CSIRO PUBLISHING

Reproduction, Fertility and Development, 2016, 28, 599–607 http://dx.doi.org/10.1071/RD14233

The effects of permeating cryoprotectants on intracellular free-calcium concentrations and developmental potential of in vitro-matured feline oocytes Jason R. Herrick A,D, Chunmin Wang B,C and Zoltan Machaty B A

National Foundation for Fertility Research, 10290 RidgeGate Cr, Lone Tree, CO 80124, USA. Department of Animal Sciences, Purdue University, Lilly Hall, 915 West State St, West Lafayette, IN 47907, USA. C Current Address: Vivere Health-Houston Surgery Center and IVF Laboratory, 2500 Fondren Rd, Suite 350, Houston, TX 77063, USA. D Corresponding author. Email: [email protected] B

Abstract. Embryos produced from vitrified feline oocytes have resulted in pregnancies, but the efficiency of oocyte vitrification in cats is still low. Our objectives were to evaluate the effects of exposing feline oocytes to ethylene glycol (EG), propanediol (PrOH) and dimethyl sulfoxide (DMSO) on changes in intracellular free-calcium concentrations ([Ca2þ]i), the time needed for enzymatic digestion of the zona pellucida (ZP), the incidence of parthenogenetic activation and degeneration and embryonic development following in vitro fertilisation (IVF). All of the chemicals tested altered [Ca2þ]i, but changes in [Ca2þ]i, resistance of the ZP to enzymatic digestion and the incidence of parthenogenetic activation (,5% for all treatments) were not affected (P . 0.05) by extracellular Ca2þ. Exposure to EG (.44.1%) and DMSO (19.7%) increased (P , 0.05) oocyte degeneration compared with control oocytes and oocytes exposed to PrOH (#2.5%). Following exposure to a combination of PrOH and DMSO (10% v/v each), blastocyst development (per cleaved embryo; 52.1%) was similar (P . 0.05) to control oocytes (64.4%). When oocytes were vitrified with PrOH and DMSO, 28.3% of surviving (intact plasma membrane) oocytes cleaved following IVF, but no blastocyst developed. When a non-permeating cryoprotectant (galactose, 0.25 M) was added to the vitrification medium, 47.7% of surviving oocytes cleaved and 14.3% developed to the blastocyst stage. Additional keywords: cat, DMSO, intracellular calcium, propanediol, vitrification. Received 3 July 2014, accepted 26 August 2014, published online 11 September 2014 Introduction Management of ex situ populations of endangered felids has become an important component of the conservation efforts for these species. Unfortunately, most of these captive populations are not sustainable due to a limited number of individuals, inadequate genetic diversity and poor reproductive success. Assisted reproductive technologies (ARTs), like in vitro fertilisation (IVF) and embryo transfer (ET), could play an important role in ensuring the sustainability of captive populations by overcoming behavioural incompatibility and reducing the need to transport animals between zoos (Swanson 2006). Combining these procedures with gamete and embryo cryopreservation enhances the potential role of ARTs by allowing an individual’s genetics to be preserved well beyond the animal’s lifespan and facilitating transport of those samples over long distances (Swanson 2006). Following cryopreservation, motility and fertilising ability of spermatozoa from numerous species of felids are sufficient for IVF and artificial insemination (AI), providing a reliable Journal compilation Ó CSIRO 2016

means of preserving the male genome (Swanson et al. 1996; Stoops et al. 2007; Herrick et al. 2010). However, an efficient procedure to preserve the genome of female felids has remained elusive. It is possible to preserve a female’s genetics by collecting oocytes, producing embryos through IVF and cryopreserving the resulting embryos, but this approach has several limitations. First, population managers must select a suitable male at the time of oocyte collection from those in the current population or those males whose spermatozoa have already been cryopreserved. Second, only a handful of zoological institutions have the personnel and laboratories to conduct ARTs. Therefore, IVF is often conducted in improvised laboratories equipped with portable versions of all necessary equipment (Swanson 2003). The ability to successfully cryopreserve feline oocytes would allow oocytes to be stored for extended periods of time until a suitable male is identified and a sperm sample is available. Similarly, oocytes could be cryopreserved within minutes of recovery and transported to a specialised laboratory for IVF where the chances of success are greatly improved. www.publish.csiro.au/journals/rfd

