Search

Very little is known about the effects of manipulating toad sperm activity in vitro, and such information is important in the development of a genetic resource bank for bufonid species. The specific objectives of this study were to: 1) identify the optimal inactivation and reactivation solutions for toad spermatozoa colleted in urine; 2) establish the length of time toad spermatozoa can be exposed to an inactivation buffer and still resume motility upon reactivation; 3) evaluate the consequence of inactivation on specific sperm characteristics; and 4) characterize the sperm mitochondria vesicle (MV) and its relationship to motility. Reactivated sperm motility was similar after inactivation in either Simplified Amphibian Ringers (SAR) solution or DeBoer's (DB) solution. Diluting the buffer by 80% with water provided the best method for reactivating sperm. Dilutions with NaCl solutions (10–50 mM) produced inferior results. SAR-inactivated spermatozoa could remain suspended up to 4 hr and still regain 25% of initial motility upon reactivation in water. Compared to the controls, sperm motility was greater (P<0.01) over time for samples treated with SAR, although forward progression was significantly lower. Furthermore, SAR treatment resulted in sperm samples with a greater number of viable, morphologically normal, and intact MVs over time. Electron microscopy and fluorescent staining confirmed that the toad sperm's MV contains a large number of active mitochondria with very few other cytoplasmic structures. Nearly all spermatozoa exhibiting motility had an intact MV, and dissociation of this structure was clearly related to motility loss. In conclusion, toad spermatozoa can be effectively inactivated and reactivated by varying the osmolality of the external solutions and, although sperm forward progression is reduced, all other characteristics are well maintained. Moreover, the increased number of spermatozoa with intact MV after inactivation suggests the process may help preserve this important structure. J. Exp. Zool. 295A:172–182, 2003. © 2003 Wiley-Liss, Inc.

This study was conducted to determine if cryopreservation and thawing reduces the quality of scimitar-horned oryx spermatozoa and thus might be responsible for sub-optimal artificial insemination (AI) efficiency. Functional capacity of frozen–thawed oryx spermatozoa was compared in a heterologous bovine in vitro fertilization (IVF) system after being prepared by four methods. Fertilizing longevity was also assessed after thawing and pre-incubating spermatozoa for 12 or 24 h before IVF. Sperm characteristics (viability, morphology, acrosomal and capacitation status) were superior for samples prepared by Percoll centrifugation and standard swim-up compared with microdrop swim-up and wash methods. Regardless of variation in sperm characteristics over time, fertilization success and embryo development were high and did not differ among treatments. Fertilization and cleavage success for spermatozoa pre-incubated for 12 h before IVF were comparable with that achieved with non-incubated spermatozoa. Even 24 h after thawing, spermatozoa were capable of fertilizing oocytes, but percentage fertilization and embryo cleavage were significantly lower than for spermatozoa pre-incubated for 12 h. Overall, functional capacity of oryx spermatozoa after thawing appears comparable with that of domestic bull spermatozoa. When used for AI, frozen—thawed oryx spermatozoa should be capable of fertilizing oocytes in females ovulating 12 or even 24 h after insemination, providing sperm transport mechanisms are adequate. The functional capacity and fertilizing longevity of oryx sperm after thawing is high, and therefore unlikely to be responsible for decreased AI efficiency in the scimitar-horned oryx. Extra keywords: antelope, artificial insemination, capacitation.