88 ENRICHMENT OF CULTURE MEDIUM WITH CROCETIN IMPROVES IN VITRO EMBRYO DEVELOPMENT IN CATTLE

2016 ◽  
Vol 28 (2) ◽  
pp. 173
Author(s):  
G. Zullo ◽  
J. E. Tamayo Palacio ◽  
C. De Canditiis ◽  
V. Longobardi ◽  
A. Salzano ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Embryo Development ◽  
Production Efficiency ◽  
Culture Medium ◽  
Culture Conditions ◽  
Active Components ◽  
Cleavage Rate ◽  
Mammalian Embryo ◽  
Morphological Criteria

The high incidence of developmental failure of bovine in vitro-produced embryos is due to suboptimal culture conditions that induce oxidative stress. Indeed, increased oxidative stress is one of the main factors affecting in vitro mammalian embryo development, decreasing the viability of IVP embryos. It is known that saffron has a powerful antioxidant capacity, mainly due to its active components crocin and crocetin. The aim of this study was to evaluate whether enriching the in vitro culture medium with crocetin improves in vitro embryo production efficiency in cattle. The range of concentrations of crocetin was chosen after a preliminary dose response trial (322 total presumptive zygotes were cultured with 0, 1, 10, and 50 μM, over 2 replicates) that showed beneficial and deleterious effects, respectively, with the lowest and highest concentration compared with the control (36.6 ± 5.6, 57.4 ± 4.5, 46.4 ± 4.4, and 6.8 ± 3.7% blastocyst rates, respectively, with 0, 1, 10, and 50 μM; P < 0.01). Therefore, the range of concentrations to test was reduced. Abattoir-derived bovine oocytes (n = 832, over 4 replicates) were in vitro matured and fertilized according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347–1355). Twenty hours after IVF, presumptive zygotes were cultured in SOF medium with 0 (control; n = 208), 1 μM (n = 208), 2.5 μM (n = 208), and 5 μM (n = 208), at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2. The embryos obtained by the end of culture (i.e. on Day 7 post-IVF) were scored for quality, based on morphological criteria, and for developmental stage, as previously described (Robertson and Nelson 2010, Manual of the IETS, 86–105). The percentages of total transferable embryos and grade 1 and 2 blastocysts were recorded. As the chronology of development is a reliable parameter to assess quality, the percentage of fast-developing embryos (i.e. hatched and expanded blastocysts) was also compared among groups. Differences among groups were analysed by ANOVA, and Tukey method was used as a post-hoc test. Data are presented as means ± s.d. The supplementation of crocetin during culture did not affect cleavage rate (74.9 ± 6.3, 76.4 ± 8.4, 81.4 ± 4.3, and 76.4 ± 8.4%, respectively, with 0, 1, 2.5, and 5 μM). However, post-fertilization embryo development improved with 1 µM crocetin compared with the control, both in terms of total embryo output (43.8 ± 4.4, 61.1 ± 5.2, 50.4 ± 6.7, and 53.3 ± 7.3%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.01) and grade 1 and 2 blastocysts (41.0 ± 3.6, 54.3 ± 5.4, 46.2 ± 6.7, and 49.4 ± 6.5%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.05), whereas no differences were observed among the other groups. Moreover, the percentage of fast developing embryos increased with 1 µM (P < 0.05) crocetin compared with the control, with no other differences recorded among groups (17.7 ± 5.8, 34.7 ± 5.7, 24.9 ± 5.1, and 28.7 ± 7.8%, respectively, with 0, 1, 2.5, and 5 μM). In conclusion, these results demonstrated a beneficial effect of low concentrations of crocetin (1 μM) during culture both on blastocyst yield and quality, as indicated by the improved chronology of embryo development.

