- Email: [email protected]
Brilliant cresyl blue staining allows the selection for developmentally competent immature feline oocytes
Accepted Manuscript Brilliant cresyl blue staining allows the selection for developmentally competent inmature feline oocytes
K. Jewgenow, L. Fernandez Gonzalez, S. Jänsch, D. Viertel, J. Zahmel PII:
S0093-691X(18)31089-6
DOI:
10.1016/j.theriogenology.2018.12.021
Reference:
THE 14813
To appear in:
Theriogenology
Received Date:
15 November 2018
Accepted Date:
09 December 2018
Please cite this article as: K. Jewgenow, L. Fernandez Gonzalez, S. Jänsch, D. Viertel, J. Zahmel, Brilliant cresyl blue staining allows the selection for developmentally competent inmature feline oocytes, Theriogenology (2018), doi: 10.1016/j.theriogenology.2018.12.021
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ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 1 2
Brilliant cresyl blue staining allows the selection for developmentally competent inmature feline oocytes
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Jewgenow K, Fernandez Gonzalez L, Jänsch S, Viertel D, Zahmel J
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Department of Reproduction Biology, Leibniz Institute for Zoo and Wildlife Research,
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PF700430, D10324 Berlin, Germany
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Correspondence should be addressed to
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Prof. K. Jewgenow, Leibniz Institute for Zoo and Wildlife Research,
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PF700430,
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D10324 Berlin Berlin, Germany.
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Email: [email protected]
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Tel. 0049 30 5168 611
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Short Title: BCB staining of feline oocytes
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ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 13
Author Contributions:
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Katarina Jewgenow designed the study, partly carried out the study, analyzed and interpreted
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the data, compiled the manuscript and finally approved of the version to be submitted.
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Lorena Fernandez Gonzalez partly carried out the study (experiment 1), analyzed and
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interpreted her data, discussed the results and critical revised the manuscript.
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Stefanie Jänsch: partly carried out the study (experiment 2,3) and analyzed her data
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Dagmar Viertel: performed the TEM analysis
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Jennifer Zahmel partly analyzed and interpreted data, discussed the results and critical revised
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the manuscript.
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All authors have approved the final article.
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ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 23
Abstract
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The outcome of in-vitro-maturation and in-vitro-fertilization of feline oocytes depends
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on the selection of high quality oocytes, and is often restricted to morphological criteria. The
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aim of this study was to test whether the Brilliant cresyl blue (BCB) staining is suitable for
27
pre-selection of feline oocytes before in-vitro-maturation.
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Cumulus-oocytes-complexes (COC) were released from domestic cat ovaries obtained after
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ovariectomy and were subjected to BCB staining. BCB+ stained oocytes were characterized
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by a violet/ pale blue staining of the ooplasma, BCB- oocytes remained unstained. Trans-
31
electron microscopy indicated for a slightly advanced stage of BCB- oocytes within the
32
maturation process. After 24 h in-vitro maturation, almost 75% of BCB+ and 21.5% of BCB-
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oocytes were able to reach metaphase II. Also, after in-vitro fertilization, significantly more
34
oocytes developed to morulae (19.2%) if oocytes were preselected for BCB staining, although
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8% of unstained COC still reached advanced embryo stages. Prolonged storage of ovaries
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before COC retrieval for 16 – 20h at 4°C was accompanied by reduced number of BCB+
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oocytes (96 of 210, 45.7%) in comparison to freshly isolated COC (151 of 225, 67.1%), and
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impaired cleavage rate (19.8%) and morula rate (9.4%) of BCB+ oocytes but the rate of
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embryos which developed to advanced stages remained unchanged (~50%).
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To conclude, BCB staining is a very useful tool to preselect immature COC of feline species
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ensuring higher developmental rates.
42 43
Keywords: feline, oocyte quality, brilliant cresyl blue,
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ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 44
1. Introduction
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Success of assisted reproduction in mammals often depends on oocyte quality
46
subjected to either in-vitro-maturation or in-vitro-fertilization. Although these assisted
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reproductive technologies both in human and animal have improved markedly it still remains
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elusive to predict embryo potential based on selected oocytes. Thus, there is a strong need for
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further refinement of existing selection methods and development of novel, robust and, non-
50
invasive procedures [1].
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Oocytes originate from individual ovarian follicles and therefore are characterized by
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a pronounced heterogeneity.
Determining the quality of oocytes is often restricted to
53
morphological analysis or to the study of cellular behaviours in the developing embryo [2].
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Although developmental and morphological information gained from microscopic assessment
55
have been positively associated with IVF outcomes (Nel-Themaat and Nagy 2011), any other
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non-invasive approach would help to identify additional parameters of oocytes, their
57
surrounding follicle cells or even ovarian status
58
competency of embryos [3].
which determine the developmental
59
The brilliant cresyl blue (BCB) test is a non-invasive approach which reflects glucose-
60
6-phosphate dehydrogenase (G6PDH) activity of the oocyte, their glutathione concentration
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and the number and quality of mitochondria [4]. Interestingly, the staining intensity for BCB
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allows identifying oocytes likely to be developmentally competent [5]. So far, it was
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effectively used in different mammalian species, including human [6], cattle [7], goats [8],
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pigs [4, 5], mice [9] and rats [10]. Thus, the aim of this study was to test the BCB staining for
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pre-selection of feline oocytes before in-vitro-maturation. We suggest that BCB staining will
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support the selection of good quality gametes obtained from old or diseased felids or after
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prolonged transportation in gamete rescue programmes [11-13], and thus, may help to
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economize efforts if the quality is insufficient for further development. Our experiences in 4
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 69
gamete rescue of different felid species from zoos indicate that often the oocyte quality is
70
compromised (Fernandez et al, 2018).
71
With the presented experiments we aimed to answer the following questions: Does BCB
72
staining allow the selection for developmentally competent feline oocytes and does BCB
73
staining reflect the quality of oocytes after prolonged storage?
74
2. Material and Methods
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All chemical reagents were purchased from Sigma–Aldrich (Taufkirchen, Germany) unless
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stated otherwise and were of the highest purity available. Ovaries and testes of domestic cats
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were obtained from the Berlin Animal Shelter after routine ovariectomy or castration.
78 79
2.1.
Collection and brilliant cresyl blue (BCB) staining of cumulus oocyte complexes
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Immediately upon arrival in the lab, ovaries were freed of surrounding tissues, washed in
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Washing Medium (WM, Medium 199 with Earle´s salts, supplemented with 3 mg/mL BSA,
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1.4 mg/mL HEPES, 0.6 mg/mL sodium lactate, 0.25 mg/mL sodium pyruvate, 0.15 mg/mL L-
83
glutamine, 0.1 mg/mL cysteine and 0.055 mg/mL gentamicin), and were sliced in 5 mL WM
84
to release cumulus-oocytes-complexes (COC). Collection and quality assessment of COC was
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performed under a stereomicroscope and only those with 3 - 4 granulosa cell layers
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surrounding the oocyte and with a homogenous, dark cytoplasm were chosen [14]. To
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evaluate the impact of prolonged storage at 4° C, some ovaries were kept refrigerated
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overnight (16 - 20 h) in HEPES-MEM, supplemented with 0.3% BSA and 1% antibiotic
89
antimycotic before isolating the oocytes by slicing.
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Good quality COC were subjected to 500 µL of BCB (13mg/L; (34 µM)) diluted in modified
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DPBS supplemented with 0.4 % BSA, 36 mg/L (0.327 mM) sodium pyruvate, 1 mg/mL and
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(5.55mM) D-(+)-Glucose, and 13 mg/L BCB for 1 hour at 38.5°C in a humidified air 5
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 93
atmosphere. Thereafter, the COCs were washed and examined under a stereomicroscope.
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According to the colour of the cytoplasma (Figure 1) two groups were created: BCB+
95
(coloured cytoplasm) and BCB– (colourless cytoplasm), respectively.
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2.2.
Transmission electron microscopy of BCB+ and BCB- stained oocytes
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After staining for BCB, cumulus-oocytes complexes were transferred to an objective slide and
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carefully mixed with pre-warmed 1% low melting agarose. After cooling for 15 min at 4°C,
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the agarose pillow around a single COC was cut to 1 mm2 pieces and transferred to
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Karnowsky solution for fixation and stored for processing by Transmission electron
101
microscopy (TEM).
