Higher Developmental Competence Research Articles

Enhancement of porcine in vitro embryonic development through luteolin-mediated activation of the Nrf2/Keap1 signaling pathway

BackgroundOxidative stress, caused by an imbalance in the production and elimination of intracellular reactive oxygen species (ROS), has been recognized for its detrimental effects on mammalian embryonic development. Luteolin (Lut) has been documented for its protective effects against oxidative stress in various studies. However, its specific role in embryonic development remains unexplored. This study aims to investigate the influence of Lut on porcine embryonic development and to elucidate the underlying mechanism.ResultsAfter undergoing parthenogenetic activation (PA) or in vitro fertilization, embryos supplemented with 0.5 µmol/L Lut displayed a significant enhancement in cleavage and blastocyst formation rates, with an increase in total cell numbers and a decrease in the apoptosis rate compared to the control. Measurements on D2 and D6 revealed that embryos with Lut supplementation had lower ROS levels and higher glutathione levels compared to the control. Moreover, Lut supplementation significantly augmented mitochondrial content and membrane potential. Intriguingly, activation of the Nrf2/Keap1 signaling pathway was observed in embryos supplemented with Lut, leading to the upregulation of antioxidant-related gene transcription levels. To further validate the relationship between the Nrf2/Keap1 signaling pathway and effects of Lut in porcine embryonic development, we cultured PA embryos in a medium supplemented with brusatol, with or without the inclusion of Lut. The positive effects of Lut on developmental competence were negated by brusatol treatment.ConclusionsOur findings indicate that Lut-mediated activation of the Nrf2/Keap1 signaling pathway contributes to the enhanced production of porcine embryos with high developmental competence, and offers insight into the mechanisms regulating early embryonic development.

Laparoscopic ovum-pick up and in vitro embryo production in gonadotropin-stimulated gilts: Preliminary results and envisioned applications

The present study was conducted to establish if laparoscopic ovum pick-up (LOPU) could be adapted to the swine species, and if the developmental competence of LOPU-sourced oocytes from peripubertal gilts could be improved by gonadotropin stimulation, by comparing with oocytes sourced from slaughtered gilts lacking hormonal stimulation. Estrus was synchronized in 34 gilts of ∼6–8 months of age by daily oral administration of 17.6 mg altrenogest for 13 days and 10 mg dinoprost IM on the last day of altrenogest. Follicular development was stimulated in all gilts with a single injection of 1250 IU eCG given 3 days before LOPU (together with the 12th dose of altrenogest). In about half of the gilts (Group eCG-hCG), 500 IU hCG were injected IM ∼72 h after eCG injection, or ∼16–18 h prior to LOPU, to initiate oocyte maturation in vivo, while the remaining animals only received eCG (Group eCG). Most gilts underwent LOPU twice alternating the gonadotropin protocol, thereby decreasing the impact of individual variation on results. Abattoir-sourced oocytes from prepubertal gilts served as Control. Following LOPU, oocytes were in vitro matured, fertilized, and cultured to the blastocyst stage following standard procedures, while oocytes collected from Group eCG-hCG gilts were considered partly matured in vivo and were matured for ∼24 h instead of ∼44 h. Embryos reaching the blastocyst stage were fixed and stained to assess quality through cell numbers. There were no significant differences in the number of follicles aspirated and cumulus-oocyte complexes (COCs) recovered between Groups eCG-hCG and eCG (22.4 and 16.9 vs. 22.6 and 17.6, P > 0.05), as well as the recovery rate (76.6 vs. 78.1, P > 0.05). Cleavage rate was not different between Group eCG-hCG, Group eCG and Control (61.1 vs. 64.4 vs. 53.4 %, P > 0.05). However, the blastocyst rate over total oocytes (32.2 vs. 36.9 vs. 11.1 %, P < 0.05), blastocyst rate over cleaved oocytes (51.8 vs. 55.1 vs. 21.2 %, P < 0.01) and the average number of cells/blastocyst (89.6 vs. 87.5 vs. 62.2, P < 0.01) were unaffected by hCG treatment in LOPU-sourced oocytes, but both LOPU groups were significantly higher than abattoir-sourced oocytes, respectively. Our results suggest LOPU may become a powerful tool for sourcing swine oocytes with higher developmental competence than abattoir-sourced oocytes and known disease status for creating swine models for human biomedical applications, as well as for accelerated genetic gain in swine breeding programs.

