scholarly journals 146COMPARISON OF EMBRYO CULTURE MEDIA FOR BLASTOCYST DEVELOPMENT AFTER INTRACYTOPLASMIC SPERM INJECTION OF IN VITRO-MATURED EQUINE OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 195
Author(s):  
Y.H. Choi ◽  
D.D. Varner ◽  
K. Hinrichs
Keyword(s):  
In Vitro Culture ◽  
Embryo Culture ◽  
Intracytoplasmic Sperm Injection ◽  
Culture Media ◽  
Polar Body ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Significant Difference

Research on in vitro culture of equine embryos has been scant, due to failure of equine in vitro fertilization to be repeatably successful. We have recently obtained high fertilization rates of equine oocytes via intracytoplasmic sperm injection (ICSI) using a piezo drill (Choi et al., 2002 Reproduction 123, 455–465). Culture of presumptive zygotes in G1.2/2.2 medium resulted in 63% cleavage and an average of 15 cells at 4d, but only 2 to 9% blastocyst development at 7 days (Choi et al., 2003 Theriogenology 59, 1219–1229). In the present study, we evaluated the effect of two different culture media, G1.3/G2.3 v. DMEM/F-12, with or without FBS, on blastocyst development after ICSI. Oocytes were collected from slaughterhouse-derived ovaries by follicular scraping and were matured in vitro for 24h in M199 with 10% FBS and 5μUmL−1 FSH. After culture, oocytes having a polar body (198/305; 65%) were fertilized by ICSI with frozen-thawed equine sperm using a piezo drill. Presumptive zygotes were cultured in 1 of 4 media: G1.3/G2.3 (which includes 0.8% BSA) with or without 10% FBS, or in DMEM/F-12 with 0.5% BSA, with or without 10% FBS. Culture was performed in microdroplets at 5μL/zygote under oil at 38.2°C in an atmosphere of 5% CO2, 5% O2 and 90% N2 for 7.5 days. In G1.3/2.3 treatments, G1.3 media were completely refreshed at 48h, zygotes were transferred to G2.3 (with or without FBS as per the first stage) at 96h, and were completely refreshed with the same media at 144h. In DMEM/F-12 treatments, media were completely refreshed every other day. Three to 5 replicates were performed in each treatment, and data were analyzed by chi-square test. There were no significant differences in cleavage rates (59–64%) among treatments. The rate of development to blastocyst, per oocyte injected, in G1.3/G2.3/BSA (1/49, 2%) was significantly lower (P<0.05) than that for the other three treatments: G1.3/2.3/BSA/FBS (9/49, 18%), DMEM/F-12/BSA (9/50, 18%), or DMEM/F-12/BSA/FBS (10/50, 20%). There was no significant difference in blastocyst development among the latter three treatments. These findings indicate that G1.3/2.3 media with BSA only do not adequately support growth of equine embryos. Development of up to 20% of injected oocytes to the blastocyst stage in G media supplemented with FBS, in DMEM/F-12/BSA or in DMEM/F-12/BSA/FBS represents the highest in vitro equine blastocyst rate in medium alone (i.e. without co-culture) yet reported. The success of DMEM/F-12 as an embryo culture medium may provide a relatively simple basis for equine in vitro culture programs. To determine whether this medium was able to support further developmental competence, we cultured equine embryos resulting from nuclear transfer of in vitro-matured oocytes in DMEM/F-12+10% FBS (without BSA). We transferred 4 resulting blastocysts to recipient mares by transcervical transfer; one pregnancy is ongoing at 230d gestation at the time of this writing. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University.

scholarly journals Impact of L-carnitine supplementation on the in vitro developmental competence and cryotolerance of buffalo embryos

2021 ◽  
pp. 3164-3169
Author(s):  
Mohamed M. M. El-Sokary ◽  
Al-Shimaa Al-H. H. El-Naby ◽  
Amal R. Abd El Hameed ◽  
Karima Gh. M. Mahmoud ◽  
T. H. Scholkamy
Keyword(s):  
Embryo Culture ◽  
In Vitro Maturation ◽  
Culture Media ◽  
Effective Concentration ◽  
Developmental Competence ◽  
Carnitine Supplementation ◽  
Oocyte Developmental Competence ◽  
High Lipid ◽  
Significant Difference

