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

Abstract

Sperm DNA fragmentation is one of the major causes of infertility; the sperm chromatin dispersion test (SCDt) evaluates this parameter and offers the advantage of species-specific validated protocol and ease of use under field conditions. The main purpose of this study was to evaluate sperm DNA fragmentation dynamics in both fresh and post-thaw bottlenose dolphin sperm using the SCDt following different cryopreservation protocols to gain new information about the post-thaw differential sperm DNA longevity in this species. Fresh and cryopreserved semen samples from five bottlenose dolphins were examined for sperm DNA fragmentation dynamics using the SCDt (Halomax(®)). Sperm DNA fragmentation was assessed immediately at collection and following cryopreservation (T0) and then after 0.5, 1, 4, 8, 24, 48 and 72 h incubation at 37°C. Serially collected ejaculates from four dolphins were frozen using different cryopreservation protocols in a TES-TRIS-fructose buffer (TTF), an egg-yolk-free vegetable lipid LP1 buffer (LP1) and human sperm preservation medium (HSPM). Fresh ejaculated spermatozoa initially showed low levels of DNA fragmentation for up to 48 h. Lower Sperm DNA fragmentation (SDF) was found in the second fresh ejaculate compared to the first when more than one sample was collected on the same day (p < 0.05); this difference was not apparent in any other seminal characteristic. While there was no difference observed in SDF between fresh and frozen-thawed sperm using the different cryopreservation protocols immediately after thawing (T0), frozen-thawed spermatozoa incubated at 37°C showed an increase in the rate of SDF after 24 h. Sperm frozen in the LP1(℗) buffer had higher levels (p < 0.05) of DNA fragmentation after 24- and 48-h incubation than those frozen in TTF or HSPM. No correlation was found between any seminal characteristic and DNA fragmentation in either fresh and/or frozen-thawed samples.

Bottlenose dolphin (Tursiops truncatus) sperm revisited: Motility, morphology and ultrastructure of fresh sperm of consecutive ejaculates

“…The quality of ejaculates from cetaceans is generally very high with high percentage motilities and few abnormal sperm (Beilis et al, 2000;Yuen et al, 2009).. Atlantic bottlenose dolphin ejaculates have been reported to be of high to very high quality with total motilities of often above 80%, high viabilities and few sperm abnormalities (Robeck and O'Brien, 2004;Robeck et al, 2013;Sánchez-Calabuig et al, 2015), which is consistent with the findings in the present study.…”

“…In some mammals a reduction in semen volume and sperm concentration was observed from the first to subsequent ejaculates (Ritar et al, 1992;Ambriz et al, 2002). In contrast, some aspects of sperm quality were greater in the second ejaculate of both Indo-Pacific (Tursiops aduncus) and Atlantic bottlenose dolphins (Yuen et al, 2009;Sánchez-Calabuig et al, 2015). Fresh Atlantic bottlenose dolphin ejaculates are in general of very high quality with excellent sperm motilities, little sperm DNA fragmentation and few abnormal sperm (Fleming et al, 1981;O'Brien and Robeck, 2006;Montano et al, 2012;Sánchez-Calabuig et al, 2015).…”

Comparison of different mathematical models to assess seasonal variations in the longevity of DNA integrity of cooled‐stored stallion sperm

“…In fact, the 5th edition of the WHO laboratory manual for the examination and processing of human semen now includes a specific section for the evaluation of DNA integrity (World Health Organization, ). These findings have been gradually supported and verified by the discoveries made in other mammals (Cortes‐Gutierrez et al, ; Gillan, Evans & Maxwell, ; Love & Kenney, ; Sanchez‐Calabuig et al, ). It has been shown that several reproductive techniques could affect the integrity of the sperm DNA in different animal species, including artificial breeding technologies in the stallion, such as sperm cooling or cryopreservation, prolonged exposure to semen extenders (Love et al, ) and handling conditions of the ejaculate (Gonzalez‐Marin, Gosalvez & Roy, ).…”

Characterization of Bottlenose Dolphin (Tursiops truncatus) Sperm Based on Morphometric Traits

“…Male dolphins provide no parental care and appear to mate promiscuously, maximizing the number of receptive females with whom they mate [ 14 ]. In this respect, male dolphins are capable of producing consecutive ejaculates while maintaining their quality [ 15 , 16 ], which requires the production of large amounts of sperm in short intervals of time. Consequently, the testicular size of this species has been adapted to be relatively large compared to body size.…”