Cryopreservation in rhinoceros—Setting a new benchmark for sperm cryosurvival

Abstract

At times when rhinoceros are fiercely poached, when some rhinoceros species are closer than ever to extinction, and when the scientific community is in debate over the use of advanced cell technologies as a remaining resort it is time to simplify and improve existing assisted reproduction techniques to enhance breeding and genetic diversity in the living populations under our care. Semen cryopreservation has been performed in all captive rhinoceros species with limited degree of success. Here we tested three freezing extenders, containing different cryoprotectants and various freezing rates for the cryopreservation of rhinoceros sperm from 14 bulls. In experiment I, semen from 9 bulls was used to determine the most suitable diluent, cryoprotectant and freezing rate for the successful cryopreservation of rhinoceros sperm. In experiment II, semen from 5 bulls was used to assess whether the removal of seminal plasma could further improve post thaw sperm quality following cryopreservation with conditions identified in Experiment I. Semen was diluted with Berliner Cryomedia, ButoCrio® or INRA Freeze®, packaged in 0.5 mL straws and frozen 3, 4, and 5 cm over liquid nitrogen (LN) vapour or directly in a dryshipper. It was found that semen extended with ButoCrio® (containing glycerol and methylformamide) and frozen 3cm over LN vapour provided the best protection to rhinoceros spermatozoa during cryopreservation. When pooled over treatments, total and progressive post thaw motility was 75.3 ± 4.2% and 68.5 ± 5.7%, respectively marking a new benchmark for the cryopreservation of rhinoceros sperm. Post thaw total and progressive motility, viability and acrosome integrity of semen diluted in ButoCrio® was significantly higher than semen extended in Berliner Cryomedia or INRA Freeze®. The removal of seminal plasma did not improve post thaw sperm survival (p > 0.05). In conclusion, the cryosurvival of rhinoceros spermatozoa was significantly improved when using a mixture of glycerol and methylformamide in combination with a fast freezing rate at 3 cm. These results describe a new protocol for the improved cryosurvival of rhinoceros spermatozoa and will enable a more successful preservation of genetic diversity between males, especially in donors whose spermatozoa may already be compromised prior to or during collection. The successful reduction of glycerol concentration in favour of methylformamide as a cryoprotectant could be a novel suggestion for the improvement of cryopreservation techniques in other wildlife species.

Rare Sperm Freezing

“…Rare sperm cryopreservation could be considered as opportunity beneficial to wildlife and endangered species preservation [70][71][72]. Breeding and assisted reproduction in animals has long-standing traditions and has been explored in husbandry for centuries.…”

Could embryonic diapause facilitate conservation of endangered species?

“…Given the rapidly declining numbers of individuals for many wildlife species, assisted reproduction technologies (ARTs) have become increasingly relevant in wildlife conservation. ART allows not only to artificially advance the number of offspring, but also provides a tool to protect the genetic quality of the remaining population, particularly of the captive held fraction of specific populations across borders [12][13][14][15][16][17].…”

tale of two islands. The reality of large-scale extinction in the early stages of the Anthropocene: a lack of awareness and appropriate action

“…This is the obvious direction in which resources and research efforts should be directed. It is early days and there are numerous difficulties in extending the practice to a wide range of species but cryogenically stored viable sperm and egg cells are already being used as a measure to conserve species close to extinction (Hermes et al, 2018) and viable somatic cells can potentially be cloned. Thus, it is not only possible to conserve and utilise genetic diversity of threatened species but preservation of viable cells offers the potential to restore species if they should become extinct (Naggs, 2017), together with associated organisms such as their gut biota.…”