Background and Aim: Despite many trials, buffalo embryos have poor cryosurvivability because of their high lipid content. L-carnitine was found to be a lipid-reducing agent when added to oocyte and embryo culture media. The study aimed to determine the most effective concentration of L-carnitine to improve the oocyte developmental competence and cryotolerance of buffalo embryos. Materials and Methods: In vitro maturation and embryo culture media were supplemented with four concentrations of L-carnitine: 0 (control), 0.25, 0.5, and 1 mM. Good-quality embryos on 7 days were vitrified using mixtures of dimethyl sulfoxide and ethylene glycol at two concentrations (3.5 and 7 M). Results: The result showed that the cleavage and morula rates were significantly (p<0.05) higher in the 0.5 mM group. Blastocyst rates were significantly (p<0.05) higher at both 0.5 and 1 mM. The rates of viable embryos directly after thawing were significantly (p<0.05) increased in the 0.5 mM group. No significant difference was found in embryos cultured for 24 h after warming among all the groups. Conclusion: The addition of L-carnitine at a concentration of 0.5 mM to the culture media improves the oocyte developmental competence and cryotolerance of buffalo embryos directly after warming but not after 24 h of culture. Nevertheless, further studies must identify how L-carnitine exerts its beneficial micromechanisms.

scholarly journals Factors affecting developmental competence of equine oocytes after intracytoplasmic sperm injection

Reproduction ◽  
2004 ◽  
Vol 127 (2) ◽  
pp. 187-194 ◽  
Author(s):  
Y H Choi ◽  
L B Love ◽  
D D Varner ◽  
K Hinrichs
Keyword(s):  
Embryo Culture ◽  
Intracytoplasmic Sperm Injection ◽  
Room Temperature ◽  
Culture Media ◽  
Developmental Competence ◽  
Glucose Medium ◽  
Factors Affecting ◽  
Nuclear Maturation ◽  
High Glucose Medium

This study was conducted to evaluate the effect of initial cumulus morphology (expanded or compact) and duration of in vitro maturation (24, 30 or 42 h) on the developmental competence of equine oocytes after intracytoplasmic sperm injection (ICSI). The effect of manipulation temperature (room temperature vs 37 °C) at the time of ICSI and concentration of glucose (0.55 vs 5.5 mM) during embryo culture was also investigated. The nuclear maturation rates of expanded (Ex) oocytes were significantly (P < 0.001) higher than those of compact (Cp) oocytes at all maturation times (61–72 vs 23–25% respectively). Forty-eight hours after ICSI of mature Ex oocytes, the rate of cleavage with normal nuclei was significantly (P < 0.05) higher for oocytes matured for 24 h than for those matured for 30 or 42 h (73 vs 57–59% respectively). For Cp oocytes, the morphologic cleavage rates for oocytes matured for 30 h were significantly higher (P < 0.05) than for those matured for 24 or 42 h (86 vs 55–61% respectively). The overall proportion of embryos having more than four normal nuclei at 48 h culture was significantly higher (P < 0.05) for Cp than for Ex oocytes. Manipulation temperature did not affect development of embryos from Ex or Cp oocytes at 96 h after ICSI. Culture in high-glucose medium significantly increased morphologic cleavage of Cp, but not Ex, oocytes (P < 0.05). Embryos from Cp oocytes had a significantly higher average nucleus number after 96-h culture than did embryos from Ex oocytes. These data indicate that developmental competence differs between Ex and Cp equine oocytes, and is differentially affected by the duration of maturation and by composition of embryo culture media.

