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

2016 ◽  
Author(s):  
Vijay Singh ◽  
A. K. Misra ◽  
Suresh Kumar ◽  
Champak Barman
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Bubalus Bubalis ◽  
Cleavage Rate ◽  
Maturation Rate ◽  
Morula Stage ◽  
The Media

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<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<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<0.05) improved with 50 μM cysteamine/ b-mercaptoethanol.

228 EFFECT OF INSULIN ON BOVINE OOCYTE MATURATION, CUMULUS CELL APOPTOSIS, AND EARLY EMBRYO DEVELOPMENT IN VITRO

2015 ◽  
Vol 27 (1) ◽  
pp. 203
Author(s):  
I. Lindgren ◽  
P. Humblot ◽  
D. Laskowski ◽  
Y. Sjunnesson
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Insulin Treatment ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Blastocyst Formation ◽  
Early Embryo Development ◽  
Maturation In Vitro

Dairy cow fertility has decreased during the last decades, and much evidence indicates that metabolic disorders are an important part of this decline. Insulin is a key factor in the metabolic challenge during the transition period that coincides with the oocyte maturation and may therefore have an impact on the early embryo development. The aim of this study was to test the effect of insulin during oocyte maturation on early embryo development by adding insulin during the oocyte maturation in vitro. In this study, abattoir-derived bovine ovaries were used and cumulus-oocyte complexes (n = 991) were in vitro matured for 22 h according to standard protocols. Insulin was added during maturation in vitro as follows: H (10 µg mL–1 of insulin), L (0.1 µg mL–1 of insulin), or Z (0 µg mL–1 of insulin). After maturation, oocytes were removed and fixed in paraformaldehyde before staining. Click-it TUNEL assay (Invitrogen, Stockholm, Sweden) was used for apoptotic staining and DRAQ5 (BioNordika, Stockholm, Sweden) for nuclear staining (n = 132). Cumulus-oocyte complexes were evaluated using laser scanning confocal microscope (Zeiss LSM 510, Zeiss, Oberkochen, Germany). Five levels of scans were used to assess oocyte maturation (MII stage) and apoptosis. Because of incomplete penetration of the TUNEL stain (3–5 layers of cumulus cells), only the outer 2 layers of the cumulus complex were investigated regarding apoptosis. Apoptotic index was calculated as apoptotic cells/total cells visualised. Remaining oocytes were fertilized and cultured in vitro until Day 8. Day 7 and Day 8 blastocyst formation was assessed as well as blastocyst stage and grade. Effect of insulin treatment on variables was analysed by ANOVA following arc sin √p transformation. Post-ANOVA comparisons between H+L group v. Z were performed by using the contrast option under GLM (Scheffé test). Results are presented as least squares means ± s.e. P-values ≤ 0.05 were considered as statistically significant. Insulin treatment during oocyte maturation in vitro had no significant effect on oocyte nuclear maturation or apoptotic index of the cumulus cells (Z: 0.052 ± 0.025, L: 0.039 ± 0.016, H: 0.077 ± 0.044, P > 0.05). No effect was seen on cleavage rates (Z: 0.85 ± 0.02, L: 0.85 ± 0.02, H: 0.89 ± 0.03, P > 0.05), but insulin treatment significantly decreased Day 7 rates from fertilized oocytes (Z: 0.19 ± 0.02, L: 0.14 ± 0.02, H: 0.12 ± 0.02, P < 0.05). This study also showed a significantly retarded developmental stage and decreased grade of blastocysts in insulin-treated groups taken together when compared with the control group (P < 0.05). In this study, no effect of insulin supplementation during in vitro maturation was seen on bovine oocyte maturation and apoptosis of cumulus cells, but blastocyst formation and development were negatively affected. Further studies are needed for understanding the relationship between the addition of insulin during maturation in vitro and impaired blastocyst formation. Insulin is a common supplement in the first phase of the first in vitro maturation medium for pig oocytes and is believed to have a beneficial effect on this species.Funding was received from Stiftelsen Nils Lagerlöfs Fond H12–0051-NLA.

