Assessment of viability and acrosomal status of Asian elephant (Elephas maximus) sperm after treatment with calcium ionophore and heparin.

Knowledge about the acrosomal status of Asian elephant (Elephas maximus) sperm is extremely limited. The objective of this study was to evaluate the viability and acrosomal status of Asian elephant sperm following induction by calcium ionophore and heparin using propidium iodide (PI) and fluorescein isothiocyanate conjugated peanut agglutinin (FITC-PNA). Semen samples were collected from elephant bulls by manual stimulation. Semen was diluted with extender, cooled to 4 degrees C and transported to a laboratory for the experiment. Sperm cells were incubated in modified Tyrode's medium containing either 1 mM calcium ionophore or 10 mg/ml heparin for 5 h at 39 degrees C. Sperm recovered at the onset (0 h), 1, 2, 3, 4 and 5 h of incubation were simultaneously assessed for the viability and acrosomal status using dual staining of FITC-PNA and PI. Results were confirmed by transmission electron microscopy. A progressive increase in the proportion of live-acrosome reacted sperm was observed within 3 h of incubation in both treatment groups which slightly decreased at 4 to 5 h of incubation. At 1 to 3 h of incubation, the percentage of live-acrosome reacted sperm induced by calcium ionophore was higher (P < 0.05) than those induced by heparin and the control. However, there were no statistical differences at 4 to 5 h of incubation. A progressive reduction of the percentage of motile sperm was observed in the control as well as both treatment groups. Sperm motility decreased sharply when they were incubated in calcium ionophore compared with incubation in heparin and control groups. These results indicate that the occurrence of live-acrosome reacted sperm in the Asian elephant was induced by calcium ionophore at a rate higher than that induced by heparin.

Semen characteristics and sperm morphology of serow (Capricornis sumatraensis).

The serow (Capricornis sumatraensis) is a critically endangered species. The objectives of this study were to evaluate ejaculate quality in captive males, and to investigate and characterize sperm morphology. Semen was collected using electroejaculation. Mean (+/-S.D.) seminal characteristics were: semen volume 2.3+/-0.8 mL, pH 7.8+/-0.4, and osmolality 329.9+/-32.9mOsmol/kg; sperm concentration 515.8+/-263.1 x 10(6) cells/mL; wave motion score (1-5) 3.9+/-0.4; motile sperm 60.5+/-22%; viable sperm 68.3+/-9.4%; morphologically normal sperm 70.8+/-19.3%; and an opacity that was yellowish to milky-white. Sperm head length, width, degree of elongation, area, and perimeter were 6.0+/-0.6 microm, 4.3+/-0.3 microm, 71.7+/-8.6%, 19.8+/-2.5 microm(2), and 17.9+/-2.1 microm. Based on these measurements, we categorized sperm head morphometry as small, medium, or large. In addition, sperm morphology was examined by light and scanning electron microscopy; overall, morphologically normal and abnormal sperm were similar to those reported for other bovidae. In summary, this study provided baseline data regarding semen characteristics of C. sumatraensis, which should be of value in the preservation of this endangered species.

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.