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

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Theriogenology 76 (2011) 267–279 www.theriojournal.com

In vitro sperm characterization and development of a sperm cryopreservation method using directional solidification in the killer whale (Orcinus orca) T.R. Robecka,*, S.A. Gearhartb, K.J. Steinmana, E. Katsumatac, J.D. Loureirod, J.K. O’Briena,e a

Sea World and Busch Gardens Reproductive Research Center, SeaWorld Parks & Entertainment, San Diego, California 92109 b SeaWorld Orlando, Orlando Florida 32821 c Kamogawa Sea World, Kamogawa, Chiba 296-0041, Japan d Mundo Marino S.A., San Clemente del Tuyu, Provincia de Buenos Aires, Argentina e Faculty of Veterinary Science, University of Sydney, NSW 2006, Australia Received 24 August 2010; received in revised form 1 February 2011; accepted 2 February 2011

Abstract Research was conducted to characterize seminal traits and to develop a sperm cryopreservation method using directional freezing (DF) for the killer whale (Orcinus orca). Experiments evaluated effects of: (i) freezing rate (SLOW, MED, FAST) by diluent (BF5F, Biladyl®, EYC) in 0.5 mL straws; and (ii) freezing method (straw or DF) by glycerol (3, 6, or 9% final concentration, v:v) on in vitro sperm quality. Fresh ejaculates (n ⫽ 161) were (mean ⫾ SD) 7.8 ⫾ 7.4 mL at 740 ⫻ 106 sperm/mL with 92.2 ⫾ 6.3% total motility (TM), 85.4 ⫾ 6.9% progressive motility (PM), 89.6 ⫾ 9.0% viability and 89.8 ⫾ 9.2% acrosome integrity. Samples frozen using straws by the MED or SLOW method were improved (P ⬍ 0.05) over FAST across all diluents. At 3 h post thaw (PT), TM, PM, Rapid motility (RM), VAP, VCL, ALH and viability for 3% and 6% glycerol were improved (P ⬍ 0.05) over 9% glycerol. Directional freezing samples at 0 h and 3 h PT, at all glycerol concentrations, displayed higher (P ⬍ 0.001) TM, PM, RM, VAP, VSL, VCL and viability /intact acrosomes (PI/FITC-PNA) than straw. These data provided the first information on ejaculate characteristics and the development of a semen cryopreservation method using DF in the killer whale. © 2011 Elsevier Inc. All rights reserved. Keywords: Delphinidae; Cetacean; Cryopreservation; Directional freezing; Genome resource bank

1. Introduction Killer whales, Orcinus orca, the physically largest member of the family Delphinidae, are the most widely distributed cetacean which can be found throughout the majority of oceans in the world [1]. Despite their wide-

* Corresponding author. Tel.: ⫹1 210 523 3294; fax: ⫹1 210 523 3299. E-mail address: [email protected] (T.R. Robeck). 0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.02.003

spread penetration of the world’s habitats, only one species of killer whale are recognized and their population status is unknown [2]. However, a potential subspecies population [2-4], the Southern Resident (SR) population, located in the waters off the state of Washington in the USA and British Columbia in Canada has recently been listed as endangered by the US Endangered Species Act [5]. The anthropogenic threats to this population, both direct (habitat competition) and indirect (environmental contaminants and climate change) are similar pressures faced by all killer whale groups worldwide [3-5].

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Due to their top position in the trophic food chain, killer whales are especially at risk from bioaccumulation of environmental contaminants [6,7]. The SR population and other killer whale groups evaluated from this region of the world are reported to contain levels of contaminants which in other marine mammals are believed to have resulted in adverse effects including reproductive suppression [8,9]. In addition, persistent organochlorine contaminants, to which killer whales are continually exposed [7], have been demonstrated in other mammalian species to have adverse effects on in vitro sperm motility [10], sperm production [11] and sperm chromatin integrity [12]. All of these effects can adversely impact fertility [10 –14]. Despite the well documented exposure of marine mammals to environmental contaminants, little information has been documented on baseline or normal reproductive physiological parameters of this species. Recent attempts at rectifying this situation have lead to information describing seasonal changes in semen production, testosterone concentrations and reproductive maturity of male killer whales [15]. However, no information was provided concerning normal ejaculate parameters including motility, concentration and morphology. In addition, an objective methodology for motility analysis, namely computer assisted motility analysis (CASA), which has been recently used in beluga (Delphinapterus leucas) [16,17] and bottlenose dolphins (Tursiops truncatus) [18], is yet to be applied to the killer whale. It is noteworthy that CASA parameters have the most promise for creating a normal database of motility characteristics, which in combination with other in vitro tests, could be used to predict fertility [19]. A simple methodology for cryopreservation of killer whale sperm has been described [20]; however, critical evaluation of extenders and freezing methods were not performed. Recent research with beluga [17] and bottlenose dolphins [18,21] demonstrated that new techniques for semen cryopreservation using directional solidification technology can have a dramatic effect on post-thaw sperm parameters, including the recovery rate of motile sperm and motility longevity. Application of this technology to optimize the post-thaw recovery of sperm may enable development of genome/ gamete resource banks for the killer whale. If populations of wild killer whales become threatened, gamete resource banks can then serve as a critical component of any long-term species survival plan [22]. In addition, ex situ population management plans that utilize AI to maintain genetic diversity can incorporate sex pre-selection

technology, using sperm sorting, to simultaneously manage the population sex ratio [23]. Sex pre-selection using sperm sorting for population sex ratio and genetic management is currently being applied on a routine basis to ex situ bottlenose dolphin populations [21–23]. However, prior to the successful application of this assisted reproductive technology (ART) to killer whales, adequate semen handling and cryopreservation techniques need to be developed. The goal of this research was to describe normal killer whale ejaculate characteristics and develop a method for long-term sperm storage that could then be used to develop a gamete resource bank for ex situ population management. The specific objectives of this research were to: (1) describe normal ejaculate characteristics including sperm motility, sperm kinematics and morphology; (2) evaluate the effects of cryodiluent and freezing rate using 0.5 mL straws on cryopreserved sperm; and (3) evaluate the effects of cryoprotectant concentrations on cryopreserved sperm using straws or directional solidification. 2. Materials and methods 2.1. Reagents and media All chemicals were of analytical grade. Disposable plastic ware was manufactured by BD Biosciences (BD, Bedford, MA, USA). Unless otherwise stated, all media components were purchased from Sigma-Aldrich (Sigma, St. Louis, MO, USA) and were prepared with tissue-grade water (Sigma or Millipore, Billerica, MA, USA). All freezing diluents containing egg yolk (free range eggs) were clarified by centrifugation for 1.5 h at 10,000 ⫻ g at 10 °C. The supernatant was filtered (0.22 ␮m; Millipore) and frozen at ⫺80 °C for a maximum of 18 mo. 2.2. Animals and experiments Semen samples (n ⫽ 173) were collected from five male killer whales from 1998 to 2010 (Table 1). Males 1 to 3 were housed at three separate SeaWorld habitats in the USA [Male 1: SeaWorld Orlando (SWO); Male 2: SeaWorld San Diego (SWSD); Male 3: SeaWorld San Antonio (SWSA)] that contained a minimum of 19,000 m3 of manufactured salt water. Male 4: Kamogawa SeaWorld (KSW; Kamogawa, Japan) and Male 5: Mundo Marino (MUN; San Clemente, Argentina) were housed in natural salt water habitats that ranged in size from 4,800 m3 to 6,000 m3. All habitats were outdoors with water temperatures either con-

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Table 1 Description of animals used and samples collected during this study. Male

Facilitya

Birth date

Weight (kg)

Length (cm)

Date range of ejaculates collected

No. samples

Calves sired

1 2 3 4 5

SWO SWSD SWSA KSW MUN

1982b 1977b 1993c 1985b 1989d

5,325 4,225 3,088 3,150 3,500

670 602 570 575 620

1998–2008 2002–2010 2010 2009 2010

n ⫽ 63 n ⫽ 72 n⫽6 n⫽8 n ⫽ 24

13 1 1 1 1

a

b c d

SWO, SeaWorld Orlando; SWSD, SeaWorld San Diego; SWSA, SeaWorld San Antonio; KSW, Kamogawa Sea World; MUN, Mundo Marino. Wild-born male. Age was estimated from total body length at capture. Captive born. Wild-born male. Age was estimated from total body length at stranding.

trolled from 11.1 to 12.8 °C (SWO, SWSA, SWSD), or ambient from 11 to 20 °C (KSW, MUN). Animals were fed a diet of frozen-thawed whole fish which contained some or all of the following fish: Herring, Clupea harengus; Columbia River smelt, Thaleichthys pacificus; Atka mackerel, Pleurogrammus azonus; and pink salmon, (Oncorhynchus gorbuscha), or for Male 5 Corvina rubia (Micropogonia furnieri), Saraca (Brevoortia aurea), and Pescadilla real (Macrodon ancylodon), at approximately 2 to 3% of their body weight per day. Males 1 to 4 were housed in mixed male and female groups of various age classes. Male 5, a stranded male, was housed with a female bottlenose dolphin (Tursiops truncatus). 2.3. Semen collection The animals were trained for voluntary semen collection, as previously described [24]. Briefly, the animals were trained to present their penis while in dorsal recumbency adjacent to the edge of the pool. Prior to training, and if available, the male was sexually stimulated by a receptive animal. Once the male appeared sexually stimulated (as assessed by display of courtship behavior and partial extrusion of penis from the genital groove), operant conditioning using a positive reinforcement schedule was commenced. During training sessions, ejaculation occurred at variable intervals before, during or after presentation of the penis. Semen was collected into a sterile 125 mL cylindrical plastic collection container (Nalgene®, Nalge Nunc Intl., Rochester, NY, USA) or a sterile 2.96 L WHIRL-PAK bag (Nasco, Fort Atkinson, WI, USA). During a training session, collections were performed 1 to 4 times in succession until an ejaculate or combined ejaculates contained adequate numbers of viable sperm required for evaluation, and experimentation.

