Morphological, biochemical and functional studies to evaluate bovine oocyte vitrification

Journal Pre-proof Morphological, biochemical and functional studies to evaluate bovine oocyte vitrification C. Gutnisky, S. Morado, T. Gadze, A. Donato, G. Alvarez, G. Dalvit, P. Cetica PII:

S0093-691X(19)30536-9

DOI:

https://doi.org/10.1016/j.theriogenology.2019.11.037

Reference:

THE 15272

To appear in:

Theriogenology

Received Date: 1 April 2019 Revised Date:

26 November 2019

Accepted Date: 29 November 2019

Please cite this article as: Gutnisky C, Morado S, Gadze T, Donato A, Alvarez G, Dalvit G, Cetica P, Morphological, biochemical and functional studies to evaluate bovine oocyte vitrification, Theriogenology (2019), doi: https://doi.org/10.1016/j.theriogenology.2019.11.037. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

Revised

1

Morphological, biochemical and functional studies to evaluate bovine

2

oocyte vitrification

3

Gutnisky C.1,2, Morado S. 1, Gadze T. 1, Donato A. 1, Alvarez G. 1,2, Dalvit

4

G. 1 and Cetica P. 1,2

5

1

6

Investigación y Tecnología en Reproducción Animal (INITRA) and 2 Unidad Ejecutora

7

de Investigaciones en Producción Animal (INPA, UBA-CONICET), Buenos Aires,

8

Argentina.

9

Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Instituto de

Corresponding author:

10

Cynthia Gutnisky

11

Cátedra de Química Biológica

12

Facultad de Ciencias Veterinarias

13

Universidad de Buenos Aires

14

Chorroarín 280

15

C1427CWO Buenos Aires

16

ARGENTINA

17

Tel/FAX: 0054-11-45248452

18

[email protected]

19 20

Abstract

21

The aim of the present study was to evaluate the effect of vitrification on

22

morphological, biochemical and functional parameters of matured bovine oocytes at

23

different recovery times. To this end, matured bovine oocytes were vitrified using

24

the Cryotech® kit (a minimum-volume system) and then incubated in maturation

25

medium for different post-warming durations (0 h, 3 h or 21 h). Morphology,

26

viability and biochemical parameters were assessed at each time point mentioned

27

above and the recovery of the metaphase plate was analyzed at 2 h, 3 h and 4 h post-

28

warming. The vitrification-warming process did not affect the viability or

29

morphology of oocytes at any time point. However, the recovery of the metaphase

30

plate occurred mostly between 3 and 4 h rather than at 2 h after warming (P<0.05).

31

Both control and vitrified-warmed oocytes showed changes in cytosolic oxidative

32

activity, quantification of active mitochondria, reactive oxygen species (ROS) levels

33

and redox status at the different time points studied (P<0.05). However, differences

34

between control and vitrified-warmed oocytes were found only in the quantification

35

of active mitochondria and ROS production (P<0.05). Finally, in vitro fertilization

36

and embryo culture were carried out as functional studies to establish whether

37

vitrification-warming affected oocyte competence, and a significant decrease was

38

found both in the cleavage rate and embryo development (P<0.05). We concluded

39

that major improvements in oocyte vitrification, at list with Cryotech® kit, are still

40

needed to avoid variations in oocyte metabolism which could contribute to the

41

reduction in the developmental competence of bovine oocytes.

42 43

Key words: cryopreservation, oocyte, Cryotech®, mitochondria, redox state

44 45

1. Introduction

46

Vitrification has become a technique routinely used in most human fertility

47

clinics to cryopreserve embryos and oocytes. However, this method is still not

48

commonly applied in animal breeding programs. Briefly, vitrification involves ultra-

49

rapid cooling in small volumes of highly viscous solutions containing relatively high

50

cryoprotectant concentrations, which results in the formation of a glass-like state both

2

51

inside and outside the cell, thus avoiding crystallization. Although at long exposure

52

times these cryoprotectants at high concentrations could induce cellular injuries, mainly

53

due to toxicity and osmotic stress [1], vitrification has improved the survival rates of

54

bovine embryos respect to the slow-freezing technique [2-4], becoming a possible

55

alternative for their cryopreservation. However, no satisfactory results have yet been

56

achieved for the bovine female gamete [5]. Although many advances have been made

57

regarding the cryopreservation of mature and immature bovine oocytes, vitrification is

58

still not considered an established procedure.

59

Vitrified oocytes may present several abnormalities related to a decrease in

60

their competence, including alterations in their cytoskeleton, an increase in

61

cytoplasmic Ca++ ion, and the hardening of the zona pellucida [6-8]. In line with

62

these findings, after vitrification, the secondary structure of proteins suffers

63

rearrangements, indicated by the increase in the β-sheet content at the expense of

64

the α-helices, as well as the lipid and carbohydrate configuration of the zona

65

pellucida [7]. Additionally, mitochondrial damages and alterations in their

66

distribution and function have been observed [9, 10]. On the other hand, oocyte

67

vitrification-warming leads to cell abnormalities that might be reversible, being the

68

post-warming recovery crucial for the improvement of the process. In mice, it has

69

been demonstrated that the incubation for 2 or 3 h results in higher percentages of

70

normal meiotic spindles than the incubation for only 1 h [11].

71

Several reports have demonstrated that vitrification may disturb the reduction-

72

oxidation (redox) status, reduce glutathione (GSH) content and increase reactive oxygen

73

species (ROS) levels, resulting in damage to biomolecules such as DNA, proteins and

74

membrane lipids and leading to mitochondrial dysfunction, which may induce apoptotic

75

responses and reduce cleavage and embryo viability [12-14]. Since GSH is related to the

3

76

reduction of disulfide bonds in the sperm nucleus and promotes its decondensation

77

inside the male pronucleus during fertilization, alterations in GSH content during

78

vitrification may affect cleavage by preventing the formation of the male pronucleus

79

[15, 16]. However, more studies about the changes in biochemical parameters after

80

oocyte vitrification need to be carried out.

