Maturation, fertilisation and culture of bovine oocytes and embryos in an individually identifiable manner: a tool for studying oocyte developmental competence

The ability to successfully culture oocytes and embryos individually would facilitate the study of the relationship between follicle parameters and oocyte developmental competence, in order to identify markers of competent oocytes, as well as the ability to use small numbers of oocytes from an individual donor such as when ovum pick-up is carried out. Using a total of 3118 oocytes, the aim of the present study was to develop a system capable of supporting the development of immature bovine oocytes to the blastocyst stage in an individually identifiable manner. Initially, post-fertilisation embryo culture in the Well-of-the-Well (WOW) system, on the cell adhesive Cell-Tak or in polyester mesh was tested and shown to result in similar development to embryos cultured in standard group culture. The results demonstrate that it is possible to culture bovine oocytes to the blastocyst stage in an individually identifiable manner in all three culture systems with comparable success rates. This permits the localisation and identification of individual embryos throughout preimplantation development in vitro while retaining the developmental benefits of group culture. In terms of ease of preparation and use, culture in isolation within the strands of a polyester mesh is preferable.

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

Human Reproduction ◽

10.1093/humrep/deab130.218 ◽

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 > 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

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Oocyte Competence Biomarkers Associated With Oocyte Maturation: A Review

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.710292 ◽

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.

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

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab327 ◽

2006 ◽

Vol 18 (2) ◽

pp. 271 ◽

Cited By ~ 1

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.

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346 BOVINE EMBRYO DEVELOPMENT AFTER IVM/IVF/IVC OF OOCYTES STORED FOR 22 H IN VARIOUS MEDIA

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab346 ◽

2007 ◽

Vol 19 (1) ◽

pp. 288

Author(s):

C. Kubota ◽

T. Kojima ◽

T. Nagai ◽

X. Tian ◽

X. Yang

Keyword(s):

Subsequent Development ◽

Industrial Applications ◽

Blastocyst Stage ◽

Developmental Competence ◽

Control Group ◽

Bovine Embryo ◽

Becton Dickinson ◽

In Vitro Storage ◽

Bovine Oocytes

The timing of IVM–IVF–IVC is restricted by the onset of oocyte maturation, and sometimes oocytes must be treated at midnight. If we could regulate the timing of IVM of oocytes without decreasing their developmental competence, the IVM–IVF–IVC system could be a more applied technology. The present study was performed to examine the effects of in vitro storage of bovine oocytes in simple media prior to maturation culture to manipulate the start of IVM. Bovine follicular fluid (bFF), Dulbecco's PBS (PBS), M199 Earle salts (M199), and Earle salts supplemented with 5 mM NaHCO3 (M199A) were used as the fundamental media, after an addition of antibiotics, for in vitro storage of bovine cumulus–oocyte complexes (COCs) collected from ovaries obtained at the slaughterhouse. The fundamental media except for bFF were supplemented with 10&percnt; fetal bovine serum (FBS) or 1 mg mL−1 polyvinyl alcohol (PVA). COCs were collected from follicles (3–8 mm in diameter) and washed twice in each medium; then approximately 50 COCs were submerged in 1 mL of each medium in cryotubes (Falcon #2812, 2.5 mL; Becton Dickinson Labware, Lincoln, NJ, USA), which were stored in a container kept at 38.5°C for 22 h under air-closed condition (in vitro storage: IVS). Subsequently, the stored COCs were in vitro-matured (IVM) for 22 h in M199 with 10&percnt; FBS and 20 µg mL−1 estradiol, fertilized (IVF), and cultured in CR1aa (IVC) for examination of their development to the blastocyst stage (Kubota et al. 1998 Mol. Reprod. Dev. 51, 281–286). Fresh oocytes without IVS were used as controls. The nuclear status of oocytes after IVS–IVM was compared to that of control oocytes by aceto-orcein stain. Their developmental rates to the blastocyst stage after IVM–IVF–IVC were compared between experimental and control groups. The experiment was repeated more than 3 times, and results were statistically analyzed using Student's t-test. When bFF and PBS supplemented with FBS or PVA were used for IVS, the rates of survived COCs after IVS and the development to the blastocyst stage after IVM–IVF–IVC (bFF (n = 87): 0&percnt;, 0&percnt;; PBS/FBS (n = 72): 84&percnt;, 1&percnt;; and PBS/PVA (n = 81): 89&percnt;, 6&percnt;, respectively) were significantly lower than those of the control group (n = 406; 97&percnt; and 29&percnt;, respectively). On the other hand, when M199A supplemented with FBS or PVA was used for IVS, the survival rate after IVS and the developmental rate to the blastocyst stage after IVS–IVM–IVF (M199A/FBS (n = 97): 82&percnt;, 28&percnt;; and M199A/PVA (n = 111): 98&percnt;, 31&percnt;, respectively) did not differ from those of the control group. After IVS, cumulus expansion was not seen and most of the oocyte nuclei reached the GVBD stage. These results suggest that the nuclear maturation progress of bovine oocytes can be regulated for at least 22 h in M199A without any deleterious influence on the number of oocytes surviving at an immature state after the storage and their subsequent development to the blastocyst stage after IVM–IVF–IVC. The delayed maturation allows a flexible fertilization schedule which is advantageous in research and industrial applications.

