Bovine oocyte vitrification using the Cryotop method: Effect of cumulus cells and vitrification protocol on survival and subsequent development

Cryobiology 61 (2010) 66–72

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Bovine oocyte vitrification using the Cryotop method: Effect of cumulus cells and vitrification protocol on survival and subsequent development q X.L. Zhou a,b, A. Al Naib a, D.W. Sun a, P. Lonergan a,* a b

School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland University of Shanghai for Science and Technology, Shanghai 200093, China

a r t i c l e

i n f o

Article history: Received 27 January 2010 Accepted 10 May 2010 Available online 25 May 2010 Keywords: Bovine oocyte Cryopreservation IVF Embryo development

a b s t r a c t The ability to successfully cryopreserve mammalian oocytes has numerous practical, economical and ethical benefits, which may positively impact animal breeding programs and assisted conception in humans. However, oocyte survival and development following vitrification remains poor. The aim of the present study was (1) to evaluate the effect of the presence of cumulus cells on the outcome of vitrification of immature (GV) or mature (MII) bovine oocytes, (2) to compare empirical and theoretical vitrification protocols, and (3) to assess the effect of adding ice blockers to vitrification media on survival and development competence of bovine oocytes following vitrification using the Cryotop method. In Experiment 1, cumulus-enclosed and partially-denuded GV and MII oocytes were vitrified in 15% EG + 15% Me2SO + 0.5 M sucrose in two steps. In Experiment 2, GV oocytes were vitrified either as above or using theoretical modeling based on permeability and osmotic tolerance characteristics in 30% EG + 11.4% trehalose in three steps or 40% EG + 11.4% trehalose in four steps. In Experiment 3, GV oocytes were vitrified in media supplemented or not with 1 of 2 ice blockers (21st Century Medicine, Fontana, CA) 1% X-1000, 1% Z-1000 or both in three steps. In Experiment 1, the survival, cleavage and blastocyst rate of cumulusenclosed oocytes was significantly higher than those of partially-denuded oocytes when vitrified at the GV stage (93.8% vs. 81.3%, 65.8% vs. 47.3%, 11.3% vs. 4.0%, respectively, P < 0.05). However, no significant effect of cumulus cover was detected between the two groups when vitrified at MII (93.0% vs. 91.8%, 35.2% vs. 36.8%, 5.0% vs. 4.4%, respectively). Furthermore, cumulus-enclosed oocytes vitrified at the GV stage exhibited significantly higher developmental competence than those vitrified at the MII stage (P < 0.05). In Experiment 2, there were no significant differences in the survival, cleavage and blastocyst rate among three protocols (86.0% vs. 92.8% vs. 91.2%, 44.8% vs. 54.4% vs. 45.6%, 5.0% vs. 5.4% vs. 4.0%, respectively). However, cleavage and blastocyst rate were significantly lower (P < 0.05) than non-vitrified control oocytes. In Experiment 3, the presence of ice blockers did not alter the cleavage rate or blastocyst development (P > 0.05). In conclusion, cumulus-enclosed GV bovine oocytes survived vitrification and subsequently developed at higher rates than MII oocytes using Cryotop method and conventional IVF procedure. Theoretical analysis of permeability characteristics and tolerance limits could not explain the low developmental competence of vitrified oocytes. Ó 2010 Elsevier Inc. All rights reserved.

Introduction Successful cryopreservation of mammalian oocytes has numerous practical, economical and ethical benefits, which may positively impact animal breeding programs and assisted conception in humans. Vitrification is a process of cryopreservation during which solidification of a solution occurs without the formation of ice crystals. This phenomenon requires either rapid cooling rates or the use

q Statement of funding: This research was funded by Science Foundation Ireland, the National Science Foundation of P.R.C. (50906057, 50776060), the Shanghai Rising-Star Program (07QA14042) of P.R.C. and NCET-07-0559. * Corresponding author. Fax: +353 1 6288421. E-mail address: [email protected] (P. Lonergan).

