Cryopreservation of rabbit semen comparing the effects of different cryoprotectants

The low survival of sperm after freezing is a major drawback for the widespread use of frozen semen in artificial insemination programs for livestock animals such as the rabbit, in which sperm cryopreservation has only been used for experimental purposes [1]. During cryopreservation, sperm cells undergo stress such as changes in the osmotic balance and temperatures during cooling, freezing, and rewarming. These changes lead to ice crystal formation, which is among the main biophysical factors which cause sperm death [2]. Cryoprotective agents (CPAs) that permeate the cell membrane are needed to increase membrane fluidity and partially dehydrating the cell, lowering the freezing point, and thus reducing the number and size of intracellular ice crystals formed. However, the paradox is that CPAs themselves can have a toxic effect on sperm (membrane destabilization, protein and enzyme denaturation) and this effect is related directly to the concentration used and the time of cell exposure [3]. Added to the freezing medium, nonpermeating cryoprotective substances such as proteins, or amino acids and sugars, acting mainly as osmoprotectants, can mitigate the cryodamage caused by permeating CPAs. At similar concentrations, these substances are less toxic than permeable CPAs, inhibit ice growth and help the sperm to stabilize internal solute concentrations under osmotic stress, and this reduces the amount of penetrating CPAs needed [3].
According to the freezing rate, sperm cryopreservation techniques can be divided into two main categories: slow freezing (conventional freezing) and ultrarapid freezing
(vitrification or similar state to vitrification). Conventional freezing involves a step-wise reduction in temperature, but ice crystals that form within the cell can have extremely deleterious effects, such that a balance needs to be found between the beneficial and toxic effects of permeating CPAs. Alternatively, vitrification is a