Effect of 9-cis retinoic acid, and, colony-stimulating factor 2 and interleukin 6 cytokines on in vitro maturation and embryo development of OPU-derived Murrah buffalo (Bubalus bubalis) oocytes

Effect of 9-cis retinoic acid, and, colony-stimulating factor 2 and interleukin 6 cytokines on in vitro maturation and embryo development of OPU-derived Murrah buffalo (Bubalus bubalis) oocytes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of 9-cis retinoic acid, and, colony-stimulating factor 2 and interleukin 6 cytokines on in vitro maturation and embryo development of OPU-derived Murrah buffalo (Bubalus bubalis) oocytes Ekta Verma, Prahlad Singh, Narinder Singh, Vijay Singh Malik, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7823204/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Reproductive inadequacy in buffaloes remains a major constraint to the efficacy of assisted reproductive technologies (ART), particularly in vitro embryo production (IVEP). This study investigated the effects of 9-cis retinoic acid (9-cisRA) during in vitro maturation (IVM) and cytokines (CSF2 and IL6) during in vitro culture (IVC) on enhancing the developmental competence of ovum pick-up (OPU) derived buffalo oocytes. In Experiment 1, oocytes were subjected to IVM supplemented with or without 9-cisRA. In Experiment 2, presumptive zygotes were cultured with CSF2, IL6, or their combination. Oocyte developmental competence was comprehensively evaluated based on cumulus cell expansion, cleavage rate, blastocyst yield, embryo quality grade, and developmental stage distribution. Data from Experiment 1 were analyzed using an independent t -test, while Experiment 2 data were subjected to one-way ANOVA followed by Tukey’s post-hoc test. The results demonstrated that 9-cisRA supplementation during IVM significantly enhanced ( P < 0.05) cumulus expansion (84.6% vs. 61.0%), cleavage rate (65.7% vs. 52.1%), blastocyst yield (31.4% vs. 19.1%), and the proportion of excellent/good quality embryos (54.4% vs. 30.0%). Furthermore, 9-cisRA-treated groups yielded a higher proportion of advanced-stage blastocysts, including expanded (54.5%) and hatched (27.3%) blastocysts. In contrast, cytokine supplementation yielded only modest and non-significant improvements across all measured parameters. These findings identify 9-cisRA supplementation during IVM as a highly effective strategy to improve buffalo IVEP outcomes, while the application of CSF2 and IL6 during embryo culture requires further dose and timing optimization to ascertain their potential benefits. Buffalo Ovum pick-up (OPU) 9-cis retinoic acid CSF2 IL6 in vitro embryo culture Figures Figure 1 Figure 2 Introduction Buffaloes (Bubalus bubalis) are indispensable to the agricultural economy of India and other Asian regions, serving as a primary source of high-quality milk, meat, and draught power. However, buffalo productivity is severely constrained by inherent reproductive challenges, including seasonality, silent estrus, and limited ovarian reserve. These factors culminate in a notably lower efficiency of in vitro embryo production (IVEP) compared to cattle, creating a significant bottleneck for genetic improvement programs (Gasparrini, 2002 ; Baruselli et al., 2018 ). Consequently, refining IVEP protocols by targeting key biological pathways is paramount to enhancing oocyte developmental competence and overall reproductive output in this species. Retinoids, the biologically active derivatives of vitamin A, are critical signalling molecules in reproduction. They mediate their effects primarily through retinoic acid receptors (RARs) and retinoid X receptors (RXRs), regulating fundamental processes of follicular development, oocyte nuclear and cytoplasmic maturation, and, embryonic morphogenesis (Ross et al., 2000 ). A key isomer of retinoid, 9-cis retinol, has been shown to enhance oocyte competence by mitigating oxidative stress and improving epigenetic regulation. Studies in cattle report that retinol supplementation during in vitro maturation (IVM) significantly improves cleavage rates and blastocyst yield (Gomez et al., 2003; Gad et al., 2018 ). The optimal application and efficacy of specific retinoids like 9-cis retinol in buffalo IVEP systems warrants a detailed investigation. Post-fertilization the embryo culture conditions are critical in addition to the oocyte maturation factors. Cytokines, such as granulocyte-macrophage colony-stimulating factor (CSF2) and interleukin 6 (IL6), are vital embryokines secreted by the reproductive to promote trophoblast cell proliferation, enhance metabolic activity, suppress apoptosis, and improve embryo viability and quality (Dobbs & Hansen, 2018 ; Ealy et al., 2021 ). CSF2 and IL6 benefits are established in mouse and cattle models, Role of CSF2 and IL6 in supporting the development of buffalo embryos, known to be highly sensitive to in vitro conditions, remains poorly defined and represents a significant knowledge gap. Based on the established roles of retinoids in oocyte maturation and embryokines in post-fertilization development, it is hypothesized that the sequential supplementation of 9-cis retinol during IVM followed by CSF2 and IL6 during in vitro culture (IVC) would act synergistically to enhance the developmental competence of buffalo oocytes and the quality of resulting embryos. In, Experiment 1 aim was to determine the individual effect of 9-cis retinol supplementation during IVM on oocyte maturation and subsequent embryo development. In Experiment 2 we investigated the effects of CSF2 and IL6, individually and in combination, during IVC, on embryos derived from both retinol-pretreated and standard IVM oocytes, to evaluate potential additive or synergistic benefits. 2. Materials and Methods 2.1 Selection of Animals Healthy pluriparous Murrah buffaloes (Experiment 1: n = 5, Experiment 2: n = 4)4–6 Years of age, maintained at the Dairy Farm of Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, India was incorporated in the study. All experimental procedures were approved by the Institutional Animal Ethics Committee (IAEC), GADVASU (Approval No. GADVASU/2024/AIEC/74/07), at its 74th meeting on 28 June 2024, in accordance with CPCSEA guidelines. Animals were housed under loose housing systems with free access to feed and water. 2.2 Ovum pick-up and oocyte classification Six ovum pick-up (OPU) Sessions per buffalo were conducted. Follicular aspiration was performed under transvaginal epidural anaesthesia, using a 7.5 MHz ultrasound probe equipped with a needle guide and connected to a vacuum pump (70 mmHg). Follicular fluid was collected into pre-warmed tubes and transported to the laboratory. Cumulus–oocyte complexes (COCs) were recovered under a stereo-microscope and classified as Grade A, B, C (culturable) or D (non-culturable) based on cumulus investment and ooplasm appearance. Only culturable oocytes were used for in vitro embryo production (IVEP). 2.3 Experimental design Two independent experiments were conducted to evaluate the effects of different supplements on oocyte maturation and subsequent embryonic development: Experiment 1 : To determine the influence of 9-cis retinoic acid (9-cisRA) on oocyte development competence. Experiment 2 : To assess the impact of cytokine supplementation [colony-stimulating factor 2 (CSF2), interleukin-6 (IL6), or CSF2 + IL6] during in vitro culture (IVC) of embryos. 2.4 In vitro maturation (IVM) OPU derived buffalo were matured in TCM-199 supplemented with 10% fetal bovine serum (FBS), 10 IU/ml luteinizing hormone, 0.5 µg/ml follicle-stimulating hormone, 1 µg/ml estradiol-17β, 50 µg/ml gentamicin, and 5 nM 9-cisRA. Control group had no 9-cisRA in the culture media. Cultures were maintained in 60 µl drops under mineral oil at 38.5°C in 6% CO₂ for 22–24 h. In vitro maturation was assessed by cumulus expansion of COCs. 2.5 In vitro fertilization (IVF) Frozen–thawed semen from proven Murrah bulls was processed using a Percoll gradient. Matured oocytes were co-incubated with capacitated spermatozoa (2 × 10⁶/ml) for 18 h in IVF medium (Vitrogen) supplemented with penicillamine, hypotaurine, epinephrine, and heparin. 2.6 In vitro culture (IVC) In Experiment 1, presumptive zygotes derived from oocytes matured with or without 9-cis retinoic acid (9-cisRA) were cultured in commercially available IVC medium (Vitrogen, India) under mineral oil at 38.5°C, 6% CO₂, 5% O₂, and 89% N₂. In Experiment 2, presumptive zygotes were cultured in CR1aa medium supplemented with essential amino acids and 3 mg/ml BSA. In treatment groups, IVC drops were further supplemented with CSF2 (10 ng/ml), IL6 (10 ng/ml), or their combination. Control cultures contained CR1aa medium without cytokines. Embryos were monitored for cleavage (day 3) and blastocyst formation (days 6). 2.7 Embryo evaluation Embryo quality was assessed according to the International Embryo Technology Society (IETS) manual, classifying blastocysts as excellent/good (Code 1), fair (Code 2), poor (Code 3), or dead/degenerating (Code 4). Developmental stages were recorded as blastocyst (Code 6), expanded blastocyst (Code 7), or hatched blastocyst (Code 8). 2.8 Statistical analysis Data were analyzed using SPSS software (version 20, IBM Corp., Armonk, NY). Values were expressed as mean ± SEM. Differences between groups were tested using independent-samples t -test or one-way ANOVA, followed by post hoc comparisons. A probability level of p < 0.05 was considered statistically significant. 