2.4. Evaluation of ejaculate and in vitro sperm characteristics Ejaculates were held at room temperature (24 °C) and processed within 30 min after collection. Ejaculate concentration, volume, pH (indicator strips; EM Science, Gibbstown, NJ), osmolality (Advanced Instruments Inc., Norwood, MA), sperm motility parameters, viability (plasma membrane integrity), acrosomal status and morphology were determined. Only ejaculates free of contamination with saltwater or urine (osmolality ⱕ 375 mOsm/kg) were considered normal and were used for experiments. Access to computer assisted motility analyzer (CASA, HTM-IVOS Version 12.2, Hamilton Thorne, Beverly, MA, USA) became available from 2007 onwards at one of the research locations (SWSD) where Male 2 was housed, and sperm kinematics were thereby determined on fresh samples from this male, and on frozen-thawed samples from Males 2 and 4 as described in subsequent sections. All subjective fresh sperm motility evaluations were made under bright-field optics (Olympus, Tokyo, Japan). The percentage of motile sperm was estimated to the nearest 5% by analyzing 4 to 5 fields of diluted sperm sample (1:25, sperm: Egg-yolk citrate (Modified EYC [25]: 2.9% Na citrate [dehydrate], 20% egg yolk (v:v), 50 ␮g/mL gentamycin sulfate), warmed to 35 °C and placed on heated slides (35 °C, ⫻ 400 magnification). Total motility (TM), total progressive motility (PM) and kinetic rating (KR, 0 to 5 scale, 0 ⫽ no movement, 5 ⫽ rapid forward progressive movement) were subjectively determined. For data analysis and sample comparisons, these values were then transformed into a sperm motility index (SMI, modified from previous [26]): SMI ⫽ PM ⫻ KR

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Sperm kinematics as determined by CASA were performed as previously described for beluga [17]. Briefly, semen was diluted with a modified RAM diluent ([27] 3.6% Tris, 0.1% Fructose, 2.0% citric acid (v:v), 0.06 mg/mL penicillin G; 0.05 mg/mL streptomycin) to a concentration of 15 to 25 ⫻ 106 sperm/mL and 8 ␮L was transferred to a glass slide and overlaid with a 22 ⫻ 22 mm glass coverslip for analysis at 35 °C. For assessment of sperm using CASA, the RAM diluent was used for sample dilution because it was found to clarify egg yolk based media without reducing sperm motility or causing hyper-activated motility [Robeck TR, O’Brien JK, unpublished]. After 2 to 3 min, a total of 5 to 10 microscopic fields representing a minimum of 400 sperm were randomly selected and examined per sample for calculation of the following motility parameters: total motility (TM, %); progressive motility (PM, %); average pathway velocity (VAP, ␮m/s); straight line velocity (VSL, ␮m/s); curvilinear velocity (VCL, ␮m/s); rapid motility (RAP, VAP ⬎ 25 ␮m/s, %); straightness of sperm movement: VSL/VAP (STR, %); linearity (LIN, %); beat cross frequency (BCF, Hz); and amplitude of lateral head displacement (ALH, ␮m). The instrument settings were 30 frames at a frame rate of 60 frames/s, minimum contrast was 60, minimum cell size (pixels) was 15; for progressive cells the VAP was ⬎ 20 ␮m/s and STR was ⬎ 70%. Sperm with a VAP ⬎ 5 ␮m/s were considered motile. A minimum of 100 sperm/sample (X 1,000 magnification) were used to evaluate sperm plasma membrane integrity (viability), percent intact acrosome and morphology. Viability was determined using a live dead exclusion stain (eosin-nigrosin [E/N], IMV International Corp., Maple Grove, MN, USA). For acrosome and morphology analysis, a smear of sperm samples was fixed with formal saline, stained with Spermac® Stain (Minitube of America, Verona, WI, USA) and assessed as previously described [17,28]. 2.5. Study 1: Effect of cryodiluent and freezing rate using 0.5 mL straws on in vitro sperm characteristics The effect of three diluents and three freezing rates on in vitro sperm characteristics (TM, PM, SMI, viability, and acrosomal status) was examined (3 ⫻ 3 factorial). Diluents were as follows: (i) Biladyl® (BLD; Minitube of America, Verona, WI , USA; Fraction A: 2.42% Tris, 1.38% citric acid, 1% fructose, 20% egg yolk (v:v), 0.5 mg/mL tylosin, 2.5 mg/mL gentamycin, 1.5 mg/mL lincomycin, and 3 mg/mL spectinomycin), (ii) modified BF5F [12] (1.2% Tes, 0.2% Tris, 1.6% Glucose, 1.6% Fructose, 20 % egg yolk (v:v), antibi-

otics as for EYC), and (iii) EYC. Biladyl® was evaluated for its ease of use (available as a commercial bovine diluent) and because preliminary cryopreservation trials with this media demonstrated that sperm frozen with this media were fertile [21]. The BF5F, originally developed for the boar as BF5 [29], was chosen because of its widespread use in domestic and exotic species [26], including the beluga [16,17]. And finally EYC, one of the original bovine extenders [30], was chosen because of its simplicity and its positive results with Pacific white-sided dolphin (Lagenorhynchus obliquidens) sperm [31]. Six ejaculates (Males 1, 3, and 5, two ejaculates/ male), held at 24 °C, were divided into three fractions (v:v), and slowly diluted 1:1 (v:v) with BF5F, BLD and EYC (at 24 °C). After cooling to 5 °C at ⫺0.12 °C/min, samples were slowly diluted 1:1 (v:v) over 30 min with their respective diluent glycerolated to 12% at 5 °C and equilibrated for 1 h. A preliminary study of glycerol toxicity indicated that 6% final glycerol was an effective concentration when combined with Biladyl® [20]. Sperm suspensions were then loaded into 12 straws (0.5 mL) per diluent (n ⫽ 4 straws/treatment) and frozen by one of three freezing protocols: 1) SLOW: straws were held at 13.5 cm above the level of the liquid nitrogen in vapor for 10 min, then plunged into liquid nitrogen; 2) MEDIUM: straws were held at 13.5 cm above the level of the liquid nitrogen in the vapor for 5 min, dropped to 4.5 cm above the liquid nitrogen for 5 min, then plunged into the liquid nitrogen; 3) FAST: straws were held at 4.5 cm above the liquid for 10 min, then plunged into the liquid nitrogen. Cooling rates (initiation to just prior to plunging) during freezing were determined using a thermocouple probe secured inside the middle of an unsealed straw containing diluent (n ⫽ 3 per freeze method). Temperature readings on the thermocouple were recorded every 5 s over the course of each treatment. For thawing, straws (n ⫽ 2 per extender/ freezing rate combination) were placed in a water bath at 35 °C and shaken vigorously for 30 s. After thawing, samples were diluted 1:1 (v:v) with pre-warmed EYC (35 °C) and evaluated at 0 h (TM, PM, SMI, viability) and at 2.5 h (TM, PM SMI) post-thaw (PT). 2.6. Study 2. The effect of glycerol concentration and freezing method (conventional straw freezing and directional freezing technology) on in vitro sperm characteristics The effect of glycerol concentration (3, 6, and 9% v:v final concentration) and freezing method: straw (STW) and directional freezing (DF), on in vitro sperm

