Ultrastructural alterations of frozen-thawed Asian elephant (Elephas maximus) spermatozoa

Intact plasma and acrosome membranes and functional mitochondria following cryopreservation are important attributes for the fertilizing ability of spermatozoa. In the present study, functional and ultrastructural changes of Asian elephant spermatozoa after cryopreservation either in TEST + glycerol or HEPT + dimethyl sulphoxide (DMSO) were evaluated by fluorescent techniques and electron microscopy. Sperm frozen in TEST + glycerol had higher proportion of sperm with intact plasma (49.1 +/- 9.2% vs. 30.9 +/- 3.9%) and acrosomal (53.7 +/- 4.9% vs. 35.8 +/- 6.1%) membranes, as well as active mitochondria (57.0 +/- 7.2% vs. 42.0 +/- 5.0%) than those cryopreserved in HEPT + DMSO. The results obtained from electron microscopy were similar to those obtained by fluorescence microscopy. The percentage of normal spermatozoa was higher when spermatozoa were frozen in TEST + glycerol than those frozen in HEPT + DMSO (31.8 +/- 5.6 vs. 28.5 +/- 6.4). The ultrastructural alterations revealed by transmission electron microscopy could be classified as (i) distension of plasma membrane, while the acrosome was swollen; (ii) disruption or loss of plasma membrane, while acrosome was swollen with distended outer acrosomal membrane; (iii) disruption or loss of plasma and outer acrosomal membrane with leakage of acrosome content; (iv) extensive vesiculation of plasma and outer acrosomal membrane and leakage of acrosome content; (v) a complete loss of both plasma membrane and outer acrosomal membrane; and (vi) swelling of mitochondria. These findings suggest that the freezing and thawing procedure caused structural damage to elephant spermatozoa, especially in the plasma membrane, acrosome and mitochondria. Fluorescence and electron microscopic evaluations are potentially a powerful tool in the analysis of elephant spermatozoa after freezing and thawing.