TCM-199 Medium Research Articles

185 OVIDUCTAL CO-CULTURE CELL DID NOT REDUCE THE RATE OF CHROMOSOMAL ABNORMALITIES IN IN VITRO-PRODUCED BOVINE EMBRYOS

One of the major causes of embryo failure on in vitro-produced cattle embryos is their increased rate of chromosomal abnormalities. It was demonstrated that they can be increased by the pre- and post-environmental conditions of culture, such as the culture media and supplementation and culture atmosphere among others. Furthermore, it was suggested that the percentage of chromosomal abnormalities detected could be used as an indirect methodology to evaluate the embryo stress during culture. The use of oviduct cells in co-culture was employed as a technique that improves the embryo quality in different culture systems in cattle, probably by modulating the variations in the embryo environment or detoxifying the culture media. For that reason, we aimed to determine if the use of an oviducal co-culture system could reduce the percentage of chromosomal abnormalities in in vitro-produced cattle embryos. Oviducal cells were obtained from abattoir oviducts, and plated and cultured in TCM-199 media following standard procedures. Cumulus-oocyte complexes were also obtained from the abattoir and matured in a standard media (TCM199, with glutamine, pyruvate, FSH, LH, and antibiotics) for 20 h (38.5°C, 5% CO2). Matured oocytes were fertilized in IVF-TALP with 1 × 106 spermatozoa/mL (previously selected through a 45–90% Percoll™ gradient) for 18 h using the same conditions. Presumptive zygotes (n = 653) were divided into 2 groups and cultured in the mSof medium for 72 h, with (SOV) or without (SOF) the addition of oviducal cells. In total, 108 embryos were successfully karyotyped following our standard laboratory techniques. Chromosomal numerical abnormalities were evaluated by direct observation at 1250× magnification in a bright-field microscope using a simple Giemsa staining. The percentage of diploid embryos (54/58 in SOV and 47/53 in SOF) and abnormal embryos (4/58 in SOV and 6/53 in SOF) were nonsignificant (P > 0.05; Z-score test for 2 population proportions). Our results suggest that the use of oviducal cells as a co-culture supplementation in in vitro-produced cattle embryos do not improve the percentage of chromosomal abnormalities detected.

187 9-cis RETINOIC ACID IMPROVES MATURATION RATE AND ALTERS GENE EXPRESSION OF IN VITRO-MATURED OOCYTES IN EGYPTIAN BUFFALO

Retinoic acid, a metabolite of vitamin A, regulates oocyte maturation through multiple mechanisms, including gene expression modulation or preventing oxidative stress. Effects of retinoic acid during oocyte maturation have been reported in several species; however, there have been no studies illustrating these effects in buffalo. Therefore, the objective of this study was to investigate the influence of 9-cis retinoic acid (9-cisRA), an active metabolite of vitamin A, on maturation rate and gene expression during in vitro maturation of buffalo oocytes. Cumulus-oocyte complexes (n = 360) were aspirated from surface follicles of Buffalo ovaries collected from local abattoirs and transported to the laboratory in physiological saline (0.9% NaCl) containing antibiotics (100 µg mL−1 of streptomycin sulfate and 100 IU mL−1 of penicillin) and maintained at 30°C. Grade A cumulus-oocyte complexes (evenly granulated cytoplasm and surrounded by multiple layers of cumulus cells) were randomly divided into 4 groups (90 oocytes/group) and allocated in TCM-199 medium supplemented with 10% fetal bovine serum, 0.2 mM sodium pyruvate, 50 μg mL−1 of gentamycin, and 10 μg mL−1 of FSH and contained 0 (control), 5, 50, or 200 nM of 9-cisRA for maturation. After 24 h, maturation rate was calculated as a percentage based on polar body extrusion. In addition, gene expression patterns were analysed for antioxidant related genes (SOD1, CAT, GPX4, HOMX1, and PRDX1) and oocyte quality-related genes (GDF9 and BMP15) using quantitative real-time PCR with GAPDH as a housekeeping gene. Fold changes (FC) were calculated using ΔΔCt method (FC ≥2; P < 0.05). The results showed that maturation rate (based on the extrusion of polar body) was significantly higher in 5 nM 9-cisRA oocyte group (49.4 ± 2.1%) compared with the control group (35 ± 1.8%); in contrast, the 200 nM 9-cisRA oocyte group showed the lowest maturation rate (27.2 ± 2.7%). However, the 50 nM 9-cisRA oocyte group showed no significant differences (31.2 ± 3.8%) compared with control group .Oocytes treated with 5 and 50 nM 9-cisRA during in vitro maturation showed significant up-regulation of SOD1 (3.4 and 3.08 FC), CAT (2.7 and 1.8 FC), and HOMX1 (4.5 and 4 FC), and significant down-regulation of BMP15 (−3.7 and −3.6 FC), respectively, compared with the control group. Moreover, GPX4, PRDX1, and GDF9 genes were highly expressed in the 50 nM compared with the control group (13.2, 10.4, and 1.8 FC, respectively). In contrast, the 200 nM 9-cisRA group showed significant down-regulation of CAT (−60.3 FC), GDF9 (−2.5 FC), and BMP15 (−9.7 FC) compared with the control group. In conclusion, these results suggested that a low concentration of 9-cisRA (5 nM) in maturation media can improves maturation rate of buffalo oocytes and up-regulates the expression of oxidative stress response-related genes.

