The effects of permeating cryoprotectants on intracellular free-calcium concentrations and developmental potential of in vitro-matured feline oocytes

2016 ◽  
Vol 28 (5) ◽  
pp. 599 ◽  
Author(s):  
Jason R. Herrick ◽  
Chunmin Wang ◽  
Zoltan Machaty
Keyword(s):  
Enzymatic Digestion ◽  
Blastocyst Stage ◽  
Intracellular Free Calcium ◽  
In Vitro Fertilisation ◽  
Free Calcium ◽  
Blastocyst Development ◽  
Oocyte Vitrification ◽  
Developmental Potential ◽  
Parthenogenetic Activation

Embryos produced from vitrified feline oocytes have resulted in pregnancies, but the efficiency of oocyte vitrification in cats is still low. Our objectives were to evaluate the effects of exposing feline oocytes to ethylene glycol (EG), propanediol (PrOH) and dimethyl sulfoxide (DMSO) on changes in intracellular free-calcium concentrations ([Ca2+]i), the time needed for enzymatic digestion of the zona pellucida (ZP), the incidence of parthenogenetic activation and degeneration and embryonic development following in vitro fertilisation (IVF). All of the chemicals tested altered [Ca2+]i, but changes in [Ca2+]i, resistance of the ZP to enzymatic digestion and the incidence of parthenogenetic activation (<5% for all treatments) were not affected (P > 0.05) by extracellular Ca2+. Exposure to EG (>44.1%) and DMSO (19.7%) increased (P < 0.05) oocyte degeneration compared with control oocytes and oocytes exposed to PrOH (≤2.5%). Following exposure to a combination of PrOH and DMSO (10% v/v each), blastocyst development (per cleaved embryo; 52.1%) was similar (P > 0.05) to control oocytes (64.4%). When oocytes were vitrified with PrOH and DMSO, 28.3% of surviving (intact plasma membrane) oocytes cleaved following IVF, but no blastocyst developed. When a non-permeating cryoprotectant (galactose, 0.25 M) was added to the vitrification medium, 47.7% of surviving oocytes cleaved and 14.3% developed to the blastocyst stage.

scholarly journals 324SPHINGOSINE-1-PHOSPHATE PROTECTS CULTURED BOVINE OOCYTES FROM PHYSIOLOGICALLY RELEVANT THERMAL STRESS

2004 ◽  
Vol 16 (2) ◽  
pp. 282 ◽  
Author(s):  
Z. Roth ◽  
P.J. Hansen
Keyword(s):  
Thermal Stress ◽  
Heat Shock ◽  
Blastocyst Stage ◽  
Caspase Activity ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
Oocyte Competence ◽  
Cell Death Pathway