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In recent years, tremendous advances have been made in oocyte vitrification (Kuwayama et al. 2005; Mullen and Fahy 2012; Vajta 2013), allowing oocytes from some species, most notably humans, to be cryopreserved with little or no loss in embryonic viability after warming (Cobo et al. 2008; Noyes et al. 2009; Sole´ et al. 2013). Initial attempts to vitrify feline oocytes have been very promising, including the production of kittens (Tharasanit et al. 2011; Pope et al. 2012), but fertilisation and embryonic development of vitrified feline oocytes are still reduced compared with non-vitrified controls. A likely explanation for the reduced viability after vitrification and warming is the limited number of studies (relative to rodents, livestock and humans) on the vitrification of feline oocytes. In addition, the few studies involving vitrification of feline oocytes have utilised protocols developed for the oocytes and embryos of other species with minimal modifications. Such proven protocols provide an important starting point when developing techniques for other species, but the potentially profound differences between species are often ignored (Comizzoli et al. 2012). All vitrification procedures utilise one or more chemical cryoprotectants (CPs) that are capable of permeating the oocyte’s membrane to displace water within the oocyte and reduce the possibility of intracellular ice formation (Rall 1987). However, exposing oocytes to these CPs at the concentrations necessary to achieve vitrification can have several adverse effects on the oocyte, including hardening of the zona pellucida, parthenogenetic activation, reduced embryonic development and cell death (Larman et al. 2006; Aye et al. 2010; Succu et al. 2011; Szurek and Eroglu 2011). Therefore, an important first step in the development of species-specific vitrification protocols is the identification of one or more CPs with minimal effects on cellular homeostasis and oocyte viability. In other species, exposure to CPs can alter the intracellular concentration of free calcium ([Ca2þ]i) within the oocyte, which can lead to zona hardening, parthenogenetic activation and loss of oocyte viability (Takahashi et al. 2004; Larman et al. 2006; Fujiwara et al. 2010; Succu et al. 2011). Interestingly, these changes in [Ca2þ]i are dependent on the type of CP used and the concentration of Ca2þ in the extracellular medium (Takahashi et al. 2004; Larman et al. 2006). Our first objective was to determine if three of the most commonly used CPs (ethylene glycol (EG), propanediol (PrOH) and dimethyl sulfoxide (DMSO)) affect [Ca2þ]i in feline oocytes and if these changes are affected by the presence or absence of extracellular Ca2þ. In addition, zona hardening (resistance to enzymatic digestion), degeneration (fragmentation or cellular lysis) and the incidence of parthenogenetic activation were also examined in oocytes exposed to these CPs in the presence or absence of Ca2þ. Based on the results of these experiments, we identified two CPs (DMSO and PrOH) that were further evaluated for effects on the developmental competence of oocytes following CP exposure and IVF. Finally, oocytes exposed to DMSO and PrOH were vitrified (with and without a non-permeating CP) to establish ‘proof of principle’ that DMSO and PrOH were effective for vitrification of feline oocytes.

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Materials and methods Chemicals and reagents Unless stated otherwise, all chemicals and reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). In vitro maturation Domestic cat ovaries were recovered from local veterinary clinics following routine ovariohysterectomies of females $6 months old. Surgeries were performed for the purpose of population control and the clinic’s standard protocols were not altered in order to collect ovarian tissue. Immediately after excision, ovaries were placed in 9.0 mg mL1 NaCl containing 50 mg mL1 gentamicin and maintained at ,58C until processing (2 to 3 h). After removing excess tissue, ovaries were repeatedly sliced in a 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)-buffered (20 mM HEPES and 5 mM NaHCO3) version of feline optimised culture medium (FOCMH; Herrick 2014) containing 4.0 mg mL1 bovine serum albumin (BSA, EmbryoGro; MP Biomedicals, Solon, OH, USA) and 40 U mL1 heparin. Cumulus–oocyte complexes containing multiple, compact layers of cumulus cells and an oocyte with a uniformly dark cytoplasm were washed in FOCMH and placed in maturation medium (five COCs per 50-mL drop under Ovoil; Vitrolife, Englewood, CO, USA). The medium used for maturation was bicarbonate-buffered (25 mM NaHCO3) feline optimised culture medium (FOCM; Herrick et al. 2007) containing 6.0 mM glucose and supplemented with 0.5 minimum essential medium (MEM) essential amino acids, 1.0 MEM vitamins, 0.6 mM cysteine, 0.1 mM cysteamine, 10 mg mL1 insulin, 5.5 mg mL1 transferrin, 5.0 ng mL1 selenium and 4.0 mg mL1 BSA (Herrick 2014). Cumulus–oocyte complexes were cultured in 7.5% CO2 in air (38.78C) to compensate for the elevation of our laboratory (,1830 m above sea level), which is similar to 6.0% CO2 (media pH ¼ 7.2 to 7.3) at sea level. After 24 h, cumulus cells were removed in the presence of hyaluronidase (,500 U mL1; 0.5 mg mL1) by shaking vigorously with a vortex mixer or aspirating through a narrow-bore glass pipette. Assessment of intracellular free Ca21 concentrations Following in vitro maturation and cumulus cell removal, oocytes were loaded with the Ca2þ indicator dye, fura-2, by incubation in FOCMH medium containing 2 mM fura-2 AM (acetoxymethyl ester form of the dye) and 0.02% pluronic F-127 (both from Invitrogen, Carlsbad, CA, USA) for 45 min. Measurements of [Ca2þ]i were made in a glass-bottom chamber that had been treated with Cell-Tak adhesive (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol. A 25-mL drop of protein-free FOCMH medium was made in the centre of the chamber and the oocytes were then transferred into the droplet and allowed to adhere to the bottom. Additional FOCMH with protein (4.0 mg mL1 BSA) was then added to the chamber for a total volume of 360 mL. Changes in [Ca2þ]i were monitored by measuring the emitted fluorescence (at 510 nm) of fura-2 excited intermittently (every 10 s) at 340 and 380 nm using a dual-wavelength fluorescence imaging system (InCyt Im2; Intracellular Imaging, Inc., Cincinnati, OH, USA).

