Oocyte developmental competence is reduced in sows during the seasonal infertility period

2010 ◽  
Vol 22 (8) ◽  
pp. 1222 ◽  
Author(s):  
M. Bertoldo ◽  
P. K. Holyoake ◽  
G. Evans ◽  
C. G. Grupen
Keyword(s):  
Follicular Fluid ◽  
Late Summer ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Progesterone Concentration ◽  
Developmental Potential ◽  
Oocyte Developmental Competence ◽  
The Mean ◽  
Time Of Year ◽  
Seasonal Infertility

The modern domestic sow exhibits a period of impaired reproductive performance during the late summer and early autumn months, known as ‘seasonal infertility’. A reduction in farrowing rate due to pregnancy loss is the most economically important manifestation of seasonal infertility. The aim of the present study was to determine whether there are changes in oocyte developmental competence associated with season. Ovaries were collected in pairs from sows sourced from commercial piggeries and slaughtered 4 days after weaning during winter and summer–autumn. Following oocyte IVM and parthenogenetic activation, the ability of oocytes from large follicles to form blastocysts was greater in winter (54.94 ± 6.11%) than in summer (21.09 ± 5.59%). During winter, the proportion of oocytes developing to the blastocyst stage from large follicles was significantly higher (54.94 ± 6.11%) than those oocytes from small follicles (23.17 ± 6.02%). There was no effect of season on the proportion of oocytes developing to the blastocyst stage from small follicles. There was no effect of follicle size on blastocyst formation from those oocytes recovered during summer. Blastocysts derived from small follicles during summer had the lowest number of cells (24.25 ± 1.48) compared with blastocysts derived from large follicles during winter (37.5 ± 1.3; P < 0.05). The mean progesterone concentration in follicular fluid collected from small follicles was greater in winter than summer (1235.55 ± 164.47 v. 701.3 ± 115.5 nmol L–1, respectively; P < 0.001). The mean progesterone concentration in the follicular fluid of large follicles was also greater in winter than in summer (2470.9 ± 169.1 v. 1469.2 ± 156.5 nmol L–1, respectively; P < 0.001). Regression analysis revealed a positive correlation between progesterone concentration and oocyte developmental competence. The results indicate that porcine oocytes fail to reach their full developmental potential during the period of seasonal infertility, suggesting that the pregnancy losses observed at this time of year may be due to reduced oocyte developmental competence.

129. REDUCED OOCYTE DEVELOPMENTAL COMPETENCE DURING THE PERIOD OF SEASONAL INFERTILITY IN PIGS

2009 ◽  
Vol 21 (9) ◽  
pp. 48
Author(s):  
M. Bertoldo ◽  
P. K. Holyoake ◽  
G. Evans ◽  
C. G. Grupen
Keyword(s):  
Linear Mixed Model ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Seasonal Effects ◽  
Cumulus Expansion ◽  
Developmental Potential ◽  
Parthenogenetic Activation ◽  
Oocyte Developmental Competence ◽  
Generalised Linear Mixed Model ◽  
Seasonal Infertility

Reduced farrowing rate due to early pregnancy loss is a manifestation of seasonal infertility in pigs. It has been hypothesised that the early disruption of pregnancy is due to poor oocyte developmental competence. The aim of this study was to determine if there are seasonal differences in oocyte developmental competence. Ovaries were collected from sows slaughtered 4 days after weaning. Cumulus-oocyte complexes (COCs) recovered from small (3–4mm) and large (5–8mm) antral follicles were morphologically graded and subjected to parthenogenetic activation following in vitro maturation (IVM) during the winter (n = 1419) and summer (n = 2803). Cumulus expansion was assessed subsequent to IVM. Data were analysed using a generalised linear mixed model in GenStat release 10. There was an effect of season on oocyte grade, with a larger proportion of oocytes collected in summer being graded suitable for IVM, compared with winter oocytes (P<0.05). A larger proportion of COCs had expanded cumulus during the winter than in the summer, which suggested that the preovulatory LH surge had already occurred. There was a season x follicle size interaction affecting cumulus expansion (P<0.05). There were no seasonal effects on the proportion of oocytes reaching metaphase II or cleaving after parthenogenetic activation. However, the proportion of oocytes from large follicles that developed to the blastocyst stage was higher in winter than in summer (55% vs 23%; P<0.05). There was no effect of season on the proportion of oocytes developing to the blastocyst stage from small follicles. However, during summer there was a reduction in the cell number of blastocysts derived from small follicles (P<0.05). Our results suggest that porcine oocytes are less able to reach their full developmental potential during the period of seasonal infertility, and that the associated pregnancy losses are due to reduced oocyte developmental competence.

