μM Cysteamine Research Articles

Kültür Medyumuna İlave Edilen Antioksidanların Blastosist Gelişim Oranlarına Etkisi

The present study, it was aimed to determine the effect of antioxidants added to culture media on blastocyst development rates in in vitro embryo production. The material of the study consisted of oocytes collected from the ovaries taken from the slaughterhouse. Cumulus oocyte complexes (COC) were collected and classified under a stereomicroscope. Oocytes included in the study were subjected to maturation and fertilization stages. Probable zygotes were transferred to the culture (IVC) containing antioxidants (L-ergothionine 100 μM (n: 163), Vitamin E 100 μM (n: 151) Cysteamine 50 μM (n: 154) and were cultured in a tri gas incubator (Hera Cell- 6% O2%, 6%CO2, 88%N). Blastocyst rates and embryo quality were determined on the 6th and 7th days in culture medium. Differences in IVMFC stages were evaluated by chi-square test. 966 oocytes were collected from 162 ovaries collected from the slaughterhouse. It was determined that the number of oocytes per ovary was 5,96, and the number of A and B quality oocytes was 4.26. It was determined that 655 (94.93%) of a total of 690 oocytes undergoing in vitro maturation were mature. The cleavage rates of the groups were 83.44%; 80.79%; 79.87%, and 83.96%, respectively. 140 (21.37%) blastocysts were obtained from 655 oocytes taken into the culture stage and the blastocyst rates in the groups were 33.13%; 8.61%; 7.79%, and 32.62%, respectively. As a result of the study, it was determined that the rates of blastocysts in the L-ergothioneine added the group was similar to the control group, but the blastocyst rates decreased significantly in the cysteamine and Vitamin E added groups. It was thought that this decrease might have been affected by the dose of antioxidants or the adequacy of oocyte development

THE EFFECTIVITY OF CYSTEAMINE SUPPLEMENTATION ON IMPROVING THE IN VITRO FERTILIZATION RATE OF SHEEP OOCYTES

The purpose of this study was to evaluate the effectiveness of cysteamine supplementation on the maturation medium and/or in vitro fertilization medium with regards to improving the normal fertilization rate of sheep oocytes, which are characterized by the formation of two pronuclei. Grade A and B oocytes were matured in medium-199 with 0.3% bovine serum albumin (BSA), 10 IU/mL follicle stimulating hormone (FSH), 10 IU/mL human chorionic gonadotrophin (hCG), and 50 μg/mL gentamicin added for 24 hours in a 5% CO2 incubator at 39° C. The treatment group was divided into the following groups: a control group with no cysteamine (P1), a group with 100 μM cysteamine in the maturation medium (P2), a group with 100 μM cysteamine in the fertilization medium (P3), and a group with 100 μM cysteamine both in the maturation and fertilization medium (P4). The fertilization was carried out by incubating sperm-oocytes for 12 hours and then staining them with 1% aceto-orcein to observe the formation of a pronucleus. Normal fertilization rates obtained by each treatment group were 56.5% (P1), 57.1% (P2), 57.8% (P3), and 59.9% (P4) with no significant difference (P0.05) between groups. It was concluded that 100 μM cysteamine supplementation in both the maturation medium and fertilization medium was not able to increase the normal fertilization rate of sheep oocytes.

Effects of different concentrations of eugenol in maturation medium on bovine oocytes, oxidative status and preimplantation embryos

Context Eugenol is a bioactive compound with defined attributes, including a role in reducing oxidative stress. Therefore, it is an interesting candidate for in vitro embryo production that has an environment that favours the formation of reactive oxygen species. Aim The objective of the study was to evaluate different concentrations of eugenol in bovine oocytes during in vitro maturation to observe the oxidative status and embryonic development. Methods Oocytes were allocated into five groups: control (without antioxidant), 100 μM cysteamine, 83 μM eugenol (E83), 100 μM eugenol and 120 μM eugenol. Three experiments were performed with 23 replicates. Oocytes were evaluated for metaphase II, first polar body, cytoplasmic maturation, cumulus cell expansion and viability. Oxidative status was measured by mitochondrial membrane potential, reactive oxygen species and glutathione. Oocytes were artificially activated and cultured in vitro. Key results The presence of eugenol at 83 μM improved the metaphase II rate compared with all treatments (P < 0.05). E83 and 100 μM cysteamine improved first polar body extrusion, cumulus cell expansion, viability and mitochondrial aggregation rates (P < 0.05). All antioxidant treatments resulted in increased cytoplasmic maturation and decreased mitochondrial membrane potential (P < 0.05). Reactive oxygen species levels decreased, and glutathione levels increased with E83 and 100 μM cysteamine treatments (P < 0.05). Finally, the E83 group increased the cleavage rates, embryo development, number of expanded blastocysts and number of blastomeres (P < 0.05). Conclusions A low concentration of eugenol (83 μM) is sufficient to generate a significant effect, attenuating the oxidative status and optimising i n vitro maturation and embryo development. Implications This study provides information of a new natural antioxidant with defined concentration, 83 μM eugenol, representing a lower cost alternative capable of improving the efficiency of in vitro embryo production in cattle.