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The ratio of emission intensities after 340 nm and 380 nm excitation is indicative of [Ca2þ]i. Each measurement began by recording the baseline [Ca2þ]i for 2 min, then 40 mL CP was added to the chamber to make a 10% CP solution and [Ca2þ]i were measured for another 2 min. Then 50 mL CP was added to make a 20% solution. After a 2-min measurement, 150 mL CP was added to the chamber to make a 40% solution and [Ca2þ]i were recorded for an additional 2 min. The same experiment was also performed in Ca2þ-free FOCMH medium, which was prepared without any CaCl2. Trace amounts of Ca2þ that may have been present as contamination from other reagents were considered negligible. Measurements were repeated several times using 9–17 different oocytes for each CP with or without Ca2þ in the medium. Assessment of hardening of the zona pellucida Immediately after [Ca2þ]i measurements, the oocytes were recovered from the chamber and washed through 20%, 10% and 0% CP in FCOMH medium (1 min in each solution). These oocytes were then placed in FOCMH medium containing 5 mg mL1 pronase to remove the zona pellucida. Oocytes were observed with a dissecting microscope (,75–100 magnification) and the time needed to completely dissolve the zona was recorded for each oocyte. Assessment of parthenogenetic activation (pronuclear formation) and degeneration after CP exposure Oocytes were sequentially exposed (2 min per concentration, 378C) to solutions containing 10% (v/v), 20%, 10% and 0% DMSO, PrOH or EG in FOCMH with or without 2.0 mM Ca2þ (present as CaCl2-2H2O) to mimic the stepwise addition and dilution of CPs that occurs during vitrification and warming. Control oocytes were maintained in FOCMH with or without Ca2þ for at least 10 min. Exposed oocytes were then transferred to 20-mL drops of FOCM under Ovoil, cultured (7.5% CO2, 6.5% O2 (,5% at sea level), 38.78C) for 14 to 16 h and evaluated for gross signs of degeneration, including fragmentation (numerous cytoplasmic fragments of various sizes) and lysis (visibly damaged plasma membrane) with a standard dissecting microscope. Oocytes were then transferred to a glass slide in a minimum amount of medium and compressed under a coverslip supported by drops of petroleum jelly and paraffin wax. The coverslip was secured in place with small drops of rubber cement and the slide was placed in fixative (6 parts 100% ethanol : 3 parts glacial acetic acid : 1 part chloroform) for at least 24 h (Herrick et al. 2003). Chromatin was stained with 1% (w/v) orcein in 45% acetic acid (v/v in H2O) and the presence or absence of pronuclei was evaluated with phase-contrast microscopy (400). Assessment of embryonic development after CP exposure Only PrOH (20%, v/v), DMSO (20%) and a combination of PrOH and DMSO (10% each) were tested given the effects of EG exposure on degeneration. In addition, all media used during CP exposure contained 2.0 mM Ca2þ. Oocytes were sequentially exposed (378C) to solutions containing 2.5% (v/v; 1 min), 5% (1 min), 10% (30 s), 20% (30 s), 10% (30 s), 5% (1 min) and

