- Email: [email protected]
Embryo development after vitrification of immature and in vitro-matured equine oocytes
Journal Pre-proof Embryo development after vitrification of immature and in vitro-matured equine oocytes Daniel Angel, Heloisa Siqueira Canesin, Joao Gatto Brom-de-Luna, Sergio Morado, Gabriel Dalvit, Diana Gomez, Natalia Posada, Osvaldo Bogado Pascottini, Rodrigo Urrego, Katrin Hinrichs, Isabel Catalina Velez PII:
S0011-2240(19)30516-4
DOI:
https://doi.org/10.1016/j.cryobiol.2020.01.014
Reference:
YCRYO 4173
To appear in:
Cryobiology
Received Date: 30 October 2019 Revised Date:
10 January 2020
Accepted Date: 17 January 2020
Please cite this article as: D. Angel, H.S. Canesin, J.G. Brom-de-Luna, S. Morado, G. Dalvit, D. Gomez, N. Posada, O.B. Pascottini, R. Urrego, K. Hinrichs, I.C. Velez, Embryo development after vitrification of immature and in vitro-matured equine oocytes, Cryobiology (2020), doi: https://doi.org/10.1016/ j.cryobiol.2020.01.014. 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. © 2020 Published by Elsevier Inc.
1
Embryo development after vitrification of immature and in vitrovitro-matured equine oocytes
2
Daniel Angelaf*, Heloisa Siqueira Canesinb, Joao Gatto Brom-de-Lunab, Sergio Moradoc, Gabriel Dalvitc,
3
Diana Gomezd, Natalia Posadad, Osvaldo Bogado Pascottinief, Rodrigo Urregoa, Katrin Hinrichsb† and
4
Isabel Catalina Veleza†
5
a
6
Universidad CES, Medellin, Antioquia, 050021, Colombia.
7
b
8
77843-4466, United States.
9
c
Research Group in Animal Sciences - INCA-CES, School of Veterinary Medicine and Animal Production,
College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX
Area of Biochemistry, Institute of Research and Technology in Animal Reproduction (INITRA), School of
10
Veterinary Sciences, Universidad de Buenos Aires, Ciudad de Buenos Aires, Buenos Aires, C1427CWO,
11
Argentina.
12
d
13
e
14
University of Antwerp, Wilrijk, Belgium.
15
f
16
University, Merelbeke, Belgium.
17
*
18
E-mail address: [email protected] (D. Angel)
19
† Drs. Hinrichs and Velez should be considered joint senior authors
20
Institute of Human Fertility – INSER, Medellin, Antioquia, Colombia.
Department of Veterinary Sciences, Gamete Research Center, Veterinary Physiology and Biochemistry,
Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent
Corresponding author. Calle 5A # 35 -113, Medellin, Antioquia, 050021, Colombia.
21
Abstract
22
Effects of meiotic stage and cumulus status on development of equine oocytes after vitrification was
23
evaluated. Immature oocytes with corona radiata (IMM); in vitro-matured oocytes with corona radiata
24
(MAT CR+); and in vitro-matured oocytes denuded of cumulus (MAT CR-) were vitrified using the
25
Cryotech® method. Warming medium was equilibrated either in 5% CO2 or Air. IMM oocytes underwent
26
in vitro maturation after warming. Recovery, survival, and maturation rates, and cleavage and blastocyst
27
rates after ICSI, were evaluated. Recovery was higher for oocytes warmed in CO2- than Air-equilibrated
28
medium (86±3 vs. 76.9±4%, respectively). Maturation for all vitrified-warmed oocyte treatments
29
(37±6.5 to 45.9±5.8%) was not different from control (50±4.1%), except for MAT CR- CO2 (20.3±4.6%).
30
Cleavage for MAT CR- CO2 and Air groups was similar to control (67.7±12.1, 71.4±8.1, and 78±5.3%,
31
respectively). One blastocyst was produced (MAT CR+ CO2), representing the first equine blastocyst
32
reported after vitrification of an in vitro-matured oocyte.
33 34 35
Keywords: blastocyst; cumulus cells; equine oocyte; ICSI; meiotic stage; vitrification.
36
Multiple factors influence the success of oocyte vitrification, such as species, maturation status,
37
presence or absence of cumulus cells, vitrification protocols, type of cryoprotectant agents (CPAs), and
38
vitrification devices [1]. In the horse, these factors remain poorly studied. The best developmental result
39
reported for equine oocyte vitrification (40% blastocyst rate) was achieved using in vivo-matured
40
oocytes [2]; however, because superovulation is not effective in the horse, this approach has low
41
potential for application. Most equine research has been performed in germinal vesicle (GV)-stage
42
oocytes, which simplifies application, but results have been poor [3–5]. Vitrification of in vitro-matured
43
(IVM) oocytes may provide better post-warming developmental competence than does vitrification of
44
immature oocytes.
45
To the best of our knowledge, only one report had assessed embryo development from equine
46
oocytes vitrified at the metaphase II (MII) stage after in vitro maturation (IVM); in that report, no
47
blastocysts were obtained [6].
