{'fraction_non_alphanumeric': 0.03035463834902825, 'fraction_numerical': 0.02123822881186135, 'mean_word_length': 5.105504587155964, 'pattern_counts': {'":': 0, '<': 16, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Effect of roscovitine on developmental competence of small follicle-derived buffalo oocytes

Background & objectives: The lower recovery of competent oocytes in buffalo species limits the commercialization of in vitro embryo production technology in field condition. In this context, pre-maturation of small follicle (SF)-derived oocytes with meiotic inhibition may be a promising alternative to obtain more number of competent oocytes. Thus, the present study was conducted with an objective to enhance the developmental potential of less competent SF-derived buffalo oocytes. Methods: All the visible follicles (used for aspiration) from buffalo ovaries were divided into two categories: large follicle (LF) (follicles having diameter ≥6 mm) and SF (follicles of diameter <6 mm). The competence of LF and SF oocytes was observed in terms of brilliant cresyl blue (BCB) staining, cleavage rate, blastocyst rate and relative gene expression of oocyte and blastocyst competence markers. Thereafter, less competent SF oocytes were treated with 0, 12.5, 25, 50 and 100 mM doses of roscovitine (cyclin-dependent kinase inhibitor) to enhance their developmental potential. Results: Based on parameters studied, LF oocytes were found to be more competent than SF oocytes. Pre-maturation incubation of SF oocytes with roscovitine reversibly arrested oocyte maturation for 24 h to ensure the proper maturation of less competent oocytes. A significantly higher number of BCB-positive oocytes were noted in roscovitine-treated group than SF group. Cleavage and blastocyst rates were also higher in roscovitine-treated group. The relative messenger RNA expression of oocyte (GDF9, BMP15, GREM1, EGFR, PTGS2 and HAS2) as well as blastocyst (INF-τ, GLUT1 and POU5F1) competence markers was significantly greater in roscovitine-treated group relative to SF group. Again, on comparison with LF group, these parameters depicted a lower value in the treatment group. Interpretation & conclusions: The findings of this study has revealed that pre-maturation incubation of SF-derived oocytes with 25 μM roscovitine can improve its developmental competence and thus can be utilized to get maximum number of competent oocytes for better commercialization of in vitro embryo production technology in buffalo.

Substantial research has been conducted for the improvement of culture requirements of buffalo in vitro embryo production (IVEP) 1 . The major concern is the lower oocytes recovery compared to other species 2 which is one of the major constraints in the commercialization of buffalo IVEP technology in field conditions 3 . The acquisition of developmental competence is a sequential process which occurs along with the follicular growth in ruminants. This developmental process includes both nuclear and cytoplasmic maturation 4 . Thus, the fully matured oocytes having complete follicular information in the form of messenger RNA (mRNA) or proteins must be collected for improvement in bubaline in vitro culture 5 . If oocytes are collected before the acquisition of adequate information, developmental potential of embryo decreases 6 . There are some important events occurring in the oocyte during the late follicular growth, which are essential to achieve full developmental competence 7 . This has already been proved as cumulus-oocyte complexes (COCs) derived from large follicle (LF, >6 mm) of adult cattle have better cytoplasmic maturation and show higher developmental competence, whereas small follicle (SF, <6 mm)-derived oocytes are less competent due to inadequate cytoplasmic maturation 8 . Along with bovine species, improved blastocyst rate through selection of competent oocytes from LF has also been reported in bubaline 9 , caprine 10 , ovine 11 , porcine 7 and humans 12 . Hence, blastocyst rate can be improved either by selecting more competent LF oocytes or by ensuring cytoplasmic maturation of SF oocytes.
In most of the mammals, oocytes are maintained at germinal vesicle (GV) stage until pre-ovulatory luteinizing hormone (LH) surge. During this period of meiotic arrest, oocytes undergo morphological and biochemical changes to achieve developmental competence 13 . However, when oocytes are removed from follicle, they spontaneously resume nuclear maturation with impaired oocyte capacitation and result in lower rate of embryo development 13 . Competence of SF-derived oocytes may be enhanced by providing sufficient pre-maturation incubation for a specific period of time 14 in the presence of meiotic inhibitors such as roscovitine, cycloheximide, 6-dimethylaminopurine and butyrolactone. Amongst all meiotic inhibitors, roscovitine is an effective and reversible inhibitor of cyclin-dependent kinase 2 15 , capable of arresting the cells in late G1 and G2/M cell cycle transition 16 . It has less detrimental effects on oocyte developmental competence than other inhibitors 14 and has been used effectively to reversibly block the nuclear maturation of oocytes for certain time period in bovine 14,16,17 , equine 6 , ovine 18 and porcine 19 . Moreover, porcine embryos obtained from oocytes pre-cultured with roscovitine developed to term, making its introduction desirable in assisted reproductive technology programmes 20 .
These studies reported the reversible inhibitory effect of roscovitine in pooled oocytes derived from the visible ovarian follicles. The present study was carried out to investigate the effect of roscovitine on in vitro maturation of SF-derived buffalo oocytes and further development to the blastocyst stage.