P–219 mtDNA content in bovine cumulus cells does not predict oocytés developmental competence

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Á Martíne. Moro ◽  
I Lamas-Toranzo ◽  
L González-Brusi ◽  
A Pérez-Gómez ◽  
P Bermejo-Álvarez
Keyword(s):  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Developmental Competence ◽  
Success Rates ◽  
Developmental Potential ◽  
Mtdna Sequence

Abstract Study question Does cumulus cell mtDNA content correlate with oocyte developmental potential in the bovine model? Summary answer The relative amount of mtDNA content did not vary significantly in oocytes showing different developmental outcomes following IVF What is known already Cumulus cells are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Previous studies have positively associated oocytés mtDNA content with developmental potential in both animal models and women. However, it remains debatable whether mtDNA content in cumulus cells could be used as a proxy to infer oocyte developmental potential. Study design, size, duration Bovine cumulus cells were allocated into three groups according to the developmental potential of the oocyte: 1) oocytes developing to blastocysts following IVF (Bl+Cl+), 2) oocytes cleaving following IVF but arresting their development prior to the blastocyst stage (Bl-Cl+), and 3) oocytes not cleaving following IVF (Bl-Cl-). Relative mtDNA content was analysed in 40 samples/group, each composed by the cumulus cells from one cumulus-oocyte complex (COC). Participants/materials, setting, methods Bovine cumulus-oocyte complexes were obtained from slaughtered cattle and individually matured in vitro (IVM). Following IVM, cumulus cells were removed by hyaluronidase treatment, pelleted, snap frozen in liquid nitrogen and stored at –80 ºC until analysis. Cumulus-free oocytes were fertilized and cultured in vitro individually and development was recorded for each oocyte. Relative mtDNA abundance was determined by qPCR, amplifying a mtDNA sequence (COX1) and a chromosomal sequence (PPIA). Statistical differences were tested by ANOVA. Main results and the role of chance Relative mtDNA abundance did not differ significantly (ANOVA p &gt; 0.05) between the three groups exhibiting different developmental potential (1±0.06 vs. 1.19±0.05 vs. 1.11±0.05, for Bl+Cl+ vs. Bl-Cl+ vs. Bl-Cl-, mean±s.e.m.). Limitations, reasons for caution Experiments were conducted in the bovine model. Although bovine folliculogenesis, monoovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, caution should be taken when extrapolating these data to humans. Wider implications of the findings: The use of molecular markers for oocyte developmental potential in cumulus cells could be used to enhance success rates following single-embryo transfer. Unfortunately, mtDNA in cumulus cells was not found to be a good proxy for oocyte quality. Trial registration number Not applicable

scholarly journals Oocyte Competence Biomarkers Associated With Oocyte Maturation: A Review

2021 ◽  
Vol 9 ◽  
Author(s):  
Batara Sirait ◽  
Budi Wiweko ◽  
Ahmad Aulia Jusuf ◽  
Dein Iftitah ◽  
R. Muharam
Keyword(s):  
Oocyte Maturation ◽  
Blastocyst Stage ◽  
Current Approach ◽  
Developmental Competence ◽  
Matrix Remodeling ◽  
Oocyte Competence ◽  
Nuclear Maturation ◽  
Oocyte Developmental Competence ◽  
Cumulus Oocyte Complex