344 EFFECTS OF BMP4 AND ITS INHIBITOR, NOGGIN, ON OOCYTE MATURATION AND DEVELOPMENT OF BOVINE PREIMPLANTING EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 328
Author(s):  
I. La Rosa ◽  
R. Fernandez y Martín ◽  
D. A. Paz ◽  
D. F. Salamone
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cell Number ◽  
Bmp Signaling ◽  
Control Group ◽  
Cleavage Rate ◽  
Exact Test ◽  
Student’S T

BMP4 regulates different events during development in all vertebrates and Noggin is one of its powerful inhibitors that blocks BMP4 interaction with its receptors (Groppe et al. 2002). In this work, the effect of these factors on bovine oocyte maturation and subsequent embryo development has been investigated. COCs were aspirated from abattoir ovaries and in vitro-matured for 22 h or 24 h in a 5% CO2 humidified atmosphere at 39°C in TCM containing 0.6% BSA, 2 mM FSH, 10 mM cysteamine, 1% antibiotic and 1% pyruvate, control group (C), plus 100 ng mL-1 of BMP4 (B), or 100 ngmL-1 of Noggin (NOG). Oocytes were stained with Hoechst 33342 and classified by their nuclear stage. Effects on embryo development were investigated for embryos produced by parthenogenic activation (PA) and IVF For PA, denuded oocytes were chemically activated in 5 μM ionomycine for 4 min, and immediately incubated in 1.9 mM of 6-dimethilaminopurine for 3 h. For IVF, frozen-thawed semen was centrifuged and resuspended in Bracket and Oliphant (BO) solution and incubated with 22 h matured COCs for 5 h. Embryos were cultured in CR2 medium free of serum and co-culture. Cleavage and blastocyst formation were registered at Day 2 and 9 respectively. Fischer’s exact test was used and P ≤ 0.05 was considered significant. Nuclear progression was not affected by maturation treatments [% of MII: 79.4(C, n = 102), 72.4 (B, n = 98), 80.9 (NOG, n = 89)]. For PA, both factors significantly increased cleavage rates [%: 51.7 (C, n = 284), 65 (B, n = 186), 62.1 (NOG, n = 198)] while blastocyst rates were not affected [%: 8.8 (C), 7.5 (B), and 8.6 (NOG)]. On the other hand, for IVF, cleavage rate was statistically lower for Noggin group [%: 70.7 (C, n = 140), 71.3 (B, n = 157), 64 (NOG, n = 159)] while blastocyst rates were similar between groups [%: 15.7 (C), 13.4 (B), 14.5 (NOG)]. Any of the added factors affected cell number of the embryos at Day 2. Blastocysts did not differ in the number of cells at Day 9 (Student’s t-test was used) neither for PA [mean ± SD: 100 ± 33 (C, n = 9), 88 ± 14 (B, n = 3) and 68 ± 8,(NOG, n = 3)] nor for IVF [mean ± SD: 90 ± 24 (C, n = 9), 132 ± 18 (B, n =4) and 99 ± 8 (NOG, n = 3)]. It is noticeable that addition of these factors during in vitro maturation showed different effects on subsequent embryo development depending on whether the embryos were PA or IVF. Probably, these responses represent differences in the BMP signaling system between these embryos which could be associated with different imprinting pattern. Further experiments are needed to elucidate clearly the mechanisms implicated. To our knowledge, this is the first work to study BMP4 inhibition during bovine in vitro maturation. To “Merlo” and “Nueva Escocia” Slaughterhouses

205 EFFECT OF OSTEOPONTIN ON CLEAVAGE AND BLASTOCYST RATES IN BUFFALO SPECIES (BUBALUS BUBALIS)

2009 ◽  
Vol 21 (1) ◽  
pp. 200
Author(s):  
S. Di Francesco ◽  
E. Mariotti ◽  
M. Rubessa ◽  
G. Campanile ◽  
R. Di Palo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cumulus Cells ◽  
Bubalus Bubalis ◽  
The Other ◽  
Control Group ◽  
Single Chain ◽  
Cleavage Rate ◽  
Chi Square ◽  
Vitro Fertilization