0011-2240/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2010.05.002

of concentrated cryoprotectant solutions [36]. After the technique was first used in embryology by Rall and Fahy [31] on mouse embryos, considerable advancements have been achieved and offspring have been produced using frozen-thawed oocytes in various species, e.g. cow [39], horse [24], human [7]. In bovine oocyte vitrification, the most successful reports are those of Vajta et al. [34], Dinnyes et al. [10], Chian et al. [8] using open pulled straws (OPS), solid surface vitrification (SSV) and Cryotops, respectively. However, the overall success in terms of the subsequent ability to complete embryo development is still not satisfactory for most species. One factor that could affect oocyte quality following vitrification is the presence or absence of cumulus cells around the oocyte prior to cryopreservation. Several lines of evidence indicate that surrounding cumulus cells play a fundamental role in the maturation

X.L. Zhou et al. / Cryobiology 61 (2010) 66–72

process and full development competence [9,22]. In addition, it has been reported that cumulus cells are beneficial to oocyte survival after cryopreservation [16]; for example, the presence of cumulus cells may minimize the release of cortical granules and prevent premature zona hardening, thus maintaining fertilization capacity of cryopreserved oocytes [40]. However, Chian et al. [8] reported that bovine oocytes matured without cumulus cells had a higher survival rate after vitrification. Moreover, the rate of embryo development to the 8-cell stage in cumulus-cells free oocytes was significantly higher than that of cumulus cell-intact oocytes. The need to maintain cumulus cells during cryopreservation of oocytes is still a matter of debate. Considering that the functions of cumulus cells are different in immature (GV) and mature (MII) oocytes, it is necessary to determine the effects of cumulus cells on bovine oocyte vitrification at both the GV and the MII stage, respectively. A wide variety of approaches have been used to improve and optimize methods of bovine oocyte vitrification. The majority of studies tend to use ‘‘trial-and-error” to examine the effects of various protective compounds [14,29], macromolecular supplements [6,13], and to compare different cooling and warming conditions. However, in recent years, the development of bovine vitrification seemed to reach a plateau phase and researchers achieved similar survival and developmental rates by different methods. At the same time, other studies make use of rigorous mathematical formulations to define and describe the behavior of oocytes at low temperature, as well as to design CPA addition and dilution procedures in a calculated manner [15,26,28]. Following this idea, we have focused on the fundamental characteristics, i.e. membrane permeability, osmotic tolerance limits and toxic tolerance limits of bovine oocytes in our earlier work [41]. Water permeability (Lp), solute permeability (Ps), and activation energies for Lp and Ps (Ea) were determined using a two parameter model. Osmotic tolerance limits and toxic tolerance limits were determined based on oocyte developmental competence following exposure to different CPAs. Based on all the information, three-step or four-step CPA addition and two-step CPA dilution protocols were designed. Cell dynamic modeling was performed with a computer program to ensure any volume change the oocytes undergo is kept within tolerable limits while minimizing the amount of time that oocytes are exposed to cryoprotectant. To verify the theoretical analysis, the efficacy of empirical and theoretical vitrification protocols need to be compared. Ice growth and recrystallization are considered to be important factors in determining vitrification outcomes. Synthetic ice blockers, which specifically inhibit the formation/emergence of ice nuclei and ice crystal growth, have recently been used to supplement vitrification solutions [11,27]. Unlike conventional cryoprotectants that inhibit freezing by interacting with water, ice blockers are believed to bind to the surface of growing ice crystals and inhibit the addition of any further water molecules in specific planes of growth [44]. This selective attraction to surfaces of ice growth permits ice blockers to exert significant effects even while present at very low concentrations. Small quantities of ice blocker can therefore modify the number and size of ice crystals and thereby change the vitrification tendency of a solution without adding additional toxicity [42]. The commercially available ice blockers are SuperCool X-1000 and SuperCool Z-1000. The SuperCool X-1000 is a copolymer of polyvinyl alcohol (PVA) of mean molecular mass 2000 Da, with 20% of the hydroxyls replaced by acetate groups. The SuperCool Z-1000 is a copolymer of polyglycerol (PGL) of mean molecular mass 750 Da [43]. The combination of SuperCool X-1000 and SuperCool Z-1000 has been used as an additive in solutions developed for kidney vitrification [11]. The SuperCool X-1000 has also been beneficial in a vitrification solution for tissue-engineered bone [27]. However, to our knowledge, there are no reports of the application of ice blockers in vitrification of mammalian oocytes.