71 MELATONIN IMPROVES PORCINE IN VITRO MATURATION VIA SONIC HEDGEHOG SIGNALLING

Melatonin (N-acetyl-5-methoxytryptamine) is the hormone synthesised from the mammalian pineal gland, which has an antioxidant property and regulates physiological processes such as cellular metabolism. It is well known that melatonin affects in vitro maturation of oocytes and embryonic development in many species. However, limited information is available on the underlying beneficial effects of melatonin. Sonic Hedgehog (Shh) signalling is important for follicular development, oocyte maturation, and embryo development. To elucidate the relationship between melatonin and Shh signalling, we designed an experiment with the following three groups: (1) control, (2) melatonin, and (3) melatonin with cyclopamine (smoothened inhibitor) during porcine in vitro maturation. Porcine ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory at 28 to 32°C. The contents of follicles 3 to 6 mm in diameter were recovered by aspiration with an 18 G needle. Cumulus–oocyte complexes were pooled and cultured in TCM-199 medium for 44 h. The aim of this study was to evaluate the effects of melatonin (10−9 M) with or without cyclopamine (2 μM) on cumulus cell expansion (a total of 432 cumulus–oocyte complexes were used in 3 replicates), embryo development after parthenogenetic activation (a total of 432 oocytes were used in 4 replicates). Moreover, we detected gene expression related to cumulus expansion, oocyte maturation, and hedgehog signalling in cumulus cells and oocyte. Results indicated that melatonin treatment significantly increased cumulus expansion index (3.75 ± 0.02 v. 3.51 ± 0.03 and 3.59 ± 0.05, respectively; P < 0.05) and blastocyst formation rates (30.4 ± 2.4 v. 21.9 ± 2.2 and 20.0 ± 2.2, respectively; P < 0.05) compared with control and melatonin with cyclopamine. In addition, the expression of cumulus expansion-related genes (Ptgs1, Ptgs2, Has2, Ptx-3, and Tnfaip6) and hedgehog signalling-related genes (Shh, Pthc1, Smo, and Gli-1) in cumulus cells were up-regulated in melatonin treatment compared with control and melatonin with cyclopamine. Similarly, the expressions of oocyte maturation-related genes (GDF9 and BMP15) in porcine oocytes were up-regulated in melatonin treatment compared with control and melatonin with cyclopamine. In conclusion, Shh signalling mediated melatonin to improve porcine cumulus cell expansion, oocyte maturation, and subsequent embryo development. Further studies are needed to evaluate the effect of melatonin on protein levels of Shh signalling. Statistical analyses were performed using SPSS 22.0 (SPSS Inc., Chicago, IL, USA). All data were tested for normality and homoscedasticity and then subjected to one-way ANOVA, followed by Duncan’s multiple range test (when the variances were assumed to be equal) or Dunnet’s T3 test (when the variances were assumed to be unequal) to determine differences among experimental groups. All results are expressed as means ± SEM; P-values < 0.05 were considered to be statistically significant. This study was supported by Ministry of Trade, Industry and Energy (#10048948), Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry, and Fisheries (#311011–05–4-SB010, #114059–03–2-SB010), National Research Foundation (2016M3A9B6903410), China Scholarship Council (CSC, No. 2015–3022), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.

Expression of fluorescent reporter protein was not obtained in ovine embryos produced through in vitro fertilization-sperm mediated gene transfer (IVF-SMGT)

Sperm mediated gene transfer (SMGT) has been reported to be a powerful tool for producing transgenic animals on a mass scale using spermatozoa as vectors for exogenous DNA. In this study the possibility of using in vitro fertilization (IVF) −SMGT to produce transgenic ovine embryos was investigated for the first time. For this purpose, sperm were obtained from the epididymal testicle areas of 4 rams and different concentration of DNA (0.4, 0.8 and 1.6μg) and TurboFect (0.25, 0.5, 1 and 2μg) were used for sperm transfection (1×106). Enhanced-GFP-expressing vector pEGFP-N1 was used as the carrier. In order to evaluate the performance of transgenic sperm, in vitro fertilization technology was used. After the preparing oocytes received from the ovaries of slaughterhouse origin, oocytes with more than three layers of granulosa and uniform cytoplasm were selected and matured in TCM-199 medium containing 10% fetal calf serum, follicle stimulating hormone (FSH) (5μg/ml), β-17 estradiol (1μg/ml) and sodium pyruvate 0.81 (Mm). Bracket and Oliphant’s (BO) medium and modified Charles Rosenkrans medium with amino acids (mCR2aa) were used for in vitro fertilization and culture, respectively. Results showed that transfected sperm with different concentrations of DNA and TurboFect carrier were unable to transfer the GFP gene to in vitro matured oocytes as the GFP gene was not expressed in neither zygotes nor morula stage embryos. Further optimization for SMGT improvement such as different transfection reagent and method like electroporation, using antioxidants in transfection medium to overcome the apoptosis and also separation of transfected sperm from untransfected ones before insemination have been suggested.