102
After washing in PBS, the samples were treated with osmium tetroxide, dehydrated in
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increasing concentrations of ethanol and embedded in Epon 812. Semi-thin sections of 1 µm
104
were performed and stained with toluidine blue for pre-selection. Ultrathin sections (70 nm)
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of the selected tissue part were stained with uranyl acetate, followed by the application of lead
106
citrate. The COC present in the selected samples were analyzed and electromicrographies
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were made with magnifications between 1,250x and 11,000x using a FEI TecnaiSpiritBT
108
device (120 kV; FEI Deutschland GmbH).
109
Oocytes were evaluated for ultrastructure by assessing the integrity of the cytoplasmic
110
membrane, the presence and density of mitochondria, organelles and vacuoles.
111
2.3.
In vitro maturation of cat oocytes
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The procedure of in vitro maturation and fertilization of domestic cat oocytes was
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performed as previously described by [15, 16]. In brief, isolated COC were washed two times
114
in WM and cultured for maturation. Maturation proceeded at 38.5 °C and 5% CO2 in a
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humidified air atmosphere for 24 h and was performed in WM supplemented with human
116
0.02 IU LH/mL (L6420) and 0.05IU human pituitary FSH/mL (Ferring, Kiel, Germany). Each 6
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 117
COC was kept separately in 20 µL medium covered under mineral oil (Reproline Medical
118
GmbH, Rheinbach, Germany) in a 6 cm petri dish. For experiment 1, the COCs were freed
119
manually of attached cumulus cells by repeated pipetting and placed on an objective slide.
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After air drying, the slide was transferred to alcohol (96% ethanol) and was kept at 4°C until
121
analysis. Following staining of oocytes with propidium iodide (1.0 mg/ mL, 1:100 in PBS;
122
Thermo Fisher Scientific) the maturation stage was assessed under a fluorescence microscope
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(Axiovert 200M, Carl Zeiss Microscopy GmbH, Jena, Germany). The configuration of DNA
124
within the oocytes allowed to determine meiotic progress during the culture (presence of GV,
125
breakdown of GV membrane, metaphase I, metaphase II accompanied by a polar body).
126
Parthenogenic activation was considered when cleavage occurred without fertilization. For all
127
other experiments, COC were washed and fertilized in vitro (see below).
128
2.4.
In fertilization of cat oocytes
129
Testes were maintained at 4 °C without any medium for 24h before processing. Fresh
130
epididymal sperm cells were isolated as described before [17]. In brief, tissues were sliced
131
with a scissors in culture medium M199 (HEPES modification) at room temperature (20–
132
23°C) and sperm suspension was flushed through a 30-µm filter (Sysmex Partec GmbH,
133
Görlitz, Germany). After centrifugation at ~500 × g for 5 min, the pellet was re-suspended in
134
a small volume of M199 and sperm concentration was adjusted to 4 x 106 sperm/ mL for in
135
vitro fertilization.
136
In vitro fertilization was performed in 20 μL of WM medium supplemented with 2.2 IU/mL
137
heparin at 38.5 °C and 5 % CO2 for 18 h. The oocytes were fertilized with a final epididymal
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sperm concentration of 1×106 motile sperm/mL.
139
2.5.
Culture of embryos
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COC were freed of attached cumulus cells after 18 hours of gamete co-incubation by
141
repeated pipetting using 155 µm stripper tip micropipette (The Stripper®, BioTipp,
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Waterford, Ireland). Putative zygotes were transferred singly to 20 µL embryo culture
143
medium covered under mineral oil in a 6 cm petri dish. Embryo culture medium was prepared
144
from Ham´s F-10 and supplemented with 5% FCS, 0.05 mg/ mL gentamycin, 0.11 mg/mL
145
sodium pyruvate, and 0.075 mg/mL L-glutamine. Culture was performed at 38.5°C in 5%
146
CO2 and 5% O2 humidified atmosphere. Evaluation of embryo development was performed
147
every 24 hours. On day five of culture, all morulae were vitrified (another project). Oocytes
148
which did not cleave till day 2 of culture and all embryos not reaching morula stage at day
149
five were fixed by placing them on an objective slide, air dried, fixed in ethanol and stained
150
with PI (see above). The number of nuclei representing embryo blastomeres was counted.
151
2.6.
Statistical analysis
152
For comparison of maturation, fertilization and cleavage rates (embryo development), two-
153
tailed contingence tables were analysed with Fisher's exact test. P levels < 0.05 were
154
considered as significant. Statistical analysis was performed by using the statistical program
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InStat3 (GraphPad Software, Inc., California, USA); blox plot was created with Sigma plot.
156 157
3. Results
158
3.1.
159
Morphological characterization of brilliant cresyl blue positive and negative feline COC
160 161
BCB+ stained oocytes were characterized by a violet/ pale blue staining of the ooplasma,
162
which was slightly overlaid by the lipids within the cytoplasma (Figure 1). Trans-electron
163
microscopy indicated for only minor differences between BCB+ and BCB- feline COC
164
(Figure 2). BCB+ oocytes were characterized by equally sized vacuoles throughout the 8
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 165
cytoplasma, accumulation of round mitochondria in the cortical region and regular
166
distribution of cortical granules beneath plasma membrane (Figure 2 A,B). In BCB- oocytes
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we found more frequently a peripheral migration of the nucleus and formation of perivitelline
168
space (Figure 2C). Vesicles were different in size and appearance, with membrane inclusions.
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These vesicles were partly extruded into perivitelline space (Figure 2 D). Mitochondria
170
tended to be elongated and cortical granules were unequally distributed in the cortex region of
171
cytoplasma (Figure 2D).
172
3.2.
Does brilliant cresyl blue staining allow for the selection of developmentally competent feline oocytes?
173 174
Table 1 summarizes the experiment on comparing the maturation rate in vitro determined by
175
staining the nuclear structure of oocytes after 24 hours culture. There was a significant shift
176
towards higher maturation in BCB-positive oocytes. In this group, almost 75% of oocytes
177
were able to precede maturation indicated by metaphase II plate and extrusion of the first
178
polar body. With glucose-6-phosphate dehydrogenase activity (BCB-negative oocytes) almost
179
half of the COC remained at GV stage, and only 21.5% reached metaphase II.
180
In a second experiment, oocytes were fertilized after maturation and embryo culture was
181
performed for 5 days. Embryo development was assessed by cleavage rate and number of
182
embryos reaching morula at day 5 of culture. Again, there was a significant increase of
183
oocytes which underwent maturation (60.0% vs. 37.5%) for BCB+ and BCB- oocytes,
184
respectively, and cleaved embryos developed to morulae (54%) if oocytes were preselected
185
for BCB staining, although still 30.7% of embryos (8% of unstained COC) were able to reach
186
advanced embryo stages (Table 2).
187 188
3.3.
Does brilliant cresyl blue staining reflect the quality of oocytes after prolonged storage? 9
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 189
In a third experiment ovaries were obtained from a local animal clinic after
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ovariohysterectomia and were processed the same day (fresh) or were stored at 4° C until that
191
next morning (16 – 20 h holding). There was a high variation in the number of high quality
192
COC obtained per ovary (median 5, range 1 - 8), including the number of BCB+ (median 4,
193
range 0 – 7), which represented 67.1% of all isolated COC (151 of 225; Figure 2). Storage of
194
ovaries over night was not accompanied by reduction of good quality COC (median 5, range 2
195
– 15), but the number of BCB+ oocytes per ovary was compromised (median 3, range 0 – 7)
196
with only 45.7% of all COC being stained (96 of 210; Figure 2).
197
Developmental capacity of oocytes was also reduced after prolonged storage for 16 - 20 h at
198
4°C (Table 3). The cleavage rate of BCB+ COC was significantly higher for the fresh group
199
(37.1%) compared to BCB+ after storage (19.8%). This difference was not statistically
200
evident for morula rates, although there was a tendency towards less morulae (18.9% vs.
201
9.4%). Within the BCB- groups no difference in their developmental capacity was found.
202
Independently from ovarian storage, half of the cleaved embryos develop until morula stage,
203
if they originated from BCB+ COC, whereas in the BCB- groups only about 25% of embryos
204
developed further.