48
The influence of cumulus cells on the success of oocyte vitrification is uncertain [5,7,8]. In
49
humans, clinical vitrification is performed with denuded MII oocytes. However, for GV-stage oocytes,
50
presence of cumulus cells is crucial for subsequent normal maturation [8]. Moreover, cumulus cells may
51
protect the meiotic spindle in vitrified oocytes [7]. For these reasons, we evaluated the effects of
52
meiotic stage and presence of cumulus, as well as warming solution equilibration, on development of
53
equine oocytes after vitrification. Furthermore, saline-based solutions become alkaline during freezing
54
[9] thus it is possible that oocytes may benefit from being warmed in a more acidic medium, thus we
55
compared the 5% CO2- vs. Air-equilibrated warming medium.
56
All chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) and media from
57
Invitrogen (Carlsbad, CA, USA). Equine cumulus-oocyte complexes (COCs) were recovered from
58
slaughterhouse-derived ovaries and processed at the laboratory at Universidad CES, Colombia. The COCs
59
were held at room temperature overnight for 17 to 24 h in 1-mL borosilicate glass vials (Thermo Fisher
60
Scientific, Waltham, MA, USA) containing commercial embryo holding media (Vigro, Bioniche, Belleville,
61
ON, Canada) as previously described [3]. The next morning, COCs were randomly divided into three
62
groups, immature oocytes with corona radiata for immediate vitrification (IMM); COCs which were
63
matured in vitro, and then vitrified either with an intact corona radiata (MAT CR+); or denuded of
64
cumulus (MAT CR-).
65
Oocytes assigned to the IMM group were pipetted to remove outer cumulus cells, leaving
66
corona radiata, then vitrified immediately. All oocytes assigned to MAT treatments were cultured for 28
67
to 30 h in 150-µL droplets of maturation medium (M199 with Earle's salts, supplemented with 5 mU
68
FSH/mL (Sioux Biochemical Inc., Sioux Center, IA), 10% fetal bovine serum and 25 mg
69
gentamycin/mL),with a maximum of 15 oocytes per droplet, under light white mineral oil at 38.2 °C in
70
5% CO2 as previously described [10]. After culture, these oocytes were divided into two groups which
71
were either partially denuded to CR, or completely denuded of cumulus by pipetting in a solution of
72
0.05% hyaluronidase, then vitrified. Oocytes were not selected for morphology or maturation status
73
before vitrification.
74
Vitrification and warming were performed using the Cryotech® method, with the supplied
75
proprietary solutions, as previously described [10]. Briefly, oocytes were equilibrated for 10 to 15 min in
76
a solution containing ethylene glycol (EG) and dimethyl sulfoxide (DMSO) in Minimum Essential Media
77
(MEM) with hydroxypropyl cellulose (HPC) and no protein supplement. The oocytes were moved to a
78
vitrification solution containing EG, DMSO, and trehalose in MEM + HPC and loaded onto the device
79
supplied with the kit (3 to 5 oocytes per device) and the sample was quickly immersed into liquid
80
nitrogen. The devices were left in the liquid nitrogen until all oocytes had been vitrified, then the tips
81
were covered with a protective cap at the end. The time between the placement of oocytes in the
82
vitrification solution and the immersion of the device into the liquid nitrogen was 60 to 90 sec. IMM
83
oocytes were processed at room temperature (~22 °C), while MAT oocytes were processed at 37 °C.
84
The vitrified oocytes were transported in liquid nitrogen to the Equine Embryo Laboratory at
85
Texas A&M University. Each vitrification group was randomly divided to be warmed in warming solution
86
previously equilibrated for two hours in air or 5% CO2 in air. The warming solution consisted of MEM +
87
trehalose + HPC at 37 °C. After 1 min, oocytes were moved for 3 min to the kit’s diluent solution
88
consisting of MEM + trehalose + HPC, and washed twice in different wells in washing solution, consisting
89
of MEM + HPC, for 5 min and 1 min, respectively.This resulted in IMM Air, IMM CO2, MAT CR+ Air, MAT
90
CR+ CO2, MAT CR- Air, and MAT CR- CO2 treatments. After warming, IMM oocytes were subjected to
91
IVM for 28 to 30 h. MAT oocytes were incubated for 2 to 4 h in maturation medium as post-warming
92
culture. After IVM or post-warming culture, oocytes still having corona radiata (IMM and MAT CR+
93
groups) were denuded of cumulus using solution of 0.05% hyaluronidase, then oocytes with polar
94
bodies were assigned to ICSI and embryo culture, as described by Salgado et al. (2018) [11]. Sperm
95
immobilization and injection were performed using a piezo drill. To control for the ICSI and embryo
96
culture procedures, oocytes were recovered via transvaginal ultrasound-guided follicular aspiration from
97
a herd of 15 mares at the Equine Embryo Laboratory (Texas A&M University) as previously described
98
[12]. The control oocytes were held overnight, cultured for IVM, and subjected to ICSI and embryo
99
culture as for vitrified-warmed oocytes. Vitrified oocytes were warmed two to three times per week for
100
four weeks (12 replicates). One replicate with control oocytes was performed each week that vitrified-
101
warmed oocytes were processed. Frozen-thawed semen from the same fertile stallion was used for all
102
control and treatment replicates. Experimental design is depicted in Figure 1.