3. Results 3.1 Experiment 1: 9-cisRA trial 3.1.1 Follicular aspiration and oocyte recovery A total of 279 follicles were aspirated across six OPU sessions in five buffaloes, yielding 209 oocytes with an overall recovery rate of 74.9%. Of these, 155 (74.2%) were culturable, Grades A, B, C (Table 1 ). Table 1 Follicle aspiration and oocyte recovery Parameter n Mean ± SEM Follicles aspirated 279 13.95 ± 0.92 COCs retrieved 209 10.45 ± 0.96 Oocyte recovery rate (%) 74.9 - Culturable oocytes 155 7.75 ± 1.09 • No. of buffaloes = 5 • Values presented as Mean ± SEM are calculated per OPU session per animal 3.1.2 COCs expansion of OPU derived buffalo oocytes The proportion of COCs showing complete expansion was significantly higher in the 9-cisRA group than control (84.6% vs. 61.0%; p < 0.01) (Table 2 ). Table 2 Cumulus expansion [n, Mean SEM, (%)] of OPU derived buffalo oocytes matured in TCM199 supplemented with 9-cisRA Group IVM media Replicates (no.) COCs cultured (n) COC Expansion Full A (%) Partial B (%) No C (%) Group 1 TCM199 + 9 cis retinoic acid 6 78 66 11 ± 0.81 (84.6) a 5 0.83 ± 0.30 (6.41) b 7 1.16 ± 0.16 (8.97) b Group 2 TCM199 (control) 6 77 47 7.8 ± 0.70 (61.03) b 17 2.8 ± 0.30 (22.07) a 12 2 ± 0.25 (15.58) a • Complete expansion: Number of COCs A out of n • Partial expansion: Number of COCs B out of n • No expansion: Number of COCs C out of n Values in a column with different superscripts differ significantly (p < 0.05). 3.1.3 Cleavage and blastocyst development Supplementation with 9-cisRA during IVM significantly improved cleavage (65.7% vs. 52.1%) and blastocyst yield (31.4% vs. 19.1%) compared with controls ( p < 0.05) (Table 3 ). Table 3 Developmental competence parameters (Mean ± SEM) of buffalo oocytes matured with 9-cis retinoic acid (9-cisRA) Group Replicates COCs Matured Oocytes Fertilized Putative zygotes Cleavage Rate Blastocyst Rate (no.) n, Mean ± SEM (%) a b c d e Group 1 6 78 73 12.16 ± 0.87 (93.58) 70 11.66 ± 0.80 (89.74) 48 a 8 ± 0.85 (65.75) 22 a 3.6 ± 0.33 (31.4) Group 2 6 77 71 11.83 ± 0.70 (92.20) 68 11.33 ± 0.61 (88.31) 37 b 6.61 ± 0.65 (52.11) 13 b 2.16 ± 0.16 (19.1) a = Total COCs matured in each group b = COCs fertilized out of a (%=n/a*100) c = Putative zygotes out of a (%=n/a*100) d = Cleavage Rate out of b (%= n/b*100) e = Blastocyst rate out of c (n/c*100) Values across a row with different superscripts differ significantly (p < 0.05). 3.1.4 Embryo quality and staging The proportion of excellent/good embryos (Code 1) was significantly higher in the 9-cisRA group than in control (54.4% vs. 30.0%; p < 0.05). More embryos developed to expanded and hatched blastocyst stages with 9-cisRA treatment (Table 4 ). Table 4 Embryo quality and stage distribution [n, Mean ± SEM, (%)] (Experiment 1). Group Code 1 Code 2 Code 3 Code 4 Stage 6 Stage 7 Stage 8 9-cisRA 12 2 ± 0.25 (54.4) 8 1.33 ± 0.33 (36.6) 2 0.33 ± 0.21 (9.09) 0 4 0.66 ± 0.33 (18.2) 12 2 ± 0.25 (54.5) 6 1.00 ± 0.36 (27.3) Control 4 0.66 ± 0.21 (30) 6 1 ± 0.25 (46.2) 1 0.16 ± 0.1 (7.7) 2 0.33 ± 0.21 (15.4) 4 0.66 ± 0.33 (30.8) 7 1.16 ± 0.16 (53.8) 2 0.33 ± 0.21 (15.4) *Embryo grading and staging codes: Code 1 = Excellent/Good; Code 2 = Fair; Code 3 = Poor; Code 4 = Dead/Degenerating; Code 6 = Blastocyst; Code 7 = Expanded blastocyst; Code 8 = Hatched blastocyst. Code 5 was not evaluated. As shown in Fig. 1 , supplementation of IVM medium with 9-cisRA significantly increased cleavage, blastocyst formation, and the proportion of Code 1 embryos compared with control. 3.2 Experiment 2: Cytokine trial 3.2.1 Follicular aspiration and oocyte recovery In four buffaloes, 157 follicles were aspirated across 4 OPU sessions, yielding 127 oocytes with a recovery rate of 80.8%. Among them, 101 were culturable (Table 5 ). Table 5 Follicle aspiration and oocyte recovery (Experiment 2: Cytokines). Parameter n Mean ± SEM Follicles aspirated 157 9.81 ± 0.63 COCs retrieved 127 7.90 ± 0.62 Oocyte recovery rate (%) 80.8 - Culturable oocytes 101 6.31 ± 0.53 • No. of buffaloes = 4 • Total OPU sessions/buffalo = 4 • Values presented as Mean ± SEM are calculated per OPU session per animal 3.2.2 Oocyte developmental competence In CR1aa medium, supplementation with CSF2, IL6, or CSF2 + IL6 produced numerical but statistically non-significant differences in cleavage and blastocyst yield compared with controls (Table 6 ). Table 6 Effect of cytokine supplementation during IVC (Experiment 2). Group Total COCs Maturation Rate COCs Fertilized Putative zygotes Cleavage Rate Blastocyst Rate n, Mean ± SEM, (%) N a b c d e CSF2 (10ng/ml) 25 21 5.25 ± 0.55 (84) 23 5.75 ± 0.72 (92) 22 5.5 ± 0.57 (88) 12 3 ± 0.47 (52.1) 6 1.5 ± 0.33 (27.2) IL6 (100ng/ml) 25 20 5 ± 0.47 (80) 24 6 ± 0.47 (96) 22 5.5 ± 0.75 (88) 14 3.5 ± 0.74 (58.3) 8 2 ± 0.47 (36.3) CSF2(10ng/ml) + IL6(100ng/ml) 25 19 4.75 ± 0.86 (76) 24 6 ± 0.81 (96) 21 5.25 ± 0.86 (84) 13 3.25 ± 0.86 (54.1) 7 1.75 ± 0.55 (33.3) Control 25 21 5.25 ± 0.86 (84) 25 6.25 ± 0.85 (100) 22 5.5 ± 1.00 (88) 16 4 ± 0.66 (64) 8 2 ± 0.47 (36.3) *N = Total COCs cultured in each group; a = COCs matured out of N; b = COCs fertilized out of N; c = putative zygotes cultured out of N; d = Cleavage rate calculated from b; e = Blastocyst rate calculated from c. No significant differences observed ( p > 0.05). 3.2.3 Embryo quality and staging Cytokine supplementation during IVC produced minor variations in embryo quality and stage distribution, but no significant differences compared with controls (Table 7 ). Table 7 Embryo quality and stage distribution (Experiment 2). Group Total blastocyst Blastocyst grade Code Blastocyst stage Code Code 1 Code 2 Code 3 Code 4 Code 6 Code 7 Code 8 CSF2 6 2 (33.3) 2 (33.3) 1 (16.7) 1 (16.7) 2 (33.3) 4 (66.7) 0 IL6 9 4 (44.5) 2 (22.3) 2 (22.3) 1 (11.1) 1 (11.1) 4 (44.5) 2 (22.3) CSF2 +IL6 7 3 (42.8) 1 (14.2) 1 (14.2) 2 (28.5) 4 (57.1) 2 (28.5) 1 (14.2) Control 8 4 (50) 2 (25) 1 (12.5) 1 (12.5) 2 (25) 4 (50) 2 (25) *Embryo grading and staging codes: Code 1 = Excellent/Good; Code 2 = Fair; Code 3 = Poor; Code 4 = Dead/Degenerating; Code 6 = Blastocyst; Code 7 = Expanded blastocyst; Code 8 = Hatched blastocyst. Code 5 was not evaluated. No significant differences observed ( p > 0.05). 4. Discussion Oocyte recovery rates following transvaginal ovum pick-up (OPU) have been reported to vary widely across studies in buffalo, ranging from 35% (10–58%) (Manik et al., 2002 ) to 68% in Murrah buffaloes (Gupta et al., 2006 ) and 42% in Indian buffaloes (Manjunatha et al., 2008). In cattle, Singh et al. ( 2025 a) documented a recovery rate of 67.4% in Sahiwal cows. In the present investigation, the recovery rate was comparatively higher at 74.9%. Such variation is influenced by donor animal condition, aspiration frequency, and refinements in OPU techniques (Konrad et al., 2017 ; Sagheer et al., 2020 ). The proportion of culturable oocytes in the present study (74.2%) is comparable to, or slightly higher than, values reported previously. Using slaughterhouse-derived ovaries, Totey et al. ( 1992 ) and Das et al. ( 1996 ) reported that 65–70% of oocytes were usable for in vitro culture. Gupta et al. ( 2006 ) reported that 59% of recovered oocytes were classified as Grades A–B and 41% as Grades C–D. Manjunatha et al. (2008) noted that 64% of recovered oocytes were suitable for IVEP (Grades A–B). A recent study in Sahiwal cattle documented a recovery rate of 67.4% with grade distribution: 15.9% Grade A, 22.3% Grade B, 39.6% Grade C, and 22.3% Grade D (Singh et al., 2025 b). The predominance of Grade C oocytes aligns with the observations of Gasparrini ( 2002 ), who highlighted the influence of follicular atresia and retrieval method on buffalo oocyte quality. These findings indicate that OPU-based recovery provides a reliable source of culturable oocytes for in vitro embryo production in buffaloes. Supplementation of IVM medium with 9-cis retinoic acid significantly improved cumulus expansion, cleavage, blastocyst yield, and embryo quality. These results agree with Gad et al. ( 2018 ), who demonstrated improved maturation rates in buffalo oocytes with retinoic acid, attributed to enhanced mitochondrial function and reduced oxidative stress. Cajuday et al. ( 2012 ) also reported that retinoic acid supplementation increased cumulus expansion and metaphase II rates in a dose-dependent manner. Evidence from other species further supports this role; Atikuzzaman et al. ( 2011 ) in pigs and Liang et al. (2012) in dogs showed that 9-cis retinoic acid promoted nuclear and cytoplasmic maturation and reduced apoptosis in cumulus–oocyte complexes. The present study confirms these positive effects in buffalo, reflected in a higher proportion of good quality embryos and advanced-stage blastocysts. Supplementation of CR1aa medium with cytokines (CSF2, IL6, or their combination) did not significantly improve cleavage, blastocyst yield, or embryo quality in buffaloes. Although some numerical variations were observed (Fig. 2 ), the effects were not statistically significant, suggesting that the concentrations or timing of exposure used may not have been optimal under the present culture conditions. Interestingly, the IL6 group performed comparably to the control. These findings are in agreement with reports in cattle, where IL-6 supplementation during group embryo culture did not significantly affect cleavage or blastocyst rates (Wooldridge & Ealy, 2019 ). However, when embryos were cultured individually, IL-6 enhanced