animals Article Selection of Immature Cat Oocytes with Brilliant Cresyl Blue Stain Improves In Vitro Embryo Production during Non-Breeding Season Anna Rita Piras 1, *,† , Federica Ariu 1,† , Maria-Teresa Zedda 1 , Maria-Teresa Paramio 2 and Luisa Bogliolo 1 1 Department of Veterinary Medicine, University of Sassari, 070100 Sassari, Italy; Federica@uniss.it (F.A.); Zedda@uniss.it (M.-T.Z.); Luis@uniss.it (L.B.) 2 Departament de ciencia Animal i Dels Aliments, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain; teresa.paramio@uab.es * Correspondence: anrpiras@uniss.it † These authors contributed equally to this work.  Received: 15 July 2020; Accepted: 22 August 2020; Published: 24 August 2020  Simple Summary: The domestic cat is commonly used as a model for the development of assisted reproductive technologies, including in vitro embryo production (IVEP) in felid species. Seasonal reproduction is a feature of domestic cats as well as of several species of wild feline. Likewise, the number and the quality of blastocysts produced in in vitro systems is linked to season. Maintaining stable in vitro embryo production throughout the year is crucial not only for research purposes but also for programs aimed at protecting endangered felines. We assess whether using Brilliant Cresyl Blue (BCB) selection in addition to the classical morphological selection could improve the IVEP outcomes during non-breeding season. Blastocyst yield and quality of the embryos (hatching rate and blastocyst cell numbers) were higher after IVM/IVF in oocytes defined as BCB+ (colored cytoplasm) based on the BCB test than in oocytes only morphologically selected. Furthermore, no adverse effects on bioenergetic/oxidative status were observed in oocytes subjected to BCB staining. In conclusion, BCB test implementation in IVEP programs might ensure a steady output of domestic cat blastocysts throughout the year. Abstract: In domestic cats, the maturation, fertilization, and development potential in vitro decreases during the non-breeding season. This study aims at evaluating the efficacy of Brilliant Cresyl Blue (BCB) staining in selecting developmentally competent oocytes to be used in in vitro embryo production (IVEP) programs in order to overcome the season variability in blastocyst yield. Cumulus-oocytes complexes (COCs) collected from antral follicles of domestic cat ovaries during the anestrus phase (July to November) were selected by BCB staining and classified as BCB+ (colored cytoplasm) and BCB− (colorless cytoplasm). COCs not exposed to BCB staining were used as control. Before and after in vitro maturation mitochondrial activity and reactive oxygen species (ROS) were measured. Following in vitro fertilization, blastocyst rate, hatching rate, and blastocyst cell numbers were recorded. The results show that BCB staining did not alter the mitochondrial function and ROS production in cat oocytes. BCB+ oocytes presented a higher (p < 0.05) blastocyst rate, hatching rate, and blastocyst cell number than BCB− and control oocytes. In conclusion, BCB staining does not affect the bioenergetic/oxidative status of the oocyte while being a useful tool for selecting good quality oocytes to increase IVEP in domestic cats during non-breeding season. Keywords: BCB; blastocyst; domestic cat; in vitro maturation; mitochondria; ROS; seasonality Animals 2020, 10, 1496; doi:10.3390/ani10091496 www.mdpi.com/journal/animals Animals 2020, 10, 1496 2 of 12 1. Introduction Over the last decades, appreciable advances have been made in the development of assisted reproductive technology (ART) for feline embryos in vitro production. The possibility of using domestic cat gametes as a valuable model has allowed the improvement of in vitro embryo production (IVEP) technology that might be helpful for preserving endangered wild felids [1]. Although living offspring have been produced after the transfer of in vitro produced embryos both in domestic [2,3] and non-domestic cats [4], the efficiency of IVEP