Cysteamine in Maturation Medium Enhances Nuclear Maturation and Fertilization Rate of Sheep Oocytes In Vitro

Low nuclear maturation and fertilization rate is one obstacle in the in vitro embryo production which decrease embryo yield. This problem is presumable related with high production of reactive oxygen species (ROS) during maturation process. Glutathione (GSH) as an antioxidant is well known to overcome effect of ROS production. GSH synthesis in the cytosol part of the oocyte cytoplasm is influenced by cysteine availability. It is therefore, this research was conducted to evaluate the ability of cysteamine to provide cysteine availability as GSH precursor on the nuclear maturation and fertilization rate of sheep oocytes. Results of this experiment revealed additional cysteamine at 150 µm and 200 µm could significantly improve nuclear maturation rate. On the other side, although additional of cysteamine at 50 µm could not improve nuclear maturation rate, however 50 µm cysteamine in the maturation medium could significantly improve the fertilization rate. Based on those experiment results, it seems that the additional cysteamine might be improve not only GSH availability but also the oocyte quality which characterized the ability of pronuclear formation. This finding strongly suggested that additional cysteamine in the maturation medium could improve nuclear maturation and fertilization rate of sheep oocytes.

66 Bovine corpus luteum affects embryo developmental competence and production

This study was conducted with the aim to determine the effect of the bovine corpus luteum on in vitro embryo production. Immature cumulus-oocyte complexes (COC) were aspirated from abattoir ovaries from cows (mainly Holstein and dairy crossbred cows and heifers) with (ipsilateral; CL+) and without (contralateral; CL−) corpus luteum (CL), and from cows without CL in any of the ovaries. The average weight of the ovaries from each group was 10.4±0.25, 5.7±0.25, and 6.1±0.25g for CL+, CL−, and C, respectively. The experiment was completed within 12 replicates (100 ovaries per group). The COC were cultured in maturation medium (TCM-199 supplemented with 10% fetal bovine serum, 100µg mL−1 sodium pyruvate, 0.75mg mL−1 of L-glutamine, 4µg mL−1 of FSH-p, 100 µM cysteamine, and 250µg mL−1 of gentamicin) followed by IVF (synthetic oviducal fluid medium supplemented with 10µg mL−1 heparin) and in vitro culture (citrate synthetic oviducal fluid medium). On Day 7 after IVF, the embryos were evaluated and classified into morulae (M), early blastocysts (EB), regular blastocysts (RB), expanded blastocysts (ExB), and hatched blastocysts (HB), and the embryos with quality 1 (according to IETS criteria) were recorded. Data were analysed by logistic regression and general linear model of SAS (SAS Institute Inc., Cary, NC, USA), and means were compared by the least squares means method. Results of cleavage, embryo rate at Day 7, and rates of M+EB, RB, ExB, and HB are shown in Table 1. The number of embryos per ovary was greater (P<0.01) in CL+ (1.16±0.11) than in CL− (0.45±0.15) and C (0.55±0.15). Also, the number of embryos per cultured oocyte was significantly greater in CL+ than in CL− and C (0.27±0.02v. 0.14±0.03 and 0.15±0.03, respectively; P<0.01). The results of this study reveal that the presence of the corpus luteum in the ovary at the time of the oocyte recovery affects the developmental capacity of the bovine embryos, and such influence probably occur through intraovarian interactions. Table 1.Effect of bovine corpus luteum on embryo production

159 IN VITRO DEVELOPMENT OF CAPRINE EMBRYO USING CRYOPRESERVED BLACK BENGAL BUCK SEMEN

The purpose of this study was to check the competence of cryopreserved black Bengal buck semen to produce goat embryo through IVF. So far, cryopreserved black Bengal buck semen has not been used to produce goat embryos by IVF. For the study, fresh goat oviducts and ovaries were collected from slaughterhouse in a thermo flask containing 0.9% saline solution supplemented with antibiotic (400 IU mL−1 penicillin and 500 mg mL−1 streptomycin) at 30–35°C and transported to laboratory within 2–3 h of slaughter. Cumulus-oocyte complexes were collected from slaughterhouse ovaries, washed 5–6 times, and cultured in maturation media (TCM-199 + 10% FBS + 10 mg mL−1 FSH-P + 0.81 mM sodium pyruvate + 5% follicular fluid + 50 mg mL−1 gentamicin sulfate + 1 μg mL−1 oestradiol + 100 μM cysteamine) for 27 h in a 5% CO2 incubator at 38.5°C with maximum humidity. After 27 h of culture, cumulus cells were separated from matured oocytes by repeated pipetting using a fine pipette in fertilization Bracket and Oliphant’s (BO) medium. After removal of cumulus cells, the oocytes were transferred to acidified Tyrode’s medium for zona thinning for 52 s and were co-incubated with capacitated sperms for fertilization in fertilization BO medium at 38.5°C in 5% CO2 in air with maximum humidity. In the experiment I, freshly collected buck semen was used for IVF after processing for capacitation. In experiment II, cryopreserved buck semen straws were thawed and sperm were capacitated in vitro and used for fertilization. After 5 h of co-incubation, presumptive zygotes were washed thoroughly and cultured in embryo development medium for cleavage. Three different in vitro development media (RVCL, mSOF + 2.5% BSA, and KSOM + 0.5% BSA) were used. After 40 to 42 h, cleavage was observed and embryos were co-incubated with oviducal cells in replacement media for further development. In the fresh group, overall cleavage rates (%) were 37.76 ± 2.98, 39.60 ± 1.75, 29.01 ± 1.74 and morula formation rates (%) were 7.72 ± 3.38, 6.03 ± 1.29, 3.00 ± 3.00 in RVCL, mSOF, and KSOM media, respectively. However, in the cryopreserved group the overall cleavage rates (%) were 29.17 ± 2.56, 27.70 ± 2.31, and 24.17 ± 1.44 in RVCL, mSOF, and KSOM media, respectively, and morula formation (%) was achieved 2.93 ± 0.97 in RVCL media. These results indicate that cryopreserved black Bengal buck semen have competence to produce embryos and could be used for embryo development through IVF.

