Theriogenology xxx (2015) 1–8
Contents lists available at ScienceDirect
Theriogenology journal homepage: www.theriojournal.com
Effects of two combinations of cryoprotectants on the in vitro developmental capacity of vitrified immature porcine oocytes A. Nohalez, C.A. Martinez, M.A. Gil, C. Almiñana, J. Roca, E.A. Martinez, C. Cuello* Department of Animal Medicine and Surgery, University of Murcia, Murcia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 February 2015 Received in revised form 24 March 2015 Accepted 11 April 2015
This study evaluated two cryoprotectant (CPA) combinations, ethylene glycol (EG) þ DMSO and EG þ propylene glycol (PG), used for the vitrification of germinal vesicle (GV) porcine oocytes. In experiment 1, the equilibration of GV with the two CPA combinations increased (P < 0.05) the percentage of oocytes that degenerated after IVM (18.1 2.3% and 19.4 2.6% for EG þ DMSO and EG þ PG groups, respectively) compared with control oocytes (7.6 1.3%). However, CPAs did not affect the fertilization or developmental parameters of the embryos. In experiment 2, the percentages of live vitrified–warmed GV oocytes at 2 hours after warming (EG þ DMSO: 67.0 2.3% and EG þ PG: 57.6 2.3%) were lower than those of fresh control GV oocytes (97.3 0.7%). The percentage of degenerated oocytes after IVM was higher (P < 0.001) in vitrified–warmed oocytes (EG þ DMSO: 59.8 2.3% and EG þ PG: 56.2 2.6%) than in the control (1.6 1.3). Fertilization efficiency was higher (P < 0.05) in the EG þ PG (39.6 2.4%) and control (42.0 2.2%) groups than in the EG þ DMSO (26.3 7.7%) group. The cleavage and blastocyst formation rates of the EG þ DMSO (25.9 3.5% and 6.6 2.5%, respectively) and EG þ PG (20.2 5.4% and 4.7 1.6%, respectively) vitrification groups were lower (P < 0.001) than those observed in the control oocytes (53.4 2.7% and 31.9 1.7%, respectively). In conclusion, in the absence of vitrification, the toxic effects of both CPA combinations on the GV oocytes were minimal. Vitrification resulted in important losses in viability at each step of the in vitro embryo production procedure. However, the surviving oocytes were able to mature and be fertilized, although the fertilization efficiency in the EG þ DMSO group was lower. Blastocysts formation was similar for both CPA combinations. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Vitrification Immature oocyte Porcine Cryoprotectant
1. Introduction Successful cryopreservation of pig oocytes is crucial for the preservation and management of valuable genetic resources. In addition, oocyte cryopreservation could be important for the application of reproductive technologies, such as in vitro embryo production, genetic engineering,
* Corresponding author. Tel.: þ34 868884812; fax: þ34 868887069. E-mail address: [email protected] (C. Cuello). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.04.004
and nuclear transfer [1]. Unfortunately, oocyte cryopreservation in pigs is very difficult compared with other domestic species [2]. The greater hypothermic sensitivity of porcine oocytes has been ascribed to the large amount of cytoplasmic lipids [3]; fatty acid quantity in immature porcine oocytes is twice that in bovines and even threefold greater than in sheep [4]. Successful cryopreservation of porcine oocytes was first achieved by Isachenko et al. [5], and since then, much research has been focused on improving the cryopreservation protocols for this species. Currently, vitrification
2
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appears to be the best alternative for cryopreserving oocytes; ultrarapid vitrification procedures result in high cooling rates during vitrification, which reduces the adverse effects associated with high concentrations of cryoprotectants (CPAs) and decreases injury from chilling [3]. Development to the blastocyst stage has been achieved in mature and immature porcine oocytes that have been vitrified using the solid-surface vitrification [1,6], open pulled straw [7], and Cryotop [8] methods. The meiotic stage highly influences the ability of porcine oocytes to be vitrified, and metaphase II (MII) oocytes are traditionally preferred for vitrification. Although the survival of oocytes matured in vitro after vitrification is relatively high, their fertilization and developmental competence are seriously compromised [9,10]. Previous investigations have revealed a failure in male pronuclear formation in vitrified MII oocytes after fertilization [11]. Vitrification at the MII stage also induces parthenogenesis and reduces cytoplasmic glutathione levels [11]. In contrast, oocytes vitrified as cumulus cell–oocyte complexes (COCs) at the germinal vesicle (GV) stage display low survival rates but maintain their capacity to undergo normal fertilization [9]. In addition, vitrification at this stage may avoid spindle damage [10], which is one of the most common alterations observed in vitrified MII oocytes [12]. Although live offspring have been obtained from vitrified immature oocytes [13], the total yield of blastocyst stage embryos remains very low. One of the factors limiting the vitrification of immature oocytes is their lower permeability to CPAs compared with matured oocytes [14,15], which may result in low survival rates. Furthermore, the presence of cumulus cells affects permeability, decreasing the efficiency of vitrification protocols [16]. Denudation is not an option for addressing this problem because cumulus cells are important for the cytoplasmic maturation of oocytes [17,18] and contribute to mitochondrial functions [19]. Therefore, the selection of highly permeable combinations of CPAs should be the strategy used to overcome this limitation. Combining CPAs has the advantage of reducing the total CPA concentration needed for vitrification, which reduces the toxicity of the vitrification solution. The permeating CPAs most commonly used for oocyte cryopreservation are ethylene glycol (EG), glycerol [20], DMSO [1], propylene glycol (PG) [13], and acetamide [21]. Among them, EG, which has high permeability and low toxicity, has been shown to be the most effective CPA for porcine oocytes [22,23] and has been used alone [23] or in combination with DMSO [24,25]. Although the combination of EG and DMSO has been shown to be very effective in the vitrification of porcine embryos [26], DMSO detrimentally affects the meiotic competence of GV stage porcine [1] and murine [27] oocytes. These investigations suggest that DMSO may not be adequate for oocyte vitrification; thus, PG could be an appropriate substitute for DMSO. This CPA is more permeable than DMSO and EG in both porcine oocytes and embryos [28]. The replacement of DMSO with PG would decrease toxicity and increase the permeability of vitrification solutions, which may improve the efficiency of the vitrification of immature porcine oocytes.
Therefore, this study aimed to assess the effectiveness of two combinations of CPAs, EG þ DMSO and EG þ PG, for the vitrification of GV stage porcine oocytes. We investigated the viability, fertilization, and developmental competence of oocytes vitrified using both combinations of CPAs. The vitrification study was preceded by a CPA toxicity experiment. 2. Materials and methods All the experimental procedures used in this study were performed in accordance with Directive 2010/63/EU EEC for animal experiments and were reviewed and approved by the Ethical Committee for Experimentation with Animals of the University of Murcia, Spain (research code: 638/2012). 2.1. Chemicals and media All chemicals used in this study were purchased from Sigma–Aldrich Co. (Alcobendas, Madrid, Spain) unless indicated otherwise. The medium used to transport ovaries from the slaughterhouse to the laboratory was a 0.9% (wt/ vol) NaCl saline solution containing 70 mg/mL of kanamycin. The collection and washing of COCs were performed using a modified Dulbecco’s PBS medium (mDPBS) composed of 136.89-mM NaCl, 2.68-mM KCl, 8.1-mM Na2HPO4, and 1.46mM CaCl2.2H2O, which was supplemented with 4 mg/mL of BSA, 0.34-mM sodium pyruvate, 5.4-mM D-glucose, and 70 mg/mL of kanamycin. The oocyte maturation medium was TCM-199 (Gibco Life Technologies S.A., Barcelona, Spain) supplemented with 0.57-mM cysteine, 0.1% (wt/vol) polyvinyl alcohol (PVA), 10 ng/mL of EGF, 75 mg/mL of penicillin G potassium, and 50 mg/mL of streptomycin sulfate. The basic medium used for IVF was the same as that used by Abeydeera and Day [29], which was designated as modified Tris-buffered medium and consisted of 113.1-mM NaCl, 3mM KCl, 7.5-mM CaCl2.2H2O, 20-mM Tris (Trizma Base), 11-mM D-glucose, and 5-mM sodium pyruvate supplemented with 2-mM caffeine and 0.2% BSA. The embryo culture medium was the North Carolina State University (NCSU-23) medium [30] supplemented with 0.4% (wt/vol) BSA. All vitrification and warming solutions were chemically defined media. The basic medium for vitrification and warming (TL-PVA) was a modification of the protein-free Tyrode medium [31] composed of 124.3-mM NaCl, 3.2mM KCl, 2-mM NaHCO3, 0.34-mM KH2PO4, 10-mM sodium lactate, 0.5-mM MgCl2.6H2O, 2-mM CaCl2.2H2O, 10-mM HEPES, 0.2-mM sodium pyruvate, 12-mM sorbitol, 0.1% (wt/vol) PVA, 75 mg/mL of potassium penicillin G, and 50 mg/ mL of streptomycin sulfate. The first vitrification medium (V1) was TL-PVA containing 7.5% (v:v) of each CPA, and the second vitrification medium (V2) was TL-PVA containing 16% (v:v) of each CPA and 0.4-M sucrose. The warming medium consisted of TL-PVA supplemented with 0.13-M sucrose. 2.2. Oocyte collection and oocyte in vitro maturation Ovaries were obtained from prepubertal gilts at a local slaughterhouse (El Pozo S.A., Alhama, Murcia, Spain) and transported to the laboratory in medium at 35 C. Oocytes
A. Nohalez et al. / Theriogenology xxx (2015) 1–8