205
4. Discussion
206
As shown for other species, COC with high activity of glucose-6-phosphate dehydrogenase
207
express a higher meiotic competence and developmental potential. In our study with domestic
208
cat oocytes, the cytoplasma of about 60% of high quality COC (528 of 884 oocytes) was
209
stained after exposure to brilliant cresyl blue (BCB). The number per ovary varied between 0
210
and 7, and was reflecting the number of high-quality COC, which was also between 1 and 8
211
per ovary (Figure 2). This high variation of both morphological good quality COC and BCB+
212
oocytes might be related to the reproduction cycle of domestic cats. Throughout all
213
reproductive phases, anoestrus, follicular and luteal phase, high-quality COC can be obtained 10
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 214
[18]; the mean number reported varied between 2 - 3 per ovary [19, 20] and 20 [21]. We
215
previously reported a mean of 4 COC per ovary independently of season [22]. For domestic
216
cat in-vitro embryo production, it is clear that a strong selection for morphological criteria,
217
like dark cytoplasma and compact cumulus layer, before maturation will increase the outcome
218
of embryos after IVF [18]. Based on our data, BCB staining can be recommended as an
219
additional quality parameter for pre-selection of COC before in-vitro maturation.
220
For routine use, however, it has to be considered that BCB staining demands one hour
221
preparation time, and within the BCB- group still some embryos can be produced.
222
Unfortunately, we were not able to characterize the BCB- embryos further as by the
223
development to day 5, which is within the physiological time frame for reaching morula stage
224
[23].
225
It is also not clear why unstained COC develop to a lesser extent. BCB- or colourless
226
cytoplasma indicate for higher enzymatic activity of G6PDH and was found to be
227
characteristic for either growing oocytes or matured (MII) preovulatory oocytes [7, 24].
228
Unstained cytoplasma in fully grown, good quality COC as describe for BCB- COC, might
229
indicate for an imbalance between cytoplasmic (G6PGH activity) and nuclear (progress in
230
meiosis) maturation. Domestic cats are induced ovulators. Thus, at any time of oocyte
231
collection, a mixed population of oocytes is obtained containing also high quality COC which
232
have already missed the optimal time for ovulation. This suggestion is partly supported by our
233
TEM data, revealing that the GV of BCB- oocytes has already moved from the centre of the
234
oocyte towards periphery and that both mitochondria and corticular granulae as well the
235
perivitelline space behave as described for oocytes after the LH surge [24, 25]. Perhaps these
236
BCB- COC require less time for maturation, and need to be fertilized earlier than after 24
237
hours in-vitro maturation. Katska-Ksiakzkiewicz suggested that two "waves" of nuclear
238
maturation of cat oocytes can be distinguished. The first wave reach maturity by 17-18 h; and 11
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 239
the second wave occurs after 28-30 h of IVM [26]. By transabdominal ultrasound it was
240
shown that the growth of feline ovarian follicles was not exactly synchronous [27]. It was also
241
suggested that cytoplasmatic BCB activity indicates for synchronous nuclear and cytoplasmic
242
in vitro maturation [28].
243 244
Determination of BCB activity in oocytes before maturation has been recommended for
245
several purposes. In ewes, BCB staining was applied to compare superovulation protocols
246
based on the production of better-quality oocytes [29], or the staining was used to improve
247
IVM medium for BCB+ oocytes that can ameliorate reproductive success following in vitro
248
fertilization [30]. Chaubey at al. (2018) suggested a selective treatment of BCB- oocytes to
249
improve their maturation capacity and improve the outcome after IVF. Another broad
250
application field for BCB is the selection of developmentally competent oocytes for nuclear
251
transfer, because BCB+ oocytes were shown to express higher nuclear reprogramming
252
capacity in cloned blastocysts [31]. In addition to cloning, the choice of the best embryos to
253
transfer is still often based on subjective morphological parameters, although more
254
sophisticated parameters to select the most competent oocytes have become available [32].
255
BCB staining before in-vitro maturation allows classifying oocytes according to their future
256
developmental potential. Thus, these competent oocytes can be analysed by genomics,
257
transcriptomics, proteomics or metabolomics in contrast to BCB- oocytes. In thisway, it was
258
already determined that BCB-positive GV oocytes of pigs express a lower frequency of DNA
259
double-strand breaks, and single-oocyte sequencing data point to a potential role of specific
260
factors, like CDC5L, in porcine oocyte meiosis and early embryo development [33].
261 262
In the case of oocyte rescue programmes initiated for endangered feline species [11, 34, 35], it
263
was shown that different factors, like animal age and health as well as prolonged
264
transportation can compromise inherent oocyte quality [35]. Although usually morphological 12
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 265
good quality oocytes were selected for in-vitro maturation, in many cases no or very low
266
maturation success was achieved. To further explore the impact of prolonged transportation,
267
we stored the ovaries for 16 - 20 h before oocytes collection. As suggested, after prolonged
268
storage less BCB+ COC were obtained from ovaries. But independently from storage, embryo
269
developmental competence was unchanged, once oocytes were selected for BCB staining and
270
cleaved. This might be an important factor in decision making in the case of using (thawing)
271
semen from a rare and valuable male for IVF or ICSI. In addition to this, ovarian ageing is
272
characterized by quantitative and qualitative alteration of the ovarian oocyte reserve. There is
273
a close relationship, between the decline of oocyte quality and ageing-related mitochondrial
274
(mt) DNA instability [36]. We suggest that BCB staining, which also reflects the quality of
275
mitochondria [4], may be also used as an alternative biomarker of oocyte quality. The impact
276
of ovarian ageing on BCB staining has to be elucidated in future.
277 278
5. Conclusion
279
BCB staining is a very useful tool to preselect immature COC of feline species ensuring
280
higher developmental rates. Identifying good quality oocytes is particularly important for
281
nuclear transfer experiments and to understand physiological processes within the ovary,
282
which might result in a loss of oocyte quality.
283 284 285 286
Acknowledgements We thank the veterinary clinic of Berlin Animal Shelter for collecting and providing the
287
samples and Shauna Kehoe for English proof reading.
288
Conflict of interest
289
The authors have declared no conflicts of interest.