135 EFFECT OF L-ERGOTHIONEINE SUPPLEMENTATION DURING CULTURE ON IN VITRO EMBRYO DEVELOPMENT IN BUFFALO (BUBALUS BUBALIS)

2015 ◽  
Vol 27 (1) ◽  
pp. 160
Author(s):  
G. Zullo ◽  
A. Salzano ◽  
G. Bifulco ◽  
V. Longobardi ◽  
G. Albero ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
In Vitro Culture ◽  
Embryo Development ◽  
Production Efficiency ◽  
Cell Function ◽  
Embryo Viability ◽  
Cleavage Rate ◽  
Mammalian Embryo ◽  
Morphological Criteria

It is known that in vitro mammalian embryo development is negatively affected by the increased oxidative stress occurring under culture conditions. The oxidative damage of cell components via reactive oxygen species interferes with proper cell function. Buffalo embryos are particularly sensitive to oxidative stress because of their high lipid content (Boni et al. 1992 Acta Med. Vet. 38, 153–161). l-Ergothioneine (LE) is a powerful scavenger of hydroxyl radicals (OH) and an inhibitor of iron or copper ion-dependent generation of OH from hydrogen peroxide (H2O2). The aim of this study was to evaluate whether enriching the in vitro-culture medium with LE improves in vitro embryo production efficiency in buffalo. Abattoir-derived buffalo oocytes (n = 854, over 6 replicates) were in vitro matured and fertilized according to standard procedures (Gasparrini et al. 2006 Theriogenology 65, 275–287). Twenty hours after IVF presumptive zygotes were cultured in SOFaa supplemented by 8 mg mL–1 BSA in a controlled gas atmosphere consisting of 5% CO2, 7% O2, 88% N2, in humidified air, at 38.5°C with 0 (control; n = 214), 0.05 mM LE (n = 217), 0.1 mM LE (n = 204), and 1 mM LE (n = 219). Cleavage rate was assessed at the time of change of culture (Day 5) and the cleaved elements were cultured for a further 2 days. The embryos obtained by the end of culture, i.e. on Day 7 post-IVF, were scored for quality, based on morphological criteria, and for developmental stage, as previously described (Robertson, Nelson 2010 Manual of the International Embryo Transfer Society 86–105). The percentages of total transferable embryos and Grade 1 and 2 blastocysts in relation to cleaved oocytes were recorded. Because the chronology of development is known to be one of the most reliable parameters for assessing quality, the percentage of fast-developing embryos, i.e. hatched and expanded blastocysts, was also recorded. Data were analysed by Chi-squared test. Cleavage rate was not affected by the treatment (71.4, 66.8, 68.7, and 63.0%, respectively, with 0, 0.05, 0.1, and 1 mM LE). The total embryo output increased in groups supplemented with 0.05 and 0.1 mM LE (31.3, 42.2, 43.8, and 21.7%, respectively, with 0, 0.05, 0.1, and 1 mM LE; P < 0.05). However, the enrichment of in vitro culture with 0.1 mM LE also increased the percentage of Grade 1 and 2 blastocysts compared with the control and to 1 mM LE (21.6, 30.9, 33.9, and 21.7%, respectively, with 0, 0.05, 0.1, and 1 mM LE; P < 0.05). Likewise, 0.1 mM LE gave higher percentages of fast developing embryos than the control and 1 mM LE groups. In conclusion, these results demonstrated a beneficial effect of LE during culture on buffalo in vitro embryo development. The dose response trial indicated that the optimal concentration is 0.1 mM that also influenced the chronology of development and hence embryo viability.

52 HYALURONIC ACID IMPROVES CRYOTOLERANCE OF BUFFALO (BUBALUS BUBALIS) IN VITRO-DERIVED EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 138
Author(s):  
L. Boccia ◽  
M. Rubessa ◽  
M. De Blasi ◽  
S. Di Francesco ◽  
G. Albero ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
In Vitro Culture ◽  
Developmental Stage ◽  
Production Efficiency ◽  
Embryo Quality ◽  
Survival Rates ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Morphological Criteria