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characteristics were examined (3 ⫻ 2 factorial). Six ejaculates (Males 2 and 5; three ejaculates/male) were held at room temperature (22–24 °C), and evaluated subjectively for TM, PM, viability and acrosome status. The samples were then slowly diluted 1:1 (v:v) with BF5F and cooled to 5 °C at ⬇ ⫺0.12 °C/min. The samples were then evenly divided into three fractions and slowly diluted 1:1 over 30 min with BF5F glycerolated at three concentrations (18%, 12% and 6% glycerol, v:v, 5 °C) and equilibrated for 1 h. Sperm suspensions were then loaded into four straws (0.5 mL) per treatment (12 total straws/ejaculate) and frozen using the SLOW method outlined in Study 2 or in two hollow tubes (2 mL tubes; 6 tubes/ejaculate; IMT International, Chester, UK) for cryopreservation using a directional solidification machine (MTG-516; IMT International [32]). The hollow tube was moved through the first block (5 °C) for 45 s at a constant velocity (1 mm/s) before reaching a distance of 2 mm into the opening of a second block (⫺50 °C) and held for 30 s for initiation of seeding (rapid induction of ice nucleation from the seeding point throughout the length of the glass tube). The tube was then moved at the same velocity across the second block for 3 min before entering the collection chamber (⫺100 to ⫺110 °C) followed by transfer to liquid nitrogen. Straws (n ⫽ 12 straws per ejaculate) were thawed as described in Study 2. While four straws per replicate were thawed, two straws were combined per treatment in a 1.5 mL cryovials to allow enough semen for analysis. Sperm samples in the six cryovials (two per treatment) were then diluted 1:1 (v:v) with RAM media (at 24 °C) for analysis. Hollow tubes (n ⫽ 6 tubes per ejaculate) were thawed in air for 45 s (⫺195.0 to ⫺100 °C at 126.6 ⫾ 10 °C/min) then transferred to a 35 °C hollow tube specific water bath designed to enable uniform sample thawing over 45 s (⫺100 to 26.0 °C at 171.1 ⫾ 7.5 °C/min [21,22]) by propelling 35 °C water through the middle, and around the outside of the hollow tube simultaneously (Harmony CryoCare Activator™, IMT International Ltd, Chester, UK). Sperm suspensions from hollow tubes (n ⫽ 2 per treatment) was transferred to individual cryotubes (n ⫽ 6) and diluted as described for the STW method. Raw and pre-freeze (PF) subjective TM, PM, KR and viability (E/N) were determined. At 0 and 3 h PT, sperm kinematics were objectively assessed using CASA to determine TM, PM, RAP, VAP, VSL, VCL, STR, LIN, BCF, and ALH. Sperm viability and acrosome integrity were evaluated (0 and 3 h PT) using the stains propidium iodide (PI) fluorescein isothiocynate-

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conjugated Arachis hypogaea (peanut) agglutinin (FITC-PNA). The PI/FITC-PNA staining method used herein was a combination of different dual staining protocols [33–35]. Briefly, a 12.5 ␮L aliquot of sperm sample was transferred to a foil covered microcentrifuge tube and mixed with 1 ␮L of PI (12.5 mg/mL working stock in PBS). After 30 s, 1 ␮L of FITC-PNA (1 mg/mL working stock in PBS) was added to the solution and incubated for 1 min. Sperm were then immobilized and fixed by the addition of 1 ␮L of 2% glutaraldehyde solution (in PBS, pH 7.0 –7.4) and 12 ␮L was placed on a glass slide and covered with a 22 ⫻ 22 mm glass coverslip for evaluation within 5 min. Sperm were observed using a fluorescence microscope (BX51, Olympus, Tokyo, Japan) equipped with a 450– 490 nm band pass excitation filter and a 535 nm emission filter. Evaluations were conducted at magnification ⫻ 400 under a low bright field setting to permit visualization of non-fluorescent sperm. A total of 100 cells were classified per sample using the following staining patterns: no stain (viable cells with an intact acrosome), green staining in the acrosome region including the equatorial segment (viable cells with a damaged or reacted acrosome), red staining (non-viable cells with an intact acrosome), red and green staining (non-viable cells with a damaged or reacted acrosome). Acrosome integrity results are presented as: Viab/Intact (viable sperm with an intact acrosome) or Total acrosome intact (viable and non-viable sperm with an intact acrosome). To validate the binding of FITC-PNA with the inner acrosomal membrane of killer whale sperm, a sample cryopreserved in a 0.5 ml straw was thawed (n ⫽ 2 straws) as previously described. The PT sample was immediately assessed with the PI/FITC-PNA dual stain. Next, the sample (800 uL) was diluted with RAM medium (1:10; sperm sample:medium, v:v), centrifuged for 20 min at 800 g and the pellet was resuspended to 1 mL in RAM medium. This suspension was sealed in a 1.8 mL cryovial, plunged into liquid nitrogen and thawed at room temperature to promote acrosome loss and/or damage. The plasma and acrosome membrane status of the second PT sample was then evaluated using PI/FITC-PNA staining and results were compared to the original PT sample. 2.7. Statistical analysis All statistical analyses were performed using SigmaStat (Version 3.5; SSPS, Inc., San Rafael, CA, USA). Data within each factorial experiment, sperm motility characteristics, viability and acrosomal status, were analyzed

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using ANOVA. All pair-wise multiple-comparison procedures between means were conducted using the HolmSidak. The relationship between eosin-nigrosin and PI viability methods was examined using the Pearson Product Moment Correlation Test. A level of P ⬍ 0.05 was considered to be significant. Unless noted, data are presented as the mean ⫾ SD. 3. Results 3.1. Ejaculate and sperm characteristics For the ejaculate characterization and semen banking, 134 ejaculates were collected from three Males (Male 1–2, 5) from April 1998 to June 2010 (Table 2). Semen quality based on in vitro sperm characteristics was high, with sperm motility, viability, acrosome integrity and normal morphology all greater than 85% (Table 2). The predominant morphological abnormality was a bent flagellum (3.0 ⫾ 5.7 %) followed by degenerated head (1.5 ⫾ 1.6%: Table 2). For Male 2, 27 ejaculates were analyzed by CASA to describe fresh sperm kinematics (Table 2). 3.2. Study 1: Effect of cryodiluent and freezing rate using 0.5 mL straws on in vitro sperm characteristics Overall cooling rates for each freezing method were ⫺10.7 °C/min for SLOW, ⫺14.62 °C/min for MEDIUM and 15.2 °C/min for FAST (Fig. 1). The effect of cryodiluent and freezing rate on semen cryopreserved using 0.5 mL straws is presented in Table 3. No differences (P ⬎ 0.05) in TM, PM or SMI were observed between raw ejaculates from each male used for this experiment. This trend continued after cooling and equilibration of the samples (PF), with no differences (P ⬎ 0.05) among cryodiluent treatments for TM, PM, SMI, and LD. In addition, no effect (P ⬎ 0.05) of male was observed for any in vitro parameters at PF. At 0 h PT, no differences (P ⬎ 0.05) in sperm characteristics were observed among freezing rate methods. At 0 h PT, differences (P ⬍ 0.001) in TM, PM, and SMI were observed between all diluents with BF5F providing the best PT TM (BF5F: 50.4 ⫾ 10.8%; BLD: 41.0 ⫾ 9.1%; EYC: 21.7 ⫾ 12.9%), PM (BF5F: 44.5 ⫾ 12.1%; BLD: 36.6 ⫾ 9.6%; EYC: 19.1 ⫾ 11.1%) and SMI (BF5F: 176.3 ⫾ 59.7; BLD: 143.3 ⫾ 45.0; EYC: 73.3 ⫾ 45.2; data pooled across freezing rate treatment groups). The diluent/freezing rate combinations that resulted in the two highest 0 h PT TM and SMI were BF5F/MEDIUM (TM: 50.1 ⫾ 11.8%; SMI: 181.8 ⫾ 77.9), and BF5F/

Table 2 Characteristics of killer whale ejaculates Parameter

Combined malesa (n ⫽ 134)

Semen characteristics Ejaculate volume (mL) 7.8 ⫾ 7.4 73.9 ⫾ 50.8 Sperm concentration (⫻ 107/mL) Total sperm per ejaculate (⫻ 108) 58.0 ⫾ 81.7 Osmolality (mOsm/kg) 359.1 ⫾ 10.2 pH 7.4 ⫾ 0.3 Sperm characteristics Total motility 92.2 ⫾ 6.3 Progressive motility 85.4 ⫾ 6.9 4.7 ⫾ 0.5 Kinetic ratingb Sperm motility indexc 401.7 ⫾ 61.7 — VAP (␮m/s) VSL (␮m/s) — VCL (␮m/s) — ALH (␮m/s) — BCF (Hz) — STR (%) — LIN (%) — Viability (%) 89.6 ⫾ 9.0 Acrosome intact (%) 89.8 ⫾ 9.2 Morphologically normal (%) 90.4 ⫾ 6.8 Morphologically abnormal (%) Head Degenerate 1.5 ⫾ 1.6 Missing 0.9 ⫾ 1.1 Microcephalic 0.8 ⫾ 1.3 Macrocephalic 0.4 ⫾ 0.9 Bicephalic 0.4 ⫾ 0.7 Midpiece Cytoplasmic Droplet 0.5 ⫾ 0.7 Degenerate 0.4 ⫾ 0.8 Flagellum Bent 3.0 ⫾ 5.7 Coiled 0.3 ⫾ 0.7 Distal Drop 0.5 ⫾ 0.9 Double 0.6 ⫾ 0.8 Missing 0.5 ⫾ 0.9