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2.5% (1 min) DMSO, PrOH or DMSO and PrOH in FOCMH containing 5% (v/v) fetal calf serum (FCS; Hyclone, Waltham, MA, USA) before IVF. Preliminary experiments indicated that viability was improved when 4.0 mg mL1 BSA was replaced with 5% FCS in FOCMH. Oocyte vitrification Oocytes were vitrified using a modification of the Cryotop technique (Kuwayama et al. 2005; Pope et al. 2012). The final medium used for vitrification was FOCMH containing 5% FCS, 10% (v/v) DMSO and 10% PrOH with or without 0.25 M galactose (Checura and Seidel 2007). Oocytes were exposed (378C) to solutions containing a total concentration of 2.5% (1.25% DMSO and 1.25% PrOH; 1 min), 5% (1 min), 10% (30 s) and 20% (30 s) CP with or without 31.3 (1 min), 62.5 (1 min), 125 (30 s) and 250 mM galactose (30 s). Oocytes were expelled onto a Cryotop (Kitazato Corp., Fuji, Japan), excess medium was removed and the Cryotop was plunged into liquid nitrogen. For warming, Cryotops containing oocytes were removed from liquid nitrogen and quickly plunged into the full strength vitrification medium (5% FCS, 10% DMSO and 10% PrOH with or without 0.25 M galactose). After 30 s, oocytes were transferred through dilutions of the vitrification medium containing 10% (5% DMSO and 5% PrOH; 30 s), 5% (1 min), 2.5% (1 min) and 0% ($5 min) CP with or without 125 (30 s), 62.5 (1 min), 31.3 (1 min) and 0 mM ($5 min) galactose before moving to IVF medium. In vitro fertilisation Oocytes (10 per drop under Ovoil) were washed and placed in 45-mL drops of FOCM containing 2.0 mM CaCl2, 0.2 mM MgSO4 and 5% FCS (Herrick et al. 2013) before the initiation of sperm processing. Cryopreserved, ejaculated spermatozoa from two domestic cats of proven fertility were thawed in air for 10 s and in a 378C water bath for 30 s and washed (10 min at 300g at room temperature) with FOCMH. Spermatozoa (5  105 motile spermatozoa mL1) diluted in the IVF medium were added to each drop containing oocytes (,45 min after warming or after CP exposure) and gametes were co-incubated for 22 h in 7.5% CO2 in air. The day of IVF was considered to be Day 0 of culture. Embryo culture Loosely bound spermatozoa were removed from presumptive zygotes by gentle pipetting and the number of oocytes that had cleaved to the 2-cell stage was determined. All zygotes and embryos were washed and placed (five per 20-mL drop under Ovoil) into FOCM (4.0 mg mL1 BSA; Herrick et al. 2007) for culture (7.5% CO2, 6.5% O2, 48 h). On Day 3 of culture, the proportion of embryos that had cleaved was evaluated and all embryos (.4-cells) were moved to fresh FOCM with 5% (v/v) FCS and cultured (7.5% CO2, 6.5% O2) for 96 h. On Day 7 of culture (,168 h after IVF), the proportion of embryos that had developed to the blastocyst stage was determined. Blastocysts resulting from vitrified and warmed oocytes were also stained with Hoechst 33342 (Pursel et al. 1985) to determine total cell number and verify morphological classification as a blastocyst (Herrick 2014).

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Statistical analysis For each replicate (day of ovary collection), oocytes were randomly allocated to as many treatments as possible (incomplete block design) and each treatment was replicated three to six times depending on the experiment. For [Ca2þ]i measurements, the average baseline value was determined for each oocyte during the first 2 min before the addition of CPs and all subsequent measurements following the addition of CPs were expressed as the fold change above or below this baseline. The mean [Ca2þ]i was calculated for each time point and plotted against time of exposure to illustrate temporal changes in [Ca2þ]i following CP exposure. In contrast, the highest [Ca2þ]i observed during the observation period (peak) and the [Ca2þ]i at the conclusion of measurements were calculated for statistical comparisons between treatments (type of CP and the presence or absence of extracellular Ca2þ). Data involving continuous variables ([Ca2þ]i and time of zona pellucida dissolution) was analysed with the mixed-model procedure in SAS (SAS Institute Inc., Cary, NC, USA; Littell et al. 1996). For oocyte degeneration and embryonic development, each oocyte or embryo was scored as a 1 or 0 depending on whether or not it degenerated or achieved the desired stage of development (cleaved or blastocyst). Data was then analysed using the generalised linear mixed-model (GLIMMIX) procedure in SAS with a binomial error distribution and a probit link function (Littell et al. 1996). In all analyses, treatment was considered a fixed factor and replicate and replicate-by-treatment were included in the model as random factors. Pairwise comparisons were made using Fisher’s protected least-significant difference test and P , 0.05 was considered to be a significant difference. All values are presented as the mean  s.e.m.