Although in vitro embryo production efficiency in buffalos has greatly improved over the years, the in vitro-produced embryos show lower viability and resistance to cryopreservation. Therefore, it is necessary to optimize the in vitro culture conditions to improve embryo quality. Hyaluronic acid, a glycosaminoglican present in oviducal and uterine fluids, has been shown to successfully support in vitro development of bovine embryos (Stojkovic et al. 2002 Reproduction 124, 141–153). The aim of this study was to evaluate the influence of high concentrations of hyaluronic acid (HA) during late in vitro culture on blastocyst development, as well as on their cryotolerance after cryotop vitrification in buffalos. In vitro matured and fertilized buffalo oocytes (n = 1007) from slaughterhouse ovaries were cultured for 4 days in SOFaa supplemented by 8 mg mL–1 of BSA in a controlled gas atmosphere consisting of 5% CO2, 7% O2 and 88% N2, in humidified air, at 38.5°C. On Day 4, cleavage rate was assessed (75.2%) and all of the cleaved elements were divided into 3 different late culture groups: 8 mg mL–1 of BSA (n = 244; group A), 8 mg mL–1 of BSA supplemented by 6 mg mL–1 of HA (n = 251; group B) and 1 mg mL–1 of BSA supplemented by 6 mg mL–1 of HA (n = 262; group C). On Day 7 after IVF, embryo outcome was assessed and all of the embryos were vitrified by cryotop [De Rosa et al. 2007 Ital. J. Anim. Sci. 6 (Suppl 2), 747–750] and cultured for 24 h. The resistance to cryopreservation was evaluated by assessing the survival rate on the basis of morphological criteria and the percentage of embryos reaching a more advanced developmental stage after 24 h culture. Data were analysed by the chi-square test. No differences in blastocyst rate were recorded among groups (43.9, 44.3 and 40.0%, respectively in A, B and C groups). However, out of the total embryos, a higher percentage of Grade 1 hatched blastocysts (Robertson and Nelson 1998 Manual of the International Embryo Transfer Society 9, 103–16) was observed in group C (P < 0.05) than in groups A and B (14.3, 18.8 and 25.5% in A, B and C groups, respectively). Although the supplementation with HA did not improve the survival rates following vitrification-warming (51.1, 59.4 and 58.4% in A, B and C groups, respectively), the percentage of vitrified-warmed embryos that resumed development and reached a more advanced developmental stage after culture increased (P < 0.01) in group C (20.7, 27.7 and 37.6% in A, B and C groups, respectively). In conclusion, the addition of 6 mg mL–1 of HA, together with a limited protein source (i.e. 1 mg mL–1 of BSA), during late culture improved buffalo embryo quality, indicated by both the greater percentage of advanced-stage embryos and by the resumption of development after post-warming culture.

149 EFFECT OF RESVERATROL ANALOGUE ON DEVELOPMENT OF IN VITRO-FERTILIZED BOVINE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 183 ◽  
Author(s):  
T. A. Patrocínio ◽  
C. A. C. Fernandes ◽  
L. S. Amorim ◽  
J. R. Ribeiro ◽  
G. C. Macedo ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Culture Medium ◽  
Synthetic Analogue ◽  
Bovine Embryo ◽  
High Humidity ◽  
Cleavage Rate ◽  
Antioxidant Agents ◽  
Blastocyst Rate

Oxidative stress is one of the main effects of in vitro culture. Generation of reactive oxygen species (ROS) by embryos can be enhanced by the sub-optimal in vitro culture conditions and are associated with a delay in embryonic development. However, supplementation of culture medium with antioxidant agents can minimize the effects of ROS (Guérin et al. 2001 Hum. Reprod. Update 7, 175–189). Resveratrol is an example of a potent antioxidant, and modifications in its structure can improve its biological activity. This study evaluated the effect of AR33 (formula with patent pending), an analogue of resveratrol with high antioxidant activity, on embryo development. Bovine cumulus-oocyte complexes recovered from ovaries collected at the slaughterhouse were in vitro matured for 24 h and oocytes were in vitro fertilized for 20 h, both at 38.8°C under 5% CO2 in air and high humidity. Partially denuded presumptive zygotes were randomly distributed in 4 treatments (with 6 replicates): 0 µM (control, n = 347), 0.1 µM (n = 337), 0.5 µM (n = 277), and 2.5 µM (n = 343) of AR33. The base medium was SOFaa supplemented with 2.5% FCS and incubation conditions were 38.8°C under 5% CO2 in air and high humidity. Half of culture medium was renewed (feeding) at Day 3 and 5 post-fertilization. Cleavage was evaluated at Day 3 and blastocyst rates at Day 7 and 8 post-fertilization. Data were analysed by logistic regression considering the significance level of P < 0.05. Values are shown as mean ± SEM. Cleavage rate was higher (P < 0.05) for 2.5 µM (69.0 ± 4.4%) than for 0, 0.1, and 0.5 µM AR33 (62.1 ± 2.0%, 60.7 ± 5.9%, and 56.7 ± 5.8%, respectively). At Day 7, the blastocyst rate was similar (P > 0.05) among 0.1, 0.5, and 2.5 µM (18.1 ± 5.4%, 17.5 ± 2.9%, and 19.4 ± 3.3%, respectively) and all of them were higher (P < 0.05) than 0 µM AR33 (12.4 ± 2.5%). At Day 8, there was again no difference (P > 0.05) among 0.1, 0.5, and 2.5 µM AR33 (21.0 ± 5.0%, 18.4 ± 2.1%, and 24.6 ± 3.3%, respectively) but only 0.1 and 2.5 µM showed higher (P < 0.05) blastocyst rate than 0 µM AR33 (15.2 ± 2.5%). In conclusion, the synthetic analogue of resveratrol tested in this study can improve bovine embryo development in culture medium supplemented with 2.5% FCS under 5% CO2 in air. A concentration of 2.5 µM AR33 can be a choice for further studies. This study was supported by Fapemig, CAPES, and CNPq.