Effect of cysteine in maturation medium on embryos development by in vitro fertilization (IVF) in porcine

In this article, we studied the effect of cystein in maturation culture on glutathione concentration in oocyte and on embryos development by in vitro fertilization in porcine. 0.6 mM cysteine supplementation on TCM-199 medium has increased the amount of GSH in the oocyte. The GSH level began to increase at the 40th hour after cystein supple metation and attained the highest at the 44th hour (18.32 pmol/oocyte). After 5 days of embryos culture, a total of 1000 oocyte maturation and fertilization was obtained including 583 embryos containing 2 cells, 408 embryos containing 3-4 cells, 264 embryos containing 5-8 cells and 106 morula. In particular, fertilization rate and embryo division was the best in the oocyte maturation of the 44th hour with 200 embryos containing 2 cell (80 %), 161 embryos containing 3-4 cells (64.4 %), 114 embryos containing 5-8 cells (45.6 %) and 62 morula (24.8 %). The results showed that the time of the cysteine supplementation into medium and the time of muturation affected oocyte GSH level. In addition, embryoic development rate was also affected by maturation time and oocyte GSH level.

Effect of Culture Media on Mare Oocytes Maturation

432 mare oocytes were collected from ovaries of slaughtered animals and cultured in three mediums supplimented with hormons (FSH, LH) and nutritive substances and were cultivated for 24 hours in three media, the highest cultivation rate in three media was obatined in TCM-199 medium 53.33% (80 oocytes), followed by SOF medium, maturation rate was 39.65% (58 oocytes), but the less maturation rate (34.54%) was observed in the medium composed of TCM+ 5% SOF + 5% FCS + 1% glutamine + 1% A.A + 500 I¼l antibiotic). Oocytes were selected and classified in two quality categories (mature, degenerated).

Curcacycline A and B modulate apoptosis induced by heat stress in sheep oocytes during in vitro maturation

The main objective of the current study was to investigate the effect of Curcacycline A and B against heat stress induced apoptosis. Cumulus-oocyte complexes (COCs) were allocated in several groups were cultured at 39°C and 42°C in TCM-199 supplemented either with hormones alone, or with two cyclic peptides isolated from the latex of Jatropha curcas, namely Curcacycline A and B. In general, the results revealed a concrete reduction in the maturation rate of sheep oocytes cultured in TCM-199 medium at 42°C. At the same temperature, higher percentages of sheep oocytes cultured with Curcacycline A or A+B were matured compared with TCM-199 medium alone. Both the expression of apoptotic related-genes (Bax, Bcl-2, Caspase 3, P53 and C-myc) and the generation of reactive oxygen species (ROS) increased significantly in oocytes cultured in TCM-199 medium at 42°C. The addition of Curcacycline A and B supplements to the culture media at 42°C suppressed the effect of heat stress on the expression of apoptotic genes and on the generation of ROS. In conclusion, results indicated that the two cyclic peptides inhibited the negative effect of heat stress on meiotic maturation, expression of apoptotic genes and ROS generation in oocytes.

Efecto de la Suplementación del Medio de Maduración con Cisteamina y de Dos Medios de Cultivo (KSOMaa y SOF) en la Fecundación in vitro de Ovocitos Bovinos

Se evaluó el efecto de la cisteamina en el medio de maduración sobre la tasa de división posfecundación y el efecto de los medios de cultivo KSOMaa y SOF sobre la tasa de desarrollo embrionario in vitro hasta los 8 días posfecundación. Se utilizaron 680 ovocitos procedentes de ovarios de hembras bovinas distribuidos al azar en dos tratamientos: T1 (n=321) medio de maduración TCM-199 y T2 (n=359) medio de maduración TCM-199 adicionado con cisteamina 100 mM. Los ovocitos fueron fecundados a las 24 h con semen congelado de un solo toro y cultivados en medio KSOMaa por 18 h, evaluándose la tasa de división 72 h después. En la segunda fase, luego del primer cultivo, ambos grupos se subdividieron en dos grupos, donde el primero se cultivó con medio KSOMaa y el segundo con medio SOF hasta el día 8 posfecundación. Los resultados de la primera fase indican una tasa de división de 43.6% para el grupo control y 46.0% para el grupo suplementado con cisteamina, sin encontrar diferencia significativa. En la fase 2, se obtuvo una tasa de 18.0% de blastocistos para el tratamiento TCM-199 + KSOMaa y 20.9% para TCM-199 c/ cisteamina + KSOMaa (p<0.05), y 15.8% para TCM-199 + KSOMaa y SOF y 18.7% para TCM-199 c/ cisteamina + KSOMaa y SOF (p<0.05). Los resultados indican que la adición de cisteamina al medio de cultivo mejoró la tasa de desarrollo embrionario in vitro hasta el estadio de blastocisto en ovocitos madurados en medio TCM 199, no habiendo diferencias por el uso de los medios de cultivo.

