Buffalo Cumulus Oocyte Complexes Research Articles

LPS-Induced Mitochondrial Dysfunction Reduces Oocyte Maturation and Developmental Competence of Buffalo Embryos via ROS Mediated TLR4 Signalling.

Lipopolysaccharide (LPS) from gram-negative bacteria has reportedly been associated with infectious diseases like metritis, which has a substantial adverse effect on animal reproductive performance and causes serious financial losses for the dairy sector. The current work aimed to establish the impact of LPS on in vitro oocyte maturation and subsequent in vitro developmental competence of oocytes, as well as to investigate the explanatory molecular mechanism underlying this effect. Buffalo cumulus-oocyte complexes (COCs) were challenged with 0, 5, 10 and 20 µg/mL LPS during IVM followed by IVF and IVC. Cytoplasmic and nuclear maturation, cleavage and blastocyst rate, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP, ΔΨm) and transcript abundance of genes related to inflammation, antioxidation and apoptosis were evaluated. The maturation and subsequent embryonic development competency were found to be significantly (p≤0.05) reduced with the addition of 10 and 20µg/mL LPS to IVM media. ROS production accompanied by a decreased ΔΨm was recorded in LPS-treated oocytes in comparison to the control group (p≤0.05). Our results were further supported by the transcriptional expression of proinflammatory (TLR4, CD14 and RPS27A) and apoptotic gene (Caspase 3) which were found to be significantly increased while antioxidant genes (SOD2 and GPX1) were decreased significantly in matured oocytes and blastocyst after LPS exposure. The deleterious effects of LPS are mediated through ROS generation, which triggers inflammatory processes via the TLR4 pathway and impairs oocyte maturation and subsequent embryonic development.

Effects of MEK1/2 inhibitor U0126 on FGF10-enhanced buffalo oocyte maturation in vitro

Fibroblast growth factor 10 (FGF10) plays critical roles in oocyte maturation and embryonic development; however, the specific pathway by which FGF10 promotes in vitro maturation of buffalo oocytes remains elusive. The present study was aimed at investigating the mechanism underlying effects of the FGF10-mediated extracellular regulated protein kinases (ERK) pathway on oocyte maturation and embryonic development in vitro. MEK1/2 (mitogen-activated protein kinase kinase) inhibitor U0126, alone or in combination with FGF10, was added to the maturation culture medium during maturation of the cumulus oocyte complex. Morphological observations, orcein staining, apoptosis detection, and quantitative real-time PCR were performed to evaluate oocyte maturation, embryonic development, and gene expression. U0126 affected oocyte maturation and embryonic development in vitro by substantially reducing the nuclear maturation of oocytes and expansion of the cumulus while increasing the apoptosis of cumulus cells. However, it did not have a considerable effect on glucose metabolism. These findings suggest that blocking the MEK/ERK pathway is detrimental to the maturation and embryonic development potential of buffalo oocytes. Overall, FGF10 may regulate the nuclear maturation of oocytes and cumulus cell expansion and apoptosis but not glucose metabolism through the MEK/ERK pathway. Our findings indicate that FGF10 regulates resumption of meiosis and expansion and survival of cumulus cells via MEK/ERK signaling during in vitro maturation of buffalo cumulus oocyte complexes. Elucidation of the mechanism of action of FGF10 and insights into oocyte maturation should advance buffalo breeding. Further studies should examine whether enhancement of MEK/ERK signaling improves embryonic development in buffalo.

Transient suppression of Wnt signaling in poor-quality buffalo oocytes improves their developmental competence.

One of the most evolutionary conserved communication systems, the Wnt signaling pathway is a major gene regulatory pathway that affects the developmental competence of oocytes and regulates most embryonic developmental processes. The present study was undertaken to modulate the canonical Wnt (Wingless/integration) signaling pathway in the poor-quality (colorless cytoplasm after Brilliant Cresyl Blue staining, BCB-) buffalo cumulus-oocyte complexes (COCs) to improve their in vitro maturation (IVM) and embryo production (IVEP) rates. The expression of key Wnt pathway genes was initially assessed in the good (blue cytoplasm after Brilliant Cresyl Blue staining, BCB+) and poor quality (BCB-) buffalo COCs to establish a differential activity of the Wnt pathway. The BCB- COCs were supplemented with the Wnt pathway inhibitor, Dickkopf-related protein 1 (DKK1) and later subjected to IVM and IVEP along with the BCB+ and BCB- controls. The cumulus expansion index (CEI), rate of nuclear maturation (mean percentage of oocytes in the MII stage) and embryo production, and the expression of developmentally important genes were evaluated to assess the effect of Wnt pathway inhibition on the development competence of these poor-quality oocytes. The Wnt pathway genes exhibited a significantly higher expression (p < 0.05) in the poor-quality BCB- oocytes compared to the good-quality BCB+ oocytes during the early maturation stages. The supplementation of BCB- COCs with 100 ng/mL DKK1 effectively inhibited the expression of the key mediators of the Wnt pathway (β-catenin and dishevelled homolog 1, DVL1). DKK1 supplemented BCB- COCs exhibited significantly improved cytoplasmic and nuclear maturation indices, development rates and significantly elevated expression (p < 0.05) of genes implicated in germinal vesicle breakdown (GVBD) and embryonic genome activation (EGA) vis-à-vis BCB- control COCs. These data indicate that inhibition of the Wnt pathway during the initial course of oocyte maturation can improve the development competence of poor-quality buffalo oocytes.