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Therefore, the objectives of the present study were (1) to evaluate the effect of the presence of cumulus cells on the outcome of vitrification of immature (GV) or mature (MII) oocytes, (2) to compare empirical and theoretical vitrification protocols, and (3) to assess the effect of adding ice blockers SuperCool X-1000 and SuperCool Z-1000 to vitrification media on oocyte survival and subsequent embryonic development. Materials and methods Chemicals and supplies All chemicals were purchased from Sigma–Aldrich Ireland Ltd. (Dublin, Ireland) unless otherwise stated. Ice blocker SuperCool X-1000 and SuperCool Z-1000 were purchased from 21st Century Medicine Inc. (Fontana, CA, USA). Cryotops were purchased from Kitazato Biopharma (Fujinomiya, Japan). Collection of oocytes Bovine ovaries were obtained from a local abattoir and transported to the laboratory at 35 °C in phosphate buffered saline within 2 h of slaughter. Cumulus-oocyte complexes (COCs) were aspirated from 2 to 8 mm follicles using an 18-gauge needle attached to a 5 ml syringe. COCs with three or more layers of cumulus cells and a homogeneous cytoplasm were selected for the experiment. In vitro maturation Selected oocytes were washed three times in PBS supplemented with 36 lg/ml pyruvate, 50 lg/ml gentamycin and 0.5 mg/ml BSA. Groups of 50 COCs were placed in 500 ll maturation medium in four-well plates and cultured for 24 h at 39 °C in a 5% CO2 humidified air atmosphere. The maturation medium was Tissue Culture Medium 199 (TCM 199) supplemented with 10% Fetal Bovine Serum (FBS) and 10 ng/ml epidermal growth factor (EGF). Vitrification and warming The holding medium (HM) used for handling oocytes during vitrification and warming was HEPES-buffered TCM 199 supplemented with 20% (v/v) FBS. All manipulations were performed on a 39 °C heated stage in a warm room (25–27 °C). All the media were used at room temperature, except for the warming solution which was used at 37 °C. The equilibration protocols are presented under the section ‘Experimental Design’. A pasteur pipette were pulled in a flame and cut in half to get a suitable internal diameter about 150 lm following which 3–5 oocytes were loaded onto the film strip of a Cryotop device under a stereomicroscope. Before vitrification, almost all the solution was removed to leave only a thin layer (<0.1 ll) covering the oocytes. Then, the Cryotop was immediately submerged into liquid nitrogen vertically with rapid horizontally movements to obtain the maximum cooling rate. For warming, the Cryotop was directly inserted into 37 °C HM containing 1 M sucrose for 1 min. The warmed oocytes were transferred to diluent solution (HM containing 0.5 M sucrose) for 3 min, and then washed twice in HM for 5 min. According to the manufacturer, the cooling and warming rates of the Cryotop are 23,000 and 42,000 °C/min, respectively. In vitro fertilization Control and vitrified GV and MII oocytes were fertilized using the same conditions. All oocytes were washed four times in