81

Results regarding oocyte vitrification differ among species because of the

82

differences between the female gametes. In the human clinic, women’s oocytes have

83

been vitrified successfully, showing high survival percentages and no alterations in their

84

ultrastructure [17, 18]. This is the reason why, as explained above, the cryopreservation

85

technique is currently routinely used in most human fertility clinics. One of the

86

minimum-volume vitrification methods most used for human oocytes, due to its high

87

efficiency, is Cryotech® [19, 20]. However, this method has not yet been assessed for

88

bovine oocytes. Therefore, the aim of the present study was to evaluate the effect of

89

vitrification on morphological, biochemical and functional parameters of bovine oocytes

90

by using Cryotech® and by evaluating different post-warming durations of the in vitro

91

culture.

92 93 94 95 96

2. Materials and methods 2.1. Materials Unless specified, all chemicals and reagents were purchased from Sigma (Sigma Chemical Co., St. Louis, MO, USA).

97 98

2.2. Recovery of cumulus-oocyte complexes

99

Bovine ovaries were collected at an abattoir within 30 min after slaughter and

100

kept warm (30ºC) during the 2-h journey to the laboratory. Ovaries were washed with

101

physiological saline containing 100,000 IU L-1 penicillin and 100 mg L-1 streptomycin. 4

102

Cumulus-oocyte complexes (COCs) were recovered by aspiration of antral follicles (3 -

103

5 mm in diameter) and collected directly in the maturation medium described in item

104

2.3. COCs were washed three times before being placed in the definitive maturation

105

medium. Only oocytes surrounded by a compact and multilayered cumulus oophorus

106

were used.

107 108

2.3. In vitro maturation of cumulus-oocyte complexes

109

COCs were cultured in medium 199 (Earle’s salts, L-glutamine, 2.2 mg L-1

110

sodium bicarbonate; GIBCO, Grand Island, NY, USA) supplemented with 5% (v/v)

111

fetal bovine serum (FBS; GIBCO), 0.2 mg L-1 porcine FSH (Folltropin-V; Bioniche,

112

Belleville, Ontario, Canada), 2 mg L-1 porcine LH (Lutropin-V; Bioniche) and 50 mg

113

L-1 gentamicin sulfate under mineral oil at 39ºC for 22 h in a humidified atmosphere

114

containing 5% CO2 in air.

115 116 117

2.4. Oocyte preparation Unless specified, matured oocytes were partially denuded by repeated pipetting

118

in phosphate buffer saline (PBS) with 1 g L-1 hyaluronidase. External cumulus cells

119

were removed maintaining the corona radiata.

120 121

2.5. Oocyte vitrification

122

After 22 h maturation, partially denuded oocytes were vitrified using the

123

Cryotech® vitrification kit. Groups of five oocytes were placed in an equilibration

124

solution provided with the kit. At first, oocytes shrank due to the high osmolarity of

125

this solution, but once they fully recovered their normal morphology, they were

126

moved to the vitrification solution provided with the kit. The whole process took

5

127

between 9 and 11 min (8 to 10 min to recover their normal morphology in the

128

equilibration solution and 1 min in the vitrification solution). Oocytes were then

129

loaded in groups of five oocytes with a glass capillary onto the top of the film strip

130

supplied with the kit with a minimum volume of vitrification medium (less than 1

131

µL per oocyte) and the sample was quickly immersed into liquid nitrogen and

132

covered with a protective cap. Oocytes were maintained in liquid nitrogen for 1

133

week before warming.

134

For the warming procedure, the protective cap was removed and the film

135

strips containing the groups of five oocytes were removed from the liquid nitrogen

136

and directly introduced in the warming solution provided with the Cryotech® kit at

137

37º C for 1 min. Then, the oocytes were consecutive moved to different solutions.

138

Firstly, oocytes were placed in the dilution solution for 3 min and then moved to two

139

consecutive washing solutions provided with the kit for 5 min and 1 min,

140

respectively. After warming, oocytes were cultured in medium 199 (Earle’s salts, L-

141

glutamine, 2.2 mgL-1 sodium bicarbonate; GIBCO, Grand Island, NY, USA) as it

142

was described above.

143 144

2.6. Evaluation of the oocyte morphology and viability

145

Vitrified-warmed oocytes were evaluated at three different time points:

146

immediately after warming (0 h); at 3 h post-warming for their metabolic recovery,

147

and at 21 h post-warming, being this the time required for the formation of pronuclei

148

after fertilization. For each time point, a group of 15 oocytes was removed from the

149

medium for their evaluation and then discarded. Controls were performed using

150

matured non-vitrified oocytes left in the maturation medium during the same time

6

151

points. For morphological and biochemical evaluations, oocytes were taken from the

152

same batch of ovaries and processed separately (Fig. 1).

153 154

2.6.1. Morphological evaluation

155

The morphology of vitrified-warmed and control oocytes was evaluated

156

subjectively under a stereoscopic microscope with differential interferential contrast

157

with base on their cytoplasmic structure and volume recovery, as well as on the

158

integrity and definition of their plasma membrane, zona pellucida and perivitelline

159

space (n=98-103 oocytes for each group in three replicates). Oocytes with

160

asymmetric or irregular forms, increased perivitelline space or presence of

161

granularity in their cytoplasm were classified as abnormal.

162 163

2.6.2. Viability evaluation

164

The viability of vitrified-warmed and control oocytes was determined using

165

the fluorescein diacetate (FDA) fluorochrome. Oocytes were incubated in a solution

166

containing 0.12 µM FDA for 15 min. After incubation, they were washed twice in a

167

PBS + 0.1% polyvinyl alcohol (PVA) solution and loaded on a glass slide for their

168

observation under an epifluorescent microscope [21] (n=96 oocytes for each group,

169

i.e. control and vitrified-warmed oocytes, in three replicates).

170 171 172 173

2.7. Biochemical evaluations Vitrified-warmed and control oocytes were evaluated at the same time points described above.

174 175

2.7.1. Cytosolic oxidative status and quantification of active mitochondria

7

176 177

For these experiments, the zona pellucida was dissolved with 5 g L-1 pronase solution for 1 min.