RNA sequencing-based transcriptome analysis of granulosa cells from follicular fluid: Genes involved in embryo quality during in vitro fertilization and embryo transfer.

Granulosa cells play an important role in folliculogenesis, however, the role of RNA transcripts of granulosa cells in assessing embryo quality remains unclear. Therefore, we aims to investigate that RNA transcripts of granulosa cells be used to assess the probability of the embryonic developmental capacity. This prospective cohort study was attempted to figure out the probability of the embryonic developmental capacity using RNA sequencing of granulosa cells. Granulosa cells were collected from 48 samples in good-quality embryo group and 79 in only poor- quality embryo group from women undergoing in vitro fertilization and embryo transfer treatment. Three samples from each group were used for RNA sequencing. 226 differentially expressed genes (DEGs) were related to high developmental competence of embryos. Gene Ontology enrichment analysis indicated that these DEGs were primarily involved in biological processes, molecular functions, and cellular components. Additionally, pathway analysis revealed that these DEGs were enriched in 13 Kyoto Encyclopedia of Genes and Genomes pathways. Reverse transcription quantitative polymerase chain reaction verified the differential expression of the 13 selected DEGs. Among them,10 genes were differently expressed in the poor-quality embryo group compared to good-quality embryo group, including CSF1R, CTSH, SERPINA1, CYP27A1, ITGB2, IL1β, TNF, TAB1, BCL2A1, and CCL4. RNA sequencing data provide the support or confute granulosa expressed genes as non-invasive biomarkers for identifying the embryonic developmental capacity.

Equine in vitro produced blastocysts: relationship of embryo morphology, stage and speed of development to foaling rate.

Information on factors associated with developmental competence of equine in vitro -produced (IVP) blastocysts is lacking. To determine the relationships of stage, grade, day of development, and specific morphological parameters of equine IVP blastocysts, to pregnancy and foaling rates. Photomicrographs of 316 IVP embryos with known pregnancy outcomes were scrutinised individually by four observers. Inter-observer variation was assessed, and pregnancy outcome evaluated in relation to day of blastocyst development and assigned grade and stage. Individual component analysis was performed to determine the association of specific morphological parameters with foaling rate. Overall pregnancy rate was 76.9% and foaling rate was 56.3%. The day of embryo development did not affect pregnancy rate but significantly affected foaling rate. Embryo stage did not affect foaling rate. Embryo grade affected foaling rate only for Day-9 embryos. Some morphological features in the bovine grading system did not predict outcome in equine IVP embryos. Significant individual parameters differed between Stage 5 and Stage 6 equine blastocysts. Day of blastocyst development is the major factor related to foaling rate for equine IVP embryos. Notably, there was no effect of embryo stage on foaling rate and no evidence that prolonging culture until embryos advance in stage increases foaling rate. The standard bovine grading system is not directly applicable to equine IVP embryos; equine-specific staging and grading systems are proposed. This information will allow laboratories to identify embryos with the highest developmental competence. Use of the proposed systems will increase consistency in embryo assessment among laboratories.

COMPARISON OF LABORATORY OUTCOMES FROM DIFFERENT SPERM SOURCES USING FRESH AND FROZEN DONOR EGGS IN A SINGLE HIGH VOLUME LABORATORY

Use of frozen donor eggs for ART has increased substantially. The convenience of using frozen donor eggs in advantageous and addresses limitations present with fresh donor egg cycles. However, compared to fresh donor cycles, the number of frozen eggs provided per cycle is low. Thus, high developmental competence of frozen eggs is critical to ensure success, especially with certain sperm sources. Fertilization and blastocyst development from fresh and frozen donor eggs following injection with varying sperm sources were compared.

Transcriptional profile of cumulus associated GJA1, PTX3, PRSS35, and SERPINE2 genes with oocytes and embryonic development in water buffalo.