It was previously reported that osteopontin (OPN), an acidic single-chain phosphorylated glycoprotein found in the oviductal fluid in cattle (Gabler C et al. 2003 Reproduction 126, 721–729), is able to facilitate fertilization in this species (Gasparrini B et al. 2008 Reprod. Fertil. Dev. 20(Suppl. I), 180 abst). The present study aimed to investigate whether the addition of OPN to the fertilization medium would affect both cleavage and postfertilization embryo development in the buffalo. To assess the influence of OPN on cleavage and blastocyst rates, in vitro-matured oocytes were fertilized in modified Tyrode’s albumin lactate pyruvate medium (Lu KH et al. 1987 Vet. Rec. 121, 259–260) supplemented with penicillamine, hypotaurine, and heparin, in the presence of 0.0 (n = 258), 0.1 (n = 263), 1 (n = 261), and 10 μg mL–1 (n = 264) of OPN. In vitro fertilization was carried out with frozen–thawed spermatozoa from a bull already tested for IVF. After 20 to 22 h of co-incubation at 38.5°C and 5% CO2 in air, putative zygotes were gently pipetted to remove cumulus cells, washed, and transferred, 10 per droplet, into 20 μL of SOF medium including essential and nonessential amino acids and BSA (Tervit HR et al. 1972 J. Reprod. Fertil. 30(3), 493–497), in a controlled gas atmosphere consisting of 5% CO2, 7% O2, and 88% N2, in humidified air, at 38.5°C. The culture medium was changed on Day 5 (Day 0 = day of insemination), when cleavage rate was assessed and embryos were moved into fresh medium for an additional 2 days. On Day 7, development rates into blastocysts of superior quality were recorded. Differences in the percentages of both cleavage and blastocyst rates among groups were analyzed by chi-square test. Significantly higher cleavage rates (59.3, 70.3, 71.6, and 42.4%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. Likewise, higher blastocyst rate percentages (17.4, 27.4, 29.9, and 9.5%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. In conclusion, these results showed that addition of low concentrations of OPN in the fertilization medium improved both cleavage and postfertilization embryo development in the buffalo, whereas the higher concentration resulted in impaired late-stage embryo development.

Effect of butafosfan supplementation during oocyte maturation on bovine embryo development

Zygote ◽  
2019 ◽  
Vol 27 (05) ◽  
pp. 321-328
Author(s):  
Lucas Teixeira Hax ◽  
Joao Alveiro Alvarado Rincón ◽  
Augusto Schneider ◽  
Lígia Margareth Cantarelli Pegoraro ◽  
Letícia Franco Collares ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Nuclear Maturation

SummaryAround 60–80% of oocytes maturated in vivo reached competence, while the proportion of maturation in vitro is rarely higher than 40%. In this sense, butafosfan has been used in vivo to improve metabolic condition of postpartum cows, and can represent an alternative to increase reproductive efficiency in cows. The aim of this study was to evaluate the addition of increasing doses of butafosfan during oocyte maturation in vitro on the initial embryo development in cattle. In total, 1400 cumulus–oocyte complexes (COCs) were distributed in four groups and maturated according to supplementation with increasing concentrations of butafosfan (0 mg/ml, 0.05 mg/ml, 0.1 mg/ml and 0.2 mg/ml). Then, 20 oocytes per group were collected to evaluate nuclear maturation and gene expression on cumulus cells and oocytes and the remaining oocytes were inseminated and cultured until day 7, when blastocysts were collected for gene expression analysis. A dose-dependent effect of butafosfan was observed, with decrease of cleavage rate and embryo development with higher doses. No difference between groups was observed in maturation rate and expression of genes related to oocyte quality. Our results suggest that butafosfan is prejudicial for oocytes, compromising cleavage and embryo development.

scholarly journals Does the Apoptosis Value of Cumulus Cells Play a Role in Rescue Oocyte in Vitro Maturation?