Male 2 (n ⫽ 27)

10.6 ⫾ 6.7 51.9 ⫾ 36.6 57.1 ⫾ 61.9 358.4 ⫾ 36.6 7.3 ⫾ 0.2 97.5 ⫾ 3.3 85.9 ⫾ 5.3 4.8 ⫾ 0.5 415.7 ⫾ 52.8 259.1 ⫾ 53.9 239.7 ⫾ 53.7 316.2 ⫾ 59.9 9.7 ⫾ 2.6 33.1 ⫾ 6.2 90.9 ⫾ 3.7 75.5 ⫾ 7.2 90.7 ⫾ 5.5 86.6 ⫾ 8.9 85.3 ⫾ 1.6 2.0 ⫾ 1.6 1.7 ⫾ 1.3 1.7 ⫾ 1.7 0.0 ⫾ 0.0 0.8 ⫾ 0.9 0.5 ⫾ 0.5 0.3 ⫾ 0.5 4.4 ⫾ 6.6 0.3 ⫾ 0.7 0.8 ⫾ 1.4 1.0 ⫾ 1.2 0.1 ⫾ 0.4

Values are the mean ⫾ SD. a Combined samples from Male 1 (n ⫽ 65), Male 2 (n ⫽ 45), Male 3 (n ⫽ 24). b Kinetic rating was graded subjectively: 0 ⫽ no movement; 5 ⫽ rapid forward progression. c Sperm motility index ⫽ progressive motility ⫻ kinetic rating. VAP, average pathway velocity; VSL, straight line velocity; VCL, curvilinear velocity; ALH, amplitude of lateral head displacement; BCF, beat cross frequency; STR, straightness of sperm movement (STR (%) ⫽ VSL/VAP); LIN, linearity

SLOW (TM: 54.1 ⫾ 7.9%; SMI: 185.2 ⫾ 41.2). At 0 h PT, there were no differences (P ⬎ 0.05) in viability among diluents or freezing rate methods. The treatment combinations that resulted in the two highest 0 h PT viability were BF5F/FAST (56.5 ⫾ 16.8%) and BF5F/ SLOW (56.3 ⫾ 10.8%; Table 3).

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Fig. 1. Comparison of cooling rates (mean ⫾ SD) of 0.5 mL straws held in liquid nitrogen vapor for 10 min at three different levels above the liquid prior to plunging; SLOW: 13.5 cm, MEDIUM: 13.5 cm for 5 min, then 4.5 cm for 5 min, FAST: 4.5 cm for 10 min. The magnified area of the graph (inset) provides increased clarity of the early phase of the freezing curves.

Similar to 0 h PT, differences (P ⬍ 0.001) in TM, PM and SMI at 2.5 h PT were observed among diluents across freezing methods with BF5F once again having the highest PT TM (BF5F: 48.3 ⫾ 12.2%; BLD: 34.4 ⫾ 14.9%; EYC: 22.8 ⫾ 13.4%), PM (BF5F: 41.7 ⫾ 11.6%; BLD: 29.8 ⫾ 13.7%; EYC: 19.3 ⫾ 11.1%) and SMI ( BF5F: 156.2 ⫾ 48.4; BLD: 106.8 ⫾ 54.4; EYC: 71.0 ⫾ 42.1). However, in contrast to 0 h PT, differences (P ⬍ 0.01) in TM, PM, and SMI were observed among freezing rates across all extenders at 2.5 h PT with parameters increased for MEDIUM (TM: 39.9 ⫾ 15.1; PM: 34.8 ⫾ 13.7; SMI: 130.4 ⫾ 55.1) and SLOW (TM: 38.6 ⫾ 17.5; PM: 33.1 ⫾ 15.9; SMI: 124.1 ⫾ 62.5) increased (P ⬍ 0.01) compared to FAST (TM: 31 ⫾ 16.9; PM: 26.2 ⫾ 14.7; SMI: 91.4 ⫾ 55.5). The two diluent/freezing rate combinations with the highest motility parameters at 2.5 h PT were BF5F/SLOW (TM: 53.8 ⫾ 7.5%; PM: 46.2 ⫾ 10.2%; SMI: 175.4 ⫾ 48.6) and BF5F/MEDIUM (TM: 51.4 ⫾ 7.4%; PM: 41.7 ⫾ 11.6%; SMI: 156.2 ⫾ 48.4). No differences (P ⬎ 0.05) were observed in viability among diluents or freezing rates at 2.5 h PT (Table 3). 3.3. Study 2. The effect of glycerol concentration and freezing method (conventional straw freezing and directional freezing technology) on in vitro sperm characteristics The percentages of viable sperm for each replicate as determined by light (eosin-nigrosin) and fluorescence (PI) microscopy were correlated (n ⫽ 140; r2 ⫽ 0.6; P ⬍ 0.01). As such, only PT viability data obtained from the PI/FITC-PNA staining method were used for data analysis

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(Table 4). The PI/FITC-PNA acrosome specificity validation trials demonstrated that after staining of sperm from the frozen thawed straw, 57% were plasma membrane intact and 83% of cells were acrosome intact. Once this sample was plunged into liquid nitrogen and thawed, 3% of sperm were plasma membrane intact, and 25% of cells were acrosome intact. The FITC-PNA membrane staining patterns were consistent with the inner acrosome membrane morphology as previously described (Fig. 2 [36]). The effects of freeze-thawing using varying concentrations of glycerol (3, 6, and 9%, v:v) within two different freezing methods (STW and DF) on in vitro sperm characteristics are presented in Table 4. Sample cooling, the addition of cryoprotectant and equilibration (PF), did not affect (P ⬎ 0.05) sperm characteristics; however, TM, PM, KR, and viability all had decreasing trends with increasing glycerol concentration. Across all glycerol concentrations and times PT, DF resulted in significantly (P ⬍ 0.001) higher TM (DF: 67.7 ⫾ 15.5%; STW: 58.8 ⫾ 22.7%), PM (DF: 49.3 ⫾ 15.1%; STW: 38.5 ⫾ 17.5%), RAP (DF: 67.1 ⫾ 15.8%; STW: 58.1 ⫾ 22.8%) VAP (DF: 148.5 ⫾ 36.6 ␮m/s; STW: 114.1 ⫾ 30.8 ␮m/s), VSL (DF: 134.0 ⫾ 38.6 ␮m/s; STW: 98.7 ⫾ 31.2 ␮m/s), VCL (DF: 205.8 ⫾ 34.2 ␮m/s; STW: 179.3 ⫾ 34.1 ␮m/s) and Viab/Intact (DF: 55.2 ⫾ 9.1%; STW: 45.5 ⫾ 11.6%) when compared to STW. No interactions (P ⬎ 0.05) between glycerol concentration and freeze method were observed for any sperm characteristics. At 0 h and 3 h PT, with the exception of VSL, STR, LIN, BCF and acrosome intact, all parameters within each freezing method were affected by the concentration of glycerol (Table 4; P ⬍ 0.05). Within each freezing method at 0 h PT, VAP, VCL and RAP at 6% glycerol were increased (P ⬍ 0.05) compared with 3% and 9% glycerol. The 0 h PT TM, PM, RAP, and the 3 h PT TM, PM, RV, VAP, VCL, and ALH were similar for 3 and 6%, and better (P ⬍ 0.05) than 9% glycerol. At 0 h after thawing with 3 and 6% glycerol, the DF method was superior to the straw method for maintaining velocity parameters VAP, VSL and VCL and at 3 h after thawing VAP and VSL (Table 4; P ⬍ 0.05). In addition, at 0 and 3 h PT, using 6% glycerol, PM, LIN and Viab/Intact were greater for the DF method (Table 4; P ⬍ 0.05). For 9% glycerol at 0 and 3 h PT, all sperm characteristics were increased for the DF compared to the STW method except for ALH and Viab/Intact at 0 h PT, and RAP, ALH, STR, and LIN at 3 h PT. Overall, the treatment