Synergistic impact of α-linolenic acid and α-tocopherol on in vitro maturation and culture of buffalo oocytes.

The objective of the current study was to investigate the synergistic impact of α-Tocopherol and α-Linolenic acid (100 µM) on IVM and IVC of Nili Ravi buffalo oocytes. Oocytes were obtained from the ovaries of slaughtered buffaloes within two hours after slaughter and brought to laboratory. Buffalo cumulus oocyte complexes were placed randomly in the five experimental groups included; GROUP 1: Maturation media (MM) + 100 µM ALA (control), GROUP 2: MM + 100 µM ALA + 50μM α-Tocopherol, GROUP 3: MM + 100 µM ALA + 100μM α-Tocopherol, GROUP 4: MM + 100 µM ALA + 200 μM α-Tocopherol and GROUP 5: MM + 100 µM ALA + 300 μM α-Tocopherol under an atmosphere of 5% CO2 in air at 38.5 °C for 22-24 h. Cumulus expansion and nuclear maturation status was determined (Experiment 1). In experiment 2, oocytes were matured as in experiment 1. The matured oocytes were then fertilized in Tyrode's Albumin Lactate Pyruvate (TALP) medium for about 20 h and cultured in synthetic oviductal fluid (SOF) medium to determine effect of α-Linolenic acid (100 µM) and α-Tocopherol in IVM medium on IVC of presumptive zygotes. To study the effect of α-Linolenic acid (100 µM) in IVM media and increasing concentration of α-tocopherol in the culture media on early embryo development (Experiment 3), the presumptive zygotes were randomly distributed into the five experimental groups with increasing concentration of α-tocopherol in culture media. Higher percentage of MII stage oocytes in experiment 1(65.2±2.0), embryos at morula stage in experiment 2 (30.4±1.5) and experiment 3 (22.2±2.0) were obtained. However, overall results for cumulus cell expansion, maturation of oocyte to MII stage and subsequent embryo development among treatments remain statistically similar (P > 0.05). Supplementation of α-tocopherol in maturation media having α-Linolenic acid and/or in embryo culture media did not further enhance in vitro maturation of oocyte or embryo production.

Beneficial effects of fibroblast growth factor 10 supplementation during in vitro maturation of buffalo cumulus-oocyte complexes

Fibroblast growth factor 10 (FGF10) is an important regulator of the mammalian cumulus-oocyte complex that plays a crucial role in oocyte maturation. In this study, we investigated the effects of FGF10 supplementation on the in vitro maturation (IVM) of buffalo oocytes and its related mechanisms. During IVM, the maturation medium was supplemented with a range of concentrations of FGF10 (0, 0.5, 5, and 50 ng/mL) and the resulting effects were corroborated using aceto-orcein staining, TUNEL apoptosis assay, detection of Cdc2/Cdk1 kinase in oocytes, and real-time quantitative PCR. In matured oocytes, the 5 ng/mL-FGF10 treatment resulted in a significantly increased nuclear maturation rate, which increased the activity of maturation-promoting factor (MPF) and enhanced buffalo oocyte maturation. Furthermore, it treatment significantly inhibited the apoptosis of cumulus cells, while simultaneously promoting its proliferation and expansion. This treatment also increased the absorption of glucose in cumulus cells. Thus, our results indicate that adding an appropriate concentration of FGF10 to a maturation medium during IVM can be beneficial to the maturation of buffalo oocytes and improve the potential of embryo development.

Effect of milrinone on the meiosis resumption and cytoplasm maturation of buffalo oocytes.