2020 ◽  
pp. 1
Author(s):  
Tulay Irez ◽  
Sinem Ercan Dogan ◽  
Enver Ciraci ◽  
Saadet Busra Aksoyer ◽  
Muhammet Sait Toprak ◽  
...  
Keyword(s):  
Experimental Study ◽  
Luteinizing Hormone ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Immature Oocytes ◽  
Maturation Rate

<p><strong>OBJECTIVE:</strong> In this study, we aimed to investigate the role of the cumulus cell’s apoptosis parameter in the maturation of immature rescue oocytes. </p><p><strong>STUDY DESIGN:</strong> In this experimental study, donated immature germinal vesicle oocytes were cultured for, in vitro maturation, embryo development in matured germinal vesicle oocytes were compared with apoptotic properties of cumulus cells. </p><p><strong>RESULTS:</strong> In all of the immature oocytes after oocyte in vitro maturation, the maturation rate has been observed as 56.1% and 2PN rate as 63.0%. Afterin vitro maturation of germinal vesicle oocytes, there was no difference in apoptosis rates of the cumulus cells between mature and immature oocytes (p&gt; 0.05). The ratio of 2PN in matured germinal vesicle oocytes showing embryo development was 35.4%. A positive correlation was found between luteinizing hormone values on day 3 and E2 values during HCG days during oocyte maturation and embryo development (p=0.021, p=0.020). In addition, it has been observed that the germinal vesicle oocytes, which have completed their maturation and developed into embryos, have high E2 values during HCG days (p=0.020).</p><p><strong>CONCLUSION:</strong> In our study, it has been demonstrated that in vitro maturation in rescue oocytes from stimulated cycles, embryo development potential could not be explained by the apoptosis parameter.</p>

155 Oocyte Maturation in Lyophilized In Vitro Maturation Medium as a Method to Increase the Medium’s Shelf-Life

2018 ◽  
Vol 30 (1) ◽  
pp. 217
Author(s):  
M. Rubessa ◽  
D. Weisgerber ◽  
S. Bessler ◽  
J. Bertels ◽  
B. Harley ◽  
...  
Keyword(s):  
Shelf Life ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Freeze Dried ◽  
Maturation Rate ◽  
The Impact

The in vitro production of bovine embryos has dramatically increased in recent years, and with it the demand of stable media with a long shelf-life. In this experiment we evaluated the impact of the freeze-dried in vitro maturation (IVM) medium (Mdry) on in vitro oocyte maturation. We compared the standard IVM and the Mdry media. Medium M199 was used as base for the IVM medium. The percentage of metaphase II oocytes and embryo production were evaluated. Media solutions (10 mL) were aliquoted into 50-mL conical tubes and lyophilized to form a powder concentrate using a Genesis freeze-dryer (VirTis, Gardener, NY, USA). Lyophilization consisted of a constant cooling from 20°C to –10°C at a constant rate of 1°C/min with a 2-h hold at –10°C before sublimation at 0°C. The Mdry medium was held at –80°C for 4 months (only serum and hormones were added before the incubation). When the IVM medium was rehydrated, the pH were adjusted to 7.4. The percentage of mature oocytes was evaluated after 24 h of maturation. The oocytes were stained with Hoechst 33342, and only oocytes with metaphase and a polar body were evaluated as matured. Abattoir-derived Holstein oocytes (n = 540) were in vitro matured (25–30/well in 400 µL) and fertilized with sexed semen, according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). The oocytes were split for analysis (432 were used for IVP and 108 for maturation rate) over 6 replicates. Twenty hours after IVF, presumptive zygotes were cultured in SOF medium at 39°C with 5% CO2, 7% O2, and 88% N2. On Day 7, embryo yields were assessed. All recorded parameters were subjected to a Student’s t-test. The parameters compared were maturation rate, cleavage rate, blastocyst rate and the percentage of embryos cleaved. The α level was set at 0.05. All data were expressed as quadratic means and mean standard deviations. The results showed no differences between the 2 groups (75.9% v. 74.1%) (t = 0.37; SD = 12.69; P = 0.36; df = 5) when we compared the nuclear maturation; however, when we evaluated embryo production, we found the Mdry treatment had a higher cleavage percentage (t = 2.39; SD = 14.81; P = 0.02; df = 5) and total embryos produced (t = 2.49; SD = 5.6; P = 0.02; df = 5) compared with the control (Table 1.). These results showed that lyophilization can be a valid method to increase the shelf life of IVP media. More replicates must be done in order to understand why the freeze-dried media produced more embryos. Table 1.Mean (SD in parentheses) percentage cleavage and blastocysts

