In Vitro Embryo Culture Research Articles

A L-glutamine binding protein modified MNM structured optical fiber biosensor based on surface plasmon resonance sensing for detection of L-glutamine metabolism in vitro embryo culture

A surface plasmon resonance (SPR) optical fiber sensor with multimode-coreless-multimode (MNM) structure was developed, which modified by L-glutamine-binding protein (QBP) for detection of L-glutamine (Gln). The QBP was immobilized on the surface of gold films by chemical cross-linking and exhibited a binding affinity for L-glutamine. The conformation of QBP can be changed from the “open” to the “closed”, which led to a red-shift of the SPR peak when QBP bounded to L-glutamine. There was a good linear correlation between is a dependence of the SPR peak on and the concentration of L-glutamine concentration in the range 10–100 μM, with a sensitivity of 10.797nm/log10[Gln] for L-glutamine in the in vitro embryo culture (IVC) medium environment, and the limit of detection (LOD) is 1.187 μM. This QBP-modified MNM structure optical fiber SPR sensor provides a new idea for the developmental potential assessment of embryos in the process of in vitro embryo culture.

Zinc oxide nanoparticles functionalized with curcumin supplementation during in vitro embryo culture impaired in a concentration-dependent-manner blastocyst production in cattle.

This work investigated the effect of zinc oxide nanoparticles functionalized with curcumin (ZnO(np) + CUR) supplementation during the in vitro embryo culture (IVC) on the bovine in vitro embryo production, and the cellular antioxidant response. The cumulus-oocyte complexes (COCs) were matured, fertilized and then the presumptive zygotes were cultured in the medium in the absence (0 μM-control) or presence of different concentrations of ZnO(np) + CUR (3, 6 or 12 μM). After IVC, the embryos were destined either to assay intracellular ROS levels and mitochondrial membrane potential. The results demonstrated that only the addition of 12 μM ZnO(np) + CUR during IVC decreased intracellular ROS production and the rate of blastocyst production when compared to the control (p < .05). In conclusion, ZnO(np) + CUR addition during the IVC impaired in concentration-dependent-manner bovine in vitro embryo production.

Influences of glycine supplementation during vitrification on the developmental potential of vitrified/warmed immature dromedary camel oocytes.

Oocytes experience detrimental osmotic stress during vitrification and warming procedures because of the osmolality imbalance between the vitrification-warming fluids and the intracellular environment. Cellular osmotic homeostasis can be preserved by glycine, a powerful osmolyte with antioxidant properties. We aimed to examine the influences of supplementing glycine to the vitrification solutions (VS) on the developmental potential of vitrified/warmed immature dromedary camel oocytes following IVM/IVF and in vitro embryo culture (IVC). Cumulus oocyte complexes (COCs) were collected from dromedary camel ovaries and randomly allocated into two groups namely control (oocytes subjected directly to IVM) and vitrified (COCs were vitrified into VS supplemented with 0.0, 0.5, 1.0 or 2.0 mM glycine). For vitrification, COCs were equilibrated for 3 min in 12.5% ethylene glycol; EG plus 12.5% dimethyl sulfoxide; DMS and then they were vitrified for 60 s in VS composed of 25% EG + 25% DMSO using solid surface vitrification (SSV). Warming of vitrified oocytes was conducted in decreasing concentrations of trehalose solution. Following vitrification and warming, the morphologically viable oocytes were subjected to IVM for 36 h. Matured oocytes were then fertilized in vitro by epididymal spermatozoa and cultured for seven days. The results showed that the percentage of viable oocytes assessed by trypan blue stain was significantly higher (p ≤ .05) in the 1.0 mM glycine-supplemented group than 0.0- and 2.0-mM glycine-supplemented ones (90.0 % vs. 80.0% and 76.6%, respectively). However, no significant difference was observed between 0.5 mM glycine and other vitrified groups. Nuclear maturation rates, cleavage (48-h post-insemination; pi) and blastocyst rate (7-days pi) were significantly lower in vitrified groups than control ones (p ≤ .05). Among vitrified groups, these parameters were the highest in the 1.0 mM glycine-supplemented group. Taken together, supplementation of vitrification solutions with 1.0 mM glycine could enhance the developmental potential of vitrified/warmed immature dromedary camel oocytes.

First pilot study of maternal spindle transfer for the treatment of repeated in vitro fertilization failures in couples with idiopathic infertility

