Activation of caspase-3/7, an apoptotic-related marker, during incubation and cryopreservation of alpaca (Vicugna pacos) spermatozoa

Carlos Segura, José La Rosa, Luis Báez, Oscar E. Gómez-Quispe, Shirley Evangelista-Vargas, Jane M. Morrell, Alexei Santiani

Research output: Contribution to journalArticlepeer-review


Caspases are crucial mediators of programmed cell death (apoptosis). Apoptosis can occur in spermatozoa during spermatogenesis or epididymal transit, as well as in ejaculated spermatozoa. A high proportion of apoptotic sperm would be a poor indicator of the freezability of a raw seminal sample. Alpaca spermatozoa are notoriously difficult to freeze successfully. Therefore, the objectives of this study were to study caspase activation during incubation (37°C) of fresh alpaca spermatozoa, as well as before and after cryopreservation, to gain some insight into the mechanisms behind the vulnerability of alpaca spermatozoa. Eleven sperm samples were incubated for 4 h at 37°C (Study 1), and 23 samples were frozen using an automated system (Study 2). Caspase-3/7 activation was assessed at 0,1,2,3, and 4 h in samples incubated at 37°C (Study 1); and before/after cryopreservation (Study 2) using CellEvent™ Caspase 3/7 Green Detection Reagent and flow cytometry. The proportions of alpaca spermatozoa with caspase-3/7 activated increased (p < 0.05) after 3–4 h of incubation at 37°C; however, caspase activation was similar before and after cryopreservation (36.2 ± 11.2% vs. 36.6 ± 33.7%, p > 0.05). The high standard deviation found after freezing could be explained by the existence of two subpopulations: one subpopulation where caspase-3/7 activation decreased during cryopreservation (from 36.6 ± 9.1% to 1.5 ± 2.2%), and the other subpopulation where caspase-3/7 activation increased after cryopreservation (from 37.7 ± 13.0% to 64.3 ± 16.7%). In conclusion, after 3–4 h of incubation, caspase-3/7 activation increased in fresh alpaca sperm, whereas cryopreservation affects alpaca sperm samples in different ways.

Original languageEnglish
Pages (from-to)1005-1011
Number of pages7
JournalReproduction in Domestic Animals
Issue number7
StatePublished - Jul 2023


  • Caspase-3/7
  • alpaca
  • apoptosis
  • cryopreservation
  • spermatozoa


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