Buffalo has many excellent economic traits and it is one of the greatest potential livestock. Compared with cattle, buffalo has poorer reproductivity, it is of great significance to improve the development potential of oocytes. Buffalo oocyte in vitro maturation (IVM) has been widely used in production, but the poor development ability of bovine oocytes IVM limits the development of buffalo reproductivity. Milrinone as a phosphodiesterase inhibitor could affect the maturation of oocytes in goat and mice, but there have been few reported studies in water buffalo. To optimize buffalo oocyte in vitro maturation systems, the effects of phosphodiesterase inhibitor (milrinone) on pre-maturation culture of buffalo oocytes were investigated in this study. Buffalo cumulus-oocyte complexes (COCs) were cultured in medium with different concentrations (0, 12, 25, 50 and 100 mol/l) of milrinone for different times (0, 4, 8, 12, 16, 22 and 24 h). The results showed that the buffalo COCs nuclear maturation process could be inhibited by milrinone (25-100 mol/l) in a dose-dependent manner. The inhibitory effect of milrinone on in vitro maturation of buffalo oocytes did not decrease with the extension of time. This indicated that milrinone can be used as a nuclear maturation inhibitor during the maturation process in buffalo oocytes. In addition, milrinone can inhibit the effect of follicle stimulating hormone (FSH)-induced IVM of buffalo oocytes, but with time FSH partially eliminated the inhibition. Therefore, inhibition of milrinone on the nuclear maturation of buffalo oocytes was reversible, and buffalo oocytes can mature normally after the inhibition is lessened.

Effect of in Vitro Maturation Medium Supplementation with Chitosan Nanoparticles on The River Buffalo Cumulus-Oocyte Complexes

The present study aimed to investigate the possible effects of chitosan nanoparticles (CNPs) supplementation to the in vitro maturation medium on the expansion of cumulus cells, nuclear maturation, and relative gene expression of Superoxide dismutase-1 (SOD1), B-cell lymphoma-2 Apoptosis Regulator (BCL2), and BCL2 Associated X (BAX) in the River buffalo. Slaughterhouse-derived cumulus-oocyte complexes (COCs) became matured in vitro in the absence (control) or presence of CNPs (10, 25, and 50µg/mL). At the end of maturation, we assessed expansion rates and denuded COCs; then, some replicates were fixed and stained to determine nuclear maturation. Other replicates were vitrified until we acquired enough oocytes, thawed, and assessed gene expression. We concluded that 10µg/mL CNPs significantly increased cumulus cell expansion and nuclear maturation. Whereas 10 and 25µg/mL CNPs non-significantly increased SOD1 relative expression, BCL2/BAX ratio for 10µg/mL CNPs was significantly higher than in control, 25 and 50µg/mL CNPs groups.

Theca cell-conditioned medium added to in vitro maturation enhances embryo developmental competence of buffalo (Bubalus bubalis) oocytes after parthenogenic activation.

Theca cells (TCs) play a key role in follicular growth and atresia. TCs synthesize androgens that act as substrate for granulosa cells (GCs) aromatization to estrogens needed for oocyte maturation. However, the effects of TCs in the form of conditioned medium on in vitro maturation (IVM) and developmental competence of buffalo oocytes remain unclear. In the present study, we examined the impacts of TC-conditioned medium (TCCM) on maturation efficiency and embryo development of buffalo oocytes after parthenogenic activation (PA). Our results showed that TCCM that was collected on day 2 and added to IVM medium at a 20% proportional level (2days & 20%) exerted no significant effect on IVM rate (43.06% vs. 44.71%), but significantly (p<.05) enhanced embryo development (oocyte cleavage, 80.93% vs. 69.66%; blastocyst formation, 39.85% vs. 32.84%) of buffalo oocytes after PA compared with the control group. However, monolayer TC significantly (p<.05) promoted both maturation efficiency (48.84% vs. 44.53%) and embryo development (oocyte cleavage, 80.39% vs. 69.32%; blastocyst formation, 35.38% vs. 29.25%) of buffalo oocytes after PA compared to that in the control group. Furthermore, TCs secreted some testosterone into the conditioned medium, which significantly (p<.05) promoted the expression levels of oestrogen synthesis-related genes (CYP11A1, CYP19A1 and 17β-HSD) in buffalo cumulus-oocyte complexes (COCs). Our study indicated that TCCM (2days & 20%) did not significantly affect IVM efficiency, but enhanced embryo developmental competence of oocytes after PA principally by stimulating the secretion of testosterone and facilitating estradiol synthesis of buffalo COCs.