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Results Changes in concentrations of intracellular Ca21 after CP exposure All three of the tested cryoprotectants (EG, PrOH and DMSO) altered [Ca2þ]i in feline oocytes, (Fig. 1). Changes in [Ca2þ]i were first observed at ,180 s, or ,60 s after the concentration of CP was increased from 0 to 10%. Increasing the concentration of CP from 10 to 20% at 240 s did not cause further increases in [Ca2þ]i and varying degrees of recovery (return to baseline [Ca2þ]i) were observed between 240 and 360 s depending on the CP and the presence of extracellular Ca2þ. For example, the [Ca2þ]i of oocytes 360 s after exposed to EG was lower (closer to baseline) than that of oocytes exposed to PrOH when extracellular Ca2þ was present, but higher (further from baseline) than that of PrOH-exposed oocytes when extracellular Ca2þ was absent. The peak [Ca2þ]i during exposure was greater (P , 0.05) than that observed in control oocytes for all CPs (Fig. 2a). The [Ca2þ]i at the conclusion of exposure was increased (P , 0.05) compared with control oocytes after exposure to EG or PrOH, but was not affected by exposure to DMSO (P . 0.05; Fig. 2b). Neither peak [Ca2þ]i nor the [Ca2þ]i at the conclusion of exposure were significantly affected (P . 0.05) by the presence of Ca2þ in the extracellular medium for any of the CPs, but [Ca2þ]i was higher (0.05 , P , 0.1) after exposure to EG

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Seconds Fig. 1. Fold change in the intracellular concentration of free Ca2þ ([Ca2þ]i) over baseline (prior to chemical addition) in feline oocytes exposed to (a) dimethyl sulfoxide (DMSO), (b) ethylene glycol and (c) propanediol in the presence (n ¼ 17, 17, 14 oocytes, respectively) or absence (Ca2þ-free, n ¼ 12, 14, 14 oocytes, respectively) of extracellular Ca2þ. Chemicals were added at 2-min intervals such that oocytes were sequentially exposed to 0% (0 to 120 sec), 10% (120 to 240 sec), 20% (240 to 360 sec) and 40% (360 to 480 sec) solution of CP.

and PrOH in Ca2þ-free medium compared with media containing Ca2þ. Resistance of the zona pellucida to enzymatic digestion after CP exposure The presence of Ca2þ in the medium during CP exposure did not affect (P . 0.05) the resistance of the ZP to pronase (Fig. 3). Only exposure to PrOH resulted in a significant (P , 0.05)

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Fig. 3. Time (seconds) taken to dissolve the zona pellucida with pronase following exposure to dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propanediol (PrOH) in the presence (n ¼ 22, 19, 20 oocytes, respectively) or absence (Ca2þ-free, n ¼ 21, 15, 19 oocytes, respectively) of extracellular Ca2þ compared with oocytes that were not exposed to chemicals (Control; Ca2þ, n ¼ 22 oocytes; Ca2þ-free, n ¼ 18 oocytes). The presence Ca2þ in the extracellular medium did not affect (P . 0.05) digestion of the zona pellucida in any of the treatments. Different letters (a, b, c) indicate a significant difference between treatments.

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Fig. 2. (a) Peak and (b) final intracellular concentration of free Ca2þ (fold change over baseline, [Ca2þ]i) in feline oocytes exposed to dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propanediol (PrOH) in the presence (n ¼ 17, 17, 14 oocytes, respectively) or absence (Ca2þ-free, n ¼ 12, 14, 14 oocytes, respectively) of extracellular Ca2þ compared with oocytes not exposed to chemicals (Ca2þ, n ¼ 15 oocytes; Ca2þ-free, n ¼ 9 oocytes). The concentration of each chemical increased from 0 to 40% (by volume) during measurements. The presence of Ca2þ in the extracellular medium did not affect (P . 0.05) either endpoint. Different letters (a, b, c) indicate a significant difference between treatments.