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that can block the sphingomyelin cell-death pathway by suppressing ceramide-induced apoptosis. The present study was performed to test whether S1P protects oocytes from heat shock during in vitro maturation. Cumulus-oocyte complexes obtained by slicing follicles were placed in maturation medium with or without 50nM S1P and cultured at 38.5°C (CON) or 41°C (41C) for the first 12h of maturation. Incubation during the last 10h of maturation (22-h total maturation time), fertilization, and embryonic development were performed at 38.5°C and 5% (v/v) CO2. Blastocyst development was recorded at 8 days post-insemination (dpi) and activity of group II caspases in 8-day blastocysts was determined using a fluoroprobe, PhiPhiLux-G1D2 (OncoImmunin, Gaithersburg, MD, USA). Data were analysed by least-squares ANOVA with the GLM procedure of SAS. Percentage data were subjected to arcsin transformation before analysis. Exposure of oocytes to thermal stress during the first 12h of maturation reduced cleavage rate (P&lt;0.01) and the number of oocytes developing to the blastocyst stage (P&lt;0.04). There was a temperature x S1P interaction for cleavage rate (P&lt;0.03) because S1P blocked effects of thermal stress on cleavage rate. Without S1P, the percentage of oocytes that cleaved by 3 dpi were 83.6±2.7% and 65.8±2.7% for CON and 41C, respectively. In the presence of S1P, percent cleavage was 86.7±2.7% and 83.9±2.7% for CON and 41C, respectively. There was a trend (P=0.06) for a temperature x S1P interaction for percent oocytes developing to blastocyst stage because S1P blocked effects of heat shock on development. Without S1P, the percentages of oocytes that developed to the blastocyst stage were 28.7±3.0% and 15.2±3.0% for CON and 41C, respectively. In the presence of S1P, percent blastocysts were 24.3±3.4% and 23.9±3.0% for CON and 41C, respectively. When development was expressed as percentage of cleaved embryos, however, there were no effects of temperature, S1P, or temperature x S1P on percent development to the blastocyst stage. Blastocyst caspase activity was not affected by temperature or S1P. In summary, exposure to physiologically relevant thermal stress during the first 12h of maturation has a deleterious effect on oocyte competence and this effect can be reduced by S1P. The fact that heat shock reduced the percentage of oocytes but not the percentage of cleaved embryos that became blastocysts suggests that oocytes that survive effects of heat shock and cleave have normal potential to develop to the blastocyst stage. Moreover, since heat shock did not affect caspase activity, it is likely that blastocysts from heat-shocked oocytes have normal developmental potential, at least as determined by caspase activity. Support: BARD FI-330-2002 and USDA Grants 2002-35203-12664 and 2001-52101-11318.

Tolerance of lamb and mouse oocytes to cryoprotectants during vitrification

Zygote ◽  
2018 ◽  
Vol 27 (1) ◽  
pp. 36-45
Author(s):  
Jaqueline Sudiman ◽  
Alice Lee ◽  
Kheng Ling Ong ◽  
Wu Zi Yuan ◽  
Sarah Jansen ◽  
...  
Keyword(s):  
Cell Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cortical Granule ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Parthenogenetic Activation ◽  
Mouse Oocytes ◽  
Different Response

SummaryMouse and lamb oocytes were vitrified with, or exposed to, different cryoprotectants and evaluated for their effects on their survival and developmental competence after in vitro fertilization (IVF) and activation treatments. Control oocytes remained untreated, whilst the remainder were exposed to three different combinations of vitrification solutions [dimethyl sulfoxide (DMSO) + ethylene glycol (EG), EG only, or propanediol (PROH) + EG] and either vitrified or left unfrozen (exposed groups). Oocytes in the control and vitrified groups underwent IVF and developmental competence was assessed to the blastocyst stage. In lambs, survival rate in vitrified oocytes was significantly lower than for oocytes in the exposed groups (P <0.05). Blastocyst development was low in vitrified oocytes compared with controls (<6% vs 38.9%, P <0.01). Parthenogenetic activation was more prevalent in vitrified lamb oocytes compared with controls (P <0.05). No evidence of zona pellucida hardening or cortical granule exocytosis could account for reduced fertilization rates in vitrified lamb oocytes. Mouse oocytes demonstrated a completely different response to lamb oocytes, with survival and parthenogenetic activation rates unaffected by the vitrification process. Treatment of mouse oocytes with DMSO + EG yielded significantly higher survival and cleavage rates than treatment with PROH + EG (87.8% and 51.7% vs 32.7% and 16.7% respectively, P <0.01), however cleavage rate for vitrified oocytes remained lower than for the controls (51.7% vs 91.7%, P <0.01) as did mean blastocyst cell number (33 ± 3.1 vs 42 ± 1.5, P <0.05). From this study, it is clear that lamb and mouse show different tolerances to cryoprotectants commonly used in vitrification procedures, and careful selection and testing of species-compatible cryoprotectants is required when vitrifying oocytes to optimize survival and embryo development.

scholarly journals Morphometric, subcellular, in vitro fertilisation and embryonic developmental assessment of mouse oocytes produced by anti-inhibin serum or pregnant mare serum gonadotrophin superovulation