were collected by scraping intact healthy antral follicles (3– 6 mm in diameter) with a surgical blade, and oocytes surrounded by a compact cumulus mass with an evenly granulated and dark cytoplasm were selected. For IVM, oocytes were washed three times in maturation medium, and 70 to 80 oocytes were transferred into each well of a Nunc 4-well multidish (Nunc, Roskilde, Denmark) containing 500 mL of maturation medium covered with warm mineral oil. Oocytes were incubated at 39 C in air with 5% CO2 and 95% to 97% of relative humidity for 22 hours in the maturation medium supplemented with 10 IU/mL of eCG (Folligon; Intervet International B.V., Boxmeer, the Netherlands) and 10 IU/mL of hCG (Veterin Corion; Divasa Farmavic, S.A., Barcelona, Spain) and incubated again for another 22 hours in the same medium without hormones. 2.3. Oocyte viability assessment Oocyte viability was evaluated using fluorescein diacetate (FDA) staining as described by Mohr and Trounson [32]. A stock solution of 5 mg/mL of FDA was prepared in acetone and stored for a maximum of 3 days in polypropylene tubes at 20 C. The working solution was prepared on the day of oocyte evaluation by a 1:2000 dilution of the FDA stock solution in mDPBS to reach a final concentration of 2.5 mg/mL of FDA. The COCs were stained in 500 mL of this solution for 2 minutes at 37 C in a dark room, then washed three times in mDPBS, and individually placed in drops of 20 mL of maturation medium overlaid with warm mineral oil. Oocytes were evaluated with ultraviolet irradiation using a GFP-II filter under an inverted fluorescence microscope. Live oocytes exhibited bright green fluorescence, and FDA viability was calculated as the percentage of live oocytes of the total number of oocytes evaluated. 2.4. In vitro fertilization After maturation, IVF was performed as described by Gil et al. [33]. Briefly, oocytes were treated with 0.1% (wt/vol) hyaluronidase in NCSU-23 medium and vortexed for 2 minutes at 1660 rounds/minute to remove the cumulus cells. Denuded oocytes were washed three times in maturation medium and three times in preequilibrated fertilization medium. After washing, denuded oocytes were placed into 50-mL drops of fertilization medium in a 35 10-mm Petri dish (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ, USA) under mineral oil. The oocytes were incubated at 39 C in an atmosphere of 5% CO2 for 30 minutes until the spermatozoa were added. Semen from a mature boar was processed and cryopreserved in 0.5-mL straws as described by Roca et al. [34]. The straws were thawed in a circulating water bath at 37 C for 20 seconds. Then, 100 mL of thawed semen was washed three times by centrifugation at 1900 g for 3 minutes in 10 mL of mDPBS, and the resulting pellet was suspended in the fertilization medium. After appropriate dilution, 50 mL of this sperm suspension was added to a 50-mL drop of fertilization medium containing the oocytes. The spermatozoa:oocyte ratio was 1000:1. The oocytes were incubated with the spermatozoa for 5 hours at 39 C in a humidified atmosphere of 5% CO2.
3
2.5. Assessment of fertilization parameters Eighteen hours after insemination, some presumptive zygotes were mounted on slides, fixed for 48 to 72 hours in 25% (v:v) acetic acid in ethanol at room temperature, stained with 1% lacmoid in 45% (v:v) acetic acid, and examined under a phase-contrast microscope at 400 magnification. Oocytes were considered penetrated when they contained one or more swollen sperm heads and/or male pronuclei with their corresponding sperm tails and two polar bodies. The fertilization parameters evaluated were as follows: penetration (percentage of the number of penetrated oocytes/total oocytes inseminated), monospermy (percentage of the number of monospermic oocytes/total oocytes penetrated), number of penetrated spermatozoa/oocyte (mean number of spermatozoa in penetrated oocytes), and efficiency of fertilization (number of monospermic oocytes/total oocytes inseminated). Oocytes at the GV stage and those oocytes with a plate but no polar body (MI oocytes) were classified as immature oocytes, and oocytes with a broken oolemma or abnormal cytoplasm were considered degenerated. 2.6. In vitro culture and assessment of in vitro embryo development and total number of blastocyst cells Presumptive zygotes were cultured for 7 days in a fourwell multidish plate containing 500 mL of NCSU-23 medium covered with mineral oil. Presumptive zygotes were cultured for the first 2 days in glucose-free NCSU-23 medium supplemented with 0.33-mM pyruvate and 4.5-mM lactate and then in fresh NCSU-23 medium containing 5.5-mM glucose supplemented with 10% fetal calf serum at 39 C in humidified air with 5% CO2 and 95% to 97% relative humidity. Two days after fertilization, the cleavage rate (number of two- and four-cell embryos/total cultured embryos) was recorded. On Day 7, the blastocyst formation rate (number of blastocysts/total cleaved embryos) and the total blastocyst formation rate (number of blastocysts/total cultured embryos) were assessed. The total cell number, as an indicator of embryo quality, was evaluated by mounting each blastocyst on a slide in 4 mL of Vectashield (Vector Laboratories, Burlingame, CA, USA) containing 10 mg/mL of Hoechst 33342 under a coverslip. Samples were analyzed by fluorescence microscopy using a 330- to 380-nm excitation filter, and the total number of Hoechst-stained nuclei that displayed blue fluorescence was assessed. Only embryos without a visible perivitelline space and a clear blastocele containing more than 10 blastomeres were considered blastocysts. All blastocysts were subjected to total cell number assessment. 