203 SUPPLEMENTATION WITH LINOLENIC ACID, L-CARNITINE, ALONE OR ASSOCIATED, DURING IVM RESULTED IN DECREASE OF ROS LEVELS AND APOPTOTIC INDEX OF BOVINE IN VITRO PRODUCED EMBRYOS

Supplementation of in vitro maturation (IVM) medium with linolenic acid (ALA) has been used in order to reduce oocyte lipid content and have beneficial effects on maturation and acquisition of competence for embryonic development. Besides the effect of reducing cellular lipid content, l-carnitine (l-car) has an antioxidant effect by reducing the levels of reactive oxygen species (ROS) and protecting cells from apoptosis. However, the association of ALA and l-car has never been tested. This study was conducted to evaluate the effects of supplementation of IVM medium of bovine oocytes with ALA, l-car or the association of both (ALA+l-car) on embryonic development and blastocysts reactive oxygen species (ROS) levels and occurrence of apoptosis. Cumulus-oocyte complexes (n = 2241, in 11 replicates) were matured during 22 h at 38.5°C and 5% CO2 in air, in TCM-199 medium with bicarbonate, hormones and 10% FCS (control group), also supplemented with 100 μM ALA group; or 5 mM l-car (l-car group); or 100 μM ALA associated with 5 mM l-car (ALA+l-car group). After fertilisation (Day 0), zygotes were cultured 7 days in SOF that was supplemented with 0.5% BSA and 2.5% FCS, in 5% CO2 in air at 38.5°C. The cleavage and blastocysts rates were evaluated, respectively, at Days 3 and 7. Blastocysts were stained with 5 mM of H2DCFDA (Molecular Probes, Invitrogen, Carlsbad, CA, USA) and TUNEL (In Situ Cell Death Detection Kit, Roche Applied Science, Boston, MA, USA), to evaluate the ROS levels and the blastomers apoptotic index, respectively. The ROS (n = 115) and TUNEL (n = 102) stained blastocysts were evaluated under an epifluorescence microscope (excitation 495 nm/510–550 nm and emission 404 nm/590 nm), and the ROS levels (expressed as arbitrary fluorescence units) were measured by Q-Capture Pro image software (Q Imaging, Surrey, BC, Canada). The fluorescence intensity values were subtracted from mean values of background in the images. The variables were analysed by ANOVA followed by Tukey’s test (P < 0.05) and data are presented as mean ± s.e.m. There was no effect (P > 0.05) of the supplements during IVM on cleavage and blastocysts rates (%), respectively, for control (81.1 ± 1.8 and 29.0 ± 3.1), ALA (80.5 ± 2.1 and 29.7 ± 2.3), l-car (79.5 ± 2.8 and 29.2 ± 2.3), and ALA+l-car (82.2 ± 1.1 and 30.5 ± 2.0) groups. The oocytes supplementation resulted in a decrease (P < 0.05) in ROS levels for ALA (0.84 ± 0.04), l-car (0.85 ± 0.03) and ALA+l-car (0.82 ± 0.02) groups, compared to the Control (1.00 ± 0.05). Consequently, the percentage of apoptotic blastomeres decreased (P < 0.05) after ALA (6.9 ± 1.0%), l-car (7.5 ± 1.2%) and ALA+l-car (4.6 ± 0.7%) supplementations, unlike to the Control group (12.0 ± 1.2%). In conclusion, the supplementation with ALA, l-car or ALA+l-car during IVM did not affect the blastocyst development, but led to a reduction in ROS levels and in the apoptotic index of such blastocysts. These findings may be due to some antioxidant effect of these supplements in the oocytes and/or the produced embryos. Financial support was through FAPESP (#2012/10084–4 and #2013/07382–6).

136 COMPARISON OF NCSU-23 AND ALPHA-MINIMAL ESSENTIAL MEDIA IN THE DEVELOPMENT OF ISOLATED PORCINE PREANTRAL FOLLICLES IN VITRO

To develop a preantral follicular culture system that will support follicular growth and result in fertilizable oocytes, we conducted an experiment designed to determine the best medium for culture. In our preliminary experiment, we compared 2 common base media used for porcine oocytes: α-minimal essential medium and NCSU-23. Ovaries were collected from prepubertal gilts at a local abattoir and transported to the laboratory in saline solution (0.9% NaCl) maintained at 30–35°C. The ovaries were cut into small pieces (1–3 mm), and preantral follicles were isolated mechanically. Preantral follicles from 280 to 300 μm in diameter were collected into a small dish containing medium TCM199 (Lonza 12–117F) supplemented with 5% fetal bovine serum. The follicles were transferred from the collecting medium to the culture medium that consisted of base medium (NCSU23 or α-minimal essential medium) supplemented with 3.5 μg mL–1 of insulin, 10 μg mL–1 of transferrin, 100 μg mL–1 of l-ascorbic acid, 7.5% porcine serum, and 1.5 ng mL–1 of FSH. The follicles were randomly distributed to the different experimental treatments and cultured for 6 days in 24-well cell culture plates, with 3 follicles per well in 280 μL of culture medium. The culture was carried out at 38.5°C in 5% CO2 in air. Culture medium was changed every 2 days with freshly prepared medium. The diameters of follicles were measured every 2 days, and each follicle was photographed and evaluated at 20× magnification. Forty-two follicles per group were analysed and collected in 4 replicates. Data were statistically analysed with ANOVA using the Generalized Linear Model (GLM) procedure (SPSS, version 18, SPSS Inc., Chicago, IL, USA), where the independent variable was the sample (group and day of culture). Tukey's post hoc test was used to perform multiple comparisons; the α level was set at 0.05. All data were expressed as quadratic means with standard error of the means. Only the antrum formation was evaluated by chi-square test. The results, reported in Table 1, show that there was no statistical differences between follicle size between NCSU23 or α-minimal essential media, but at Day 6 there was a positive trend (P = 0.08). Otherwise, when we compared the size inside the groups, we observed that the preantral follicles grew more in α-minimal essential media than in NCSU23. The percentages of antrum formation were 65 v. 76% (NCSU23 and α-minimal essential media, respectively). These results support the use of α-minimal essential media because it had a positive effect on the antrum formation, and that after Day 4 some follicles could undergo a regression phase. Future studies will be necessary to evaluate the molecular status and the hormone production. Table 1.Follicle size (μm) from Day 0 to Day 6