2020 ◽  
Vol 32 (5) ◽  
pp. 474
Author(s):  
Liga Wuri ◽  
Cansu Agca ◽  
Yuksel Agca
Keyword(s):  
Fluorescence Intensity ◽  
Morphological Characteristics ◽  
In Vitro Fertilisation ◽  
Oocyte Diameter ◽  
Cortical Granules ◽  
Blastocyst Development ◽  
Developmental Potential ◽  
Mouse Oocytes ◽  
Pregnant Mare Serum Gonadotrophin

This study compared the morphometric, subcellular characteristics, in vitro fertilisation (IVF) and embryonic developmental potential of metaphase II (MII) mouse oocytes obtained from females superovulated with either anti-inhibin serum–human chorionic gonadotrophin (AIS-hCG) or pregnant mare serum gonadotrophin (PMSG)-hCG. The oocyte’s quantity, quality, zona pellucida (ZP) thickness, perivitelline space (PVS), diameter, microtubules, F-actin, cortical granules (CGs) and mitochondrial distribution were determined. Superovulation using AIS-hCG resulted in a higher numbers of oocyte/donor compared with PMSG-hCG (P=0.002). There was no difference in morphologically normal and abnormal oocytes between AIS-hCG and PMSG-hCG (P=0.425 and P=0.194, respectively). The morphometric measurements showed no difference in oocyte diameter between AIS-hCG and PMSG-hCG (P=0.289). However, the thickness of the ZP of oocytes from AIS-hCG females was decreased compared with PMSG-hCG (P&lt;0.001). The PVS of oocytes from the AIS-hCG was larger than with PMSG-hCG (P&lt;0.001). The microtubules of oocytes from both AIS-hCG and PMSG-hCG were normal, although there was an increased fluorescence intensity in the AIS-hCG oocytes (P&lt;0.001). The F-actin and CGs distribution in oocytes from both AIS-hCG and PMSG-hCG were similar (P=0.330 and P=0.13, respectively). Although the oocytes from PMSG-hCG females had homogenously distributed mitochondria, AIS-hCG oocytes showed more peripheral distribution with no differences in fluorescence intensity (P=0.137). The blastocyst development rates after IVF with fresh sperm showed no difference between AIS-hCG and PMSG-hCG (P=0.235). These data suggested that AIS-hCG superovulation produces high numbers of morphologically normal oocytes that also possess normal subcellular structures, good morphological characteristics and had high invitro embryonic developmental potential.

Comparison of glucose metabolism in in vivo- and in vitro-matured tammar wallaby oocytes and its relationship to developmental potential following intracytoplasmic sperm injection

2004 ◽  
Vol 16 (6) ◽  
pp. 617 ◽  
Author(s):  
Genevieve M. Magarey ◽  
Karen E. Mate
Keyword(s):  
Glucose Metabolism ◽  
Intracytoplasmic Sperm Injection ◽  
Tammar Wallaby ◽  
Blastocyst Stage ◽  
In Vitro Fertilisation ◽  
Cell Stage ◽  
Developmental Potential ◽  
Developmental Capacity

Although marsupial oocytes undergo nuclear maturation in vitro, there is, at present, no indication of their developmental potential, largely owing to the lack of in vitro fertilisation and related technologies for marsupials. Glucose metabolism has proven a useful indicator of oocyte cytoplasmic maturation and developmental potential in several eutherian species. Therefore, the aims of the present study were to compare: (1) the rates of glycolysis and glucose oxidation in immature, in vitro-matured and in vivo-matured tammar wallaby oocytes; and (2) the metabolic rate of individual oocytes with their ability to form pronuclei after intracytoplasmic sperm injection. The rates of glycolysis measured in immature (2.18 pmol oocyte–1 h–1), in vitro-matured (0.93 pmol oocyte–1 h–1) and in vivo-matured tammar wallaby oocytes (0.54 pmol oocyte–1 h–1) were within a similar range to values obtained in eutherian species. However, unlike the trend observed in eutherian oocytes, the glycolytic rate was significantly higher in immature oocytes compared with either in vivo- or in vitro-matured oocytes (P < 0.001) and significantly higher in in vitro-matured oocytes compared with in vivo-matured oocytes (P < 0.001). No relationship was identified between glucose metabolism and the developmental capacity of oocytes after intracytoplasmic sperm injection when assessed after 17–19 h. Oocytes that became fertilised (two pronuclei) or activated (one or more pronucleus) were not distinguished from others by their metabolic rates. Longer culture after intracytoplasmic sperm injection (e.g. blastocyst stage) may show oocyte glucose metabolism to be predictive of developmental potential; however, culture to the single-cell stage did not reveal any significant differences in normally developing embryos.