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Table 3 Effects of diluent and freezing rate on in vitro characteristics of killer whale sperm cryopreserved with straws (n ⫽ 6 ejaculates; n ⫽ 3 males (two ejaculates/male). Sperm characteristics Prefreeze (n ⫽ 6) Total Motility (%) Progressive motility (%) Kinetic rating SMI and Viability (%) 0 h post-thaw (n ⫽ 12) Fast freezing rate Total motility (%) Progressive motility (%) Kinetic rating SMI Viability (%) Medium freezing rate Total motility (%) Progressive motility (%) Kinetic rating SMI Viability (%) Slow freezing rate Total motility (%) Progressive motility (%) Kinetic rating SMI Viability (%) 2.5 h post-thaw (n ⫽ 12) Fast freezing rate Total motility (%) Progressive motility (%) Kinetic rating SMI Viability (%) Medium freezing rate Total motility (%) Progressive motility (%) Kinetic rating SMI Viability (%) Slow freezing rate Total motility (%) Progressive motility (%) Kinetic rating SMI Viability (%)

BF5F

Diluent Biladyl®

EYC

91.3 ⫾ 3.3 86.5 ⫾ 1.3 4.3 ⫾ 0.3 367.8 ⫾ 24.9 91.7 ⫾ 5.8

92.0 ⫾ 3.2 87.2 ⫾ 3.1 4.3 ⫾ 0.4 377.5 ⫾ 32.9 91.3 ⫾ 4.0

91.0 ⫾ 4.0 86.4 ⫾ 2.5 4.3 ⫾ 0.5 374 ⫾ 50.9 90.4 ⫾ 1.7

47.2 ⫾ 12.0a 44.3 ⫾ 14.5a 3.8 ⫾ 0.5 161.9 ⫾ 56.7a 56.5 ⫾ 16.8

37.0 ⫾ 6.8b 32.7 ⫾ 6.9b 3.75 ⫾ 0.3 122.9 ⫾ 28.3a 55.1 ⫾ 16.0

20.2 ⫾14.8c 17.7 ⫾ 12.4c 3.8 ⫾ 0.4 66.5 ⫾ 47.5b 49.9 ⫾ 4.5

50.1 ⫾ 11.8a 44.3 ⫾ 14.5a 4.0 ⫾ 0.5 181.8 ⫾ 77.9a 53.1 ⫾ 15.7

44.5 ⫾ 9.3a 39.7 ⫾ 0.3a 4.0 ⫾ 0.3 158.0 ⫾ 48.1a 50.9 ⫾ 14.0

24.2 ⫾ 13.4a 21.2 ⫾ 12.3b 3.7 ⫾ 0.7 82.7 ⫾ 51.3b 47.9 ⫾ 12.7

54.1 ⫾ 7.9a 46.6 ⫾ 8.1a 4.0 ⫾ 0.3 185.2 ⫾ 41.2a 56.4 ⫾ 10.8

41.5 ⫾ 9.9b 37.5 ⫾ 10.4b 3.9 ⫾ 0.5 148.9 ⫾ 50.9a 52.5 ⫾ 15.9

20.8 ⫾ 11.1c 18.3 ⫾ 9.0c 3.8 ⫾ 0.4 70.7 ⫾ 38.0b 49.6 ⫾ 7.0

46.2 ⫾ 8.4a 39.8 ⫾ 8.8a 3.6 ⫾ 0.3 144.7 ⫾ 35.0a 55.4 ⫾ 14.9

25.9 ⫾ 15.3b,1 21.8 ⫾ 13.3b,1 3.1 ⫾ 0.5 72.3 ⫾ 49.7b,1 50.8 ⫾ 15.1

20.9 ⫾ 14.7b 17.0 ⫾ 10.9b 3.4 ⫾ 0.4 57.2 ⫾ 36.7b 53.3 ⫾ 5.8

51.4 ⫾ 7.4a 44.3 ⫾ 8.7a 3.8 ⫾ 0.3 167.1 ⫾ 40.2a 49 ⫾ 16.8

42.0 ⫾ 10.4a,2 37.5 ⫾ 10.4a,2 3.6 ⫾ 0.3 137.6 ⫾ 46.8a,2 52.8 ⫾ 13.6

26.2 ⫾ 14.5b 22.8 ⫾ 12.2b 3.5 ⫾ 1.1 86.5 ⫾ 47.1b 49.1 ⫾ 7.2

53.8 ⫾ 7.5a 46.2 ⫾ 10.2a 3.8 ⫾ 0.3 175.4 ⫾ 48.6a 53.0 ⫾ 12.1

39.0 ⫾ 14.6b,2 33.3 ⫾ 13.3b,2 3.3 ⫾ 1.1 121.7 ⫾ 50.6b,2 49.8 ⫾ 14.2

22.9 ⫾ 13.7c 19.6 ⫾ 11.5c 3.8 ⫾ 0.3 75.3 ⫾ 45.3c 44.1 ⫾ 9.7

Kinetic rating of sperm was graded subjectively: 0 ⫽ no movement; 5 ⫽ rapid forward progression; SMI: Sperm motility index ⫽ progressive motility ⫻ kinetic rating. a,b,c,1,2 Values with different superscripts within each row (letters) or columns (numbers: within each time period) are different (P ⬍ 0.05).

combinations that resulted in the highest 3 h PT TM, PM, VAP and Viab/Intact, in ascending order were: STW/3% (63.5 ⫾ 16.1%; 40.8 ⫾ 13.9%; 130.9 ⫾ 26.7 ␮m/s; 52.4 ⫾ 4.0%), DF/3% (71.4 ⫾ 12.8%; 51.6 ⫾ 16.0%; 154.3 ⫾ 42.9 ␮m/s; 58.7 ⫾ 6.8%), and DF/6% (75.5 ⫾ 12.7%; 56.7 ⫾ 18.2%; 164.7 ⫾ 43.7 ␮m/s; 57.9 ⫾ 6.4%) respectively (Table 4).

4. Discussion The development of a semen cryopreservation methodology for the killer whale is high priority for longterm genetic management of the ex situ population of the species due to its small size and wide dispersion among aquaria. In addition, the procedure outlined

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Table 4 Effects of cryoprotectant concentration and freeze method (straw and directional freezing [DF]) on in vitro characteristics of killer whale sperm (n ⫽ 6 ejaculates; three ejaculates from two males). Sperm characteristic

Glycerol 3% straw

Prefreeze (n ⫽ 6)a Total Motility (%) Progressive motility (%) Kinetic ratingb Viability (%) 0 h post-thaw (n ⫽ 12)c Total motility (%) Progressive motility (%) RAP (%) VAP (␮m/s) VSL (␮m/s) VCL (␮m/s) STR (%) LIN (%) BCF (Hz) ALH (␮m) Viable/Intact (%) Total acrosome intact (%) 3 h post-thaw (n ⫽ 12)c Total motility (%) Progressive motility (%) RAP (%) VAP (␮m/s) VSL (␮m/s) VCL (␮m/s) STR (%) LIN (%) BCF (Hz) ALH (␮m) Viable/Intact (%) Total acrosome intact (%)

Glycerol 6% DF

86.7 ⫾ 4.1 81.2 ⫾ 7.9 4.5 ⫾ 0.3 91.2 ⫾ 2.9

Straw

Glycerol 9% DF

86.7 ⫾ 4.1 81.2 ⫾ 7.9 4.3 ⫾ 0.4 89.5 ⫾ 6.3

Straw

DF 84.2 ⫾ 5.8 77.8 ⫾ 8.7 3.9 ⫾ 0.5 87.8 ⫾ 6.6

71.2 ⫾ 11.0a 48.7 ⫾ 13.6a 70.5 ⫾ 11.2a 107.1 ⫾ 20.4a,d 92.6 ⫾ 19.4a 166.6 ⫾ 30.4a 83.3 ⫾ 4.2 55.7 ⫾ 8.3a,b 34.1 ⫾ 2.3a,b 6.9 ⫾ 1.7a 51.3 ⫾ 4.2a,b 93.3 ⫾ 2.2

71.4 ⫾ 11.0a 50.8 ⫾ 10.6a,b 70.9 ⫾ 11.3a,c 137.6 ⫾ 26.2b 123.3 ⫾ 26.9b 198.2 ⫾ 26.5b 84.8 ⫾ 4.2 59.0 ⫾ 7.1a,c 35.7 ⫾ 2.8a,c 7.4 ⫾ 0.7 57.0 ⫾ 4.9b 83.2 ⫾ 16.8

72.9 ⫾ 6.8a 48.7 ⫾ 7.0a 72.0 ⫾ 7.4a 130.9 ⫾ 26.7a 114.3 ⫾ 28.1a 200.1 ⫾ 26.0c 82.9 ⫾ 3.9 54.5 ⫾ 7.4b 32.5 ⫾ 3.2b 8.6 ⫾ 0.9b 46.9 ⫾ 7.3a 90.8 ⫾ 4.5

77.7 ⫾ 8.8a 56.3 ⫾ 10.2b 77.3 ⫾ 8.5a 162.5 ⫾ 31.8c 145.7 ⫾ 35.0b 224.3 ⫾ 24.1d 85.5 ⫾ 5.2 61.8 ⫾ 8.8c 35.7 ⫾ 3.4c 8.2 ⫾ 1.0 59.3 ⫾ 6.7b 93.8 ⫾ 3.8

