Abstract
The factors affecting the inter-individual differences in sperm freezability is a major line of research in spermatology. Poor sperm freezability is mainly characterised by a low sperm velocity, which in turn is associated with low fertility rates in most animal species. Studies concerning the implications of sperm morphometry on freezability are quite limited, and most of them are based on sperm head size regardless of the structural parts of the flagellum, which provides sperm motility. Here, for the first time, we determined the volumes of the flagellum structures in fresh epididymal red deer spermatozoa using a stereological method under phase contrast microscopy. Sperm samples from thirty-three stags were frozen and classified as good freezers (GF) or bad freezers (BF) at two hours post-thawing using three sperm kinetic parameters which are strongly correlated with fertility in this species. Fourteen stags were clearly identified as GF, whereas nineteen were BF. No significant difference in sperm head size between the two groups was found. On the contrary, the GF exhibited a lower principal piece volume than the BF (6.13 µm3 vs 6.61 µm3, respectively, p = 0.006). The volume of the flagellum structures showed a strong negative relationship with post-thawing sperm velocity. For instance, the volume of the sperm principal piece was negatively correlated with sperm velocity at two hours post-thawing (r = −0.60; p<0.001). Our results clearly show that a higher volume of the sperm principal piece results in poor freezability, and highlights the key role of flagellum size in sperm cryopreservation success.
Highlights
It is well known that the cryopreservation process negatively affects the viability and fertility of reproductive cells
Since the variables of sperm velocity (VAP, VCL, VSL, ALH, and sperm motility index (SMI)) were highly correlated among themselves, we reduced the number of predictor variables using principal component analysis (PCA) to obtain an overall sperm velocity at 0 and 2 hours post-thawing, respectively
Our results clearly show that sperm with a higher principal piece volume freeze worse, that is, the good freezers’’ (GF) have a lower principal piece volume than the bad freezers’’ (BF)
Summary
It is well known that the cryopreservation process negatively affects the viability and fertility of reproductive cells. There are several factors caused by cryopreservation protocols that alter sperm integrity, such as a change in the temperature of the diluents [1]; osmotic and toxic stresses induced by cryoprotectants [1,2]; formation/reshaping of intracellular ice during freezing and thawing [3]; and dissolution of ice in the extracellular environment [2] All of these factors induce sperm volumetric changes, plasma membrane alterations, flagellum morphological defects, as well as decrease mitochondrial membrane potential, sperm motility, viability, and fertility [2,4,5,6,7,8,9,10]. It has been demonstrated that these consistent inter-individual variations in sperm freezability are genetically determined [12]
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