2.7. Vitrification and warming Vitrification was performed according to the method described by Berthelot et al. [35] and modified by Cuello et al. [26]. Oocytes were handled in media at 39 C. Groups of five to seven oocytes were washed twice in TL-PVA and sequentially equilibrated in V1 for 3 minutes and V2 for 1 minute for vitrification. During the final equilibration, oocytes were placed in a 1-mL drop and loaded in the
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narrow end of a super open pulled straw (SOPS; Minitüb, Tiefenbach, Germany) by capillary action. Subsequently, straws containing the oocytes were plunged horizontally into liquid nitrogen. After storage in liquid nitrogen for 2 weeks, the straws were removed and warmed by the onestep warming method [36,37]. Briefly, the straws were vertically submerged in one well of a four-well multidish plate containing 800 mL of warming medium and equilibrated for 5 minutes. 2.8. Experimental design 2.8.1. Experiment 1 In the first experiment, we evaluated in three replicates the toxicity of the vitrification media prepared with two different CPA combinations (EG þ DMSO or EG þ PG groups). Oocytes were subjected to equilibration in the vitrification and warming solutions as described previously, but the oocytes were not vitrified. Oocytes equilibrated in TL-PVA medium without CPAs and then in the warming solution were used as controls. Oocyte viability was examined by FDA staining 2 hours after treatment, and live oocytes selected from the control and treatment groups were matured in vitro. Oocytes with lysed cytoplasmic membranes at 44 hours of maturation were considered to be degenerated and were not selected for IVF. At 18 hours after IVF, some presumptive zygotes were fixed to assess the fertilization parameters. At this time, immature and degenerated oocytes were not considered for the calculation of penetration, monospermy, and efficiency rates. The remaining presumptive zygotes were cultured in vitro, and the developmental parameters of the embryos derived from oocytes exposed to CPAs were compared with those from control oocytes. 2.8.2. Experiment 2 The second experiment was designed to evaluate the following: (1) Oocyte viability and fertilization parameters obtained with GV oocytes vitrified using either EG þ DMSO or EG þ PG. A total of four replicates were performed. Oocytes were subjected to equilibration in vitrification media and vitrified and warmed as described previously. Nonvitrified oocytes were used as controls. The treated and control oocytes were selected and evaluated as described in experiment 1. Oocytes were fixed 18 hours after IVF to assess their fertilization parameters. (2) Embryo developmental competence of GV oocytes vitrified with either EG þ DMSO or EG þ PG. A total of four experimental replicates were performed. Oocytes were subjected to equilibration in vitrification media and vitrified and warmed as described previously. Nonvitrified oocytes were used as controls. After 44 hours of IVM, oocytes were selected for IVF as described in experiment 1. Cleavage rate, blastocyst formation, and blastocyst total cell number from the EG þ DMSO and
EG þ PG vitrification groups were compared with those derived from the nonvitrified control oocytes. 2.9. Statistical analysis Statistical analysis was performed using the IBM SPSS 19 statistics package (SPSS, Chicago, IL, USA), and differences were considered significant at P < 0.05. For statistical purposes, each drop was considered an experimental unit. The mean standard error of the mean of the binary variables (viability; matured, fertilized, and monospermic oocytes; cleaved embryos; blastocyst formation; efficiency) and continuous variables (total cell number per blastocyst) were obtained after calculating the percentage in every drop of each group and in each replicate. Significant differences between the groups were assessed by mixed linear ANOVA with treatment as a fixed effect and the replicate as a random effect. When the ANOVA revealed a significant effect, values were compared using the Bonferroni test. 3. Results 3.1. Experiment 1 The FDA viability of the oocytes after 2 hours of equilibration in EG þ DMSO– and EG þ PG–based media was very high (95.7 0.4% and 95.6 1.1%, respectively) but lower (P < 0.01) than that observed in the control group (99.5 0.8%). Higher (P < 0.05) proportions of live oocytes from the EG þ DMSO (18.1 2.3%) or EG þ PG (19.4 2.6%) groups were degenerated at 44 hours of maturation compared with those in the control group (7.6 1.3%). At 18 hours after IVF, the numbers of immature oocytes (