In the present study, the potential of different groups of cumulus-oocyte complexes (COC's) for in vitro maturation (IVM) and embryonic development was assessed in two groups of COC's of water buffalo. Further, the expression pattern of cumulus-associatedGJA1,PTX3, PRSS35,andSERPINE2genes and their effects on embryonic development was analyzed. Slaughterhouse-derived buffalo COC's were graded into groups A and B. An equal number of 410 COC's were taken in both groups. IVM was carried out using Slaughterhouse-derived buffalo epididymis. A remarkable degree of cumulus expansion was noticed in group A (92.68%) as compared to group B (81.25%) oocytes. On in vitro fertilization (IVF) and embryo culture, group A produced a significantly higher rate of cleavage and blastocyst (92.682 ± 0.7179% and 42.682 ± 0.9683%) as compared to group B (85.365 ± 0.7608% and 31.707 ± 0.9688%). Also, the transcriptional analysis of cumulus-associated genes revealed significantly higher expression in group A as compared to group B. It was revealed that oocytes having good cumulus mass had a higher developmental potential. Based on differential gene expression of cumulus-associated genes, different quality of COC's, and the resultant embryos after IVF, it was concluded that these genes could be used as a marker for predicting the developmental competence of the oocytes. We concluded that morphologically good quality of COC's had a higher developmental competence, and also the differential expressions of cumulus-associated genes in cumulus cells and embryos. So, we can conclude that these genes could be used as marker genes for predicting the developmental competence of buffalo's oocytes.

Mouse in vivo-derived late 2-cell embryos have higher developmental competence after high osmolality vitrification and -80°C preservation than IVF or ICSI embryos.

Mammalian embryos are most commonly cryopreserved in liquid nitrogen; however, liquid nitrogen is not available in special environments, such as the International Space Station (ISS), and vitrified embryos must be stored at −80°C. Recently, the high osmolarity vitrification (HOV) method was developed to cryopreserve mouse 2-cell stage embryos at −80°C; however, the appropriate embryo is currently unknown. In this study, we compared the vitrification resistance of in vivo-derived, in vitro fertilization (IVF)-derived, and intracytoplasmic sperm injection (ICSI)-derived mouse 2-cell embryos against cryopreservation at −80°C. The ICSI embryos had lower survival rates after warming and significantly lower developmental rates than the in vivo and IVF embryos. Further, IVF embryos had a lower survival rate after warming, but a similar rate to the in vivo embryos to full-term development. This result was confirmed by simultaneous vitrification of in vivo and IVF embryos in the same cryotube using identifiable green fluorescent protein-expressing embryos. We also evaluated the collection timing of the in vivo embryos from the oviduct and found that late 2-cell embryos had higher survival and developmental rates to full-term than early 2-cell embryos. Some early 2-cell embryos remained in the S-phase, whereas most late 2-cell embryos were in the G2-phase, which may have affected the tolerance to embryo vitrification. In conclusion, when embryos must be cryopreserved under restricted conditions, such as the ISS, in vivo fertilized embryos collected at the late 2-cell stage without long culture should be employed.

Comparing mRNA and sncRNA profiles during the maternal-to-embryonic transition in bovine IVF and scNT embryos†.

Production of embryos with high developmental competence by somatic cell nuclear transfer (scNT) is far less efficient than for in vitro fertilized (IVF) embryos, likely due to an accumulation of errors in genome reprogramming that results in aberrant expression of RNA transcripts, including messenger RNAs (mRNA) and, possibly, microRNAs (miRNA). Thus, our objectives were to use RNAseq to determine the dynamics of mRNA expression in early developing scNT and IVF embryos in the context of the maternal-to-embryonic transition (MET) and to correlate apparent transcriptional dysregulation in cloned embryos with miRNA expression profiles. Comparisons between scNT and IVF embryos indicated large scale transcriptome differences, which were most evident at the 8-cell and morula stages for genes associated with biological functions critical for the MET. For two miRNAs previously identified as differentially expressed in scNT morulae, miR-34a and miR-345, negative correlations with some predicted mRNA targets were apparent, though not widespread among the majority of predicted targets. Moreover, although large-scale aberrations in expression of mRNAs were evident during the MET in cattle scNT embryos, these changes were not consistently correlated with aberrations in miRNA expression at the same developmental stage, suggesting that other mechanisms controlling gene expression may be involved.