To gain insights into the technical feasibility of maternal spindle transfer (MST) applied in the context of repeated invitro fertilization (IVF) failures for the treatment of idiopathic infertility. A prospective pilot study. IVF center. Twenty-five infertile couples with multiple previous unsuccessful IVF cycles (range, 3-11), no previous pregnancy, and no history of mitochondrial DNA (mtDNA) disease participated. The study focused on women <40 years, with previous IVF attempts characterized by a pattern of low fertilization rates and/or impaired embryo development. Couples with severe male-factor infertility were not eligible. Oocyte donors with previous successful IVF outcomes were matched with patients according to standard practice. We performed MST by transferring metaphase II spindles from the patients' oocytes into the previously enucleated donor oocytes, followed by intracytoplasmic sperm injection, invitro embryo culture, blastocyst biopsy, and vitrification. Only euploid blastocysts were considered for embryo transfer. Outcome measures included oocyte fertilization, blastocyst development, clinical pregnancy and live birth, incidence of mitochondrial carryover and potential mtDNA reversal, as well as general health of the children born. Twenty-eight MST cycles produced 6 children (19 embryo transfers, 7 clinical pregnancies). Pediatric follow-up of the children, performed at intervals from birth to 12-24 months of age, revealed their development to be unremarkable. DNA fingerprinting confirmed that the nuclear DNA of MST children was inherited from both parents, without any contribution from the oocyte donor. For 5 of the children, mtDNA was derived almost exclusively (>99%) from the donor. However, 1 child, who had similarly low mtDNA carryover (0.8%) at the blastocyst stage, showed an increase in the maternal mtDNA haplotype, accounting for 30% to 60% of the total at birth. This pilot study provides the first insights into the feasibility of applying MST for patients with idiopathic infertility and repeated IVF failures. Reconstructed oocytes produced embryos capable of implanting, developing to term and producing apparently healthy newborns/children. However, claims concerning the efficacy of MST with respect to infertility treatment would be premature considering the limitations of this study. Importantly, mtDNA reversal was detected in one child born after MST, a finding with possible implications for mitochondrial replacement therapies. Pilot trial registry number, ISRCTN11455145. The date of registration: 20/02/2018. The date of enrolment of the first patients: 18/03/2018.

Bovine In Vitro Oocyte Maturation and Embryo Production Used as a Model for Testing Endocrine Disrupting Chemicals Eliciting Female Reproductive Toxicity With Diethylstilbestrol as a Showcase Compound.

Endocrine disrupting chemicals (EDCs) can interfere with normal hormonal action and regulation. Exposure of women to EDCs has been associated with adverse reproductive health outcomes. The assays currently used to identify EDCs that elicit female reproductive toxicity lack screening tests that address effects on the maturation of oocytes, a process that enables them to be fertilized and develop into embryos. Here, a screening method employing the bovine model of in vitro oocyte maturation and embryo production is described. Endpoints explored address important events in oocyte maturation and developmental competence acquisition. To test the method, the effects of the known human EDC diethylstilbestrol (DES; an estrogen receptor agonist) were evaluated in a range of concentrations (10–9 M, 10–7 M, 10–5 M). Bovine oocytes were exposed to DES during in vitro maturation (IVM) or embryos were exposed during in vitro embryo culture (IVC). The endpoints evaluated included nuclear maturation, mitochondrial redistribution, cumulus cell expansion, apoptosis, and steroidogenesis. DES-exposed oocytes were fertilized to record embryo cleavage and blastocyst rates to uncover effects on developmental competence. Similarly, the development of embryos exposed to DES during IVC was monitored to assess the impact on early embryo development. Exposure to 10–9 M or 10–7 M DES did not affect the endpoints addressing oocyte maturation or embryo development. However, there were considerable detrimental effects observed in oocytes exposed to 10–5 M DES. Specifically, compared to vehicle-treated oocytes, there was a statistically significant reduction in nuclear maturation (3% vs 84%), cumulus expansion (2.8-fold vs 3.6-fold) and blastocyst rate (3% vs 32%). Additionally, progesterone and pregnenolone concentrations measured in IVM culture media were increased. The screening method described here shows that bovine oocytes were sensitive to the action of this particular chemical (i.e., DES), albeit at high concentrations. In principle, this method provides a valuable tool to assess the oocyte maturation process and early embryo development that can be used for reproductive toxicity screening and possibly EDC identification. Further studies should include EDCs with different mechanisms of action and additional endpoints to further demonstrate the applicability of the bovine oocyte model for chemical risk assessment purposes and EDC identification.