119 LEPTIN ENHANCED THE DEVELOPMENT OF BUFFALO (BUBALUS BUBALIS) EMBRYO CULTURED IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 159 ◽  
Author(s):  
Y. Q. Lu ◽  
D. N. Ye ◽  
M. Zhang ◽  
S. S. Lu ◽  
K. H. Lu
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Bubalus Bubalis ◽  
Development Rate ◽  
Blastocyst Development ◽  
Mediterranean Countries ◽  
Technical Factors ◽  
Development In Vitro

Buffalo is an important livestock resource in many Asian and Mediterranean countries. In vitro embryo production (IVEP) and transfer of the embryos to produce calves with high genetic merit would be of great interest in buffalo species. The efficiency of the IVEP in buffalo is low compared to that in bovine. It may be due to the reproductive physiology of buffalo or the technical factors in IVEP procedures. Recent research revealed that supplementation of leptin in the in vitro culture (IVC) medium could significantly increase embryo development (2005 Mol. Cell Endocrinol. 229, 141–147; 2006 Reproduction 132, 247–256). In this study, the effect of leptin on buffalo embryo development in vitro was assessed by supplementation of the leptin into the IVC medium. Methods: Buffalo oocytes were aspirated form 2 to 6 mm follicles from slaughterhouse ovaries and washed in TCM199 and once more with in vitro maturation (IVM) medium (TCM199, 5% ECS, 15 μg mL–1 FSH). Oocytes with compact cumulus cells were matured in IVM medium at 38.5°C, 5% CO2 for 22–22 h. The frozen–thawed buffalo sperm underwent a centrifugation in Percoll gradient to remove the dead sperm. Ten to 15 matured oocytes were added to a drop of 40 μL modified Tyrode’s medium supplemented with 0.6% BSA, 2.0 mm caffeine and 20 μg mL–1 Heparin. Concentration of sperm added into the fertilization medium was 1 to 2 million per mL. Eight to 10 h after insemination, the presumptive zygotes were transferred to IVC medium (TCM199, 10% newborn cow serum) supplemented with 0 ng mL–1 (control), 10 ng mL–1, 100 ng mL–1 or 500 ng mL–1 of leptin. Cleavage and blastocyst development rate was recorded on Day 2 and Day 6 to 8 after insemination. The experiment was repeated 10 times, and a total of 831 oocytes were used with the IVF procedures. The results revealed that the cleavage rates in the group of 0 ng mL–1, 10 ng mL–1, 100 ng mL–1 and 500 ng mL–1 of leptin were 50.1 ± 3.5%, 55.0 ± 1.3%, 50.0 ± 1.8% and 52.9 ± 2.2%, respectively. No statistical difference was observed regarding cleavage rates between treatments (P > 0.05). The percentage of oocytes developing to blastocysts in the group of 10 ng mL–1 and 100 ng mL–1 leptin were 26.1 ± 1.5% and 23.5 ± 1.2%, respectively, significantly higher than that of 17.5 ± 2.1% in the control (P < 0.05). The blastocyst development rate in the group of 500 ng mL–1 leptin was 20.9 ± 1.4%, less than that of 10 ng mL–1 (P < 0.05). In conclusion, the results of this study indicated that supplementation of leptin in the IVC medium could enhance the blastocyst development in buffalo species and the optimal concentration of leptin in the present procedures was 10 ng mL–1. This work was jointly supported by National Science and Technology Supporting Program (No. 2006BAD04A18), Guangxi Science Foundation (0832012) and Guangxi University Key Research Program (No. 2005ZD05).