124 COMPARISON OF TWO EMBEDDING SYSTEMS FOR FOLLICULAR CULTURE

Recent research has focused on developing secondary, preantral follicles as a new way to collect female gametes to develop in vitro or to study folliculogenesis. Secondary follicles are abundant in ovaries of almost all females, and can be isolated from the ovarian cortex of fresh or cryopreserved tissue. These follicles must be matured to obtain mature oocytes, but the mechanism for early follicle development is not well understood. Currently, culture techniques are being developed to promote the growth and quality of these follicles. The aim of this preliminary study was to compare 2 embedding systems for porcine follicles to improve preantral follicle culture. We collected secondary preantral follicles isolated from porcine ovaries recovered from the abattoir. Ovaries were transported to our laboratory in saline solution (0.9% NaCl) and cut into smaller fragments with a scalpel. Follicles were further mechanically isolated with a 23 1/2-gauge needle and placed into a dish with medium TCM199 plus Hepes (Lonza 12–117F) supplemented with 5% fetal bovine serum (FBS). Secondary follicles less than 300 µm were selected. Furthermore, only clear follicles with dark oocytes and no antrum were used. The selected follicles were randomly divided into 5 different treatment groups: 1) embedded in 0.5% alginate gel; 2) 1.0% alginate gel; 3) 1.0% agarose gel; 4) 2.0% agarose gel; and 5) control. Three follicles were placed in each well with 280 µL of α-minimal essential medium supplemented with 3.5 μg mL–1 of insulin 10 μg mL–1 of transferrin, 100 μg mL–1 of l-ascorbic acid, and 7.5% porcine serum and cultured for 4 days at 39°C at 5% CO2. Media (140 µL) from each well was changed on Day 2 and saved for metabolic and functional analysis. Initial diameters and measurements on Day 2 and 4 were taken to analyse dimensional growth. We evaluated 6 follicles per group per replicate, for a total of 3 replicates (18 per group). All recorded parameters were subjected to a 2-way ANOVA using the procedure of the Generalized Linear Model (SPSS, 18th version, SPSS Inc., Chicago, IL, USA). Independent variable was the result of the balance between the size on Day 2 and 0 and the size on Day 4 and 0 time of observation. Data were normally distributed. Least squares means tests were used to perform statistical multiple comparisons. The α level was set at 0.05. All data were expressed as quadratic means and mean standard errors. The results in the Table 1 show that at Day 2 there was no difference between groups. However, after 4 days of development, the group agar 2% and alginate 1% showed a statistical difference when compared with the control. These results suggest that there is a positive effect when the follicles are embedded during culture. Table 1.Growth of Follicles (in μm) since Day 0 in different embedding systems

58 EFFECT OF LYSOPHOSPHATIDIC ACID ON PORCINE OOCYTE IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER PARTHENOGENETIC ACTIVATION AND IVF