Establishment of a Wisent (Bison bonasus) Germplasm Bank

Simple SummaryThe wisent (European bison) is a protected species. For this reason, we undertook the use of biotechnologies—such as in vitro maturation of oocytes, in vitro fertilization of matured oocytes, in vitro culture of embryos, and embryo vitrification—to establish a wisent embryo bank. The competencies of the vitrified embryos were tested by transferring the warming embryos to cattle (interspecies embryo transfer). The pregnancy was confirmed biochemically and using USG, and although the fetuses were resorbed, the embryos’ competence for development was demonstrated. The results of these studies open the way for the cryoconservation of wisent germplasm.The wisent, or European bison (Bison bonasus), belongs to the same family (Bovidae) as the American bison and domestic cattle. The wisent is the largest mammal in Europe, and is called the “Forest Emperor”. The wisent is listed as “Vulnerable” on the IUCN Red List, and is protected by international law. Achievements in reproductive biotechnology have opened new possibilities for the cryoconservation of the wisent germplasm. Therefore, this research aimed to improve a strategy for the protection and preservation of the European bison through the creation of a wisent germplasm bank, based on the following procedures: isolation and in vitro maturation (IVM) of oocytes, in vitro fertilization (IVF) of matured oocytes, in vitro embryo culture (IVC), and embryo cryopreservation. Wisent ovaries were isolated from females outside the reproductive season, and eliminated from breeding for reasons other than infertility. Cumulus–oocyte complexes (COCs) were isolated from follicles greater than 2 mm in diameter and matured for 24 h and 30 h. After IVM, COCs were fertilized in vitro with wisent sperm. The obtained wisent zygotes, based on oocytes matured for 24 h and 30 h, were cultured for 216 h. Embryos at the morula and early blastocyst stages were vitrified and then warmed and transferred to interspecies recipients (Bos taurus). USG and biochemical tests were used to monitor pregnancies. This study obtained embryos in the morula and early blastocyst stages only after oocytes were fertilized and matured for 30 h. On average, per oocyte donor, 12.33 ± 0.5 COCs were isolated, and only 9.33 ± 0.61 COCs were qualified for in vitro maturation (75.68%), while 9.16 ± 0.48 COCs were matured (84.32%). On average, per donor, 5.5 ± 0.34 embryos were cleaved (59.96%) after 48 h post-fertilization (hpf), and 3.33 ± 0.21 achieved the eight-cell stage (36.52%) after 96 hpf, while 1 ± 0.21 morula and early blastocyst stages (10.71%) were achieved after 216 hpf. A total of six embryos (one morula and five early blastocysts) were obtained and vitrified; after warming, five of them were interspecies transferred to cattle (Bos taurus). On day 41 after fertilization, 3 out of 5 pregnancies were detected based on USG, P4, and PAG tests. However, no pregnancy was observed on day 86 after fertilization, indicating embryo resorption. This study shows that obtaining wisent embryos in vitro, and subsequent cryopreservation to create a wisent embryo bank, can be applied and implemented for the wisent protection program.

32 An approach to invitro oocyte maturation and fertilization in threatened Saharan Dorcas gazelle (Gazella dorcas osiris) using frozen-thawed epididymal sperm cells

The Dorcas gazelle (Gazella dorcas), in the family Bovidae, is one of the smallest species of the genus Gazella. Its population is decreasing and it is classified as vulnerable by the International Union of Conservation of Nature Red List of Threatened Species. The subspecies Saharan Dorcas gazelle (G. dorcas osiris), originating in the central-western Saharan and Sahel regions, is almost extinct in the wild because of over-hunting. Thereon, assisted reproductive techniques (ARTs) could help in its population management. Here, we performed invitro maturation (IVM) of oocytes recovered from an adult female of Saharan Dorcas gazelle (10 years old) to attempt IVF with frozen-thawed epididymal sperm cells from an adult male (9 years old) of the same subspecies, following protocols for ARTs in cattle. Epididymal sperm was obtained after postmortem examination by cannulation of vas deferens with commercial semen extender Gent A (Minitub). Motility features were assessed using a computer-assisted sperm analysis system (Proiser SL), and viability was evaluated using eosin/nigrosin staining. After collection, viability (88.3%), total (17.8%) and progressive (8.7%) motility were assessed. A total of 820.5×106 sperm cells was obtained and diluted (1:1) in a semen extender with permeable cryoprotectants (Gent B, Minitub). Then, the sample was refrigerated in straws (200×106 mL−1; 0.5mL) at 4°C for 1h and exposed to liquid nitrogen vapors (LN2) for 10min before being directly plunged into and kept on LN2. A total of 35 oocytes were recovered by slicing, 27 of which were selected for IVM (24h, 38.5°C, 5% CO2 in humidified air). Motile sperm were obtained by centrifugation of thawed semen (10min, 1000×g) by performing a density gradient (BoviPure, Nidacon). Post-thawing viability (72.9%) and total (14%) and progressive (4%) motility were evaluated just before IVF. After IVM, 17 oocytes were incubated for IVF for 20h with 1×106 thawed sperm cells mL−1 (although the first polar body extrusion was observed only in 5 of the 17 oocytes before IVF; 29.4%). Also, 7 oocytes selected for IVM were vitrified and kept for the genetic bank, and 3 were fixed. Thereafter, all oocytes that underwent IVF were incubated for invitro embryo culture (38.5°C, 5% CO2, 5% O2 in humidified air) to avoid discarding any potential zygote. Cleavage was assessed at 48h post-IVF. From those, only 2 oocytes successfully achieved IVF out of the 5 that showed first polar body extrusion (40%), although cleavage was asymmetric and embryos did not progress. The rest of oocytes (12) did not achieved IVM, IVF, or cleavage. Results suggest that optimal protocols and appropriate individualized conditions for vulnerable nondomestic ungulates are required for successful ARTs. Future efforts are needed to improve preservation strategies for the gametes/embryo conservation of endangered species. JG and MRC contributed equally to this work. JG is supported by AGAUR (2018 FI_B 00236) and MRC by FPU (Training for Academic Staff) (FPU15/06029).