Material & Methods
The study was conducted in the division of Physiology and Climatology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, India. On the basis of diameter, ovarian follicles were categorized as LF (≥6 mm) and SF (<6 mm) 21,22 . The study was undertaken as three individual experiments: (i) comparative analysis of developmental competence of LF and SF oocytes, (ii) determination of doses of roscovitine to improve the developmental competence of SF-derived oocytes, and (iii) comparison between the developmental correlates of SF, LF and roscovitinetreated SF group.
The best dose of roscovitine was decided based on the following criteria: (i) inhibitory effect of roscovitine (0, 12.5, 25, 50 and 100 µM) treatment for 24 h on nuclear maturation; (ii) reversibility of roscovitine on nuclear maturation after 24 h maturation in roscovitinefree medium; (iii) cleavage rate; and (iv) blastocyst rate. Thereafter, the developmental competence of LF, SF and 25 µM roscovitine-treated COCs was compared on the basis of brilliant cresyl blue (BCB) staining, cleavage rate, blastocyst rate, relative gene expression of oocyte competence markers in cumulus cells and denuded oocytes and blastocyst competence markers.

In vitro embryo production
Oocyte retrieval and grading: Buffalo (Bubalus bubalis) ovaries collected from local abattoir were transported to the laboratory in pre-warmed normal saline solution (NSS) supplemented with 50 μg/ml gentamycin sulphate (Sigma, USA) at 37-39°C, within 2-3 h of slaughter. Ovaries were washed with NSS to remove dirt, blood, extra tissues, etc. The follicular fluid was aspirated separately from LF (≥6 mm) and SF (<6 mm) using 18-gauge needle fitted with 5 ml syringe containing oocyte collection medium [OCM consisting of TCM-199 HEPES modified (Sigma)]. Three mg/ml bovine serum albumin (BSA) (Sigma) 21,22 . All COCs with more than two layers of compact cumulus layers and homogeneous granular ooplasm were used for the study 21,22 .
The remaining two-thirds of the COCs were kept in inhibitor-free medium for additional 24 h period to resume the nuclear maturation.
Brilliant cresyl blue (BCB) staining to select competent oocyte: COCs derived from LF (n=97), SF (n=110) and 25 µM roscovitine-treated (n=107) groups were subjected for BCB staining as per the defined protocol 21,23 . The COCs were divided into two groups depending on the colour of cytoplasm; BCB-positive COCs were those with any degree of blue coloration, whereas BCB-negative COCs were without any blue coloration of the cytoplasm (Fig. 2). The percentage of BCB-positive and BCB-negative oocytes was recorded.
In vitro maturation: After 24 h of roscovitine pre-maturation incubation, COCs were rinsed 4-5 times in maturation medium to avoid the carry-over effect of roscovitine into the final drop of maturation medium. Pre-mature oocytes were subjected to in vitro maturation (IVM) to allow the resumption of meiosis, whereas oocytes derived from LF and control group were directly kept for IVM (no roscovitine treatment) in 50 μl droplets of maturation medium for 24 h at 38.5°C and 5 per cent CO 2 in the air with maximum relative humidity.
In vitro fertilization: In vitro fertilization (IVF) was done as per the modified protocol of Pandey et al 21 .
In vitro matured COCs of different groups were subjected to IVF. The frozen buffalo bull semen straws  Institute (ICAR-NDRI), Karnal, India. The semen was washed twice in Tyrode's albumin lactate pyruvate (FerTALP) medium supplemented with 0.2 mM sodium pyruvate, 6 mg/ml BSA and 20 μg/ml heparin (Sigma) and centrifuged at 70 g for 10 min. The pellet formed was suspended in FerTALP, and progressively motile sperm from the supernatant solution was placed as 50 μl droplets having a final concentration of 2×10 6 spermatozoa/ml. The matured COCs (10-15) were washed, introduced to the semen droplets overlaid with the mineral oil and co-incubated for 18 h with 5 per cent CO 2 in air at 38.5°C with maximum relative humidity.
In vitro embryo culture : In vitro embryo culture was done as per the protocol described by Bhardwaj et al 23 .
Medium was replaced at alternate day till blastocyst development or day 8 post-IVF. Group of healthy blastocysts has been depicted in Fig. 3.