Influence of Epidermal Growth Factor with Cysteamine on in -Vitro Buffalo Embryo Development

FOR IMPROVING embryo development in buffalo, two experiments were conducted. The first one was carried out to evaluate the different concentrations (0, 5, 25, 50 ng/ml) of epidermal growth factors (EGF) on developmental competence of buffalo oocytes. The selected oocytes were cultured in the four concentrations of EGF. The embryo cleavage rate was significantly higher in oocytes exposed to 5, 25, 50 ng/ml EGF than control. There were no significant difference in four groups (0, 5, 25, 50 ng/ml EGF) in the rate of morula. But, better cleavage and blastocyst rates were observed at 5 ng/ml EGF. In the second experiment, the additive effect of 5 ng/ml EGF with 50 µM cysteamine on maturation and embryo development was studied. Oocytes were collected, matured and cultured in three groups. In the first group, media supplemented with 5 ng/ml EGF + 50 µM cysteamine combination. In the second group, media supplemented with EGF. The third group was supplemented with cysteamine. There was a significant increase in cleavage rate in combination group than EGF (P< 0.05) and cysteamine (P< 0.01) groups. But there was no significant difference in cleavage rate between EGF and cysteamine. The morula percentage was nearly similar in the three groups. But blastocyst rate was significantly (P<0.05) higher in combination group than cysteamine. Thus better cleavage and blastocyst rate were observed at combination group. It is concluded that, the addition of 5 ng/ml EGF in buffalo oocytes during in vitro culture was the best concentration for embryo developmental competence. Higher cleavage and blastocyst rate were achieved by combination of epidermal growth factor and antioxidant.

Cysteamine and b-mercaptoethanol supplementation improves the in vitro embryo development in buffaloes (Bubalus bubalis)

The objective of the present experiment was to investigate the effect of cysteamine and b-mercaptoethanol supplementation on in -vitro maturation, cleavage of oocytes and development of embryo in buffalo (Bubalus bubalis). Oocytes were aspirated from abattoir ovarian follicles of 3-10 mm diameter followed by maturation in the media in vitro containing cysteamine/b-mercaptoethanol (treatment) and without antioxidant (control). Matured oocytes were co-incubated with sperm (approx.1×106/ml) of Murrah bull in mSOF medium using heparin (10 μg/ml). After 22 h of oocyte-sperm incubation, fertilized oocytes were stripped of cumulus cells and cultured in mSOF medium for 8 days to study embryo development. The oocyte maturation rate improved significantly (P&lt;0.05) following addition of 50 or 100 μM of cysteamine and 10, 50 and 100 μM of b- mercaptoethanol (ME), respectively as compared to control. The cleavage rate was found to be significantly (P&lt;0.05) higher at 50 and 100 μM of cysteamine and at all concentrations of b-mercaptoethanol as compared to control and development of embryos to morula stage was significantly (P&lt;0.05) improved with 50 μM cysteamine/ b-mercaptoethanol.

Influence of cysteamine supplementation during in vitro culture of early stage caprine embryos on blastocyst production

The objective of this study was to evaluate the effect of cysteamine supplementation on embryo development in mCR2 media. The COCs (1251) were matured in TCM–199 medium containing FSH (5µg/ml), LH (10µg/ml), follicular fluid (10%), FBS (10%) with 3 mg/ml BSA for 27h at 38.5°C and 5% CO2 in an incubator. Matured oocytes were co-cultured with 1×106 spermatozoa/ml collected from a Sirohi buck in fertTALP (10% FBS+ 4mg/ml BSA and 10 µg/ml heparin) for 18 h in incubation. After 18 h of sperm-oocytes, co-incubation of oocytes with sperms were washed in embryo development medium to remove sperm cells adhered to zonapellucida. Presumptive zygotes (1,171) were selected and randomly divided into 2 groups. Group 1, the presumptive zygotes (610) were cultured in mCR2aa as a control for 12 days. Group 2, the presumptive zygotes (561) were culture in mCR2aa medium supplemented with 100 µM cysteamine for 10 days. The percentage of cleavage, morula, and blastocyst production in groups 1 and 2 were 36.39% and 31.71% (cleavage); 21.62% and 30.89% (morula); 4.95% and 8.98% (blastocyst) respectively. In conclusion, results indicated that the addition of cysteamine to the IVC medium stimulates caprine embryo development and blastocyst production.

Influence of vascular endothelial growth factor and Cysteamine on in vitro bovine oocyte maturation and subsequent embryo development.