Oocyte developmental competence is one of the determining factors that influence the outcomes of an IVF cycle regarding the ability of a female gamete to reach maturation, be fertilized, and uphold an embryonic development up until the blastocyst stage. The current approach of assessing the competency of an oocyte is confined to an ambiguous and subjective oocyte morphological evaluation. Over the years, a myriad of biomarkers in the cumulus-oocyte-complex has been identified that could potentially function as molecular predictors for IVF program prognosis. This review aims to describe the predictive significance of several cumulus-oocyte complex (COC) biomarkers in evaluating oocyte developmental competence. A total of eight acclaimed cumulus biomarkers are examined in the study. RT-PCR and microarray analysis were extensively used to assess the significance of these biomarkers in foreseeing oocyte developmental competence. Notably, these biomarkers regulate vital processes associated with oocyte maturation and were found to be differentially expressed in COC encapsulating oocytes of different maturity. The biomarkers were reviewed according to the respective oocyte maturation events namely: nuclear maturation, apoptosis, and extracellular matrix remodeling, and steroid metabolism. Although substantial in vitro evidence was presented to justify the potential use of cumulus biomarkers in predicting oocyte competency and IVF outcomes, the feasibility of assessing these biomarkers as an add-on prognostic procedure in IVF is still restricted due to study challenges.

scholarly journals MicroRNAs from Extracellular Vesicles Secreted by Bovine Embryos as Early Biomarkers of Developmental Competence

2020 ◽  
Vol 21 (23) ◽  
pp. 8888
Author(s):  
Bárbara Melo-Baez ◽  
Yat S. Wong ◽  
Constanza J. Aguilera ◽  
Joel Cabezas ◽  
Ana C. F. Mançanares ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Fatty Acid Biosynthesis ◽  
Culture Media ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Target Cells ◽  
Bovine Embryos ◽  
Developmental Potential ◽  
Maternal Communication

During early development, embryos secrete extracellular vesicles (EVs) that participate in embryo–maternal communication. Among other molecules, EVs carry microRNAs (miRNAs) that interfere with gene expression in target cells; miRNAs participate in embryo–maternal communication. Embryo selection based on secreted miRNAs may have an impact on bovine breeding programs. This research aimed to evaluate the size, concentration, and miRNA content of EVs secreted by bovine embryos with different developmental potential, during the compaction period (days 3.5–5). Individual culture media from in vitro–produced embryos were collected at day 5, while embryos were further cultured and classified at day 7, as G1 (conditioned-culture media by embryos arrested in the 8–16-cells stage) and G2 (conditioned-culture media by embryos that reached blastocyst stages at day 7). Collected nanoparticles from embryo conditioned culture media were cataloged as EVs by their morphology and the presence of classical molecular markers. Size and concentration of EVs from G1 were higher than EVs secreted by G2. We identified 95 miRNAs; bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were upregulated in G1, whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were downregulated. The most significant upregulated pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The characteristics of EVs secreted by bovine embryos during the compaction period vary according to embryo competence. Embryos that reach the blastocyst stage secrete fewer and smaller vesicles. Furthermore, the loading of specific miRNAs into the EVs depends on embryo developmental competence.

scholarly journals Predictive value of bovine follicular components as markers of oocyte developmental potential

2014 ◽  
Vol 26 (2) ◽  
pp. 337 ◽  
Author(s):  
Satoko Matoba ◽  
Katrin Bender ◽  
Alan G. Fahey ◽  
Solomon Mamo ◽  
Lorraine Brennan ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Cumulus Cell ◽  
Principal Component ◽  
Transcript Abundance ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Potential ◽  
Thecal Cells ◽  
Total Fatty Acids