100 SEQUENTIAL VERSUS SINGLE-STEP MEDIUM FOR RHESUS MACAQUE EMBRYO CULTURE

2016 ◽  
Vol 28 (2) ◽  
pp. 180
Keyword(s):  
Embryonic Development ◽  
Embryo Culture ◽  
Culture Media ◽  
Blastocyst Stage ◽  
Single Step ◽  
Blastocyst Development ◽  
Equal Variance ◽  
Significant Difference ◽  
Health Studies

The nonhuman primate (NHP) is a valuable translational model for human health studies and is widely used to investigate pre-implantation embryo development. Central to these investigations is the dependency on in vitro embryo culture (IVC). Since 2001, the single-step hamster embryo culture medium (HECM) has been the accepted standard for NHP IVC. With recent advances in formula optimization for IVC in human clinics, a re-examination of optimal NHP IVC media is warranted. Thus, two types of commercially available IVC media routinely used in human applications were compared with HECM-9: Global (single-step; LifeGlobal Group, Guilford, CT, USA), and Quinns Advantage (sequential; SAGE, Trumbull, CT, USA). Normally cycling, adult rhesus monkeys (n = 3) underwent controlled ovarian stimulations, and follicles were aspirated via laparoscope. Recovered ova were fertilized in vitro and the resultant zygotes (n = 138) were cultured for 9 days in HECM-9, Global, or Quinns with 10% protein supplement at 37.5°C in humidified tri-gas (6% CO2, 5% O2, and 89% N). Single-step media (HECM-9 and Global) were refreshed every two days while embryos were cultured for Days 1–3 in Quinns Advantage Cleavage medium without being replaced and in Quinns Advantage Blastocyst medium for Days 4–9 with medium changes every 2 days. Embryos were observed for cleavage, compaction, and blastocyst development. Proportional data with equal variance and normal distribution were analysed by one-way ANOVA, and significance was determined post-hoc by the Holm-Sidak method with P < 0.05. Developmental stage data ± s.e.M are presented in Table 1; a change in superscript indicates a significant difference within the column. There was no difference in embryonic cleavage or morula compaction between the three culture media evaluated, indicating no obvious differences in their effects on embryonic development 1 to 3 days after fertilization. However, a greater proportion of blastocysts developed in Global medium compared with HECM-9, and though it was not statistically different, embryos cultured in Global tended to reach the blastocyst stage more frequently than those in Quinns. Although not significant due to large variances in each group, blastocyst expansion also tended to occur more frequently in Global medium than in HECM-9 or Quinns. Taken together, these data indicate that single-step Global is as supportive of early embryonic development as HECM-9 but is better formulated to facilitate later stage differentiation and would be better suited for use in updated standard NHP IVC protocols. Table 1.Cleavage, compaction, blastocysts, and expansion of embryos in HECM-9, Global, and Quinns media

125 INTRACYTOPLASMIC SPERM INJECTION (ICSI)-BASED MOUSE EMBRYO ASSAY: CHOICE OF EMBRYO CULTURE SYSTEM OUTWEIGHS THE EFFECT OF FERTILIZATION PROCEDURE ON EMBRYO DEVELOPMENT

2013 ◽  
Vol 25 (1) ◽  
pp. 209
Author(s):  
C. Schwarzer ◽  
T. C. Esteves ◽  
S. Le Gac ◽  
V. Nordhoff ◽  
S. Schlatt ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Mouse Embryo ◽  
Embryo Culture ◽  
Intracytoplasmic Sperm Injection ◽  
Culture Medium ◽  
Culture Media ◽  
Blastocyst Formation ◽  
The Absolute ◽  
Mouse Embryo Assay

