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

Abstract

Over the last two decades, there have been significant advances in the use of assisted reproductive technology for genetic and reproductive management of captive dolphin populations, including evaluation of sperm DNA quality. This study validated a customized sperm chromatin dispersion test (SCDt) for the bottlenose dolphin (Tursiops truncatus) as a means of assessing sperm DNA damage both in the field and in the laboratory. After performing the SCDt, two different sperm morphotypes were identified: (i) sperm with fragmented DNA showed large haloes of dispersed DNA fragments emerging from a compact sperm nucleoid core and (ii) sperm containing non-fragmented DNA displayed small compact haloes surrounded by a dense core of non-dispersed DNA and protein complex. Estimates of sperm DNA fragmentation by means of SCDt were directly comparable to results obtained following a two-tailed comet assay and showed a significant degree of correlation (r = 0.961; p < 0.001). This investigation also revealed that the SCDt, with minor modifications to the standard protocol, can be successfully conducted in the field using a LED florescence microscopy obtaining a high correlation (r = 0.993; p = 0.01) between the data obtained in the laboratory and in the field.

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

“…Recently, we have conducted a validation experiment on DNA fragmentation of dolphin spermatozoa from multiple ejaculates of frozen–thawed semen based one individual and found a high correlation between the two‐tail comet assay and SCDt (Sánchez‐Calabuig et al. ); this work revealed that the SCDt, with minor modifications to the standard protocol, can be readily performed in the field as in the laboratory.…”

“…Indeed, the SCDt is the only technique to date that has been specifically customized and validated for dolphin spermatozoa in terms of protein lysis for assessing damaged DNA; the SCDt can also be conducted in a standard andrology laboratory or in the field (Sánchez‐Calabuig et al. ). In fact, it is very important to standardize all sperm evaluation techniques, especially for wildlife species, as access to these animals is limited and samples obtained are highly valuable.…”

“…All animals were trained for voluntary semen collection by the Sea World Gold Coast staff as previously described (Keller ; Robeck and O'Brien ; Sánchez‐Calabuig et al. ). Care was taken to avoid any contamination of the ejaculate with sea water.…”

“…The main advantage of SCDt with respect to other SDF tests is the species-specific nature of the protocol (following validation) and its ease of use, especially its field applicability. Recently, we have conducted a validation experiment on DNA fragmentation of dolphin spermatozoa from multiple ejaculates of frozen-thawed semen based one individual and found a high correlation between the two-tail comet assay and SCDt (S anchez-Calabuig et al 2014); this work revealed that the SCDt, with minor modifications to the standard protocol, can be readily performed in the field as in the laboratory.…”

“…Semen collection procedures were approved by the University of Queensland Animal Ethics Committee (SAFS/133/11). All animals were trained for voluntary semen collection by the Sea World Gold Coast staff as previously described (Keller 1986;Robeck and O'Brien 2004;S anchez-Calabuig et al 2014). Care was taken to avoid any contamination of the ejaculate with sea water.…”