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fertilization medium, and then transferred in groups of 50 into four-well dishes containing 250 ll of fertilization medium (Tyrode’s medium with 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate and 6 mg/ml fatty acid-free BSA). In addition, 10 lg/ml heparin–sodium salt (184 U/ml heparin; Calbiochem, San Diego, CA) was added. Motile spermatozoa were obtained by centrifugation of frozen–thawed semen (Dairygold A.I. Station, Mallow) on a discontinuous Percoll (Pharmacia, Uppsala) density gradient (2.5 ml 45% (v/v) Percoll over 2.5 ml 90% (v/v) Percoll) at 700g for 9 min at room temperature. Viable spermatozoa collected at the bottom of the 90% fraction were washed in Hepes-buffered Tyrode’s medium and pelleted by centrifugation at 100g for 5 min. The spermatozoa were counted in a haemocytometer and diluted in the appropriate volume of fertilization medium to give a concentration of 2  106 spermatozoa/ml. A 250 ll aliquot of this suspension was added to each fertilization well to obtain a final concentration of 1  106 spermatozoa/ml. The plates were incubated for 20 h at 39 °C under an atmosphere of 5% CO2 in air with maximum humidity. In vitro culture At approximately 20 h post-insemination, presumptive zygotes were denuded by vortexing and washed four times in PBS before they were randomly assigned to groups. Zygotes were transferred in groups of 25 to 25 ll culture droplets of synthetic oviductal fluid medium (SOF) under mineral oil. The dishes were incubated in a 5% CO2, 5% CO2 and 90% N2 humidified atmosphere at 39 °C. Oocyte survival was evaluated on the basis of the integrity of the oocyte membrane and the zona pellucida together with the discoloration of the cytoplasm at Day 1 after insemination. Cleavage rates were recorded at Day 2 after insemination and the proportion of oocytes developing to the blastocyst stage was assessed at Day 8 after insemination. Experimental design Experiment 1: Effect of cumulus cells on vitrification of GV and MII bovine oocytes Immediately after collection, COCs were randomly divided into two groups: (1) control COCs and (2) COCs partially denuded of cumulus cells by gentle pipetting in PBS at 37 °C. Cumulus-enclosed and partially-denuded oocytes (3–5) were suspended in equilibration solution (HM containing 7.5% (v/v) ethylene glycol (EG) and 7.5% (v/v) dimethylsulfoxide (Me2SO)) for 15 min to allow initial shrinkage and recovery. Following equilibration, they were transferred to the vitrification solution (HM containing 15% (v/v) EG, 15% (v/v) Me2SO and 0.5 M sucrose) for 45–60 s. Then the oocytes were loaded onto Cryotop for vitrification. Just after the vitrification procedure, warming was started with a different group in each replicate. As a control, half of the cumulus-enclosed and partially-denuded oocytes were placed in HM during vitrification at 37 °C for 45 min. After warming, all oocytes were transferred to maturation medium and then submitted to the same IVM, IVF, IVC procedures as described above. The survival, cleavage and blastocyst rates were compared between groups. For MII bovine oocytes, following 24 h maturation, matured COCs were randomly divided into two groups: (1) control oocytes covered with expanded cumulus cells or (2) oocytes partially denuded of cumulus cells by gentle pipetting in PBS as above. The cumulus-enclosed and partially-denuded matured oocytes were equilibrated, vitrified and warmed as described above. As a control, half of the cumulus-enclosed and partially-denuded matured oocytes were suspended in HM during vitrification at 37 °C for 45 min. After warming, oocytes of four groups were transferred in fertilization medium and then submitted to the same IVF, IVC

procedures as described above. The survival, cleavage and blastocyst rates were compared between groups. Experiment 2: Comparison of empirical and theoretical vitrification protocols Immediately after collection, COCs were randomly divided into four groups. (1) Control group: COCs suspended in HM during vitrification. (2) Empirical two-step: COCs equilibrated, vitrified and warmed as described in Experiment 1. (3) Theoretical three-step: COCs incubated in the first equilibration solution (HM containing 10% (v/v) EG) for 1 min, and then transferred to the second equilibration solution (HM containing 20% (v/v) EG and 6% (w/v) trehalose) for 30 s. Finally, the COCs were transferred to vitrification solution (HM containing 30% (v/v) EG and 11.4% (w/v) trehalose) and loaded onto the Cryotop device for vitrification. The time between the contact of the oocytes with the concentrated CPA solution and vitrification did not exceed 20 s. (4) Theoretical four-step: COCs incubated in the first equilibration solution (HM containing 10% (v/v) EG) for 1 min, and then transferred to the second equilibration solution (HM containing 20% (v/v) EG and 4% (w/v) trehalose) for 40 s, the third equilibration solution (HM containing 30% (v/v) EG and 8% (w/v) trehalose) for 25 s. Finally, the COCs were transferred to vitrification solution (HM containing 40% (v/v) EG and 11.4% (w/v) trehalose) and loaded onto the Cryotop device for vitrification. The time between the contact of the oocytes with the concentrated CPA solution and the freezing did not exceed 12 s. After warming, oocytes in the four groups were transferred in maturation medium and then submitted to the same IVM, IVF, IVC procedures as described above. The survival, cleavage and blastocyst rates were compared in each group. Experiment 3: Effect of ice blocker X-1000 and Z-1000 Immediately after collection, COCs were randomly divided into five groups. (1) Control group: COCs suspended in HM during vitrification. (2) Basic media: COCs incubated, vitrified and warmed with the Theoretical three-step procedure described in Experiment 2. (3) Basic media + X-1000: COCs treated as in the basic media group, except vitrification solution was supplemented with 1% (v/v) X-1000. (4) Basic media + Z-1000: COCs treated as the basic media group, except vitrification solution was supplemented with 1% (v/v) Z-1000. (5) Basic media + X-1000 + Z-1000: COCs treated as the basic media group, except the vitrification solution was supplemented with 1% (v/v) X-1000 and 1% (v/v) Z-1000. After warming, all oocytes were transferred in maturation medium and then submitted to the same IVM, IVF, IVC procedures as described above. The survival, cleavage and blastocyst rates were compared in each group. Statistical analysis The data for survival, cleavage and blastocyst rates were expressed as mean ± SD and analyzed using one-way ANOVA. Differences were considered significant at a level of P < 0.05. Results Experiment 1 For GV stage bovine oocytes, the survival, cleavage and blastocyst rate of cumulus-enclosed vitrified oocytes were significantly higher than that of partially-denuded vitrified and control oocytes (Table 1). Vitrification of cumulus-enclosed oocytes led to significant reductions in survival, cleavage and blastocyst rate compared to controls (P < 0.05). Similarly, the survival, cleavage and blastocyst rate of partially-denuded oocytes was significantly reduced