scholarly journals 275FOLLICULAR ATRESIA IN SMALL, NON-FSH-DEPENDENT BOVINE FOLLICLES IS ASSOCIATED WITH INCREASED DEVELOPMENTAL POTENTIAL OF OOCYTES FOLLOWING IVP

2004 ◽  
Vol 16 (2) ◽  
pp. 258
Author(s):  
H. Irving-Rodgers ◽  
S. Morris ◽  
R. Collett ◽  
K. Catanzariti ◽  
T. Peura ◽  
...  
Keyword(s):  
Basal Lamina ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Early Cleavage ◽  
Blastocyst Development ◽  
Chi Square ◽  
Developmental Potential ◽  
Chi Square Analysis

Oocytes from small, non-FSH-dependent follicles are associated with reduced developmental competence following in vitro embryo production (IVP) compared to oocytes from larger follicles. It has been suggested that, for small follicles, oocytes derived from atretic follicles are more developmentally competent than those from healthy follicles (Blondin P and Sirard MA, 1995 Mol. Reprod. Dev. 41, 54–62). Little is known of the characteristics of small follicles that support developmentally competent oocytes. Here we examine the development to blastocyst stage of oocytes collected from histologically-assessed bovine 2–5mm follicles. Ovaries were obtained at a local abattoir;; 4 follicles were dissected from each ovary and oocytes were recovered. A section of each follicle wall was taken and fixed in 2.5% glutaraldehyde for histological assessment of the follicle and characterization of the morphology of the follicular basal lamina by electron microscopy (Irving-Rodgers HF and Rodgers RJ, 2000 J. Reprod. Fert. 118, 221–228). Oocytes recovered from follicles underwent IVP utilizing a novel single IVP system. Oocytes were matured for 24h (10μL per COC) in TCM199, supplemented with FSH, hCG, FCS, cysteamine and pyruvate. Mature oocytes were inseminated with 1×106 sperm mL−1 for an additional 24h using Bovine Fertilization Medium (10μL per COC;; Cook, Australia). Following insemination, putative zygotes were stripped of remaining cells and placed within individual micro-wells prepared in 1% agar in Bovine Early Cleavage Medium, Cook, Australia. The agar (350μL) was prepared within wells of a 4-well plate and small plugs of agar were removed to form micro-wells. The agar was over-laid with 450μL of Early Cleavage Medium and 250μL mineral oil, and equilibrated overnight before putative zygotes were placed individually within micro-wells. Culture was performed under 7% O2, 6% CO2, and 87% N2 at 39°C. On Day 5 following insemination, fetal calf serum (final concentration 10% v/v) was added to facilitate blastocyst development. Blastocyst formation was assessed on Day 8. A total of 211 oocytes were cultured and 69% were from healthy follicles;; 67 oocytes (32%) had developed to the blastocyst stage by Day 8. Forty-three percent of oocytes recovered from atretic follicles (28/65) had developed to the blastocyst stage by Day 8, as compared to only 27% (39/146) oocytes recovered from healthy follicles, this difference was significant (P&lt;0.05, chi-square analysis). Seventy-eight percent (14/18) of oocytes from healthy follicles with additional follicular basal lamina material (Irging-Rodgers HF and Rodgers RJ, 2000 J. Reprod. Fert. 118, 221–228) failed to develop, whereas only 44% (4/9) of oocytes from healthy follicles with a normal basal lamina failed to develop (P&gt;0.08). The present study finds a direct association between the follicle morphology and oocyte maturational potential within non-FSH dependent follicles, revealing that high levels of development (&gt;40%) can be obtained from atretic follicles. Furthermore, differences between healthy follicles may also contribute to developmental variation.