178

The RedoxSensor Red and Mitotracker Green dual stain was used to evaluate

179

the cytosolic oxidative activity and to quantify active mitochondria of vitrified-

180

warmed and control oocytes. Cells were incubated for 30 min in the dark, at a final

181

concentration of 1 nM RedoxSensor Red and 0.5 nM Mitotracker Green, then

182

washed twice in PBS + 0.1% PVA and loaded on a glass slide for their observation

183

under an epifluorescent microscope. Microphotographs were taken and oocyte

184

fluorescence intensity was analyzed with IMAGE J software [22] (n=30–40 oocytes

185

for each treatment in three replicates; Fig. 2). No autofluorescence was detected

186

without either of the two fluorochromes with their respective filters.

187 188

2.7.2. Determination of reactive oxygen species levels

189

Denuded oocytes were incubated with 5 µM 2’,7’-dichlorodihydro-

190

fluorescein diacetate (DCHFDA) solution in PBS + 0.1% PVA for 30 min. To

191

analyze esterase activity, a group of oocytes was incubated in a solution containing

192

0.12 µM FDA for 15 min. After the exposure of both groups of oocytes to their

193

respective fluorochrome, they were washed twice in a PBS + 0.1% PVA solution

194

and loaded on a glass slide for their observation using an epifluorescent microscope.

195

Microphotographs were taken and oocyte fluorescence intensity was analyzed using

196

IMAGE J software. The ratio between DCHFDA fluorescence intensity for each

197

oocyte and the mean FDA fluorescence intensity for each treatment was used as an

198

estimate of ROS production per oocyte [23] (n=30–40 oocytes for each treatment

199

group in three replicates; Fig. 2).

200

8

2.7.3. Determination of the redox state

201

The

202

oocyte

redox

state

was

determined

measuring

NAD(P)H

203

autofluorescence using a blue filter (excitation 405 nm, emission 420-520 nm).

204

Microphotographs were taken and the fluorescence intensity was analyzed with

205

IMAGE J software [24] (n=30–40 oocytes for each treatment in three replicates; Fig.

206

2).

207 208 209

2.8. Oocyte functional studies 2.8.1. Analysis of metaphase II plate recovery

210

Before they may be activated by in vitro fertilization (IVF) or chemical

211

activation, vitrified-warmed oocytes must first recover their metaphase II chromosome

212

configuration. So, to study metaphasic plate recovery time, vitrified-warmed oocytes

213

were incubated for 2, 3 or 4 h in the same medium used for oocyte maturation. After

214

incubation, denuded oocytes were fixed in 40 mg L-1 paraformaldehyde solution for 1 h.

215

Finally, the fixed oocytes were stained with 10 mg L-1 Hoechst 33342 solution for 15

216

min. The oocyte nuclear status was observed at a x400 magnification, using 330–380

217

nm (excitation) and 410 nm (emission) filters for a Jenamed II epifluorescence

218

microscope (Carl Zeiss Jena, Buenos Aires, Argentina) to establish the time point when

219

most of the metaphase plates appear [25]. The nuclear material that was not condensed

220

as metaphase II was considered as unrecovered (Fig. 3) (n=20-26 in two replicates).

221 222

2.8.2. In vitro fertilization and embryo development

223

To study the competence of in vitro matured vitrified-warmed oocytes, we

224

analyzed their ability to be fertilized and develop to blastocyst stage. To this end, three

225

groups of in vitro matured oocytes were fertilized: oocytes surrounded by cumulus cells

9

226

(IVF control), partially denuded oocytes (control) and vitrified-warmed partially

227

denuded oocytes (treatment). All the groups were incubated for 3 additional hours

228

before insemination to allow the metaphase II plate of vitrified oocytes to recover.

229

IVF was performed using frozen–thawed semen from a Holstein bull of proven

230

fertility. Semen was thawed at 37ºC in a water bath, then resuspended in modified

231

synthetic oviductal fluid (mSOF) (sodium chloride 107.7 mM, potassium chloride 7.16

232

mM, potassium monobasic phosphate 1.19 mM, calcium chloride 1.71 mM, magnesium

233

chloride 0.49 mM, sodium bicarbonate 25.07 mM, sodium lactate 3.30 mM, and sodium

234

pyruvate 0.30 mM) [26], centrifuged twice at 500 g for 5 min and then resuspended in

235

fertilization medium to a final concentration of 2×106 motile spermatozoa mL−1 after the

236

swim-up procedure [27], each well was inseminated with 5 x 105 motile spermatozoa.

237

Sperm concentration was determined by hemocytometry using a Neubauer chamber,

238

and an average of the count of both chambers was used for each determination. Motility

239

was evaluated three times by the same observer by using an optical microscope

240

equipped with a thermal stage at 37ºC. Fertilization was performed in 500 µL IVF-

241

mSOF (mSOF supplemented with bovine serum albumin (BSA) 3 g L-1 and heparin

242

10000 U L-1) under mineral oil at 39ºC, in 5% CO2 in air for 20 h. Putative zygotes were

243

denuded by repeated pipetting and placed in 500 µL in vitro culture (IVC)-mSOF,

244

consisting of mSOF supplemented with 30 mL L−1 amino acid MEM (GIBCO), 10 mL

245

L−1 non-essential amino acid MEM (GIBCO), 2 mmol-glutamine, 6 g L−1 BSA and 5%

246

(v/v) FBS (GIBCO), under mineral oil at 39ºC in a humidified atmosphere with 90%

247

N2:5% CO2:5% O2 for 24 h. The proportion of cleaved embryos after 48 h was

248

evaluated by the number of embryos that presented two or more blastomeres.

249

In vitro embryo development was performed in IVC-mSOF, under mineral oil at

250

39ºC in a humidified atmosphere with 90% N2:5% CO2:5% O2, renewing the culture

10

251

medium every 48 h. The proportion of blastocysts produced was determined on days 7

252

and 8 following insemination (n=73–98 for each treatment in three replicates).