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Table 1 Survival rate and in vitro embryo development of bovine GV stage cumulus-enclosed and partially-denuded oocytes vitrified-warmed using the Cryotop method (four replicates). Oocytes treated

N

Cumulus-enclosed control Partially-denuded control Cumulus-enclosed vitrified Partially-denuded vitrified

141 118 177 143

Survived, n (%)A a

141 (100 ± 0.0) 117 (99.3 ± 0.8)a,b 166 (93.8 ± 2.5)b 117 (81.3 ± 3.6)c

Cleaved, n (%)B 121 89 108 56

Blastocyst, n (%)B a

(86.3 ± 1.9) (75.8 ± 3.9)a,b (65.8 ± 5.6)b (47.3 ± 4.0)c

a

47 (33.8 ± 1.8) 14 (11.5 ± 4.2)b 19 (11.3 ± 1.7)b 4 (4.0 ± 2.3)c

Blastocyst/cleavage (%) (39.5 ± 2.2)a (14.8 ± 5.2)b (18.0 ± 3.5)b (7.8 ± 4.5)b

Values with different superscripts in the same column are significantly different (P < 0.05). A The percentages were based on the number of oocytes treated. B The percentages were based on the number of oocytes survived.

(P < 0.05) following vitrification compared to controls. In the control groups, the survival rate and cleavage rate were not significantly different, but the blastocyst rate of partially-denuded oocytes was significantly lower (P < 0.05) than those of cumulusenclosed control. For MII stage bovine oocytes, no significant differences were detected between cumulus-enclosed vitrified oocytes and partiallydenuded vitrified oocytes in survival, cleavage or blastocyst rate (Table 2). Although the survival rate was not different between cumulus-enclosed vitrified and control oocytes, the cleavage rate, blastocyst rate and blastocyst/cleaved oocytes of cumulus-enclosed vitrified oocytes were significantly lower (P < 0.05) than those of cumulus-enclosed control. Similarly, the survival rate of partially-denuded vitrified oocytes was not different to controls. However, the cleavage rate, blastocyst rate and blastocyst/cleaved of partially-denuded vitrified oocytes were significantly lower (P < 0.05) than those of control oocytes. Partial removal of cumulus cells resulted in a decrease (P < 0.05) in the cleavage rate and blastocyst rate compared to cumulus-enclosed control. Cumulus-enclosed oocytes vitrified at the GV stage exhibited a significantly higher cleavage rate and blastocyst rate than those vitrified at MII stage (P < 0.05). Based on this result, cumulus-enclosed GV oocytes were used in the following experiments. Experiment 2 The survival rates of bovine oocytes vitrified by theoretical three-step and theoretical four-step were not different to that of control oocytes, whereas survival following the empirical two-step was significantly lower (P < 0.05) than the control (Table 3).

No significant differences were detected among bovine oocytes vitrified by empirical two-step, theoretical three-step and theoretical four-step protocol in terms of cleavage rate or blastocyst formation rate; however, all were significantly lower (P < 0.05) than the control.

Experiment 3 The survival of oocytes vitrified in media supplemented with 1% X-1000, 1% Z-1000 and 1% X-1000 + 1% Z-1000 were not different to the control (Table 4). The cleavage rate was not different amongst the four treatment groups; however, all were lower than the control (Table 4). The blastocyst formation rate was unaffected by the presence of the ice blockers, however, when both were used in combination, no embryos developed to blastocyst stage (Table 4).