both cleavage and blastocyst formation and nearly doubled inner cell mass (ICM) cell numbers, highlighting its role in improving embryo quality rather than overall yield (Wooldridge & Ealy, 2019 ; Sang et al., 2020 ). Further evidence shows that IL-6 activates the JAK/STAT3 pathway, increasing ICM and primitive endoderm cell numbers, thereby supporting lineage specification and post-culture survival even without changes in total blastocyst numbers (Wooldridge et al., 2021). These findings suggest that IL-6 may primarily act to enhance competence and post-transfer potential, rather than consistently boosting blastocyst production. Similarly, CSF2 supplementation did not significantly influence buffalo embryo development in this study. By contrast, bovine studies have demonstrated that CSF2 can improve blastocyst development and survival when administered during specific developmental windows (Loureiro et al., 2009 ; Whitty et al., 2025 ). Benefits reported include higher hatching rates, improved ICM numbers, and reduced apoptosis. Comparative evidence also supports the conserved action of colony-stimulating factors across species: granulocyte-colony stimulating factor (G-CSF) improved embryo quality in pigs (Cai et al., 2023 ); GM-CSF supplementation enhanced blastocyst competence in sheep (Rooke et al., 2005 ); and in humans, GM-CSF improved blastocyst development, morphology, and reduced apoptosis (Sjöblom et al., 1999 ). In the combined CSF2 + IL6 group, cleavage and blastocyst rates did not exceed those obtained with IL6 alone, indicating no additive benefit. In fact, performance was slightly lower, suggesting possible interactions or overlapping pathways. Since both cytokines converge on survival and lineage allocation mechanisms, concurrent supplementation without optimized dosage or timing may blunt individual effects. Taken together, these results indicate that while cytokine supplementation did not significantly enhance cleavage or blastocyst yield in buffaloes, evidence from cattle and other species supports the roles of CSF2 and IL6 in promoting embryo competence, cell survival, and lineage specification. Further buffalo-specific studies with refined concentrations, treatment windows, and larger datasets are necessary to clarify their potential in improving in vitro embryo production outcomes. These results indicate that 9-cis retinoic acid supplementation during IVM represents a practical approach to improve developmental competence of buffalo oocytes. In contrast, cytokine supplementation during IVC under the present conditions did not produce measurable benefit. 5. Conclusion Supplementation of in vitro maturation medium with 9-cis retinoic acid significantly improved buffalo oocyte competence, as indicated by higher cumulus expansion, cleavage, blastocyst yield, and embryo quality. In contrast, supplementation of in vitro culture medium with CSF2, IL6, or their combination did not result in significant improvements under the present conditions. These findings suggest that 9-cis retinoic acid during IVM may serve as a useful refinement to enhance buffalo IVEP efficiency, while the role of cytokines requires further investigation with larger datasets and optimized culture conditions. Declarations Ethical Statement All experimental procedures were approved by the Institutional Animal Ethics Committee (IAEC), Guru Angad Dev Veterinary and Animal Sciences University (GADVASU) (Approval No. GADVASU/2024/AIEC/74/07), at its 74th meeting on 28 June 2024, in accordance with CPCSEA guidelines. Competing Interests: The authors declare that they have no financial interests related to this work. No financial or non-financial interests that could be perceived to influence the research, within or outside the last three years, are associated with this manuscript. Author Contribution Sr.AuthorContribution1.Ekta VermaPerformed research, Analyzed data, Wrote the paper2.Prahlad SinghConceived of or designed study, Analyzed data, Contributed new methods or models, edited and Wrote the paper3.Narinder SinghPerformed research, Contributed new methods or models4.Vijay Singh MalikConceptualization of the research problem5.Shashi Kant MahajanConceptualization of the research problem6.Khushpreet SinghPerformed research Acknowledgement The authors are grateful to Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, for providing the facilities and support to carry out this research. The authors also sincerely acknowledge the Director, Livestock Farms, GADVASU, Ludhiana, for extending valuable assistance in obtaining and managing the experimental animals used in this study. Data Availability The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. 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Indian J Anim Reprod 46:30–35 Singh K, Singh N, Mavi GK, Honparkhe M, Shekhar A, Singh P (2025) The effect of L-carnitine supplementation during in vitro maturation on oocyte maturation and cleavage rates in Sahiwal cows. Indian J Anim Reprod 46:44–49 Sjöblom C, Wikland M, Robertson SA (1999) Granulocyte–macrophage colony-stimulating factor promotes human blastocyst development in vitro. Hum Reprod 14:3069–3076 Totey SM, Singh G, Taneja M, Pawshe CH, Talwar GP (1992) In vitro maturation, fertilization and development of follicular oocytes from buffalo (Bubalus bubalis). J Reprod Fertil 95:597–607 Whitty AL, Jennings SK, Lyons HE, Thompson JG, Mcpherson NO, Dunning KR, Nottle MB (2025) Addition of granulocyte-macrophage colony-stimulating factor during in vitro oocyte maturation improves cattle in vitro embryo production. Theriogenology ;117589 Wooldridge LK, Ealy AD (2019) Interleukin-6 increases inner cell mass numbers in bovine embryos. BMC Dev Biol 19:2 Wooldridge LK, Ealy AD (2021) Interleukin-6 promotes primitive endoderm development in bovine blastocysts. BMC Dev Biol 21:3 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7823204","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":536662501,"identity":"7fecad9e-cb3f-4c94-a6f4-273f8a2070e7","order_by":0,"name":"Ekta Verma","email":"","orcid":"","institution":"Guru Angad Dev Veterinary and Animal Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Ekta","middleName":"","lastName":"Verma","suffix":""},{"id":536662502,"identity":"738931c6-4e80-4056-a0c5-55280a354b53","order_by":1,"name":"Prahlad Singh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIiWNgGAWjYHACNoYHDMwghgGIkAMRBx4Q0pKApMUYrCWBFC2JDSASnxZz6cPPHiTUWDPIux/eJvHjz+H0+WGHHwJtsZPTbcCuxbIvzdwg4Vg6g+GZtDLJ3rbDuRtvpxkAtSQbmx3ArsXgDIOZRALbYQbDhhxjA94GoJbZCSAtBxK34dTC/k0i4R9QS/8bY8M/QIcZzk7/QEALj5lEYtthBnmJHMPHPGyHE+Slc/DbYtnDUyaR2JfOYyDxrPCxbFu64QbpnIIDCQa4/WLOw75N4sM3azn5/uQNB9/8sZaXn52++cOHCjs5nN6H0jwGMAUQhgE2xahaGOQb0BmjYBSMglEwCqAAAD8TYdlwaBlIAAAAAElFTkSuQmCC","orcid":"","institution":"Guru Angad Dev Veterinary and Animal Sciences University","correspondingAuthor":true,"prefix":"","firstName":"Prahlad","middleName":"","lastName":"Singh","suffix":""},{"id":536662503,"identity":"b530eb38-0727-43e3-9c65-6bc0f4ced163","order_by":2,"name":"Narinder Singh","email":"","orcid":"","institution":"Guru Angad Dev Veterinary and Animal Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Narinder","middleName":"","lastName":"Singh","suffix":""},{"id":536662504,"identity":"3965d565-860d-4f91-98a8-1add96191d42","order_by":3,"name":"Vijay Singh Malik","email":"","orcid":"","institution":"Guru Angad Dev Veterinary and Animal Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Vijay","middleName":"Singh","lastName":"Malik","suffix":""},{"id":536662505,"identity":"e5dab680-bc81-4d2f-afcb-6543318ec8c4","order_by":4,"name":"Shashi Kant Mahajan","email":"","orcid":"","institution":"Guru Angad Dev Veterinary and Animal Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Shashi","middleName":"Kant","lastName":"Mahajan","suffix":""},{"id":536662506,"identity":"b832ca5b-adeb-4d0f-9da2-5c2c2ed3e113","order_by":5,"name":"Khushpreet Singh","email":"","orcid":"","institution":"Guru Angad Dev Veterinary and Animal Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Khushpreet","middleName":"","lastName":"Singh","suffix":""}],"badges":[],"createdAt":"2025-10-10 06:23:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7823204/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7823204/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94723610,"identity":"c9935266-234c-4df5-9f96-8efd3373c604","added_by":"auto","created_at":"2025-10-30 06:04:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":57461,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCleavage, Blastocyst, and Code 1 Embryo Rates with 9-cisRA Supplementation\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7823204/v1/b9e0398d3ea8f43ae99c2e9f.png"},{"id":94723609,"identity":"0a34cd9c-dced-47f9-a13a-a416a1ee48cd","added_by":"auto","created_at":"2025-10-30 06:04:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":62920,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCleavage and Blastocyst Rates Following Cytokine Supplementation\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7823204/v1/bdcf202a4006920d5767b12a.png"},{"id":94723612,"identity":"ba278c75-275d-49da-88ff-d89d7dff61d4","added_by":"auto","created_at":"2025-10-30 06:04:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1254182,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7823204/v1/ddca6524-8e63-4054-b48b-8cf06723e748.