Prediction of oocyte quality using mRNA transcripts screened by RNA sequencing of human granulosa cells

Research questionCan RNA transcripts of granulosa cells be used to assess oocyte quality? The possibility of predicting the developmental competence of oocytes by RNA sequencing analysis of granulosa cells was evaluated. DesignGranulosa cell samples were collected from 29 women undergoing assisted reproductive technology treatment and divided into two groups: 14 samples from the high blastocyst rate group and 15 from the low blastocyst rate group. Ten samples from each group were selected for RNA sequencing. ResultsA total of 129 differentially expressed genes associated with high developmental competence of oocytes were identified. COL1A2, renin and COL1A1 were selected and further examined by quantitative real-time polymerase chain reaction (qRT-PCR). Expression levels of COL1A2 and renin by qRT-PCR were consistent with the results of RNA sequencing. ConclusionRNA sequencing data could provide novel marker genes for the non-invasive evaluation of oocyte quality and embryo developmental competence.

Induction of autophagy protects against extreme hypoxia-induced damage in porcine embryo.

In the mammalian female reproductive tract, physiological oxygen tension is lower than that of the atmosphere. Therefore, to mimic in vivo conditions during in vitro culture (IVC) of mammalian early embryos, 5% oxygen has been extensively used instead of 20%. However, the potential effect of hypoxia on the yield of early embryos with high developmental competence remains unknown or controversial, especially in pigs. In the present study, we examined the effects of low oxygen tension under different oxygen tension levels on early developmental competence of parthenogenetically activated (PA) and in vitro-fertilized (IVF) porcine embryos. Unlike the 5% and 20% oxygen groups, exposure of PA embryos to 1% oxygen tension, especially in early-phase IVC (0-2 days), greatly decreased several developmental competence parameters including blastocyst formation rate, blastocyst size, total cell number, inner cell mass (ICM) to trophectoderm (TE) ratio, and cellular survival rate. In contrast, 1% oxygen tension did not affect developmental parameters during the middle (2-4 days) and late phases (4-6 days) of IVC. Interestingly, induction of autophagy by rapamycin treatment markedly restored the developmental parameters of PA and IVF embryos cultured with 1% oxygen tension during early-phase IVC, to meet the levels of the other groups. Together, these results suggest that the early development of porcine embryos depends on crosstalk between oxygen tension and autophagy. Future studies of this relationship should explore the developmental events governing early embryonic development to produce embryos with high developmental competence in vitro.

Randomised controlled trial on the effect of clomiphene citrate and gonadotropin dose on ovarian response markers and IVF outcomes in poor responders.