is still low compared to the level achieved in farm animals [5]. Among the multitude of factors which may affect the developmental potential of cat oocytes following in vitro maturation (IVM) and fertilization (IVF) [6–9], the influence of season and ovarian status has previously been highlighted [10–12]. Under natural conditions, the free-ranging domestic cat is a seasonally polyestrous long-day species. In the northern hemisphere, the breeding season begins in January or February, with the highest incidence of estrus in February and March, and ends between June and November [13]. Spindler and Wildt [10] first reported that the time of the year has a different impact on the quality of domestic cats oocytes. In vitro embryo development up to the blastocyst stage after IVM\IVF of domestic cat oocytes is significantly reduced during the non-breeding season compared to the reproductive season [10–12]. The selection of immature cumulus oocyte complexes (COCs) for IVM in cat, as well as in other species, is routinely based on the microscopical assessment of morphological features [9] such as cumulus thickness, ooplasm compactness and homogeneity, and oocytes sizes [9]. However, evidence shows that the morphological criteria do not really reflect the oocyte ability to develop. Consistently, morphological high-quality oocytes retrieved from domestic cats during the non-breeding season displayed a reduced ability to reach nuclear maturation, to be fertilized, and to develop to the blastocyst stage in vitro [10]. Understanding the mechanism that induces a reduction of oocyte quality during the non-breeding season is still limited. It has been suggested that seasonal variation in follicle stimulating hormone (FSH) levels and receptors [12,14,15] or the lack of antioxidant capacity of the cumulus-oocyte complex might be responsible for reduced oocyte developmental competence [12]. Brilliant Cresyl Blue (BCB) staining is a non-invasive method that has been used in different animal species [16], including in humans [17], for identifying the quality of oocytes retrieved from antral follicles [18]. Several studies in different animal species demonstrated that in vitro maturation, cleavage, and development to blastocyst after IVF were significantly higher in BCB+ selected oocytes [18–23]. Similar findings have recently been obtained for feline oocytes [24]. BCB staining allowed selecting domestic cat oocytes likely to be developmentally competent [24]. Indeed, domestic cat oocytes with low G6PDH activity respond to IVM\IVF with high nuclear maturation and development to the morula stage [24]. However, in the abovementioned study, the oocytes were collected from cat ovaries independently of the time of the year, and the resulting data were not separated by months or seasons. Despite the overall positive results obtained with BCB selection, the utility of this test is still controversial. Some studies reported no significant differences in developmental competence between BCB+ and unstained control oocytes [25,26]. Recent evidences do not recommended using this test for oocyte screening before IVM, as BCB is toxic and has a deleterious effect on oocytes [27–29]. This study was conceived to test whether the selection of immature oocytes retrieved from domestic cat ovaries during the non-breeding season, by using a combination of COC morphology and BCB staining, would be helpful in selecting developmentally competent oocytes, thus enhancing the in vitro fertilization outcome. The possibility of classifying oocytes according to their developmental potential is particularly important to understand oocyte biology and might help to reduce the negative impact of season on in vitro embryo production especially in the case of wild felines that die unexpectedly during the anestrus period. Animals 2020, 10, x 3 of 12 Animals 2020, 10, 1496 3 of 12 2. Materials and Methods All chemical 2. Materials reagents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and Methods unless otherwise specified. All chemical reagents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) unless otherwise specified. 