The objective of this study was to investigate the effect of VEGF and Cysteamine during in vitro maturation (IVM) of bovine oocytes on GSH content and developmental competence. For this purpose, experiments were designed to evaluate the effect of 0, 100, 300, and 500 ng/mL VEGF in IVM medium on: GSH content in oocytes and cumulus cells (Exp. 1) and subsequent embryo development (Exp. 2). Also, influence of adding 500 ng/mL VEGF and 100 μM Cysteamine to IVM medium on GSH content in oocytes and cumulus cells (Exp. 3) and oocyte developmental capacity (Exp. 4) were evaluated. Oocytes were matured in: a) Control; b) VEGF 0-3 h; c) Cysteamine 4-24 h; d) VEGF 0-3 h + Cysteamine 4-24 h; and e) VEGF + Cysteamine 24 h. The results showed that: i) VEGF did not alter GSH content in oocytes and cumulus cells; (ii) supplementation of 300 and 500 ng/mL VEGF increased blastocyst yield; (iii) the presence of VEGF + Cysteamine simultaneously during 24 h improved GSH content but not embryo development; and (iv) the presence of VEGF during the first 3 h + Cysteamine from 4 to 24 h increased GSH concentrations and subsequent embryo development. In conclusion, the addition of VEGF and Cysteamine in two sequential steps to maturation medium result in an improvement of cytoplasmic maturation, with a positive impact on oocyte developmental capacity by increasing the efficiency of in vitro blastocyst production. However, the effect was detrimental when both VEGF and Cysteamine were present during 24 of IVM.

227 SUPPLEMENTATION WITH CYSTEINE, CYSTEAMINE, AND CATALASE DURING IN VITRO MATURATION PROTECTS BOVINE PRE-IMPLANTATION EMBRYOS FROM ANTI-DEVELOPMENTAL ACTIONS OF MENADIONE

Menadione (MD), a naphthoquinone used as a vitamin K source in animal feed, can generate reactive oxygen species (ROS) and cause apoptosis. Hence, we examined whether MD reduces development of pre-implantation bovine embryos by inducing ROS production and apoptosis and tested the hypothesis that the anti-developmental actions of MD would be reduced by supplementation of the in vitro maturation (IVM) medium with antioxidants (a mixture of 0.6 mM cysteine + 100 μM cysteamine + 100 UI mL–1 catalase). Cumulus-oocyte complexes (COC; n = 3334; 50 per well in 11 replications) were matured in IVM medium (TCM-199 with bicarbonate, hormones, and 10% FCS) for 22 h. After fertilization, presumptive zygotes were cultured in synthetic oviduct fluid medium (SOF) with 2.5% FCS and 0.5% BSA up to 7 days (Day 0 = IVF). On Day 6, MD was included in the SOF medium (0 μM: Control; or 5.0 μM: MD5) during 24 h. All cultures were conducted at 38.5°C in 5% CO2 in air. In a second experiment, COC were matured in TCM-199 supplemented with or without the mixture of antioxidants (Antiox; only during IVM) and with or without MD at Day 6 in a 2 × 2 factorial design (Control, Control/Antiox, MD5, MD5/Antiox). The cleavage and blastocysts rates were evaluated on Days 3 and 7, respectively. A sample of the blastocysts (n = 1112) was stained for TUNEL (In Situ Cell Death Detection Kit, Roche, Indianapolis, IN, USA) to detect the apoptotic cells or with 5 μM H2DCFDA (Molecular Probes, Canada) to evaluate the ROS levels. Stained embryos were evaluated under an epifluorescence microscope and the images of embryos stained with H2DCFDA were analysed by Q-Capture Pro image software for determining the fluorescent intensity. The means (±s.e.m.) were compared by ANOVA followed by Tukey's test (P &lt; 0.05). In the first experiment, and the cleavage rates were similar (P &gt; 0.05) for Control (80.5% ± 0.9) and MD5 groups (78.9% ± 1.2), but the blastocyst rates were lower (P &lt; 0.05) in the MD5 group (25.9% ± 2.1) when compared with Control (36.4% ± 1.5). The rate of apoptotic cells in blastocysts was higher (P &lt; 0.05) in the MD5 group (19.2% ± 0.7) than in Control (10.0% ± 0.5), as well as the fluorescence intensity for ROS quantification (0.7 ± 0.1 v. 1.1 ± 0.1, respectively, for Control and MD5 groups). In an effort to rescue the impaired embryonic development, a mixture of antioxidants was added to the IVM medium and the results were transitional in response in which MD5/Antiox (28.3% ± 3.5) was intermediate (P &gt; 0.05) between the Controls [Control (38.2% ± 2.5) and Control/Antiox (34.7% ± 1.9) groups] and the MD5 (21.4% ± 2.9) group. Principal effects evaluated were the presence of MD (without: 35.9% ± 2.3 v. with: 24.8% ± 2.3; P &lt; 0.05) or Antiox (without: 28.4% ± 2.3 v. with: 32.3% ± 2.3; P &gt; 0.05). In conclusion, addition of MD to embryonic culture reduces the blastocyst development and increases intracellular levels of ROS and the occurrence of apoptosis. Anti-developmental actions of MD on embryonic development can be partially blocked by treating oocytes with a mixture of antioxidants during IVM.Financial support was provided by FAPESP (2012/10083-8 and 2013/07382-6).

