Oral Presentation ESA-SRB-ANZBMS 2024 in conjunction with ENSA

Drying for a cause: quality, PLCz localization, and freeze-drying of koala epididymal spermatozoa recovered postmortem  (#39)

Patricio Palacios Benitez 1 , Jiaqi Zhao 2 , Rhiannon J. Gurkin 1 , Yolande Campbell 3 , Stephen D. Johnston 3 4 , Katerina Damyanova 5 6 , Tessa Lord 5 6 , Andres Gambini 1 4
  1. School of Agriculture and Food Sustainability, The University of Queensland, Gatton, Queensland, Australia
  2. School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia
  3. School of the Environment, The University of Queensland, Brisbane, Queensland, Australia
  4. School of Veterinary Science, The University of Queensland, Gatton, Queensland, Australia
  5. Discipline of Biological Sciences, The University of Newcastle, Callaghan, New South Wales, Australia
  6. Infertility and Reproduction Program, Hunter Medical Research Institute, New Lambton Heights,, New South Wales, Australia

Koalas are iconic Australian marsupials facing numerous threats, including habitat loss and disease, which have led to declining populations. Preserving their genetic material is crucial for future conservation efforts. This study aimed to assess koala epididymal spermatozoa's concentration, motility, viability, and morphology recovered at different postmortem time intervals. Additionally, we sought to characterize the localization of the phospholipase-C-zeta (PLCz) and the resilience of koala sperm cells to freeze-drying. Samples were collected from euthanised koalas and refrigerated at 5 ºC for 24, 48, 72, and 96 hours postmortem. Epididymal spermatozoa were recovered by mincing the cauda epididymis followed by incubation for 10 minutes at 35 ºC in phosphate-buffered media. Sperm concentration and quality parameters were determined using established methods (1-2). Fixed spermatozoa were stained for PLCz immunofluorescence using a rabbit polyclonal antibody (3). Sperm lyophilization was performed following described protocols (4). Results revealed no significant differences in sperm concentration or quality postmortem (n=18, Table 1), except for the number of head morphotype III (Figure 1). Immunofluorescence detected PLCz to be present in almost all tails and in 89.00 ± 3.05% of heads in fresh spermatozoa (mean ± SEM, n=3). After reconstituting lyophilized sperm stored for a month at 5 ºC, no motility was observed, but 6.00 ± 1.16% had an intact membrane. However, the proportion of sperm with head-localized PLCz significantly dropped to 20.00 ± 3.60% (mean ± SEM, Fisher's exact test, n=3). Our results indicate that epididymal koala sperm maintain their quality for up to 96 hours postmortem, providing a sufficient window for sample processing and preservation. We report the PLCz expression pattern in marsupial sperm and show that while some sperm survive lyophilization, PLCz localization is altered. These findings contribute to novel genetic preservation strategies for koala sperm and enhance understanding of the role of PLCz in marsupial reproduction.66b5a807d7ab3-SRB+ABSTRACT_original_page-0002.jpg

  1. Johnston, S. D., MacCallum, C., Blyde, D., McClean, R., Lisle, A., & Holt, W. V. (2006). An investigation into the similarities and differences governing the cryopreservation success of koala (Phascolarctos cinereus: goldfuss) and common wombat (Vombatus ursinus: shaw) spermatozoa. Cryobiology, 53(2), 218-228.
  2. Hulse, L., Beagley, K., Larkin, R., Nicolson, V., Gosálvez, J., & Johnston, S. (2021). The effect of Chlamydia infection on koala (Phascolarctos cinereus) semen quality. Theriogenology, 167, 99-110.
  3. Arroyo-Salvo, C., Rio, S., Bogetti, M. E., Plaza, J., Miragaya, M., Yaneff, A., Davio, C., Fissore, R., Gervasi, M. G., Gambini, A., & Perez-Martinez, S. (2024). Effect of bicarbonate and polyvinyl alcohol on in vitro capacitation and fertilization ability of cryopreserved equine spermatozoa. Andrology (Oxford). https://doi.org/10.1111/andr.13667
  4. Choi YH, Varner DD, Love CC, Hartman DL, Hinrichs K. Production of live foals via intracytoplasmic injection of lyophilized sperm and sperm extract in the horse. Reproduction. 2011 Oct;142(4):529-38. doi: 10.1530/REP-11-0145. Epub 2011 Aug 16. PMID: 21846810.