Abstract
Simple SummaryEfficient collection and cryosurvival of semen from threatened wildlife species is key for the success of artificial reproductive technologies (ARTs). The viscous nature of ejaculates often collected from species such as rhinoceros, elephant, hippopotamus and primate, render the majority of spermatozoa collected useless and is therefore wasted. The enzyme papain has been used to reduce the viscosity of camelid semen but has yet to be tested in wildlife species. Therefore, the current study investigated the ability of papain to improve the yield and quality of spermatozoa collected from viscous fractions of the rhinoceros ejaculate during cryopreservation. Papain increased the quantity of useable spermatozoa collected from ejaculates, as well as the motility prior to freezing. It also improved the post-thaw motility, velocity, linearity and straightness of samples compared to spermatozoa frozen from the sperm-rich fraction of the ejaculate. There was no detrimental effect on membrane characteristics or DNA integrity. These results show that treating rhinoceros ejaculates with papain is able to salvage valuable spermatozoa and improve survival post-thaw, ultimately increasing the success of ARTs and creation of biobanks for the maintenance and survival of threatened species.The preservation of rhinoceros semen is vital for captive breeding programs. While successful collection and cryopreservation of rhinoceros semen has been reported, the volume and quality of semen produced is often low due to the high viscosity associated with ejaculates collected via electroejaculation. Reducing semen viscosity would enable access to previously unusable spermatozoa from viscous fractions and could improve quality post-thaw. The enzyme papain successfully reduced the viscosity of camelid semen but has yet to be tested in wildlife species. This study assessed the influence of papain on the in vitro quality of rhinoceros spermatozoa during cryopreservation using advanced semen assessment. In experiment 1, the motility of spermatozoa from the viscous fraction of an ejaculate, either untreated or treated with papain and its inhibitor E-64 prior to cryopreservation, was assessed post-thaw. In experiment 2, spermatozoa from papain-treated viscous fractions were compared to spermatozoa frozen from untreated sperm-rich fractions pre-freeze, as well as after 0, 1.5 and 3 h of incubation post-thaw (37 °C). Papain significantly increased the quantity of spermatozoa collected from ejaculates, as well as the motility prior to freezing. Papain also improved the post-thaw motility, velocity, linearity and straightness of samples compared to sperm-rich samples, with no detriment to sperm viability, lipid membrane disorder, production of ROS or DNA integrity (p < 0.05). Results show the benefit of supplementing rhinoceros spermatozoa with papain prior to cryopreservation on sperm cryosurvival and demonstrates the potential of using papain to improve the success of cryopreservation protocols, not only for the rhinoceros, but also for other wildlife species.
Highlights
Current numbers of white, black, greater one-horned, Sumatran and Javan rhinoceros in situ of 10000, 3100, 2200, 30 and 18 adult individuals, respectively [1], emphasise the importance of captive breeding programs for the maintenance of genetic diversity and survival of these species [2,3,4]
When a standard 20 μm Leja Computer-Assisted Sperm Analysis (CASA) slide was loaded with 3 μL of the viscous sperm fraction, it took 17 s for the chamber to load
To the best of our knowledge, the current study reports, for the first time, the advanced membrane and molecular characteristics of white rhinoceros spermatozoa, assessed post-thaw using flow cytometry
Summary
Black, greater one-horned, Sumatran and Javan rhinoceros in situ of 10000, 3100, 2200, 30 and 18 adult individuals, respectively [1], emphasise the importance of captive breeding programs for the maintenance of genetic diversity and survival of these species [2,3,4] It highlights the need for continued development of advanced assisted reproductive technologies (ARTs) to increase the likelihood of success. The presence of seminal plasma has been investigated during the freezing protocol, with no significant improvements in motility post-thaw noted, if seminal plasma was removed via centrifugation from sperm-rich fractions prior to cryopreservation [11] Despite these major developments, the use and success of cryopreservation protocols in the rhinoceros is limited by the quality and quantity of the original ejaculate produced [12]. While the development of ultrasonography and species-specific stimulation probes for EJ have resulted in a greater understanding of rhinoceros reproductive anatomy [13,15,16], creating greater consistency in the number of successful collections, the variability in semen quality associated with these collections is often still high [12]
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