103
All statistical analyses were performed using R-core (version 3.6.1; R Core Team, Vienna,
104
Austria). The oocyte/zygote/embryo was considered as the unit of interest. Generalized mixed effects
105
models were used to test the effect of vitrification method (control vs. IMM vs. MAT CR+ vs. MAT CR-)
106
on maturation, cleavage, and blastocyst rates. Warming method (Air vs. CO2) was forced into each
107
model to test its effect on recovery, survival, maturation, cleavage, and blastocyst rates. For all the
108
models the replicate was set as a random effect. Results are expressed as least squares means and
109
standard errors. The differences between treatment groups were assessed using the Tukey’s post hoc
110
test. The significance and tendency levels were set at P < 0.05 and P < 0.1, respectively.
111
The number of oocytes that were fitted in the statistical analysis to calculate the recovery,
112
survival, maturation, cleavage, and blastocyst rates are described in Supplementary Tables 1 and 2. The
113
pH of warming medium was measured after two hours of equilibration, it was 7.2 for Air and 7.0 for 5%
114
CO2. Figure 2 shows the recovery, survival, maturation, and cleavage rates of vitrified (IMM, MAT CR+,
115
and MATR CR-) oocytes warmed in Air- or CO2-equilibrated medium. Recovery rate (number of vitrified
116
oocytes located after warming/ number initially vitrified) was greater (P < 0.05) for MAT CR+ warmed in
117
CO2-equilibrated medium than for all Air-equilibrated groups. The maturation rate was lower in MAT CR-
118
warmed in CO2-equilibrated medium than MAT CR- warmed in Air- or MAT CR+ warmed in CO2-
119
equilibrated medium (P < 0.05). Figure 3 shows the maturation, cleavage, and blastocyst rates of
120
vitrified oocytes (IMM, MAT CR+, and MATR CR-) either warmed in Air- or CO2-equilibrated medium
121
compared to control (non-vitrified) oocytes. The maturation rate was greater in control than MAT CR-
122
oocytes (P < 0.05). However, the maturation rate was not different among control and IMM and MAT
123
CR+ oocytes (P ˃ 0.05). The cleavage rate was similar between control and MAT CR - oocytes (P ˃ 0.05)
124
but it was greater in control than IMM and MAT CR+ oocytes P < 0.05). MAT CR+ warmed in CO2
125
equilibrated medium produced one blastocyst (1/51; 1.9%); this blastocyst rate was lower than injected
126
control oocytes (14/60; 23.3% blastocysts per injected oocyte; P < 0.001).
127
Notably, 7 to 28 % of oocytes were not recovered after warming, and empty zonas or fragments
128
were not found after thorough search of the warming medium and vitrification device. This might
129
indicate that losses occurred during plunging or storage of the device. The higher recovery rate
130
observed in MAT CR+ is probably associated with greater adherence to the vitrification device by the
131
intercellular matrix of the expanded cumulus.
132
The vitrification method we utilized has been successful for human oocytes, and a similar
133
method was successful in yielding blastocysts from vitrified equine in vivo-matured oocytes [2]. We
134
found that this method was associated with excellent oocyte survival (93 to 98%). Oocytes vitrified,
135
denuded, and warmed in medium with lower pH (MAT CR- CO2) presented significantly lower
136
maturation rates than MAT CR- Air and MAT CR+ CO2, despite the fact that they were cultured for
137
maturation concurrently before vitrification. It is not clear if this reflects variation in the rate of MII
138
before this group was vitrified, or degeneration of the oocytes after vitrification. We hypothesize that
139
stress induced by low pH plus the lack of cumulus cells, which help to protect against pH change within
140
the oocyte, might lead to oocyte degeneration [13,14]. Except for MAT CR- CO2, maturation rates of
141
vitrified-warmed oocytes were broadly comparable to those for the control. IMM oocytes matured at
142
rates equivalent to control, despite having only corona radiata, in accordance with a previous report [8].
143
The rate of maturation achieved after vitrification compares favorably with that reported in some
144
previous publications [11,12]. However, the failure of vitrified-warmed oocytes to support
145
developmental competence is compatible with previous findings in the horse [8,2,3], and underlines the
146
fact that evaluation of vitrification techniques in equine oocytes must include assessment of blastocyst
147
production.
148
The highest cleavage rate in this study was in MAT CR- oocytes and the only blastocyst came
149
from MAT CR+. This suggests that vitrification of MII oocytes may provide better developmental
150
competence than does vitrification at the GV stage. This contrasts with the results of Tharasanit et al.
151
(2006), who, using a short vitrification protocol, reported higher cleavage in oocytes vitrified in the
152
immature state (28-34%) than for those vitrified after IVM (4-16%) [12].
153
The effect of presence of cumulus cells during vitrification is unclear [9,7]. Tharasanit et al.
154
(2009) showed that the presence of multiple layers of cumulus cells protected spindle integrity during
155
the vitrification of mature equine oocytes [11]. Nevertheless, in cattle, Ortiz-Escribano et al. (2018)
156
reported that maturation was lower after vitrification of intact COCs than for oocytes vitrified with CR
157
only, which they attributed to impaired exchange of CPAs [8]. In horses, we found no difference in
158
survival or cleavage rates between mature oocytes vitrified with or without CR, suggesting no effect of
159
the CR on CPA exchange rates. In this experiment, we produced the first blastocyst obtained from an
160
equine oocyte vitrified at MII after IVM. However, the vitrification protocols studied here are not yet
161
efficient; more research is needed to optimize this procedure for equine oocytes.