Results
The present study was conducted to analyze the effect of roscovitine on the developmental potential of SF-derived less competent oocytes, as during the initial experiments, the higher competence of LF oocytes was noted in comparison to SF oocytes.

Comparison of developmental competence between large follicle and small follicle (LF and SF):
LFderived oocytes were found to be developmentally more competent in terms of higher BCB-positive oocyte (LF-75.2% and SF-29.09%), cleavage rate and blastocyst rate than the SF-derived oocytes. The relative mRNA study also revealed higher developmental potential of LF oocytes than SF oocytes in terms of upregulated expression of the competence markers such as GDF9 and BMP15 in oocyte; GREM1, PTGS2, HAS2 and EGFR in cumulus cells and interferon-t, GLUT1 and POU5F1 (Figs. 4-6).

Selection of best dose of roscovitine:
Different concentrations of roscovitine were tested to select the best dose. First, the inhibition of cumulus expansion after pre-maturation incubation was determined. Roscovitine effectively arrested the cumulus cells' expansion in a dose-dependent manner. The number of COCs showing cumulus expansion was apparently lowest in 100 µM followed by 50 µM and 25 µM groups than 12.5 µM group. Thus, the maximum inhibition of cumulus expansion was observed in 100 µM roscovitine group, followed by 50 and 25 µM, and the least inhibitory effect was noted in 12.5 µM group (Fig. 7).
Second, data of the dose-dependent inhibitory effect of roscovitine were observed. Concentrations of 25, 50 and 100 µM roscovitine exhibited higher percentage of oocytes at GV and GVBD stages than control and 12.5 µM roscovitine (Table I). A comparatively smaller proportion of oocytes reached MII stage with these concentrations of roscovitine. Third, the reversible effect of roscovitine in the oocytes was observed through nuclear staining after 24 h of inhibitor-free maturation. All the groups depicted non-significant difference amongst themselves, but the concentration of 100 µM roscovitine revealed significantly (P<0.05) less number of MII oocytes in comparison to other groups (Table II). Finally, the cleavage and blastocyst rates of different groups were analyzed, and data are provided in Table III. The data depicted significantly (P<0.05)     higher cleavage rate in 25 and 50 µM roscovitinetreated groups; however, no significant difference observed amongst control and 12.5 and 100 µM roscovitine-treated groups. A significantly (P<0.05) higher blastocyst rate was observed in 25 and 50 µM roscovitine-treated groups than control and 12.5 µM roscovitine-treated groups which reduced significantly (P<0.05) in 100 µM roscovitine-treated group though it was significantly (P<0.05) higher than the control and 12.5 µM roscovitine-treated group (Table III).
On the basis of the above findings, 25 µM roscovitine was selected as the minimum effective dose for the improvement of developmental competence of oocytes derived from the SF.
Comparison of best-selected dose of roscovitine with large follicle (LF)-derived oocytes: COCs pre-treated with 25 µM roscovitine were compared with LF-derived COCs in terms of the BCB staining, cleavage rate, blastocyst rate and relative mRNA expression of oocyte and blastocyst competence markers. BCB staining revealed that mean per cent of BCB-positive oocytes was significantly (P<0.05) higher in LF (75.26%) and 25 µM roscovitine-treated (64.49%) groups than SF (29.09%) group. However, no difference was noted between LF and treatment groups. The cleavage and blastocyst rate was significantly higher (P<0.05) in 25 µM roscovitinetreated group than the SF group but was significantly lower (P<0.05) than the LF group (data not shown).
PCR reaction and agarose gel electrophoresis confirmed predicted amplification of primers of respective genes. The relative mRNA expression of oocyte competence markers in denuded oocytes (GDF9 and BMP15) and cumulus cells (GREM1, EGFR, PTGS2 and HAS2 are given in Figs. 4 and 5, respectively. The mRNA expression of GDF9 and BMP15 transcript was significantly higher (P<0.05) in denuded oocytes of roscovitine-treated group than LF and SF groups, whereas no significant difference was observed in GDF9 and BMP15 expression between the LF and SF groups (Fig. 4). The mRNA expression level of GREM1, PTGS2, HAS2 and EGFR in cumulus cells was significantly higher (P<0.05) in roscovitinetreated group than LF and SF groups. Similarly, a significantly higher (P<0.05) expression of GREM1, PTGS2 and EGFR was recorded in LF group than SF group, whereas no difference was noticed in the expression of HAS2 transcript (Fig. 5).
The relative mRNA expression of blastocyst competence markers has been provided in Fig. 6. GLUT1 and POU5F1 revealed a significantly higher (P<0.05) expression in LF group, followed by roscovitine-treated group than the SF group, whereas INF-τ expression was insignificant amongst the groups.