26 Season affects cryotolerance of in vitro-produced buffalo embryos

Buffaloes are tendentially short-day breeders, and seasonality is one of the main factors affecting the feasibility of ovum pickup and in vitro embryo production technology in this species. An improvement of oocyte developmental competence during decreasing daylight months was previously reported in Italian Mediterranean buffalo (Di Francesco et al. 2011 Anim. Reprod. Sci. 123, 48-53). The aim of this work was to evaluate whether season also affects embryo quality and cryotolerance. Abattoir-derived buffalo cumulus-oocyte complexes were collected during the breeding season, characterised by decreasing daylight length (n=349 over 6 replicates), and the non-breeding season, characterised by increasing daylight length (n=770 over 12 replicates). Buffalo cumulus-oocyte complexes were in vitro matured, fertilized, and cultured according to standard procedures (Di Francesco et al. 2011 Anim. Reprod. Sci. 123, 48-53). The embryos obtained by the end of culture (i.e. on Day 7 post-IVF) were scored for quality and developmental stage, and the percentages of total transferable embryos (tight morulae and blastocysts) were recorded. Embryos (n=107 and 110 in the breeding and non-breeding seasons, respectively) were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide, and 0.5M sucrose (Boccia et al. 2013 Ital. J. Anim. Sci. 12, 492-496). Warming was carried out by plunging the cryotop strip into a 0.25M sucrose solution and transferring the embryos into 0.15M sucrose for 5min. Embryos were then washed and cultured in SOF for 24h to evaluate post-culture viability. The resistance to cryopreservation was evaluated by assessing the survival rate, on the basis of morphological criteria, and development rate (i.e. the percentage of embryos that resumed their development and reached a more advanced developmental stage) after 24h post-warming culture. Data were analysed by Student’s t-test. Both cleavage (82.8±4.3v. 73.1±1.7 in the breeding and non-breeding seasons, respectively; P&amp;lt;0.05) and blastocyst (32.9±3.5v. 18.3±1.7 in the breeding and non-breeding seasons, respectively; P&amp;lt;0.01) rates increased during the breeding season, confirming previous observations. Due to the different efficiency, a higher number of replicates was required during the non-breeding season to obtain an equal number of embryos. In addition, a seasonal effect was recorded on embryo quality, indicated by poorer cryotolerance of in vitro-produced buffalo embryos during the non-breeding season. Indeed, both survival (94.6±2.7% and 74.0±5.5% in the breeding and non-breeding seasons, respectively; P&amp;lt;0.01) and development (67.3±7.6% and 40.0±7.2% in the breeding and non-breeding seasons, respectively; P&amp;lt;0.01) rates of vitrified blastocysts decreased after 24h post-warming culture in the non-breeding season. These findings suggest that the reduced developmental competence of buffalo oocytes during the non-breeding season may also lead to lower blastocyst quality. This is in contrast to the evidence in cattle that embryo quality is mainly determined by culture conditions, whereas blastocyst production depends on oocyte quality.

In vitro production of early stage buffalo embryos in modified synthetic oviductal fluid (mSOF) medium

The objective of the present study was to observe the developmental rates and the stage of development since fertilization of in vitro produced early stage buffalo embryos. Buffalo cumulus-oocyte complexes (COC’s) obtained from slaughterhouse ovaries were matured and fertilized in vitro. The fertilized oocytes (400) were then cultured in modified synthetic oviductal fluid (mSOF) medium containing bovine serum albumin (BSA) and fetal bovine serum (FBS) and evaluated for the developmental stages of preimplantation early stage embryos up to morula on 48 h, 72 h, 96 h and 7th day post fertilization. Highest percentage of 8 cell embryos were obtained followed by 16 cell, 2 cell, 4 cell and morula at the end of in vitro culture (IVC). Similarly, the mean embryo percent of 2 cell, 4 cell, 8 cell, 16 cell and morula stage at the end of IVC was 8.20±1.28, 6.50±0.97, 12.19±1.03, 11.83±0.96 and 5.27±0.87. The overall embryos developed at the end of IVC were 43.98±2.11%. The percentage of oocytes which arrested and subsequently degenerated were 56.02% till day 7 post-insemination. It can be concluded that early stage buffalo embryos till morulae can be produced by using modified synthetic oviductal fluid (mSOF) medium as embryo development medium but for further development, the supplementation of different growth factors required.

