation where slow freezing offers considerable advantages compared with vitrification. In contrast, the overwhelming majority of published data prove that the latest vitrification methods are more efficient and reliable than any version of slow freezing. Application of the proper vitrification methods increases the efficiency of long-term storage of stem cells and opens new perspectives in cryopreservation of oocytes, both for IVF and somatic cell nuclear transfer. However, lack of support from regulatory authorities, and conservative approachs regarding novel techniques can slow down the implementation of vitrification. The opinion of the authors is that vitrification is the future of cryopreservation. The public have the final say in whether they want and allow this future to arrive.

https://www.rbmojournal.com/article/S1472-6483(10)61091-7/pdf

Are programmable freezers still needed in the embryo laboratory? Review on vitrification

Improving cryopreservation systems.

Sperm cells are the endpoint of male spermatogenesis and have particular anatomic and metabolic features. Sperm cryopreservation and storage currently require liquid nitrogen or ultralow refrigeration methods for long or short term storage, which requires routine maintenance and extensive space requirements. Conserving sperms have several purposes such as artificial reproductive technologies (ART), species conservation and clinical medicine. The combinations of storage temperature, cooling rate, chemical composition of the extender, cryoprotectant concentration, reactive oxygen species (ROS), seminal plasma composition and hygienic control are the key factors that affect the life-span of spermatozoa. Sperm preservation protocols vary among animal species owing to their inherent particularities that change extenders used for refrigeration and freezing. Extenders for freezing sperm cells contain buffers, carbohydrates (glucose, lactose, raffinose, saccharose and trehalose), salts (sodium citrate, citric acid), egg yolk and antibiotics. The use of different cryoprotectants, like trehalose or glycerol, as well as different concentrations of egg yolk and other constituents in semen extenders are being studied in our laboratory. Several cooling rates have been tested to freeze sperm cells. The use of faster rates (15-60 degrees C/min) gives rise to best sperm survivals after freezing-thawing, but more studies are needed to find the adequate cooling rates for each animal species. Sheep and goat males of some native breeds are being used in studies performed in EZN. Semen from those males has been frozen and stored as part of the Portuguese Animal Germplasm Bank. In small ruminants, individual variations in the quality of frozen semen have been observed, suggesting specific differences in sperm susceptibility to freezing methods, particularly obvious in goat males. Best quality frozen semen from small ruminants is being used in cervical artificial insemination studies aiming to increase productive parameters in selected flocks.

Cryopreservation of domestic animal sperm cells

New trends in gamete's cryopreservation

Successful pregnancies with directional freezing of large volume buck semen

Post-mortem semen cryopreservation and characterization in two different endangered gazelle species (Gazella gazella and Gazella dorcas) and one subspecies (Gazella gazelle acaiae).

To achieve the ultimate goal of both cryosurgery and cryopreservation, a thorough understanding of the processes responsible for cell and tissue damage is desired. The general belief is that cells are damaged primarily due to osmotic effects at slow cooling rates and intracellular ice formation at high cooling rates, together termed the "two factor theory." The present study deals with a third, largely ignored component--mechanical damage. Using pooled bull sperm cells as a model and directional freezing in large volumes, samples were frozen in the presence or absence of glass balls of three different diameters: 70-110, 250-500, and 1,000-1,250 microm, as a means of altering the surface area with which the cells come in contact. Post-thaw evaluation included motility at 0 h and after 3 h at 37 degrees C, viability, acrosome integrity, and hypoosmotic swelling test. Interactions among glass balls, sperm cells, and ice crystals were observed by directional freezing cryomicroscopy. Intra-container pressure in relation to volume was also evaluated. The series of studies presented here indicate that the higher the surface area with which the cells come in contact, the greater the damage, possibly because the cells are squeezed between the ice crystals and the surface. We further demonstrate that with a decrease in volume, and thus increase in surface area-to-volume ratio, the intra-container pressure during freezing increases. It is suggested that large volume freezing, given that heat dissipation is solved, will inflict less cryodamage to the cells than the current practice of small volume freezing.

Do physical forces contribute to cryodamage

The objective of the following paper is to describe a new technology for large volume and double freezing of semen in 12 mL test tubes Semen from two different bulls was frozen with a new technique using 12 mL test tubes and was refrozen after thawing in mini straws All freezing was done in a "Multi thermal gradient" (MTG) freezing apparatus, which moves the container at a constant velocity (V) through a thermal gradient (G) producing a controlled cooling rate B = (G) x (V) Each of the two bulls ejaculated were evaluated for post thaw motility in the lab and then in a field trial which was carried out in a split sample mode We inseminated 105 cows after a double freezing/thawing cycle, and another 123 cows were inseminated with semen frozen in mini-straws and a conventional method The results showed a 75 +/- 5% post thaw motility after freezing a 12 mL test tube and 50 +/- 5% after a second freezing/thawing in mini-straws, respectively Controlled vapour freezing showed a 60 +/- 10% post thaw motility The results of the field trial showed a pregnancy rate of 44% (47/105) for the double freezing group in comparison to 455% (56/123) for the controlled group These results can be beneficial for large volume freezing, and therefore for bull semen cryobanking in a large volume which will be followed by second freezing in a regular insemination volume

https://rnd.edpsciences.org/articles/rnd/pdf/2002/07/R2603Arav.pdf

Haim Gacitua

Papers

New trends in gamete's cryopreservation

Successful pregnancies with directional freezing of large volume buck semen

Post-mortem semen cryopreservation and characterization in two different endangered gazelle species (Gazella gazella and Gazella dorcas) and one subspecies (Gazella gazelle acaiae).

Do physical forces contribute to cryodamage

Successful pregnancies in cows following double freezing of a large volume of semen.