41.4 ⫾ 25.7b 26.8 ⫾ 17.2c 40.6 ⫾ 25.4b 104.5 ⫾ 23.1d 90.9 ⫾ 23.7a 167.0 ⫾ 25.6a 83.3 ⫾ 5.3a 52.8 ⫾ 7.2b 32.4 ⫾ 3.6b 7.5 ⫾ 1.3 38.3 ⫾ 13.6c 86.1 ⫾ 6.8

59.0 ⫾ 16.5c 43.7 ⫾ 10.9d 58.3 ⫾ 16.8c 146.1 ⫾ 26.0b 133.3 ⫾ 28.9b 197.7 ⫾ 23.7b 87.3 ⫾ 5.2b 63.9 ⫾ 8.4c 35.4 ⫾ 2.4c 7.2 ⫾ 1.2 51.8 ⫾ 12.4 91.5 ⫾ 3.6

66.0 ⫾ 15.3a 40.8 ⫾ 13.9a 65.6 ⫾ 15.3a 130.9 ⫾ 26.7a 114.3 ⫾ 28.1a 200.0 ⫾ 26a 82.9 ⫾ 3.9 54.5 ⫾ 7.4a 32.5 ⫾ 3.2 8.6 ⫾ 0.9a 52.4 ⫾ 4.0a 92.5 ⫾ 2.5

71.4 ⫾ 12.8a 51.6 ⫾ 16.0a,b 70.8 ⫾ 13.2a 154.3 ⫾ 42.9b 139.4 ⫾ 45.7b 211.3 ⫾ 39.7a 84.9 ⫾ 6.6 60.9 ⫾ 11.0b 37.2 ⫾ 2.6 7.4 ⫾ 0.7a 58.7 ⫾ 6.8a,c 93.1 ⫾ 5.5

63.5 ⫾ 16.1a 40.3 ⫾ 16.1a 63.0 ⫾ 16.0a 126.3 ⫾ 35.5a 108.9 ⫾ 38.2a 197.7 ⫾ 34.7a 80.8 ⫾ 8.1 52.1 ⫾ 10.0a 35.9 ⫾ 3.1 7.9 ⫾ 0.9a 46.3 ⫾ 4.3b 91.5 ⫾ 2.6

75.5 ⫾ 12.7a 56.7 ⫾ 18.2b 75.0 ⫾ 12.8a 164.7 ⫾ 43.7b 148.8 ⫾ 46.8b 222.1 ⫾ 39.5a 85.5 ⫾ 7.6 63.1 ⫾ 12.2b 37.4 ⫾ 2.6 7.5 ⫾ 1.0a 57.9 ⫾ 6.4c 93.3 ⫾ 3.1

37.8 ⫾ 27.2b 25.9 ⫾ 21.1c 36.8 ⫾ 27.1b 99.1 ⫾ 38.6a,c 85.8 ⫾ 38.8a 159.7 ⫾ 41.5b 82.3 ⫾ 7.2 51.7 ⫾ 9.5a,c 34.9 ⫾ 4.1 6.5 ⫾ 1.1b 38.2 ⫾ 14.7d 88.6 ⫾ 5.7

51.4 ⫾ 13.6c 36.9 ⫾ 14.3a 50.5 ⫾ 13.4b 125.8 ⫾ 34.6a,c 113.1 ⫾ 37.9b 181.4 ⫾ 32.3c 85.0 ⫾ 6.8 58.4 ⫾ 10.8b,c 37.0 ⫾ 2.3 6.7 ⫾ 1.1b 52.6 ⫾ 13.9c,d 91.5 ⫾ 3.7

Values are the mean ⫾ SD. aValues determined by subjective analysis. bKinetic rating of sperm was graded subjectively: 0 ⫽ no movement; 5 ⫽ rapid forward progression. cValues determined using computer assisted motility analyses (CASA). RAP, rapid motility; VAP, average pathway velocity; VSL, straight line velocity; VCL, curvilinear velocity; ALH, amplitude of lateral head displacement; BCF, beat cross frequency; STR, straightness of sperm movement (STR(%) ⫽ VSL/VAP); LIN, linearity. Viable/Intact, viable sperm with an intact acrosome. a-d Values without a common superscript within the same row are different (P ⬍ 0.05).

herein could be applied toward gamete rescue efforts of wild killer whales that die after stranding or that are killed incidentally. Gamete resource banks have become an integral part of conservation programs for many endangered and non-endangered wildlife [22,37]. The future application of such sperm resources to species management and conservation rely on the development of optimized, species-specific sperm preservation protocols. As such, the results described herein are the first description of killer whale ejaculate characteristics, sperm characteristics (morphology, motility, plasma membrane, and acrosome integrity), and an optimized sperm cryopreservation method. Consistent with data presented from Male 1 in a parallel investigation on the development of AI in killer

whales [20], semen collected from all males in the present study was of high quality, with high progressive motility (⬎ 85%), acrosome integrity (⬃90%), and normal morphology (⬃90%). The mean ejaculate volume (⬃7 mL) recorded in the present study is lower than that originally reported (13.2 mL [20]), but for the following reasons both results are not thought to accurately reflect normal ejaculate potential for this species. Firstly, some ejaculate is lost during the technically difficult collection procedure, and ejaculate volumes are, to some extent, a function of how well trained a particular male is to the methodology. And secondly, a recent report [38] described semen collection in 2 bottlenose dolphins utilizing a novel artificial vagina which resulted in ejaculates (n ⫽ 192) with similar

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NonViab-Int

NonViab-NonInt

Viab-NonInt NonViab-NonInt Viab-Int

Fig. 2. Micrograph of killer whale sperm illustrating the four staining patterns after incubation with PI/FITC-PNA (⫻ 400 original magnification): a viable spermatozoon with an intact acrosome (Viab-Int); a viable spermatozoon with a damaged/reacted acrosome (ViabNonInt); a non-viable spermatozoon with an intact acrosome (NonViab-Int); a non-viable spermatozoon with a damage/reacted acrosome (NonViab-NonInt).

concentration, but 4 to 5 times the volume reported in the former study. This work supports the theory that despite the ability to collect semen on a regular basis, current methods are unable to provide adequate stimulation necessary to obtain the full ejaculatory potential of killer whales. A medium to slow freezing rate across all extenders provided the best 2.5 h PT, TM, SMI, and viability. These results were different to those which have been reported for bottlenose dolphins where a fast freezing rate (⬎ ⫺100 °C/min) resulted in the highest quality sperm PT [28]. The differences in the optimum freezing rate between these two species may be associated with the different freezing methodologies used (freezing using a controlled rate freezer [28] versus freezing manually over liquid nitrogen vapour). The species-specific freezing rates may also be a reflection of increased membrane sensitivity to freezing and thawing processes of killer whale sperm [39]. Evaluation of the differences in the membrane lipid composition and thus its fluidity or theoretical resistance to rapid changes in osmotic pressure may be worth investigating [40]. The results from the straw freezing trial demonstrated the superiority of BF5F in maintaining PT qual-

ity when compared to Biladyl® and EYC. Interestingly, of all examined diluents, EYC was best able to maintain in vitro sperm characteristics at 4 °C, but was unable to maintain PT quality parameters (TM, SMI and Viability), regardless of freezing rate. While all the diluents tested comprised a buffer(s) (sodium citrate, TES or Tris, sodium bicarbonate, HEPES), EYC differs in that it does not contain any sugars (fructose or glucose). While fructose has been found to be metabolized by bovine semen [41] and its addition may be important for long term liquid storage (⬎ 24 h), sugars are most important for their cryoprotectant effect during freezing [42]. This effect is believed to be related to their ability to replace water molecules at the polar binding sites of the sperm membrane, which in turn is hypothesized to stabilize cell membranes during critical phase transition periods [43]. The addition of sugars alone may explain why the extender with the highest content of sugar (BF5F) provided the best PT results. Another sugar, trehalose, a non-permeable sugar, has been used as a successful cryoprotectant in the beluga [17] and other species (goat [44]; dog [45]). Further research with different types of sugars alone or in combination with glycerol may be useful at improving the PT recovery of killer whale sperm reported herein. The glycerol concentration within the directional freezing (DF) or straw freezing method had a significant impact on the PT motility parameters TM, PM, RAP, VAP, VCL and ALH with 3 and 6% glycerol being superior to 9%. However, 6% glycerol was superior to 3% in RV, VAP, and VCL. These data suggest that whereas higher glycerol (⬎ 6 %) concentrations negatively impact motility, this cryoprotectant is able to optimize PT motility parameters within a prescribed range (3 to 6%) PT. This optimal glycerol concentration range is compatible with that used in two other delphinids (bottlenose dolphin [28]; Pacific white-sided dolphin [31]). In contrast, due to the extreme membrane sensitivity of sperm from beluga (family Monodontidae), optimum PT quality was accomplished only by replacing glycerol with the non-penetrating cryoprotectant trehalose [17]. Similar to our results, glycerol concentrations at or above 9% have been shown to be detrimental to sperm quality [46]. The mechanism for this toxicity has been related to both osmotic changes across the cell membrane and the binding to and disruption of cell membrane structure [46,47]. The most effective concentration of glycerol for cryopreservation is intricately related to inter-species membrane differences, diluent composition, and thawing rate [25,48,49]. Future re-