Discussion For many years, vitrification has been considered a promising alternative to slow cooling for bovine oocyte cryopreservation. Several methods have been developed to minimize the likelihood of ice forming during the vitrification procedure, including evaluating cryoprotectants with differing composition, antinucleating and antifreeze compounds, minimizing the cryoprotectant volume and evaluating different cooling strategies. [1,18,19,21,23,25,30,34] According to the data published by different research groups, the Cryotop technique is at present one of the most efficient methods for cryopreservation of oocytes [35] and were therefore, used to vitrify bovine oocytes in the present study.

Table 2 Survival rates and in vitro embryo development of bovine MII stage cumulus-enclosed and partially-denuded oocytes vitrified-warmed by Cryotop method (five replicates). Oocytes treated

N

Survived, n (%)A

Cumulus-enclosed control Partially-denuded control Cumulus-enclosed vitrified Partially-denuded vitrified

130 122 158 167

126 119 147 153

(96.8 ± 1.5)a (97.4 ± 1.1)a (93.0 ± 2.3)a (91.8 ± 2.4)a

Cleaved, n (%)B 115 92 51 57

(91.6 ± 2.5)a (78.4 ± 4.7)b (35.2 ± 4.6)c (36.8 ± 3.2)c

Blastocyst, n (%)B

Blastocyst/cleavage (%)

45 (35.6 ± 2.8)a 32 (27.2 ± 2.3)b 7 (5.0 ± 4.3)c 7 (4.4 ± 1.4)c

(39.0 ± 4.1)a (34.6 ± 2.7)a,b (12.6 ± 9.7)b,c (10.8 ± 3.5)c

Values with different superscripts in the same column are significantly different (P < 0.05). A The percentages were based on the number of oocytes treated. B The percentages were based on the number of oocytes survived.

Table 3 Survival rates and in vitro embryo development of bovine GV stage cumulus-enclosed oocytes vitrified with empirical and theoretical protocol by Cryotop method (five replicates). Oocytes treated Control Empirical two-step Theoretical three-step Theoretical four-step

N 115 107 121 104

Survived, n (%)A 112 92 112 96

a

(97.4 ± 1.1) (86.0 ± 2.4)b (92.8 ± 3.3)a,b (91.2 ± 5.2)a,b

Cleaved, n (%)B 88 41 61 43

a

(78.0 ± 4.0) (44.8 ± 4.2)b (54.4 ± 6.3)b (45.6 ± 3.8)b

Values with different superscripts in the same column are significantly different (P < 0.05). A The percentages were based on the number of oocytes treated. B The percentages were based on the number of oocytes survived.

Blastocyst, n (%)B

Blastocyst/cleavage (%)

30 (26.2 ± 3.2)a 4 (5.0 ± 2.6)b 6 (5.4 ± 2.3)b 4 (4.0 ± 1.3)b

(33.4 ± 3.0)a (12.2 ± 6.1)b (10.2 ± 4.6)b (9.6 ± 2.8)b

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Table 4 Survival rates and in vitro embryo development of bovine GV stage cumulus-enclosed oocytes vitrified with different ice blocker media by Cryotop method (five replicates). Oocytes treated Control Basic media Basic media + X-1000 Basic media + Z-1000 Basic media + X-1000 + Z-1000

N 110 114 111 110 114

Survived, n (%)A 104 98 97 93 101

a

(94.4 ± 1.9) (86.0 ± 2.7)b (88.4 ± 2.9)a,b (83.6 ± 4.8)a,b (88.0 ± 3.6)a,b

Cleaved, n (%)B 77 38 36 37 43

a

(74.6 ± 3.2) (38.0 ± 3.2)b (37.2 ± 2.4)b (40.0 ± 3.0)b (41.4 ± 6.7)b

Blastocyst, n (%)B a

24 (23.0 ± 10.4) 2 (2.0 ± 1.3)b,c 3 (2.8 ± 1.2)b 2 (2.2 ± 1.4)b,c 0 (0)c

Blastocyst/cleavage (%) (31.2 ± 1.9)a (6.2 ± 4.1)b,c (7.8 ± 3.2)b (4.8 ± 3.0)b,c (0)c

Values with different superscripts in the same column are significantly different (P < 0.05). A The percentages were based on the number of oocytes treated. B The percentages were based on the number of oocytes survived.