Does the gonadotropin (GN) starting dose and the addition of clomiphene citrate (CC) during the early follicular phase influence oocyte yield in poor responders undergoing ovarian stimulation for IVF treatment? The number of retrieved oocytes was similar regardless of the starting dose of GN (150 versus 450 IU) with or without the addition of CC (100 mg from Day 3 to 7 versus placebo). ART in poor responders is a challenge for patients and clinicians. So far, randomised controlled studies addressing interventions have shown that neither the GN dose nor the addition of oral medication has any significant effect on the clinical outcome of ART in poor responders. There is limited knowledge about the effect of GN starting dose in combination with CC during the early follicular phase of ovarian stimulation on ovarian response markers and ART outcome. This single-centre randomised double-blinded clinical trial was conducted from August 2013 until November 2017. Using the Bologna criteria, 220 of 2288 patients (9.6%) were identified as poor responders and 114 eligible participants underwent ovarian stimulation in a GnRH-antagonist protocol for ART. The participants were equally randomised to one of four treatment arms: Group A (n = 28) received 100 mg CC (Day 3-7) and a starting dose of 450 IU HMG, Group B (n = 29) received 100 mg CC and a starting dose of 150 IU HMG, Group C (n = 30) received placebo and a starting dose of 450 IU HMG and Group D (n = 27) received placebo and a starting dose of 150 IU HMG. Serum levels of FSH, LH, estradiol and progesterone were measured on Day 1 and 5 and on the day of ovulation induction. Available embryos were cultured up to the blastocyst stage and were always transferred in the same cycle. The primary outcome was the number of oocytes collected after ovarian stimulation. Other outcome measures were response to ovarian stimulation, embryo development and obstetrical outcome. All study participants (n = 114) fulfilled at least two of the Bologna criteria for poor responders. Median age of the study population was 38.5 years. There were 109 patients who underwent oocyte retrieval. The number of oocytes retrieved was similar among the groups (±SD; 95% confidence intervals); A: 2.85 (±0.48; 2.04-3.98), B: 4.32 (±0.59; 3.31-5.64); C: 3.33 (±0.52; 2.45-4.54); D: 3.22 (±0.51; 2.36-4.41); P overall = 0.246. However, ovarian stimulation with 150 IU plus CC resulted in a higher number of blastocysts compared to ovarian stimulation with 450 IU plus CC (±SD; 95% confidence intervals); A: 0.83 (±0.15; 0.58-1.2), B: 1.77 (±0.21; 1.42-2.22); P overall = 0.006. Mean FSH serum levels were lower in the groups with a starting dose of 150 IU. Adding CC did not affect mean serum FSH levels. There were no differences in estradiol concentrations among the groups. Endometrial thickness was lower in the groups receiving CC. The overall live birth rate (LBR) was 12.3%, and the cumulative LBR was 14.7%. The trial was powered to detect differences in neither the number of blastocysts nor the LBR, which would be the preferable primary outcome of interventional trials in ART. We found that ovarian stimulation with 150 IU gonadotrophin in combination with 100 mg CC produced more blastocysts. The effect of adding CC to GN on LBR in poor responders remains to be proven in randomised trials. High GN doses (450 IU) resulted in high FSH serum levels but increased neither the estradiol levels nor the number of retrieved oocytes, implying that granulosa cell function is not improved by high FSH serum levels. Lower starting doses of GN lead to a reduction of costs of medication. The small but significant difference in blastocyst formation and the lower FSH levels in the treatment groups receiving less GN may be an indication of better oocyte quality with higher developmental competence. The costs for the HMG used for ovarian stimulation were provided by IBSA Switzerland. The study was also supported by the Repronatal Foundation, Basel, Switzerland. The authors declare no conflict of interest. NCT01577472. 13 April 2012. August 2013.

Selection based on morphological features of porcine embryos produced by in vitro fertilization: Timing of early cleavages and the effect of polyspermy.

The aim of this study was to examine whether a morphological approach is efficient for selecting high‐quality porcine embryos produced by in vitro fertilization (IVF) under high polyspermy conditions. Frozen‐thawed Meishan epididymal spermatozoa showing moderate and high polyspermy were subjected to IVF (1 × 105 sperms/ml). Under conditions of moderate polyspermy, 4‐cell embryos selected at 48 hr after IVF (single selection) and 8‐cell embryos selected at 79 hr after IVF from the collected 4‐cell embryos (double selection) showed high developmental competence. Likewise, 4‐ and 8‐cell embryos produced by IVF under high polyspermy conditions also showed high competence for development to blastocysts. However, blastocysts derived from high polyspermy conditions had significantly fewer cells than those produced under moderate polyspermy conditions. Furthermore, the frequency of nuclear and chromosomal abnormalities in 4‐ and 8‐cell embryos produced under conditions of high polyspermy was significantly (p < .05) higher in comparison to moderate polyspermy conditions. These findings suggest that although high polyspermy affects the frequency of nuclear and chromosomal anomalies in porcine IVF embryos, subsequent selection based on morphological features of 4‐ and 8‐cell embryos even under high polyspermy conditions, could be an alternative option for selecting porcine IVF embryos with high development ability.

Embryo aggregation regulates in vitro stress conditions to promote developmental competence in pigs.