2.1. Animal Selection 2.1. Animal Healthy, Selection free-ranging queens (Felis catus ≥ 1-year-old) were used in this research. The study was carried out between July and Healthy, free-ranging November queens (non-breeding (Felis catus ≥ 1-year-old)season) in Sardinia, were used Italy (40°43′33″ in this research. The study N; 8°33′19″ E). Anestrus was confirmed by visualization of morphological characteristics was carried out between July and November (non-breeding season) in Sardinia, Italy (40 43 33 N; of the ◦ ovaries 0 00 8after ovariohysterectomy. ◦ 330 1900 E). Anestrus was Only inactive confirmed by ovaries (having visualization of neither follicular morphological or luteal activity characteristics of the[10] were ovaries included in the study). after ovariohysterectomy. Only inactive ovaries (having neither follicular or luteal activity [10] were Live in included animals were not used in this study. The samples (ovaries) were collected during routine the study). ovariectomies, Live animals following theused were not basicincriteria of good this study. Theveterinary surgery practices. samples (ovaries) were collected during routine ovariectomies, following the basic criteria of good veterinary surgery practices. 2.2. Cumulus-Oocyte Complexes Collection and Morphological Selection 2.2. Cumulus-Oocyte Complexes Collection Ovaries were recovered and Morphological Selection from ovariohysterectomies carried out at the Veterinary Teaching Hospital Sassari University (Sassari, Italy). Ovaries were Ovaries were recovered from ovariohysterectomies carried out placed in at sterile 15 mL tubes the Veterinary containing Teaching PBS Hospital (Phosphate Sassari Buffered University Saline, Italy). (Sassari, Life Technologies, Ovaries were Carlsbad, placed in CA, USA)15with sterile mL penicillin (100 mg/L), tubes containing PBS streptomycin (100 mg/L) and immediately transported to the laboratory at room (Phosphate Buffered Saline, Life Technologies, Carlsbad, CA, USA) with penicillin (100 mg/L), temperature (22–25 °C). streptomycin (100 mg/L) and immediately transported to the laboratory at room temperature (22–25 ◦ C). Cumulus-oocyte complexes Cumulus-oocyte complexes(COCs) (COCs)werewere recovered recovered fromfrom the antral the antral follicles follicles by the by slicing slicing the ovarian ovarian surface in 25 mM Hepes-buffered TCM 199 supplemented with 0.1% (w/v) surface in 25 mM Hepes-buffered TCM 199 supplemented with 0.1% (w/v) polyvinyl alcohol (PVA) polyvinyl alcohol (PVA) and and antibiotics. antibiotics. The immatureThe COCs immature COCs were morphologically were morphologically classified underclassified under a a stereomicroscope, stereomicroscope, as as previously previously described by Wooddescribed and Wildtby [9].Wood Only and COCs Wildt with[9]. twoOnly COCs or more with two granulosa orlayers cell more granulosa cell layers surrounding the oocyte and with homogeneous surrounding the oocyte and with homogeneous dark cytoplasm were selected. dark cytoplasm were selected. Selected COCs Selected COCs were were randomized randomized in in two two groups: groups: (1) (1) oocytes oocytes not exposed to not exposed to BCB BCB staining staining andand directly submitted to IVM (control group); (2) oocytes submitted to the BCB test directly submitted to IVM (control group); (2) oocytes submitted to the BCB test (BCB group). (BCB group). 