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of 9-cis retinoic acid, and, colony-stimulating factor 2 and interleukin 6 cytokines on in vitro maturation and embryo development of OPU-derived Murrah buffalo (Bubalus bubalis) oocytes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBuffaloes (Bubalus bubalis) are indispensable to the agricultural economy of India and other Asian regions, serving as a primary source of high-quality milk, meat, and draught power. However, buffalo productivity is severely constrained by inherent reproductive challenges, including seasonality, silent estrus, and limited ovarian reserve. These factors culminate in a notably lower efficiency of in vitro embryo production (IVEP) compared to cattle, creating a significant bottleneck for genetic improvement programs (Gasparrini, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Baruselli et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Consequently, refining IVEP protocols by targeting key biological pathways is paramount to enhancing oocyte developmental competence and overall reproductive output in this species.\u003c/p\u003e\u003cp\u003eRetinoids, the biologically active derivatives of vitamin A, are critical signalling molecules in reproduction. They mediate their effects primarily through retinoic acid receptors (RARs) and retinoid X receptors (RXRs), regulating fundamental processes of follicular development, oocyte nuclear and cytoplasmic maturation, and, embryonic morphogenesis (Ross et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). A key isomer of retinoid, 9-cis retinol, has been shown to enhance oocyte competence by mitigating oxidative stress and improving epigenetic regulation. Studies in cattle report that retinol supplementation during in vitro maturation (IVM) significantly improves cleavage rates and blastocyst yield (Gomez et al., 2003; Gad et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The optimal application and efficacy of specific retinoids like 9-cis retinol in buffalo IVEP systems warrants a detailed investigation.\u003c/p\u003e\u003cp\u003ePost-fertilization the embryo culture conditions are critical in addition to the oocyte maturation factors. Cytokines, such as granulocyte-macrophage colony-stimulating factor (CSF2) and interleukin 6 (IL6), are vital embryokines secreted by the reproductive to promote trophoblast cell proliferation, enhance metabolic activity, suppress apoptosis, and improve embryo viability and quality (Dobbs \u0026amp; Hansen, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ealy et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). CSF2 and IL6 benefits are established in mouse and cattle models, Role of CSF2 and IL6 in supporting the development of buffalo embryos, known to be highly sensitive to in vitro conditions, remains poorly defined and represents a significant knowledge gap.\u003c/p\u003e\u003cp\u003eBased on the established roles of retinoids in oocyte maturation and embryokines in post-fertilization development, it is hypothesized that the sequential supplementation of 9-cis retinol during IVM followed by CSF2 and IL6 during in vitro culture (IVC) would act synergistically to enhance the developmental competence of buffalo oocytes and the quality of resulting embryos. In, Experiment 1 aim was to determine the individual effect of 9-cis retinol supplementation during IVM on oocyte maturation and subsequent embryo development. In Experiment 2 we investigated the effects of CSF2 and IL6, individually and in combination, during IVC, on embryos derived from both retinol-pretreated and standard IVM oocytes, to evaluate potential additive or synergistic benefits.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Selection of Animals\u003c/h2\u003e\u003cp\u003eHealthy pluriparous Murrah buffaloes (Experiment 1: n\u0026thinsp;=\u0026thinsp;5, Experiment 2: n\u0026thinsp;=\u0026thinsp;4)4\u0026ndash;6 Years of age, maintained at the Dairy Farm of Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, India was incorporated in the study. All experimental procedures were approved by the Institutional Animal Ethics Committee (IAEC), GADVASU (Approval No. GADVASU/2024/AIEC/74/07), at its 74th meeting on 28 June 2024, in accordance with CPCSEA guidelines. Animals were housed under loose housing systems with free access to feed and water.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Ovum pick-up and oocyte classification\u003c/h2\u003e\u003cp\u003eSix ovum pick-up (OPU) Sessions per buffalo were conducted. Follicular aspiration was performed under transvaginal epidural anaesthesia, using a 7.5 MHz ultrasound probe equipped with a needle guide and connected to a vacuum pump (70 mmHg). Follicular fluid was collected into pre-warmed tubes and transported to the laboratory. Cumulus\u0026ndash;oocyte complexes (COCs) were recovered under a stereo-microscope and classified as Grade A, B, C (culturable) or D (non-culturable) based on cumulus investment and ooplasm appearance. Only culturable oocytes were used for in vitro embryo production (IVEP).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Experimental design\u003c/h2\u003e\u003cp\u003eTwo independent experiments were conducted to evaluate the effects of different supplements on oocyte maturation and subsequent embryonic development:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eExperiment 1\u003c/b\u003e: To determine the influence of 9-cis retinoic acid (9-cisRA) on oocyte development competence.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eExperiment 2\u003c/b\u003e: To assess the impact of cytokine supplementation [colony-stimulating factor 2 (CSF2), interleukin-6 (IL6), or CSF2\u0026thinsp;+\u0026thinsp;IL6] during in vitro culture (IVC) of embryos.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 In vitro maturation (IVM)\u003c/h2\u003e\u003cp\u003eOPU derived buffalo were matured in TCM-199 supplemented with 10% fetal bovine serum (FBS), 10 IU/ml luteinizing hormone, 0.5 \u0026micro;g/ml follicle-stimulating hormone, 1 \u0026micro;g/ml estradiol-17β, 50 \u0026micro;g/ml gentamicin, and 5 nM 9-cisRA. Control group had no 9-cisRA in the culture media. Cultures were maintained in 60 \u0026micro;l drops under mineral oil at 38.5\u0026deg;C in 6% CO₂ for 22\u0026ndash;24 h. In vitro maturation was assessed by cumulus expansion of COCs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 In vitro fertilization (IVF)\u003c/h2\u003e\u003cp\u003eFrozen\u0026ndash;thawed semen from proven Murrah bulls was processed using a Percoll gradient. Matured oocytes were co-incubated with capacitated spermatozoa (2 \u0026times; 10⁶/ml) for 18 h in IVF medium (Vitrogen) supplemented with penicillamine, hypotaurine, epinephrine, and heparin.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 In vitro culture (IVC)\u003c/h2\u003e\u003cp\u003eIn Experiment 1, presumptive zygotes derived from oocytes matured with or without 9-cis retinoic acid (9-cisRA) were cultured in commercially available IVC medium (Vitrogen, India) under mineral oil at 38.5\u0026deg;C, 6% CO₂, 5% O₂, and 89% N₂. In Experiment 2, presumptive zygotes were cultured in CR1aa medium supplemented with essential amino acids and 3 mg/ml BSA. In treatment groups, IVC drops were further supplemented with CSF2 (10 ng/ml), IL6 (10 ng/ml), or their combination. Control cultures contained CR1aa medium without cytokines. Embryos were monitored for cleavage (day 3) and blastocyst formation (days 6).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Embryo evaluation\u003c/h2\u003e\u003cp\u003eEmbryo quality was assessed according to the International Embryo Technology Society (IETS) manual, classifying blastocysts as excellent/good (Code 1), fair (Code 2), poor (Code 3), or dead/degenerating (Code 4). Developmental stages were recorded as blastocyst (Code 6), expanded blastocyst (Code 7), or hatched blastocyst (Code 8).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e\u003cp\u003eData were analyzed using SPSS software (version 20, IBM Corp., Armonk, NY). Values were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Differences between groups were tested using independent-samples \u003cem\u003et\u003c/em\u003e-test or one-way ANOVA, followed by post hoc comparisons. A probability level of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Experiment 1: 9-cisRA trial\u003c/h2\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e3.1.1 Follicular aspiration and oocyte recovery\u003c/h2\u003e\u003cp\u003eA total of 279 follicles were aspirated across six OPU sessions in five buffaloes, yielding 209 oocytes with an overall recovery rate of 74.9%. Of these, 155 (74.2%) were culturable, Grades A, B, C (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFollicle aspiration and oocyte recovery\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFollicles aspirated\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e279\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCOCs retrieved\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e209\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOocyte recovery rate (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e74.