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search on thawing rates with cryopreserved killer whale sperm may enable improvement of the PT recovery rate reported in the current study. Across all glycerol concentrations and time-points PT, the samples frozen using the DF method demonstrated significantly better motility parameters (TM, PM, RM, VAP, VSL, VCL) when compared to the straw method. Previous studies in the bottlenose dolphin [21], beluga [17] and the white rhinoceros (Ceratotherium simum) [50] similarly demonstrated improved PT in vitro characteristics when sperm were cryopreserved using DF compared to a conventional straw methodology. The horse is the only domestic species where DF has been compared to a conventional straw cryopreservation methodology [51,52]. The initial study found no differences between freezing methods whereas the latter study (which increased stallion numbers and used different cooling methods) demonstrated improved PT motility and viability with DF. The DF system uses a linear temperature gradient system that controls seeding or nucleation and thus ice crystal front propagation [32]. Random nucleation leading to ice crystal formation, which typical occurs during the straw freezing method, is believed to cause physical damage to cells [48]. This, along with exposure to supersaturated pockets of media which cause osmotic membrane damage, are the two primary forces that are believed to affect the PT survivability of sperm [39,40]. Thus, the DF method improves PT viability by decreasing mechanical damage to sperm membranes. In addition, the linear controlled nucleation process allows for a steady decrease of temperature without experiencing a freezing point plateau, thus reducing the time sperm are exposed to a supersaturated solution [32,17]. Together, these two benefits are believed to be responsible for the significant improvement of PT in vitro killer whale sperm motility parameters using the DF compared to the straw method. Currently, the significance of differences detected by CASA, as it relates to fertility, is more difficult to explain and appears to vary between species. Typically, an analysis of multiple motility parameters, with or without concentration and other morphological tests has resulted in a significant relationship with fertility in humans [53] and bulls [19]. In addition, specific individual parameters have been identified as correlating with in vivo fertility in bulls (VSL [19]), humans (VCL, VAP, VSL and BCF [53]) and boars (VCL, VAP, VSL [17,54]). If these trends apply to killer whales, then our CASA results provided evidence, even when TM and PM are similar, that the DF methodology resulted in

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a higher quality and potential fertility of sperm PT than the straw method. Similar superiority of DF compared to straws was obtained with sorted and non-sorted dolphin sperm undergoing a single [21] or double [18] freezing cycle. In addition, DF recryopreserved sperm displayed reduced DNA fragmentation across the 24 h PT period compared to sperm frozen with straws [18]. Evaluation of the viability and acrosome status using PI/FITC-PNA showed a significant increase in viability, with no change in acrosome integrity for samples frozen by the DF compared to the straw method. This pattern was also observed in bottlenose dolphins during comparisons between the DF and the straw methods [21,18]. The use of the dual stain PI/FITC-PNA to evaluate viability and acrosome integrity has not been previously reported in killer whales and has only recently been reported in the bottlenose dolphin [18]. Due to the expense of a fluorescent microscope and remote location of many animals, evaluations of viability and acrosome integrity have traditionally relied on staining with eosin-nigrosin and Spermac®, respectively. Our results demonstrated good correlation between viability methods. Further comparison should be made between Spermac® and FITC-PNA methodologies. The current study provides a description of normal killer whale semen characteristics and sperm kinematics. Future data acquisition using the standardized procedures outlined herein could form the basis for developing fertility assessments of wild animals which could then be used for monitoring changes induced by environmental pressures over time. In addition, the DF protocol described herein resulted in significant improvement over previously published methods relying on straws [20]. The fertility of semen frozen using straws has already been demonstrated [20], and the improvements detailed herein by using the DF method should result in more efficient use of semen for AI procedures. The results also justify the development of a gamete resource bank from ex situ or in situ males, through gamete rescue techniques, to save valuable genetics for potential integration into future generations [22]. Finally, the maximized PT sperm quality opens the possibility of developing sex ratio population management techniques [23] that are currently being applied to ex situ bottlenose dolphin populations.

Acknowledgments The veterinary, animal laboratory, animal care and animal training staff at SeaWorld Orlando, San Antonio

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and San Diego and the animal staffs at Kamogawa Sea World and Mundo Marino are thanked for their consistent support. We especially thank Laura Surovik (SWO), Matt Fripp (SWSD), Brian Rokeach (SWSD), Doug Acton (SWSA), Julie Sigman (SWSA), Chris White (SWSA), Chuck Tompkins (SEA), Seiki Konno (KSW), and Jorge Rebollo (MUN) for their dedicated work toward semen collection. We also thank Sherry Dickerson (SWSA), Pam Thomas (SWSD), Melinda Tucker (SWSD) and Jacob Vandenberg (SWO) for technical support, as are SeaWorld and Busch Gardens Reproductive Research Center staff, Michelle Morrisseau and Gisele Montano. We thank Alex Ziolkowski and Hamilton Thorne for providing digital camera equipment used for Figure 2. We thank Brad Andrews (SeaWorld Parks and Entertainment, SEA), Kazutoshi Arai (KSW) and Jose Mendez (MUN) for their support of this project. This research was conducted under the NMFS permit number 116-1691, was funded by SEA, KSW and MUN and is a SeaWorld Technical contribution number 2010-03-T.

[10]

[11]

[12]

[13]

[14]

[15]

[16]

References [1] Rice DW. Marine mammals of the world. Systematics and distribution. Society for Marine Mammalogy Special Publication 1998;4:1–231. [2] Taylor BL, Baird R, Barlow J, Dawson SM, Ford J, Mead JG, Notarbartolo di Sciara G, Wade P, Pitman RL. 2008. Orcinus orca. In: IUCN 2010. IUCN Red List of Threatened Species. Version 2010.2. Available at: http://www.iucnredlist.org. Accessed 19 July 2010. [3] Bigg MA. An Assessment of killer whale (Orcinus orca) stocks off Vancouver Island, British Columbia. Rep Int Whal Comm 1982;32:655– 66. [4] Olesiuk, PF, Bigg MA, Ellis GE. Life history and population dynamics of resident killer whales (Orcinus orca) in the coastal waters of British Columbia and Washington State. Rep Int Whal Comm 1990;12:209 – 43. [5] National Marine Fisheries Service. 2008. Recovery Plan for Southern Resident Killer Whales (Orcinus orca). National Marine Fisheries Service, Northwest Region, Seattle, Washington. [6] O’Shea TJ. Environmental contaminants and marine mammals. In: Biology of marine mammals, Reynolds JE, Rommel SA (Eds.) Smithsonian Institution Press, Washington, D.C. 1999, p. 485– 63. [7] De Guise SD, Beckmen KB, Holladay SD. Contaminants and marine mammal immunotoxicology and pathology. In: Vos JG, Bossart GD, Fournier M, O’Shea TJ, editors. Toxicology of Marine Mammals. Taylor and Francis, New York, NY, 2003, p. 38 –54. [8] Ross PS, Ellis GM, Ikonomou MG, Barrett-Lennard LG, Addison RF. High PCB concentrations in free-ranging Pacific killer whales, Orcinus orca: effects of age, sex and dietary preference. Mar Pollution Bull 2000;40:504 –15. [9] Reijnders, P. J. H. Reproductive and developmental effects of environmental organochlorines on marine mammals. In: Vos