Discussion
Many studies 6,14,[16][17][18][19] have already been conducted to enhance the developmental competence of oocytes by the use of various nuclear inhibitors including roscovitine, but none conducted specifically to enhance the developmental competence of SF-derived oocytes. Hence, the present study was undertaken to enhance the developmental competence of SF-derived oocytes by pre-maturation incubation with roscovitine. During the study, the effect of the different concentrations of roscovitine on developmental competence of SFderived buffalo oocytes was evaluated in terms of reversible nuclear maturation inhibition, embryo development and relative gene expression of oocyte and blastocyst competence markers, with its further comparison with LF-derived oocytes.
Cumulus expansion was better after 24 h of in vitro maturation in LF oocytes than SF oocytes; also, more number of BCB-positive oocytes was obtained from LF in comparison to the SF group. A similar result has been reported in ovine species 26 . BCB staining has already been employed as a non-invasive, indirect method for selection of more competent oocytes in various species 1,27,28 . The present study also reported a significantly higher cleavage and blastocyst rate in LF oocytes than those of SF-derived oocytes. A relationship between follicle size and oocyte quality has been demonstrated in several species. This is due to the greater storage of the transcripts and proteins in the cytoplasm of LF oocytes 29 .
Results of roscovitine treatment revealed a dosedependent inhibition of meiotic resumption, and this meiotic inhibition was reversible for all the doses of roscovitine. However, a concentration of 25 µM was found to be the minimum effective dose at which about 75 per cent of the oocytes were arrested at GV stage. The results of the present study were supported by previous studies 14,17 which reported that 25 µM roscovitine maintained the cattle oocytes at GV stage for 24 h. Albarracin et al 16 found 50 µM roscovitine as the best dose for calf oocytes. There are reports in equine 6 where oocyte nuclear maturation was inhibited with 50 µM roscovitine, whereas in swine, 80 µM roscovitine 30 and 50 µM roscovitine 19 were the best doses to inhibit the meiotic resumption. Further, Zhang et al 31 concluded that 25 µM roscovitine improved the developmental competence of porcine oocyte. In ovine, 75 µM roscovitine was found to be efficient to reversibly block the meiotic resumption without any detrimental effect on development and quality of in vitro-produced embryos 18 .
BCB staining and embryo development rate was analyzed in roscovitine-treated group. The dose of 25 µM depicted better BCB staining and embryo development and thus selected as best roscovitine dose because it was the minimum dose which effectively arrested the oocyte meiotic division and significantly improved the in vitro developmental potential of SF-derived oocytes as well as embryos. It has been demonstrated that bovine oocytes arrested with roscovitine and butyrolactone I (BL-I) are competent to undergo normal early development and embryo derived from these oocytes developed into normal foetuses up to day 27 32 . Kaedi et al 33 found an enhanced blastocyst formation rate of reconstructed embryos derived from oocytes pre-cultured with roscovitine. In the present study, roscovitine pretreatment was given only to the SF-derived oocytes, which had a compromised developmental competence, and thus, during the pre-maturation incubation, these might have attained better cytoplasmic maturation and in turn revealed greater competence than untreated SF oocytes.
The enhanced relative expression of competence markers such as GDF9 and BMP15 in oocyte and GREM1, PTGS2 and EGFR in cumulus cells depicted the role of 25 µM roscovitine in enhancing oocyte competence. Lequarre et al 34 also reported the increased mRNA expression in roscovitinetreated bovine oocytes. This may define the process of neotranscription, or the addition of poly(A) tails to transcripts not yet adenylated, or both in the GVarrested oocytes. This effect of roscovitine is transient as it is no longer detectable after 24 h of maturation 31 . Hence, the significantly upregulated expression of the studied transcripts in roscovitine-treated group may be related to the neotranscription or increased polyadenylation.
Further, the blastocyst derived from LF and roscovitine-treated SF group showed significantly higher expression of blastocyst competence markers (GLUT1 and POU5F1) than SF group, which suggested that roscovitine treatment enhanced the developmental potential of SF-derived oocytes in terms of blastocysts number as well as its quality.
Hence, it is concluded from the study that 25 µM roscovitine effectively improved the developmental potential of less competent SF oocytes as it enhanced the mRNA expression pattern of competence-related molecules. It provides a useful method for ensuring the availability of more number of competent oocytes for buffalo embryo transfer technology as well as an efficient tool for transportation or manipulation of oocytes at the onset of maturation.