Developmental and molecular responses of buffalo (Bubalus bubalis) cumulus–oocyte complex maturedin vitrounder heat shock conditions

SummaryTo investigate the effects of physiologically relevant heat shock during oocyte maturation, buffalo cumulus-oocyte complexes (COCs) were cultured at 38.5°C (control) or were exposed to 39.5°C (T1) or 40.5°C (T2) for the first 6 h of in vitro maturation (IVM), followed by 38.5°C through the next 18 h/IVM and early embryonic development up to the blastocyst stage. Gene expression analysis was performed on selected target genes (HSF-1, HSF-2, HSP-70, HSP-90, BAX, p53, SOD1, COX1, MAPK14) in denuded oocytes and their isolated cumulus cells resulting from control COCs as well as from COCs exposed to a temperature of 39.5°C (T1). The results indicated that heat shock significantly (P < 0.01) decreased the maturation rate in T1 and T2 cells compared with the control. After in vitro fertilization (IVF), cleavage rate was lower (P < 0.01) for oocytes exposed to heat stress, and the percentage of oocytes arrested at the 2- or 4-cell stage was higher (P < 0.01) than that of the control. The percentage of oocytes that developed to the 8-cell, 16-cell or blastocyst stage was lower (P < 0.01) in both T1 and T2 groups compared with the control group. mRNA expression levels for the studied genes were decreased (P < 0.05) in treated oocytes (T1) except for HSP-90 and HSF-1, which were increased. In cumulus cells isolated from COCs (T1), the expression for the target genes was upregulated except for BAX, which was downregulated. The results of this study demonstrated that exposure of buffalo oocytes to elevated temperatures for 6 h severely compromised their developmental competence and gene expression.

Induced cumulus expansion of poor quality buffalo cumulus oocyte complexes by Interleukin-1beta improves their developmental ability.

The present study was conceived with the aim of modulating the cumulus expansion characteristics of poor quality (BCB-) buffalo oocyte complexes (COCs) in order to improve their fertilization outcomes. BCB- COCs were subjected to in vitro maturation (IVM) in presence of Interleukin-1 beta (IL-1β) along with BCB- (control) and good quality (BCB+) COCs. Results were assessed morphologically, by scanning electron microscopy (SEM) and by expression analysis of cumulus expansion related genes. Also, numbers of zona pellucida bound spermatozoa were counted and development rates of oocytes were monitored under different groups. Expression of versican isoforms and ADAMTS-1 was observed to be significantly different between cumulus cells of BCB+ and BCB- COCs. Upon IL-1β supplementation, ADAMTS-1 expression increased in BCB- COCs along with corresponding cumulus expansion rates. SEM analysis also revealed improved cumulus expansion in IL-1β supplemented BCB- COCs. HAS2 and TNFAIP-6 were significantly up-regulated after IL-1β supplementation while PTGS2 expression remained unaffected. Significantly more numbers of sperms crossed the cumulus barrier, especially in 100 ng/mL IL-1β supplemented COCs. Besides, cleavage and blastocyst development rates were also improved upon IL-1β addition. We concluded that IL-1β supplementation in IVM medium can improve cumulus expansion and development ability of poor quality buffalo oocytes.

Effect of exogenous progesterone on cumulus characteristics of buffalo oocytes by allowing passage of more number of sperm through cumulus but not essentially fertilization

Objective: To understand the level of progesterone (P4) in different quality of buffalo cumulus oocyte complexes (COCs) and further to evaluate the effect of exogenous P4 supplementation on maturation and subsequent developmental ability of poor quality brilliant cresyl blue (BCB- ) COCs. Methods: Progesterone secreted by different quality of buffalo oocytes was estimated by enzyme linked immunosorbent assay and the concentration differences were translated into P4 doses to be incorporated in the maturation medium of BCB-ve COCs followed by expression analysis of genes involved in the cumulus expansion, extracellular matrix disintegration and progesterone receptor signalling. In addition, the study also evaluated the effect of exogenous P4 on sperm-cumulus interaction. Results: More than 10-fold up- regulated expression of progesterone receptor in P4 supplemented oocytes signified that P4 might be acting predominantly through this receptor. Also, exogenous P4 supplementation had significant effect on transcatheter arterial chemoembolization protease regulated by P4- progesterone receptor pathway which in turn had an important role in extracellular matrix disintegration. On the contrary, cumulus expansion genes HAS2, TNFAIP6, AREG were not altered upon P4 supplementation. Also, it was observed that P4 addition did facilitate passage of significantly more number of spermatozoa through P4 treated cumulus cells. Further, incorporation of different doses of P4 did not improve significantly the cleavage and blastocyst rates of BCB-ve COCs. Conclusions: Different qualities of buffalo COCs secrete substantially diverse levels of P4, and its supplementation has a role in oocyte maturation via modulation of cumulus characteristics but perhaps not fertilization.