2.3. Brilliant Cresyl Blue (BCB) Staining The BCB group included COCs washed three times in modified PBS (mPBS: PBS supplemented with 1 g/L glucose, glucose, 36 mg/L sodium sodium pyruvates, 0.5 g/L bovine serum albumin (BSA) and 0.05 g/L gentamicin) ◦ C in a humidified air gentamicin) and andincubated incubatedwith with13 µMofofBCB 13µM BCBdiluted inin diluted mPBS mPBSforfor 4545min at 38.5 min at 38.5 °C in a humidified atmosphere [22]. Following air atmosphere BCB incubation, [22]. Following COCs wereCOCs BCB incubation, examined wereand sorted under examined anda stereomicroscope sorted under a stereomicroscope (Olympus (Olympus IX 40, Segrate, IXaccording Italy) 40, Segrate, to Italy) oocytesaccording to oocytes cytoplasm cytoplasm coloration (Figure 1) coloration into two (Figure groups 1) into(colored BCB+ two groups BCB+ (colored cytoplasm) and BCB−cytoplasm) (colorlessand BCB− (colorless cytoplasm). COCs werecytoplasm). washed COCs werein three times washed mPBS three times before IVM.in mPBS before IVM. Figure 1. High quality cumulus oocyte complexes, collected from ovaries of anestrus cats, stained by Brilliant Cresyl Blue (BCB). Animals 2020, 10, 1496 4 of 12 2.4. Experimental Design Firstly, we evaluated the effect of BCB staining on: (i) meiotic competence of BCB and control oocytes and (ii) mitochondrial activity and the level of intracellular reactive oxygen species (ROS) of the oocytes immediately after BCB selection and after in vitro maturation (Experiment 1). This analysis explored whether dye exposure can have potentially damaging effects on meiotic progression and on oocytes bioenergetic/oxidative status. A previous study [29] reported that BCB staining negatively affects the redox state in porcine oocytes. In Experiment 2, the developmental competence of BCB+ and BCB−, and control oocytes subjected to IVM\IVF were evaluated. Embryonic cleavage, blastocyst formation and hatching and blastocyst cell number were recorded. 2.5. Measurement of Mitochondria Activity and Reactive Oxygen Species (ROS) Levels Mitochondrial membrane potential and intracellular ROS levels of oocytes were measured through staining with MitoTracker Orange CMTMRos (Molecular Probes, Eugene, OR, USA) and 20 ,70 -dichlorodihydrofluorescein diacetate (H2 DCF-DA, Molecular Probe, Eugene, OR, USA), respectively. Briefly, denuded immature and mature (selected on the basis of polar body extrusion) oocytes were washed three times in PBS with 0.1% BSA and were incubated for 30 min in PBS with 3% BSA containing 200 nM MitoTracker Orange CMTMRos at 38.5 ◦ C under 5% CO2 . After the first incubation, the oocytes were washed again in PBS with 0.1% BSA and incubated for 30 min in PBS with 0.1% BSA and 10 µM H2 DCFDA [30]. After washing in PBS with 0.1% BSA, the oocytes were fixed for 60 min at 38 ◦ C in 2% paraformaldehyde and stained for 5 min with 1 µM Hoechst 33,342 solution. All procedures were carried out while paying attention to reducing the exposure of the sample to light in order to avoid photo-bleaching. The oocytes were placed in Eppendorf tubes containing 1 mL of PBS with 0.1% PVA at 4 ◦ C in the dark until their analysis. The oocytes were transferred to a glass slide into a 5 µL drop of PBS and glycerol (3:1 v/v) solution, covered with a coverslip, supported by four droplets of Vaseline and sealed with nail varnish. Mitochondria activity and ROS level measurement were performed by a confocal laser-scanning fluorescence microscope (Leica TCS SP5 CLSM), equipped with 543 nm He/Ne, 488 nm Argon and 405 nm diode lasers and recorded on a host computer. For mitochondrial evaluation, samples were observed with a helium/neon laser ray at 543 nm to detect MitoTracker Orange CMTMRos (excitation 554 nm). The fluorescence signal of DCF, indicating the ROS level, was measured with an argon-ion laser ray at 488 nm (excitation 460 nm). Images were taken at equatorial plane with 40x magnification under mineral oil. The setting of scanning conditions with respect to laser energy, signal detection, offset, filters and pinhole size were maintained constant for all image acquisitions [30]. Acquisition and image analysis were performed by using Leica LAS lite 170 Image software. In each individual oocyte, a circle region of interest (ROI) delimiting the cytoplasm area of the oocyte was selected and fluorescence intensity was measured and reduced by compensation for the background fluorescence. Intensities of fluorescence were normalized to those of control value. 