The follicle is a unique micro-environment within which the oocyte can develop and mature to a fertilisable gamete. The aim of this study was to investigate the ability of a panel of follicular parameters, including intrafollicular steroid and metabolomic profiles and theca, granulosa and cumulus cell candidate gene mRNA abundance, to predict the potential of bovine oocytes to develop to the blastocyst stage in vitro. Individual follicles were dissected from abattoir ovaries, carefully ruptured under a stereomicroscope and the oocyte was recovered and individually processed through in vitro maturation, fertilisation and culture. The mean (± s.e.m.) follicular concentrations of testosterone (62.8 ± 4.8 ng mL–1), progesterone (616.8 ± 31.9 ng mL–1) and oestradiol (14.4 ± 2.4 ng mL–1) were not different (P > 0.05) between oocytes that formed (competent) or failed to form (incompetent) blastocysts. Principal-component analysis of the quantified aqueous metabolites in follicular fluid showed differences between oocytes that formed blastocysts and oocytes that degenerated; l-alanine, glycine and l-glutamate were positively correlated and urea was negatively correlated with blastocyst formation. Follicular fluid associated with competent oocytes was significantly lower in palmitic acid (P = 0.023) and total fatty acids (P = 0.031) and significantly higher in linolenic acid (P = 0.036) than follicular fluid from incompetent oocytes. Significantly higher (P < 0.05) transcript abundance of LHCGR in granulosa cells, ESR1 and VCAN in thecal cells and TNFAIP6 in cumulus cells was associated with competent compared with incompetent oocytes.

196 A NEW APPROACH TO PREDICT MOUSE EMBRYONIC DEVELOPMENTAL COMPETENCE USING A TIME-LAPSE SYSTEM AND THE 'SHORTEST HALF' analysis procedure

2007 ◽  
Vol 19 (1) ◽  
pp. 214 ◽  
Author(s):  
S. Yavin ◽  
A. Aroyo ◽  
Z. Roth ◽  
A. Arav
Keyword(s):  
Embryonic Development ◽  
Time Lapse ◽  
Time Frame ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Embryo Morphology ◽  
Cell Stage ◽  
Developmental Potential ◽  
Additional Tool ◽  
Embryonic Cleavage

Embryonic development is a dynamic process in which embryo morphology may change immensely within several hours. Therefore, identifying and selecting embryos with the highest probability of developing and achieving a pregnancy is a major challenge. The timing of embryonic cleavage may serve as an additional indicator for the identification of quality embryos. The aim of this study was to characterize the cleavage timing of mouse embryos and to identify the stage that is most indicative of blastocyst formation. Mated mice (CB6F1) were sacrificed 20 h after hCG administration; putative zygotes were recovered and cultured (50 embryos in each 20-µL drop of M16) in a time-lapse system (EmbryoGuard; IMT, Ltd., Ness-Ziona, Israel) inside the incubator. The time-lapse system was programmed to take photos at half-hour intervals such that culture dishes were not removed from the incubator. The ‘shortest half’ statistical procedure of JMPIN (SAS Institute, Inc., Cary, NC, USA) was utilized to evaluate the period during which at least 50% of the embryonic population cleaves within the shortest time frame. Captured images made it possible to search along the time axis for the densest 50% of cleavage observations. Developing embryos were categorized into 3 groups according to the time of cleavage after hCG administration: before, during, and after the ‘shortest half’ for each developmental stage. Two hundred thirty putative zygotes cleaved and created 2-cell-stage embryos, of which 55 arrested at various stages and 175 progressed to the blastocyst stage. During embryonic development, cleavage timing appeared to become less uniform and the ‘shortest half’ became longer for each successive cell division: Whereas the shortest period in which 50% of the 2-cell-stage embryos cleaved was a 2-h interval, cleavage into the 4-cell, 8-cell, and blastocyst stages took 2.5, 3.5, and 5 h, respectively. The ‘short half’ for the first cleavage appears to be a predictive time frame for subsequent embryonic development, because cleavage was closely synchronized with 80% of the embryos developing to the blastocyst stage. Note that only a small number of embryos were actually cleaving early, while the ‘shortest half’ consisted of 50% of the embryonic population. Moreover, late-cleaving embryos in the 2-cell stage expressed inferior developmental potential relative to those that cleaved within the ‘shortest half’ (see Table 1). In summary, 2-cell-stage embryos that cleaved within the ‘shortest half’ seemed to be better synchronized and consequently more competent than the rest of the embryonic population. Embryonic cleavage timing using the ‘shortest half’ parameter can be considered a biological indicator of embryo potential. It may be useful as an additional tool for selecting embryos for transfer and cryopreservation. Table 1. Cleavage timing distribution into the 2-cell stage according to the shortest half