94 USE OF β-MERCAPTOETHANOL AND CYSTEINE FOR IN VITRO MATURATION AND CULTURE OF SHEEP EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 159
Author(s):  
J. Pradiee ◽  
L. L. Viana ◽  
E. C. S. Santos ◽  
A. Gonçalves ◽  
R. G. Mondadori ◽  
...  
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
In Vitro Production ◽  
Embryo Viability ◽  
Negative Effects ◽  
Cleavage Rate ◽  
Chi Squared ◽  
Antibiotic Solution ◽  
The Media

During in vitro production (IVP), embryos are sensitive to suffering negative effects from catabolites, such as reactive oxygen species (ROS). Under physiological conditions, the action of the ROS is blocked by antioxidants such as glutathione, but glutathione's concentration is reduced during the main steps of the IVP process. The objective of the present study is to evaluate the effect of the supplementation of the media for in vitro maturation (IVM) and in vitro culture (IVC) with β-mercaptoethanol and cysteine on the rates of embryo development and viability after vitrification in open pulled straws (OPS). Ten IVP routines were conducted for IVP, using ovaries form pubertal sheep collected in a slaughterhouse. The ovaries were kept in a saline/antibiotic solution at 30°C during transport to the laboratory. The cumulus oophurus–oocytes complexes (COC) selected for IVM were allocated to 2 treatments: T1 (control), including no antioxidants in the IVM and IVC media (n = 676); and T2, including 50 μM β-mercaptoethanol and 600 μM cysteine, in the IVM and IVC media (n = 729). The IVM was conducted using the TCM 199 medium including oestradiol, FSH, LH, pyruvate, heat inactivated sheep serum and antibiotics, for 22 to 24 h. Sperm selection was conducted by swim-up in medium with tris-glucose-citric acid with fresh semen. For IVF, conducted for 18 to 22 h, 1 × 106 spermatozoa per mL were used in SOF medium including 2% heat-inactivated sheep serum. Both IVM and IVF were conducted with incubation with 5% CO2 at 39°C with saturated humidity. After IVF, the probable zygotes were denuded and cultured for 8 days in SOF medium with 0.4% BSA, at 39°C, in bags with 3 gases (5% CO2, 90% N2 and 5% O2). The criteria considered for embryo viability were: cleavage rate at Day 2 (cleaved/inseminated), embryo development at Day 7 (blastocysts/cleaved) and the reexpansion rate 24 h post-vitrification. Such frequencies were compared between treatments by the chi-squared test. The cleavage rate did not differ (P > 0.05) for T1 (60.3%) and T2 (64.3%). The rate of embryro development at Day 7 was also similar (P > 0.05) for T1 (33.6%) and T2 (36.6%). The reexpansion rate for T1 (76.9%) and T2 (54.1%) were also similar (P > 0.05). Thus, supplementation of IVM and IVC media with β-mercaptoethanol and cysteine presented no effect in the development and viability of vitrified sheep embryos. CAPES, MARFRIG Group.

212 EFFECT OF CANINE OVIDUCT CELLS AND CUMULUS CELLS CO-CULTURE ON IN VITRO MATURATION OF PORCINE OOCYTES AND EMBRYO DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 237
Author(s):  
S. H. Lee ◽  
H. J. Oh ◽  
G. A. Kim ◽  
M. J. Kim ◽  
Y. B. Choi ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cumulus Cells ◽  
In Vitro Development ◽  
First Polar Body ◽  
Vitro Development ◽  
And Control

