284 IN VITRO FUNCTION OF FROZEN - THAWED BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) SPERM UNDERGOING SORTING AND RECRYOPRESERVATION

Abstract

Artificial insemination (AI) using sex-selected sperm of bottlenose dolphins is currently used for the reproductive and social management of captive populations, but distance of males to the sorting facility represents a limitation of the procedure. Sorting and recryopreservation of previously frozen–thawed (FSF) sperm would facilitate the global application of this technology. Although a calf has been produced using FSF sperm (O’Brien et al. 2009 Theriogenology 71, 98–107), a comprehensive examination of the in vitro quality of such samples is needed. The objective was to compare the in vitro quality of nonsorted (CNTR) and sorted (FSF) dolphin sperm before and after recryopreservation using straw (STR) and directional freezing (DF) methods. At all assessment intervals, sperm were evaluated for 1) motility parameters with computer-assisted sperm analysis (CASA); 2) plasma membrane integrity (viability) and acrosome integrity using propidium iodide/fluorescein isothiocyanate-labeled peanut agglutinin (PI/FITC-PNA) staining and 3) DNA denaturation using the sperm chromatin structure assay (SCSA). Semen from 3 ejaculates × 3 males was cryopreserved by DF. After thawing, samples were divided into CNTR and FSF. The CNTR sperm were recryopreserved using STR and DF methods with assessments performed after the first thaw (PT1) and before recryopreservation (PF2). The FSF sperm were prepared for sorting using a density gradient centrifugation (DGC) method, stained with Hoechst 33342, sorted (SX MoFlo®, Dako, Fort Collins, CO, USA), then recryopreserved using STR and DF methods. The FSF sperm were assessed post-PT1, post-DGC, post-stain, post-sort, and at PF2. After the second thaw (PT2), CNTR and FSF samples were diluted (1:0.1, vol/vol) with Androhep Enduraguard™ (AE; Minitube of America, Verona, WI, USA), incubated at room temperature, and assessed at 0, 6, 12, 18, and 24 h PT2. The PT1 samples retained high proportions of their PF1 total motility (TM) and progressive motility (PM) (mean ± SD; 87.9 ± 7.3% and 92.2 ± 5.9%, respectively). The FSF sperm had improved (ANOVA; P < 0.05) motility (TM, PM, VAP, VCL, VSL) and viability at PF2 compared with PF1. The FSF sperm recryopreserved using DF had higher (P < 0.05) motility over the 24-h post-thaw incubation period compared with STR. The CNTR sperm DNA fragmentation remained unchanged throughout the process. The DNA fragmentation of FSF samples increased after staining (P < 0.05), then decreased during the PT2 incubation period, stabilising at lower values (P < 0.05) than CNTR from 6 to 24 h PT2. This unusual pattern indicates a possible interaction between Hoechst 33342 and acridine orange. After recryopreservation, the viability of FSF sperm was higher (P < 0.05) than that of CNTR sperm. Results indicate that bottlenose dolphin sperm undergoing cryopreservation, sorting, and recryopreservation are of adequate quality for use in AI.