162 163
Funding
164
This research was supported by the Clinical Equine ICSI Program at Texas A&M University; the Link
165
Equine Research Endowment Fund - Texas A&M University; CES University; the University of Buenos
166
Aires; and INSER – Instituto de Fertilidad Humana.
167 168
Acknowledgements
169
The authors thank the group from the Reproductive Biotechnology Laboratory, Veterinary faculty of CES
170
University, during the development of part of the project with slaughterhouse-derived COCs. Osvaldo
171
Bogado Pascottini was granted by Fonds voor Wetenschappelijk Onderzoek–Vlaanderen (FWO, Research
172
Foundation, Flanders) under project number 12Y5220N.
173 174 175 176
References
177
[1]
178 179
A. Arav, Cryopreservation of oocytes and embryos., Theriogenology. 81 (2014) 96–102. https://doi.org/10.1016/j.theriogenology.2013.09.011.
[2]
L.J. Maclellan, J.E. Stokes, K.A. Preis, P.M. Mccue, E.M. Carnevale, Vitrification, warming, ICSI and
180
transfer of equine oocytes matured in vivo, Anim. Reprod. Sci. 121 (2010) 260–261.
181
https://doi.org/10.1016/j.anireprosci.2010.04.157.
182
[3]
H.S. Canesin, J.G. Brom-de-Luna, Y.H. Choi, I. Ortiz, M. Diaw, K. Hinrichs, Blastocyst development
183
after intracytoplasmic sperm injection of equine oocytes vitrified at the germinal-vesicle stage,
184
Cryobiology. 75 (2017) 52–59. https://doi.org/10.1016/j.cryobiol.2017.02.004.
185
[4]
H.S. Canesin, J.G. Brom-de-Luna, Y.-H. Choi, A.M. Pereira, G.G. Macedo, K. Hinrichs, Vitrification
186
of germinal-vesicle stage equine oocytes: Effect of cryoprotectant exposure time on in-vitro
187
embryo production, Cryobiology. (2018). https://doi.org/10.1016/j.cryobiol.2018.01.001.
188
[5]
N. Ortiz-Escribano, O. Bogado Pascottini, H. Woelders, L. Vandenberghe, C. De Schauwer, J.
189
Govaere, E. Van den Abbeel, T. Vullers, C. Ververs, K. Roels, M. Van De Velde, A. Van Soom, K.
190
Smits, An improved vitrification protocol for equine immature oocytes, resulting in a first live
191
foal, Equine Vet. J. 50 (2018) 391–397. https://doi.org/10.1111/evj.12747.
192
[6]
T. Tharasanit, S. Colleoni, G. Lazzari, B. Colenbrander, C. Galli, T. a E. Stout, Effect of cumulus
193
morphology and maturation stage on the cryopreservability of equine oocytes, Reproduction.
194
132 (2006) 759–769. https://doi.org/10.1530/rep.1.01156.
195
[7]
T. Tharasanit, S. Colleoni, C. Galli, B. Colenbrander, T.A.E. Stout, Protective effects of the
196
cumulus-corona radiata complex during vitrification of horse oocytes, Reproduction. 137 (2009)
197
391–401. https://doi.org/10.1530/REP-08-0333.
198
[8]
N. Ortiz-Escribano, K. Smits, S. Piepers, E. Van den Abbeel, H. Woelders, A. Van Soom, Role of
199
cumulus cells during vitrification and fertilization of mature bovine oocytes: Effects on survival,
200
fertilization, and blastocyst development, Theriogenology. 86 (2016) 635–641.
201
https://doi.org/10.1016/j.theriogenology.2016.02.015.
202
[9]
H. Watanabe, T. Otsuka, M. Harada, T. Okada, Imbalance between anion and cation distribution
203
at ice interface with liquid phase in frozen electrolyte as evaluated by fluorometric
204
measurements of pH, J. Phys. Chem. C. 118 (2014) 15723–15731.
205
https://doi.org/10.1021/jp5031653.
206
[10]
C. Gutnisky, G.M. Alvarez, P.D. Cetica, G.C. Dalvit, Cryobiology Evaluation of the Cryotech
207
Vitrification Kit for bovine embryos q, Cryobiology. 67 (2013) 391–393.
208
https://doi.org/10.1016/j.cryobiol.2013.08.006.
209
[11]
R.M. Salgado, J.G. Brom-de-Luna, H.L. Resende, H.S. Canesin, K. Hinrichs, Blastocyst Rates and
210
Kinetics of Sperm Processing After Conventional vs. Piezo-driven ICSI, J. Equine Vet. Sci. 66 (2018)
211
175. https://doi.org/10.1016/j.jevs.2018.05.067.
212
[12]
C.C. Jacobson, Y.H. Choi, S.S. Hayden, K. Hinrichs, Recovery of mare oocytes on a fixed biweekly
213
schedule, and resulting blastocyst formation after intracytoplasmic sperm injection,
214
Theriogenology. 73 (2010) 1116–1126. https://doi.org/10.1016/j.theriogenology.2010.01.013.
215
[13]
216 217 218 219
D. Fabian, Š. Čikoš, P. Rehák, J. Koppel, Do embryonic polar bodies commit suicide?, Zygote. 22 (2014) 10–17. https://doi.org/10.1017/S0967199412000159.