2.6. In Vitro Maturation and Assessment of Nuclear Maturation Cumulus-oocyte complexes from control and BCB groups were in vitro matured in NaHCO3 -TCM 199 [31] supplemented with 0.36 mM pyruvate, 2 mM glutamine, 2.2 mM calcium lactate, 1.2 mM cysteine, 4 mg/mL BSA and FSH 1 IU⁄mL and luteinizing hormone (LH) 1 IU/mL in groups of 25–35 in 650 µL of IVM medium, in a humidified atmosphere of 5% CO2 , at 38.5 ◦ C for 24 h. At the end of IVM, COCs were completely denuded of granulosa cells via gentle pipetting with a fine bore glass pipette and stained with a solution of Hoechst 33,342 (10 µg/mL) in 1:1 (v/v) glycerol/PBS [7]. The nuclear configuration was classified under an epifluorescent microscope (Olympus IX 70, Segrate, Italy) as germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), Animals 2020, 10, 1496 5 of 12 or metaphase II (MII). Oocytes looking diffusely stained through cytoplasm and with unidentifiable or invisible chromatin were considered degenerated [7]. 2.7. In Vitro Fertilization and Embryo Culture Spermatozoa were collected from epididymides of adult cats [32] following routine orchiectomy and frozen according to the procedure described by Tsutsui et al. [33]. Frozen-thawed spermatozoa were selected by the swim-up technique and used at a final concentration of 5 × 105 sperm/mL for IVF. After in vitro maturation, COCs were partially denuded by gentle pipetting and washed in synthetic oviductal fluid (SOF) containing 6 mg/mL BSA, 100 IU/mL penicillin, 50 µg/mL gentamicin [31]. COCs and spermatozoa were co-incubated in four-wells Petri dishes with 450 µL of SOF covered with mineral oil, for approximately 22 h at 38.5 ◦ C in a humidified atmosphere of 5% CO2 in air [31]. Presumptive zygotes (day 1 post IVF) were washed and cultured in 650 µL of SOF containing 4 mg/mL BSA, and 100 IU/mL penicillin and 1% MEM non-essential amino acids (IVC-1 medium). On day 3, the embryos were transferred to IVC-2 medium (SOF supplemented with 10% fetal calf serum (FCS) and 2% MEM essential amino acids (EAA), and cultured at 38.5 ◦ C in a humidified atmosphere of 5% CO2 in air for additional 4 days [31]. Embryonic cleavage was assessed at 24 h post-fertilization, blastocyst formation and hatching at 7 days post-fertilization. 2.8. Assessment of Blastocyst Cell Number The blastocyst cell number was evaluated by differential staining of the inner cell mass (ICM) and trophectoderm (TE) cell compartments [34]. Briefly, seven-day-old blastocysts were exposed to 1% Triton X-100 in 20 mM HEPES-buffered TCM 199 containing 30 mg/mL propidium iodide (PI) for 35–40 s. The blastocysts were then transferred into ice-cold ethanol for 2–5 s. Finally, the blastocysts were incubated in medium with 50% (v/v) glycerol and ethanol containing 0.1 mg/mL bis-benzimide (Hoechst 33,342) for 5 min. Embryos were subsequently transferred into a small droplet of glycerol on a glass slide and examined under epifluorescent microscope (Olympus IX70, Segrate, Italy). A digital image of each embryo was taken, and the numbers of ICM (blue) and TE (red) nuclei were counted by the microscope system (Image J 1.5Oi). 2.9. Statistical Analysis All statistical analyses were performed using STATA\IC 11.28 (StataCorp LP, Lakeway Drive, college station, TX, USA). Data on nuclear maturation, cleavage, and development to blastocyst stage were analyzed by chi-square test. The overall chi-square was calculated and found to be significant before the Fisher’s exact test was conducted to identify differences among the experimental classes. The Shapiro-Wilk test [35] was used to verify the normal distribution of data on blastocyst cell number, intracellular ROS level and mitochondria activity. Data (mean ± SEM) for blastocyst cell numbers were normally distributed and were analyzed using one-way ANOVA followed by Bonferroni correction as post-hoc test. Data (mean ± SEM) for intracellular ROS level and mitochondrial activity were not normally distributed and were analyzed with a non-parametric Kruskal-Wallis test [36]. Differences with p < 0.05 were considered as statistically significant. All experiments were replicated at least three times. 