Lysophosphatidic acid (LPA) is a signalling molecule derived from phospholipid, known to have biological activities such as stimulating cell proliferation, differentiation, and migration. The purpose of this study was to investigate the effect of LPA on in vitro maturation (IVM) of porcine oocytes and subsequent embryonic development after in vitro fertilization (IVF) and parthenogenetic activation (PA). We examined nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels and subsequent embryonic development after IVF in porcine oocytes matured in TCM-199 media containing either 0, 10, 30, or 60 μM LPA (No. of cultured oocytes 225, 225, 230, and 226, respectively). All data were compared for all groups using one-way ANOVA, followed by Duncan’s multiple range test. After 44 h of IVM, no significant difference was observed for nuclear maturation from the different (0, 10, 30, and 60 μM) LPA treatment groups (86.61, 87.48, 92.97, and 88.69%, respectively) compared with the control (P < 0.05). The 30 μM LPA treated group exhibited a significant (P < 0.05) increase in intracellular GSH levels and decrease in intracellular ROS levels compared with other LPA-treated groups. Oocytes matured with 30 μM LPA during IVM had significantly (P < 0.05) higher cleavage rates after PA (88.50%) than other LPA-treated groups (79.42, 83.87, and 83.65%, respectively). Blastocyst formation rates following PA increased up to 30 μM LPA, but the rates decreased in the 60 μM LPA-treated group (49.64, 53.28, 57.00, and 51.09%, respectively). However, a statistical difference was not observed (P < 0.05). Upon evaluation of embryonic competence following IVF, treatment with 30 μM LPA during IVM significantly increased cleavage rates (70.44%; P < 0.05) compared with 0 and 60 μM LPA-treated groups (61.51 and 65.27%, respectively). Blastocyst formation rates improved with increasing concentrations of LPA up to 30 µM; however, the rates decreased in the 60 μM LPA-treated group (26.34, 30.10, 33.26, and 27.77%, respectively). However, like the preceding PA blastocyst formation rates, a statistical difference was not observed (P < 0.05). In conclusion, treatment with 30 μM LPA during IVM improved the developmental potential of PA and IVF porcine embryos by increasing the intracellular GSH level, thereby decreasing the intracellular ROS level during oocyte maturation. This work was supported, in part, by a grant from the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ011077, PJ011288), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2013R1A2A2A04008751). These are the results of a study from the Leaders INdustry-University Cooperation Project 2015-B-0013–010118, supported by the Ministry of Education, Republic of Korea.

213 MATURATION OF OOCYTES WITH FOLLICULAR FLUID FROM GILTS CONSUMING HIGH FAT AND FRUCTOSE AFFECTS SUBSEQUENT EMBRYO DEVELOPMENT

Infertility among women has become a growing issue in the world requiring a significant number to seek treatment by means of assisted reproductive technologies. One suggested reason for the fertility issue, which is known to specifically affect oocyte quality, is the modern diet. Previously, we have demonstrated that feeding a high-fructose diet to gilts led to poor reproductive tract characteristics and infertility. In this study, pre-pubescent gilts were fed either a high-fructose; high-fat diet (HFHF), with 15% beef tallow and 35% fructose; or an industry control diet (IND). Porcine follicular fluid (pFF) collected from these gilts was introduced into in vitro maturation systems to determine whether characteristics of the follicular fluid affect oocyte competence and embryo development. Follicles from ovaries, collected at a local abattoir, were aspirated by an 18 G needle attached to a 10-mL sterile syringe. Then selected cumulus‐oocyte complexes were maturated in vitro in a TCM-199 maturation media with cysteine, glucose, sodium pyruvate, epidermal growth factor (EGF), FSH, LH, and 20% pFF from treatment groups. Additionally, another group of oocytes, labelled follicle fluid free (FFF), were maturated in TCM-199 media without pFF. Three replicate experiments were conducted using a total of 365 oocytes, 124 FFF, 121 IND, and 120 HFHF. Oocytes were denuded by exposure to 0.1% hyaluronidase and oocytes that reached metaphase II (MII) were selected for in vitro fertilisation. After 5 h of co-incubation in modified Tween medium B with milk powder (mTBM)-based IVF media, presumable zygotes were transferred to porcine zygote medium-3 (PZM-3). Blastocyst frequency was recorded on Days 5 and 6. Day 6 blastocysts were stained with Hoechst for total cell number evaluation. The frequencies of blastocyst formation among the treatment groups were compared by a chi-squared test, and total cell numbers were compared by Student's t-test. Statistical significance was defined by P < 0.05. The frequency of oocytes reaching metaphase II (MII) were observed as 77.4% FFF, 72.7% IND, and 71.7% HFHF (P > 0.05), indicating the supplementation of pFF did not affect maturation. Day 5 blastocysts were observed at frequencies of 8.3% FFF, 6.8% IND, and 4.7% HFHF and did not differ. However, frequency of Day 6 blastocysts from HFHF group was tended to be lower compared with that of other groups; 12.5% FFF, 11.4% IND, and 4.7% HFHF (P = 0.06 and P = 0.1). Average total cell number of Day 6 blastocysts observed were 41.0 ± 9.1 FFF, 36.0 ± 8.9 IND, and 48.3 ± 10.6 HFHF. The total cell number from HFHF group tended to be higher than only that of IND group (P = 0.07). Based on these results, we concluded that the follicular fluid of females consuming HFHF diets did not have impact on nuclear maturation of oocytes but might affect oocyte competency, thus resulting in detrimental effects on subsequent development of embryos, especially blastocyst formation. Further studies will help us identify more specific effects of nutrition on oogenesis and subsequent embryo development.