Analysis of the activity of oncocalyxone A (Auxemma oncocalyx) and doxorubicin on the in vitro development of porcine oocytes

Most anticancer drugs like doxorubicin (DXR) have low specificity that results in undesirable effects especially when it comes to collateral effects on reproduction. Plants are excellent sources when searching for new drugs. Auxemma oncocalyx (A. oncocalyx) and its main component Oncocalyxone A (onco A) have anti-tumoral activity and are less toxic than DXR in reproductive parameters. However, there are no studies on the action of these drugs regarding the porcine in vitro oocyte competence and embryo development. The aim of this study was to evaluate the effect of A. oncocalyx and onco A exposure during in vitro maturation (IVM) of oocytes (Experiment 1) or in vitro embryo culture (IVC) (Experiment 2) on the oocyte developmental competence. For experiment 1, COCs were distributed in IVM medium alone (control) or supplemented with DXR (0.3 g/mL), A. oncocalyx (1.2 g/mL) and onco A (1 g/mL). Then, oocytes were submitted to in vitro fertilization (IVF) and in vitro embryo culture. For experiment 2, zygotes were cultured with DXR, A. oncocalyx and onco A for 7 days. Viability, maturation, fertilization and embryo developmental parameters were evaluated in both experiments. In experiment 1; DXR, A. oncocalyx and onco A reduced (P&lt;0.05) oocyte viability and IVM efficiency. Onco A increased (P&lt;0.05) the meiotic resumption. After IVF, all drugs reduced (P&lt;0.05) viability, IVF efficiency and percentage of cleaved embryos, nevertheless, only DXR decreased the percentage of blastocyst. In experiment 2; all drugs reduced (P&lt;0.05) the percentage of penetration, but only DXR and onco A decreased (P&lt;0.05) IVF efficiency. DXR and A. oncocalyx decreased (P&lt;0.05) the percentage of cleaved embryo, but had no effect on blastocyst formation. In conclusion, the addition of DXR during IVM or IVC negatively affected the IVF efficiency and cleavage rate. In addition, the exposure of COCs to DXR only during IVM was more detrimental to oocyte viability and blastocyst formation than A. oncocalyx and onco A.

35 Ethanolic extracts of Cerrado plants in cryotolerance of invitro-produced bovine embryos

Invitro embryo culture induces excessive production of reactive oxygen species (ROS) that impair the quality of produced blastocysts, making them less cryotolerant. Supplementation of culture media with antioxidants would be an alternative to minimise these effects. The present study evaluated the effect of ethanolic extracts obtained from Brazilian Cerrado plants on the cryotolerance of invitro-produced embryos. Bovine ovaries from a slaughterhouse were used to obtain grade I and II oocytes, which were submitted to maturation, fertilization (Day 0), and invitro culture. After fertilization, zygotes were distributed into five groups: one fresh control group (GCont), cultured in high O2 tension (~21%), and four groups cryopreserved at Day 7: a control group subject to high O2 tension without extract supplementation (GDT); a group (G5%) cultured in low O2 tension (5%) without extract supplementation, and two groups cultured under high O2 tension, one supplemented with 0.01mgmL−1 of the ethanolic cagaita extract (Eugenia dysenterica; GCag) and another with 0.01mgmL−1 of murici extract (Byrsonima crassifolia; GMur). Only the expanded blastocysts (EB) of each group were submitted to the direct-transfer cryopreservation system, with slow freezing with ethylene glycol (Sanches et al. 2016 Theriogenology 85, 1147-1151). A total of 163 thawed embryos were evaluated 12, 24, and 36h post-thawing for re-expansion, hatching, and embryonic degeneration rate. In addition, after 12h of post-thaw culture, only the EB embryos were subjected to ROS measurement, quantified in confocal microscopy using 2′,7′-dichlorodihydrofluorescein diacetate, and the rate of apoptosis was obtained by the terminal deoxynucleotidyl transferase dUTP nick end labelling method. Data were analysed by analysis of variance, and the means were compared by TUKEY. A total of 133 EB were used (GCont: 20; GDT: 22; GCag: 35; GMur: 36; G5%: 20). Results are expressed as means±standard deviation. No differences were observed on re-expansion among treatments and time of culture. However, 24h post-thaw, hatching and degeneration rates differed between groups. The group GCont had a higher (P&amp;lt;0.05) hatching rate (47.5±10.3) than the GDT (14.4±6.7) and G5% (10.5±5.8) groups but was similar (P&amp;gt;0.05) to groups supplemented with extracts GCag (22.2±17.1) and GMur (17.5±17.5). Degeneration rate was higher (P&amp;lt;0.05) at 12h for the direct-transfer group (30.2±11.3) and similar among the others (GCont: 0; GCag: 16.8±12; GMur: 10.8±10.8; G5%: 11±11). Nevertheless, GCag had lower embryonic degeneration rates (20±16.7) than the others cryopreserved groups 24h after thawing (GDT: 37.7±5.5; GMur: 38.9±8.2; G5%: 41.3±14.4). Although ROS levels were similar among all groups (P&amp;gt;0.05), apoptosis rate was higher (P&amp;lt;0.05) in the GDT group (23.4±1.9) than in the GCont groups (10.6±1.3): GCag (10.8±1.1), GMur (10.1±1.1), and G5% (7.8±1.2). Therefore, supplementation of ethanolic extracts (0.01mgmL−1) of cagaita and murici improved the embryo quality by reducing the degeneration and apoptosis rates of cryopreserved embryos when compared with the group without supplementation (GDT). Such extracts may be an alternative to increase the cryotolerance of invitro-produced bovine embryos.