Full term development of normal mice after transfer of IVF embryos derived from oocytes stored at room temperature for 1 day

Zygote ◽  
2008 ◽  
Vol 16 (1) ◽  
pp. 21-27 ◽  
Author(s):  
Zi-Li Lei ◽  
Jun-Cheng Huang ◽  
Li-Hong Shi ◽  
Yi-Liang Miao ◽  
Chang-Long Nan ◽  
...  
Keyword(s):  
Mouse Embryo ◽  
Room Temperature ◽  
Blastocyst Stage ◽  
Developmental Potential ◽  
Parthenogenetic Activation ◽  
Practical Procedure ◽  
Mouse Embryo Development ◽  
17Β Estradiol ◽  
Healthy Growth

SummaryEarly studies have shown that some mouse cumulus–oocyte complexes (COCs) stored at room temperature for 24 h still retained full developmental potential. In this study, we stored denuded mouse oocytes (DOs) at room temperature (25 °C) for 24 h and activated these oocytes with 10 mM SrCl2 or fertilized the oocytes by IVF. We found that nearly half of the DOs stored at room temperature for 1 day can be fertilized normally by IVF and that two foster mothers gave birth to seven pups. Embryos from stored oocytes were cultured in CZB medium with or without 1 μg/ml 17β-estradiol (E2). The numbers of embryo that developed to morula/blastocyst stage after parthenogenetic activation and IVF were significantly increased when E2 was added to the culture (p < 0.05). These results suggest that E2 might improve mouse embryo development in vitro. The birth of seven agouti pups and their healthy growth indicated that the storage of DOs at room temperature for 1 day may be a practical procedure for mammalian reproduction.

Reduced competence of immature and mature oocytes vitrified by Cryotop method: assessment by in vitro fertilization and parthenogenetic activation in a bovine model

Zygote ◽  
2017 ◽  
Vol 25 (2) ◽  
pp. 222-230 ◽  
Author(s):  
Daiane L. Bulgarelli ◽  
Alessandra A. Vireque ◽  
Caroline P. Pitangui-Molina ◽  
Marcos F. Silva-de-Sá ◽  
Ana Carolina J. de Sá Rosa-e-Silva
Keyword(s):  
Embryo Development ◽  
Germinal Vesicle ◽  
Control Group ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Oocyte Vitrification ◽  
Vitro Fertilization ◽  
Parthenogenetic Activation ◽  
Nuclear Maturation

SummaryThis study aimed to evaluate the embryo development competence, the nuclear maturation and the viability of germinal vesicle (GV) and metaphase II (MII) oocytes vitrified by the Cryotop method. Cumulus–oocyte complexes were derived from bovine ovaries and three experiments were conducted. In Experiment 1, GV oocytes were vitrified and underwent in vitro maturation (IVM) or not and their nuclear maturation was assessed by orcein staining. In Experiment 2, GV oocytes and MII oocytes were vitrified or not and the viability was assessed by calcein/ethidium homodimer-1 staining. In Experiment 3, MII oocytes matured before or after vitrification were submitted to in vitro fertilization (IVF) and parthenogenetic activation (PA) in order to evaluate embryo development. No difference was found for the nuclear maturation rate in the GV group (50%) and the GV control group (67%; P = 0.23) and for viability rate (56%; 77%; P = 0.055, respectively). However, in the MII group (27%) viability was significantly lower than that of the MII control group (84%; P < 0.0001). The cleavage rate by IVF and PA was similar in the GV group and the MII group. In contrast, vitrified MII oocytes showed no capacity for blastocyst development after IVF or PA and vitrified GV oocytes were able to develop to blastocysts only after PA, but not after IVF. In conclusion, oocyte vitrification by the Cryotop method reduced the capacity for embryo development. Vitrification of GV oocytes, however, did not influence the capacity of meiotic nuclear maturation and they exhibited higher viability following vitrification at the MII stage.