Leptin supplementation in vitro improved developmental competence of buffalo oocytes and embryos

The aim of the present study was to determine potential role of leptin on in vitro developmental competence of buffalo oocytes and embryos. Slaughterhouse derived culture grade buffalo cumulus oocyte complexes (COCs) were matured in vitro (IVM) with leptin (10 ng/ml) or without leptin (control). In each experiment, a pool of matured COCs was used for further in vitro embryo production and another pool of COCs was used for cumulus cells and mature oocytes isolation to study the relative mRNA expression of developmentally important genes. Presumptive zygotes were cultured in embryo culture (IVC) media supplemented with leptin (10 ng/ml) or without leptin (control). Cleavage rate was higher (p < 0.05) when leptin was supplemented during IVM + IVC, both, as compared to other groups. Higher cleavage rate was observed in leptin-treated groups, though it was non-significant. Blastocyst rate was higher (p < 0.05) in all the leptin treated groups. The relative mRNA expression of LEPR (Ob-Rb), HAS2 and EGFR was significantly (p < 0.05) up-regulated and the expression of CASPASE3 was down-regulated in cumulus cells of leptin-treated groups. The expression of GDF9, BMP15, GLUT1, LEPR and CASPASE3 transcripts in leptin and non-treated oocytes did not differ. The relative mRNA expression of POU5F1and LEPR transcripts in blastocysts was higher (p < 0.05) in leptin-treated groups; the change in expression of GLUT1, INF-τ and CASPASE3 transcripts was not significant (p > 0.05). Thus, it is concluded that leptin promotes developmental competence of bubaline oocytes by modulating cumulus enabling factors and genes regulating pluripotentcy in the blastocysts.

Supplementing ±-Linolenic acid in the in vitro maturation media improves nuclear maturation rate of oocytes and early embryonic development in the Nili Ravi buffalo

The present study was conducted to investigate the effect of omega-3 poly unsaturated fatty acid (PUFA), α-linolenic acid (ALA; 18:3 n-3) on the in vitro maturation (IVM) of buffalo oocytes and subsequent embryonic development. Buffalo cumulusoocyte complexes (COCs; n = 2282) were in vitro matured in TCM-199 (0.6% fatty acid free bovine serum albumin, 0.02 Units/ml FSH, 1 µg/ml 17-β-estradiol, 10 µg/ml epidermal growth factor, 50 µg/ml gentamicin) supplemented with 0 (control), 25, 50, 100, 150 or 300 µm ALA under an atmosphere of 5% CO2 in air at 38.5oC for 22-24 h. The matured oocytes were then fertilized in Tyrode’s Albumin Lactate Pyruvate (TALP) medium and cultured in synthetic oviductal fluid (SOF) medium. Concentrations up to 100 μm ALA improves (P ≤ 0.05) the cumulus expansion compared to control. Higher percentage of oocytes reaching MII stage was observed at 50 μm and 100 μm of ALA compared to control (P ≤ 0.05). Concentrations of 150 and 300 µm ALA were detrimental both for cumulus expansion and nuclear maturation rate of buffalo oocytes. Moreover, supplementation with 100 μm ALA improved (P ≤ 0.05) cleavage rate compared to control and treatment with 50 and 100 μm ALA yielded significantly higher morulae compared to control. The results of present study indicate that the supplementation with 100 μm ALA to the IVM medium improves nuclear maturation rate of buffalo oocytes and subsequent early embryonic development.

34 PRODUCTION OF TRANSGENIC CLONED BUFFALO EMBRYOS CONTAINING OVEREXPRESSED STEAROYL Co-A DESATURASE GENE FOLLOWING EFFICIENT TRANSFECTION