Human embryo culture media, intended for assisted reproductive technologies (ARTs), are released for clinical use if they pass the mouse embryo assay (MEA). This assay prescribes that at least 70% of in vivo fertilized mouse 1-cell embryos form blastocysts, in order to grant the culture medium approval. In the fertility clinic, however, human embryos undergo more manipulation than their MEA counterparts through, for example, fertilization by intracytoplasmic sperm injection (ICSI); further, only a minority of the embryos transferred to the uterus goes on to establish gestations. In this context, we asked if the results of the MEA only depend on the type of in vitro culture, or are also affected by the method of fertilization. Superovulated B6C3F1 mouse oocytes were fertilized by ICSI using C57Bl/6 sperm. Pronuclear-stage eggs were allocated to four developmental environments: two ART culture protocols (HTF/MultiBlast, Irvine Scientific; ISM1/ISM2, Origio), standard mouse culture medium (KSOM(aa), made in-house) and the oviduct of pseudopregnant CD1 mice. As control for the invasive manipulation, pronuclear-stage eggs were generated by mating (B6C3F1 × C57Bl/6) and cultured in KSOM(aa) medium. Embryos were recovered from culture or from the CD1 uterus and scored for blastocyst formation at 96 h of development (Table 1). For these blastocysts, we determined the number of total, inner cell mass (ICM), and trophectoderm (TE) cells (Table 1) by confocal immunofluorescence microscopy (Schwarzer et al. 2012 doi:10.1093/humrep/des223). Our results show that ART culture protocols applied to mouse ICSI embryos are not equivalent in supporting blastocyst formation. Based on blastocyst rates, the ranking observed here after ICSI, reflects the ranking reported by us for IVF embryos (Schwarzer et al. 2012); that is, KSOM(aa) > HTF/MultiBlast > oviduct > ISM1/2. This similarity suggests that the effect of in vitro culture on mouse development exceeds the effect of ICSI, provided gametes are of good quality. From the analysis of cell numbers, we note that while the ICM/TE ratios are not of easy interpretation, the absolute numbers of cells in the ICM draw a clear line between the environment of the oviduct and those of culture media. Irrespective of the ICM/TE ratio, only the oviduct environment secures 8 cells in the ICM (Table 1). Soriano and Jaenisch (1986 Cell 46, 19–29) reported that 8 cells of the ICM are set aside to give rise to the body of a mouse. In summary, the current MEA is a valuable assay to assess the quality of culture medium, however, its refinement is necessary to better model the adaptive properties of embryo culture when different methods of fertilization are applied. Until the MEA is extended into postimplantation development, as we advocate (Schwarzer et al. 2012), the absolute numbers of cells in the ICM may be a better gauge of embryo quality than the blastocyst rates. Table 1.Mouse embryo assay outcomes after ICSI

102 EFFECT OF DEMECOLCINE PRE-TREATMENT ON VIABILITY, TIMING OF FIRST POLAR BODY EXTRUSION, SPINDLE CONFIGURATION, AND SUBSEQUENT DEVELOPMENT OF OVINE OOCYTES VITRIFIED AT GERMINAL VESICLE STAGE

2010 ◽  
Vol 22 (1) ◽  
pp. 210 ◽  
Author(s):  
A. R. Moawad ◽  
J. Zhu ◽  
I. Choi ◽  
K. H. S. Campbell
Keyword(s):  
Polar Body ◽  
Germinal Vesicle ◽  
Subsequent Development ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
First Polar Body ◽  
Polar Body Extrusion ◽  
And Control

Oocyte cryopreservation is a potentially valuable way of preserving female germ cells. However, to date the reported developmental competence of cryopreserved oocytes is low. The objectives of this study were to investigate the effects of demecolcine pretreatment on viability, timing of the first polar body extrusion (PBI), spindle, chromatin organization, and in vitro embryo development of ovine vitrified germinal vesicle (GV) oocytes after in vitro fertilization (IVF) and parthenogenetic activation. Cumulus-oocyte complexes (COC) aspirated from ovine ovaries collected at slaughter were selected and randomly divided into 3 groups: (1) untreated (in vitro matured, IVM) as a control, (2) vitrified (Moawad AR et al. 2009 Reprod. Fertil. Dev. 21, 135 abst), and (3) deme + vitrified (oocytes were incubated with 0.1 μg mL-1 demecolcine for 20 min before vitrification). After vitrification COC were thawed and matured in vitro for 24 h. Following IVM, oocytes from 3 groups were subsequently subjected to (1) immunostaining, (2) IVF, or (3) activation. Presumptive zygotes were cultured in vitro in SOF media for 7 days. Data were analyzed using chisquare and t-test. No significant differences (P > 0.05) were observed in survival rates between deme + vitrified (90.8%, 324/357) and vitrified (87.2%, 211/242). However, the numbers of oocytes with PBI in two vitrified groups at 18 h (20.4 and 8.5 v. 47.1%) and 24 h post IVM (51 and 43.2 v. 88.5%) were significantly lower (P < 0.01) than those in the control. Percentage of normal spindle and chromatin configuration in the two vitrified groups also significantly decreased (P < 0.05) compared with those in the control (42.5 and 41.8 v. 76.5%), whereas missing spindle in the 2 vitrified groups significantly increased (P < 0.001) compared with the controls (47.5 and 32.7 v. 3.9%). Following IVF (pi), cleavage rates at 24.48 hpi and morula development (5 days pi) were significantly lower (P < 0.001) in deme + vitrified (6.1, 43.1, and 28.5%) and vitrified groups (3.3, 30.1, and 22.9%) than control (50.4, 82.4, and 46.4%). Blastocyst development in deme + vitrified (9.8%) and control (33.6%) was significantly higher (P < 0.01) than in vitrified group (1.3%). Hatched blastocysts were observed only in deme + vitrified and control groups (4.9 v. 12.8%). In addition, post activation (pa) cleavage rates in deme + vitrified (10.3 v. 40.7%) and control (52.5 v. 76.7%) at 24 and 48 hpa were significantly higher (P < 0.05) than those in the vitrified group. Blastocyst development in deme + vitrified (4.8%) was higher than that in the vitrified group (1.8%), but not significant (P > 0.05); however, these values were still significantly lower (P < 0.001) than those in the control (24.2%). No significant differences were observed in total cell numbers per blastocyst between all the groups. Taken together, these results suggest that pretreatment of oocytes with demecolcine before vitrification could improve the developmental competence of ovine vitrified-thawed GV-stage oocytes. A. R. Moawad was supported by the Egyptian government.