P–133 Monopronuclear (1PN) embryos can derive in healthy pregnancies

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
E Brinkmann ◽  
C Demmers. va. d. Werken ◽  
L Ramos
Keyword(s):  
Live Birth Rate ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
In Vitro Fertilisation ◽  
Developmental Potential ◽  
Increased Risk ◽  
Registration Number ◽  
The Right

Abstract Study question Should 1PN embryos be considered suitable for transfer when normal development is observed at day 3 or day 5? Summary answer In IVF/ICSI cycles, 1PN zygotes are encountered in 2.7% of inseminated oocytes. Transfer of 1PN-embryos should be considered in the absence of suitable 2PN embryos. What is known already During in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) zygotes containing only a single pronucleus (monopronuclear, 1PN) are encountered in 1–7.7% of cases, while the display of two pronuclei is expected in a normally fertilised oocyte. A 1PN zygote can be of gynogenetic or androgenetic origin, but it can also be biparental. Gynogenetic and androgenetic 1PN embryos can be haploid or diploid, so a diploid 1PN embryo is not guaranteed to be normally fertilised. Generally, 1PN are discarded, as they have an increased risk for aneuploidy. However, sporadically they can develop into healthyl babies. Study design, size, duration 1PN-zygotes (n = 1287, 2.7% from all inseminated oocytes) from 1–1–2016 up to 15–12–2020 were retrospectively evaluated. The development and fate (discarded/transferred/cryopreserved) of all embryos were recorded. Embryos were evaluated at day 2, 3 or 5 of development. The policy of our unit is that, in absence of 2PN embryos, normal developed 1PN-embryos can be transferred on day 3. Supernumerary 1PN embryos can be cryopreserved at blastocyst stage. Ongoing pregnancies from fresh embryo transfers (ET) were analysed. Participants/materials, setting, methods In 946 IVF/ICSI cycles, at least one 1PN zygote was observed (total 1287 embryos). ICSI with ejaculated, PESA or TESE sperm counted for a total of 795 embryos, IVF cycles for 494 embryos. Embryo evaluation was performed using a home-made numerical algorithm: A (top embryo; 150–200 points), B (regular embryo; 100–149 points) or C (poor embryo; 0–99 points). Monopronuclear embryos always scored lower than equal developed 2PN embryos. Blastocyst evaluation was according to Gardner score. Main results and the role of chance From the 795 ICSI embryos, 49 (6.1%) were used for fresh ET (26 scored quality A or B), and a total of 60 embryos developed to blastocyst and were cryopreserved. From these 49 ICSI transfers, 4 (8.1%) ongoing pregnancies were obtained, all 4 from DET (1PN+2PN embryo), from which one twin pregnancy was confirmed. From the 494 IVF embryos, 41 (8.3%) were used for fresh ET (24 scored A or B), and 62 blastocysts were cryopreserved. A total of 9/41 (22%) ongoing pregnancies were obtained: 5 from SET (1PN) and 4 from DET (1PN+ 2PN embryo). Therefore, in only five IVF cycles a confirmed pregnancy was observed from a 1PN embryo (all A-quality embryos). Considering six ongoing pregnancies with complete certainty of monopronuclear origin from fresh tranfers could be confirmed from our retrospective data, we can conclude that although the live birth rate of these embryos is very low (around 0,5- 1.0%), they should not be discarded when development is normal and no dipronuclear embryos are present. Limitations, reasons for caution Cryo-thawing data is missing as these embryos were not differentially marked at freezing. Therefore, the cumulative pregnancies from monopronuclear embryos could be higher. Embryos were not evaluated in a time lapse system, so asynchronicity of PN formation could explain missing the right moment for evaluation, while normal fertilized. Wider implications of the findings: Notably, IVF monopronuclear embryos display a higher developmental potential than those derived from ICSI. We suggest that, in absence of dipronuclear embryos, culture to blastocyst stage before considering fresh ET or cryopreservation will help differentiate viable 1PN embryos, reducing the higher chance of genetic anomalies and miscarriages. Trial registration number N.A.