308 VERBASCOSIDE TREATMENT DURING IN VITRO MATURATION IMPROVES THE EMBRYO DEVELOPMENT OF PREPUBERTAL OVINE OOCYTES

Verbascoside (VB), a bioactive polyphenol from olive mill wastewater with known antioxidant activity, was shown to act as a pro-oxidant molecule, by impairing energy/redox status and embryo developmental competence of prepubertal ovine oocytes when added at micromolar concentrations in a continuative 24-h in vitro maturation (IVM) exposure protocol (1). The aim of the present study was to determine whether a lower (nanomolar) VB concentration and a shorter exposure time (2 v. 24 h) during IVM may improve the maturation rates of prepubertal ovine oocytes and their subsequent embryonic development in vitro. Cumulus-oocyte complexes derived from the ovaries of slaughtered 1-mo-old prepubertal sheep oocytes underwent IVM in TCM 199 with 10% oestrus sheep serum, 0.1 IU mL–1 of FSH/LH, and 100 µM cysteamine, in 5% CO2 in air at 38.5°C for 24 h. Based on our previous results (Dell'Aquila et al. 2014 Biomed. Res. Int. 2014, 878062), VB was added in the IVM medium at 1.03 nM, and 2 incubation times (24 and 2 h) were tested. In the 2-h exposure group, after 2 h of exposure to VB, oocytes were washed and cultured up to 24 h without VB. A group of oocytes were cultured in absence of VB, as controls. Matured oocytes were fertilized with frozen-thawed ram semen in SOF medium for 22 h and zygotes were cultured in vitro for 8 days. Metaphase II (MII) cleavage and blastocyst rates were analysed by Chi-squared test. Embryo quality was evaluated by staining and total cell count of the blastocyst and analysis of variance (ANOVA) was applied. Differences were considered to be significant when P &lt; 0.05. Compared to controls, VB treatment at the concentration of 1.03 nM and 24 h of exposure had no effect on MII rates (196/268, 73% v. 226/323, 70% MII/cultured oocytes; P &gt; 0.05). However, this treatment allowed to obtain significantly higher rates of cleaved embryos/MII oocytes (156/196, 80% v. 165/226, 73%; respectively; P &lt; 0.05), blastocyst yield/cleaved embryos (59/156, 38% v. 45/165, 27%, respectively; P &lt; 0.05), and total blastocyst cell numbers (108.62 ± 19.87 v. 89.61 ± 26.32, respectively; P &lt; 0.05) compared to control oocytes. The VB treatment at the same concentration but for 2 h induced only significantly higher cleavage rate (196/210, 93% v. 165/226, 73%; P &lt; 0.05). In conclusion, our results showed that VB treatment at 1.03 nM during 24 h of IVM exerted a positive effect on in vitro embryo development of prepubertal ovine oocytes by increasing the blastocyst yield and their quality. The hypothesis that VB at nanomolar concentrations may improve cumulus-oocyte energy/redox status is under investigation.The authors acknowledge support by the Regione Autonoma della Sardegna (LR 7, Agosto 2007, no. 7, CRP-17602). The authors thank Dr D. Bebbere and L. Falchi, Dept. Veterinary Medicine, Sassari, for statistical analysis.

207 EFFECT OF REPEAT HORMONE STIMULATION ON THE YIELD AND IN VITRO DEVELOPMENT OF JUVENILE CALF OOCYTES

Juvenile in vitro embryo transfer can markedly reduce animal generation intervals. The purpose of this study was to investigate the ovarian response of juvenile calves and in vitro oocyte developmental capacity after superstimulation. Experiments on calves were performed in accordance with the Animal Welfare Regulations. A total of 36 donor juvenile calves on standard nutrition and in a disease-free environment, were selected from the breeding farm of the Beijing Dairy Cattle Center. At 60 days of age, calves were randomly assigned into three groups of four calves each, replicated three times. On day 1, Group 1 received a progesterone vaginal insert (CIDR, 300 mg per device); Group 2 received a CIDR and 0.5 mg oestrogen benzoate (China); Group 3 received a CIDR, 0.5 mg oestrogen benzoate, and 50 mg progesterone (China). Then, calves were injected with FSH (Folltropin-V, Bioniche Animal Health, Belleville, ON, Canada) twice daily on days 5 (40 mg/40 mg) and 6 (30 mg/30 mg) at 12 h intervals. Cumulus–oocyte complexes (COCs) were recovered from the superstimulated calves 12 to 14 h after the final FSH treatment. COCs were considered usable unless they were damaged or had expanded cumulus layers. Usable COCs were matured in vitro for 24 h in maturation medium consisting of TCM199, 10% FBS, 10 μg mL–1 FSH, 1 μg mL–1 LH, 1 μg mL–1 E2–17β, 100 IU mL–1 penicillin, 100 μg mL–1 streptomycin, with (+Cys) or without (–Cys) 100 μM Cysteamine. Each calf oocyte was cultured in one well. The final concentration added to each fertilization drop was 5 × 106 sperm mL–1. Sperm and oocytes were co-cultured in IVF-100 medium (BO liquid+10 μg mL–1 heparin, Japan) at 38.5°C, 5% CO2 and a saturated humidity for 6 to 8 h. Blastocyst production rates were determined after 7 and 8 d of in vitro culture in CR1aa medium without the addition of cysteamine. Differences among treatments in each experiment were determined by one-way ANOVA and a multiple range test. Superstimulatory results indicated that more follicles were aspirated (63.2 per calf) and more usable oocytes were recovered (48.0 per calf) in Group 1 than in the other two groups (Group 2–45.2 and 31.8, respectively; Group 3–35.4 and 28.3, respectively; P &lt; 0.05). No difference was observed between Groups 2 and 3. Superstimulation of calves twice at 30 day intervals in Group 2 (n = 12) did not affect the number of follicles or usable oocytes (overall, 44.2 and 28.0 per calf). Maturation rates (86.5% v. 85.0%, respectively) and cleavage rates (84.4% v. 80.0%, respectively) did not differ whether cysteamine was not (–Cys; n = 318) or was (+Cys; n = 330) added to the maturation medium. However, the blastocyst rate differed significantly (12.9% v. 35.2%, respectively; P &lt; 0.01). This study established a protocol for the superstimulation of juvenile calves with an average of 48 oocytes obtained per calf. Superstimulation and surgical oocyte recovery twice at an interval of 30 days had no adverse effect on follicle development or oocyte recovery. The novelty of this research is that the blastocyst production rate of calf oocytes (35.2%) in maturation medium supplemented with cysteamine was similar to that reported in the cow.