Analysis of Ferrous on Ten-Eleven Translocation Activity and Epigenetic Modifications of Early Mouse Embryos by Fluorescence Microscopy

2016 ◽  
Vol 22 (2) ◽  
pp. 342-348 ◽  
Author(s):  
Ming-Hui Zhao ◽  
Shuang Liang ◽  
Jing Guo ◽  
Jeong-Woo Choi ◽  
Nam-Hyung Kim ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryoid Body ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Cleavage Rate ◽  
Mammalian Embryo ◽  
Preimplantation Embryo Development ◽  
Oct4 Expression ◽  
Early Mouse

AbstractIron is an essential trace element that plays important roles in the cellular function of all organs and systems. However, the function of Fe(II) in mammalian embryo development is unknown. In this study, we investigated the role of Fe(II) during preimplantation embryo development. Depletion of Fe(II) using thiosemicarbazone-24 (TSC24), a specific Fe(II) chelator, rescued quenching of the Fe(II)-sensitive fluorophore phen green-SK. Afterin vitrofertilization, TSC24 significantly reduced the cleavage rate as well as blastocyst formation. The hatch rate of blastocysts was also reduced with 1 pM TSC24 treatment (20.25±1.86 versus 42.28±12.96%,p<0.05). Blastocysts were cultured in leukemia inhibitory factor-free mouse embryonic stem cell culture medium with or without TSC24, and those with depleted Fe(II) displayed delayed attachment and lost the ability to induce embryoid body formation. To further explore the mechanism of Fe(II) in embryo development, we assessed the expression of 5-hydroxymethylcytosine (5hmC) and OCT4 in the pronuclear and blastocyst stages, respectively. We observed that Fe(II) reduced 5hmC and OCT4 expression, which could be explained by low ten-eleven translocation (TET) enzyme activity induced by TSC24 treatment. These findings demonstrate that Fe(II) is required for mammalian embryo development and that it facilitates the process via regulation of TET activity.

238 ASSOCIATION BETWEEN SPERM MORPHOLOGY AND IN VITRO EMBRYO PRODUCTION IN MICE

2011 ◽  
Vol 23 (1) ◽  
pp. 217
Author(s):  
M. Vilariño ◽  
M. Crispo ◽  
A. Pinczak ◽  
A. Menchaca
Keyword(s):  
Embryo Development ◽  
Sperm Morphology ◽  
Culture Conditions ◽  
Cleavage Rate ◽  
Fluid Medium ◽  
Vitro Fertilization ◽  
Sperm Abnormalities ◽  
Sperm Suspension ◽  
Cervical Dislocation