Resveratrol-cyclodextrin complex affects the expression of genes associated with lipid metabolism in bovine in vitro produced embryos.

Antioxidants have been widely used during in vitro production to decrease the negative effect of reactive oxygen species. It was reported that the complex resveratrol-methyl β-cyclodextrin (RV-CD) improves resveratrol's stability and bioavailability and increases its antioxidant activity. This study evaluates the effect of RV-CD during in vitro oocyte maturation (IVM) or in vitro embryo culture (IVC) on developmental competence and quantitative changes in gene expression of developmental important genes. In experiment 1, RV-CD was added to IVM media and maturation level, embryo development and oocytes, cumulus cells, and blastocysts gene expression by RT-qPCR were examined. In experiment 2, presumptive zygotes were cultured in SOF supplemented with RV-CD and embryo development and blastocysts gene expression by RT-qPCR were studied. A group without RV-CD (control- ) and a group with cyclodextrin (control+ ) were included. No differences were found in cleavage rate or blastocyst yield between groups. However, the expression of LIPE was higher in blastocysts derived from oocytes treated with resveratrol compared with control groups (p<.05). Blastocysts produced by IVC with resveratrol showed that RV-CD could modify the expression of genes related to lipid metabolism (CYP51A1, PNPLA2 and MTORC1) compared with control groups (p<.05). RV-CD in the IVM and IVC media could reduce accumulated fat by increasing lipolysis and suppressing lipogenesis of blastocysts.

Enhanced in vitro developmental competence of sheep embryos following sericin supplementation of the in vitro maturation and in vitro culture media

The objective was to determine the effects of various sericin concentrations (0, 0.1, 0.5, 1, and 2.5%) during in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on ovine embryo development. Pubertal Sanjabi ewes’ oocytes were aspirated from slaughterhouse derived ovaries and matured in vitro. Semen was collected from a healthy fertile ram, capacitated and the in vitro matured oocytes were fertilized using capacitated spermatozoa. The IVM and IVC media were supplemented with 0, 0.1, 0.5, 1 and 2.5% of sericin. Addition of 0.5% of sericin during IVM and IVC significantly increased cleavage and blastocyst rates (85.48±1.57 and 50.07±3.04, respectively) compared with 0 and 0.1% of sericin. However, a too high concentration (2.5%) of sericin during IVM and IVC decreased cleavage and blastocyst rates compared with low concentrations (P<0.05). In conclusion, appropriate concentrations (0.1 and 0.5%) of sericin promoted ovine blastocyst formation in vitro, although a high concentration of sericin suppressed embryo development.

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

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 &lt; 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

234 NEW CULTURE MEDIA AFFECTS BLASTOCYST DEVELOPMENT AND GENE EXPRESSION LEVELS IN IN VITRO-PRODUCED BOVINE EMBRYOS

The purpose was to examine effects of different media for bovine in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on blastocyst rates, morpho-kinetics, and relative abundance of mRNA of 8 genes associated with critical processes and developmental competence in the embryo. Abattoir-derived cumulus-oocyte complexes (COC) were in vitro matured (IVM) in either TCM199 [+0.5% BSA and gonadotropins (Suigonan Vet 150 I.E. mL–1)] or in a novel commercially available media (Bo-IVM), and a total of 1196 presumptive zygotes, from 4 replicates, were submitted to in vitro culture (IVC) and cultured in either SOF (+0.5% BSA) or in a novel commercially available media (Bo-IVC). Blastocyst rates and morpho-kinetics were assessed on Day 8 after fertilization. The high-quality blastocysts from each group were analysed by RT-qPCR, on single blastocysts using earlier verified primers, for BAX, BCL2L1, DNMT3A, FASN, G6PD, HSPA1A, SLC2A1, and SLC2A3. Data on blastocyst rates were analysed for statistical differences using a linear regression model, using a binary reproach and general estimating equations. One-way ANOVA was used to detect differences in the relative abundance of mRNA between groups, whereas differences between maturation and culture media were analysed by a 2-way ANOVA. Blastocyst rates in the Bo-IVM/Bo-IVC (37%), TCM199/Bo-IVC (33%), Bo-IVM/SOF (26%), and TCM199/SOF (28%) groups were significantly different from each other (P &lt; 0.0001). Specifically, the Bo-IVM/Bo-IVC group differed significantly from both SOF-cultured groups (P &lt; 0.01). Subjectively, this group also had embryos of the highest quality and most advanced development. Significantly increased levels of mRNA transcripts were found for embryos cultured in Bo-IVC for all genes (P &lt; 0.05) except BCL2L1. In conclusion, the developmental rates and gene expression of in vitro-produced bovine blastocysts were affected by the use of different culture media. Increased blastocyst rates, apparently superior embryo quality, and more abundant gene expression were achieved when blastocysts were cultured in Bo-IVC culture media compared with SOF.The project was supported by The Danish Council for Strategic Research.