Effects of gaseous atmosphere and antioxidants on the development and cryotolerance of bovine embryos at different periods ofin vitroculture

This study examined the effects of antioxidant supplementation and O2 tension on embryo development, cryotolerance and intracellular reactive oxygen species (ROS) levels. The antioxidant supplementation consisted of 0.6 mM cysteine (CYST); 0.6 mM cysteine + 100 μM cysteamine (C+C); 100 IU catalase (CAT) or 100 μM β-mercaptoethanol (β-ME) for 3 or 7 days of in vitro culture (IVC). Two O2 tensions (20% O2 [5% CO2 in air] or 7% O2, 5% CO2 and 88% N2 [gaseous mixture]) were examined. After 7 days of antioxidant supplementation, the blastocyst frequencies were adversely affected (P < 0.05) by CYST (11.2%) and C+C (1.44%), as well as by low O2 tension (17.2% and 11.11% for 20% and 7% O2, respectively) compared with the control (26.6%). The blastocyst re-expansion rates were not affected (P > 0.05) by the treatments (range, 66-100%). After 3 days of antioxidant supplementation, the blastocyst frequencies were not affected (P > 0.05) by any of the antioxidants (range, 43.6-48.5%), but they were reduced by low O2 tension (P < 0.05) (52.1% and 38.4% for 20% and 7% O2, respectively). The intracellular ROS levels, demonstrated as arbitrary fluorescence units, were not affected (P > 0.05) by antioxidant treatment (range, 0.78 to 0.95) or by O2 tension (0.86 and 0.88 for 20% and 7% O2, respectively). The re-expansion rates were not affected (P > 0.05) by any of the treatments (range, 63.6-93.3%). In conclusion, intracellular antioxidant supplementation and low O2 tension throughout the entire IVC period were deleterious to embryo development. However, antioxidant supplementation up to day 3 of IVC did not affect the blastocyst frequencies or intracellular ROS levels.

Production of the first viable ovum pick–up and in vitro embryo produced (OPU–IVEP) buffalo calf in India

Abstract This report puts on record production of the first viable calf following ovum pick-up in vitro embryo production (OPU-IVEP) technique in buffalo ( Bubalus bubalis ) in India. A total of 313 transvaginal OPU sessions were performed in six elite Murrah buffaloes and 1 171 follicles were aspirated. From these follicles, a total of 729 immature oocytes were recovered. The recovered oocytes were graded as A (59, 8.1%), B (111, 15.2%), C (293, 40.2%) and D (262, 35.9%) following standard procedures. Maturable grade oocytes (A, B, and C; 473, 68.9%) were subjected to in vitro maturation in Medium 199 supplemented with 10% FBS, 0.5 μg/mL FSH, 10 IU/mL LH, 1 μ g/mL estradiol-17 β, 20 ng/mL EGF and 50 μM cysteamine in CO 2 incubator at 39 °C temperature, 5% CO 2 and high humidity. The matured oocytes (427 out of 473; 90.3%) were co-incubated with 1×10 6 /mL buffalo sperms in modified synthetic oviductal fluid (mSOF) containing 10 ng/mL heparin for 22 hrs for in vitro fertilization. After fertilization the presumptive zygotes were stripped off of remaining cumulus cells and cultured in mSOF for 7 days to study embryonic development. A total of 278 (65.1%) oocytes cleaved after 24-36 hrs of fertilization. Out of the cleaved oocytes, 249 (89.6%), 216 (77.7%), 167 (60.1%), 139 (50.0%) and 73 (26.3%) developed up to 4-cell, 8-cell, 16-cell, morula, and blastocyst stages, respectively. A total of 29 embryos were transferred into 27 synchronized recipient buffaloes. Two of these buffaloes were found pregnant at 90 days. One of them aborted at 4.5 months of gestation (male fetus) and the other gave birth to a live male calf weighing 25 kg. Currently, the calf is normal and has attained the age of 4.5 years.

Blastocyst rate of in vitro embryo production in sheep is affected by season

It has been reported that the number and quality of in vitro produced embryos is season related. This study was conducted to assess the effect of season on cleavage, blastocyst and lambing rates of in vitro produced ovine embryos during 3 years of collection data. Ovaries of Sarda sheep were collected from a slaughterhouse. In total, 5035 oocytes were recovered and matured in TCM-199 with 4 mg/ml bovine serum albumin (BSA), 100 μM cysteamine, 0.3 mM Na pyruvate, 0.1 UI/ml recombinant follicle-stimulating hormone (r-FSH), 0.1 UI/ml recombinant luteinising hormone (r-LH), and 1 μg/ml estradiol-17β. Matured oocytes were fertilized with fresh semen in synthetic oviductal fluid (SOF) with 20% heat inactivated estrous sheep serum. The presumptive zygotes were cultured for 6-7 days (blastocyst stage) in SOF medium supplemented with 1% Basel Medium Eagle (BME), 1% Minimum Essential Medium, 1 mM glutamine and 8 mg/ml fatty acid-free BSA. The embryos produced were vitrified and a total of 165 blastocysts (80 from the breeding season and 85 from the anoestrous season) were transferred in pairs into recipient ewes during the reproductive period. There were no significant differences in cleavage rates between seasons in any of the 3 years examined (84% versus 83%, 81% versus 80% and 80% versus 79%, respectively). The blastocyst rate varied significantly between seasons in 2005 and 2007 (P < 0.05), and in 2006 (P < 0.001). There were no differences in pregnancy and lambing rates between embryos during anoestrous versus during the breeding season. In conclusion, only the blastocyst rate appeared to have been affected by season, possibly due to variation in the number of developmentally competent oocytes.