290 13
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References [1] Ajduk A, Zernicka-Goetz M. Quality control of embryo development. Mol Aspects Med. 2013;34:903-18. [2] Brayboy LM, Wessel GM. The double-edged sword of the mammalian oocyte-advantages, drawbacks and approaches for basic and clinical analysis at the single cell level. Mol Hum Reprod. 2016;22:200-7. [3] Royere D, Feuerstein P, Cadoret V, Puard V, Uzbekova S, Dalbies-Tran R, et al. [Non invasive assessment of embryo quality: proteomics, metabolomics and oocyte-cumulus dialogue]. Gynecol Obstet Fertil. 2009;37:917-20. [4] El Shourbagy SH, Spikings EC, Freitas M, St John JC. Mitochondria directly influence fertilisation outcome in the pig. Reproduction. 2006;131:233-45. [5] Fu B, Ren L, Liu D, Ma JZ, An TZ, Yang XQ, et al. Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities. AsianAustralas J Anim Sci. 2015;28:1703-12. [6] Duarte Alcoba D, Gonsales Valerio E, Conzatti M, Schneider J, Capp E, von Eye Corleta H, et al. Selection of developmentally competent human oocytes aspirated during cesarean section. J Matern Fetal Neonatal Med. 2018;31:735-9. [7] Alm H, Torner H, Lohrke B, Viergutz T, Ghoneim IM, Kanitz W. Bovine blastocyst development rate in vitro is influenced by selection of oocytes by brillant cresyl blue staining before IVM as indicator for glucose-6-phosphate dehydrogenase activity. Theriogenology. 2005;63:2194-205. [8] Abazari-Kia AH, Mohammadi-Sangcheshmeh A, Dehghani-Mohammadabadi M, Jamshidi-Adegani F, Veshkini A, Zhandi M, et al. Intracellular glutathione content, developmental competence and expression of apoptosis-related genes associated with G6PDH-activity in goat oocyte. J Assist Reprod Genet. 2014;31:313-21. [9] Wu YG, Liu Y, Zhou P, Lan GC, Han D, Miao DQ, et al. Selection of oocytes for in vitro maturation by brilliant cresyl blue staining: a study using the mouse model. Cell Res. 2007;17:722-31. [10] Alcoba DD, da Rosa Braga BL, Sandi-Monroy NL, Proenca LA, Felix Lopes RF, de Oliveira AT. Selection of Rattus norvegicus oocytes for in vitro maturation by brilliant cresyl blue staining. Zygote. 2013;21:238-45. [11] Jewgenow K, Blottner S, Lengwinat T, Meyer HH. New methods for gamete rescue from gonads of nondomestic felids. J Reprod Fertil Suppl. 1997;51:33-9. [12] Jewgenow K, Wiedemann C, Bertelsen MF, Ringleb J. Cryopreservation of mammalian ovaries and oocytes. . International Zoo Yearbook. 2011;45:124-32. [13] Johnston LA, Donoghue AM, Obrien SJ, Wildt DE. Rescue and Maturation Invitro of Follicular Oocytes Collected from Nondomestic Felid Species. Biology of Reproduction. 1991;45:898-906. [14] Hribal R, Jewgenow K, Braun BC, Comizzoli P. Influence of culture medium composition on relative mRNA abundances in domestic cat embryos. Reproduction in domestic animals = Zuchthygiene. 2013;48:245-51. [15] Waurich R, Ringleb J, Braun BC, Jewgenow K. Embryonic gene activation in in vitro produced embryos of the domestic cat (Felis catus). Reproduction. 2010;140:531-40. [16] Zahmel J, Mundt H, Jewgenow K, Braun BC. Analysis of gene expression in granulosa cells post-maturation to evaluate oocyte culture systems in the domestic cat. Reproduction in domestic animals = Zuchthygiene. 2017;52 Suppl 2:65-70. [17] Klaus C, Eder S, Franz C, Muller K. Successful Cryopreservation of Domestic Cat (Felis catus) Epididymal Sperm after Slow Equilibration to 15 or 10 degrees C. Reproduction in domestic animals = Zuchthygiene. 2016;51:195-203. 14
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[18] Wood TC, Wildt DE. Effect of the quality of the cumulus-oocyte complex in the domestic cat on the ability of oocytes to mature, fertilize and develop into blastocysts in vitro. J Reprod Fertil. 1997;110:355-60. [19] Spindler RE, Wildt DE. Circannual variations in intraovarian oocyte but not epididymal sperm quality in the domestic Cat. Biol Reprod. 1999;61:188-94. [20] Comizzoli P, Wildt DE, Pukazhenthi BS. Overcoming poor in vitro nuclear maturation and developmental competence of domestic cat oocytes during the non-breeding season. Reproduction. 2003;126:809-16. [21] Freistedt P, Stojkovic M, Wolf E. Efficient in vitro production of cat embryos in modified synthetic oviduct fluid medium: effects of season and ovarian status. Biol Reprod. 2001;65:9-13. [22] Hribal R, Jewgenow K, Braun BC, Comizzoli P. Influence of Culture Medium Composition on Relative mRNA Abundances in Domestic Cat Embryos. Reproduction in Domestic Animals. 2013;48:245-51. [23] Roth TL, Swanson WF, Wildt DE. Developmental competence of domestic cat embryos fertilized in vivo versus in vitro. Biol Reprod. 1994;51:441-51. [24] Gjorret JO, Crichton EG, Loskutoff NM, Armstrong DL, Hyttel P. Ultrastructure of oocyte maturation, fertilization, and early embryo development in vitro in the Siberian tiger (Panthera tigris altaica). Mol Reprod Dev. 2002;63:79-88. [25] Martins LR, Fernandes CB, Minto BW, Landim-Alvarenga FC, Lopes MD. Ultrastructural characteristics of non-matured and in vitro matured oocytes collected from pre-pubertal and adult domestic cat ovaries. Reproduction in domestic animals = Zuchthygiene. 2009;44 Suppl 2:251-4. [26] Katska-Ksiazkiewicz L, Rynska B, Kania G, Smorag Z, Gajda B, Pienkowski M. Timing of nuclear maturation of nonstored and stored domestic cat oocytes. Theriogenology. 2003;59:1567-74. [27] Malandain E, Rault D, Froment E, Baudon S, Desquilbet L, Begon D, et al. Follicular growth monitoring in the female cat during estrus. Theriogenology. 2011;76:1337-46. [28] Silva DS, Rodriguez P, Galuppo A, Arruda NS, Rodrigues JL. Selection of bovine oocytes by brilliant cresyl blue staining: effect on meiosis progression, organelle distribution and embryo development. Zygote. 2013;21:250-5. [29] Braganca GM, Batista R, Souza-Fabjan JMG, Alfradique VAP, Arashiro EKN, Cosentino IO, et al. Dose and administration protocol for FSH used for ovarian stimulation affect gene expression in sheep cumulus?oocyte complexes. Reprod Fertil Dev. 2018. [30] Zare Z, Abouhamzeh B, Masteri Farahani R, Salehi M, Mohammadi M. Supplementation of L-carnitine during in vitro maturation of mouse oocytes affects expression of genes involved in oocyte and embryo competence: An experimental study. Int J Reprod Biomed (Yazd). 2017;15:779-86. [31] Sadeesh EM, Fozia S, Meena K. Combined positive effect of oocyte extracts and brilliant cresyl blue stained recipient cytoplasts on epigenetic reprogramming and gene expression in buffalo nuclear transfer embryos. Cytotechnology. 2017;69:289-305. [32] Sutton-McDowall ML, Purdey M, Brown HM, Abell AD, Mottershead DG, Cetica PD, et al. Redox and anti-oxidant state within cattle oocytes following in vitro maturation with bone morphogenetic protein 15 and follicle stimulating hormone. Mol Reprod Dev. 2015;82:281-94. [33] Liu XM, Wang YK, Liu YH, Yu XX, Wang PC, Li X, et al. Single-cell transcriptome sequencing reveals that cell division cycle 5-like protein is essential for porcine oocyte maturation. J Biol Chem. 2018;293:1767-80.
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[34] Johnston LA, Donoghue AM, O'Brien SJ, Wildt DE. Rescue and maturation in vitro of follicular oocytes collected from nondomestic felid species. Biol Reprod. 1991;45:898906. [35] Fernandez-Gonzalez L, Mueller K, Jewgenow K, Zahmel J. Felid-gamete-rescue within EAZA - efforts and results in biobanking felid oocytes and sperm JZAR. [36] May-Panloup P, Boucret L, Chao de la Barca JM, Desquiret-Dumas V, Ferre-L'Hotellier V, Moriniere C, et al. Ovarian ageing: the role of mitochondria in oocytes and follicles. Hum Reprod Update. 2016;22:725-43.
398 399
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ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 400 401 402 403
Table 1. Oocytes from morphologically high quality COCs were stained in vitro before maturation with brilliant cresyl blue (BCB) and cultured separately for 24 hours. Maturation stage was determined by PI staining. Summarized are 10 experiments between November 2016 and March 2017. Stage of meiosis GV GVBD MI MII+ PB Parthenogenetic
404
5 2 19 84 3
BCB+ N = 113 (4.4%) (1.8%) (16.8%) (74.3%) (2.7%)
32 13 4 14 2
BCB– N = 65 (49.2%) (20.0%) (6.2%) (21.5%) (3.1%)
Fisher‘s exact test P < 0.0001 P < 0.0001 n.s. P < 0.0001 n.s.
BCB+: oocytes were stained blue with BCB; BCB-: oocytes remained unstained.
405 406 407 408 409 410
Table 2. Oocytes from morphologically high quality COCs were stained in vitro before maturation with brilliant cresyl blue (BCB), and subjected to in vitro fertilization and embryo culture for 5 days. Cleavage rate was determined every day and verified by PI staining. Summarized are 13 experiments between August 2017 and February 2018. Developmental stage maturation cleavage morula Morula rate per cleaved embryos
411
BCB+ N = 140 84 (60.0)% 50 (35.7%) 27 (19.2%)
BCB– N = 112 42 (37.5%) 26 (23.2%) 8 (8.2%)
54.0%
Fisher‘s exact test P < 0.001 P < 0.05 P < 0.001
30.7%
BCB+: oocytes were stained blue with BCB; BCB-: oocytes remained unstained.