Although sperm morphology affects male fertility in several species, normally, it is not considered for in vitro fertilization (IVF) programs in mice. In order to correlate sperm morphology with IVF rates in murines, a total of 3336 oocytes were used in 11 identical IVF replicates using 8-wk-old B6D2 F1 (C57BL/6 × DBA/2J) males and 3- to 4-wk-old C57BL/6J donors females. For each replicate, 10 females were injected intraperitoneally with 5 IU of eCG (Novormon, Syntex, Buenos Aires, Argentina), and 5 IU of hCG (Ovusyn, Syntex) 48 h apart. Superovulated females were killed 14 to 16 h after hCG injection. The oviducts were isolated and cumulus–oocyte complexes were recovered and introduced into each sperm suspension drop. Previously, sperm was obtained from males killed by cervical dislocation, the cauda epididymis were recovered, minced with fine scissors in equilibrated human tubal fluid medium (HTF, EmbryoMax, Chemicon International, Phillipsburg, NJ, USA), and resuspended in a total volume of 200 μL of HTF. The sperm suspension was incubated at 0.05 or 3 × 106 spermatozoa/mL concentration in equilibrated 100-μL drops of HTF under embryo tested mineral oil (Sigma, St. Louis, MO, USA), at 37°C in 5% CO2, 95% air for 1 h before insemination. To evaluate sperm morphology, a sample of each replicate was fixed with 10% formalin and observed under phase-contrast microscopy (Olympus IX81) at 100× magnification. Sperm abnormalities regarding head and neck, tail, and cytoplasmic droplet were recorded from 200 cells. Five hours after insemination, the fertilized eggs were washed and transferred in groups of 50 to 100 into 100-μL drops of equilibrated culture medium (KSOM, EmbryoMax, Chemicon International) under the same culture conditions. The number of 2- (cleavage) and 8- (development) cell embryos was scored after 24 and 72 h in culture, respectively. Statistical analysis was performed by logistic regression taking into account the effect of the sperm abnormalities, the replicate, and the sperm dose. The effect of the sperm abnormalities is shown in Table 1. Results were not significantly influenced by the replicate or the sperm dose (P > 0.05). In conclusion, sperm morphology affects cleavage and embryo development rates in mice, and it should be taken into account as a source of variation in the success of IVF. Table 1.Effect of total sperm abnormalities on cleavage rate and embryo development in mice

124 CRYOPRESERVED BOVINE OVIDUCTAL EPITHELIAL CELLS SURVIVAL, GROWTH, AND ABILITY TO SUPPORT EARLY EMBRYO DEVELOPMENT

2015 ◽  
Vol 27 (1) ◽  
pp. 154
Author(s):  
E. Corbin ◽  
A. Cordova ◽  
J. Grosbois ◽  
P. Mermillod
Keyword(s):  
Cell Culture ◽  
Epithelial Cells ◽  
Embryo Development ◽  
Culture Medium ◽  
Early Embryo ◽  
Cell Culture Medium ◽  
Cleavage Rate ◽  
Early Embryo Development ◽  
Blastocyst Rate

Previous experiments demonstrated that co-culture of bovine embryos with bovine oviducal epithelial cells (BOEC) improved blastocyst rate and quality (Cordova et al. 2014). However, the use of primary cell support for improving embryo development in vitro may introduce a higher variability of the results between different BOEC batches used, as well as sanitary risks. The use of well-controlled large batches of frozen BOEC may help to solve these problems. Therefore, the aim of the present study was to characterise the survival and functionality of frozen-thawed BOEC. Bovine oviducts attached to ovaries showing recent ovulation were collected at a local slaughterhouse during 4 replicates (3 oviducts per replicate). Epithelial cells were expelled by gentle squeezing and washed 3 times. Half of the cell pellet was diluted 100-fold in culture medium (TCM199 + 10% FCS) for culture of fresh cells. The other half was diluted 10-fold in cell freezing medium (TCM199 + 20% FCS + 10% dimethyl sulfoxide), allowed to equilibrate in this medium for 10 min, and frozen at –80°C in a container filled with isopropyl alcohol. After 4 h, the tubes were transferred into LN for at least 1 h. The tubes were then thawed (5 min in 37°C water bath), diluted 1 : 1 in cell culture medium, and centrifuged for 10 min at 100 × g. The pellet was then diluted 100× in cell culture medium. Fresh or frozen-thawed cells were seeded in 4-well NUNC plates for 7 days at 38.8°C in a humidified atmosphere with 5% CO2 in air. The medium was renewed every 48 h, and the viability of cells was assessed by calcein-AM and ethidium homodimer labelling. After 7 days of culture, the medium was replaced by SOF medium + 5% FCS, and bovine in vitro-produced zygotes were added the day after and co-cultured for 8 days at 38.8°C in a humidified atmosphere with 5% CO2 in air to evaluate embryo development. Half of the medium was renewed every 48 h. Frozen-thawed cells showed the same viability than fresh ones at Days 0 and 7 of culture and reached confluence at the same time (Day 7). Development results are shown in Table 1. Frozen and fresh cells support early embryo development at the same rate. In conclusion, the present study showed that BOEC frozen on the day of collection are equivalent to fresh BOEC in regards to their survival and proliferation and their ability to support early embryo development. At collection, the cells may face stresses that are just as considerable as freezing/thawing (temperature shock, scrapping, change of environment). This may explain why they are not affected by freezing than at collection. The differentiation status of these cells is now under analysis by immunocytochemistry. Table 1.Cleavage rate and blastocyst rate in 3 different types of culture systems