253 OXIDATIVE STRESS MAY IMPAIR OOCYTE QUALITY IN DAIRY COWS OF REPRODUCTIVE AGE WITH A REDUCED ANTRAL FOLLICLE COUNT

In dairy cattle, oocytes isolated from ovaries with a reduced antral follicle count (AFC) have a low embryonic developmental competence. This may be related to oxidative stress, as indicated by our recent finding that ovaries with reduced AFC show a defective endothelial nitric oxide synthase/nitric oxide system. To further test this hypothesis, we evaluated whether the poor developmental competence of these oocytes was possibly due 1) to an imbalance of the reduced glutathione (GSH) system, because GSH is the major antioxidant compound stored within the oocyte and protects the zygote and early embryos from oxidative damage, and 2) to reduced mitochondrial activity. Ovaries were obtained from the abattoir, and oocytes were collected from ovaries with reduced AFC, with fewer than 10 follicles of 2 to 6 mm in diameter, and aged-matched controls, with more than 10 follicles of 2 to 6 mm in diameter. Oocyte GSH content was evaluated using the 5,5′-dithio-bis(2-nitrobenzoic acid)-GSH reductase recycling micro-GSH assay before and after in vitro maturation (IVM) in the presence or absence of 100 µM cysteamine, a GSH precursor. At the same time the developmental competence after IVF was assessed. Moreover, the mitochondrial activity during IVM was evaluated in additional oocytes from the two ovarian categories by specific MitoTracker dyes (MitoTracker FM Green and MitoTracker Orange CMTMRos, Invitrogen, Carlsbad, CA, USA) and subsequent image analysis (ImageJ software). All data were analysed by ANOVA followed by Fisher’s least significant differences test, and P-values &lt;0.05 were considered significant. Experiments were repeated at least three times. Oocytes isolated from ovaries with a low AFC had a similar GSH content compared with oocytes isolated from control ovaries (n = 65 and 85, respectively; 4.31 ± 0.41 v. 4.51 ± 0.42 pmol oocyte–1). After IVM, oocytes from ovaries with reduced AFC showed a significantly lower GSH content compared with control oocytes (n = 55 and 65, respectively; 4.36 ± 0.31 v. 6.59 ± 0.39 pmol oocyte–1); however, cysteamine supplementation during IVM induced GSH accumulation similar to the control (n = 80 and 85, respectively; 9.88 ± 0.77 v. 10.45 ± 0.88 pmol oocyte–1). It is interesting that the increase in intracellular GSH content significantly improved the developmental competence of oocytes from ovaries with a reduced AFC (n = 196 and 201, respectively; 20.1 ± 2.9% v. 6.2 ± 1.6%), although the blastocyst rate remained lower than the control either with or without cysteamine (n = 218 and 212, respectively; 33.3 ± 3.8% and 34.2 ± 2.4%). Further, immature oocytes from ovaries with a low AFC showed a reduced mitochondrial membrane potential compared with control oocytes (n = 13 and 18, respectively; 1.74 ± 1.19 v. 2.22 ± 1.72, calculated as the ratio between the fluorescence of active and total mitochondria), whereas at the end of IVM, it declined in both categories at a comparable level (n = 17 and 24, respectively; 1.19 ± 0.10 and 1.30 ± 0.06). Our data confirmed the hypothesis that both the GSH imbalance and defective mitochondrial activity contribute to the limited developmental competence of oocytes from ovaries with a reduced AFC. This work was supported by Dote ricerca applicata-FSE, Regione Lombardia, Italy (VL, IT).

Effect of cysteamine supplementation during in vitro culture of early stage bovine embryos on blastocyst rate and quality

The aim of this study was to evaluate whether the addition of cysteamine to the in vitro culture media enhances the yield, hatching rate, total cell number and inner cell mass/total cell number ratio of bovine embryos. A total of 933 bovine oocytes collected from ovaries of 60 slaughtered donors were subjected to in vitro maturation and in vitro fertilization. Following fertilization, embryos were cultured in synthetic oviductal fluid without glucose. After 24 h embryos were transferred into synthetic oviductal fluid with 1.5 mM glucose and 0 (control), 50, 100 and 200 µM of cysteamine. After 48 h, the embryos were transferred into synthetic oviductal fluid with glucose but without cysteamine and cultured until Day 9. The number of cleaved embryos on Day 2, the total number of blastocysts on Day 7 and the number of hatched blastocysts on Day 9 were calculated. Differential staining of inner cell mass and trophectoderm cells of blastocysts were performed on Day 7 and Day 9 of in vitro culture. Supplementation of in vitro culture media with 100 µM cysteamine increased the blastocyst yield (P &lt; 0.05) without affecting the hatching rate. Furthermore, the embryos cultured in the presence of 100 µM cysteamine had significantly higher number of inner cell mass cells (P &lt; 0.05) and the proportion of inner cell mass cells (P &lt; 0.05) compared with the controls. The results of the present study demonstrated that the addition of 100 µM cysteamine to the in vitro culture media improved blastocyst production rate and enhance embryo quality, which could lead to the improvement of the in vitro culture system for bovine embryos.