327 OOCYTE-SECRETED FACTORS DIRECTLY AFFECT OOCYTE DEVELOPMENTAL COMPETENCE DURING IN VITRO MATURATION OF THE BOVINE CUMULUS - OOCYTE COMPLEX

2006 ◽  
Vol 18 (2) ◽  
pp. 271 ◽  
Author(s):  
T. S. Hussein ◽  
R. B. Gilchrist ◽  
J. G. Thompson
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
Paracrine Factors ◽  
Cell Functions ◽  
Oocyte Developmental Competence ◽  
Secreted Factors

Paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) regulate a broad range of cumulus cell functions including proliferation, differentiation, and apoptosis. The capacity of oocytes to regulate their own microenvironment by OSFs may in turn contribute to oocyte developmental competence. The aim of this study was to determine if OSFs have a direct influence on bovine oocyte developmental competence during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were obtained by aspiration of >3-mm follicles from abattoir-derived ovaries. IVM was conducted in Bovine VitroMat (Cook Australia, Eight Mile Plains, Brisbane, Australia) supplemented with 0.1 IU/mL rhFSH for 24 h under 6% CO2 in air at 38.5�C. In the first experiment, COCs were co-cultured with denuded oocytes (DOs, 5/COC in 10 �L) beginning at either 0 or 9-h of IVM. To generate the 9-h DO group, COCs were first cultured intact for 9-h and then denuded. In the second experiment, specific OSFs, recombinant bone morphogenetic protein-15 (BMP-15) and growth differentiation factor 9 (GDF-9), were prepared as partially purified supernatants of transfected 293H cells, and used as 10% v/v supplements in Bovine VitroMat. Treatments were: (1) control (no supplement), (2) BMP-15, (3) GDF-9, (4) BMP-15 and GDF-9, and (5) untransfected 293H control. Following maturation, in vitro production of embryos was performed using the Bovine Vitro system (Cook Australia) and blastocysts were examined on Day 8 for development. Developmental data were arcsine-transformed and analyzed by ANOVA, followed by Tukey's test. Cell numbers were analyzed by ANOVA. Co-culturing intact COCs with DOs from 0 or 9 h did not affect cleavage rate, but increased (P < 0.001) the proportion of cleaved embryos that reached the blastocyst stage post-insemination (50.6 � 1.9 and 61.3 � 1.9%, respectively), compared to COCs cultured alone (40.7 � 1.4%). Therefore, paracrine factors secreted by DOs increased the developmental competence of oocytes matured as COCs. OSFs also improved embryo quality, as co-culture of COCs with DOs (0 or 9 h) significantly increased total cell (156.1 � 1.3 and 159.1 � 1.3, respectively) and trophectoderm (105.7 � 1.3 and 109.8 � 0.4, respectively) numbers, compared to control COCs (total = 148 � 1.2, trophectoderm = 98.2 � 0.8, P < 0.001). BMP-15 alone or with GDF-9 also significantly (P < 0.001) increased the proportion of oocytes that reached the blastocyst stage post insemination (57.5 � 2.4% and 55.1 � 4.5%, respectively), compared to control (41.0 � 0.9%) and 293H-treated (27.1 � 3.1%) COCs. GDF-9 also increased blastocyst yield (49.5 � 3.9%) but not significantly. These results are the first to demonstrate that OSFs, and particularly BMP-15 and GDF-9, directly affect bovine oocyte developmental competence. These results have far-reaching implications for improving the efficiency of IVM in domestic species and human infertility treatment, and support the role of OSF production by oocytes as a diagnostic marker for developmental competence.