412 413 414 415 416 417 418
Table 3. Oocytes from morphologically high quality COCs obtained from ovaries on the same day of surgery or after holding ovaries for 16 – 20 h at 4°C were stained in vitro before maturation with brilliant cresyl blue (BCB), and subjected to in vitro fertilization and embryo culture for 5 days. Cleavage –and developmental stage was determined every day and verified by final PI staining. Summarized are 10 experiments August to September 2017 and February to August 2018. Fresh
419 420 421 422
Developmental stage cleavage morula Morula rate per cleaved embryos
BCB+ N = 175 65a (37.1%) 33 (18.9%) 50.8%
BCB– N = 135 34 (25.2%) 8 (5.9%) 23.5%
Holding for 16 - 20h BCB+ BCBN = 96 N = 114 19b (19.8%) 20 (17.5%) 9 (9.4%) 5 (4.4%) 47.4%
25.0%
BCB+: oocytes were stained blue with BCB; BCB-: oocytes remained unstained. a,b – superscripts indicate for a significant difference (Fishers exact test; P = 0.0038)
17
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 423
Figure legends:
424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440
Figure 1: High quality cumulus oocyte complexes obtained from ovaries of domestic cats stained for glucose-6-phosphate dehydrogenase (G6PDH) activity by brilliant cresyl blue. Figure 2: Transelectron microscopic section through oocytes from BCB+ COC (A, B) and from a BCB- COC (C, D). Cg – corticular granulae, Ga – Golgi apparatus, Ld – lipid droplet, Mx- mitochondria, Nc – nucleus, pvs – perivitelline space, V – vesicle, Zpzona pellucida. Figure 3: Boxplot (median, 25- and 75-percentiles) of good quality COCs and BCB+ COCs isolated per ovary on the same day after surgery or following day after 16 – 20 h storage of ovaries at 4° C (holding). Summarized are date from 15 experiments from August to September 2017 and February to August 2018.
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ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 441 442
Figure 1:
443 444
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ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 445
Figure 2:
446 447
20
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 448
Figure 3:
449 16 14
n oocytes per ovary
12 10 8 6 4 2 0
fresh
450
n = 225
BCB+ fresh n = 151
holding n = 210
BCB+ holding n = 96
21
ACCEPTED MANUSCRIPT Jewgenow et al. BCB staining of feline oocytes Highlights
1. BCB staining is a very useful tool to preselect immature COC of feline species before maturation. 2. Selection for BCB+ COC before in vitro maturation ensures higher developmental rates after fertilization. 3. Prolonged storage of ovaries at 4°C results in less BCB+ oocytes isolated from ovaries.
1
K. Jewgenow, L. Fernandez Gonzalez, S. Jänsch, D. Viertel, J. Zahmel PII:
S0093-691X(18)31089-6
DOI:
10.1016/j.theriogenology.2018.12.021
Reference:
THE 14813
To appear in:
Theriogenology
Received Date:
15 November 2018
Accepted Date:
09 December 2018
Please cite this article as: K. Jewgenow, L. Fernandez Gonzalez, S. Jänsch, D. Viertel, J. Zahmel, Brilliant cresyl blue staining allows the selection for developmentally competent inmature feline oocytes, Theriogenology (2018), doi: 10.1016/j.theriogenology.2018.12.021
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ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 1 2
Brilliant cresyl blue staining allows the selection for developmentally competent inmature feline oocytes
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Jewgenow K, Fernandez Gonzalez L, Jänsch S, Viertel D, Zahmel J
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Department of Reproduction Biology, Leibniz Institute for Zoo and Wildlife Research,
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PF700430, D10324 Berlin, Germany
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Correspondence should be addressed to
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Prof. K. Jewgenow, Leibniz Institute for Zoo and Wildlife Research,
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PF700430,
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D10324 Berlin Berlin, Germany.
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Email: [email protected]
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Tel. 0049 30 5168 611
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Short Title: BCB staining of feline oocytes
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ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 13
Author Contributions:
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Katarina Jewgenow designed the study, partly carried out the study, analyzed and interpreted
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the data, compiled the manuscript and finally approved of the version to be submitted.
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Lorena Fernandez Gonzalez partly carried out the study (experiment 1), analyzed and
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interpreted her data, discussed the results and critical revised the manuscript.
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Stefanie Jänsch: partly carried out the study (experiment 2,3) and analyzed her data
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Dagmar Viertel: performed the TEM analysis
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Jennifer Zahmel partly analyzed and interpreted data, discussed the results and critical revised
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the manuscript.
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All authors have approved the final article.
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ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 23
Abstract
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The outcome of in-vitro-maturation and in-vitro-fertilization of feline oocytes depends
25
on the selection of high quality oocytes, and is often restricted to morphological criteria. The
26
aim of this study was to test whether the Brilliant cresyl blue (BCB) staining is suitable for
27
pre-selection of feline oocytes before in-vitro-maturation.
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Cumulus-oocytes-complexes (COC) were released from domestic cat ovaries obtained after
29
ovariectomy and were subjected to BCB staining. BCB+ stained oocytes were characterized
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by a violet/ pale blue staining of the ooplasma, BCB- oocytes remained unstained. Trans-
31
electron microscopy indicated for a slightly advanced stage of BCB- oocytes within the
32
maturation process. After 24 h in-vitro maturation, almost 75% of BCB+ and 21.5% of BCB-
33
oocytes were able to reach metaphase II. Also, after in-vitro fertilization, significantly more
34
oocytes developed to morulae (19.2%) if oocytes were preselected for BCB staining, although
35
8% of unstained COC still reached advanced embryo stages. Prolonged storage of ovaries
36
before COC retrieval for 16 – 20h at 4°C was accompanied by reduced number of BCB+
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oocytes (96 of 210, 45.7%) in comparison to freshly isolated COC (151 of 225, 67.1%), and
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impaired cleavage rate (19.8%) and morula rate (9.4%) of BCB+ oocytes but the rate of
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embryos which developed to advanced stages remained unchanged (~50%).
40
To conclude, BCB staining is a very useful tool to preselect immature COC of feline species
41
ensuring higher developmental rates.
42 43
Keywords: feline, oocyte quality, brilliant cresyl blue,
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ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 44
1. Introduction
45
Success of assisted reproduction in mammals often depends on oocyte quality
46
subjected to either in-vitro-maturation or in-vitro-fertilization. Although these assisted
47
reproductive technologies both in human and animal have improved markedly it still remains
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elusive to predict embryo potential based on selected oocytes. Thus, there is a strong need for
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further refinement of existing selection methods and development of novel, robust and, non-
50
invasive procedures [1].
51
Oocytes originate from individual ovarian follicles and therefore are characterized by
52
a pronounced heterogeneity.
Determining the quality of oocytes is often restricted to
53
morphological analysis or to the study of cellular behaviours in the developing embryo [2].
54
Although developmental and morphological information gained from microscopic assessment
55
have been positively associated with IVF outcomes (Nel-Themaat and Nagy 2011), any other
56
non-invasive approach would help to identify additional parameters of oocytes, their
57
surrounding follicle cells or even ovarian status
58
competency of embryos [3].
which determine the developmental
59
The brilliant cresyl blue (BCB) test is a non-invasive approach which reflects glucose-
60
6-phosphate dehydrogenase (G6PDH) activity of the oocyte, their glutathione concentration
61
and the number and quality of mitochondria [4]. Interestingly, the staining intensity for BCB
62
allows identifying oocytes likely to be developmentally competent [5]. So far, it was
63
effectively used in different mammalian species, including human [6], cattle [7], goats [8],
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pigs [4, 5], mice [9] and rats [10]. Thus, the aim of this study was to test the BCB staining for
65
pre-selection of feline oocytes before in-vitro-maturation. We suggest that BCB staining will
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support the selection of good quality gametes obtained from old or diseased felids or after
67
prolonged transportation in gamete rescue programmes [11-13], and thus, may help to
68
economize efforts if the quality is insufficient for further development. Our experiences in 4
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 69
gamete rescue of different felid species from zoos indicate that often the oocyte quality is
70
compromised (Fernandez et al, 2018).
71
With the presented experiments we aimed to answer the following questions: Does BCB
72
staining allow the selection for developmentally competent feline oocytes and does BCB
73
staining reflect the quality of oocytes after prolonged storage?
74
2. Material and Methods
75
All chemical reagents were purchased from Sigma–Aldrich (Taufkirchen, Germany) unless
76
stated otherwise and were of the highest purity available. Ovaries and testes of domestic cats
77
were obtained from the Berlin Animal Shelter after routine ovariectomy or castration.
78 79
2.1.