Embryo aggregation is a useful method to produce blastocysts with high developmental competence to generate more offspring in various mammals, but the underlying mechanism(s) regarding the beneficial effects are largely unknown. In this study, we investigated the effects of embryo aggregation using 4-cell stage embryos in in vitro developmental competence and the relationship of stress conditions in porcine early embryogenesis. We conducted aggregation using the well of the well system and confirmed that aggregation using two or three embryos was useful for obtaining blastocysts. Aggregated embryos significantly improved developmental competence, including blastocyst formation rate, blastomere number, ICM/TE ratio, and cellular survival rate, compared to non-aggregated embryos. Investigation into the relationship between embryo aggregation and stress conditions revealed that mitochondrial function increased, and oxidative and endoplasmic reticulum (ER)-stress decreased compared to 1X (non-aggregated embryos) blastocysts. In addition, 3X (three-embryo aggregated) blastocysts increased the expression of pluripotency, anti-apoptosis, and implantation related genes, and decreased expression of pro-apoptosis related genes. Therefore, these findings indicate that embryo aggregation regulates in vitro stress conditions to increase developmental competence and contributes to the in vitro production of high-quality embryos and the large-scale production of transgenic and chimeric pigs.

87 Analyzing metabolomic profile of bovine IVF and somatic cell nuclear transfer embryos through Raman spectroscopy

While knowledge of early embryo development and viability has continually increased, there is still a need for noninvasive methods to identify embryos with the highest chances of development to term when transferred. The most widely used technique, morphological assessment, is highly subjective and limited by personnel experience. Spent media from invitro culture could be used as a valuable noninvasive marker for embryo quality assessment. Raman spectroscopy has proven to be a powerful tool for identifying the molecular characteristics of spent culture media by measuring vibration of chemical bonds, allowing for metabolomic profiling of varying stages and quality of embryos. It is well documented that embryos produced through somatic cell nuclear transfer (SCNT) result in lower pregnancy rates and higher incidences of pregnancy loss when compared with embryos produced through IVF (Heyman et al. 2002 Biol. Reprod. 66, 1-13). This study was designed to examine differences in spent media between bovine embryos produced by IVF and SCNT. The SCNT embryos with a metabolomic profile more similar to IVF embryos may have a higher developmental competence. Bovine cumulus-oocyte complexes (COCs) were retrieved from abattoir-derived ovaries and matured for 21h in maturation medium. After IVM, COCs were either used for IVF or SCNT and cultured in 50μL droplets of synthetic oviductal fluid medium + fetal bovine serum in groups of 45 from Day 0-5. On Day 5, embryos that had reached morula stage were placed in individual droplets of 13μL of synthetic oviductal fluid + bovine serum albumin until Day 7. All embryos were cultured at 38.5°C and 5% CO2. On Day 7 embryos were assessed for developmental stage and quality and 10μL of medium from individual culture drops was collected for Raman spectroscopy. Samples were loaded on an MgF2 optical window, dried, and analysed using a 785nm near-infrared laser in the spectral range of 600 to 1800cm−1. Raw Raman data were first pre-processed by baseline correction (asymmetric least-squares smoothing) and normalization. Principal component analysis and partial least squares were then applied to reduce data dimensions. The score of the most significant principal components from principal component analysis and the optimum number of scores from partial least squares were used for linear discriminant analysis. Spent media samples from 4 SCNT embryos, 3 IVF embryos, and 3 empty media samples were analysed, with 50 spectra obtained from each sample. Preliminary data showed grouping of medium containing embryos developed to blastocyst from medium containing embryos arrested at morula or empty medium. We also saw grouping of medium containing SCNT embryos from medium containing IVF embryos within both the morula and blastocyst stage from empty medium. This shows evidence of metabolomic differences between embryos of different developmental potential and embryos produced by IVF and SCNT. Further investigation of the Raman profile of these groups can display specific differences in chemical components and help to identify candidate genes causing differing metabolism of these groups.

