Effect of Cryopreservation Methods and Precryopreservation Storage on Bottlenose Dolphin (Tursiops truncatus) Spermatozoa1

Abstract

Research was conducted to develop an effective method for cryopreserving bottlenose dolphin (Tursiops truncatus) semen processed immediately after collection or after 24-h liquid storage. In each of two experiments, four ejaculates were collected from three males. In experiment 1, three cryopreservation methods (CM1, CM2, and CM3), two straw sizes (0.25 and 0.5 ml), and three thawing rates (slow, medium, and fast) were evaluated. Evaluations were conducted at collection, prefreeze, and 0-, 3-, and 6-h postthaw. A sperm motility index (SMI; total motility [TM] x % progressive motility [PPM] x kinetic rating [KR, scale of 0-5]) was calculated and expressed as a percentage MI of the initial ejaculate. For all ejaculates, initial TM and PPM were greater than 85%, and KR was five. At 0-h postthaw, differences in SMI among cryopreservation methods and thaw rates were observed (P < 0.05), but no effect of straw size was observed. In experiment 2, ejaculates were divided into four aliquots for dilution (1:1) and storage at 4 degrees C with a skim milk- glucose or a N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES)-TRIS egg yolk solution and at 21 degrees C with a Hepes-Tyrode balanced salt solution (containing bovine albumin and HEPES) (TALP) medium or no dilution. After 24 h, samples were frozen and thawed (CM3, 0.5-ml straws, fast thawing rate) at 20 x 10(6) spermatozoa ml(-1) (low concentration) or at 100 x 10(6) spermatozoa ml(-1) (standard concentration). The SMI at 0-h postthaw was higher for samples stored at 4 degrees C than for samples stored at 21 degrees C (P < 0.001), and at 6-h postthaw, the SMI was higher for samples frozen at the standard concentration than for samples frozen at the low concentration (P < 0.05). For both experiments, acrosome integrity was similar across treatments. In summary, a semen cryopreservation protocol applied to fresh or liquid-stored semen maintained high levels of initial ejaculate sperm characteristics.

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

“…Simultaneous assessment of plasma membrane and acrosome integrity using the PI/FITC-PNA method, validated herein for use with dolphin spermatozoa, will be useful for future studies on sperm biology in this species. Previous studies have illustrated the robust nature of the bottlenose dolphin acrosome during cryopreservation and sorting processes (Robeck & O'Brien 2004, O'Brien & Robeck 2006. Such findings were confirmed in the present study whereby !5% of spermatozoa, across all treatments, presented a damaged/reacted acrosome.…”

“…Longitudinal studies enabled the prediction of ovulation and characterization of female reproductive cycles through transabdominal ultrasonic evaluation of the ovary (Brook et al 2000) and urinary hormone measurement (Robeck et al 2005). These techniques were combined with sperm cryopreservation (Robeck & O'Brien 2004) and hysteroscopic insemination, which resulted in the first bottlenose dolphin calf being born by way of AI (Robeck et al 2005). Sex-sorted spermatozoa obtained using flow cytometry (Johnson et al 1989) combined with AI led to the first pre-sexed bottlenose dolphin having been born in 2005 at SeaWorld San Diego (O'Brien & Robeck 2006).…”

Heterologous murine and bovine IVF using bottlenose dolphin (Tursiops truncatus) spermatozoa

“…), were analyzed to determine chromatin condensation and stability. Samples were collected, processed, frozen and thawed as previously described [22]. The degree of chromatin condensation was evaluated by assessing propidium iodide (PI) uptake, a fluorescent …”

“…All the experiments were performed in M A N U S C R I P T A C C E P T E D proven fertility (33 years old and 220 kg weight) housed at the Zoo Aquarium, (Madrid, Spain) and trained for voluntary semen collection [21,22]. Immediately after collection, sperm samples were processed and analyzed for standard parameters.…”

“…Immediately after collection, sperm samples were processed and analyzed for standard parameters. Ejaculates with total motility higher than 80%, normal morphology higher than 85% and sperm concentration higher than 400×10 6 spermatozoa/mL were selected and frozen as 120 previously described [22]. Bovine semen was collected by artificial vagina from a reproductive mature proven breeding bull (Asturgen, Gijón, Spain).…”