Gene expression in sheep cumulus-oocyte complexes meiotically inhibited with roscovitine

The majority of mammalian oocytes destined for in vitro maturation (IVM) have not undergone all molecular and structural changes necessary for competence acquisition to support the fertilization and early embryogenesis. In this context, different methods able to provide a transient arrest of meiosis resumption have been tested in order to improve the in vitro developmental potential of oocytes. Based on that, our study aimed to evaluate the effect of temporary meiosis inhibition using roscovitine on gene expression in sheep oocytes and cumulus cells. For this, cumulus-oocyte complexes (COCs) were cultured for 6h in modified TCM199 medium with (Rosco) and without (Control) 75μM roscovitine. Subsequently, they were in vitro matured for a further 18h in inhibitor-free TCM199 medium supplemented with gonadotropins. At 0, 6 and 24h of culture, nuclear status of oocytes and expression of selected genes were evaluated by Hoescht staining and qRT-PCR, respectively. The analysis of oocyte chromatin organization revealed that roscovitine efficiently inhibited the meiosis of sheep oocytes for 6h and its action was completely reversed after 18h of in vitro maturation in inhibitor-free medium. Besides, no detrimental effect on cumulus expansion was observed. The expression profile of most investigated genes in cumulus cells (PTX3, GREM1, GLUT1, PTGS2, ALK5, ALK6) and oocytes (ZAR1, NLRP5, SOD1, BMP15, GDF9) was similar between Control and Rosco treatments and the ratio BCL2/BAX was maintained in both cell types even in the presence of roscovitine. These results indicate that reversible meiotic arrest promoted by roscovitine, at the concentration and exposure time studied, neither impaired nor improved the expression of investigated genes in sheep oocytes and cumulus cells. Moreover, the efficiency of temporary meiotic arrest and the absence of deleterious effect on COCs suggest that roscovitine provides a useful method for transportation or manipulation of sheep oocytes at onset of maturation. However, further investigations are necessary to evaluate the benefits of roscovitine treatment on in vitro development of sheep embryos and its effects on cellular ultrastructure.

Development of Intergeneric Canine Embryo Using Bovine and Porcine Oocyte as Cytoplasm Recipient

This study wa conducted to increase the efficiency of canine embryo production by intergeneric somatic cell nuclear transfer (SCNT) technology. The effect of oocyte recipient for development of intergeneric canine somatic cell cloning embryos were examined. Bovine and porcine cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries and matured in TCM-199 medium depend on species. Adult dog fibroblasts collected from 3.5 years old Afghandhound dog, and cell between passage 1 and passage 10 used for intergeneric somatic cell cloning using bovine and porcin oocytes as oocyte cytolplasm donor. The result showed that oocytes from bovine and porcine can de-differentiated canine nucleus and no different between bovine and porcine oocyte in fusion and embryo development in vitro. Canine intergeneric cloned embryos developed to morula stages in vitro.

The effect of temperature during liquid storage of in vitro–matured bovine oocytes on subsequent embryo development

The aim of the present study was to optimize the temperature for the temporal storage of matured bovine oocytes. In vitro–matured bovine oocytes were preserved in HEPES-buffered TCM199 medium supplemented with 10% newborn calf serum at different temperatures (4 °C, 15 °C, 25 °C, and 38.5 °C) for 20 hours. Embryo development and blastocyst quality after in vitro fertilization, cytoplasmic ATP and glutathione levels in oocytes, and the frequency of apoptotic oocytes were compared among storage groups and a control group without storage. Among the storage groups, those at 25 °C and 38.5 °C showed the highest rates of blastocyst development (19.3% and 24.5%, respectively) compared with those stored at 4 °C and 15 °C (8.5% and 14.9%, respectively); however, blastocyst formation rates in all storage groups were lower than that in the control group (39.8%; P < 0.05). Storage at 38.5 °C and 15 °C was associated with reduced cell numbers in resultant blastocysts compared with the control and the 25 °C storage groups. Storage at 4 °C reduced metabolic activity of oocytes characterized by their lower ATP levels compared with the other groups. Storage for 20 hours significantly reduced the glutathione content in oocytes in all groups in a similar manner, irrespective of the temperature. Storage at 4 °C or 15 °C but not at 25 °C and 38.5 °C significantly increased the percentage of apoptotic oocytes compared with the control group. In conclusion, 25 °C was found to be the most suitable temperature for the temporal storage of matured bovine oocytes regarding both the developmental competence of oocytes and the quality of resultant blastocysts.

Effect of hormones, follicular fluid, serum and media on in vitro maturation of porcine oocyte

The present study was conducted to evaluate the effect of hormones, follicular fluid, serum and media on in vitro maturation (IVM) of porcine oocyte. The procedures of IVM could be used for basic research purposes or commercial utilization. The IVM of oocyte is a critical step in in-vitro embryo production and it needs to be carried out in a precise manner under optimal conditions for subsequent fertilization and embryo development. For this purpose ovaries were collected from slaughtered pigs from an abattoir. Ovaries (1,232) were aspirated in 52 trials for IVM and fertilization for this study. Dulbecco Modified Eagle's medium (DMEM) or tissue culture medium (TCM-199) supplemented with gentamycin (50μl/ml), sodium pyruvate (100 mmol) and 10% serum, viz. estrus sow serum (ESS) or fetal calf serum (FCS) with or without porcine follicular fluid (PFF) and hormones such as pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) were used. The maturation was assessed by loosening of cumulus cells, enlargement of peri-viteline space or extrusion of first polar body and examination of metaphase II by aceto-orcein stain. The results revealed that DMEM with ESS yielded 53% while TCM+ESS yielded 45% maturation rates. The replacement of ESS with FCS resulted in 48.50 and 35.33% maturation in DMEM and TCM-199 medium, respectively. The addition of PFF in DMEM+ESS improved the maturation rate and was found better (74.55%) than TCM-199+ESS (65.35%). However, the replacement of ESS by FCS in either DMEM orTCM-199+PFF did not differ significantly. It can be concluded from the present study that use of DMEM+ESS+PFF along with hormones PMSG + luteinizing hormone (LH) for first 20-24 h followed by hormone free medium for next 20-22 h i.e. 42-44 h gave optimum (81.5%) in-vitro maturation rates in pigs. DMEM with ESS and PFF with hormone (PMSG/HCG) yielded highest maturation (81.50%) rate than TCM-199 with ESS, PFF and hormone (PMSG/HCG) which showed maturation rate of 76.59%.