Contents lists available at ScienceDirect
Theriogenology journal homepage: www.theriojournal.com
Effects of two combinations of cryoprotectants on the in vitro developmental capacity of vitrified immature porcine oocytes A. Nohalez, C.A. Martinez, M.A. Gil, C. Almiñana, J. Roca, E.A. Martinez, C. Cuello* Department of Animal Medicine and Surgery, University of Murcia, Murcia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 February 2015 Received in revised form 24 March 2015 Accepted 11 April 2015
This study evaluated two cryoprotectant (CPA) combinations, ethylene glycol (EG) þ DMSO and EG þ propylene glycol (PG), used for the vitrification of germinal vesicle (GV) porcine oocytes. In experiment 1, the equilibration of GV with the two CPA combinations increased (P < 0.05) the percentage of oocytes that degenerated after IVM (18.1 2.3% and 19.4 2.6% for EG þ DMSO and EG þ PG groups, respectively) compared with control oocytes (7.6 1.3%). However, CPAs did not affect the fertilization or developmental parameters of the embryos. In experiment 2, the percentages of live vitrified–warmed GV oocytes at 2 hours after warming (EG þ DMSO: 67.0 2.3% and EG þ PG: 57.6 2.3%) were lower than those of fresh control GV oocytes (97.3 0.7%). The percentage of degenerated oocytes after IVM was higher (P < 0.001) in vitrified–warmed oocytes (EG þ DMSO: 59.8 2.3% and EG þ PG: 56.2 2.6%) than in the control (1.6 1.3). Fertilization efficiency was higher (P < 0.05) in the EG þ PG (39.6 2.4%) and control (42.0 2.2%) groups than in the EG þ DMSO (26.3 7.7%) group. The cleavage and blastocyst formation rates of the EG þ DMSO (25.9 3.5% and 6.6 2.5%, respectively) and EG þ PG (20.2 5.4% and 4.7 1.6%, respectively) vitrification groups were lower (P < 0.001) than those observed in the control oocytes (53.4 2.7% and 31.9 1.7%, respectively). In conclusion, in the absence of vitrification, the toxic effects of both CPA combinations on the GV oocytes were minimal. Vitrification resulted in important losses in viability at each step of the in vitro embryo production procedure. However, the surviving oocytes were able to mature and be fertilized, although the fertilization efficiency in the EG þ DMSO group was lower. Blastocysts formation was similar for both CPA combinations. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Vitrification Immature oocyte Porcine Cryoprotectant
1. Introduction Successful cryopreservation of pig oocytes is crucial for the preservation and management of valuable genetic resources. In addition, oocyte cryopreservation could be important for the application of reproductive technologies, such as in vitro embryo production, genetic engineering,
* Corresponding author. Tel.: þ34 868884812; fax: þ34 868887069. E-mail address: [email protected] (C. Cuello). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.04.004
and nuclear transfer [1]. Unfortunately, oocyte cryopreservation in pigs is very difficult compared with other domestic species [2]. The greater hypothermic sensitivity of porcine oocytes has been ascribed to the large amount of cytoplasmic lipids [3]; fatty acid quantity in immature porcine oocytes is twice that in bovines and even threefold greater than in sheep [4]. Successful cryopreservation of porcine oocytes was first achieved by Isachenko et al. [5], and since then, much research has been focused on improving the cryopreservation protocols for this species. Currently, vitrification
2
A. Nohalez et al. / Theriogenology xxx (2015) 1–8
appears to be the best alternative for cryopreserving oocytes; ultrarapid vitrification procedures result in high cooling rates during vitrification, which reduces the adverse effects associated with high concentrations of cryoprotectants (CPAs) and decreases injury from chilling [3]. Development to the blastocyst stage has been achieved in mature and immature porcine oocytes that have been vitrified using the solid-surface vitrification [1,6], open pulled straw [7], and Cryotop [8] methods. The meiotic stage highly influences the ability of porcine oocytes to be vitrified, and metaphase II (MII) oocytes are traditionally preferred for vitrification. Although the survival of oocytes matured in vitro after vitrification is relatively high, their fertilization and developmental competence are seriously compromised [9,10]. Previous investigations have revealed a failure in male pronuclear formation in vitrified MII oocytes after fertilization [11]. Vitrification at the MII stage also induces parthenogenesis and reduces cytoplasmic glutathione levels [11]. In contrast, oocytes vitrified as cumulus cell–oocyte complexes (COCs) at the germinal vesicle (GV) stage display low survival rates but maintain their capacity to undergo normal fertilization [9]. In addition, vitrification at this stage may avoid spindle damage [10], which is one of the most common alterations observed in vitrified MII oocytes [12]. Although live offspring have been obtained from vitrified immature oocytes [13], the total yield of blastocyst stage