The effects of brain-derived neurotrophic factor and metformin on in vitro developmental competence of bovine oocytes

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 187-193 ◽  
Author(s):  
So Gun Hong ◽  
Goo Jang ◽  
Hyun Ju Oh ◽  
Ok Jae Koo ◽  
Jung Eun Park ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Embryo Development ◽  
Neurotrophic Factor ◽  
Brain Derived Neurotrophic Factor ◽  
Early Embryo ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Significant Difference

SummaryBrain-derived neurotrophic factor (BDNF) signalling via tyrosine kinase B receptors may play an important role in ovarian development and function. It has been reported that metformin elevates the activity of Tyrosine kinase receptors and may amplify BDNF signalling. The objective of this study was to investigate the effect of BDNF during in vitro maturation (IVM) and/or in vitro culture (IVC) (Experiment 1), and to evaluate the collaborative effect of BDNF and metformin treatment on the developmental competence of bovine in vitro fertilized (IVF) embryos (Experiment 2). In Experiment 1, BDNF, which was added to our previously established IVM systems, significantly increased the proportions of MII oocytes at both 10 ng/ml (86.7%) and 100 ng/ml (85.4%) compared with the control (64.0%). However, there was no statistically significant difference in blastocyst development between the control or BDNF-supplemented groups. In Experiment 2, in order to investigate the effect of BDNF (10 ng/ml) and/or metformin (10−5 M) per se, TCM-199 without serum and hormones was used as the control IVM medium. The BDNF (48.3%) and BDNF plus metformin (56.5%) significantly enhanced the proportions of MII oocytes compared with the control (34.4%). Although, BDNF or metformin alone had no effect in embryo development, BDNF plus metformin significantly improved early embryo development to the 8–16-cell stage compared with the control (16.5 vs. 5.5%). In conclusion, the combination of BDNF and metformin may have a collaborative effect during the IVM period. These results could further contribute to the establishment of a more efficient bovine in vitro embryo production system.

scholarly journals 253 COMPARISON OF THREE DIFFERENT IVF PROTOCOLS FOR BOVINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 277
Author(s):  
S. Romo ◽  
J. Pryor ◽  
D.D. Varner ◽  
K. Hinrichs ◽  
C.R. Looney
Keyword(s):  
Embryo Culture ◽  
Culture Media ◽  
Sperm Concentration ◽  
Vitro Fertilization ◽  
Group 3 ◽  
Group 2 ◽  
The Given ◽  
Sperm Separation ◽  
Group 1