Moringa oleifera extract modulates the expression of fertility related genes and elevation of calcium ions in sheep oocytes

Success of in vitro maturation of mammalian oocytes and subsequently embryo production are normally affected by several factors such as hormonal alterations and culture conditions including nutrients or natural extracts. The leaves of Moringa oleifera are a good source for both macro- and micronutrients, and rich source of β-carotene, protein, vitamin C, calcium, and potassium. Gene expression techniques have become a powerful tool to analyze the relative abundance of transcripts related to quality of oocytes. In the present study, the relationship between mRNA expression profiles of maturation related genes [Cyclin B, cell division control (CDC2, CD44), extracellular signal-regulated kinases (ERK2) and c-mos genes], progression of meiosis and calcium concentration of sheep oocytes was evaluated. To carry out this study cumulus–oocyte complexes (COCs) aspirated from sheep ovaries were used. COCs were allocated in several groups cultured in TCM-199 medium without or with hormones plus different concentration of M. oleifera extract (20, 50 and 100μg/ml). The results indicated that the relative abundance of the maturation related genes was affected by the concentration of M. oleifera extract and meiotic progression. Moreover, sheep oocytes cultured with hormones plus M. oleifera extract expressed high levels of the [Ca2+] concentration. The results suggest that M. oleifera combined with hormone supplementations improved the rate of maturation of sheep oocytes and could have acted as a promoter to induce mRNA expression and synthesis of essential protein, e.g. MPF, for the maturational processes.

A comparative study of parthenogenetic activation and in vitro fertilization of in vitro matured caprine oocytes.

The aim of the study was to compare the parthenogenetic activation and in vitro fertilization (IVF) of in vitro matured caprine oocytes. A total of 881 cumulus-oocyte complexes (COC's) were collected from 243 ovaries. Oocytes were matured in TCM-199 medium containing eCG (20 IU/ml), hCG (20 IUµg/ml), oestradiol-17β (1 µg/ml), BSA embryo tested (3 mg/ml) supplemented with 10% fetal bovine serum at 38.5°C and 5% CO2 in an incubator under humidified air for 27 h. Based on cumulus expansion, the maturation rate was 86.86%. Morphological matured oocytes (n=749) were selected, denuded and randomly divided into two groups. Group 1 (n=223) in vitro matured oocytes activated with 5 µm calcium ionophore for 5 min and cultured in mCR2aa medium containing 5 mM DMAP for 4 h. After 4 h of DMAP treatment, the presumptive zygotes were washed and cultured in the embryo culture medium. Group 2 (n=526) in vitro matured oocytes processed for IVF in mTALP using fresh semen of a fertile pure bred adult Sirohi buck and in vitro culture in mCR2aa medium. Development of putative zygotes was observed every 24 h till day 9 post activation or fertilization under inverted phase contrast microscope. The cleavage rate, morula and blastocyst percentage in groups 1 and 2 were 67.36%, 23.07% and 9.23%, and 30.99%, 19.63% and 9.82%, respectively. The results indicated that the cleavage rate was comparatively higher following parthenogenetic activation with ionomycin/6-DMAP than IVF.

In vitro maturation of nude caprine oocytes recovered during oocyte collection

The present study was undertaken to investigate the effect of granulosa cell co-culture, granulosa monolayer and hormone on in vitro maturation of nude caprine oocytes. Nude oocytes (644) having evenly granulated cytoplasm were recovered by puncture technique. The oocytes were divided into 6 groups and matured in TCM-199 medium supplemented with 10% fetal bovine serum (FBS) and granulosa cell co-culture/ granulosa monolayer/ hormones. After maturation (27h), the oocytes were fixed for 15 min in 2.5% (w/v) glutaraldehyde in phosphate buffer saline,washed twice with PBS, stained with 0.1mg/ml DAPI in PBS and mounted on slides. The evaluation of nuclear status was done by epiflourescence microscope by observing either a polar body release or presence of two shiny chromatin spots. The maturation rate of oocytes in group 1, 2, 3, 4, 5 and 6 were 23.47, 32.40, 31.77, 26.00, 38.09 and 51.37%, respectively. The results of the study indicated that TCM-199 supplemented with hormones and coculture granulosa cells significantly increases the maturation rate of in vitro matured nude caprine oocytes.