Stearoyl-CoA desaturase (SCD) is a rate-limiting enzyme that catalyses the synthesis of monounsaturated fatty acids and polyunsaturated fatty acids from saturated fatty acids, which are components of triglycerides, wax esters, cholesteryl esters, and membrane phospholipids. Alterations in phospholipid composition have been implicated in a variety of diseases including obesity and the associated metabolic syndrome. SCD also magnifies the conjugated linoleic acid (CLA) content in milk; CLA is a natural fat element, having reputed therapeutic health values including anti-carcinogenic properties. In light of this fact, this study was designed to amplify the levels of SCD at the gene level. In order to achieve the enhanced expression of SCD gene, combination of techniques were used. The somatic cells (fetal fibroblast) culture were established from ear pinnae obtained from bovine fetus procured from the abattoir and were cultured in basal medium, comprising DMEM with 10% fetal bovine serum, 1X NEAA and 1X PS antibiotics. These isolated cultured cells were transfected with a gene construct carrying buSCD gene (pAcGFPN1-buSCD) as BLGP-buSCD-BLG3′UTR-CMVP-EGFP-SV40. The buffalo fetal fibroblast cells were transfected using 3 methods: Nucleofection, Fugene and Lipofection. The successful transfection, as confirmed by PCR and Southern hybridisation, proved Nucleofection to be more efficient in transfecting cells among the techniques used, which were further maintained and selected by Geneticin (G418). These selected transfected cells were then used for nuclear transfer. Somatic cell nuclear transfer (SCNT) has provided an efficient pathway for the production of transgenic animals. Buffalo cumulus–oocyte complexes (COCs) were collected from ovaries collected from abattoir and matured in TCM-199 supplemented with 10% FBS, 5 µg mL−1 FSH, and 1 µg mL−1 β-oestradiol for 21 h in CO2 incubator at 5% CO2 in air and 38.5°C temperature with &gt;95% relative humidity. After 21 h, these COCs were denuded and subjected to zona removal. These zona-free oocytes were manually enucleated using microsurgical blades and 2 enucleated oocytes were fused with a transgenic cell using electro cell manipulator. Further, these reconstructed embryos were activated using calcium ionophore and cultured in IVC media thereafter for 8 days. The developmental competence rate as recorded on Day 8 was 53.26 ± 1.73%, 69.87 ± 6.24%, 62.99 ± 7.15%, 42.71 ± 5.05% and 28.00 ± 3.33% for 2-cell, 4-cell, 8–16 cell, morula, and blastocyst, respectively. When observed under fluorescence microscope, the embryos showed successful expression of GFP, which can be further used for animal production or further research analysis. In conclusion, amplified SCD at gene level will result in a boost to the dairy sector as well ameliorating human health due to its crucial role in anti-cancer, anti-diabetic, reduced cardio-vascular disease, and improved immune responses.

Retrieval and Recovery Rate of Buffalo (Bubalus bubalis) Oocytes Through Aspiration Technique

The objective of the present study was to retrieve good and excellent quality of oocytes from the ovaries of buffalo having unknown reproductive history by using the aspiration technique. The retrieval as well as recovery percent was recorded. Total nine hundred and twenty ovaries were collected during the period of study, on an average 20 ovaries were collected per slaughter. Buffalo cumulus-oocyte complexes (COC's) were retrieved by aspiration technique and further graded on the basis of cellular investment and homogenicity in to grade A, B, C and D. The mean recovery rate (i.e. no. of oocytes/ovary) was 2.20 ± 0.08, while that of grade A, B, A+B were 0.82 ± 0.03, 0.53 ± 0.03 and 1.35 ± 0.06, respectively. The mean retrieval percent of Grade A, B and A+B COC's were 37.10 ± 0.67, 23.74 ± 0.62 and 60.83 ± 0.84, respectively. The present finding led to the conclusion that aspiration technique seems to be a very promising technique for retrieval of developmentally competent oocytes from surface follicles and not from the deeper cortex which contained developing oocytes. By using aspiration technique, less amount of tissue debris obtained which may otherwise have adverse effect on the oocytes maturation and subsequent development of poor quality of embryos in vitro.

Seasonal variations in developmental competence and relative abundance of gene transcripts in buffalo (Bubalus bubalis) oocytes

