In vitro sperm characterization and development of a sperm cryopreservation method using directional solidification in the killer whale (Orcinus orca)

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

“…Motility parameters were evaluated using CASA (HamiltonThorne, HTM-IVOS Version 12.2; Holt et al 2007) in a manner similar to that previously described for beluga (Delphinapterus leucas;O'Brien et al 2008) and killer whales (Orcinus orca; Robeck et al 2011). Semen was diluted with Androhep Enduraguard (AE; Minitube of America, Verona, WI, USA) to a concentration of 15-25!10 6 spermatozoa/ml.…”

“…For all other samples, membrane status and acrosome integrity were measured simultaneously using the stains PI (Sigma 287075, Sigma-Aldrich) and FITC-PNA (Sigma L-7381, Sigma-Aldrich; Graham et al 1990, Pena et al 2001. The PI/FITC-PNA staining method used herein was a combination of different dual staining protocols (Graham et al 1990, Szasz et al 2000, Pena et al 2001, as previously described for killer whales (Robeck et al 2011). Briefly, a 12.5-ml aliquot of sperm sample was transferred to a foil-covered microcentrifuge tube and mixed with 1 ml of PI (12.5 mg/ml working stock in PBS).…”

“…The sperm suspension was placed in a container with 200 ml of room temperature water and cooled to 5 8C over a 1.5-2-h period (K0.2 8C/min), and then diluted with Part B extender (2:1, semen:Part B) previously cooled to 5 8C in a stepwise manner over 30 min (three steps of 25, 25 and 50% volume) to obtain a final concentration of 200-300!10 6 spermatozoa/ml and 3% glycerol. One hour after the last addition of Part B, the sperm suspension was transferred to 9-ml hollow glass tubes (at 5 8C, IMT International, Chester, UK) for cryopreservation using a directional solidification machine (MTG-516, IMT, Ness, Ziona, Israel) as described previously for dolphins (O'Brien & Robeck 2006), beluga (O'Brien & Robeck 2010) and killer whales (Robeck et al 2011). Briefly, the hollow tubes moved through the first block of the machine (5 8C) for 45 s at a constant velocity (1 mm/s) before reaching a 2-mm distance inside of the second block (K50 8C), where it was held for 60 s for initiation of ice crystal formation (rapid induction of ice nucleation from the seeding point throughout the length of the glass tube).…”

An Inventory of Peer-reviewed Articles on Killer Whales (Orcinus orca) with a Comparison to Bottlenose Dolphins (Tursiops truncatus)

“…Research conducted on health problems caused by contaminants, reproductive physiology, genetics, and hormone levels has contributed significantly to the care of captive animals and to an increased awareness of the plight of animals in their natural habitat and many of these studies have been conducted with wild and captive populations (killer whales: health: LaMere et al, 2009;Robeck et al, 2015;St. Leger et al, 2009;physiology: Worthy, Worthy, Yochem, & Dold, 2013;reproduction, Asper, Young, & Walsh, 1988;Robeck et al, 2009). Even research on aspects of cognition and sociality of bottlenose dolphins has been conducted in both wild and captive settings and resulted in information that facilitated more species-appropriate forms of enrichment and social groupings for captive animals (Fabienne & Helen, 2012;Waples & Gales, 2002).…”

Genome Banking for Vertebrates Wildlife Conservation

“…Too often direct adaptation is not possible and either adjustments to protocols or complete revision are required, forcing researchers to settle for small animal study population, at times comprised of a single animal (e.g. Robeck et al, 2011), and samples that are hard to come by. As in the case of oocytes, slow freezing and vitrification are currently used for embryo cryopreservation.…”