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCulturable oocytes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e155\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003e\u0026bull; No. of buffaloes\u0026thinsp;=\u0026thinsp;5\u003c/p\u003e\u003cp\u003e\u0026bull; Values presented as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM are calculated per OPU session per animal\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e3.1.2 COCs expansion of OPU derived buffalo oocytes\u003c/h2\u003e\u003cp\u003eThe proportion of COCs showing complete expansion was significantly higher in the 9-cisRA group than control (84.6% vs. 61.0%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCumulus expansion [n, Mean SEM, (%)] of OPU derived buffalo oocytes matured in TCM199 supplemented with 9-cisRA\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eIVM media\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eReplicates (no.)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCOCs\u003c/p\u003e\u003cp\u003ecultured\u003c/p\u003e\u003cp\u003e(n)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eCOC Expansion\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eFull\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eA (%)\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003ePartial\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eB (%)\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eNo\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eC (%)\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTCM199 +\u003c/p\u003e\u003cp\u003e9 cis retinoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e66\u003c/p\u003e\u003cp\u003e11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003c/p\u003e\u003cp\u003e(84.6) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5\u003c/p\u003e\u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e\u003cp\u003e(6.41) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7\u003c/p\u003e\u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003cp\u003e(8.97) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTCM199\u003c/p\u003e\u003cp\u003e(control)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e47\u003c/p\u003e\u003cp\u003e7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e\u003cp\u003e(61.03) \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e17\u003c/p\u003e\u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e\u003cp\u003e(22.07) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e12\u003c/p\u003e\u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003cp\u003e(15.58) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003e\u0026bull; Complete expansion: Number of COCs A out of n\u003c/p\u003e\u003cp\u003e\u0026bull; Partial expansion: Number of COCs B out of n\u003c/p\u003e\u003cp\u003e\u0026bull; No expansion: Number of COCs C out of n\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eValues in a column with different superscripts differ significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e3.1.3 Cleavage and blastocyst development\u003c/h2\u003e\u003cp\u003eSupplementation with 9-cisRA during IVM significantly improved cleavage (65.7% vs. 52.1%) and blastocyst yield (31.4% vs. 19.1%) compared with controls (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDevelopmental competence parameters (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM) of buffalo oocytes matured with 9-cis retinoic acid (9-cisRA)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReplicates\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCOCs Matured\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOocytes\u003c/p\u003e\u003cp\u003eFertilized\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePutative zygotes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCleavage Rate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eBlastocyst Rate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(no.)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c7\" namest=\"c4\"\u003e\u003cp\u003en, Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ee\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e73\u003c/p\u003e\u003cp\u003e12.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003c/p\u003e\u003cp\u003e(93.58)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e70\u003c/p\u003e\u003cp\u003e11.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/p\u003e\u003cp\u003e(89.74)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/p\u003e\u003cp\u003e(65.75)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003cp\u003e(31.4)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e71\u003c/p\u003e\u003cp\u003e11.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e\u003cp\u003e(92.20)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e68\u003c/p\u003e\u003cp\u003e11.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e\u003cp\u003e(88.31)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e37\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e6.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e\u003cp\u003e(52.11)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e2.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003cp\u003e(19.1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003ea\u0026thinsp;=\u0026thinsp;Total COCs matured in each group\u003c/p\u003e\u003cp\u003eb\u0026thinsp;=\u0026thinsp;COCs fertilized out of a (%=n/a*100)\u003c/p\u003e\u003cp\u003ec\u0026thinsp;=\u0026thinsp;Putative zygotes out of a (%=n/a*100)\u003c/p\u003e\u003cp\u003ed\u0026thinsp;=\u0026thinsp;Cleavage Rate out of b (%= n/b*100)\u003c/p\u003e\u003cp\u003ee\u0026thinsp;=\u0026thinsp;Blastocyst rate out of c (n/c*100)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eValues across a row with different superscripts differ significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e3.1.4 Embryo quality and staging\u003c/h2\u003e\u003cp\u003eThe proportion of excellent/good embryos (Code 1) was significantly higher in the 9-cisRA group than in control (54.4% vs. 30.0%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). More embryos developed to expanded and hatched blastocyst stages with 9-cisRA treatment (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEmbryo quality and stage distribution [n, Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, (%)] (Experiment 1).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCode 1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCode 2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCode 3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCode 4\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eStage 6\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eStage 7\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eStage 8\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9-cisRA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12\u003c/p\u003e\u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003cp\u003e(54.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003cp\u003e(36.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003cp\u003e(9.09)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4\u003c/p\u003e\u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003cp\u003e(18.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e12\u003c/p\u003e\u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003cp\u003e(54.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6\u003c/p\u003e\u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e\u003cp\u003e(27.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003cp\u003e(30)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003cp\u003e1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003cp\u003e(46.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003cp\u003e(7.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003cp\u003e(15.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4\u003c/p\u003e\u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003cp\u003e(30.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7\u003c/p\u003e\u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003cp\u003e(53.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003cp\u003e(15.4)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e\u003cp\u003e*Embryo grading and staging codes: Code 1\u0026thinsp;=\u0026thinsp;Excellent/Good; Code 2\u0026thinsp;=\u0026thinsp;Fair; Code 3\u0026thinsp;=\u0026thinsp;Poor; Code 4\u0026thinsp;=\u0026thinsp;Dead/Degenerating; Code 6\u0026thinsp;=\u0026thinsp;Blastocyst; Code 7\u0026thinsp;=\u0026thinsp;Expanded blastocyst; Code 8\u0026thinsp;=\u0026thinsp;Hatched blastocyst. Code 5 was not evaluated.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, supplementation of IVM medium with 9-cisRA significantly increased cleavage, blastocyst formation, and the proportion of Code 1 embryos compared with control.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Experiment 2: Cytokine trial\u003c/h2\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1 Follicular aspiration and oocyte recovery\u003c/h2\u003e\u003cp\u003eIn four buffaloes, 157 follicles were aspirated across 4 OPU sessions, yielding 127 oocytes with a recovery rate of 80.8%. Among them, 101 were culturable (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFollicle aspiration and oocyte recovery (Experiment 2: Cytokines).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFollicles aspirated\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e157\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCOCs retrieved\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e127\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOocyte recovery rate (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e80.