It is generally accepted that cumulus-oocyte communication via an intact corona radiata is necessary for oocytes to attain full cytoplasmic maturation during IVM and improve fertilization rates during IVF [32,37]. In preliminary studies (data not shown) we established that the cleavage rate of denuded (GV and MII) bovine oocytes was significantly reduced compared to cumulus-enclosed oocytes, and almost no denuded bovine oocytes developed up to blastocyst stage after in vitro fertilization. In this study, we therefore partially denuded the cumulus cells by gentle pipetting, leaving 1–2 layers of cells on the surface of each oocyte. Little attention has been paid to the consequences of the presence or absence of cumulus cells on the survival of GV bovine oocytes following cryopreservation. In other species, the effect of cumulus cells on immature oocytes following vitrification was evaluated by the maturation rate, spindle and chromatin quality. Tharasanit et al. [33] reported that cumulus removal from equine oocytes prior to IVM or vitrification resulted in reduced meiotic competence, MII spindle and chromatin quality. They concluded that while the cumulus does protect immature equine oocytes during vitrification it does so by mechanisms other than support during maturation. In contrast, Bogliolo et al. [3] reported that immature ovine oocytes vitrified without cumulus cells showed a significantly higher survival and meiotic maturation rate than those with cumulus cells, and no differences in spindle and chromatin organization between two groups were observed. In the present study, we vitrified immature cumulus-enclosed and partially-denuded bovine oocytes and evaluated the effects by the outcomes after IVF procedure. The results indicate that the survival, cleavage and blastocyst rate of cumulus-enclosed vitrified oocytes are significantly higher than that of partially-denuded vitrified oocytes. The role of the cumulus cells during vitrification of MII oocytes remains controversial. Some investigators reported that cumulus presence would protect MII oocytes against vitrification-induced damage. Kuwayama et al. [20] reported higher survival and blastocyst rate for cumulus-enclosed than cumulus-free human MII oocytes after vitrification. Tharasanit et al. [33] found that cumulus-enclosed equine MII oocytes preserved their meiotic spindle and chromatin quality better during vitrification than denuded oocytes. Conversely, other investigators believed cumulus cells are not necessary or detrimental to MII oocytes during vitrification. Zhang et al. [46] found no difference in the development of vitrified ovine MII oocytes with or without cumulus cells. Gasparrini et al. [12] reported that the presence of cumulus cells severely reduced the cleavage rate of MII buffalo oocytes following vitrification. In the present study, we vitrified mature cumulus-enclosed and partially-denuded bovine oocytes and evaluated the survival and development competence after IVF. No significant differences were detected between vitrified cumulus-enclosed and partiallydenuded oocytes in the survival, cleavage and blastocyst rate. The possible explanation is the cumulus was detrimental to vitrification, which comprises the benefits of cumulus in IVF procedure. From another point of view, the intracytoplasmic sperm injection

technique rather than conventional IVF has been used to achieve fertilization, which can circumvent the detrimental effects of removing the cumulus on subsequent zona penetrability. Moreover, the oocytes were easier to handle for vitrification using a Cryotop when the oocytes were denuded completely from their cumulus cells. Therefore, cumulus removal prior to vitrification is at present a standard practice during cryopreservation of MII human oocytes. The cell cycle stage during meiosis appears to affect the results of bovine oocyte vitrification due to varying sensitivity to cooling procedures. Chilling injury is reported to be higher in vitrified immature oocytes, owing to low membrane stability and susceptibility of the cytoskeleton. However, an increase in chromosomal abnormality has been observed in vitrified mature oocytes, owing to alterations in the meiotic spindle [3]. Although numerous studies have been conducted to vitrify bovine oocytes at different meiotic stages: germinal vesicle (GV) [1,5,17,38,39,45] and metaphase II (MII) [6,8,10,13,14,29,34], the optimal meiotic stage is still not clear because of various devices and solutions adopted by different research groups. In the present study, we compared the efficiency of oocyte vitrification at two meiotic stages using the same vitrification protocol. The results indicate that cumulus-enclosed oocytes vitrified at the GV stage exhibited a significantly higher cleavage and blastocyst rate than those vitrified at MII. This may be due to the increase in volume associated with cumulus expansion during maturation. It may also be due to the higher water permeability (Lp) and solute permeability (Ps) of MII than GV bovine oocytes as previously reported [41]. That means the changes of cell volume and intracellular CPA concentrations are more severe in MII than GV bovine oocytes during CPA addition and dilution process, which make it more sensitive. Theoretical analysis of membrane transport characteristics of oocytes has been considered to be an efficacious approach to optimization of oocyte cryopreservation, because it can design the vitrification protocol in a rational manner and reduce the times of replicate experiment greatly. In our earlier work [41], water permeability (Lp), solute permeability (Ps) and activation energies (Ea) for Lp and Ps were determined for GV bovine oocytes in the presence of EG. They were 0.11 ± 0.013 lm/min/atm, 0.0033 ± 0.00023 cm/min, 3.68 kcal/mol and 6.84 kcal/mol, respectively. The osmotic tolerance limits tests indicated that the safe volume changes of GV bovine oocytes were from 150% to 46% of their volume. Toxicity tolerance limits tests suggested that Me2SO are more toxic than EG, and the exposure time to 30% and 40% EG should be less than 1 min. Integrating all the information together, three-step or four-step CPA addition and two-step CPA dilution protocols were presented. Volumetric response and the corresponding intracellular cryoprotectant concentration simulation showed the shrinkage and expansion did not exceed the safe limits for volume excursion and at the end of each addition and dilution period, EG had almost reached its equilibrium concentration. In the present study, we tested the efficiency of our theoretical design protocol and compared it with the often used empirical protocol for the first