3. Results A total of 88 ovaries were included in the experiments. Eight hundred twenty-seven COCs were selected, of which 241 COCs were used as the control group and 591 COCs were exposed to BCB test. After BCB staining, 356 COCs (60.2%) were classified as BCB+ and 230 COCs (27.8%) as BCB−. Of these oocytes, 212 were used to evaluate the meiotic configuration, 147 for the evaluation of mitochondrial activity and ROS levels, and 468 for in vitro embryo production. Animals 2020, 10, 1496 6 of 12 3.1. Effects of BCB Staining on Nuclear Maturation Table 1 summarizes the results of the meiotic progression of the COCs stained with BCB before in vitro maturation. Table 1. Meiotic progression of cat oocytes obtained from ovaries of anestrus cats selected by Brilliant Cresyl Blue (BCB). Total Nuclear Chromatin Configuration n (%) Degenerated n (%) Group Oocytes n GV GVBD MI\TI MII Control 78 7 (8.9) a 6 (7.7) 6 (7.7) 50 (64.1) a 9 (11.5) BCB+ 80 10 (12.5) a 5 (6.2) 4 (5.0) 54 (67.5) a 7 (8.7) BCB− 54 15 (27.8) b 6 (11.1) 2 (3.7) 20 (37.0) b 11 (20.4) Total BCB 134 25 (18.6) ab 11 (8.2) 6 (4.5) 74 (55.2) a 18 (13.4) abDifferent superscript letters within the same column indicate significant difference (p < 0.05). GV: Germinal vesicle; GVBD: Germinal vesicle breakdown; MI: Metaphase I; TI: Telophase I; MII: Metaphase II. No significant difference in nuclear maturation and degeneration rate were observed between oocytes exposed to BCB staining (BCB+ plus BCB−) and unexposed oocytes. The percentage of oocytes that progressed to metaphase II stage was similar between control and BCB+ groups and lower (p < 0.05) in BCB− oocytes compared to control and BCB+. No difference was found in the degeneration rate among groups. The percentage of BCB− oocytes at GV stage (27.8%) was higher (p < 0.05) than that of control (8.9%) and BCB+ (12.5%) oocytes. The rate of oocytes that resumed meiosis (GVBD and MI/TI stage) did not differ among groups. 3.2. Effects of BCB Staining on Mitochondria Activity and Reactive Oxygen Species (ROS) Levels Mitochondria activity of BCB exposed (BCB+ plus BCB−) oocytes immediately after staining (n = 57; 0.95 ± 0.07 pixels\oocyte) and after in vitro maturation (n = 39; 0.96 ± 0.05 pixels\oocyte) was similar to the respective counterpart of control groups (n = 29; 1 ± 0.09 and n = 22; 1 ± 0.04 pixels\oocyte, p > 0.05; Figure 2A). Brilliant Cresyl Blue exposure did not affect intracellular ROS level in immature (n = 57; 1.18 ± 0.16 pixels\oocyte) and in in vitro matured (n = 39; 0.93 ± 0.04 pixels\oocyte) oocytes compared to control oocytes (n = 29; 1 ± 0.15 and n = 22; 1 ± 0.04 pixels\oocyte, respectively, p > 0.05; Figure 2B). 10, x Animals 2020, 7 of 12 Figure 2.2.Mitochondria Figure Mitochondriaactivity (A)(A) activity andand intracellular reactive intracellular oxygen reactive speciesspecies oxygen (ROS) level (B)level (ROS) in domestic (B) in cat domestic cat oocytes immediately after Brilliant Cresyl Blue (BCB) staining and after inValues oocytes immediately after Brilliant Cresyl Blue (BCB) staining and after in vitro maturation. vitro are expressed maturation. as mean Values ± standardaserror are expressed meanof± the mean error standard (SEM). Immature: of the mean (SEM).germinal vesicle;germinal Immature: in vitro matured: vesicle; inmetaphase II; total vitro matured: BCB: BCB+ metaphase plus BCB: II; total BCB−.BCB+ Totalplus BCBBCB−. groupTotal was compared BCB grouptowascontrol group. compared Symbol * indicate significant difference (p < 0.05). to control group. Symbol * indicate significant difference (p < 0.05). Mitochondria 3.3. Effect activityonimmediately of BCB Selection afterProduction In Vitro Embryo staining was not statistically different among the control (n = 29; 1 ± 0.09 pixels\oocyte), BCB+ (n = 34; 0.98 ± 0.11 pixels\oocyte) and BCB− (n = 23; 0.92 ±The 0.10results