Collection and brilliant cresyl blue (BCB) staining of cumulus oocyte complexes
80
Immediately upon arrival in the lab, ovaries were freed of surrounding tissues, washed in
81
Washing Medium (WM, Medium 199 with Earle´s salts, supplemented with 3 mg/mL BSA,
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1.4 mg/mL HEPES, 0.6 mg/mL sodium lactate, 0.25 mg/mL sodium pyruvate, 0.15 mg/mL L-
83
glutamine, 0.1 mg/mL cysteine and 0.055 mg/mL gentamicin), and were sliced in 5 mL WM
84
to release cumulus-oocytes-complexes (COC). Collection and quality assessment of COC was
85
performed under a stereomicroscope and only those with 3 - 4 granulosa cell layers
86
surrounding the oocyte and with a homogenous, dark cytoplasm were chosen [14]. To
87
evaluate the impact of prolonged storage at 4° C, some ovaries were kept refrigerated
88
overnight (16 - 20 h) in HEPES-MEM, supplemented with 0.3% BSA and 1% antibiotic
89
antimycotic before isolating the oocytes by slicing.
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Good quality COC were subjected to 500 µL of BCB (13mg/L; (34 µM)) diluted in modified
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DPBS supplemented with 0.4 % BSA, 36 mg/L (0.327 mM) sodium pyruvate, 1 mg/mL and
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(5.55mM) D-(+)-Glucose, and 13 mg/L BCB for 1 hour at 38.5°C in a humidified air 5
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 93
atmosphere. Thereafter, the COCs were washed and examined under a stereomicroscope.
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According to the colour of the cytoplasma (Figure 1) two groups were created: BCB+
95
(coloured cytoplasm) and BCB– (colourless cytoplasm), respectively.
96
2.2.
Transmission electron microscopy of BCB+ and BCB- stained oocytes
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After staining for BCB, cumulus-oocytes complexes were transferred to an objective slide and
98
carefully mixed with pre-warmed 1% low melting agarose. After cooling for 15 min at 4°C,
99
the agarose pillow around a single COC was cut to 1 mm2 pieces and transferred to
100
Karnowsky solution for fixation and stored for processing by Transmission electron
101
microscopy (TEM).
102
After washing in PBS, the samples were treated with osmium tetroxide, dehydrated in
103
increasing concentrations of ethanol and embedded in Epon 812. Semi-thin sections of 1 µm
104
were performed and stained with toluidine blue for pre-selection. Ultrathin sections (70 nm)
105
of the selected tissue part were stained with uranyl acetate, followed by the application of lead
106
citrate. The COC present in the selected samples were analyzed and electromicrographies
107
were made with magnifications between 1,250x and 11,000x using a FEI TecnaiSpiritBT
108
device (120 kV; FEI Deutschland GmbH).
109
Oocytes were evaluated for ultrastructure by assessing the integrity of the cytoplasmic
110
membrane, the presence and density of mitochondria, organelles and vacuoles.
111
2.3.
In vitro maturation of cat oocytes
112
The procedure of in vitro maturation and fertilization of domestic cat oocytes was
113
performed as previously described by [15, 16]. In brief, isolated COC were washed two times
114
in WM and cultured for maturation. Maturation proceeded at 38.5 °C and 5% CO2 in a
115
humidified air atmosphere for 24 h and was performed in WM supplemented with human
116
0.02 IU LH/mL (L6420) and 0.05IU human pituitary FSH/mL (Ferring, Kiel, Germany). Each 6
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 117
COC was kept separately in 20 µL medium covered under mineral oil (Reproline Medical
118
GmbH, Rheinbach, Germany) in a 6 cm petri dish. For experiment 1, the COCs were freed
119
manually of attached cumulus cells by repeated pipetting and placed on an objective slide.
120
After air drying, the slide was transferred to alcohol (96% ethanol) and was kept at 4°C until
121
analysis. Following staining of oocytes with propidium iodide (1.0 mg/ mL, 1:100 in PBS;
122
Thermo Fisher Scientific) the maturation stage was assessed under a fluorescence microscope
123
(Axiovert 200M, Carl Zeiss Microscopy GmbH, Jena, Germany). The configuration of DNA
124
within the oocytes allowed to determine meiotic progress during the culture (presence of GV,
125
breakdown of GV membrane, metaphase I, metaphase II accompanied by a polar body).
126
Parthenogenic activation was considered when cleavage occurred without fertilization. For all
127
other experiments, COC were washed and fertilized in vitro (see below).
128
2.4.
In fertilization of cat oocytes
129
Testes were maintained at 4 °C without any medium for 24h before processing. Fresh
130
epididymal sperm cells were isolated as described before [17]. In brief, tissues were sliced
131
with a scissors in culture medium M199 (HEPES modification) at room temperature (20–
132
23°C) and sperm suspension was flushed through a 30-µm filter (Sysmex Partec GmbH,
133
Görlitz, Germany). After centrifugation at ~500 × g for 5 min, the pellet was re-suspended in
134
a small volume of M199 and sperm concentration was adjusted to 4 x 106 sperm/ mL for in
135
vitro fertilization.
136
In vitro fertilization was performed in 20 μL of WM medium supplemented with 2.2 IU/mL
137
heparin at 38.5 °C and 5 % CO2 for 18 h. The oocytes were fertilized with a final epididymal
138
sperm concentration of 1×106 motile sperm/mL.
139
2.5.
Culture of embryos
7
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COC were freed of attached cumulus cells after 18 hours of gamete co-incubation by
141
repeated pipetting using 155 µm stripper tip micropipette (The Stripper®, BioTipp,
142
Waterford, Ireland). Putative zygotes were transferred singly to 20 µL embryo culture
143
medium covered under mineral oil in a 6 cm petri dish. Embryo culture medium was prepared
144
from Ham´s F-10 and supplemented with 5% FCS, 0.05 mg/ mL gentamycin, 0.11 mg/mL
145
sodium pyruvate, and 0.075 mg/mL L-glutamine. Culture was performed at 38.5°C in 5%
146
CO2 and 5% O2 humidified atmosphere. Evaluation of embryo development was performed
147
every 24 hours. On day five of culture, all morulae were vitrified (another project). Oocytes
148
which did not cleave till day 2 of culture and all embryos not reaching morula stage at day
149
five were fixed by placing them on an objective slide, air dried, fixed in ethanol and stained
150
with PI (see above). The number of nuclei representing embryo blastomeres was counted.
151
2.6.
Statistical analysis
152
For comparison of maturation, fertilization and cleavage rates (embryo development), two-
153
tailed contingence tables were analysed with Fisher's exact test. P levels < 0.05 were
154
considered as significant. Statistical analysis was performed by using the statistical program
155
InStat3 (GraphPad Software, Inc., California, USA); blox plot was created with Sigma plot.
156 157
3. Results
158
3.1.
159
Morphological characterization of brilliant cresyl blue positive and negative feline COC
160 161
BCB+ stained oocytes were characterized by a violet/ pale blue staining of the ooplasma,
162
which was slightly overlaid by the lipids within the cytoplasma (Figure 1). Trans-electron
163
microscopy indicated for only minor differences between BCB+ and BCB- feline COC
164
(Figure 2). BCB+ oocytes were characterized by equally sized vacuoles throughout the 8
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 165
cytoplasma, accumulation of round mitochondria in the cortical region and regular
166
distribution of cortical granules beneath plasma membrane (Figure 2 A,B). In BCB- oocytes
167
we found more frequently a peripheral migration of the nucleus and formation of perivitelline
168
space (Figure 2C). Vesicles were different in size and appearance, with membrane inclusions.
169
These vesicles were partly extruded into perivitelline space (Figure 2 D). Mitochondria
170
tended to be elongated and cortical granules were unequally distributed in the cortex region of
171
cytoplasma (Figure 2D).
172
3.2.
Does brilliant cresyl blue staining allow for the selection of developmentally competent feline oocytes?
173 174
Table 1 summarizes the experiment on comparing the maturation rate in vitro determined by
175
staining the nuclear structure of oocytes after 24 hours culture. There was a significant shift
176
towards higher maturation in BCB-positive oocytes. In this group, almost 75% of oocytes
177
were able to precede maturation indicated by metaphase II plate and extrusion of the first
178
polar body. With glucose-6-phosphate dehydrogenase activity (BCB-negative oocytes) almost
179
half of the COC remained at GV stage, and only 21.5% reached metaphase II.
180
In a second experiment, oocytes were fertilized after maturation and embryo culture was
181
performed for 5 days. Embryo development was assessed by cleavage rate and number of
182
embryos reaching morula at day 5 of culture. Again, there was a significant increase of
183
oocytes which underwent maturation (60.0% vs. 37.5%) for BCB+ and BCB- oocytes,
184
respectively, and cleaved embryos developed to morulae (54%) if oocytes were preselected
185
for BCB staining, although still 30.7% of embryos (8% of unstained COC) were able to reach
186
advanced embryo stages (Table 2).