Recently, the development of commercially available defined media and sperm centrifugation gradients has offered new possibilities for increasing the efficiency of commercial in vitro fertilization (IVF) systems. The objective of this study was to compare three different IVF protocols using two different separation gradients, two fertilization media, and two embryo culture media, as follows: Group 1. sperm separation (SS): Percoll (Sigma, St. Louis, MO, USA), fertilization medium (FM): TALP-Fert (TFM), embryo culture media (ECM): G1/G2 (version 3, Vitrolife, Englewood, CO, USA). Group 2. SS: Percoll, FM: Bovine vitro Fert (Cook, Brisbane, Australia), ECM: Bovine vitro Blast/Bovine vitro Cleave (Cook); and Group 3. SS: EquiPure (Nidacon, Spectrum Technologies, Healdsburg, CA, USA), FM: TFM, ECM: G1/G2. Oocytes were obtained from slaughterhouse ovaries and matured in vitro (Looney et al. 1994 Theriogenology 41, 67). IVF was conducted using frozen/thawed semen from one bull. Semen was separated by centrifugation at 700g for 30 min in the given density gradients; Percoll was used in a 45% to 90% gradient. Sperm viability after separation was assessed by fast-green/eosin stain (Sigma). IVF was carried out in 0.5 mL of the given fertilization medium supplemented with PHE1 and heparin (10 μg/mL), in humidified 5% CO2 in air atmosphere at 38.7°C. Final sperm concentration in the IVF wells was 1 × 106/mL. In Experiment 1, a total of 368 oocytes (2 replicates) were fixed and stained (Hoechst 33342, Sigma) 24 h post-IVF to assess sperm penetration (Group 1, n = 128, Group 2, n = 108, Group 3, n = 132). In Experiment 2, a total of 400 embryos (2 replicates) were cultured in 0.5 mL of the given culture medium under mineral oil in a 5% O2, 5% CO2, 90% N2 atmosphere at 38.7°C with high humidity for 112 h before fixation and staining. Embryos in Groups 1 (n = 129) and 3 (n = 139) and Group 2 (n = 132) were changed to G2 and Cleave media, respectively, at 84 h. Sperm separation with Percoll yielded lower numbers of sperm (average sperm concentration after separation of 218 vs. 383 × 106 for EquiPure; P < 0.05), but resulted in higher total motility (60% vs. 41%, respectively; P < 0.05) and higher viability (93% vs. 70%, respectively; P < 0.05) of separated sperm. In Experiment 1, rates of normal fertilization were significantly lower for Group 3 (58%) than for Groups 1 and 2 (74% and 77%, respectively, P < 0.05). In Experiment 2, rates of development to <8, 9 to 16, and >16 cells at 112 h were not significantly different among groups (43, 48, and 46% for Group 1; 22, 18, and 31% for Group 2; and 35, 34, and 23% for Group 3, respectively; P > 0.1). These results indicate that the commercial separation medium, EquiPure, may be associated with lowered sperm motility, viability, and fertilization rates when compared to a standard medium (Percoll) for bovine sperm separation. Commercial fertilization and embryo culture media (Bovine vitro Fert, Cleave, and Blast) provided equivalent embryo development to that currently in use by our laboratory (TFM, G1/G2).

80 DEVELOPMENT OF A SYNTHETIC MEDIUM FOR THE IN VITRO CULTURE OF BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 154 ◽  
Author(s):  
D. Moreno ◽  
A. Neira ◽  
L. Dubreil ◽  
L. Liegeois ◽  
S. Destrumelle ◽  
...  
Keyword(s):  
Growth Factors ◽  
Embryo Culture ◽  
Disease Transmission ◽  
Culture Medium ◽  
Culture Media ◽  
Synthetic Medium ◽  
Wilcoxon Test ◽  
Blastocyst Development ◽  
Negative Effect