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time. The results showed that the survival, cleavage and blastocyst rate of vitrified bovine oocytes using theoretical three-step and theoretical four-step were comparable to those reported in literature, but no significant differences with empirical two-step. That is not to say that the determination of fundamental biophysical characteristics of oocytes is not worthwhile. On the contrary, they contribute enormously to understanding the complex physiology and cell biology of the reproductive cells of mammalian species. Soon after the original discovery of polar fish proteins (AFPs), Fahy and co-workers [44] proposed creating synthetic ice blocker specifically designed to bind to nucleators and nascent ice crystals in a manner similar to that of natural antifreeze proteins. Up to now, several specific molecules and polymers were proposed and added to vitrification solutions. SuperCool X-1000 and SuperCool Z-1000 are the most used products among them, which have been reported as an additive in solution developed for kidney and tissue-engineered bone vitrification [11,27]. In the area of embryo cryopreservation, Cabrita et al. [4] vitrified gilthead seabream embryos in Me2SO-based vitrification solutions by macrotubes (1 ml), they found ice recrystallization was avoided during thawing in almost all samples containing 2% X-1000 as additive, in which the percentage of embryo with intact morphology was significantly higher. In the present study, we report for the first time the effect of ice blockers on the bovine oocytes, however, the results indicate that the survival rate and development competence of bovine oocytes vitrified in solutions supplemented with or without X-1000 and/or Z-1000 by Cryotop method are not significantly different. Ice blockers did not affect the survival rate and developmental competence of vitrified bovine oocytes. The similar results were obtained by Badrzadeh et al [2]. They studied the effect of different ice blocker media on vitrification/thawing of mouse embryos. Eight cells mouse embryos were vitrified in four groups, EG, EG + X1000, EG + Z-1000, EG + X-1000 + Z-1000, using V-kim closed system. The results showed that combination of ice blocker X-1000 and Z-1000 resulted in significant higher survival rate of mouse embryos after vitrification/thawing process. No significant difference was noted in blastocyst formation rate between the experimental groups. The possible explanation for the different results obtained above depends on the function of ice blockers during vitrification. When the vitrification systems are large volume, such as organs, engineered tissues and macrotubes, in which a large quantity of nucleators exit, ice blockers can suppress nucleation and recrystallization by binding to nucleators in solutions during vitrification and warming. Therefore, the ice growth was inhibited and damage to the systems was reduced. Nevertheless, when the vitrification systems are small volume, such as Cryotop and V-kim, the small amount of liquid and ultra-fast cooling rate make the extent of vitrification high enough, the main obstacle in these systems is not nucleators. Ice blockers cannot exert effects to vitrification process. From this point of view, it can be speculated that ice blockers are more beneficial to vitrification system of large volume than small volume. Furthermore, the results of combination of the two ice blocker agents depend on the appropriate proportion of each agent in the mixture [43]. In this study, the combination of the two ice blocker agents inhibit blastocyst development maybe because 1% X-1000 and 1% Z-1000 is not the ideal balance, which shows less effective than either agent alone. In conclusion, cumulus-enclosed GV bovine oocytes survived vitrification and subsequently developed at higher rates than MII oocytes using the Cryotop method and conventional IVF procedures. Theoretical analysis of permeability characteristics and tolerance limits alone may not be sufficient to improve vitrification protocols. Ice blockers had no effect on bovine oocyte vitrification.

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