187 188
3.3.
Does brilliant cresyl blue staining reflect the quality of oocytes after prolonged storage? 9
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 189
In a third experiment ovaries were obtained from a local animal clinic after
190
ovariohysterectomia and were processed the same day (fresh) or were stored at 4° C until that
191
next morning (16 – 20 h holding). There was a high variation in the number of high quality
192
COC obtained per ovary (median 5, range 1 - 8), including the number of BCB+ (median 4,
193
range 0 – 7), which represented 67.1% of all isolated COC (151 of 225; Figure 2). Storage of
194
ovaries over night was not accompanied by reduction of good quality COC (median 5, range 2
195
– 15), but the number of BCB+ oocytes per ovary was compromised (median 3, range 0 – 7)
196
with only 45.7% of all COC being stained (96 of 210; Figure 2).
197
Developmental capacity of oocytes was also reduced after prolonged storage for 16 - 20 h at
198
4°C (Table 3). The cleavage rate of BCB+ COC was significantly higher for the fresh group
199
(37.1%) compared to BCB+ after storage (19.8%). This difference was not statistically
200
evident for morula rates, although there was a tendency towards less morulae (18.9% vs.
201
9.4%). Within the BCB- groups no difference in their developmental capacity was found.
202
Independently from ovarian storage, half of the cleaved embryos develop until morula stage,
203
if they originated from BCB+ COC, whereas in the BCB- groups only about 25% of embryos
204
developed further.
205
4. Discussion
206
As shown for other species, COC with high activity of glucose-6-phosphate dehydrogenase
207
express a higher meiotic competence and developmental potential. In our study with domestic
208
cat oocytes, the cytoplasma of about 60% of high quality COC (528 of 884 oocytes) was
209
stained after exposure to brilliant cresyl blue (BCB). The number per ovary varied between 0
210
and 7, and was reflecting the number of high-quality COC, which was also between 1 and 8
211
per ovary (Figure 2). This high variation of both morphological good quality COC and BCB+
212
oocytes might be related to the reproduction cycle of domestic cats. Throughout all
213
reproductive phases, anoestrus, follicular and luteal phase, high-quality COC can be obtained 10
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 214
[18]; the mean number reported varied between 2 - 3 per ovary [19, 20] and 20 [21]. We
215
previously reported a mean of 4 COC per ovary independently of season [22]. For domestic
216
cat in-vitro embryo production, it is clear that a strong selection for morphological criteria,
217
like dark cytoplasma and compact cumulus layer, before maturation will increase the outcome
218
of embryos after IVF [18]. Based on our data, BCB staining can be recommended as an
219
additional quality parameter for pre-selection of COC before in-vitro maturation.
220
For routine use, however, it has to be considered that BCB staining demands one hour
221
preparation time, and within the BCB- group still some embryos can be produced.
222
Unfortunately, we were not able to characterize the BCB- embryos further as by the
223
development to day 5, which is within the physiological time frame for reaching morula stage
224
[23].
225
It is also not clear why unstained COC develop to a lesser extent. BCB- or colourless
226
cytoplasma indicate for higher enzymatic activity of G6PDH and was found to be
227
characteristic for either growing oocytes or matured (MII) preovulatory oocytes [7, 24].
228
Unstained cytoplasma in fully grown, good quality COC as describe for BCB- COC, might
229
indicate for an imbalance between cytoplasmic (G6PGH activity) and nuclear (progress in
230
meiosis) maturation. Domestic cats are induced ovulators. Thus, at any time of oocyte
231
collection, a mixed population of oocytes is obtained containing also high quality COC which
232
have already missed the optimal time for ovulation. This suggestion is partly supported by our
233
TEM data, revealing that the GV of BCB- oocytes has already moved from the centre of the
234
oocyte towards periphery and that both mitochondria and corticular granulae as well the
235
perivitelline space behave as described for oocytes after the LH surge [24, 25]. Perhaps these
236
BCB- COC require less time for maturation, and need to be fertilized earlier than after 24
237
hours in-vitro maturation. Katska-Ksiakzkiewicz suggested that two "waves" of nuclear
238
maturation of cat oocytes can be distinguished. The first wave reach maturity by 17-18 h; and 11
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 239
the second wave occurs after 28-30 h of IVM [26]. By transabdominal ultrasound it was
240
shown that the growth of feline ovarian follicles was not exactly synchronous [27]. It was also
241
suggested that cytoplasmatic BCB activity indicates for synchronous nuclear and cytoplasmic
242
in vitro maturation [28].
243 244
Determination of BCB activity in oocytes before maturation has been recommended for
245
several purposes. In ewes, BCB staining was applied to compare superovulation protocols
246
based on the production of better-quality oocytes [29], or the staining was used to improve
247
IVM medium for BCB+ oocytes that can ameliorate reproductive success following in vitro
248
fertilization [30]. Chaubey at al. (2018) suggested a selective treatment of BCB- oocytes to
249
improve their maturation capacity and improve the outcome after IVF. Another broad
250
application field for BCB is the selection of developmentally competent oocytes for nuclear
251
transfer, because BCB+ oocytes were shown to express higher nuclear reprogramming
252
capacity in cloned blastocysts [31]. In addition to cloning, the choice of the best embryos to
253
transfer is still often based on subjective morphological parameters, although more
254
sophisticated parameters to select the most competent oocytes have become available [32].
255
BCB staining before in-vitro maturation allows classifying oocytes according to their future
256
developmental potential. Thus, these competent oocytes can be analysed by genomics,
257
transcriptomics, proteomics or metabolomics in contrast to BCB- oocytes. In thisway, it was
258
already determined that BCB-positive GV oocytes of pigs express a lower frequency of DNA
259
double-strand breaks, and single-oocyte sequencing data point to a potential role of specific
260
factors, like CDC5L, in porcine oocyte meiosis and early embryo development [33].
261 262
In the case of oocyte rescue programmes initiated for endangered feline species [11, 34, 35], it
263
was shown that different factors, like animal age and health as well as prolonged
264
transportation can compromise inherent oocyte quality [35]. Although usually morphological 12
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 265
good quality oocytes were selected for in-vitro maturation, in many cases no or very low
266
maturation success was achieved. To further explore the impact of prolonged transportation,
267
we stored the ovaries for 16 - 20 h before oocytes collection. As suggested, after prolonged
268
storage less BCB+ COC were obtained from ovaries. But independently from storage, embryo
269
developmental competence was unchanged, once oocytes were selected for BCB staining and
270
cleaved. This might be an important factor in decision making in the case of using (thawing)
271
semen from a rare and valuable male for IVF or ICSI. In addition to this, ovarian ageing is
272
characterized by quantitative and qualitative alteration of the ovarian oocyte reserve. There is
273
a close relationship, between the decline of oocyte quality and ageing-related mitochondrial
274
(mt) DNA instability [36]. We suggest that BCB staining, which also reflects the quality of
275
mitochondria [4], may be also used as an alternative biomarker of oocyte quality. The impact
276
of ovarian ageing on BCB staining has to be elucidated in future.
277 278
5. Conclusion
279
BCB staining is a very useful tool to preselect immature COC of feline species ensuring
280
higher developmental rates. Identifying good quality oocytes is particularly important for
281
nuclear transfer experiments and to understand physiological processes within the ovary,
282
which might result in a loss of oocyte quality.
283 284 285 286
Acknowledgements We thank the veterinary clinic of Berlin Animal Shelter for collecting and providing the
287
samples and Shauna Kehoe for English proof reading.
288
Conflict of interest
289
The authors have declared no conflicts of interest.