embryos remains very low. One of the factors limiting the vitrification of immature oocytes is their lower permeability to CPAs compared with matured oocytes [14,15], which may result in low survival rates. Furthermore, the presence of cumulus cells affects permeability, decreasing the efficiency of vitrification protocols [16]. Denudation is not an option for addressing this problem because cumulus cells are important for the cytoplasmic maturation of oocytes [17,18] and contribute to mitochondrial functions [19]. Therefore, the selection of highly permeable combinations of CPAs should be the strategy used to overcome this limitation. Combining CPAs has the advantage of reducing the total CPA concentration needed for vitrification, which reduces the toxicity of the vitrification solution. The permeating CPAs most commonly used for oocyte cryopreservation are ethylene glycol (EG), glycerol [20], DMSO [1], propylene glycol (PG) [13], and acetamide [21]. Among them, EG, which has high permeability and low toxicity, has been shown to be the most effective CPA for porcine oocytes [22,23] and has been used alone [23] or in combination with DMSO [24,25]. Although the combination of EG and DMSO has been shown to be very effective in the vitrification of porcine embryos [26], DMSO detrimentally affects the meiotic competence of GV stage porcine [1] and murine [27] oocytes. These investigations suggest that DMSO may not be adequate for oocyte vitrification; thus, PG could be an appropriate substitute for DMSO. This CPA is more permeable than DMSO and EG in both porcine oocytes and embryos [28]. The replacement of DMSO with PG would decrease toxicity and increase the permeability of vitrification solutions, which may improve the efficiency of the vitrification of immature porcine oocytes.
Therefore, this study aimed to assess the effectiveness of two combinations of CPAs, EG þ DMSO and EG þ PG, for the vitrification of GV stage porcine oocytes. We investigated the viability, fertilization, and developmental competence of oocytes vitrified using both combinations of CPAs. The vitrification study was preceded by a CPA toxicity experiment. 2. Materials and methods All the experimental procedures used in this study were performed in accordance with Directive 2010/63/EU EEC for animal experiments and were reviewed and approved by the Ethical Committee for Experimentation with Animals of the University of Murcia, Spain (research code: 638/2012). 2.1. Chemicals and media All chemicals used in this study were purchased from Sigma–Aldrich Co. (Alcobendas, Madrid, Spain) unless indicated otherwise. The medium used to transport ovaries from the slaughterhouse to the laboratory was a 0.9% (wt/ vol) NaCl saline solution containing 70 mg/mL of kanamycin. The collection and washing of COCs were performed using a modified Dulbecco’s PBS medium (mDPBS) composed of 136.89-mM NaCl, 2.68-mM KCl, 8.1-mM Na2HPO4, and 1.46mM CaCl2.2H2O, which was supplemented with 4 mg/mL of BSA, 0.34-mM sodium pyruvate, 5.4-mM D-glucose, and 70 mg/mL of kanamycin. The oocyte maturation medium was TCM-199 (Gibco Life Technologies S.A., Barcelona, Spain) supplemented with 0.57-mM cysteine, 0.1% (wt/vol) polyvinyl alcohol (PVA), 10 ng/mL of EGF, 75 mg/mL of penicillin G potassium, and 50 mg/mL of streptomycin sulfate. The basic medium used for IVF was the same as that used by Abeydeera and Day [29], which was designated as modified Tris-buffered medium and consisted of 113.1-mM NaCl, 3mM KCl, 7.5-mM CaCl2.2H2O, 20-mM Tris (Trizma Base), 11-mM D-glucose, and 5-mM sodium pyruvate supplemented with 2-mM caffeine and 0.2% BSA. The embryo culture medium was the North Carolina State University (NCSU-23) medium [30] supplemented with 0.4% (wt/vol) BSA. All vitrification and warming solutions were chemically defined media. The basic medium for vitrification and warming (TL-PVA) was a modification of the protein-free Tyrode medium [31] composed of 124.3-mM NaCl, 3.2mM KCl, 2-mM NaHCO3, 0.34-mM KH2PO4, 10-mM sodium lactate, 0.5-mM MgCl2.6H2O, 2-mM CaCl2.2H2O, 10-mM HEPES, 0.2-mM sodium pyruvate, 12-mM sorbitol, 0.1% (wt/vol) PVA, 75 mg/mL of potassium penicillin G, and 50 mg/ mL of streptomycin sulfate. The first vitrification medium (V1) was TL-PVA containing 7.5% (v:v) of each CPA, and the second vitrification medium (V2) was TL-PVA containing 16% (v:v) of each CPA and 0.4-M sucrose. The warming medium consisted of TL-PVA supplemented with 0.13-M sucrose. 2.2. Oocyte collection and oocyte in vitro maturation Ovaries were obtained from prepubertal gilts at a local slaughterhouse (El Pozo S.A., Alhama, Murcia, Spain) and transported to the laboratory in medium at 35 C. Oocytes
A. Nohalez et al. / Theriogenology xxx (2015) 1–8