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 160EFFECTS OF EXTRACELLULAR ION CONCENTRATIONS ON IN VITRO DEVELOPMENT OF DOMESTIC CAT IVF EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 202 ◽  
Author(s):  
W.F. Swanson ◽  
A.L. Manharth ◽  
J.B. Bond ◽  
H.L. Bateman ◽  
R.L. Krisher ◽  
...  
Keyword(s):  
Culture Media ◽  
Ionic Composition ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Vitro Development

Domestic cat embryos typically are cultured in media formulated for somatic cells or embryos from rodents or livestock species. Under these conditions, blastocyst development has been inconsistent and delayed relative to embryos grown in vivo, and embryo viability following transfer has been low. Our goal is to systematically define the culture requirements of the feline embryo to improve embryo development and viability. The objective of this study was to determine the ionic (NaCl, KCl, KH2PO4, and CaCl2:MgSO4) preferences of domestic cat IVF embryos. Anestral female cats were injected (i.m.) with 150IU eCG followed 84h later by 100IUhCG. Oocytes were recovered via laparoscopic follicular aspiration approximately 24h post-hCG injection (Day 0). Semen was collected from one of two males by means of an artificial vagina and washed once in HEPES-buffered IVF medium. Mature cumulus-oocyte complexes were co-incubated with 2.5–5×105 motile sperm mL−1 in IVF medium (100mM NaCl, 4.0mM KCl, 1.0mM KH2 PO4, 2.0mM CaCl2, 1.0mM MgSO4-7H2O, 25.0mM NaHCO3, 3.0mM glucose, 0.1mM pyruvate, 6.0mM L-lactate, 1.0mM glutamine, 0.1mM taurine, 1×MEM nonessential amino acids, 50μgmL−1 gentamicin, and 4.0mgmL−1 BSA) for 19 to 22h in 6% CO2 in air (38.7°C). Cumulus cells were removed and embryos cultured (8–11 embryos/50μL drop; 6% CO2, 5% O2, 89% N2, 38.7°C) in media containing 100.0 or 120.0mM NaCl, 4.0 or 8.0mM KCl, 0.25 or 1.0mM KH2PO4, and 1.0mM:2.0mM or 2.0mM:1.0mM CaCl2:MgSO4 (2×2×2×2 factorial design). The remaining components of the culture medium were identical to the IVF medium (but w/o gentamicin). Development to the blastocyst stage by Day 6, metabolism (glycolysis and pyruvate) of each blastocyst, and final cell number (Hoechst 33342 staining) of all embryos were evaluated. Final cell number of cleaved embryos and development to the blastocyst stage were analyzed using analysis of variance in the GLIMMIX macro of SAS. A total of 236 oocytes were inseminated, yielding 128 cleaved embryos (54%), including 6 blastocysts (4.7% of cleaved embryos). Cell number was not (P&gt;0.05) affected by NaCl, KCl, or KH2PO4 concentrations, but tended (P=0.057) to be higher after culture in 2.0mM:1.0mM CaCl2:MgSO4. Treatments did not significantly affect (P&gt;0.05) development to the blastocyst stage, but numerically more blastocysts were produced in 100.0mM NaCl (4/6), 8.0mM KCl (5/6), or 1.0mM KH2PO4 (5/6). Both CaCl2:MgSO4 ratios resulted in 3 blastocysts. Blastocysts contained 61.08±5.1 (mean±SEM, n=6) cells and actively metabolized glucose (glycolysis, 3.7±0.8pmol/embryo/3h or 0.06±0.01pmol/cell/3h) and pyruvate (0.75±0.27pmol/embryo/3h or 0.013±0.005pmol/cell/3h). These results suggest that the ionic composition of culture media influences the in vitro development of cat IVF embryos. (Supported by NIH grant RR15388.)

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