253 254

2.8.3. Parthenogenetic activation

255

Next, to study the activation capacity of in vitro matured vitrified-warmed

256

oocytes independently of spermatozoa, we analyzed their ability to be chemically

257

activated. To this end, matured fresh oocytes and vitrified-warmed oocytes were

258

completely denuded and then incubated in TALP (sodium chloride 114 mM, potassium

259

chloride 3.2 mM, sodium monobasic phosphate 0.3 mM, calcium chloride 2 mM,

260

magnesium chloride 0.5 mM, sodium bicarbonate 25.00 mM, sodium lactate 10 mM,

261

and sodium pyruvate 0.20 mM) supplemented with 3 g L-1 BSA and 5 µM ionomycin

262

for 5 min and then in mSOF added with 2 mM 6-dimethylaminopurine for 3 h as

263

described by Grupen et al. [28]. Oocytes were then washed and placed in IVF-mSOF

264

under mineral oil in a humidified atmosphere with 90% N2: 5% CO2: 5% O2 for 45 h.

265

The percentage of cleaved embryos was determined as described above (n= 114 oocytes

266

for each treatment, in two replicates).

267 268

2.9. Experimental design

269

For the morphological, viability and biochemical evaluation, COCs were

270

randomly divided into two groups: control and vitrified-warmed oocytes, whereas for

271

the functional studies, COCs were randomly divided in three groups after maturation:

272

control and vitrified-warmed oocytes, and an additional group representing an IVF

273

control consisting of COCs completely surrounded by cumulus cells. For the

274

morphological, viability and biochemical evaluation, control and vitrified-warmed

275

oocytes came from the same batch of ovaries but were processed at different times

11

276

because of the extra time needed by the vitrification-warming process. For the

277

functional studies, control oocytes came from a different batch of ovaries to synchronize

278

the warming time with the completion of maturation (Fig. 1).

279 280

2.10. Statistical analysis

281

The cytosolic oxidative status, quantification of active mitochondria, ROS

282

production and redox state values were expressed as mean ± S.E.M. and interactions

283

were analyzed by two-way ANOVA, using post-hoc general contrasts for

284

comparison among treatments (2x3 factorial analysis). All statistical tests were

285

performed using the InfoStat software (Universidad de Córdoba, Córdoba,

286

Argentina, see http://www.infostat.com.ar).

287

Metaphase II plate recovery, cleavage and embryo development rates were

288

compared using a chi-square analysis for non-parametric data. P<0.05 was

289

considered significant.

290 291 292

3. Results 3.1. Evaluation of the oocyte morphology and viability

293

At 0 (n= 28) and 3 h (n=43) of incubation, all the vitrified-warmed oocytes

294

studied conserved a normal morphology, preserving their plasma membrane

295

integrity, a reconstructed perivitelline space and a uniform cytoplasm, while at 21 h

296

of incubation, 94% (n=32) of the oocytes remained with the normal characteristics

297

described above. No differences were observed between vitrified-warmed oocytes

298

and controls (n = 98; 0 h =30; 3 h = 34; 21 h= 34 oocytes, in three replicates).

299

Regarding their survival capacity, vitrified-warmed oocytes showed 100%

300

viability for the three time points studied. No differences were observed between

12

301

controls and vitrified oocytes (n = 192; 0 h =32; 3 h = 32; 21 h= 32 oocytes for each

302

group, in three replicates).

303 304 305 306

3.2. Biochemical evaluations 3.2.1. Cytosolic oxidative status and quantification of active mitochondria

307

Differences in oxidative activities were observed in both groups at the

308

different time points studied. A decrease in oxidative activity was recorded in the

309

vitrified-warmed group at 3 and 21 h and in the control group at 3 h compared with

310

0 h (P<0.05), while no differences were observed between treatments (Fig. 4a)

311

(mean ± SEM: control 0 h 2.59 x 106± 2.56 x 105 vs. vitrified-warmed oocytes 0 h

312

2.85 x 106± 2.91 x 105; control 3 h 1.73 x 106± 1.24 x 105 vs. vitrified-warmed

313

oocytes 3 h 1.84 x 106± 1.89 x 105; control 21 h 2.12 x 106± 1.87 x 105 vs. vitrified-

314

warmed oocytes 21 h 1.84 x 106± 1.89 x 105).

315

As regards the quantification of active mitochondria, significant differences

316

were observed along the time points studied and between treatments. The control

317

group showed an increase in the quantification of active mitochondria at 21 h respect

318

to that at 3 h, while vitrified-warmed oocytes showed a decrease at 3 h and 21 h

319

compared to that at 0 h (P<0.05). Also, an increase in the quantification of active

320

mitochondria was observed at 0 and 3 h in vitrified-warmed oocytes compared with

321

their respective controls (P<0.05) (Fig. 4b) (mean ± SEM: control 0 h 1.28 x 107 ±

322

7.28 x 105 vs. vitrified-warmed oocytes 0 h 1.64 x 107± 1.10 x 106; control 3 h 1.08

323

x 107± 5.02 x 105 vs. vitrified-warmed oocytes 3 h 1.35 x 107± 9.30 x 105; control 21

324

h 1.38 x 107± 1.54 x 106 vs. vitrified-warmed oocytes 21 h 1.35 x 107± 1.08 x 106).

325

13

326

3.2.2. Reactive oxygen species levels

327

Vitrified-warmed and control oocytes showed variations in ROS production

328

at the different time points studied. ROS levels were higher in vitrified-warmed

329

oocytes than in the control oocytes at 0 h, and higher in control oocytes at 0 h than

330

in control ones at 3 h and 21 h (P<0.05) (Fig. 5) (mean ± SEM: control 0 h 521.1 ±

331

37.75 vs. vitrified-warmed oocytes 0 h 617.37 ± 69.43; control 3 h 339.29 ± 25.12

332

vs. vitrified-warmed oocytes 3 h 244 ± 10.17; control 21 h 4.15 ± 26.87 vs. vitrified-

333

warmed oocytes 21 h 259.32 ± 19.85).

334 335

3.2.3. Redox state

336

An increase in the redox state at 21 h was observed in both control and

337

vitrified-warmed oocytes (P<0.05), but no differences were detected between

338

treatments (Fig. 6) (mean ± SEM: control 0 h 3.35 x 106± 1.96 x 105 vs. vitrified-

339

warmed oocytes 0 h 4.03 x 106± 3.60 x 105; control 3 h 3.61 x 106± 2.49 x 105 vs.

