Evaluation of motility, membrane status and DNA integrity of frozen–thawed bottlenose dolphin (Tursiops truncatus) spermatozoa after sex-sorting and recryopreservation

Abstract

Artificial insemination (AI) with sex-sorted frozen-thawed spermatozoa has led to enhanced management of ex situ bottlenose dolphin populations. Extended distance of animals from the sorting facility can be overcome by the use of frozen-thawed, sorted and recryopreserved spermatozoa. Although one bottlenose dolphin calf had been born using sexed frozen-thawed spermatozoa derived from frozen semen, a critical evaluation of in vitro sperm quality is needed to justify the routine use of such samples in AI programs. Sperm motility parameters and plasma membrane integrity were influenced by stage of the sex-sorting process, sperm type (non-sorted and sorted) and freezing method (straw and directional) (P!0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P!0.05) motility parameters over a 24-h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of non-sorted spermatozoa, as assessed by the sperm chromatin structure assay (SCSA), was high and remained unchanged throughout freeze-thawing and incubation processes. Though a possible interaction between Hoechst 33342 and the SCSA-derived acridine orange was observed in stained and sorted samples, the proportion of sex-sorted, recryopreserved spermatozoa exhibiting denatured DNA was low (6.6G4.1%) at 6 h after the second thawing step and remained unchanged (PO0.05) at 24 h. The viability of sorted spermatozoa was higher (P!0.05) than that of non-sorted spermatozoa across all time points after recryopreservation. Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI.

Effect of Cryopreservation on the Sperm DNA Fragmentation Dynamics of the Bottlenose Dolphin (Tursiops truncatus)

“…These results are in agreement with the SCSA ® technique performed on same species following recryopreservation of sperm samples (Montano et al. ), where the rate of DNA fragmentation in the sperm immediately after thawing remained low (6.6 ± 4.1%) after 6 h and remained unchanged (p > 0.05) after 24‐h incubation at 37°C. No data exist for SDF evaluation in this species using other techniques than the SCSA.…”

“…The recent listing of this species as protected (Appendix II of Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), http://www.iucnredlist.org/details/22563/0 consulted on September 2013) has further exacerbated the issue of genetic management and heightened the importance of developing methods for assessment of male fertility, reproductive management (Montano et al. ) and the establishment of a frozen genetic resource bank (Robeck and O'Brien ; Robeck et al. ).…”

“…Montano et al. () have recently described SCSA analysis of the dolphin spermatozoa following sex‐sorting and recryopreservation; these authors reported that the quality of sperm DNA of non‐sorted spermatozoa was high and remained unchanged throughout freeze–thawing and incubation processes and that while a possible interaction between Hoechst 33342 and the SCSA‐derived acridine orange was observed in stained and sorted samples, the proportion of sex‐sorted, recryopreserved spermatozoa exhibiting denatured DNA was low at 6 h after the second thawing step and remained unchanged at 24 h.…”

Validation of a Field Based Chromatin Dispersion Assay to Assess Sperm DNA Fragmentation in the Bottlenose Dolphin (Tursiops truncatus)

“…Our observations of SDF performed with the five males examined in this study are in accordance with a previous study of frozen‐thawed dolphin spermatozoa using the SCSA technique (Montano et al. ) where the level of DNA fragmentation immediately after thawing was low (6.6 ± 4.1%). When the sperm DNA fragmentation was evaluated in male 1 after incubation at 37°C, there was a higher increase compared to the results in the cited study where sperm DNA fragmentation remained unchanged after 24 h of incubation.…”

“…Sperm DNA fragmentation has recently been assessed in the bottlenose dolphin using the SCSA (Montano et al. ). The SCDt was firstly developed for humans (Chohan et al.…”