In vitro developmental competence of prepubertal goat oocytes cultured with recombinant activin-A

The present study was designed to evaluate the effect of activin-A during the in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on nuclear maturation, blastocyst yield and blastocyst quality of prepubertal goat oocytes. In Experiment 1, three groups of oocytes were used during the IVM of prepubertal goat oocytes to determine the optimal concentration of recombinant human activin-A added to the maturation medium. Cumulus–oocyte complexes were matured in an IVM medium containing 0, 10 and 100 ng/ml (groups A0, A10 and A100), fertilized and in vitro cultured using standard procedures. In Experiment 2, the addition of 10 ng/ml activin-A at IVM (A10A0), IVC (A0A10) or IVM+IVC (A10A10) was studied and compared with the control group (A0A0). Results of the first experiment demonstrated that the addition of activin-A yielded similar percentages of maturation (⩽71.0%) and blastocyst formation rates (⩽24.9%) than the control group (A0). Experiment 2 showed that exposure of prepubertal goat oocytes to an IVC medium containing 10 ng/ml activin-A (A0A10) significantly increased the rates of development to the blastocyst stage, as compared with the control group (A0A0) (19.5±2.21% v. 13.1±2.37%, respectively; P<0.05). With regard to the blastocyst quality, total number of cells, inner cell mass (ICM) and trophectoderm of prepubertal goat embryos produced in the presence of activin-A did not differ significantly among experimental groups. In summary, these results indicate that supplementation of the IVC medium with activin-A enhances embryo development of prepubertal goat oocytes.

Embryo Culture Challenge: Microbial Contamination

The success rate of embryo production critically depends on several factors. One of the pivotal factors is a technique used for the whole procedure of in vitro fertilization (IVF) and in vitro embryo culture (IVC) (1, 2). Besides the technique selected for IVF / in vitro embryo production (IVEP), there is an important consideration called IVF/IVEP culture dishes disinfection (3). For many years, microbial contamination of embryo culture dishes has caused several deficiencies in the success rate of assisted reproductive technique (ART) laboratory. Despite precise culture conditions, strict trafficking regulations, rigorous discipline of aseptic technique and applying culture medium supplemented with antibiotics including Penicillin and Streptomycin, a dramatic increase in the number of infections was recorded routinely worldwide (4, 5). There is a great demand to overcome the abovementioned issue in human fertility & sterility clinics and laboratory procedures. Results of intensive studies regarding the identification of the main sources of such contamination reveled that Escherichia coli (E. coli) and Candida species have caused more than 59% and 26% of these contaminations, respectively (6-8). Among E. coli species, 74% showed a great resistant to both Penicillin and Streptomycin in the culture medium. On the other hand, 24% of E. coli appeared to be resistant to both antibiotics. “Infections in IVF culture dishes are mainly caused by bacterial strains insensitive to the antibiotics used or due to yeast colonization by Candida species, which frequently reside in the vagina” (6, 9). IVF culture dishes catch E. coli/Candida either via ejaculation which is the paternal source of infection obtained from semen samples, and / or maternal sources. The maternal source of contamination is either ovum pick up procedure (OPU) or vaginal environment (10). In a report, identification of the contaminating micro-organisms revealed that some of the materials utilized in the culture procedure such as “mineral oil”, which is used to cover the fertilization/culture drops, were infected with Aspergilles terreus. Therefore, not only, a great variation has been observed in case of the origin of culture dishes infections, but also there would be a great number of bacterial / fungal species transferred by the infecting sources to the culture dishes (6). However, as mentioned before, many of the infectious bacterial or fungal colonies are insensitive to antibiotics (6). Therefore, it would be an obligation to develop an efficient and simple prevention method to reduce the high rate of culture dishes contaminations. Animal biotechnologist and other scientists who are involved in IVEP aspired to reduce the mentioned deficiency caused by these microorganisms. Disinfection procedure of IVF/IVC culture dishes involved two main obstacles, in which there were complications; selection of an appropriate and efficient antibiotic as well as wide variation of bacterial / fungal sensitivity to the selected antibiotic. Diverse techniques have been suggested to overcome the current problem, such as different combinations of antibiotics added to culture mediums and / or antibiotic administration to oocyte and sperm donors. Getting to work all these directions, a great number of bacterial / fungal contaminations are yet being reported. On the other hand, the current knowledge has a huge doubt about side effects of antibiotics administrated to patients with subfertility and / or added to embryo culture medium, on oocyte / embryo competence and survival (6, 9). In recent years some laboratories such as University of Tehran IVF/IVC laboratory has employed and proven the efficiency of applying a specific regiment of Ultra Violet lamp (UV) along with non-toxic disinfectant gaseous phase “Ozone (O3)” (physicochemical disinfection method (PDM)). The results showed that besides administrating and supplementing culture mediums with common antibiotics for disinfection of IVF/IVC culture medium, the new horizon in physicochemical disinfection procedure could also be promising in this context; through the applications of PDM. Results revealed that, when this regiment was applied, no infection was observed through 2367 IVF treatment cycles. The same combination of UV and O3 recently has been applied in an incubator (Simple Embryo Culture Chamber (SECC)) designed by Dadashpour Davachi et al. (unpublished data) to protect all kinds of cell and embryo culture dishes against infecting microorganism. Results showed that using SECC compared to other commercial incubators led to a dramatic decrease in culture dishes contamination (Table 1). In SECC only before putting the culture dishes in the chamber the UV lamp was on. It would be demonstrated that many kinds of the infecting microorganism are introduced to culture dishes via external sources such as mineral oil, tissue culture medium and other reagents. Applying different UV and O3 regiment in IVF / IVC laboratory would be promising to reduce antibiotic consumption in IVEP. There is no acknowledgment. Implication for health policy/practice/research/medical education: Embryo production laboratory and animal biotechnologist may use the presented information Authors’ Contribution: The authors have participated equally in manuscript preparation. Financial Disclosure: There is no financial disclosure.