[14]
G. FitzHarris, J.M. Baltz, Regulation of intracellular pH during oocyte growth and maturation in mammals, Reproduction. 138 (2009) 619–627. https://doi.org/10.1530/rep-09-0112.
S0011-2240(19)30516-4
DOI:
https://doi.org/10.1016/j.cryobiol.2020.01.014
Reference:
YCRYO 4173
To appear in:
Cryobiology
Received Date: 30 October 2019 Revised Date:
10 January 2020
Accepted Date: 17 January 2020
Please cite this article as: D. Angel, H.S. Canesin, J.G. Brom-de-Luna, S. Morado, G. Dalvit, D. Gomez, N. Posada, O.B. Pascottini, R. Urrego, K. Hinrichs, I.C. Velez, Embryo development after vitrification of immature and in vitro-matured equine oocytes, Cryobiology (2020), doi: https://doi.org/10.1016/ j.cryobiol.2020.01.014. 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. © 2020 Published by Elsevier Inc.
1
Embryo development after vitrification of immature and in vitrovitro-matured equine oocytes
2
Daniel Angelaf*, Heloisa Siqueira Canesinb, Joao Gatto Brom-de-Lunab, Sergio Moradoc, Gabriel Dalvitc,
3
Diana Gomezd, Natalia Posadad, Osvaldo Bogado Pascottinief, Rodrigo Urregoa, Katrin Hinrichsb† and
4
Isabel Catalina Veleza†
5
a
6
Universidad CES, Medellin, Antioquia, 050021, Colombia.
7
b
8
77843-4466, United States.
9
c
Research Group in Animal Sciences - INCA-CES, School of Veterinary Medicine and Animal Production,
College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX
Area of Biochemistry, Institute of Research and Technology in Animal Reproduction (INITRA), School of
10
Veterinary Sciences, Universidad de Buenos Aires, Ciudad de Buenos Aires, Buenos Aires, C1427CWO,
11
Argentina.
12
d
13
e
14
University of Antwerp, Wilrijk, Belgium.
15
f
16
University, Merelbeke, Belgium.
17
*
18
E-mail address: [email protected] (D. Angel)
19
† Drs. Hinrichs and Velez should be considered joint senior authors
20
Institute of Human Fertility – INSER, Medellin, Antioquia, Colombia.
Department of Veterinary Sciences, Gamete Research Center, Veterinary Physiology and Biochemistry,
Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent
Corresponding author. Calle 5A # 35 -113, Medellin, Antioquia, 050021, Colombia.
21
Abstract
22
Effects of meiotic stage and cumulus status on development of equine oocytes after vitrification was
23
evaluated. Immature oocytes with corona radiata (IMM); in vitro-matured oocytes with corona radiata
24
(MAT CR+); and in vitro-matured oocytes denuded of cumulus (MAT CR-) were vitrified using the
25
Cryotech® method. Warming medium was equilibrated either in 5% CO2 or Air. IMM oocytes underwent
26
in vitro maturation after warming. Recovery, survival, and maturation rates, and cleavage and blastocyst
27
rates after ICSI, were evaluated. Recovery was higher for oocytes warmed in CO2- than Air-equilibrated
28
medium (86±3 vs. 76.9±4%, respectively). Maturation for all vitrified-warmed oocyte treatments
29
(37±6.5 to 45.9±5.8%) was not different from control (50±4.1%), except for MAT CR- CO2 (20.3±4.6%).
30
Cleavage for MAT CR- CO2 and Air groups was similar to control (67.7±12.1, 71.4±8.1, and 78±5.3%,
31
respectively). One blastocyst was produced (MAT CR+ CO2), representing the first equine blastocyst
32
reported after vitrification of an in vitro-matured oocyte.
33 34 35
Keywords: blastocyst; cumulus cells; equine oocyte; ICSI; meiotic stage; vitrification.
36
Multiple factors influence the success of oocyte vitrification, such as species, maturation status,
37
presence or absence of cumulus cells, vitrification protocols, type of cryoprotectant agents (CPAs), and
38
vitrification devices [1]. In the horse, these factors remain poorly studied. The best developmental result
39
reported for equine oocyte vitrification (40% blastocyst rate) was achieved using in vivo-matured
40
oocytes [2]; however, because superovulation is not effective in the horse, this approach has low
41
potential for application. Most equine research has been performed in germinal vesicle (GV)-stage
42
oocytes, which simplifies application, but results have been poor [3–5]. Vitrification of in vitro-matured
43
(IVM) oocytes may provide better post-warming developmental competence than does vitrification of
44
immature oocytes.
45
To the best of our knowledge, only one report had assessed embryo development from equine
46
oocytes vitrified at the metaphase II (MII) stage after in vitro maturation (IVM); in that report, no
47
blastocysts were obtained [6].
48
The influence of cumulus cells on the success of oocyte vitrification is uncertain [5,7,8]. In
49
humans, clinical vitrification is performed with denuded MII oocytes. However, for GV-stage oocytes,
50
presence of cumulus cells is crucial for subsequent normal maturation [8]. Moreover, cumulus cells may
51
protect the meiotic spindle in vitrified oocytes [7]. For these reasons, we evaluated the effects of
52
meiotic stage and presence of cumulus, as well as warming solution equilibration, on development of
53
equine oocytes after vitrification. Furthermore, saline-based solutions become alkaline during freezing
54
[9] thus it is possible that oocytes may benefit from being warmed in a more acidic medium, thus we
55
compared the 5% CO2- vs. Air-equilibrated warming medium.