58 HISTONE DEACETYLASES INHIBITOR, SCRIPTAID, IS BENEFICIAL TO THE REPROGRAMMING OF SOMATIC NUCLEI FOLLOWING NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 129
Author(s):  
J. G. Zhao ◽  
J. W. Ross ◽  
Y. H. Hao ◽  
D. M. Wax ◽  
L. D. Spate ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Histone Deacetylases ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Untreated Group ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos

Somatic cell nuclear transfer (SCNT) is a promising technology with potential applications in both agriculture and regenerative medicine. The reprogramming of differentiated somatic nuclei into totipotent embryonic state following NT is not efficient and the mechanism is currently unknown. However, accumulating evidence suggests that faulty epigenetic reprogramming is likely to be the major cause of low success rates observed in all mammals produced through SCNT. It has been demonstrated that increased histone acetylation in reconstructed embryos by applying histone deacetylases inhibitor (HDACi) such as trychostatin A (TSA) significantly enhanced the developmental competence in several species in vitro and in vivo. However TSA has been known to be teratogenic. Compared with TSA, Scriptaid is a low toxic but more efficient HDACi (Su GH et al. 2000 Cancer Res. 60, 3137–3142). The objectives of this study were: 1) to investigate and optimize the application Scriptaid to the NT using Landrace fetal fibroblast cells (FFCs) as donor; 2) investigate the effect of increased histone acetylation on the developmental competence of reconstructed embryos from NIH mini inbred FFCs in vitro and in vivo. The reconstructed embryos were treated with Scriptaid at different concentrations (0 nm, 250 nm, 500 nm and 1000 nm) after activation for 14 to 16 h. IVF embryos without treatment were produced as an additional control. Developmental rates to the 2-cell and blastocyst stage were determined. Developmental potential was determined by transferring Day 1 NT zygotes to the oviducts of surrogates on the day of, or one day after, the onset of estrus. Experiments were repeated at least 3 times and data were analyzed with chi-square tests using SAS 6.12 program (SAS institute, Inc., Cary, NC, USA). The percentage blastocyst of cloned embryos using Landrace FFCs as donors treated with 500 nm Scriptaid was the highest and was significantly higher than untreated group (25% v. 11%, P < 0.05). Percent cleaved was not different among four treatment groups. We used 500 nm Scriptaid for 14 to 16 h after activation for all subsequent experiments. Developmental rate to the blastocyst stage was significantly increased in cloned embryos derived from NIH mini inbred FFCs after treating with Scriptaid (21% v. 9%, P < 0.05), while the blastocyst rate in IVF group was 30%. Embryo transfer (ET) results showed that 5/6 (Transferred embryos No. were 190, 109, 154, 174, 152, and 190, respectively) surrogates (83%) became pregnant resulting in 2 healthy piglets from 2 litters (recipients received 190 and 154 embryos, respectively) in the Scriptaid treatment group, while no pregnancies were obtained in the untreated group from 5 ET (Embryos transferred No. are 140, 163, 161, 151 and 151, respectively). These results suggest that 500 nm Scriptaid treatment following activation increase both the in vitro and in vivo development of porcine SCNT embryos from NIH mini inbred FFCs and the hyperacetylation might actually improve reprogramming of the somatic nuclei after NT. Funding from the National Institutes of Health National Center for Research Resources RR018877.