340

vitrified-warmed oocytes 3 h 3.89 x 105± 1.89 x 105; control 21 h 5.40 x 106± 5.56 x

341

105 vs. vitrified-warmed oocytes 21 h 4.91 x 106± 5.90 x 105).

342 343 344

3.3 Oocyte functional studies 3.3.1 Metaphase II plate recovery analysis

345

The metaphase II configuration recovered 3 hours after warming (69.2%). No

346

further improvement was found after 4 hours of incubation (61.5%), while 2 hours of

347

incubation post-warming proved to be insufficient (10 %; P<0.05; Table 1).

Metaphase II (%) Unrecovered nuclear material n

2 hours

3 hours

4 hours

2 (10)a 18 20

18 (69.2)b 8 26

16 (61.5)b 10 26

348 14

349

Table 1. Metaphase II plate recovery of vitrified-warmed oocytes incubated for 2, 3 and

350

4 hours. Numbers in parentheses are percentages. Different superscript letters indicate

351

significant differences in the percentage of oocytes at different time points (n=20-26 per

352

group in two replicates, P<0.05).

353 354

3.4. In vitro fertilization and embryo development

355

Significant differences between the three previously described groups (IVF

356

control, control and treatment) were observed in the cleavage and blastocyst rates,

357

with vitrified-warmed oocytes showing a decrease in both parameters (cleavage

358

rates for IVF control, control and vitrified-warmed oocytes: 79.6%, 57.4% and

359

31.18%, respectively; blastocyst rates for IVF control, control and vitrified-warmed

360

oocytes: 35.5%, 18.2% and 8.8%, respectively) (P<0.05; Table 2).

IVF Control Control Vitrified-warmed

Number of oocytes used for IVF

Number (%) of cleaved embryos

Number of embryos (%) developed to blastocyt stage

118 115 125

94 (79.6) a 66 (57.4) b 39 (31.18) c

42 (35.5) a 21 (18.2) b 11 (8.8) c

361 362

Table 2. Cleavage and blastocyst rates following IVF. Numbers in parentheses are

363

percentages. Different superscript letters indicate significant differences in the

364

percentage of cleaved embryos and number of embryos developed to blastocyst on days

365

2 and 7- 8 respectively, P<0.05).

366 367

3.5. Parthenogenetic activation

368

Parthenogenetic activation was performed to analyze the cause of the

369

reduction in the cleavage rate observed in vitrified-warmed oocytes after IVF.

370

Denuded oocytes (controls) and vitrified-warmed denuded oocytes were chemically 15

371

activated and the cleavage rate was evaluated after 48 hours in both groups, showing

372

no significant differences (control: 55.4 % vs. vitrified-warmed oocytes: 50 %; n=

373

57 oocytes per group, in two replicates).

374 375

4.

Discussion

376

Although in the last decade many studies have been conducted to improve

377

embryo and oocyte vitrification, this technique is not currently used for oocytes in

378

animal breeding programs. Our goal was to study the effect of bovine oocyte

379

vitrification using the Cryotech® method on morphological, biochemical and

380

functional parameters at different recovery times to assess the ability of oocytes to

381

recover from the damages induced by vitrification.

382

By analyzing ultrastructural morphological parameters, several authors have

383

previously concluded that vitrification may affect nuclear morphology [29],

384

microtubule formation [30], cytoplasmic organization [29, 31], and membrane

385

integrity [32]. So, in the present study, we first determined oocyte morphology and

386

viability in matured vitrified and control bovine oocytes along 21 h of extended

387

incubation and observed no differences between the groups studied, thus

388

demonstrating that Cryotech® did not affect oocyte macromorphology and viability.

389

We then evaluated biochemical parameters in control and vitrified oocytes.

390

Although we found no differences in the cytosolic oxidative activity between

391

groups, vitrified oocytes presented an increase in the quantification of active

392

mitochondria at 0 and 3 h after warming. This increase may be a consequence of a

393

higher energy requirement for the reorganization of organelles, cytoplasm and redox

394

potential after oocyte vitrification-warming. In previous studies on porcine oocyte in

395

vitro maturation, we observed that the quantification of active mitochondria is

16

396

modulated by ATP or AMP [22]. Similarly, by evaluating mitochondrial activity

397

with Mitotracker Red CMXRos, Succu et al. described high mitochondrial activity

398

and low ATP content after warming of vitrified oocytes [33].

399

As regards ROS levels, some reports have described an increase in this

400

parameter in vitrified oocytes after warming [33-35]. We also found an increase in

401

ROS production at 0 h for the vitrified group, probably because of the oxidative

402

stress produced by the vitrification-warming process. However, these levels did not

403

persist in time and were continued with a significant decrease after 3 h of incubation

404

post-warming, reaching lower levels than the control and persisting at 21 h. The

405

decrease in ROS levels, cytosolic oxidative activity and quantification of active

406

mitochondria observed in both groups coincides with that observed in a previous

407

study, in which non-activated oocytes presented decreasing levels of both ROS and

408

cytosolic oxidative activity throughout 24 h post-maturation [36]. This suggests that

409

this behavior is related to oocytes going through an ageing process.

410

The redox state, which was determined by NAD(P)H autofluorescence,

411

showed no differences between vitrified and control groups. A peak at 21 h post-

412

warming was recorded for both control and vitrified oocytes, which might result

413

from a quieter metabolic state [37]. This statement is supported by the results of a

414

previous study in bovine oocytes, which suggested that tricarboxylic acid cycle

415

activity would be related to NAD(P)H levels [24].