In the majority of media for embryo culture, 2 of typical components used are FCS or BSA; however, the presence of FCS in the culture medium has been shown to have a negative effect on embryo quality and the use of animal-derived proteins in culture media increases the risks of disease transmission through in vitro embryo production. The aim of this study was to develop an in vitro embryo culture medium free from FCS and BSA, but with the addition of various growth factors and cytokines (GF-CYK: IGF-I, IGF-II, bFGF, LIF, GM-CSF) 50 ng mL–1 and (TGF-β1) 100 ng mL–1 supplemented with hyaluronan (HA) and recombinant albumin (RA). Bovine oocytes (n = 1043, 6 replicates) from abattoir ovaries were matured in TCM-199 medium with 60 μg mL–1 penicillin, 60 μg mL–1 streptomycin, and 10 ng mL–1 EGF for 24 h at 39°C and 5% CO2 in humidified air. Afterward, the oocytes were fertilized in IVF-TALP medium with 6 mg mL–1 fatty acid-free BSA and 1.7 IU mL–1 heparin for 18 h under the same conditions. After fertilization, presumptive zygotes were divided into two groups and cultured in 30 μL droplets of SOF supplemented with (1) 0.4% BSA + 5 μg mL–1 insulin, 5 μg mL–1 transferrin, and 5 ng mL–1 selenium (ITS) as a control; or (2) GF-CYK + 0.5 mg mL–1 HA + 0.15% RA (M1). Droplets were preserved under mineral oil in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 at 39°C. Blastocyst development and blastocyst diameter was observed at 7 and 8 days post-fertilization (dpf). Developmental and diameter data were analysed using the Wilcoxon test by using R software. The blastocyst rates were not significantly different between the control and M1 medium: at 7 dpf (22.9% ± 4.8 and 30.2% ± 3.0), and at 8 dpf (29.6% ± 5.1 and 37.4% ± 2.0 respectively; P > 0.05). The blastocyst diameter obtained with the M1 medium was significantly greater (P < 0.05) than that of the control at 7 dpf (173.3 μm ± 4.9 and 157.2 μm ± 4.1, respectively); however, no significant differences were observed at 8 dpf (190.3 μm ± 5.2 and 179.7 μm ± 5.3, respectively). In conclusion, the FCS- and BSA-free medium with GF-CYK, HA, and RA (M1) showed a comparable development rate to the control medium at 7 and 8 dpf. These growth factors and cytokines in association with hyaluronan and recombinant albumin have a synergistic action by promoting an increase in the blastocyst diameter at 7 dpf. This is fully synthetic method of embryo culture; it presents a valuable tool to reduce the risks of disease transmission via embryo transfer.

44 PRODUCTION OF LIVE PIGLETS AFTER CRYOPRESERVATION OF IMMATURE PORCINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 136
Author(s):  
T. Somfai ◽  
K. Kikuchi ◽  
K. Yoshioka ◽  
F. Tanihara ◽  
H. Kaneko ◽  
...  
Keyword(s):  
Polar Body ◽  
Petri Dish ◽  
Developmental Competence ◽  
Basic Medium ◽  
High Survival ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Vitrification Solution

Development to term of vitrified porcine follicular oocytes is reported in the present study. Immature cumulus-oocyte complexes (COC) were collected from slaughtered prepubertal gilts and were vitrified according to our method published recently (Somfai et al. 2013 J. Reprod. Dev., in press). Briefly, after pretreatment with 7.5 μg mL–1 of cytochalasin B (CB) for 30 min in modified NCSU-37 (a basic medium, BM) at 38.5°C, groups of 88 to 121 COC were equilibrated in a mixture of 2% ethylene glycol (EG), 2% propylene glycol (PG), and 7.5 μg mL–1 CB for 13 to 15 min. Then, COC were washed in vitrification solution (17.5% EG, 17.5% PG, 5% polyvinyl pyrrolidone, and 0.3 M trehalose in BM) and then dropped with 2 μL of vitrification solution onto the surface of aluminum foil floating on liquid nitrogen (LN2). Microdroplets (each containing 10–25 COC) were transferred into cryotubes. After storage in LN2 for 2 to 4 weeks, the oocytes were warmed by dropping the microdroplets directly into 2.5 mL of warming solution (0.4 M trehalose in BM) kept in a 35-mm Petri dish on a 42°C hotplate for less than 1 min. Then, the warming dish was placed on a 38°C hotplate and COC were consecutively transferred for 1-min periods into BM containing 0.2, 0.1, or 0.05 M trehalose at 38°C. The COC were matured in vitro for 44 h using porcine oocyte medium (POM) supplemented with 10% follicular fluid (Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213). Then, oocytes were denuded, and their live/dead status and nuclear maturation were determined by their morphology and the presence of the first polar body, respectively. To assess their developmental competence, vitrified and non-vitrified (control) oocytes were in vitro fertilized (IVF; Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) and then in vitro cultured in porcine zygote medium-5 (PZM-5; Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213). Blastocyst rates were recorded on Days 5, 6, and 7 of culture (Day 0 = the day of IVF). The experiment was replicated 4 times. Data were analysed with 1-way ANOVA and the Tukey test. The results revealed that 86.4% (364/424) of oocytes survived after vitrification, which was significantly lower (P < 0.05) than that of controls [100% (326/326)]. Live oocytes in vitrified and control groups did not differ statistically in terms of nuclear maturation (63.9 v. 65.3%). Blastocyst rates of surviving vitrified oocytes were significantly lower compared with controls on Days 5 (2.4 v. 12.7%), 6 (4.8 v. 17.6%), and 7 (5.6 v. 18.4%). To test their ability to develop to term, 16 and 27 blastocysts on Day 5 developing from vitrified COC were transferred into 2 recipients. Both recipients became pregnant and farrowed a total of 10 live piglets (4 and 6 piglets, respectively). These data demonstrate that large groups of immature porcine oocytes could be cryopreserved by this method showing high survival and maturation rates. Furthermore, despite a low rate of blastocyst development, transfer of Day-5 blastocysts generated from vitrified oocytes resulted in piglet production for the first time in the world. Partially supported by JSPS and HAS under the Japan-Hungary Research Cooperative Program.

