Cryopreservation‐Induced Changes in Protamine Levels and DNA Fragmentation in Alpaca Spermatozoa

Cryopreservation is known to destabilise spermatozoa and is associated with deficiencies in protamine levels and increased DNA fragmentation, which can reduce fertility in various species. The objective of this study was to evaluate the impact of cryopreservation on protamine levels and DNA fragmentation in alpaca spermatozoa. A total of 108 testicles/epididymides were collected from a slaughterhouse and sperm were recovered from the cauda epididymis. Only samples meeting the criteria of > 10 g in weight, > 3 cm in length, > 30% motility, and > 50 million spermatozoa/mL were processed. Sixty samples (n = 60) were suitable for cryopreservation: 30 were used to assess protamine levels, and 30 to evaluate DNA fragmentation. Assessments were conducted both before and after cryopreservation using imaging flow cytometry. Protamine levels were assessed with chromomycin A3 (CMA3, 0.25 mg/mL), where fluorescence inversely correlates with protamination levels. The TUNEL assay was used to analyse DNA fragmentation, following fixation with 0.4% formaldehyde and permeabilisation with 0.8% Triton X-100. Results showed a significant decrease in CMA3 mean fluorescence after cryopreservation (288.19 ± 145.53 mFL vs. 68.54 ± 51.25 mFL, p < 0.05) and an increase in DNA fragmentation (2.98 ± 2.39 vs. 9.45 ± 15.43, p < 0.05). In conclusion, cryopreservation decreases CMA3 fluorescence, related to a possible increase in protamination, and increases DNA fragmentation in alpaca spermatozoa.