416

Finally, functional studies were carried out to evaluate oocyte competence

417

after the vitrification-warming process. It has been described that vitrification affects

418

the normal oocyte spindle configuration [6, 11]. Therefore, we studied the

419

metaphase II recovery time and found that around 3 and 4 h of incubation post-

420

warming are necessary to achieve a higher recovery rate. As regards oocyte

17

421

competence, we observed that although vitrified oocytes could cleave after

422

fertilization, the cleavage rate diminished respect to that of controls and,

423

subsequently, embryo development was adversely affected by this procedure. We

424

also observed that the partial denudation of the oocytes, done before IVF in the

425

control group, negatively affected the cleavage rate and embryo development,

426

indicating that the low rates observed are a result of both the vitrification-warming

427

procedure and partial denudation. In line with these results, Hwang and Hochi

428

reviewed that the integrity of cumulus cells after vitrification is an important factor

429

to harvest cytoplasmically matured oocytes [38]. Several groups have reported the

430

possibility of cryopreserving bovine oocytes, but subsequent embryo development in

431

most of these studies has remained low, ranging from 0 to 15% [33, 39-44]. Only

432

few authors have reached higher rates with other vitrification methods, but also with

433

lower blastocyst rates respect to fresh embryos [30, 45]. Although we did not

434

observe macrostructural changes in vitrified-warmed oocytes, we cannot discard

435

ultrastructural changes that could affect developmental competence, as suggested by

436

other authors [29-31]. On the other hand, Cryotech® has successfully been used in

437

the vitrification of human oocytes, but there are important differences in the

438

characteristics of bovine and human oocytes that must be taken into account. Bovine

439

oocytes have a higher content of lipids than human oocytes, which makes them more

440

sensitive to cryopreservation [46]. Moreover, bovine oocytes are particularly

441

sensitive to cytoskeleton damages, such as alterations to the meiotic spindle, as a

442

consequence of cryopreservation [6, 47]. When subjected to parthenogenetic

443

activation, no differences were observed in the cleavage rates between control and

444

vitrified-warmed oocytes. This suggests that the interaction between vitrified-

18

445

warmed oocytes and spermatozoa could negatively be affected by the vitrification

446

process, as reported by other authors [48].

447

In conclusion, this study shows that it is possible to vitrify bovine oocytes,

448

given that their morphology and viability could be maintained through the

449

vitrification–warming process and 21 h in vitro post warming incubation. Our results

450

also suggest that the metabolic changes observed in bovine oocytes as a

451

consequence of vitrification and warming, represented mainly by variations in the

452

quantification of active mitochondria and levels of ROS production after warming,

453

could be in part responsible for their diminished developmental competence.

454

Therefore, the protocol must still be improved to avoid variations in oocyte

455

metabolism that could reduce their developmental competence.

456 457

5. Acknowledgments

458

This work was supported by a grant of the University of Buenos Aires (UBACyT

459

20020130100693BA 2014/2017), Argentina.

460

6. Bibliography

461

[1] Wang Y, Xiao Z, Li L, Fan W, Li SW. Novel needle immersed vitrification: a

462

practical and convenient method with potential advantages in mouse and human ovarian

463

tissue cryopreservation. Hum Reprod 2008;23:2256-65.

464

[2] Gutnisky C, Alvarez GM, Cetica PD, Dalvit GC. Evaluation of the Cryotech

465

Vitrification Kit for bovine embryos. Cryobiology 2013;67:391-3.

466

[3] Do VH, Walton S, Catt S, Taylor-Robinson AW. A comparative analysis of the

467

efficacy of three cryopreservation protocols on the survival of in vitro-derived cattle

468

embryos at pronuclear and blastocyst stages. Cryobiology 2017;77:58-63.

469

[4] Gupta A, Singh J, Dufort I, Robert C, Dias FCF, Anzar M. Transcriptomic

470

difference in bovine blastocysts following vitrification and slow freezing at morula

471

stage. PLoS ONE 2017;12:e0187268.

472

[5] Saragusty J, Arav A. Current progress in oocyte and embryo cryopreservation by

473

slow freezing and vitrification. Reproduction 2011;141:1-19. 19

474

[6] Morato R, Izquierdo D, Paramio MT, Mogas T. Embryo development and structural

475

analysis of in vitro matured bovine oocytes vitrified in flexipet denuding pipettes.

476

Theriogenology 2008;70:1536-43.

477

[7] Rusciano G, De Canditiis C, Zito G, Rubessa M, Roca MS, Carotenuto R, et al.

478

Raman-microscopy investigation of vitrification-induced structural damages in mature

479

bovine oocytes. PLoS ONE 2017;12:e0177677.

480

[8] Wang N, Hao HS, Li CY, Zhao YH, Wang HY, Yan CL, et al. Calcium ion

481

regulation by BAPTA-AM and ruthenium red improved the fertilisation capacity and

482

developmental ability of vitrified bovine oocytes. Sci Rep 2017;7:10652.

483

[9] Yan CL, Fu XW, Zhou GB, Zhao XM, Suo L, Zhu SE. Mitochondrial behaviors in

484

the vitrified mouse oocyte and its parthenogenetic embryo: effect of Taxol pretreatment

485

and relationship to competence. Fertil Steril 2010;93:959-66.

486

[10] Zhao XM, Fu XW, Hou YP, Yan CL, Suo L, Wang YP, et al. Effect of vitrification

487

on mitochondrial distribution and membrane potential in mouse two pronuclear (2-PN)

488

embryos. Mol Reprod Dev 2009;76:1056-63.

489

[11] Chen SU, Lien YR, Cheng YY, Chen HF, Ho HN, Yang YS. Vitrification of

490

mouse oocytes using closed pulled straws (CPS) achieves a high survival and preserves

491

good patterns of meiotic spindles, compared with conventional straws, open pulled

492

straws (OPS) and grids. Hum Reprod 2001;16:2350-6.

493

[12] Castillo-Martin M, Bonet S, Morato R, Yeste M. Supplementing culture and

494

vitrification-warming media with l-ascorbic acid enhances survival rates and redox

495

status of IVP porcine blastocysts via induction of GPX1 and SOD1 expression.

496

Cryobiology 2014;68:451-8.

497

[13] Dehghani-Mohammadabadi M, Salehi M, Farifteh F, Nematollahi S, Arefian E,

498

Hajjarizadeh A, et al. Melatonin modulates the expression of BCL-xl and improve the

499

development of vitrified embryos obtained by IVF in mice. J of Assist Reprod Genet

500

2014;31:453-61.