74 Treatment with Melatonin During In Vitro Culture Enhances Porcine Parthenogenetically Activated Embryo Development

2018 ◽  
Vol 30 (1) ◽  
pp. 175
Author(s):  
G. A. Kim ◽  
J.-X. Jin ◽  
S. Lee ◽  
A. Taweechaipaisankul ◽  
B. C. Lee
Keyword(s):  
Oxidative Stress ◽  
Embryo Development ◽  
Developmental Competence ◽  
Free Radical Scavengers ◽  
Control Group ◽  
Cleavage Rate ◽  
Cell Numbers ◽  
Significant Difference ◽  
Blastocyst Rate

Melatonin and its metabolites are powerful antioxidants and free radical scavengers. Because porcine embryos are vulnerable to oxidative stress in vitro, the addition of various protective chemicals to the culture medium, including melatonin, has been explored. The aim of this study was to investigate the effect of melatonin on in vitro developmental competence of porcine parthenogenetically activated (PA) embryos. Immature cumulus–oocyte complexes (COC) were collected and cultured in medium comprising TCM-199 supplemented with 10 ng mL−1 epidermal growth factor, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 5 μL mL−1 insulin, transferrin selenium solution 100×, 10% porcine follicular fluid, 10 IU mL−1 eCG, and 10 IU mL−1 hCG for 44 h. Then, COC were denuded and PA with electrical stimulation, and PA embryos were cultured in porcine zygote medium 5 (PZM-5) supplemented with melatonin at increased concentrations (10−9, 10−7, 10−5 M) at 39°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 for 7 days. Subsequent embryo development, including cleavage rate, blastocyst rate, and blastocyst cell numbers, was compared between groups (mean no. of embryos; control, 27.14; 10−9 M, 28.86; 10−7 M, 27.71; 10−5 M, 26.43). The experiments were repeated 7 times for each treatment group. Statistical analyses of all data were performed using one-way ANOVA with Dunn’s multiple comparison test. Results are expressed as the mean ± SEM and all differences were considered significant at P < 0.05. No apparent effect on cleavage rate of melatonin treatment of various concentrations was noted. Blastocyst cell number did not show any significant difference between groups. However, the potential of PA oocytes to develop into blastocysts was significantly higher in the group supplemented with 10−9 M melatonin compared with the control group (35.44 ± 3.84 v. 24.71 ± 1.59) and other melatonin treated groups (10−5 M, 21.35 ± 2.82; 10−7 M, 24.01 ± 2.31; P < 0.05). These indicated that treatment with 10−9 M melatonin in embryo culture might reduce the oxidative stress properly compared with other concentrations, which results in improvement of blastocyst rate formation. In conclusion, treatment with 10−9 M melatonin positively promoted the blastocyst formation rate of porcine PA embryos with no beneficial effects on their blastocyst cell numbers or cleavage rate. This study was supported by the National Research Foundation (#2015R1C1A2A01054373; 2016M3A9B6903410), Research Institute for Veterinary Science and the BK21 PLUS Program.