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCulturable oocytes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e101\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003e\u0026bull; No. of buffaloes\u0026thinsp;=\u0026thinsp;4\u003c/p\u003e\u003cp\u003e\u0026bull; Total OPU sessions/buffalo\u0026thinsp;=\u0026thinsp;4\u003c/p\u003e\u003cp\u003e\u0026bull; Values presented as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM are calculated per OPU session per animal\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e3.2.2 Oocyte developmental competence\u003c/h2\u003e\u003cp\u003eIn CR1aa medium, supplementation with CSF2, IL6, or CSF2\u0026thinsp;+\u0026thinsp;IL6 produced numerical but statistically non-significant differences in cleavage and blastocyst yield compared with controls (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffect of cytokine supplementation during IVC (Experiment 2).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTotal COCs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMaturation Rate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCOCs\u003c/p\u003e\u003cp\u003eFertilized\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePutative zygotes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCleavage Rate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eBlastocyst Rate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e\u003cp\u003e\u003cb\u003en, Mean\u003c/b\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;\u003cb\u003eSEM, (%)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eN\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003ea\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eb\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003ec\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003ed\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ee\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCSF2\u003c/p\u003e\u003cp\u003e(10ng/ml)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e21\u003c/p\u003e\u003cp\u003e5.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e\u003cp\u003e(84)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e23\u003c/p\u003e\u003cp\u003e5.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e\u003cp\u003e(92)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e22\u003c/p\u003e\u003cp\u003e5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e\u003cp\u003e(88)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12\u003c/p\u003e\u003cp\u003e3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e\u003cp\u003e(52.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6\u003c/p\u003e\u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003cp\u003e(27.2)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIL6\u003c/p\u003e\u003cp\u003e(100ng/ml)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003cp\u003e5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e\u003cp\u003e(80)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24\u003c/p\u003e\u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e\u003cp\u003e(96)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e22\u003c/p\u003e\u003cp\u003e5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003c/p\u003e\u003cp\u003e(88)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14\u003c/p\u003e\u003cp\u003e3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e\u003cp\u003e(58.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8\u003c/p\u003e\u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e\u003cp\u003e(36.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCSF2(10ng/ml)\u0026thinsp;+\u0026thinsp;IL6(100ng/ml)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19\u003c/p\u003e\u003cp\u003e4.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003c/p\u003e\u003cp\u003e(76)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24\u003c/p\u003e\u003cp\u003e6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003c/p\u003e\u003cp\u003e(96)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e21\u003c/p\u003e\u003cp\u003e5.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003c/p\u003e\u003cp\u003e(84)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13\u003c/p\u003e\u003cp\u003e3.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003c/p\u003e\u003cp\u003e(54.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7\u003c/p\u003e\u003cp\u003e1.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e\u003cp\u003e(33.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e21\u003c/p\u003e\u003cp\u003e5.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003c/p\u003e\u003cp\u003e(84)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e25\u003c/p\u003e\u003cp\u003e6.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003c/p\u003e\u003cp\u003e(100)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e22\u003c/p\u003e\u003cp\u003e5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e\u003cp\u003e(88)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e16\u003c/p\u003e\u003cp\u003e4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e\u003cp\u003e(64)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8\u003c/p\u003e\u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e\u003cp\u003e(36.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003e*N\u0026thinsp;=\u0026thinsp;Total COCs cultured in each group; a\u0026thinsp;=\u0026thinsp;COCs matured out of N; b\u0026thinsp;=\u0026thinsp;COCs fertilized out of N; c\u0026thinsp;=\u0026thinsp;putative zygotes cultured out of N; d\u0026thinsp;=\u0026thinsp;Cleavage rate calculated from b; e\u0026thinsp;=\u0026thinsp;Blastocyst rate calculated from c.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eNo significant differences observed (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e3.2.3 Embryo quality and staging\u003c/h2\u003e\u003cp\u003eCytokine supplementation during IVC produced minor variations in embryo quality and stage distribution, but no significant differences compared with controls (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEmbryo quality and stage distribution (Experiment 2).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003cp\u003eblastocyst\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e\u003cp\u003eBlastocyst grade Code\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e\u003cp\u003eBlastocyst stage Code\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eCode 1\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eCode 2\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eCode 3\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eCode 4\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eCode 6\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003eCode 7\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eCode 8\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCSF2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003cp\u003e(33.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003cp\u003e(33.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003cp\u003e(16.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003cp\u003e(16.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003cp\u003e(33.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4\u003c/p\u003e\u003cp\u003e(66.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIL6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003cp\u003e(44.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003cp\u003e(22.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003cp\u003e(22.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003cp\u003e(11.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003cp\u003e(11.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4\u003c/p\u003e\u003cp\u003e(44.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003cp\u003e(22.3)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCSF2\u003c/p\u003e\u003cp\u003e+IL6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003cp\u003e(42.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003cp\u003e(14.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003cp\u003e(14.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003cp\u003e(28.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4\u003c/p\u003e\u003cp\u003e(57.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003cp\u003e(28.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003cp\u003e(14.2)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003cp\u003e(50)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003cp\u003e(25)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003cp\u003e(12.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003cp\u003e(12.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003cp\u003e(25)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4\u003c/p\u003e\u003cp\u003e(50)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003cp\u003e(25)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e\u003cp\u003e*Embryo grading and staging codes: Code 1\u0026thinsp;=\u0026thinsp;Excellent/Good; Code 2\u0026thinsp;=\u0026thinsp;Fair; Code 3\u0026thinsp;=\u0026thinsp;Poor; Code 4\u0026thinsp;=\u0026thinsp;Dead/Degenerating; Code 6\u0026thinsp;=\u0026thinsp;Blastocyst; Code 7\u0026thinsp;=\u0026thinsp;Expanded blastocyst; Code 8\u0026thinsp;=\u0026thinsp;Hatched blastocyst. Code 5 was not evaluated.