Hot season is a major constraint to production and reproduction in buffaloes. The present work aimed to investigate the effect of season on ovarian function, developmental competence, and the relative abundance of gene expression in buffalo oocytes. Three experiments were conducted. In experiment 1, pairs of buffalo ovaries were collected during cold season (CS, autumn and winter) and hot season (HS, spring and summer), and the number of antral follicles was recorded. Cumulus oocyte complexes (COCs) were aspirated and evaluated according to their morphology into four Grades. In experiment 2, Grade A and B COCs collected during CS and HS were in vitro matured (IVM) for 24 hours under standard conditions at 38.5 °C in a humidified air of 5% CO2. After IVM, cumulus cells were removed and oocytes were fixed, stained with 1% aceto-orcein, and evaluated for nuclear configuration. In vitro matured buffalo oocytes harvested during CS or HS were in vitro fertilized (IVF) using frozen-thawed buffalo semen and cultured in vitro to the blastocyst stage. In experiment 3, buffalo COCs and in vitro matured oocytes were collected during CS and HS, and then snap frozen in liquid nitrogen for gene expression analysis. Total RNA was extracted from COCs and in vitro matured oocytes, and complementary DNA was synthesized; quantitative Reverse Transcription-Polymerase Chain Reaction was performed for eight candidate genes including GAPDH, ACTB, B2M, GDF9, BMP15, HSP70, and SOD2. The results indicated that HS significantly (P < 0.01) decreased the number of antral follicles and the number of COCs recovered per ovary. The number of Grade A, B, and C COCs was lower (P < 0.05) during HS than CS. In vitro maturation of buffalo oocytes during HS significantly (P < 0.01) reduced the number of oocytes reaching the metaphase II stage and increased the percentage of degenerated oocytes compared with CS. Oocytes collected during HS also showed signs of cytoplasmic degeneration. After IVF, cleavage rate was lower (P < 0.01) for oocytes collected during HS, and the percentage of oocytes arrested at the two-cell stage was higher (P < 0.01) than oocytes IVF during CS. Oocytes matured during CS showed a higher (P < 0.01) blastocyst rate than those matured during HS. Also, COCs recovered in HS showed significant (P < 0.05) upregulation of HSP70 mRNA expression compared with those recovered in CS. For in vitro matured oocytes, CS down regulated the transcript abundance of ACTB and upregulated GAPDH and HSP70 mRNA levels compared with HS condition. In conclusion, HS could impair buffalo fertility by reducing the number of antral follicles and oocyte quality. In vitro maturation of buffalo oocytes during HS impairs their nuclear and cytoplasmic maturation, fertilization, and subsequent embryo development to the morula and blastocyst stages. This could be in part because of the altered gene expression found in COCs and in vitro matured oocytes.

Co-culture embedded in cumulus clumps promotes maturation of denuded oocytes and reconstructs gap junctions between oocytes and cumulus cells

The present study was undertaken to establish an effective method for in vitro maturation (IVM) of denuded oocytes (DOs) by simulating the ovarian three-dimensional status in vivo using buffalo ovarian tissues or cumulus cells, so as to provide a model for investigating the mechanisms of oocyte maturation. Buffalo cumulus-oocyte complexes from ovaries taken at slaughter were denuded by pipetting, and then allocated randomly into four groups for IVM by direct culture in maturation medium (M1, control group), co-culture with a monolayer of cumulus cells (M2), embedded in cumulus cell clumps (M3) and ovarian tissue (M4) for 24 h. The nuclear maturation of DOs was assessed by the extrusion of the first polar body and the cytoplasmic maturation was evaluated by subsequently developmental capacity after parthenogenetic activation. More DOs matured to MII (56.89%) and developed to blastocysts (25.75%) when they were matured in vitro with M3 in comparison with DOs matured in vitro with M1 (45.14 and 15.97%) and M4 (40.48 and 13.49%). Further detection of gap junctions by injecting Lucifer yellow directly into cytoplasm of matured DOs with adherent cumulus cells and scanning with confocal microscope showed that Lucifer yellow were found in nine out of 11 the adherent cumulus cells in M3, indicating that the gap junctions between oocytes and cumulus cells was reconstructed in vitro. These results indicate that co-culture of DOs embedded in cumulus cell clumps can improve their nuclear and cytoplasmic maturation of DOs, possibly through the reconstruction of gap junctions in vitro.

Ultrastructure of in vitro oocyte maturation in buffalo (Bubalus bubalis)

The objective of the present study was to describe ultrastructural changes in the nucleus and cytoplasmic organelles during in vitro maturation (IVM) of buffalo cumulus-oocyte complexes (COCs). The structures were collected by ovum pick-up (OPU). Some COCs, removed from maturation medium at 0, 6, 12, 18 and 24 h, were processed for transmission electron microscopy. The average number of COCs collected by OPU/animal/session was 6.4, and 44% of them were viable. Immature oocytes had a peripherally located nucleus, Golgi complex and mitochondrial clusters, as well as a large number of coalescent lipid vacuoles. After 6 h of IVM, the oocyte nucleus morphology changed from round to a flatter shape, and the granulosa cells (GC) lost most of their contact with zona pellucida (ZP). At 12 h the first polar body was extruded and the aspect of lipid droplet changed to dark, probably denoting lipid oxidation. Cortical granules were clearly visible at 18 h of maturation, always located along the oocyte periphery. At 24 h of IVM the number of cortical granules increased. Ultrastructure studies revealed that: (1) immature oocytes have a high lipid content; (2) the perivitelline space (PS) increases during IVM; (3) Golgi complexes and mitochondrial clusters migrate to oocyte periphery during IVM; (4) 6 h of IVM are enough to lose contact between GC and ZP; (5) the oocyte lipid droplets' appearance changes between 6 and 12 h of IVM.