56
All chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) and media from
57
Invitrogen (Carlsbad, CA, USA). Equine cumulus-oocyte complexes (COCs) were recovered from
58
slaughterhouse-derived ovaries and processed at the laboratory at Universidad CES, Colombia. The COCs
59
were held at room temperature overnight for 17 to 24 h in 1-mL borosilicate glass vials (Thermo Fisher
60
Scientific, Waltham, MA, USA) containing commercial embryo holding media (Vigro, Bioniche, Belleville,
61
ON, Canada) as previously described [3]. The next morning, COCs were randomly divided into three
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groups, immature oocytes with corona radiata for immediate vitrification (IMM); COCs which were
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matured in vitro, and then vitrified either with an intact corona radiata (MAT CR+); or denuded of
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cumulus (MAT CR-).
65
Oocytes assigned to the IMM group were pipetted to remove outer cumulus cells, leaving
66
corona radiata, then vitrified immediately. All oocytes assigned to MAT treatments were cultured for 28
67
to 30 h in 150-µL droplets of maturation medium (M199 with Earle's salts, supplemented with 5 mU
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FSH/mL (Sioux Biochemical Inc., Sioux Center, IA), 10% fetal bovine serum and 25 mg
69
gentamycin/mL),with a maximum of 15 oocytes per droplet, under light white mineral oil at 38.2 °C in
70
5% CO2 as previously described [10]. After culture, these oocytes were divided into two groups which
71
were either partially denuded to CR, or completely denuded of cumulus by pipetting in a solution of
72
0.05% hyaluronidase, then vitrified. Oocytes were not selected for morphology or maturation status
73
before vitrification.
74
Vitrification and warming were performed using the Cryotech® method, with the supplied
75
proprietary solutions, as previously described [10]. Briefly, oocytes were equilibrated for 10 to 15 min in
76
a solution containing ethylene glycol (EG) and dimethyl sulfoxide (DMSO) in Minimum Essential Media
77
(MEM) with hydroxypropyl cellulose (HPC) and no protein supplement. The oocytes were moved to a
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vitrification solution containing EG, DMSO, and trehalose in MEM + HPC and loaded onto the device
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supplied with the kit (3 to 5 oocytes per device) and the sample was quickly immersed into liquid
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nitrogen. The devices were left in the liquid nitrogen until all oocytes had been vitrified, then the tips
81
were covered with a protective cap at the end. The time between the placement of oocytes in the
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vitrification solution and the immersion of the device into the liquid nitrogen was 60 to 90 sec. IMM
83
oocytes were processed at room temperature (~22 °C), while MAT oocytes were processed at 37 °C.
84
The vitrified oocytes were transported in liquid nitrogen to the Equine Embryo Laboratory at
85
Texas A&M University. Each vitrification group was randomly divided to be warmed in warming solution
86
previously equilibrated for two hours in air or 5% CO2 in air. The warming solution consisted of MEM +
87
trehalose + HPC at 37 °C. After 1 min, oocytes were moved for 3 min to the kit’s diluent solution
88
consisting of MEM + trehalose + HPC, and washed twice in different wells in washing solution, consisting
89
of MEM + HPC, for 5 min and 1 min, respectively.This resulted in IMM Air, IMM CO2, MAT CR+ Air, MAT
90
CR+ CO2, MAT CR- Air, and MAT CR- CO2 treatments. After warming, IMM oocytes were subjected to
91
IVM for 28 to 30 h. MAT oocytes were incubated for 2 to 4 h in maturation medium as post-warming
92
culture. After IVM or post-warming culture, oocytes still having corona radiata (IMM and MAT CR+
93
groups) were denuded of cumulus using solution of 0.05% hyaluronidase, then oocytes with polar
94
bodies were assigned to ICSI and embryo culture, as described by Salgado et al. (2018) [11]. Sperm
95
immobilization and injection were performed using a piezo drill. To control for the ICSI and embryo
96
culture procedures, oocytes were recovered via transvaginal ultrasound-guided follicular aspiration from
97
a herd of 15 mares at the Equine Embryo Laboratory (Texas A&M University) as previously described
98
[12]. The control oocytes were held overnight, cultured for IVM, and subjected to ICSI and embryo
99
culture as for vitrified-warmed oocytes. Vitrified oocytes were warmed two to three times per week for
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four weeks (12 replicates). One replicate with control oocytes was performed each week that vitrified-
101
warmed oocytes were processed. Frozen-thawed semen from the same fertile stallion was used for all
102
control and treatment replicates. Experimental design is depicted in Figure 1.
103
All statistical analyses were performed using R-core (version 3.6.1; R Core Team, Vienna,
104
Austria). The oocyte/zygote/embryo was considered as the unit of interest. Generalized mixed effects
105
models were used to test the effect of vitrification method (control vs. IMM vs. MAT CR+ vs. MAT CR-)
106
on maturation, cleavage, and blastocyst rates. Warming method (Air vs. CO2) was forced into each
107
model to test its effect on recovery, survival, maturation, cleavage, and blastocyst rates. For all the
108
models the replicate was set as a random effect. Results are expressed as least squares means and
109
standard errors. The differences between treatment groups were assessed using the Tukey’s post hoc
110
test. The significance and tendency levels were set at P < 0.05 and P < 0.1, respectively.