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eNo significant differences observed (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOocyte recovery rates following transvaginal ovum pick-up (OPU) have been reported to vary widely across studies in buffalo, ranging from 35% (10\u0026ndash;58%) (Manik et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) to 68% in Murrah buffaloes (Gupta et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and 42% in Indian buffaloes (Manjunatha et al., 2008). In cattle, Singh et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003ea) documented a recovery rate of 67.4% in Sahiwal cows. In the present investigation, the recovery rate was comparatively higher at 74.9%. Such variation is influenced by donor animal condition, aspiration frequency, and refinements in OPU techniques (Konrad et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sagheer et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe proportion of culturable oocytes in the present study (74.2%) is comparable to, or slightly higher than, values reported previously. Using slaughterhouse-derived ovaries, Totey et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) and Das et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) reported that 65\u0026ndash;70% of oocytes were usable for in vitro culture. Gupta et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) reported that 59% of recovered oocytes were classified as Grades A\u0026ndash;B and 41% as Grades C\u0026ndash;D. Manjunatha et al. (2008) noted that 64% of recovered oocytes were suitable for IVEP (Grades A\u0026ndash;B). A recent study in Sahiwal cattle documented a recovery rate of 67.4% with grade distribution: 15.9% Grade A, 22.3% Grade B, 39.6% Grade C, and 22.3% Grade D (Singh et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003eb). The predominance of Grade C oocytes aligns with the observations of Gasparrini (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), who highlighted the influence of follicular atresia and retrieval method on buffalo oocyte quality. These findings indicate that OPU-based recovery provides a reliable source of culturable oocytes for \u003cem\u003ein vitro\u003c/em\u003e embryo production in buffaloes.\u003c/p\u003e\u003cp\u003eSupplementation of IVM medium with 9-cis retinoic acid significantly improved cumulus expansion, cleavage, blastocyst yield, and embryo quality. These results agree with Gad et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), who demonstrated improved maturation rates in buffalo oocytes with retinoic acid, attributed to enhanced mitochondrial function and reduced oxidative stress. Cajuday et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) also reported that retinoic acid supplementation increased cumulus expansion and metaphase II rates in a dose-dependent manner. Evidence from other species further supports this role; Atikuzzaman et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) in pigs and Liang et al. (2012) in dogs showed that 9-cis retinoic acid promoted nuclear and cytoplasmic maturation and reduced apoptosis in cumulus\u0026ndash;oocyte complexes. The present study confirms these positive effects in buffalo, reflected in a higher proportion of good quality embryos and advanced-stage blastocysts.\u003c/p\u003e\u003cp\u003eSupplementation of CR1aa medium with cytokines (CSF2, IL6, or their combination) did not significantly improve cleavage, blastocyst yield, or embryo quality in buffaloes. Although some numerical variations were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the effects were not statistically significant, suggesting that the concentrations or timing of exposure used may not have been optimal under the present culture conditions.\u003c/p\u003e\u003cp\u003eInterestingly, the IL6 group performed comparably to the control. These findings are in agreement with reports in cattle, where IL-6 supplementation during group embryo culture did not significantly affect cleavage or blastocyst rates (Wooldridge \u0026amp; Ealy, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, when embryos were cultured individually, IL-6 enhanced both cleavage and blastocyst formation and nearly doubled inner cell mass (ICM) cell numbers, highlighting its role in improving embryo quality rather than overall yield (Wooldridge \u0026amp; Ealy, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sang et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Further evidence shows that IL-6 activates the JAK/STAT3 pathway, increasing ICM and primitive endoderm cell numbers, thereby supporting lineage specification and post-culture survival even without changes in total blastocyst numbers (Wooldridge et al., 2021). These findings suggest that IL-6 may primarily act to enhance competence and post-transfer potential, rather than consistently boosting blastocyst production.\u003c/p\u003e\u003cp\u003eSimilarly, CSF2 supplementation did not significantly influence buffalo embryo development in this study. By contrast, bovine studies have demonstrated that CSF2 can improve blastocyst development and survival when administered during specific developmental windows (Loureiro et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Whitty et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Benefits reported include higher hatching rates, improved ICM numbers, and reduced apoptosis. Comparative evidence also supports the conserved action of colony-stimulating factors across species: granulocyte-colony stimulating factor (G-CSF) improved embryo quality in pigs (Cai et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e); GM-CSF supplementation enhanced blastocyst competence in sheep (Rooke et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2005\u003c/span\u003e); and in humans, GM-CSF improved blastocyst development, morphology, and reduced apoptosis (Sj\u0026ouml;blom et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the combined CSF2\u0026thinsp;+\u0026thinsp;IL6 group, cleavage and blastocyst rates did not exceed those obtained with IL6 alone, indicating no additive benefit. In fact, performance was slightly lower, suggesting possible interactions or overlapping pathways. Since both cytokines converge on survival and lineage allocation mechanisms, concurrent supplementation without optimized dosage or timing may blunt individual effects.\u003c/p\u003e\u003cp\u003eTaken together, these results indicate that while cytokine supplementation did not significantly enhance cleavage or blastocyst yield in buffaloes, evidence from cattle and other species supports the roles of CSF2 and IL6 in promoting embryo competence, cell survival, and lineage specification. Further buffalo-specific studies with refined concentrations, treatment windows, and larger datasets are necessary to clarify their potential in improving in vitro embryo production outcomes.\u003c/p\u003e\u003cp\u003eThese results indicate that 9-cis retinoic acid supplementation during IVM represents a practical approach to improve developmental competence of buffalo oocytes. In contrast, cytokine supplementation during IVC under the present conditions did not produce measurable benefit.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eSupplementation of in vitro maturation medium with 9-cis retinoic acid significantly improved buffalo oocyte competence, as indicated by higher cumulus expansion, cleavage, blastocyst yield, and embryo quality. In contrast, supplementation of in vitro culture medium with CSF2, IL6, or their combination did not result in significant improvements under the present conditions. These findings suggest that 9-cis retinoic acid during IVM may serve as a useful refinement to enhance buffalo IVEP efficiency, while the role of cytokines requires further investigation with larger datasets and optimized culture conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthical Statement\u003c/h2\u003e\u003cp\u003eAll experimental procedures were approved by the Institutional Animal Ethics Committee (IAEC), Guru Angad Dev Veterinary and Animal Sciences University (GADVASU) (Approval No. GADVASU/2024/AIEC/74/07), at its 74th meeting on 28 June 2024, in accordance with CPCSEA guidelines.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting Interests:\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no financial interests related to this work. No financial or non-financial interests that could be perceived to influence the research, within or outside the last three years, are associated with this manuscript.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSr.AuthorContribution1.Ekta VermaPerformed research, Analyzed data, Wrote the paper2.Prahlad SinghConceived of or designed study, Analyzed data, Contributed new methods or models, edited and Wrote the paper3.Narinder SinghPerformed research, Contributed new methods or models4.Vijay Singh MalikConceptualization of the research problem5.Shashi Kant MahajanConceptualization of the research problem6.Khushpreet SinghPerformed research\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors are grateful to Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, for providing the facilities and support to carry out this research. The authors also sincerely acknowledge the Director, Livestock Farms, GADVASU, Ludhiana, for extending valuable assistance in obtaining and managing the experimental animals used in this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. All relevant data supporting the findings of this study will be shared in accordance with Springer Nature\u0026rsquo;s data sharing policies and institutional requirements.