271 EFFECT OF MARE AGE ON OOCYTE MORPHOLOGY AND DEVELOPMENTAL COMPETENCE AFTER INTRACYTOPLASMIC SPERM INJECTION

2008 ◽  
Vol 20 (1) ◽  
pp. 215
Author(s):  
J. L. Altermatt ◽  
T. K. Suh ◽  
J. E. Stokes ◽  
L. F. Campos-Chillon ◽  
E. M. Carnevale
Keyword(s):  
Embryo Development ◽  
Zona Pellucida ◽  
Intracytoplasmic Sperm Injection ◽  
Pregnancy Rates ◽  
Developmental Competence ◽  
Vitelline Membrane ◽  
Computer Assisted ◽  
Developmental Potential ◽  
Oocyte Morphology ◽  
Oocyte Developmental Competence

Reduced fertility in aged mares is associated with delayed early embryo development and lower pregnancy rates, potentially related to oocyte developmental competence. Human oocyte morphology has been associated with developmental potential, although comparative evidence is lacking in the mare. Exogenous FSH may be beneficial in obtaining more oocytes; however, effects on oocyte morphology and competence are unknown. Objectives were to determine if zona pellucida thickness (ZPT), ooplasm volume (OV), and perivitelline space volume (PVSV) were related to mare age or FSH treatment and to cleavage, blastocyst, and pregnancy rates after intracytoplasmic sperm injection (ICSI). Cycles with and without eFSH treatment were alternated; eFSH treatments began in diestrus with a cohort of follicles ≥20 mm. Oocytes were collected by transvaginal aspiration from follicles >30 mm from young (4 to 9 years) and old (>20 years) mares at 20 to 24 h after administration of recombinant eLH. Oocytes were cultured for 18 h in TCM-199 at 38.5�C in 6% CO2 in air. Sperm were injected 40 � 1 h after eLH, using frozen sperm from a single ejaculate. Presumptive zygotes were incubated in Dulbecco's modified Eagle's medium/F12 + 10% fetal calf serum at 38.5�C in 5% CO2, 5%O2, and 90% N2. Cleavage (≥2 cells) was recorded 48 h after ICSI. Blastocysts considered viable (formation before 9 d and good quality) were transferred nonsurgically into recipients 3 to 7 days after ovulation. Only pregnancies of fetuses with heart beats were included. Morphological parameters of oocytes (old, n = 40; young, n = 37) were obtained from photographic images taken at ICSI and analyzed by computer-assisted measurement using digital calipers (Spot Software, Diagnostic Instruments, Inc., Sterling Heights, MI, USA). Zona pellucida thickness was averaged from 2 measurements 90� to 180� apart. Ooplasm volume was calculated (4/3πr3) from the average of 2 diameters of the ooplasm 90� apart; and PVSV was calculated as the difference of the vitelline membrane volume and that of the volume at the inner volume of the ZP calculated as an oblate spheroid (4/3πa2b) from the average of 2 diameters. Zona pellucida thickness, OV, and PVSV were analyzed using 2-way ANOVA for main effects of age and treatment and 3-way ANOVA by adding cleavage as a factor. Zona pellucida thickness was less (P = 0.007) for old compared with young (least squares mean SEM of 11.4 � 0.2 and 12.3 � 0.2 µm, respectively) with no effect on cleavage, blastocyst, or pregnancy rates. Ooplasm volume was not different (P = 0.14) between old and young (309 036 � 5373 and 320 544 � 5639 µm3, respectively) and did not affect cleavage, blastocyst, or pregnancy rates. The PVSV was greater (P = 0.001) in old compared with young (157 505 � 10 853 and 102 161 � 11 388 µm3, respectively) and may be related to the lower cleavage (P = 0.03), blastocyst (P = 0.02), and pregnancy (P = 0.05) rates. Treatment with FSH had no effect (P > 0.1) on morphology or embryo development. In this study, ZPT and PVSV differed with mare age and could be of predictive value for oocyte developmental competence.