95 SHEEP ZYGOTES CULTURED WITH HYALURONAN, BOVINE SERUM ALBUMIN, OR SERUM AND THEIR EFFECTS ON PRENATAL AND POSTNATAL EMBRYO PRODUCTION AND DEVELOPMENT

Mammalian pre-implantation embryos are sensitive to their environment and numerous publications suggest that post-fertilization culture is the most critical period affecting embryo quality and subsequent embryo development. This study was conducted to examine the viability, twining, sex ratio and birthweight (BW) after transfer of fresh and vitrified in vitro-produced sheep embryos derived from oocytes cultured in a semi- or defined medium supplemented with fatty-acid–free bovine serum albumin (BSA) with or without hyaluronan (HA) versus fetal bovine serum (FBS) as a control. A total of 649 oocytes were matured in TCM-199 containing 2 mM glutamine, 4 mg mL–1 BSA, 100 μM cysteamine, 0.3 mM sodium pyruvate, 1 μg mL–1 oestradiol-17β and fertilized with fresh semen prepared with SOF, enriched with 20% oestrus sheep serum. Presumptive zygotes were cultured for 48 h in SOF supplemented with 1% BME-essential and 1% MEM-nonessential amino acids, 1 mM glutamine and 8 mg mL–1 BSA. On the 3rd day of culture, presumptive zygotes were divided randomly into 3 groups: (1) BSA+HA, medium supplemented with 8 mg mL–1 BSA and 6 mg mL–1 HA, (2) BSA, medium supplemented with 8 mg mL–1 BSA only, or (3) control, medium supplemented with 5% charcoal-stripped fetal bovine serum (FBS). On the 5th day of culture, media were replaced accordingly in all groups. Embryos that reached the expanded blastocyst stage by 6 or 7 days/group were recorded and transferred as fresh or vitrified. The cleavage rate, blastocyst formation, pregnancies (PR) at 40, 60 and 80 days, lambing, twinning, sex ratio and BW were all recorded. Among all groups, no significant differences were observed in cleavage and blastocyst rates. The proportion of zygotes that developed to blastocysts at 6 days was significantly higher after culture with BSA+HA vs BSA and control (67/92, 72.8%), (46/118, 39.0%) and (29/72, 40.3%), respectively. Compared with the control group, vitrified BSA blastocysts were associated with increased (P &lt; 0.05) PRs after 60 and 80 days and lambing rates. In comparison, there were no significant differences in pregnancy and lambing rate after transfer of fresh blastocysts. There was a positive correlation between age of embryo at transfer and higher BW. The mean BWs were higher after transfer of 7-day vs 6-day embryos (3.95 and 3.37 kg), respectively. However, there were no significant differences in twining and sex ratio after transfer fresh/or vitrified treated embryos versus control group. In conclusion, the results indicate that sheep zygotes cultured with BSA+HA had an increased development to blastocysts at 6 days, but the rate of blastocyst formation was not increased, PR and lambing rate were comparable with the BSA and control groups. In addition, transfer of vitrified embryos produced in the presence of BSA significantly increased the PR at 60, 80 days and lambing rate versus control group.

91 INTERACTION OF OXYGEN TENSION AND CYSTEAMINE SUPPLEMENTATION DURING IN VITRO CULTURE OF BOVINE EMBRYOS

Reactive oxygen species damage early mammalian embryos, so culture of bovine in vitro-produced (IVP) embryos at 5% O2 is clearly superior to 20% O2. The thiol compound cysteamine is an antioxidant and improves in vitro blastocyst production when added to in vitro maturation (IVM) medium. The purpose of this study was to investigate supplementation of IVP embryo culture medium with cysteamine at different oxygen tensions. Bovine ovaries from feedlot heifers were collected from a local abattoir and cumulus–oocyte complexes (COC) were aspirated from 3- to 8-mm follicles. COC were matured in maturation medium with 100μM cysteamine (Reprod. Fertil. Dev. 18, 585) for 23 h in a humidified incubator at 38.5°C in 5% CO2 in air. COC at 23 h of maturation were co-incubated with sperm for 18 h. Cumulus cells were then removed and presumed zygotes were cultured in our chemically defined culture medium system (Reprod. Fertil. Dev. 18, 585) in a 2 × 2 factorial arrangement: with or without 50 μM cysteamine × atmospheres of either 5% O2, 5% CO2, 90% N2, or 5% CO2 in air. There were no treatment differences (P &gt; 0.01) in percentages of oocytes cleaving or reaching the 8-cell stage (Table 1). However, blastocyst production rates were lower (P &lt; 0.01) in the group cultured without cysteamine at 20% O2 compared with all the other groups. Adding cysteamine for embryo culture at 20% O2 resulted in blastocyst rates similar to those cultured at 5% O2 with or without cysteamine. Cysteamine was not beneficial at 5% O2. Table 1.Cysteamine supplementation during in vitro culture of bovine embryos