Cryopreservation of domestic animal sperm cells

Abstract

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.

Effect of low-density lipoproteins, spermatozoa concentration and glycerol on functional and motility parameters of bull spermatozoa during storage at 4 °C

“…3 However, the temperature reduction induces membrane stress; probably because of phase changes within the lipid bilayer that affects the functional state of membranes. 16 The results of this study show that sperm motility, plasma membrane integrity and acrosome integrity decreased with increasing length of storage.…”

“…13,14 Male animals can be classified as 'good or bad freezers' depending on the aptitude of their semen to support freezing with little cryodamage. 3 Often the selection of the reproductive bulls is based on the capacity of semen to support freezing. Thus, many bulls of high genetic value cannot be selected to produce frozen doses.…”

“…15 However, there are no studies carried out on the bull concerning the success of refrigeration when egg yolk is replaced by LDLs in the presence or absence of other cryoprotective agents such as glycerol, or on the effects on sperm motility and membrane functionality. The purpose of this study was to assess the effect of bovine semen dilution with an LDL extender on spermatozoal motility and plasma membrane functionality after refrigeration to 4 uC, using Trisegg yolk-glycerol (TEG) as control, Tris-egg yolk without glycerol (TE) as a second control, 8% LDL with glycerol (LDL 1 ) and LDL without glycerol (LDL 2 ) at two different dilutions: 80310 6 sperm per ml (normally recommended for frozen semen 3,16 ) and 240310 6 sperm per ml (predilution recommended by Benson et al, 17 the greater volume of seminal plasma helps to maintain semen quality at 4 uC). Glycerol is generally used for freezing semen; however, it was tested here to determine whether the use of LDL extenders 'ready to use for freezing semen' containing glycerol will be toxic and detrimental for semen at 4 uC.…”

Rapid thawing human sperm does not affect basic parameters in normozoospermic men: a double-blind prospective study

“…Many sperm thawing protocols for animals use high heating levels (above 50º C) and obtain good motility recovery rates (2)(3)(4).…”

Potato freeze‑thaw solution enhances immune function and antitumor activity in?vivo

“…Second, the freeze-thaw method causes cells to swell and shrink and break up ultimately due to the ice crystals formed in the freezing process. Several freeze-thaw cycles may be required to facilitate cell membrane breakage and the release of cell components (29). Methodological modification is ongoing for maximizing the PFTS bioactivity.…”