342 DISTINCT EFFECTS OF FOLLICULAR FLUID ON CUMULUS CELL APOPTOSIS AND PORCINE OOCYTE DEVELOPMENTAL COMPETENCE

2007 ◽  
Vol 19 (1) ◽  
pp. 286
Author(s):  
C. G. Grupen ◽  
T. S. Hussein ◽  
S. J. Schulz ◽  
D. T. Armstrong
Keyword(s):  
Oxidative Stress ◽  
Follicular Fluid ◽  
Cell Apoptosis ◽  
Polar Body ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Potent Inducer ◽  
Oocyte Developmental Competence ◽  
Cytoplasmic Maturation

Supplementing medium with follicular fluid (FF) during in vitro maturation (IVM) enhances the developmental competence of porcine oocytes, indicating that factors present in FF are beneficial to cytoplasmic maturation. Previous findings suggest that porcine FF contains high levels of superoxide dismutase activity and exerts a beneficial effect on cytoplasmic maturation by protecting oocytes from oxidative stress (Tatemoto et al. 2004 Biol. Reprod. 71, 1150–1157). Since oxidative stress is a potent inducer of apoptosis, the aim of the present study was to examine the temporal effects of FF during IVM on cumulus cell apoptosis and oocyte developmental competence. Ovaries of prepubertal pigs were collected from a local abattoir and antral follicles, 3 to 7 mm in diameter, were aspirated. Cumulus–oocyte complexes (COCs) with at least 3 uniform layers of compact cumulus cells (CCs) were recovered, washed, and transferred to maturation medium (MM) with or without 25% FF. At 22 h of IVM, COCs from each group were washed and transferred to fresh MM with or without 25% FF, forming 4 groups: -FF/-FF, -FF/+FF, +FF/-FF, and +FF/+FF. Cohorts of COCs were TUNEL stained at 22 and 44 h of IVM using the In Situ Cell Death Detection kit (Roche Diagnostics, Castle Hill, NSW, Australia) according to the manufacturer&apos;s instructions, and apoptotic CCs were visualized using confocal microscopy. Oocytes denuded at 44 h, that had a polar body, were treated with ionomycin and 6-dimethylaminopurine to induce parthenogenetic development, and were cultured for 7 days in NCSU-23 medium at 38.5&deg;C in 5&percnt; O2, 5&percnt; CO2, and 90&percnt; N2. Data were subjected to ANOVA and Tukey&apos;s post-hoc test. At 22 h of IVM, the presence of FF reduced the proportion of apoptotic CCs in COCs (2.1&percnt; vs. 4.6&percnt;). COCs matured with FF from 22 to 44 h of IVM had much lower proportions of apoptotic CCs (&plus;FF/&plus;FF: 0.9&percnt;; &minus;FF/&plus;FF: 2.6&percnt;) compared with those matured without FF (&plus;FF/&minus;FF: 10.3&percnt;; &minus;FF/&minus;FF: 17.8&percnt;). The rate of maturation to the metaphase-II stage was greater when oocytes were matured with FF from 0 to 22 h of IVM (&minus;FF/&minus;FF: 68.6&percnt;; &minus;FF/&plus;FF: 72.8&percnt;; &plus;FF/&minus;FF: 89.2&percnt;; &plus;FF/&plus;FF: 86.2&percnt;). Maturation without FF for the entire IVM interval reduced the proportion of activated oocytes that formed blastocysts compared with the other groups (&minus;FF/&minus;FF: 25.1&percnt;; &minus;FF/&plus;FF: 44.6&percnt;; &plus;FF/&minus;FF: 46.6&percnt;; &plus;FF/&plus;FF: 47.3&percnt;). Despite a 4-fold difference in the proportion of apoptotic CCs between COCs of the &plus;FF/&minus;FF and &minus;FF/&plus;FF groups, exposure to FF for the first or second half of IVM was as beneficial to oocyte developmental competence as exposure to FF for the entire IVM interval. This suggests that the protective effect of FF in reducing oxidative stress on oocytes during IVM is distinct from the effect on oocyte developmental competence.

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