290 13
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[18] Wood TC, Wildt DE. Effect of the quality of the cumulus-oocyte complex in the domestic cat on the ability of oocytes to mature, fertilize and develop into blastocysts in vitro. J Reprod Fertil. 1997;110:355-60. [19] Spindler RE, Wildt DE. Circannual variations in intraovarian oocyte but not epididymal sperm quality in the domestic Cat. Biol Reprod. 1999;61:188-94. [20] Comizzoli P, Wildt DE, Pukazhenthi BS. Overcoming poor in vitro nuclear maturation and developmental competence of domestic cat oocytes during the non-breeding season. Reproduction. 2003;126:809-16. [21] Freistedt P, Stojkovic M, Wolf E. Efficient in vitro production of cat embryos in modified synthetic oviduct fluid medium: effects of season and ovarian status. Biol Reprod. 2001;65:9-13. [22] Hribal R, Jewgenow K, Braun BC, Comizzoli P. Influence of Culture Medium Composition on Relative mRNA Abundances in Domestic Cat Embryos. Reproduction in Domestic Animals. 2013;48:245-51. [23] Roth TL, Swanson WF, Wildt DE. Developmental competence of domestic cat embryos fertilized in vivo versus in vitro. Biol Reprod. 1994;51:441-51. [24] Gjorret JO, Crichton EG, Loskutoff NM, Armstrong DL, Hyttel P. Ultrastructure of oocyte maturation, fertilization, and early embryo development in vitro in the Siberian tiger (Panthera tigris altaica). Mol Reprod Dev. 2002;63:79-88. [25] Martins LR, Fernandes CB, Minto BW, Landim-Alvarenga FC, Lopes MD. Ultrastructural characteristics of non-matured and in vitro matured oocytes collected from pre-pubertal and adult domestic cat ovaries. Reproduction in domestic animals = Zuchthygiene. 2009;44 Suppl 2:251-4. [26] Katska-Ksiazkiewicz L, Rynska B, Kania G, Smorag Z, Gajda B, Pienkowski M. Timing of nuclear maturation of nonstored and stored domestic cat oocytes. Theriogenology. 2003;59:1567-74. [27] Malandain E, Rault D, Froment E, Baudon S, Desquilbet L, Begon D, et al. Follicular growth monitoring in the female cat during estrus. Theriogenology. 2011;76:1337-46. [28] Silva DS, Rodriguez P, Galuppo A, Arruda NS, Rodrigues JL. Selection of bovine oocytes by brilliant cresyl blue staining: effect on meiosis progression, organelle distribution and embryo development. Zygote. 2013;21:250-5. [29] Braganca GM, Batista R, Souza-Fabjan JMG, Alfradique VAP, Arashiro EKN, Cosentino IO, et al. Dose and administration protocol for FSH used for ovarian stimulation affect gene expression in sheep cumulus?oocyte complexes. Reprod Fertil Dev. 2018. [30] Zare Z, Abouhamzeh B, Masteri Farahani R, Salehi M, Mohammadi M. Supplementation of L-carnitine during in vitro maturation of mouse oocytes affects expression of genes involved in oocyte and embryo competence: An experimental study. Int J Reprod Biomed (Yazd). 2017;15:779-86. [31] Sadeesh EM, Fozia S, Meena K. Combined positive effect of oocyte extracts and brilliant cresyl blue stained recipient cytoplasts on epigenetic reprogramming and gene expression in buffalo nuclear transfer embryos. Cytotechnology. 2017;69:289-305. [32] Sutton-McDowall ML, Purdey M, Brown HM, Abell AD, Mottershead DG, Cetica PD, et al. Redox and anti-oxidant state within cattle oocytes following in vitro maturation with bone morphogenetic protein 15 and follicle stimulating hormone. Mol Reprod Dev. 2015;82:281-94. [33] Liu XM, Wang YK, Liu YH, Yu XX, Wang PC, Li X, et al. Single-cell transcriptome sequencing reveals that cell division cycle 5-like protein is essential for porcine oocyte maturation. J Biol Chem. 2018;293:1767-80.
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[34] Johnston LA, Donoghue AM, O'Brien SJ, Wildt DE. Rescue and maturation in vitro of follicular oocytes collected from nondomestic felid species. Biol Reprod. 1991;45:898906. [35] Fernandez-Gonzalez L, Mueller K, Jewgenow K, Zahmel J. Felid-gamete-rescue within EAZA - efforts and results in biobanking felid oocytes and sperm JZAR. [36] May-Panloup P, Boucret L, Chao de la Barca JM, Desquiret-Dumas V, Ferre-L'Hotellier V, Moriniere C, et al. Ovarian ageing: the role of mitochondria in oocytes and follicles. Hum Reprod Update. 2016;22:725-43.
398 399
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ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 400 401 402 403
Table 1. Oocytes from morphologically high quality COCs were stained in vitro before maturation with brilliant cresyl blue (BCB) and cultured separately for 24 hours. Maturation stage was determined by PI staining. Summarized are 10 experiments between November 2016 and March 2017. Stage of meiosis GV GVBD MI MII+ PB Parthenogenetic
404
5 2 19 84 3
BCB+ N = 113 (4.4%) (1.8%) (16.8%) (74.3%) (2.7%)
32 13 4 14 2
BCB– N = 65 (49.2%) (20.0%) (6.2%) (21.5%) (3.1%)
Fisher‘s exact test P < 0.0001 P < 0.0001 n.s. P < 0.0001 n.s.
BCB+: oocytes were stained blue with BCB; BCB-: oocytes remained unstained.
405 406 407 408 409 410
Table 2. Oocytes from morphologically high quality COCs were stained in vitro before maturation with brilliant cresyl blue (BCB), and subjected to in vitro fertilization and embryo culture for 5 days. Cleavage rate was determined every day and verified by PI staining. Summarized are 13 experiments between August 2017 and February 2018. Developmental stage maturation cleavage morula Morula rate per cleaved embryos
411
BCB+ N = 140 84 (60.0)% 50 (35.7%) 27 (19.2%)
BCB– N = 112 42 (37.5%) 26 (23.2%) 8 (8.2%)
54.0%
Fisher‘s exact test P < 0.001 P < 0.05 P < 0.001
30.7%
BCB+: oocytes were stained blue with BCB; BCB-: oocytes remained unstained.
412 413 414 415 416 417 418
Table 3. Oocytes from morphologically high quality COCs obtained from ovaries on the same day of surgery or after holding ovaries for 16 – 20 h at 4°C were stained in vitro before maturation with brilliant cresyl blue (BCB), and subjected to in vitro fertilization and embryo culture for 5 days. Cleavage –and developmental stage was determined every day and verified by final PI staining. Summarized are 10 experiments August to September 2017 and February to August 2018. Fresh
419 420 421 422
Developmental stage cleavage morula Morula rate per cleaved embryos
BCB+ N = 175 65a (37.1%) 33 (18.9%) 50.8%
BCB– N = 135 34 (25.2%) 8 (5.9%) 23.5%
Holding for 16 - 20h BCB+ BCBN = 96 N = 114 19b (19.8%) 20 (17.5%) 9 (9.4%) 5 (4.4%) 47.4%
25.0%
BCB+: oocytes were stained blue with BCB; BCB-: oocytes remained unstained. a,b – superscripts indicate for a significant difference (Fishers exact test; P = 0.0038)
17
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 423
Figure legends:
424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440
Figure 1: High quality cumulus oocyte complexes obtained from ovaries of domestic cats stained for glucose-6-phosphate dehydrogenase (G6PDH) activity by brilliant cresyl blue. Figure 2: Transelectron microscopic section through oocytes from BCB+ COC (A, B) and from a BCB- COC (C, D). Cg – corticular granulae, Ga – Golgi apparatus, Ld – lipid droplet, Mx- mitochondria, Nc – nucleus, pvs – perivitelline space, V – vesicle, Zpzona pellucida. Figure 3: Boxplot (median, 25- and 75-percentiles) of good quality COCs and BCB+ COCs isolated per ovary on the same day after surgery or following day after 16 – 20 h storage of ovaries at 4° C (holding). Summarized are date from 15 experiments from August to September 2017 and February to August 2018.
18
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 441 442
Figure 1:
443 444
19
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 445
Figure 2:
446 447
20
ACCEPTED MANUSCRIPT revised Jewgenow et al. BCB staining of feline oocytes 448
Figure 3:
449 16 14
n oocytes per ovary
12 10 8 6 4 2 0
fresh
450
n = 225
BCB+ fresh n = 151
holding n = 210
BCB+ holding n = 96
21
ACCEPTED MANUSCRIPT Jewgenow et al. BCB staining of feline oocytes Highlights
1. BCB staining is a very useful tool to preselect immature COC of feline species before maturation. 2. Selection for BCB+ COC before in vitro maturation ensures higher developmental rates after fertilization. 3. Prolonged storage of ovaries at 4°C results in less BCB+ oocytes isolated from ovaries.
1