were collected by scraping intact healthy antral follicles (3– 6 mm in diameter) with a surgical blade, and oocytes surrounded by a compact cumulus mass with an evenly granulated and dark cytoplasm were selected. For IVM, oocytes were washed three times in maturation medium, and 70 to 80 oocytes were transferred into each well of a Nunc 4-well multidish (Nunc, Roskilde, Denmark) containing 500 mL of maturation medium covered with warm mineral oil. Oocytes were incubated at 39 C in air with 5% CO2 and 95% to 97% of relative humidity for 22 hours in the maturation medium supplemented with 10 IU/mL of eCG (Folligon; Intervet International B.V., Boxmeer, the Netherlands) and 10 IU/mL of hCG (Veterin Corion; Divasa Farmavic, S.A., Barcelona, Spain) and incubated again for another 22 hours in the same medium without hormones. 2.3. Oocyte viability assessment Oocyte viability was evaluated using fluorescein diacetate (FDA) staining as described by Mohr and Trounson [32]. A stock solution of 5 mg/mL of FDA was prepared in acetone and stored for a maximum of 3 days in polypropylene tubes at 20 C. The working solution was prepared on the day of oocyte evaluation by a 1:2000 dilution of the FDA stock solution in mDPBS to reach a final concentration of 2.5 mg/mL of FDA. The COCs were stained in 500 mL of this solution for 2 minutes at 37 C in a dark room, then washed three times in mDPBS, and individually placed in drops of 20 mL of maturation medium overlaid with warm mineral oil. Oocytes were evaluated with ultraviolet irradiation using a GFP-II filter under an inverted fluorescence microscope. Live oocytes exhibited bright green fluorescence, and FDA viability was calculated as the percentage of live oocytes of the total number of oocytes evaluated. 2.4. In vitro fertilization After maturation, IVF was performed as described by Gil et al. [33]. Briefly, oocytes were treated with 0.1% (wt/vol) hyaluronidase in NCSU-23 medium and vortexed for 2 minutes at 1660 rounds/minute to remove the cumulus cells. Denuded oocytes were washed three times in maturation medium and three times in preequilibrated fertilization medium. After washing, denuded oocytes were placed into 50-mL drops of fertilization medium in a 35 10-mm Petri dish (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ, USA) under mineral oil. The oocytes were incubated at 39 C in an atmosphere of 5% CO2 for 30 minutes until the spermatozoa were added. Semen from a mature boar was processed and cryopreserved in 0.5-mL straws as described by Roca et al. [34]. The straws were thawed in a circulating water bath at 37 C for 20 seconds. Then, 100 mL of thawed semen was washed three times by centrifugation at 1900 g for 3 minutes in 10 mL of mDPBS, and the resulting pellet was suspended in the fertilization medium. After appropriate dilution, 50 mL of this sperm suspension was added to a 50-mL drop of fertilization medium containing the oocytes. The spermatozoa:oocyte ratio was 1000:1. The oocytes were incubated with the spermatozoa for 5 hours at 39 C in a humidified atmosphere of 5% CO2.
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2.5. Assessment of fertilization parameters Eighteen hours after insemination, some presumptive zygotes were mounted on slides, fixed for 48 to 72 hours in 25% (v:v) acetic acid in ethanol at room temperature, stained with 1% lacmoid in 45% (v:v) acetic acid, and examined under a phase-contrast microscope at 400 magnification. Oocytes were considered penetrated when they contained one or more swollen sperm heads and/or male pronuclei with their corresponding sperm tails and two polar bodies. The fertilization parameters evaluated were as follows: penetration (percentage of the number of penetrated oocytes/total oocytes inseminated), monospermy (percentage of the number of monospermic oocytes/total oocytes penetrated), number of penetrated spermatozoa/oocyte (mean number of spermatozoa in penetrated oocytes), and efficiency of fertilization (number of monospermic oocytes/total oocytes inseminated). Oocytes at the GV stage and those oocytes with a plate but no polar body (MI oocytes) were classified as immature oocytes, and oocytes with a broken oolemma or abnormal cytoplasm were considered degenerated. 2.6. In vitro culture and assessment of in vitro embryo development and total number of blastocyst cells Presumptive zygotes were cultured for 7 days in a fourwell multidish plate containing 500 mL of NCSU-23 medium covered with mineral oil. Presumptive zygotes were cultured for the first 2 days in glucose-free NCSU-23 medium supplemented with 0.33-mM pyruvate and 4.5-mM lactate and then in fresh NCSU-23 medium containing 5.5-mM glucose supplemented with 10% fetal calf serum at 39 C in humidified air with 5% CO2 and 95% to 97% relative humidity. Two days after fertilization, the cleavage rate (number of two- and four-cell embryos/total cultured embryos) was recorded. On Day 7, the blastocyst formation rate (number of blastocysts/total cleaved embryos) and the total blastocyst formation rate (number of blastocysts/total cultured embryos) were assessed. The total cell number, as an indicator of embryo quality, was evaluated by mounting each blastocyst on a slide in 4 mL of Vectashield (Vector Laboratories, Burlingame, CA, USA) containing 10 mg/mL of Hoechst 33342 under a coverslip. Samples were analyzed by fluorescence microscopy using a 330- to 380-nm excitation filter, and the total number of Hoechst-stained nuclei that displayed blue fluorescence was assessed. Only embryos without a visible perivitelline space and a clear blastocele containing more than 10 blastomeres were considered blastocysts. All blastocysts were subjected to total cell number assessment. 