129 DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES AFTER IN VITRO MATURATION AND IN VITRO CULTURE UNDER COMPARATIVE OXYGEN TENSION

2011 ◽  
Vol 23 (1) ◽  
pp. 169
Author(s):  
J. T. Kang ◽  
M. Atikuzzaman ◽  
D. K. Kwon ◽  
S. J. Park ◽  
S. J. Kim ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Oxygen Tension ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Mrna Abundance ◽  
Developmental Competence ◽  
Multiple Genes ◽  
Oxygen Concentrations

The in vitro developmental abilities of porcine oocytes are generally increasing steadily at a similar ratio to those of in vivo embryos. However, it has been suggested that the in vitro culture system for the development of porcine embryos is not optimal. In this study, we investigated the effect of 2 oxygen concentrations (5 and 20%) on porcine embryo development during in vitro maturation and in vitro culture and analyzed differences in gene expression of resulting blastocysts. Oocytes were recovered by aspiration of slaughterhouse ovaries and then matured in tissue culture medium (TCM) 199 supplemented with 10% porcine follicular fluid (pFF), epidermal growth factor (EGF), insulin, pyruvate, cystine, and gonadotropin. Matured oocytes were then activated parthenogenetically, cultured in PZM-3 media for 7 days. In vitro maturation (M group) of oocytes was carried out under two oxygen concentration (5 and 20%) in terms of nuclear maturation (polar body extrusion; Exp. 1). The developmental differences between 5% oxygen culture group and 20% oxygen culture group during in vitro culture (C group) of embryos after parthenogenetic activation was investigated in terms of first cleavage and blastocyst formation (Exp. 2). Relative mRNA abundance of multiple genes in blastocysts was analyzed for transcript abundance of genes related with metabolism (GLUT1, LDHA), oxidative response (MnSOD, GPX1), apoptosis (BAX, Bcl2), and developmental competence (CCNB1, IGF2R; Exp. 3). The results show there were no significant differences in maturation rate between 2 oxygen concentrations during in vitro maturation (83 v. 86%). It was thought that cumulus cells surrounding oocytes might have attenuated oxidative stress, but number of resulting blastocysts were (P < 0.05) increased in 5% IVC group when compared with 20% IVC group (18.67 v. 14.09%, respectively). Moreover, the M20C5 group (23.01%) had a beneficial effect on in vitro culture compared with M5C5 (14.32%), M5C20 (10.30%), and M20C20 (17.88%) groups. Total cell numbers were not significantly different among groups. According to mRNA abundance data of multiple genes, each group altered the expression of genes in various patterns. Therefore, it could be concluded that high oxygen tension during in vitro maturation and low oxygen tension during in vitro culture might alter the expression of multiple genes related to oocyte competence and improve (P < 0.05) embryo development, but not blastocyst quality. This study was supported by MKE (#2009-67-10033839, #2009-67-10033805), NRF (#M10625030005-508-10N25), BK21 for Veterinary Science, IPET (#109023-05-1-CG000), and Hanhwa L&C.

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