Use of a novel polydimethylsiloxane well insert to successfully mature, culture and identify single porcine oocytes and embryos

The objective of this study was to evaluate the efficacy of a novel polydimethylsiloxane (PDMS) well-insert system for oocyte in vitro maturation (IVM) and in vitro embryo culture (IVC) in pigs. The PDMS well inserts, consisting of multiple microwells with connecting microchannels, resulted in equivalent blastocyst development compared with standard microdrop culture for IVC. These PDMS well inserts were then evaluated for IVM or IVC in a rocking versus static environment. The rocking environment during both oocyte IVM and embryo culture had detrimental effects on oocyte and embryo development compared with a static environment. Importantly, blastocyst development of oocytes and embryos cultured in the PDMS well inserts in the static environment was equivalent to that of standard microdrops. Further analysis of transcript abundance in blastocysts produced from these different environments revealed that the PDMS well-insert system may produce more viable embryos. In conclusion, this PDMS well-insert system can successfully mature oocytes and culture embryos in an individually-identifiable manner without compromising, and perhaps enhancing, developmental potential.

Effects of ghrelin on developmental competence and gene expression of in vitro fertilized ovine embryos

The objective was to determine the effects of various ghrelin concentrations (0, 10, 50, and 250 ng/mL) during in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on ovine embryo development and expression patterns of genes involved in transcription regulation (OCT4), glucose transport (GLUT1), pregnancy recognition (IFNT), and ghrelin receptor (GHSR-1a). Rates of cleavage and blastocyst formation were decreased when oocytes were matured with 250 ng/mL ghrelin compared with 0, 10, or 50 ng/mL ghrelin (P < 0.05). Addition of 50 ng/mL ghrelin during IVC or during IVM and IVC significantly increased blastocyst rates (35.3% and 36.7% vs. 26.9% and 26.8%, respectively) and total cell numbers per blastocyst (110.4 and 108.3 vs. 81.2 and 77.4) compared with 0 and 10 ng/mL ghrelin. However, a high concentration (250 ng/mL) of ghrelin during IVM and IVC decreased cleavage, blastocyst rate, and total cell number of blastocyst compared with low concentrations (P < 0.05). Relative abundances of GLUT1 and IFNT transcripts were higher in blastocysts treated with 50 ng/mL ghrelin during IVC compared with other concentrations (P < 0.05). Expression of GHSR-1a was higher when 10 ng/mL ghrelin was added during IVM (0.079) or during IVM and IVC (0.053) compared with other treatments. However, addition of ghrelin at higher concentrations (50 or 250 ng/mL) reduced relative abundances of GHSR-1a transcripts (0.032 and 0.039, P < 0.05). In conclusion, appropriate concentrations of ghrelin promoted ovine blastocyst formation in vitro and increased expression of GLUT1, IFNT, and GHSR-1a genes in blastocysts, although a high concentration of ghrelin suppressed embryo development.

136 EFFECTS OF INSULIN-LIKE GROWTH FACTOR I (IGF-1) ON THE DEVELOPMENT AND APOPTOSIS OF PREIMPLANTATION BUFFALO (BUBALUS BUBALIS) EMBRYOS