In oestrus stage, canine oocytes surrounded by cumulus cells undergo maturation in oviduct for 3 days after ovulation. We hypothesised that canine cumulus cells (cCC) and canine oviduct cells (cOC) in oestrus stage might affect the maturation of oocyte and embryo development. Therefore, the present study was aimed to compare the effects of cCC and cOC co-culture system on oocyte in vitro maturation and embryo in vitro development. cCC were separated from cumulus‐oocyte complex (COC) in ovary from bitches in oestrus phase. cOC were collected from oviduct flushing of bitches in oestrus phase. Both cCC and cOC were cultured and cryopreserved until use for co-culture. In the first experiment, the effect of co-culture using cCC and cOC on porcine oocyte in vitro maturation (IVM) were investigated. The porcine COC were randomly cultured in different co-culture groups as follows: 1) co-culturing with cCC for 42 h, 2) co-culturing with cOC for 42 h, and 3) culturing in absence of cCC or cOC. After IVM, extrusion of the first polar body was observed under a microscope. In the second experiment, the matured oocytes with the first polar body derived from each group were activated with electrical stimulus. Parthenotes were cultured in porcine zygote medium-5 (PZM-5) for 7 days at 39°C, 5% CO2 and O2 in a humidified atmosphere. The embryo developmental competence was estimated by assessing the in vitro development under microscope. The third experiment was to evaluate the reactive oxygen species (ROS) levels in each supernatant medium obtained from cCC and cOC co-culture group after IVM using a OxiselectTM ROS ELISA Assay kit. Last, analysis of genes (MAPK1/3, SMAD2/3, GDF9 and BMP15) expression in cCC and cOC co-cultured with porcine COC using real-time PCR is in progress. As results, IVM rate of cOC group (91.19 ± 0.45%) was significantly higher than that of cCC and control group (86.50 ± 0.61% and 79.81 ± 0.82%; P < 0.05). Also, cOC groups expressed the highest efficiency in cleavage rate, blastocyst formation rate, and the total cell number in blastocyst (P < 0.05). In ROS levels, cOC group (555 ± 7.77 nM) were significantly lower than cCC and control groups (596.8 ± 8.52 nM and 657.8 ± 11.34 nM). The present study demonstrated that co-culture with cOC improved the in vitro oocyte maturation and the in vitro development rate of porcine embryos. The ROS level decreased in cOC co-culture would have beneficial influence on oocytes maturation. For further study, we will investigate the relation between gene expression related to oocyte maturation and the co-culture results. This research was supported by a global PhD Fellowship Program through NRF funded by the Ministry of Education (NRF-20142A1021187), RDA (#PJ010928032015), IPET (#311011–05–4-SB010, #311062–04–3-SB010), Research Institute for Veterinary Science, and the BK21 plus program.

Effect of nano-selenium and nano-zinc particles during in vitro maturation on the developmental competence of bovine oocytes

2018 ◽  
Vol 58 (11) ◽  
pp. 2021 ◽  
Author(s):  
B. R. Abdel-Halim ◽  
Nermeen A. Helmy
Keyword(s):  
Dna Damage ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Expansion Rate ◽  
Control Group ◽  
Nano Zinc ◽  
Bovine Oocytes

The objectives of the current study were to evaluate the effects of supplemental nano-selenium (NSe) and nano-zinc oxide (NZn-O) particles during in vitro maturation (IVM) on DNA damage of cumulus cells, glutathione (GSH) concentration in bovine oocytes, subsequent embryo development and re-expansion rate of vitrified warmed blastocysts. The current study was conducted on bovine ovaries obtained from a local abattoir and transported to the laboratory in sterile phosphate buffer saline with antibiotics at 37°C, within 1 h after slaughter. Ovaries were pooled, regardless of stage of the oestrous cycle of the donor. Only cumulus-intact complexes with evenly granulated cytoplasm were selected for IVM. Experimental design included the following: Experiment 1 studied the effect of addition of 1.0 µg/mL NSe or NZn-O to IVM medium on DNA damage of cumulus cells; Experiment 2 evaluated the effects of NSe or NZn-O on intracellular glutathione in oocytes and cumulus cells; in Experiment 3, the development of oocytes matured in IVM medium supplemented with 1.0 µg/mL NSe or NZn-O was investigated; and in Experiment 4, the effects of adding 1.0 µg/mL NSe and NZn-O to in vitro fertilisation media on vitrified oocytes and embryos were investigated. The DNA damage in cumulus cells decreased with supplemental NSe and NZn-O at concentration of 1 µg/mL in the IVM medium (180.2 ± 21.4, 55.8 ± 4.3 and 56.6 ± 3.9 for the control and NSe and NZn-O groups respectively). Total GSH concentrations increased following supplementation with 1 µg/mL NSe and 1 µg/mL NZn-O, compared with the control group. Re-expansion rate of vitrified warmed blastocysts in experimental media containing NSe and NZn-O with ethylene glycol was higher than that of the control. In conclusion, providing NSe and NZn-O during oocyte maturation significantly increased both intracellular GSH concentration and DNA integrity of cumulus cells. Optimal embryo development was partially dependent on the presence of NSe and NZn-O during IVM. NSe and NZn-O during oocyte maturation act as a good cryoprotective agents of vitrified, warmed blastocysts.

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