111
The number of oocytes that were fitted in the statistical analysis to calculate the recovery,
112
survival, maturation, cleavage, and blastocyst rates are described in Supplementary Tables 1 and 2. The
113
pH of warming medium was measured after two hours of equilibration, it was 7.2 for Air and 7.0 for 5%
114
CO2. Figure 2 shows the recovery, survival, maturation, and cleavage rates of vitrified (IMM, MAT CR+,
115
and MATR CR-) oocytes warmed in Air- or CO2-equilibrated medium. Recovery rate (number of vitrified
116
oocytes located after warming/ number initially vitrified) was greater (P < 0.05) for MAT CR+ warmed in
117
CO2-equilibrated medium than for all Air-equilibrated groups. The maturation rate was lower in MAT CR-
118
warmed in CO2-equilibrated medium than MAT CR- warmed in Air- or MAT CR+ warmed in CO2-
119
equilibrated medium (P < 0.05). Figure 3 shows the maturation, cleavage, and blastocyst rates of
120
vitrified oocytes (IMM, MAT CR+, and MATR CR-) either warmed in Air- or CO2-equilibrated medium
121
compared to control (non-vitrified) oocytes. The maturation rate was greater in control than MAT CR-
122
oocytes (P < 0.05). However, the maturation rate was not different among control and IMM and MAT
123
CR+ oocytes (P ˃ 0.05). The cleavage rate was similar between control and MAT CR - oocytes (P ˃ 0.05)
124
but it was greater in control than IMM and MAT CR+ oocytes P < 0.05). MAT CR+ warmed in CO2
125
equilibrated medium produced one blastocyst (1/51; 1.9%); this blastocyst rate was lower than injected
126
control oocytes (14/60; 23.3% blastocysts per injected oocyte; P < 0.001).
127
Notably, 7 to 28 % of oocytes were not recovered after warming, and empty zonas or fragments
128
were not found after thorough search of the warming medium and vitrification device. This might
129
indicate that losses occurred during plunging or storage of the device. The higher recovery rate
130
observed in MAT CR+ is probably associated with greater adherence to the vitrification device by the
131
intercellular matrix of the expanded cumulus.
132
The vitrification method we utilized has been successful for human oocytes, and a similar
133
method was successful in yielding blastocysts from vitrified equine in vivo-matured oocytes [2]. We
134
found that this method was associated with excellent oocyte survival (93 to 98%). Oocytes vitrified,
135
denuded, and warmed in medium with lower pH (MAT CR- CO2) presented significantly lower
136
maturation rates than MAT CR- Air and MAT CR+ CO2, despite the fact that they were cultured for
137
maturation concurrently before vitrification. It is not clear if this reflects variation in the rate of MII
138
before this group was vitrified, or degeneration of the oocytes after vitrification. We hypothesize that
139
stress induced by low pH plus the lack of cumulus cells, which help to protect against pH change within
140
the oocyte, might lead to oocyte degeneration [13,14]. Except for MAT CR- CO2, maturation rates of
141
vitrified-warmed oocytes were broadly comparable to those for the control. IMM oocytes matured at
142
rates equivalent to control, despite having only corona radiata, in accordance with a previous report [8].
143
The rate of maturation achieved after vitrification compares favorably with that reported in some
144
previous publications [11,12]. However, the failure of vitrified-warmed oocytes to support
145
developmental competence is compatible with previous findings in the horse [8,2,3], and underlines the
146
fact that evaluation of vitrification techniques in equine oocytes must include assessment of blastocyst
147
production.
148
The highest cleavage rate in this study was in MAT CR- oocytes and the only blastocyst came
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from MAT CR+. This suggests that vitrification of MII oocytes may provide better developmental
150
competence than does vitrification at the GV stage. This contrasts with the results of Tharasanit et al.
151
(2006), who, using a short vitrification protocol, reported higher cleavage in oocytes vitrified in the
152
immature state (28-34%) than for those vitrified after IVM (4-16%) [12].
153
The effect of presence of cumulus cells during vitrification is unclear [9,7]. Tharasanit et al.
154
(2009) showed that the presence of multiple layers of cumulus cells protected spindle integrity during
155
the vitrification of mature equine oocytes [11]. Nevertheless, in cattle, Ortiz-Escribano et al. (2018)
156
reported that maturation was lower after vitrification of intact COCs than for oocytes vitrified with CR
157
only, which they attributed to impaired exchange of CPAs [8]. In horses, we found no difference in
158
survival or cleavage rates between mature oocytes vitrified with or without CR, suggesting no effect of
159
the CR on CPA exchange rates. In this experiment, we produced the first blastocyst obtained from an
160
equine oocyte vitrified at MII after IVM. However, the vitrification protocols studied here are not yet
161
efficient; more research is needed to optimize this procedure for equine oocytes.
162 163
Funding
164
This research was supported by the Clinical Equine ICSI Program at Texas A&M University; the Link
165
Equine Research Endowment Fund - Texas A&M University; CES University; the University of Buenos
166
Aires; and INSER – Instituto de Fertilidad Humana.
