Increasing the Yield and Cryosurvival of Spermatozoa from Rhinoceros Ejaculates Using the Enzyme Papain

Abstract

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.

Impact of papain on the treatment of raw diluted dromedary semen

“…It is yet unclear how papain works to enhance the quality of the camel semen that is collected. Rickard et al (2022) consider that semen viscosity is not related to the volume of plasma generated but to the inclusion or amount of a particular protein. Previous studies in alpaca have shown that papain targets glycosylated proteins within the seminal fluid instead of glycosaminoglans (Kershaw et al, 2013).…”

“…In camelids, glycoproteins released by the bulbourethral glands are responsible for the trapping of spermatozoa within the camelid uterus (Apichela et al., 2014). It is highly probable that papain, by focusing on glycoproteins, decreased the viscosity of ejaculates (Rickard et al., 2022).…”

Correlation between Post‐Thaw Spermatozoa Quality of the Endangered Javan Banteng with OPN Gene Expression

“…Interestingly, the value of spermatozoa acrosomal integrity in our study was relatively high compared to several species that had been previously reported. The percentage of post-thaw acrosomal integrity in rhinos was reported to be 80.00% [ 49 ] and 68.00% [ 53 ], whereas in bison it was 71.13%–76.56% [ 54 ]. Here, we also noticed that spermatozoa acrosomal integrity in both samples used in our study showed a considerably high percentage regardless of the low post-thaw spermatozoa motility and viability.…”

Resurrecting biodiversity: advanced assisted reproductive technologies and biobanking

“…Spermatozoa cryopreservation is an example of just one ART that can facilitate a living cryobank capable of aiding in the genetic management of endangered species and has been achieved in many species (covered in-depth elsewhere, for example, amphibians: Browne et al 2019 ; fish: Asturiano et al 2017 , Xin et al 2017 ; mammals: Swanson et al 2007 , Rickard et al 2022 ; avians: Asano & Tajima 2017 , Cardoso et al 2020 ). Storing genes in the form of spermatozoa is particularly beneficial as spermatozoa are continuously replenished haploid cells that can be collected from numerous genetically diverse representatives of a species.…”