scholarly journals 265EFFECT OF SPECIFIC GRAVITY OF CULTURE MEDIUM ON IN VITRO EMBRYO DEVELOPMENT IN BUFFALO

2004 ◽  
Vol 16 (2) ◽  
pp. 253
Author(s):  
D.S. Arathy ◽  
S. Ashis ◽  
G.T. Sharma ◽  
A.C. Majumdar ◽  
M.S. Chauhan
Keyword(s):  
Specific Gravity ◽  
In Vitro Culture ◽  
Culture Medium ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Cell Stage

In buffalo the success rate of transferable quality embryo production through in vitro procedure is very low as compared to cattle. Sub optimal culture conditions and physical conditions such as specific gravity of the culture medium may lead to a reduced rate of transferable buffalo embryo production from the oocytes matured and fertilized in vitro (Palta &amp; Chauhan,1998 Reprod. Fertil. Dev. 10, 379–391). This experiment was therefore conducted to find out the role of specific gravity of the IVC medium on the development rate of the buffalo embryos in vitro. Follicles of slaughter house ovaries were aspirated and the collected oocytes with cumulus-oocytes complexes (COCs) were cultured in TCM-199 medium supplemented with 10% fetal calf serum, 10% buffalo follicular fluid and 0.5μgmL−1 FSH in 5% CO2 incubator at 38.5°C. The matured oocytes were then inseminated with frozen-thawed buffalo semen suspended in BO medium. After 42h of post-inseminations the cleavage rates were evaluated. The 2–4 cell-cleaved eggs (Day 2 of post-insemination) were randomly divided and cultured for eight days in vitro in 1) modified synthetic fluid (mSOF)+0.8 %BSA (control), 2) mSOF+0.8 % BSA+gelatin (1mgmL−1) 3) mSOF+0.8% BSA+1mgmL−1 gelatin+10ngmL−1 epidermal growth factor (EGF). Supplementation of gelatin increased the specific gravity of the mSOF medium from 0.9658±0.009 to 1.0331±0.013 without any change in pH (7.4). The development of embryos to the 8–16 cell-stage on day 4 of in vitro culture were significantly higher (P&lt;0.05) in mSOF+0.8% BSA+1mgmL−1 gelatin (81.8%; 27/33) than that in mSOF + 0.8% BSA (75.7%; 28/37) and mSOF+0.8% BSA+10ng/mL EGF (68.7%; 22/32). When these embryos were further cultured for another four days (Day 8), the development of transferable quality embryos (morula/blastocyst) was 42.4% (14/33), 48.7% (18/37) and 46.9% (15/32), respectively. Supplementation of gelatin increased the cleavage of eggs up to the 8–16 cell-stage embryo, but did not significantly enhance the rate of development to the morula/blastocyst stage in comparison to control and EGF-supplemented group. However, the percentage of transferable quality embryos was slightly lower in the gelatin-added group but not statistically significant than other groups. The study concluded that increase in specific gravity of the in vitro culture medium enhanced initial cleavage rate but did not have any role in transferable embryo production in buffalo.

scholarly journals Supplementation of Follicle Stimulating Hormon Into In vitro Maturation Medium to Increase Oocytes Maturation and 4 Cell Stadium Embryo Development of Bligon Goat

2018 ◽  
Vol 42 (2) ◽  
Author(s):  
Yanuar Achadri ◽  
Sigit Bintara ◽  
Diah Tri Widayati
Keyword(s):  
Tissue Culture ◽  
Embryo Development ◽  
Embryo Culture ◽  
In Vitro Maturation ◽  
Culture Medium ◽  
Tissue Culture Medium ◽  
Cleavage Rate ◽  
Maturation Medium ◽  
Phosphate Buffered Saline