The aim of this study was to explore the effects of insulin-like growth factor-I (IGF-1) on the development and apoptosis of preimplantation buffalo (Bubalus bubalis) embryos derived from IVF or somatic cell nuclear transfer (SCNT) in order to improve the quality of in vitro embryo culture (IVC). Buffalo oocytes collected from ovaries at slaughter were cultured in the maturation medium (TCM-199 + 26.2 mmol L–1 NaHCO3 + 5 mmol L–1 HEPES + 5% FBS) for 22–24 h, and fertilized in vitro, or enucleated and reconstructed for SCNT. Embryos were then cultured in the culture medium (CM: TCM-199 + 3% FBS) supplemented with different concentrations of IGF-1. Blastocyst development was evaluated after 7 days of culture. A total of 1566 oocytes were used in this study. The experimental data were analyzed using two-way ANOVA, P &lt; 0.05 was considered to be statistically significant. The results showed that the cleavage rates of IVF or SCNT embryos cultured with 0, 10, 50, or 100 ng mL–1 IGF-I, were not significantly different (P &gt; 0.05). However, the blastocyst rate of IVF embryos cultured with 50 ng mL–1 IGF-1 was significantly increased compared to the 0 ng mL–1 group (35.1 v. 23.0%; P &lt; 0.05), but not significantly different among the 0, 10, and 100 ng mL–1 groups (23.0 v. 28.2 and 26.5%; P &gt; 0.05). In the same line, more SCNT embryos could develop to the blastocyst stage when cultured in the CM supplemented with 50 ng mL–1 IGF-I by comparison with the 0 ng mL–1 group (32.3 v. 20.2%; P &lt; 0.05), but the blastocyst development decreased with 100 ng mL–1 (32.3 v. 21.4%; P &lt; 0.05). Apoptosis and total cell number (TCN) of IVF/SCNT blastocysts were respectively detected by TUNEL or Hoechst 33342 staining. By comparison with the 0 ng mL–1 group, the TCN of IVF/SCNT blastocysts was significantly increased (IVF: 91.7 ± 6.9 v. 108.7 ± 3.9, SCNT: 76.3 ± 5.6 v. 92.8 ± 3.9; P &lt; 0.05) and the apoptotic index was obviously decreased (IVF: 3.9 ± 0.7 v. 2.5 ± 0.7; 7.2 ± 0.5 v. 2.9 ± 0.5; P &lt; 0.05) when the embryos were cultured in the CM with 50 ng mL–1 IGF-I. The result of RT-qPCR analysis showed that the mRNA expression level of the anti-apoptotic bcl-2 gene was distinctly enhanced, while the mRNA expression level of the pro-apoptotic bax gene was remarkably reduced in IVF/SCNT embryos cultured with 50 ng mL–1 IGF-I by comparison with the 0 ng mL–1 group (P &lt; 0.05). These results demonstrated that supplementing CM with 50 ng mL–1 IGF-1 could improve the developmental competence of buffalo embryos, increase the TCN of blastocysts and decrease their apoptotic index, probably by down-regulating the mRNA level of pro-apoptotic bax gene and up-regulating the mRNA level of anti-apoptotic bcl-2 gene. This work was funded by the China High Technology Development Program (2011AA100607), China Natural Science Foundation (31072033), and Guangxi Science Foundation (2012GXNSFFA060004).

Effect of leptin supplementation during in vitro oocyte maturation and embryo culture on bovine embryo development and gene expression patterns

Leptin is a metabolic hormone related to body condition and nutritional status that influences fertility in assisted reproductive technologies modulating oocyte and embryo quality. The aim of the present study was to establish the effect of various leptin concentrations (0, 10, 100 ng/mL) during in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on bovine embryo development and quality in terms of gene expression. The relative mRNA abundance of the genes encoding solute carrier family 2 (facilitated glucose transporter) member 1 (SLC2A1), Bcl-2-associated X protein (BAX), placenta-specific 8 (PLAC8), aldo-keto reductase family 1 member B1 (AKR1B1) and leptin receptor (LEPR) were determined on Day 7 blastocysts by qRT-PCR. Cleavage rate (P < 0.005) and blastocyst yield (P = 0.05) was significantly lower when cumulus-oocyte complexes (COCs) were matured with 100 ng/mL leptin compared to 0 or 10 ng/mL leptin. No significant effect of different concentrations of leptin added during IVC on blastocyst yield was observed. The presence of 100 ng/mL leptin in both IVM and IVC further decreased cleavage rate (P < 0.005) and blastocyst yield compared to the control group without leptin (P = 0.05) and those supplemented with 10 ng/mL leptin or FCS (P < 0.005). There was no evidence of any leptin-induced difference in the relative transcript abundance of SLC2A1, BAX and PLAC8 genes in Day 7 blastocysts. Expression of AKR1B1 was significantly lower in blastocysts from COCs matured with 100 ng/mL leptin compared to those matured with 0 or 10 ng/mL leptin (P < 0.005). LEPR expression was up regulated when leptin concentration was increased from 0 ng/mL during IVM to 10 ng/mL during IVC, but it was down-regulated in the opposite situation (P < 0.005). In conclusion, high leptin concentrations possibly related to obesity seem to be more detrimental rather than the absence of this hormone for preimplantation embryo survival; this effect is independent of LEPR gene expression and it does not influence expression of SLC2A1, BAX and PLAC8 genes in Day 7 blastocysts.

Profile and regulation of annexin II expression during early embryogenesis in cattle

The presence of annexin II (Ann-II) during the initial stages of bovine embryo development and the regulation of Ann-II expression by retinol and insulin-like growth factor I (IGF-I) were studied. Bovine embryos at different stages of development were produced in vitro on Synthetic Oviductal Fluid (SOF) medium (control group), SOF supplemented with retinol (retinol group; 0.1ng/ml), or IGF-I (IGF-I group; 10ng/ml). The embryos were processed for mRNA extraction, cDNA production and polymerase chain reaction (PCR) using Ann-II-specific oligonucleotides. Ann-II was detected in all stages of early embryo development, except for the 16-cell stage. The blastocyst rates were significantly higher (P&lt;0.05) in the group supplemented with retinol (37.8%, 45/119) during in vitro embryo culture (IVC) than in those cultured in SOF (20.5%, 24/117) or SOF with IGF-I (25.8%, 24/93). Semiquantitative analysis of Ann-II expression in embryos produced in medium supplemented with IGF-I or retinol revealed a lower expression of this gene when compared with embryos cultured in SOF (P&lt;0.05). The Ann-II expression was not different in embryos cultured in the presence of retinol and IGF-I. The presence of retinol increased the production of embryos in vitro by decreasing the expression of Ann-II in early-stage of bovine embryo.