Characterization and control of oocyte large-scale chromatin configuration in different cattle breeds

Differences in reproductive physiology between cattle breeds may help to explain distinct responses to assisted reproductive techniques and to define breed-specific protocols with improved efficiency. Germinal vesicle (GV) stage oocytes are characterized by increasing levels of chromatin compaction enclosed within the nucleus (graded from GV0 to GV3), associated with different developmental competence. The first objective of this study was to characterize chromatin configuration of GV stage oocytes recovered by OPU at random days of the estrous cycle from Nelore (Bos indicus) and Holstein (Bos taurus) cows. In Nelore 90% of the oocytes presented advanced stages of chromatin compaction associated with higher developmental competence (GV2 and GV3), while in Holstein, only 65% of the oocytes were at these stages. Then, aiming to obtain a more homogeneous population of oocytes in Holstein, we tested two synchronization protocols combining aspiration of all visible follicles at a random day (day 0), two IM injections of FSH 12 h apart on day 2, and OPU on day 4 (OPU/D4) or 5 (OPU/D5). The protocol OPU/D4 provided around 45% of the oocytes with low chromatin compaction (GV1), while the protocol OPU/D5 provided 70% of the oocytes at GV2 and 20% at GV3. Finally, we assessed the effects of a culture system known to prevent meiotic resumption on chromatin configuration of the GV2 enriched oocyte population obtained with the protocol OPU/D5. After 9 h of culture most oocytes transited from GV2 to GV3, with 90% of the oocytes at GV3 stage. This study demonstrates differences between Nelore and Holstein cows regarding patterns of chromatin configuration that may account for their different performance in IVM/IVF. In addition, it provides novel references for the design of protocols aiming to regulate oocyte quality before IVM for the optimization of IVF outcomes.

Genes regulating programmed cell death are significantly upregulated in porcine immature oocytes

Abstract Correct maturation of the oocyte is crucial for further fertilization and embryogenesis. It comprises of both nuclear and cytoplasmic maturation, during which the proteins, nutrients and mRNAs are assembled. Cumulus cells are connected with the oocyte via gap-junctions, which enable bi-directional transfer of molecules, forming cumulus-oocyte complex (COC). The expression pattern in CCs is thought to resemble the genes expressed in the oocyte. The CCs surrounding the gamete of high developmental competence have an increased expression of apoptotic markers. Therefore, our aim in this study was to determine whether any apoptosis-related genes are upregulated in porcine oocytes before or after IVM. We isolated COCs from 45 pubertal crossbred gilts, performed brilliant cresyl blue (BCB) staining and analyzed the gene expression pattern in oocytes before and after IVM with the use of microarray analysis. The results include 419 differentially expressed transcripts, 25 of which belong to „regulation of apoptosis” and „regulation of cell death” GO BP terms. This set of genes includes BCLAF1, EIF2AK3, KLF10, MIF, MAP3K1, NOTCH2, TXNIP and APP, all of which have been upregulated in immature porcine oocytes. Our results suggest that they play part in porcine oocyte maturation and could be used as potential markers of female gamete’s developmental competence. This knowledge could serve as a basis to improve ART in pigs.

Follicular factors determining the developmental competence of porcine oocyte.

PurposeThis study aimed to examine the relationship between granulosa cells (GCs), number of follicles, and the ability of follicular fluid to support in vitro growth of oocytes.MethodsThe culture medium was supplemented with follicular fluid (FF) collected from GC‐rich ovaries and GC‐poor ovaries, and its effect on in vitro growth and quality of oocytes derived from early antral follicles (EAFs) was assessed.ResultsGC‐rich FF treatment enhanced oocyte growth and augmented changes in the chromatin configuration and lipid content of oocytes when compared to oocytes treated with GC‐poor FF. Moreover, oocytes treated with GC‐rich FF had a higher ability to progress to the blastocyst stage, than oocytes derived from large antral follicles (3‐5 mm in diameter). In addition, supplementation of the culture medium with either GC‐rich or GC‐poor FF enhanced histone acetylation in oocytes grown in vitro.ConclusionGC‐rich FF contains key factors that support in vitro oocyte growth; hence, oocytes grown in GC‐rich FF medium had high developmental competence, which was comparative to the oocytes grown in vivo.

Acquisition of developmental competence and in vitro growth culture of bovine oocytes.