The study was carried out to investigate the effect of follicle stimulating hormon (FSH) into in vitro maturation medium to increase oocytes maturation and 4 cell stadium embryo development of Bligon goat. Goat ovaries were obtained from a slaughterhouse and transported to the laboratory in a flask of NaCl at temperature of 31 – 34°C. Oocytes were aspirated from 2 – 6 mm of follicles into a 3 mL syringe (23G needle) that contained Dulbecco’s Phosphate-Buffered Saline. Oocytes were divided into three groups, i.e tissue culture medium (TCM) with FSH supplementation 0, 50, and 100 IU/mL. Oocytes were put into those medium and incubated on 39°C, 5% CO2, and 95% humidity for 24 hours. Matured oocytes were fertilized with capacitated frozen thawed-semen and incubated on 39°C, 5% CO2, and 95% humidity for 5 hours. Fertilized oocytes were washed for 3 times in TCM and incubated in the same condition for embryo culture. The data of FSH supplementation and embryo development were analyzed using randomized completely one way classification. The results showed that the percentages of mature oocytes from FSH supplementation 0, 50, and 100 IU/mL were 70,48±23,22, 78,48±15,80, and 80,29±12,86%, respectively. Cleavage rate of the two cells stage were 36,00±14,22, 44,00±33,94, and 57,45±31,78%, respectively, and for the 4 cells stage were 27,33±22,04, 35,33±40,73, and 39,45±20,38%. It is concluded that supplementation of FSH in the maturation medium could not increase the percentages of in vitro maturation and embryo development.

Bovine non-competent oocytes (BCB–) negatively impact the capacity of competent (BCB+) oocytes to undergo in vitro maturation, fertilisation and embryonic development

Zygote ◽  
2015 ◽  
Vol 24 (2) ◽  
pp. 245-251 ◽  
Author(s):  
M.B. Salviano ◽  
F.J.F. Collares ◽  
B.S. Becker ◽  
B.A. Rodrigues ◽  
J.L. Rodrigues
Keyword(s):  
Embryonic Development ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
The Other ◽  
Control Group ◽  
Cleavage Rate ◽  
Morphological Evaluation ◽  
Group 3

SummaryCompetent oocyte selection remains a bottleneck in the in vitro production (IVP) of mammalian embryos. Among the vital assays described for selecting competent oocytes for IVP, the brilliant cresyl blue (BCB) test has shown consistent results. The aim of the first experiment was to observe if oocytes directly submitted to IVM show similar cleavage and blastocyst rates as those obtained with oocytes maintained under the same in vitro conditions as the oocytes that undergo the BCB test. Bovine cumulus–oocyte complexes (COCs) were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised grouped into three groups: (1) directly submitted to IVM; (2) oocytes submitted to the BCB test without the addition of BCB stain (BCB control group); and (3) submitted to the BCB test. The results showed that oocytes directly submitted to IVM reached similar cleavage (48/80 – 60%) and embryonic development rates to the blastocyst stage (10/48 – 21%) as the results obtained with the BCB control group oocytes (45/77 – 58% and 08/45 – 18%, respectively). The aim of the second experiment was to determine the cleavage and blastocyst rates obtained from BCB+ oocytes undergoing IVM in the presence of BCB– oocytes at a ratio of 10:1. COCs were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised into two groups that were submitted to IVM either directly (1: control group) or submitted to the BCB test prior to IVM. After the BCB test, the COCs were classified as either BCB+ (blue cytoplasm) or BCB– (colourless cytoplasm) and then divided into four experimental groups: (2) BCB+; (3) BCB–; and (4) BCB+ matured in same IVM medium drop as (5) BCB– at a ratio of 10:1. After IVM (24 h), oocytes from the different experimental groups were submitted to in vitro fertilisation (IVF) and in vitro culture (IVC) under the same culture conditions until they reached the blastocyst stage (D7). With regards to the cleavage rate (48 h after IVF), only group 3 (102/229 – 44%) differed (P < 0.05) from the other groups [1 (145/241 – 60%); 2 (150/225 – 67%); 4 (201/318 – 63%) and 5 (21/33 – 63%)]. On day 7, the embryos from group 2 (BCB+) achieved the highest blastocyst rate (46/150 – 31%) (P < 0.05) when compared with the embryo development capacity of the other experimental groups (1: 31/145 – 21%; group 3: 17/102 – 17%; group 4: 46/201 – 23%; and group 5: 2/21 – 10%). In conclusion, submitting BCB+ oocytes that were separated from BCB– oocytes to IVM increases the rate of embryonic development to the blastocyst stage when compared to the control group, BCB– oocyte group, BCB+ paracrine group and BCB– paracrine group. The presence of non-competent oocytes during IVM, even in low proportion (1:10), reduces the capacity of competent oocytes to undergo embryo development and achieve blastocyst stage during IVC.

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