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hardening of the ZP, while DMSO and EG actually softened and weakened (P , 0.05) the ZP of feline oocytes (Fig. 3).

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Parthenogenetic activation and degeneration after CP exposure The incidence of parthenogenetic activation was ,5% for all treatments (data not shown). However, exposure to EG (44.1– 52.4%) and DMSO (19.7%) caused a significant (P , 0.05) proportion of oocytes to degenerate within 16 h of exposure to CP compared with control oocytes (#1.9%) and oocytes exposed to PrOH (#2.5%; Fig. 4).

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Developmental competence of oocytes after CP exposure For control (maintained in FOCMH without CP exposure) and treated oocytes, $55% of oocytes cleaved following IVF and $26% of oocytes developed to the blastocyst stage (P . 0.05; Fig. 5). When blastocyst development was evaluated as the proportion of cleaved embryos, the combination of PrOH and DMSO (52.1%) was not different from control oocytes (64.4%; P . 0.05).

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Fig. 4. Incidence of degeneration (fragmentation or lysis) within 16 h of exposure to dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propanediol (PrOH) in the (a) presence (n ¼ 63, 60, 60 oocytes, respectively) or (b) absence (Ca2þ-free, n ¼ 43, 44, 44 oocytes, respectively) of extracellular Ca2þ compared with oocytes not exposed to chemicals (Control; Ca2þ, n ¼ 55 oocytes; Ca2þ-free, n ¼ 54 oocytes). Different letters (a, b, c) indicate a significant difference between treatments.

Developmental competence of oocytes after vitrification When oocytes were vitrified with a combination of PrOH and DMSO (10% each), 74.2% of oocytes retained an intact plasma

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membrane after warming. Of these oocytes, 28.3% cleaved following IVF. However, no embryos developed to the blastocyst stage. When a non-permeating CP (galactose, 0.25 M) was added to the vitrification medium, a similar percentage of oocytes retained an intact plasma membrane after warming (P . 0.05, 69.8%), but 47.7% of the oocytes cleaved and 14.3% of cleaved embryos developed to the blastocyst stage (Table 1, Fig. 6).

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Fig. 5. Development of feline oocytes exposed to dimethyl sulfoxide (DMSO, 20%; n ¼ 59 oocytes), propanediol (PrOH, 20%; n ¼ 64 oocytes) or DMSO and PrOH (10% each; n ¼ 67 oocytes) compared with oocytes not exposed to chemicals (Control, n ¼ 120 oocytes) following in vitro fertilisation and embryo culture (7 days). NS or the same letter (a) above columns indicates no significant difference (P . 0.05) between treatments.

Table 1. Developmental potential of feline oocytes vitrified with 10% DMSO and 10% PrOH with or without galactose (0.25 M) following warming and IVF Galactose (M) 0 0.25

Vitrified (n) 62 63

Cleaved Blastocyst SurvivedA (n, %) (n, % per survived) (n, % per cleaved) 46 (74.2%) 44 (69.8%)

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A Oocytes retained an intact plasma membrane, based on visual observations, after warming.

Fig. 6. Blastocyst produced from an oocyte that was vitrified in the presence of 10% (v/v) DMSO, 10% PrOH and 0.25 M galactose stained with Hoechst 33342 on Day 7 after insemination to determine cell number (n ¼ 167). Scale bar represents approximately 125 mm.