Recently, the demand of transferable embryos in cattle industry is increasing, and the number of embryos produced in vitro is also increasing in the world. Although oocytes are collected from individual elite cattle by ovum-pick up (OPU) and used for in vitro production (IVP) of embryos, the cattle are mono-ovulatory animal. It means that most of oocytes collected from ovaries are destined to degenerate. To improve the IVP efficiency, we should predict the developmental competence of oocytes correctly and culture them by the suitable way. In addition, in vitro production of bovine oocytes by in vitro growth (IVG) culture system will become a candidate of supply source of oocytes for IVP. If we can produce high competent oocytes by IVG, IVP efficiency will be improved and the genetic improvement of cattle will be dramatically accelerated. In the review, I introduce our researches related to oocyte morphology, the developmental competence, and the production of oocytes having high developmental competence by IVG culture.

Phosphoric acid and phosphorylation levels are potential biomarkers indicating developmental competence of matured oocytes.

Here, we aimed to identify biomarkers for mice oocyte maturation in metaphase II in vivo and in situ using Raman spectroscopy. Principal component analysis of 324 Raman data points of oocytes at Phase I, II, III, and IV showed that the phosphoric acid concentration uniformly increased in oocytes with higher developmental competence than in oocytes at other maturation stages, and proteins were more phosphorylated. The maturation phases were successfully predicted by linear discriminant analysis with high accuracy (90.7%) using phosphoric molecular information mentioned above. Furthermore, detections of higher concentration of unsaturated fatty acids in overmatured oocytes indicated that a decline in metabolic activity due to overmaturation induced a surplus of these lipid components. Upon assessing invasiveness by laser irradiation, about 50% irradiated oocytes progressed to morula and blastocyst stages in good conditions. Thus, Raman spectroscopy holds promise in evaluating oocyte maturation and quality based on molecular information in infertility treatment.

162 Developmental competence of bovine oocytes matured in defined medium

Routinely, bovine oocytes are matured in medium supplemented with 10% fetal bovine serum (FBS). However, due to the undefined nature of FBS and biosafety concerns, a standardized protein supplementation alternative is needed. We hypothesised that oocytes matured in defined medium supplemented with 5% serum substitute (DM) would have higher developmental competence than those matured in FBS-supplemented medium (Control). In 6 replicates, in vitro-produced embryos were produced during the summer season by aspirating cumulus-oocyte complexes from 2- to 8-mm follicles of abattoir ovaries (LAB-COC), or aspirated from FSH-stimulated Holstein and Jersey donors (OPU-COC, n=20; 53 sessions). The LAB-COC (n=1307) and OPU-COC (n=679) were matured for 23h in control medium or DM supplemented with pyruvate and gonadotropins at 6% CO2 in air, fertilized with semen from 1 of 3 or 15 bulls, respectively, and cultured in SCF2 at 38.5°C, 6% CO2, 5% O2 and 89% N2. On Day 7.5 post-fertilization, blastocyst rates were evaluated, and embryos (n=4/replicate) from each group of LAB-COC were stained with 1µg mL−1 Nile Red for lipid quantification, 5 µM Cell Rox® Green (Thermo Fisher Scientific, Waltham, MA, USA) for reactive oxygen species (ROS) assessment and 300 nM Mitotracker Red CMX-Rosamine (Thermo Fisher Scientific) for mitochondrial polarity. Images were obtained with confocal microscopy and fluorescent intensity (AFU) was measured by Image J software (National Institutes of Health, Bethesda, MD, USA) adjusted per cell number. Data were analysed by one-way ANOVA using SAS software (SAS Institute Inc., Cary, NC, USA). Results (Table 1) indicate similar cleavage and blastocyst rates and mitochondrial polarity for embryos from LAB-COC in Control v. DM (P&amp;gt;0.05). However, ROS and lipid content were higher in Control than in DM (P&amp;lt;0.05). For OPU-COC, DM resulted in similar cleavage and blastocyst rates and embryos produced per OPU session compared with Control (P&amp;gt;0.05). Results suggest that maturing oocytes (LAB and OPU) in DM maintain developmental competence, and the resulting embryos had lower lipid content and ROS than those matured in Control medium. Cryopreservation studies and evaluation of pregnancy rates are ongoing. Table 1.Developmental competence of oocytes matured in control or defined medium with 5% serum substitute (DM)