501

[14] Zhao XM, Hao HS, Du WH, Zhao SJ, Wang HY, Wang N, et al. Melatonin inhibits

502

apoptosis and improves the developmental potential of vitrified bovine oocytes. J Pineal

503

Res 2016;60:132-41.

504

[15] Sutovsky P, Schatten G. Depletion of glutathione during bovine oocyte maturation

505

reversibly blocks the decondensation of the male pronucleus and pronuclear apposition

506

during fertilization. Biol Reprod 1997;56:1503-12.

20

507

[16] Trapphoff T, Heiligentag M, Simon J, Staubach N, Seidel T, Otte K, et al.

508

Improved cryotolerance and developmental potential of in vitro and in vivo matured

509

mouse oocytes by supplementing with a glutathione donor prior to vitrification. Mol

510

Hum Reprod 2016;22:867-81.

511 512 513

[17] Segovia Y, Victory N, Peinado I, García Valverde LM, García M, Aizpurua J, Monzó A, Gómez- Torres MJ. Ultrastructural characteristics of human oocytes vitrified before and after in vitro maturation. J Reprod Dev 2017; 63(4): 377-382.

514 515 516

[18] Stimpfel M, Vrtacnik- Bokal E, Virant- Klun. No difference in mitochondrial distribution is observed in human oocytes after cryopreservation. Arch Gynecol Obstet 2017; 296: 373-381.

517 518

[19] Gandhi G, Kuwayama M, Kagalwala S, Pangerkar P. Appendix A: Cryotech® Vitrification Thawing. Methods Mol Biol 2017; 1568:281-295.

519

[20] Cryotech® September, 2019. www.cryotechlab.com.

520

[21] Alvarez GM, Dalvit GC, Achi MV, Miguez MS, Cetica PD. Immature oocyte

521

quality and maturational competence of porcine cumulus-oocyte complexes

522

subpopulations. Biocell 2009;33:167-77.

523

[22] Alvarez GM, Casiró S, Gutnisky C, Dalvit GC, Sutton Mc-Dowall ML, Thompson

524

J., Cetica PD. Implications of glycolytic and pentose phosphate pathways on the

525

oxidative status and active mitochondria of the porcineoocyte during IVM.

526

Theriogenology 2016;86:2096-2106.

527

[23] Morado SA, Cetica PD, Beconi MT, Dalvit GC. Reactive oxygen species in bovine

528

oocyte maturation in vitro. Reprod Fertil Dev 2009;21:608-14.

529

[24] Sutton-McDowall ML, Purdey M, Brown HM, Abell AD, Mottershead DG, Cetica

530

PD, et al. Redox and anti-oxidant state within cattle oocytes following in vitro

531

maturation with bone morphogenetic protein 15 and follicle stimulating hormone. Mol

532

Reprod Dev 2015;82:281-94.

533

[25] Gutnisky C, Dalvit GC, Thompson JG, Cetica PD. Pentose phosphate pathway

534

activity: effect on in vitro maturation and oxidative status of bovine oocytes. Reprod

535

Fertil Dev 2014;26:931-42.

536

[26] Takahashi Y, First NL. In vitro development of bovine one-cell embryos: Influence

537

of glucose, lactate, pyruvate, amino acids and vitamins. Theriogenology 1992;37:963-

538

78.

539

[27] Hallap T, Haard M, Jaakma U, Larsson B, Rodriguez-Martinez H. Does cleansing

540

of frozen-thawed bull semen before assessment provide samples that relate better to

541

potential fertility? Theriogenology 2004;62:702-13. 21

542

[28] Grupen CG, Mau JC, McIlfatrick SM, Maddocks S, Nottle MB. Effect of 6-

543

dimethylaminopurine on electrically activated in vitro matured porcine oocytes. Mol

544

Reprod Dev 2002;62:387-96.

545

[29] Luna HS, Ferrari I, Rumpf R. Influence of stage of maturation of bovine oocytes at

546

time of vitrification on the incidence of diploid metaphase II at completion of

547

maturation. Anim Reprod Sci 2001;68:23-8.

548

[30] Chasombat J, Nagai T, Parnpai R, Vongpralub T. Pretreatment of in vitro matured

549

bovine oocytes with docetaxel before vitrification: Effects on cytoskeleton integrity and

550

developmental ability after warming. Cryobiology 2015;71:216-23.

551

[31] Palmerini MG, Antinori M, Maione M, Cerusico F, Versaci C, Nottola SA, et al.

552

Ultrastructure of immature and mature human oocytes after cryotop vitrification. J

553

Reprod Develop 2014;60:411-20.

554

[32] Succu S, Leoni GG, Bebbere D, Berlinguer F, Mossa F, Bogliolo L, et al.

555

Vitrification devices affect structural and molecular status of in vitro matured ovine

556

oocytes. Mol Reprod Dev 2007;74:1337-44.

557

[33] Succu S, Gadau SD, Serra E, Zinellu A, Carru C, Porcu C, et al. A recovery time

558

after warming restores mitochondrial function and improves developmental competence

559

of vitrified ovine oocytes. Theriogenology 2018;110:18-26.

560

[34] Gupta MK, Uhm SJ, Lee HT. Effect of vitrification and beta-mercaptoethanol on

561

reactive oxygen species activity and in vitro development of oocytes vitrified before or

562

after in vitro fertilization. Fertil Steril 2010;93:2602-7.

563

[35] Ren L, Fu B, Ma H, Liu D. Effects of mechanical delipation in porcine oocytes on

564

mitochondrial distribution, ROS activity and viability after vitrification. Cryo letters.

565

2015;36:30-6.

566

[36] Morado S, Cetica P, Beconi M, Thompson JG, Dalvit G. Reactive oxygen species

567

production and redox state in parthenogenetic and sperm-mediated bovine oocyte

568

activation. Reproduction 2013;145:471-8.

569

[37] Madrid Gaviria S, Morado SA, López Herrera A, Restrepo Betancur G, Urrego

570

Álvarez RA, Echeverri Zuluaga J, et al. Resveratrol supplementation promotes recovery

571

of lower oxidative metabolism after vitrification and warming of in vitro produced

572

bovine embryos. Reprod Fertil Devel 2019;31:521-8.