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

JG, Bossart GD, Fournier M, O’Shea TJ, editors. Toxicology of Marine Mammals. Taylor and Francis, New York, NY, 2003, p. 55– 66. Campagna C, Guillemette C, Paradis R, Sirard M, Ayotte P, Bailey JL. An Environmentally Relevant Organochlorine Mixture Impairs Sperm Function and Embryo Development in the Porcine Model. Biol Reprod 2002;67:80 –7. Oliva A, Spira A, Multigner L. Contribution of environmental factors to the risk of male infertility. Human Reprod 2001;16: 1768 –76. Spanò1 M, Toft G, Hagmar L, Eleuteri1 P, Rescia1 M, RignellHydbom A, Tyrkiel E, Zvyezday V, Bonde JP, INUENDO. Exposure to PCB and p,p’-DDE in European and Inuit populations:impact on human sperm chromatin integrity. Hum Reprod 2005;20:3488 –99. Holt C, Holt WV, Moore HDM, Reed HCB, Curnock RM. Objectively measured boar sperm motility parameters correlate with the outcomes of on-farm inseminations: results of two fertility trials. J Androl 1997;18:312–23. Evenson DP, Wixon R. Clinical aspects of sperm DNA fragmentation detection and male infertility. Theriogenology 2006; 65:979 –91. Robeck TR, Monfort SL. Characterization of male killer whale (Orcinus orca) sexual maturation and reproductive seasonality. Theriogenology 2006;66:242–50. O’Brien JK, Steinman KJ, Schmitt T, Robeck TR. Semen collection, characterization and artificial insemination in the beluga (Delphinapterus leucas) using liquid-stored spermatozoa. Reprod Fert Dev 2008;20:770 – 83. O’Brien JK, Robeck TR. Preservation of beluga (Delphinapterus leucas) spermatozoa using a trehalose-based cryodiluyent and directional freezing technology. Reprod Fertil Dev 2010;22:653– 63. Montano GM, Kraemer DC, Love CC, Robeck TR, O’Brien JK. In vitro function of frozen-thawed bottlenose dolphin (Tursiops truncatus) spermatozoa undergoing sorting and recryopreservation. Proc Int Embryo Trans Soc 2011;23:240 [Abstract]. Gillan L, Kroetsh T, Maxwell WMC, Evans G. Assessment of in vitro sperm characteristic in relation to fertility in dairy bulls. 2008;103:210 – 4. Robeck TR, Steinman KJ, Gearhart S, Reidarson TR, McBain JF, Monfort SL. Reproductive physiology and development of artificial insemination technology in killer whales (Orcinus orca). Biol Reprod 2004;71:650 – 60. O’Brien JK, Robeck TR. Development of sperm sexing and associated assisted reproductive technology for sex preselection of captive bottlenose dolphins (Tursiops truncatus). Reprod Fert Dev 2006;18:319 –29. O’Brien JK, Robeck TR. The value of ex situ cetacean populations in understanding assisted reproductive technology for ex situ and in situ species management and conservation efforts. International J Comp Psychol 2010;23:227– 48. O’Brien JK, Steinman KJ, Robeck TR. Application of sperm sorting and associated reproductive technology for wildlife management and conservation. Theriogenology 2009;71:98 – 107. Fripp M, Rokeach B, Robeck T, O’Brien J. Objective assessment of a training program to facilitate semen collection from killer whales (Orcinus orca). Proceedings International Marine Animal Trainers Association. 2005;33:41 [Abstract]. Robbins RK, Saacke RG, Chandler JE. Influence of freeze rate, thaw rate and glycerol on acrosome retention and survival of

T.R. Robeck et al. / Theriogenology 76 (2011) 267–279

[26]

[27] [28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37] [38]

[39]

bovine spermatozoa frozen in French straws. J Anim Sci 1976;42:145–54. Howard JG, Bush M, de Vos V, Schiewe MC, Pursel VG, Wildt DE. Influence of cryoprotective diluent on post-thaw viability and acrosomal integrity of spermatozoa of the African elephant (Loxodonta africana). J Reprod Fertil 1986;78:295–306. Evans, G. and Maxwell, W. M. C. Salamon’s Artificial Insemination of Sheep and Goats. Butterworths, Sydney, 1987. Robeck TR, O=Brien JK. Effect of cryopreservation methods and pre-cryopreservation storage on bottlenose dolphin (Tursiops truncatus) spermatozoa. Biol Reprod 2004;70:1340 – 8. Pursel VG, Johnson LA. Freezing of boar spermatozoa: fertilizing capacity with concentrated semen and a new thawing procedure. J Anim Sci 1975;40:99 –102. Salisbury GW, Fuller HK, Willett EL. Preservation of bovine spermatozoa in yolk– citrate diluent and field results from its use. J Dairy Sci 1941;24:905–10. Robeck TR, Steinman KJ, Greenwell M, Ramirez K, Van Bonn W, Yoshioka M, Katsumata E, Dalton L, Osborn S, O=Brien JK. Seasonality, estrous cycle characterization, estrus synchronization, semen cryopreservation, and artificial insemination in the Pacific white sided-dolphin (Lagenorhynchus obliquidens). Reproduction 2009;138:391– 405. Arav A, Yavin S, Zeron Y, Natan D, Dekel I, Gacitua H. New trends in gamete’s cryopreservation. Mol Cell Endocrinol 2002; 187:77– 81. Graham JR, Kunze E, Hammerstedt RH. Analysis of sperm cell viability, acrosomal integrity, and mitochondrial function using flow cytometry. Biol Reprod 1990;43:55– 64. Szasz F, Sirivaidyapong S, Cheng FP, Voorhout WF, Marks A, Colenbrander B, Solti AL, Gadella BM. Detection of calcium ionophore induced membrane changes in dog sperm as a simple method to predict the cryopreservability of dog semen. Mol Reprod Dev 2000;55:89 –98. Pena AI, Johannisson A, Linde-Forsberg C. Validation of flow cytometry for assessment of viability and acrosomal integrity of dog spermatozoa and for evaluation of different methods of cryopreservation. J Reprod Fertil Suppl 2001;57:371– 6. Miller DL, Styer EL, Decker SJ, Robeck T. Ultrastructure of the spermatozoa from three odontocetes, a killer whale (Orcinus orca), a Pacific white-sided dolphin (Lagenorhynchus obliquidens) and a beluga (Delphinapterus leucas). Anatomia Histologia Embryologia 2002;31:1–11. Wildt, D. Rescuing endangered animals with assisted reproductive technology. Sex Reprod Menopause 2009;7:21–5. Schultz B, Greven H. Bottlenose dolphin (Tursiops truncatus) semen collection using an artificial vagina. Proc Eur Assoc Aquat Med 2010;38:25 [Abstract]. Mazur P. Freezing of living cells:mechanisms and implications. Am J Physiol 1984;247:125– 42.

279

[40] Johnson LA, Weitze KF, Fiser P, Maxwell WMC. Storage of boar semen. Anim Reprod Sci 2000;62:143–72. [41] Mann T. Studies on the metabolism of semen 3. Fructose as a normal constituent of seminal plasma. Site of formation and function of fructose in semen. Biochem J 1946;40:481–91. [42] Pickett BW, Berndtson WE, Sullivan JJ. Influence of seminal additives and packaging systems on fertility of frozen bovine spermatozoa. J Anim Sci 1978;47:12– 47. [43] Woelders H, Matthijs A, B Engel. Effects of trehalose and sucrose, osmolality of the freezing medium, and cooling rate on viability and intactness of bull sperm after freezing and thawing. Cryobiology 1997;35:93–105. [44] Aisen EG, Medina VH, Venturino A. Cryopreservation and post-thawed fertility of ram semen frozen in different trehalose concentrations. Theriogenology 2002;57:1801– 8. [45] Yildz C, Kaya A, Aksoy M, Tekeli T. Influence of sugar supplementation of the extender on motility, viability and acrosomal integrity of dog spermatozoa during freezing. Theriogenology 2000;54:579 – 85. [46] Fahy G. The revelance of cryoprotectant “toxicity” to cryobiology. Cryobiology 1986;23:1–13. [47] Murdoch RN, Jones RC. The effects of glycerol on the metabolism and ultrastructure of boar spermatozoa. J Reprod Fert 1978;54:419 –22. [48] Darin-Bennett A, White IG. Influence of cholesterol content of mammalian spermatozoa on susceptibility to cold shock. Cryobiology 1977;14:466 –70. [49] Parks JE, Graham JK. Effects of cryopreservation procedures on sperm membranes. Theriogenology 1992;38:209 –22. [50] Reid CE, Hermes R, Blottner S, Goeritz F, Wibbelt G, Walzer C, Bryant BR, Portas TJ, Streich WJ, Hildebrandt TB. Splitsample comparison of the directional and liquid nitrogen vapour freezing method on post-thaw semen quality in white rhinoceroses (Ceratotherium simum simum and Ceratotherium simum cottoni). Theriogenology 2009;71:275–91. [51] Zirkler HK, Gerbes K, Klug E, Sieme H. Cryopreservation of stallion semen collected from good and poor freezers using a directional freezing device (Harmony CryoCare-Multi Thermal Gradient 516). Anim Reprod Sci 2005;89:291– 4. [52] Saragusty J, Gacitua H, Pettit MT, Arav A. Directional freezing of equine semen in large volumes. Reprod Dom Anim 2007; 42:610 –5. [53] Mortimer ST. A critical review of the physiological importance and analysis of sperm movement in mammals. Hum Reprod Update 1997,3:403–39. [54] Agarwal A, RK Sharma, DR Nelson. New Semen Quality Scores Developed by Principal Component Analysis of Semen Characteristics. J Andrology 2003;24:343–52.