167 168
Acknowledgements
169
The authors thank the group from the Reproductive Biotechnology Laboratory, Veterinary faculty of CES
170
University, during the development of part of the project with slaughterhouse-derived COCs. Osvaldo
171
Bogado Pascottini was granted by Fonds voor Wetenschappelijk Onderzoek–Vlaanderen (FWO, Research
172
Foundation, Flanders) under project number 12Y5220N.
173 174 175 176
References
177
[1]
178 179
A. Arav, Cryopreservation of oocytes and embryos., Theriogenology. 81 (2014) 96–102. https://doi.org/10.1016/j.theriogenology.2013.09.011.
[2]
L.J. Maclellan, J.E. Stokes, K.A. Preis, P.M. Mccue, E.M. Carnevale, Vitrification, warming, ICSI and
180
transfer of equine oocytes matured in vivo, Anim. Reprod. Sci. 121 (2010) 260–261.
181
https://doi.org/10.1016/j.anireprosci.2010.04.157.
182
[3]
H.S. Canesin, J.G. Brom-de-Luna, Y.H. Choi, I. Ortiz, M. Diaw, K. Hinrichs, Blastocyst development
183
after intracytoplasmic sperm injection of equine oocytes vitrified at the germinal-vesicle stage,
184
Cryobiology. 75 (2017) 52–59. https://doi.org/10.1016/j.cryobiol.2017.02.004.
185
[4]
H.S. Canesin, J.G. Brom-de-Luna, Y.-H. Choi, A.M. Pereira, G.G. Macedo, K. Hinrichs, Vitrification
186
of germinal-vesicle stage equine oocytes: Effect of cryoprotectant exposure time on in-vitro
187
embryo production, Cryobiology. (2018). https://doi.org/10.1016/j.cryobiol.2018.01.001.
188
[5]
N. Ortiz-Escribano, O. Bogado Pascottini, H. Woelders, L. Vandenberghe, C. De Schauwer, J.
189
Govaere, E. Van den Abbeel, T. Vullers, C. Ververs, K. Roels, M. Van De Velde, A. Van Soom, K.
190
Smits, An improved vitrification protocol for equine immature oocytes, resulting in a first live
191
foal, Equine Vet. J. 50 (2018) 391–397. https://doi.org/10.1111/evj.12747.
192
[6]
T. Tharasanit, S. Colleoni, G. Lazzari, B. Colenbrander, C. Galli, T. a E. Stout, Effect of cumulus
193
morphology and maturation stage on the cryopreservability of equine oocytes, Reproduction.
194
132 (2006) 759–769. https://doi.org/10.1530/rep.1.01156.
195
[7]
T. Tharasanit, S. Colleoni, C. Galli, B. Colenbrander, T.A.E. Stout, Protective effects of the
196
cumulus-corona radiata complex during vitrification of horse oocytes, Reproduction. 137 (2009)
197
391–401. https://doi.org/10.1530/REP-08-0333.
198
[8]
N. Ortiz-Escribano, K. Smits, S. Piepers, E. Van den Abbeel, H. Woelders, A. Van Soom, Role of
199
cumulus cells during vitrification and fertilization of mature bovine oocytes: Effects on survival,
200
fertilization, and blastocyst development, Theriogenology. 86 (2016) 635–641.
201
https://doi.org/10.1016/j.theriogenology.2016.02.015.
202
[9]
H. Watanabe, T. Otsuka, M. Harada, T. Okada, Imbalance between anion and cation distribution
203
at ice interface with liquid phase in frozen electrolyte as evaluated by fluorometric
204
measurements of pH, J. Phys. Chem. C. 118 (2014) 15723–15731.
205
https://doi.org/10.1021/jp5031653.
206
[10]
C. Gutnisky, G.M. Alvarez, P.D. Cetica, G.C. Dalvit, Cryobiology Evaluation of the Cryotech
207
Vitrification Kit for bovine embryos q, Cryobiology. 67 (2013) 391–393.
208
https://doi.org/10.1016/j.cryobiol.2013.08.006.
209
[11]
R.M. Salgado, J.G. Brom-de-Luna, H.L. Resende, H.S. Canesin, K. Hinrichs, Blastocyst Rates and
210
Kinetics of Sperm Processing After Conventional vs. Piezo-driven ICSI, J. Equine Vet. Sci. 66 (2018)
211
175. https://doi.org/10.1016/j.jevs.2018.05.067.
212
[12]
C.C. Jacobson, Y.H. Choi, S.S. Hayden, K. Hinrichs, Recovery of mare oocytes on a fixed biweekly
213
schedule, and resulting blastocyst formation after intracytoplasmic sperm injection,
214
Theriogenology. 73 (2010) 1116–1126. https://doi.org/10.1016/j.theriogenology.2010.01.013.
215
[13]
216 217 218 219
D. Fabian, Š. Čikoš, P. Rehák, J. Koppel, Do embryonic polar bodies commit suicide?, Zygote. 22 (2014) 10–17. https://doi.org/10.1017/S0967199412000159.
[14]
G. FitzHarris, J.M. Baltz, Regulation of intracellular pH during oocyte growth and maturation in mammals, Reproduction. 138 (2009) 619–627. https://doi.org/10.1530/rep-09-0112.