573

[38] Hwang I, Hochi S. Recent progress of cryopreservation of bovine oocyte. Biomed

574

Res Int 2014; Article ID 570647.

22

575

[39] Diez C, Duque P, Gomez E, Hidalgo CO, Tamargo C, Rodriguez A, et al. Bovine

576

oocyte vitrification before or after meiotic arrest: effects on ultrastructure and

577

developmental ability. Theriogenology 2005;64:317-33.

578

[40] Prentice JR, Singh J, Dochi O, Anzar M. Factors affecting nuclear maturation,

579

cleavage and embryo development of vitrified bovine cumulus-oocyte complexes.

580

Theriogenology 2011;75:602-9.

581

[41] Prentice-Biensch JR, Singh J, Mapletoft RJ, Anzar M. Vitrification of immature

582

bovine cumulus-oocyte complexes: effects of cryoprotectants, the vitrification

583

procedure and warming time on cleavage and embryo development. Reprod Biol

584

Endocrinol 2012;10:73.

585

[42] Bulgarelli DL, Vireque AA, Pitangui-Molina CP, Silva-de-Sa MF, de Sa Rosa

586

ESACJ. Reduced competence of immature and mature oocytes vitrified by Cryotop

587

method: assessment by in vitro fertilization and parthenogenetic activation in a bovine

588

model. Zygote. 2017;25:222-30.

589

[43] Ortiz-Escribano N, Smits K, Piepers S, Van den Abbeel E, Woelders H, Van Soom

590

A. Role of cumulus cells during vitrification and fertilization of mature bovine oocytes:

591

Effects on survival, fertilization, and blastocyst development. Theriogenology

592

2016;86:635-41.

593

[44] Spricigo JF, Morais K, Ferreira AR, Machado GM, Gomes AC, Rumpf R, et al.

594

Vitrification of bovine oocytes at different meiotic stages using the Cryotop method:

595

assessment of morphological, molecular and functional patterns. Cryobiology

596

2014;69:256-65.

597

[45] Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, et al. Open

598

Pulled Straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and

599

embryos. Mol Reprod Dev 1998;51:53-8.

600

[46] Somfai T, Kikuchi K, Nagai T. Factors affecting cryopreservation of porcine

601

oocytes. J Reprod Dev 2012; 58(1): 17-24.

602 603 604

[47] Bogliolo L, Ariu F, Fois S, Rosati I, Zedda MT, Leoni G, Succu S, Pau S, Ledda S. Morphological and biochemical analysis of immature ovine oocytes vitrified with or without cumulus cells. Theriogenology 2017; 68(8): 1138-49.

605

[48] Wiesak T, Wasielak M, Zlotkowska A, Milewski R. Effect of vitrification on the

606

zona pellucida hardening and follistatin and cathepsin B genes expression and

607

developmental competence of in vitro matured bovine oocytes. Cryobiology

608

2017;76:18-23. 23

609

24

Caption of the figures Fig 1. Scheme of the experimental design showing the three groups involved in the work and the asynchrony between them: Control, Vitrified-warmed and IVF control oocytes. Fig 2: Representative photographs of fresh and vitrified-warmed oocytes (x120) used for biochemical evaluations. (a) to (c) fresh oocytes at 0, 3 and 21h, (A) to (C) vitrifiedwarmed oocytes at 0, 3 and 21h stained with RedoxSensor Red CC1; (d) to (f) fresh oocytes at 0, 3 and 21h, (D) to (F) vitrified-warmed oocytes at 0, 3 and 21h stained with Mitotracker Green; (g) to (i) fresh oocytes at 0, 3 and 21h, (G) to (I) vitrified-warmed oocytes at 0, 3 and 21h stained with DCHFDA; (j) to (l) fresh oocytes at 0, 3 and 21h, (J) to (L) vitrified-warmed oocytes at 0, 3 and 21h showing NAD(P)H autofluorescence. Figure 3. A. Unrecovered nuclear material and B. Metaphase II plate, recovered after 3 h incubation post warming. Arrow indicate the presence of the first polar body and * indicate metaphase II chromatin configuration. Magnification (x100). Fig. 4 a, b. Cytosolic oxidative status (a) and active mitochondria (b) of control (white bars) and vitrified- warmed oocytes (grey bars) incubated for 0, 3 and 21 h post warming. Data are the mean ± s.e.m. (n=30–40 COCs for each treatment in three replicates). Bars of the same colour with different letters differ significantly (P<0.05). The asterisks indicate significant differences between treatments at the same time point. Fig. 5. Reactive oxygen species (ROS) of control (white bars) and vitrified- warmed oocytes (grey bars) incubated for 0, 3 and 21 h post warming. Data are the mean ± s.e.m. (n=30–40 COCs for each treatment in three replicates). Bars of the same colour with different letters differ significantly (P<0.05). The asterisks indicate significant differences between treatments at the same time point. Fig. 6. Redox state of control (white bars) and vitrified- warmed oocytes (grey bars) for 0, 3 and 21 h post warming. Data are the mean ± s.e.m. (n=30–40 COCs for each treatment in three replicates). Bars of the same colour with different letters differ significantly (P<0.05).

Highlights

•

Oocyte vitrification with Cryotech® kit does not affect oocyte morphology nor viability.

•

No differences in oxidative activity were found between control and vitrifiedwarmed oocytes at each studied time point, while differences between the studied time points were observed. Quantification of active mitochondria was higher in the vitrified- warmed oocytes than control oocytes at 0 and 3 h after warming. ROS levels were higher at 0h in the vitrified- warmed oocytes than in control oocytes. Oocyte vitrification and partial denudation affect the cleavage and blastocyst rates, showing a decrease in these parameters.

• • •

Author contributions

Gutnisky Cynthia: Methodology, investigation, formal analysis, writing original draft, reviewing, editing and visualization. Morado Sergio: Methodology, investigation, reviewing, editing and visualization. Gadze Tomas: Investigation. Donato Antonella: Investigation. Alvarez Gabriel: Investigation. Dalvit Gabriel: Supervision, project administration, funding acquisition. Cetica Pablo: Supervision, project administration, funding acquisition, reviewing, editing and visualization.