Although some vitrified feline oocyte are capable of producing pregnancies following warming and IVF, the development of vitrified–warmed oocytes remains low compared with nonvitrified oocytes and there is still much room for improvement in the efficiency of this technique. It is our hypothesis that the low success rate of vitrification is due, at least in part, to the use of protocols developed for oocytes from other species. By assessing several responses (changes in [Ca2þ]i, ZP hardening, parthenogenetic activation, degeneration and embryonic development following IVF) following exposure to three commonly used CPs (EG, PrOH and DMSO), DMSO and PrOH were identified as having minimal effects on the viability of feline oocytes. In other species, there is an interaction between CP exposure and the concentration of Ca2þ in the medium (Takahashi et al. 2004; Larman et al. 2006; Fujiwara et al. 2010; Succu et al. 2011). Larman et al. (2006, 2007) demonstrated that exposing murine oocytes to EG, DMSO and PrOH caused an increase in [Ca2þ]i. Interestingly the source of the Ca2þ resulting in this increase was dependent on the CP. Ethylene glycol and PrOH only induced an increase in [Ca2þ]i when Ca2þ was present in the medium, suggesting an influx of extracellular Ca2þ. In contrast, exposure to DMSO increased [Ca2þ]i even when the medium was devoid of Ca2þ, indicating the release of Ca2þ from intracellular stores. In oocytes from mice and sheep these increases in [Ca2þ]i have been associated with hardening of the zona pellucida and parthenogenetic activation of the oocytes (Larman et al. 2006; Succu et al. 2011). Interestingly, feline oocytes demonstrated some remarkable differences from oocytes of these other species. First, the increases in [Ca2þ]i induced by EG, PrOH and DMSO were all due to the release of Ca2þ from intracellular pools, since the presence or absence of Ca2þ in the medium did not affect the change in [Ca2þ]i. Second, these changes in [Ca2þ]i did not result in hardening of the zona pellucida. In fact, exposure to EG and DMSO actually reduced the time needed for enzymatic digestion of the zona pellucida. Finally, the incidence of parthenogenetic activation (,5%) was much lower than that reported in other species exposed to CPs, even though the amount of Ca2þ released (,2 greater than baseline) was similar to that achieved with activation protocols for feline oocytes (Wang et al. 2009). Although the effects of EG, PrOH and DMSO on [Ca2þ]i were similar, the effects of these chemicals on oocyte viability varied greatly between CPs and from what has been reported in other species. In mice, humans and pigs, oocyte viability and subsequent embryo development are improved when EG is the only CP present during vitrification compared with vitrification

Effects of cryoprotectants on feline oocytes

in the presence of PrOH alone (Szurek and Eroglu 2011; Taniguchi et al. 2011; Seet et al. 2013). As a result of these and other studies, EG, alone or in combination with DMSO, is used extensively for vitrification of oocytes and embryos from a variety of species with excellent results. However, just the opposite was observed in feline oocytes. Exposure to PrOH did not affect the incidence of oocyte degeneration, while ,20% of oocytes degenerated after exposure to DMSO and ,50% degenerated after exposure to EG. Similar results were reported by Comizzoli et al. (2004) following exposure of germinalvesicle-stage feline oocytes to 1.5 M PrOH (,11% v/v) or EG at 258C. The proportion of oocytes with a normal metaphase II spindle after in vitro maturation (IVM), embryonic development after IVF and blastocyst cell number were all improved when oocytes were exposed to PrOH. Despite these findings of Comizzoli et al. (2004), most of studies on the vitrification of feline oocytes, including those with high rates of blastocyst development (Merlo et al. 2008; Cocchia et al. 2010) and pregnancies (Tharasanit et al. 2011; Pope et al. 2012) have used EG, alone or in combination with DMSO. However, the production of viable embryos in media containing EG or DMSO should not be interpreted as an indication that these CPs are optimal, just that some embryos survive and are capable of development. Even in the present study, not all EG-exposed oocytes degenerated after exposure. In fact, ,50% of EG-exposed oocytes appeared to survive based on gross morphology, but no attempt was made to conduct IVF with these oocytes. It is also possible that other differences between studies, including the temperature and duration of CP exposure (Larman et al. 2007; Szurek and Eroglu 2011), the composition of the medium used to dilute the CPs (Wusteman et al. 2008; Zander-Fox et al. 2013), the rate of CP exposure (Tharasanit et al. 2011) or the type and concentration of macromolecule present (Checura and Seidel 2007; Mikołajewska et al. 2012), influenced the oocyte’s response to the CPs. It would be interesting to compare the efficiency of those protocols that have produced pregnancies or kittens following the direct substitution of PrOH and DMSO for EG or DMSO. Due to the high incidence of degeneration induced by exposure to EG, experiments evaluating the effects of CP exposure on embryonic development focussed on DMSO and PrOH. Two other modifications were made to the experimental protocol used for CP exposure for experiments involving IVF. First, oocytes were exposed to CPs in a total of four steps (2.5%, 5%, 10% and 20% by volume), instead of the two-step protocol used in previous experiments. Preliminary studies indicated that a slower, stepwise exposure greatly improved IVF success (data not shown), which is in agreement with studies in other species (Wang et al. 2010), as well as a study of immature feline oocytes (Tharasanit et al. 2011). Another important change for these experiments was the use of a combination of CPs. By using two or more CPs, the concentration of each can be reduced to avoid concentration-dependent effects of these chemicals (Szurek and Eroglu 2011; Seet et al. 2013). Using the four-step CP exposure, the proportion of oocytes that cleaved and developed to the blastocyst stage was not different between oocytes exposed to DMSO or PrOH and control oocytes. Interestingly, there was a trend (P ¼ 0.051) for

