Acrosome reaction and cryopreservation of dog spermatozoa

The use of AI in dogs has been limited by the lack of effective and reliable means of cryopreservation of semen and by the poor correlation between traditional methods of post-thaw assessment of semen quality and fertility. In order to address these problems, the present study focuses upon methods of cold storage and cryopreservation of dog spermatozoa by undertaking comparative evaluations of post-thaw motility and in vitro induction of acrosome reaction. Split-ejaculate protocols were used to compare the effect of storage at +4°C and cryopreservation upon (i) the maintenance of spermatozoa! motility and (ii) spontaneous or A23187-induced acrosome reactions during incubation at 39°C (in 5% CO2 in the humidified air) for 60 or 120 min. The assessments of samples were made by BrightField, Phase Contrast (PC), Differential Interference Contrast (DIC), Scanning (SEM) and Transmission (TEM) Electron Microscopy. The interaction between the process of glycerolisation and the presence of seminal plasma is one of the key limitors for success in cryopreservation of dog spermatozoa. Interactions between the effects of removal of seminal plasma (by centrifugation), dilution rate, the temperature at which glycerolisation took place and the concentration of glycerol upon survival of spermatozoa t +4°C were studied in a series of split-ejaculate experiments. Spermatozoa were suspended in Tris-fructose-citric acid extender containing 20% (v/v) egg yolk and 8% (v/v) glycerol at +4°C for 48 h. Survival was assessed as the percentage of spermatozoa displaying progressive motility. Survival of spermatozoa was higher (P<0.05) after glycerolisation at +4°C than at the room temperature. At dilution rates of 1: 1 and 1: 2 (semen: extender), the survival was higher (P<0.05) in samples that were centrifuged and glycerolised at +4°C than the samples that were neat and glycerolised at the room temperature. While at the dilution rate of 1: 16 it was higher (P<0.05) in samples that were neat plus glycerolised at +4°C than all samples that were glycerolised at the room temperature. Concentrations of glycerol that were >2% (v/v) resulted in lower (P<0.05) survival than at lower concentrations. Following the initial stage of the investigations, the optimisation and validation of a method for in vitro induction of acrosome reactions were required. Suspensions of spermatozoa in TALP medium were incubated in the presence of a logarithmic. series of concentrations of the calcium ionophore, A23187. Induction of acrosome reactions was assessed by bright-field (using naphthol yellow S/aniline blue stain, NA) and phase contrast (PC) microscopy. Using these methods, it was determined that incubation in the presence of 1 μM/1 A23187 for a period of 30-45 min was optimal for inducing acrosome reactions in fresh semen. It was also noted that the assessments of acrosome reactions by using NA staining, were highly correlated with PC microscopy. In consequence, the simple procedure of NA staining might be an acceptable alternative to PC microscopy for use in the field. Subsequently, the effect of chilling and glycerolisation upon in vitro induction of acrosome reactions by A23187 was assessed. Acrosome reactions were studied as these have been described in the literature as providing accurate bioassay of spermatozoal functionality in vivo. Acrosome reactions were assessed by using DIC microscopy. The acrosomal integrity was impaired after chilling, which accelerated the A23187-induced acrosome reaction such that a lower concentration (0.1 μM/1) of A23187 was also effective to induce the reaction within 60 min of incubation. However, the presence of 2% glycerol (v/v, final) in standard Tris extender, containing 20% egg yolk, did not significantly affect the sequence of acrosome reaction. The optimal freezing regimen (from +4°C to -120°C) was determined by using a programmable biological freezer in a series of experiments, in which various cooling rates were combined in a Latin square designs. Semen was diluted in standard Tris extender containing 20% egg yolk and 2% glycerol (v/v, final) and packed in 0.25 ml French paiettes (straws). The optimal cooling regimen was -0.5°C/min from +4°C to -9°C, -40°C/min to -20°C, -100°C/min to -120°C, followed by direct immersion of the straws in liquid nitrogen. Changes in temperatures within an individual straw were continuously measured and these data were found to be highly correlated with the eventual post-thaw motility of frozen-thawed spermatozoa. Although freezing and thawing resulted in major acrosomal deterioration, there were no significant differences between freezing regimens on the basis of in vitro induction of acrosome reactions, as assessed by DIC microscopy. Finally, ultrastructural studies, using SEM and TEM, upon chilled (as `ready to freeze') and frozen-thawed spermatozoa subjected to A23187-induced acrosome reaction demonstrated that freeze-thawing provoked the acrosome reaction such that, with TEM (i) the plasma membrane was usually damaged or missing, (ii) the acrosomal changes (including the loss of acrosomal content, as seen by decondensation and swelling) except vesiculation of the acrosomal membranes, exceeded to the equatorial segment and (iii) a further damage occurred to the post-acrosomal region. In summary, these results show that semen should be; (i) centrifuged for dilutions of <1: 8, (ii) diluted at 1: 8 in Trisfructose-citric acid extender containing 20% egg yolk, (iii) glycerolised at +4°C at a final concentration of 2% glycerol (v/v), (iv) cooled at -0.5°C/min from +4°C to -9°C, at -40°C/min to -20°C and at -100°C/min to -120°C, followed by direct immersion of the straws in liquid nitrogen for cryopreservation and (i) introduced to 1 μM/1 A23187 in TALP, (ii) incubated for at least 30-45 min for induction of acrosome reaction in vitro and, thereby, demonstrated that optimisation of cryopreservation and in vitro induction of acrosome reaction of dog spermatozoa are possible.