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAtikuzzaman M, Koo OJ, Kang JT, Kwon DK, Park SJ, Kim SJ, G\u0026oacute;mez MNL, Oh HJ, Hong SG, Jang G, Lee BC (2011) The 9-cis retinoic acid signaling pathway and its regulation of prostaglandin-endoperoxide synthase 2 during in vitro maturation of pig cumulus cell\u0026ndash;oocyte complexes and effects on parthenogenetic embryo production. Biol Reprod 84:1272\u0026ndash;1281\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaruselli PS, Soares JG, Bayeux BM, Silva JC, Mingoti RD, Carvalho NA (2018) Assisted reproductive technologies (ART) in water buffaloes. Anim Reprod 15(Suppl 1):971\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCai R, Xu X, Zhang L, Huang Y (2023) Granulocyte colony-stimulating factor improves in vitro maturation and quality of porcine oocytes derived from poor-quality follicles. Animals 13:273\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCajuday LA, Herrera AA, Duran DH (2012) Effect of retinoic acid on the development of water buffalo embryos in vitro. Philipp J Vet Anim Sci 38:2\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDas GK, Jain GC, Solanki VS, Tripathi VN (1996) Efficacy of various collection methods for oocyte retrieval in buffalo. Theriogenology 46:1403\u0026ndash;1411\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDobbs KB, Hansen PJ (2018) Regulation of bovine preimplantation embryo development by colony-stimulating factor 2: Review of mechanisms and implications for improving pregnancy success. Reprod Fertil Dev 30:147\u0026ndash;156\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEaly AD, Speckhart SL, Wooldridge LK (2021) Cytokines that serve as embryokines in cattle. Animals 11:2313\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGad A, Hamed SA, Khalifa M, Amin A, El-Sayed A, Swiefy SA, El-Assal S (2018) Retinoic acid improves maturation rate and upregulates the expression of antioxidant-related genes in in vitro matured buffalo (Bubalus bubalis) oocytes. Int J Vet Sci Med 6:279\u0026ndash;285\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGasparrini B (2002) In vitro embryo production in buffalo species: State of the art. Theriogenology 57:237\u0026ndash;256\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eG\u0026oacute;mez E, Royo LJ, Duque P, Carneiro G, Hidalgo C, Goyache F, D\u0026iacute;ez C (2003) 9-cis-retinoic acid during in vitro maturation improves development of the bovine oocyte and increases midkine but not IGF-I expression in cumulus\u0026ndash;granulosa cells. Mol Reprod Dev 66:247\u0026ndash;255\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGupta V, Manik RS, Chauhan MS, Singla SK, Akshey YS, Palta P (2006) Repeated ultrasound-guided transvaginal oocyte retrieval from cyclic Murrah buffaloes (Bubalus bubalis): Oocyte recovery and quality. Anim Reprod Sci 91:89\u0026ndash;96\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKonrad J, Cl\u0026eacute;rico G, Garrido MJ, Taminelli G, Yuponi M, Yuponi R, Sansinena M (2017) Ovum pick-up interval in buffalo (Bubalus bubalis) managed under wetland conditions in Argentina: Effect on follicular population, oocyte recovery, and in vitro embryo development. Anim Reprod Sci 183:39\u0026ndash;45\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLoureiro B, Bonilla L, Block J, Fear JM, Bonilla AQ, Hansen PJ (2009) Colony-stimulating factor 2 (CSF-2) improves development and posttransfer survival of bovine embryos produced in vitro. Endocrinology 150:5046\u0026ndash;5054\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eManik RS, Chauhan MS, Singla SK, Palta P (2002) Transvaginal ultrasound-guided aspiration of follicles from Indian buffaloes (Bubalus bubalis) with reproductive problems. Anim Reprod Sci 69:223\u0026ndash;229\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eManjunatha BM, Ravindra JP, Gupta PSP, Devaraj M, Nandi S (2009) Effect of breeding season on in vivo oocyte recovery and embryo production in non-descriptive Indian river buffaloes (Bubalus bubalis). Anim Reprod Sci 111:376\u0026ndash;383\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRooke J, Ewen M, McEvoy T, Entrican G, Ashworth C (2005) Effect of inclusion of serum and granulocyte\u0026ndash;macrophage colony stimulating factor on secretion of interferon-τ during the in vitro culture of ovine embryos. Reprod Fertil Dev 17:513\u0026ndash;521\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoss SA, McCaffery PJ, Drager UC, De Luca LM (2000) Retinoids in embryonal development. Physiol Rev 80:1021\u0026ndash;1054\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSagheer M, Ullah F, Arshad U, Saleem M, Nawaz M, Sarwar Z, Ahmad N (2020) Effect of photoperiodicity and methods of follicular wave emergence on follicle turnover, recovery and quality of oocytes, and early in-vitro developmental competence of embryos using ovum pick-up in Nili-Ravi buffaloes: Preliminary evidence. Theriogenology 157:508\u0026ndash;516\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSang L, Ortiz W, Xiao Y, Estrada-Cortes E, Jannaman EA, Hansen PJ (2020) Actions of putative embryokines on development of the preimplantation bovine embryo to the blastocyst stage. J Dairy Sci 103:11930\u0026ndash;11944\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSeekford ZK, Wooldridge LK, Ealy AD (2021) Interleukin-6 supplementation improves post-transfer embryonic and fetal development of in vitro-produced bovine embryos. 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Hum Reprod 14:3069\u0026ndash;3076\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTotey SM, Singh G, Taneja M, Pawshe CH, Talwar GP (1992) In vitro maturation, fertilization and development of follicular oocytes from buffalo (Bubalus bubalis). J Reprod Fertil 95:597\u0026ndash;607\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWhitty AL, Jennings SK, Lyons HE, Thompson JG, Mcpherson NO, Dunning KR, Nottle MB (2025) Addition of granulocyte-macrophage colony-stimulating factor during in vitro oocyte maturation improves cattle in vitro embryo production. Theriogenology ;117589\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWooldridge LK, Ealy AD (2019) Interleukin-6 increases inner cell mass numbers in bovine embryos. BMC Dev Biol 19:2\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWooldridge LK, Ealy AD (2021) Interleukin-6 promotes primitive endoderm development in bovine blastocysts. BMC Dev Biol 21:3\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Buffalo, Ovum pick-up (OPU), 9-cis retinoic acid, CSF2, IL6, in vitro embryo culture","lastPublishedDoi":"10.21203/rs.3.rs-7823204/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7823204/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eReproductive inadequacy in buffaloes remains a major constraint to the efficacy of assisted reproductive technologies (ART), particularly in vitro embryo production (IVEP). This study investigated the effects of 9-cis retinoic acid (9-cisRA) during in vitro maturation (IVM) and cytokines (CSF2 and IL6) during in vitro culture (IVC) on enhancing the developmental competence of ovum pick-up (OPU) derived buffalo oocytes. In Experiment 1, oocytes were subjected to IVM supplemented with or without 9-cisRA. In Experiment 2, presumptive zygotes were cultured with CSF2, IL6, or their combination. Oocyte developmental competence was comprehensively evaluated based on cumulus cell expansion, cleavage rate, blastocyst yield, embryo quality grade, and developmental stage distribution. Data from Experiment 1 were analyzed using an independent \u003cem\u003et\u003c/em\u003e-test, while Experiment 2 data were subjected to one-way ANOVA followed by Tukey\u0026rsquo;s post-hoc test. The results demonstrated that 9-cisRA supplementation during IVM significantly enhanced (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) cumulus expansion (84.6% vs. 61.0%), cleavage rate (65.7% vs. 52.1%), blastocyst yield (31.4% vs. 19.1%), and the proportion of excellent/good quality embryos (54.4% vs. 30.0%). Furthermore, 9-cisRA-treated groups yielded a higher proportion of advanced-stage blastocysts, including expanded (54.5%) and hatched (27.3%) blastocysts. In contrast, cytokine supplementation yielded only modest and non-significant improvements across all measured parameters. These findings identify 9-cisRA supplementation during IVM as a highly effective strategy to improve buffalo IVEP outcomes, while the application of CSF2 and IL6 during embryo culture requires further dose and timing optimization to ascertain their potential benefits.\u003c/p\u003e","manuscriptTitle":"Effect of 9-cis retinoic acid, and, colony-stimulating factor 2 and interleukin 6 cytokines on in vitro maturation and embryo development of OPU-derived Murrah buffalo (Bubalus bubalis) oocytes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-30 06:04:16","doi":"10.21203/rs.3.rs-7823204/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"24e960ff-79f2-4bcb-ad51-b000c1da7a5f","owner":[],"postedDate":"October 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-30T06:04:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-30 06:04:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7823204","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7823204","identity":"rs-7823204","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}