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improved cleavage following exposure to DMSO. However, when embryonic development was expressed as a proportion of cleaved embryos, there was a trend (P ¼ 0.06) for reduced development following exposure to either DMSO or PrOH alone. In contrast, the combination of two CPs allowed a similar proportion of cleaved embryos to develop to the blastocyst stage (52.1%) as observed in the control oocytes (64.4%). Although vitrification media typically contain 30 to 40% (v/v) CPs, the concentration of CPs is not the only variable that affects the probability of successful vitrification. Increasing the cooling and warming rates, increasing the viscosity of the medium or decreasing the sample volume will all increase the likelihood of vitrification (Yavin and Arav 2007). In the original description of embryo vitrification, Rall and Fahy (1985) used media containing 20.5% (w/v) DMSO and 10% (w/v) PrOH, as well as acetamide and poly(ethylene glycol), to vitrify mouse embryos, but cooling and warming rates were ,25008C min1 and embryos were contained in 45 mL medium within a plastic straw. In contrast, Seki and Mazur (2012) reported cooling and warming rates of 69 250 and 117 5008C min1, respectively, for a Cryotop in which embryos were contained in ,0.1 mL of medium. Seki and Mazur (2012) went on to show that with these parameters, mouse oocytes would survive vitrification even if the vitrification medium, and therefore the concentrations of CPs, was diluted by 50%. Based on these findings, we attempted to vitrify feline oocytes with reduced concentrations of DMSO and PrOH. Aside from the study of Pope et al. (2012) in which .90% of vitrified oocytes survived and 53% of surviving oocytes cleaved, most studies of feline oocytes have reported survival rates of ,50% and cleavage rates of 20–30% (Murakami et al. 2004; Merlo et al. 2008; Cocchia et al. 2010; Tharasanit et al. 2011). In our study, oocyte survival after warming (intact plasma membrane ,75%) and cleavage following IVF (,30%) were within the range of these other studies, even though we utilised lower concentrations of CPs (20% by volume) compared with other studies of feline oocytes (30 to 40% CPs). Unfortunately, none of the oocytes vitrified with DMSO and PrOH were capable of developing to the blastocyst stage. However, the addition of galactose (0.25 M) instead of sucrose increased the proportion of vitrified oocytes that cleaved to 47.7% and allowed 14.3% of those embryos to develop to the blastocyst stage. This was not surprising given the reported benefits of including sugars in the vitrification media used for oocytes of other species. Mono- or di-saccharides can buffer against osmotic changes involved with CP exposure and can alter the vitrification properties of the medium, increasing the temperature at which vitrification occurs (McWilliams et al. 1995; Kuleshova et al. 1999). Sucrose is perhaps the most commonly used sugar for vitrification in other species, but Murakami et al. (2004) reported that exposing feline oocytes to 0.5 M sucrose can be inhibitory to development. Although other studies of feline oocytes have used similar concentrations of sucrose without the negative effects reported by Murakami et al. (2004), galactose (0.25 M) was used in the present study based on the successful use of this sugar for vitrification of bovine oocytes (Checura and Seidel 2007) and beneficial effects in other cell types (Chaytor et al. 2012).

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In conclusion, we have identified a combination of two CPs, DMSO and PrOH, that maintain the viability of mature feline oocytes following exposure alone and support adequate embryonic development following vitrification and warming. Importantly, development of even a couple of embryos to the blastocyst stage was a significant achievement considering that only one of the many factors (type of CP) that affect the success of vitrification was evaluated. However, the presence of a sugar in the vitrification medium appears to be critical for blastocyst development. Evaluating different types and concentrations of both mono- and di-saccharides may be an important next step in optimising vitrification protocols for feline oocytes. For example, trehalose was recently shown to maintain germinal vesicle membrane integrity following oocyte desiccation and storage at 48C, suggesting that this sugar may have tremendous potential for vitrification (Graves-Herring et al. 2013). Further improvements will likely depend on the optimisation of additional aspects of the vitrification protocol, including the composition of the medium (Wusteman et al. 2008; Mikołajewska et al. 2012; Zander-Fox et al. 2013), the rate of CP addition or removal (Wang et al. 2011) as well as the warming rate (Seki and Mazur 2012). Acknowledgements The authors would like to thank the Animal Protective League, The Feline Fix, Spay Today and Northern Colorado Friends of Ferals for their generous assistance with this research. This work was supported by the Morris Animal Foundation (D10ZO-824A).

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