2.7. Vitrification and warming Vitrification was performed according to the method described by Berthelot et al. [35] and modified by Cuello et al. [26]. Oocytes were handled in media at 39 C. Groups of five to seven oocytes were washed twice in TL-PVA and sequentially equilibrated in V1 for 3 minutes and V2 for 1 minute for vitrification. During the final equilibration, oocytes were placed in a 1-mL drop and loaded in the
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narrow end of a super open pulled straw (SOPS; Minitüb, Tiefenbach, Germany) by capillary action. Subsequently, straws containing the oocytes were plunged horizontally into liquid nitrogen. After storage in liquid nitrogen for 2 weeks, the straws were removed and warmed by the onestep warming method [36,37]. Briefly, the straws were vertically submerged in one well of a four-well multidish plate containing 800 mL of warming medium and equilibrated for 5 minutes. 2.8. Experimental design 2.8.1. Experiment 1 In the first experiment, we evaluated in three replicates the toxicity of the vitrification media prepared with two different CPA combinations (EG þ DMSO or EG þ PG groups). Oocytes were subjected to equilibration in the vitrification and warming solutions as described previously, but the oocytes were not vitrified. Oocytes equilibrated in TL-PVA medium without CPAs and then in the warming solution were used as controls. Oocyte viability was examined by FDA staining 2 hours after treatment, and live oocytes selected from the control and treatment groups were matured in vitro. Oocytes with lysed cytoplasmic membranes at 44 hours of maturation were considered to be degenerated and were not selected for IVF. At 18 hours after IVF, some presumptive zygotes were fixed to assess the fertilization parameters. At this time, immature and degenerated oocytes were not considered for the calculation of penetration, monospermy, and efficiency rates. The remaining presumptive zygotes were cultured in vitro, and the developmental parameters of the embryos derived from oocytes exposed to CPAs were compared with those from control oocytes. 2.8.2. Experiment 2 The second experiment was designed to evaluate the following: (1) Oocyte viability and fertilization parameters obtained with GV oocytes vitrified using either EG þ DMSO or EG þ PG. A total of four replicates were performed. Oocytes were subjected to equilibration in vitrification media and vitrified and warmed as described previously. Nonvitrified oocytes were used as controls. The treated and control oocytes were selected and evaluated as described in experiment 1. Oocytes were fixed 18 hours after IVF to assess their fertilization parameters. (2) Embryo developmental competence of GV oocytes vitrified with either EG þ DMSO or EG þ PG. A total of four experimental replicates were performed. Oocytes were subjected to equilibration in vitrification media and vitrified and warmed as described previously. Nonvitrified oocytes were used as controls. After 44 hours of IVM, oocytes were selected for IVF as described in experiment 1. Cleavage rate, blastocyst formation, and blastocyst total cell number from the EG þ DMSO and
EG þ PG vitrification groups were compared with those derived from the nonvitrified control oocytes. 2.9. Statistical analysis Statistical analysis was performed using the IBM SPSS 19 statistics package (SPSS, Chicago, IL, USA), and differences were considered significant at P < 0.05. For statistical purposes, each drop was considered an experimental unit. The mean standard error of the mean of the binary variables (viability; matured, fertilized, and monospermic oocytes; cleaved embryos; blastocyst formation; efficiency) and continuous variables (total cell number per blastocyst) were obtained after calculating the percentage in every drop of each group and in each replicate. Significant differences between the groups were assessed by mixed linear ANOVA with treatment as a fixed effect and the replicate as a random effect. When the ANOVA revealed a significant effect, values were compared using the Bonferroni test. 3. Results 3.1. Experiment 1 The FDA viability of the oocytes after 2 hours of equilibration in EG þ DMSO– and EG þ PG–based media was very high (95.7 0.4% and 95.6 1.1%, respectively) but lower (P < 0.01) than that observed in the control group (99.5 0.8%). Higher (P < 0.05) proportions of live oocytes from the EG þ DMSO (18.1 2.3%) or EG þ PG (19.4 2.6%) groups were degenerated at 44 hours of maturation compared with those in the control group (7.6 1.3%). At 18 hours after IVF, the numbers of immature oocytes (