obtained after pixels\oocyte) groups.in vitro Afterfertilization and embryo in vitro maturation, culture of the mitochondria oocytes activity retrieved was lower infrom the the ovaries of anestrus cats selected by BCB staining are reported in Table 2. The cleavage rate was similar between BCB+ and control groups. BCB− oocytes cleaved at lower percentage (11.0%) compared to control (28.6%) and BCB+ (28.6%) oocytes (p < 0.05). The percentage of blastocyst formation and hatching blastocysts (Figure 3A) was higher in the BCB+ group than in the control and BCB− groups. The number of total and TE cells of blastocysts Animals 2020, 10, 1496 7 of 12 BCB− (n = 17; 0.73 ± 0.06 pixels\oocyte) group than in the BCB+ (n = 22; 0.98 ± 0.11 pixels\oocyte) and control (n = 22; 1 ± 0.04 pixels\oocyte) groups (p < 0.05, Figure 2A). Intracellular ROS levels in BCB− oocytes were Figure higher immediately 2. Mitochondria afterand activity (A) staining (n = 23; intracellular 1.76 ±oxygen reactive 0.21 pixels\oocyte) and lower species (ROS) level (B) in after in vitro = 0.8 ± 0.05 pixels\oocyte) compared to controls (n = domestic cat oocytes immediately after Brilliant Cresyl Blue (BCB) staining and after inand maturation (n 17; 29; 1 ± 0.18 n = 22; vitro 1 ± 0.04 pixels\oocyte, respectively) and BCB+ (n = 34; 0.89 ± 0.17 and n = 22; 1.02 ± 0.05 maturation. Values are expressed as mean ± standard error of the mean (SEM). Immature: germinal pixels\oocyte, respectively) vesicle; in oocytes (p < 0.05, vitro matured: Figure II; metaphase 2B). total BCB: BCB+ plus BCB−. Total BCB group was compared to control group. Symbol * indicate significant difference (p < 0.05). 3.3. Effect of BCB Selection on In Vitro Embryo Production 3.3. Effect of BCB obtained The results Selection after on Inin Vitro Embryo vitro Production fertilization and embryo culture of the oocytes retrieved from the ovaries Theofresults anestrus cats selected obtained after inby BCBfertilization vitro staining areand reported embryoin Table 2. of culture Thethecleavage oocytesrate was similar retrieved from between BCB+ and control groups. BCB− oocytes cleaved at lower percentage (11.0%) compared the ovaries of anestrus cats selected by BCB staining are reported in Table 2. The cleavage rate was to control (28.6%) similar betweenand BCB+ BCB+ (28.6%) and controloocytes (p 0.05) Blastocyst than in control and Hatched Cell BCB− oocytes. Number (Mean ± BCB− SEM) group Oocytes Blastocysts (%/n) presented the lowest (p >(%)0.05) total(%/n) and TE cell number (Figure 3B). No significant Total TEdifferencesICM were found in ICM numbers Control 112 32among (28.6) groups a (Table 2). 4 (3.6) 12 (10.7) a a 197.1 ± 8,7 a 152.6 ± 8.5 a 44.4 ± 2.6 BCB+ 220 63 (28.6) a 44 (20.0) b 26 (11.8) b 237.8 ± 12.5 b 185.8 ± 9.5 b 51.7 ± 3.8 BCB− 136 b 10 (7.3) a 2 (1.5) a 130 ± 13.7 c 104.4 ± 13.6 c 37.7 ± 5.2 15(11.0) Table 2. Embryonic development after in vitro maturation and fertilization of domestic cat oocytes abc Different superscript letters within the same column indicate significant difference (p < 0.05). ICM, inner cell obtained from ovaries of anestrus cats by Brilliant Cresyl Blue (BCB) staining. mass; TE, trophectoderm. Cleaved Blastocyst Hatched Blastocysts Cell Number (Mean ± SEM) Group No.Oocytes (%) (%/n) (%/n) The percentage of blastocyst formation and hatching blastocysts (Figure 3A)TE Total was higher ICMin the Control 112 32 (28.6) a 12 (10.7) a 4 (3.6) a 197.1 ± 8,7 a 152.6 ± 8.5 a 44.4 ± 2.6 BCB+ group than in the control and BCB− groups. The number of total and TE cells of blastocysts BCB+ 220 63 (28.6) a 44 (20.0) b 26 (11.8) b 237.8 ± 12.5 b 185.8 ± 9.5 b 51.7 ± 3.8 developed BCB− from 136 BCB+ oocytes 15(11.0) b was higher 10 (7.3) a (p > 0.05) than 2 (1.5) a in control and BCB− 130 ± 13.7 c oocytes. BCB− group 104.4 ± 13.6 c 37.7 ± 5.2 presented the lowest abc Different (p > 0.05) superscript letterstotal and within theTE cellcolumn same number (Figure indicate 3B). Nodifference significant significant (p