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325RETINOID-DEPENDENT POLY(A) MRNA CONTENTS IN BOVINE OOCYTES PREMATURED AND/OR MATURED IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab325 ◽

2004 ◽

Vol 16 (2) ◽

pp. 282

Author(s):

L.J. Royo ◽

A. Rodriguez ◽

A. Gutierrez-Adan ◽

C. Diez ◽

E. Moran ◽

...

Keyword(s):

Cumulus Cells ◽

Defined Medium ◽

Developmental Competence ◽

Mrna Levels ◽

Atp Production ◽

Beneficial Effects ◽

Oocyte Developmental Competence ◽

Grant Support ◽

Bovine Oocytes

Retinoic acid (RA) can induce cell differentiation and plays a role in controlling events within the cell cycle, but little is known of RA post-transcriptional modifications in the oocyte. Bovine oocyte and cumulus cells express most of RA receptors, and the presence of 9-cis-RA during in vitro prematuration and maturation (IVM) improves oocyte developmental competence (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003 Reproduction 125, 409–416). This work analyzes the mRNA stability in bovine oocytes during in vitro prematuration and/or maturation. Cumulus-oocyte complexes (COCs) were cultured in defined medium with polyvinyl alcohol (DM). Those COCs undergoing prematuration were cultured for 24h in DM with 25μM roscovitine. For IVM, COCs were cultured in DM containing pFSH, LH and E2 for 24h, and some prematured COCs were then allowed to mature. Incubations were made at 39°C in 5% CO2 in air and high humidity. Within experiments, COCs were cultured with 5nM 9-cis-RA, in 1% ethanol (both as a vehicle and as an inhibitor of endogenous RA synthesis), 3% ethanol, 5% ethanol and untreated. Groups of 10 COCs per treatment were cultured, and oocytes detached from cumulus cells were analyzed. Poly(A) mRNA quantification was based on the pyrophosphorylation property of the DNA polymerase (Klenow). ATP production was measured by luminometric assay as a function of numbers of poly(A) tails. Data (4 replicates) were analyzed by ANOVA and Duncan’s test (v,x,y,zP<0.01; a,bP<0.05), and poly(A) mRNA (pg oocyte−1) was expressed as LSM±SE. After prematuration, poly(A) mRNA contents differed between 9-cis-RA (125.7±4.8x) and untreated (95.5±4.8y) oocytes, as compared to 1% ethanol (72.2±4.8z) and immature (71.5±4.8z) oocytes. After IVM, untreated oocytes (23.0±2.2v) showed the lowest poly(A) mRNA amount, and poly(A) mRNA in 9-cis-RA (36.2±2.2y) basically equalled that in 1% ethanol (35.2±2.2y), while 3% (44.5±2.2yz) and 5% ethanol (52.0±2.2z) increased poly(A) mRNA levels. All groups of matured oocytes showed poly(A) mRNA contents lower than in immature (71.5±4.8x). After prematuration+maturation, poly(A) mRNA values were 34.2±2.2v (untreated+untreated), 36.5±2.2v (9-cis-RA+untreated), 49.5±2.2xa (untreated+9-cis-RA), 41.0±2.2vxb (9-cis-RA+9-cis-RA) and 59.0±2.2y (untreated+1% ethanol). Levels of poly(A) mRNA from prematured+matured oocytes were again lower than in immature (71.5±4.8x). Our study shows that beneficial effects of RA on the oocyte developmental competence can be represented in part as a gain in the quality of mRNAs stored. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175).

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205.The effect of FSH concentration during IVM and gamete co-incubation length during IVF on the development of unstimulated prepubertal ewe oocytes

Reproduction Fertility and Development ◽

10.1071/srb04abs205 ◽

2004 ◽

Vol 16 (9) ◽

pp. 205 ◽

Cited By ~ 2

Author(s):

K. M. Morton ◽

W. M. C. Maxwell ◽

G. Evans

Keyword(s):

Blastocyst Stage ◽

Developmental Competence ◽

Blastocyst Formation ◽

In Vitro Development ◽

Oocyte Collection ◽

Chi Squared ◽

Development In Vitro ◽

Stage 1 ◽

Vitro Development

The developmental competence of prepubertal oocytes can be increased by the administration of gonadotrophins prior to oocyte collection (1); but this is not possible with abattoir-sourced oocytes, and modifications to the IVP system may increase in vitro development. Experiments were conducted to determine the effects of FSH concentration (10, 20 or 60 μg mL-1) during IVM (5 replicates) and gamete co-incubation length (short: 2-3 h, long: 18-20 h) during IVF (6 replicates) on subsequent embryonic development. For both experiments ovaries were collected from prepubertal lambs (16-24 weeks) slaughtered at an abattoir and embryos produced in vitro (1). Data were analysed by chi-squared test. Oocyte cleavage at 48 hours post-insemination (hpi) was higher for oocytes matured in medium containing 20 (60/77; 77.9%) and 60 (56/73; 76.7%) than 10 μg mL-1 (40/67; 59.7%) FSH. Blastocyst formation (% cultured oocytes) on Day 7 (Day 0 = IVF) was higher for oocytes matured with 20 (31/77; 40.3%) than 10 (16/67; 23.9%) or 60 μg mL-1 (20/73; 27.4%). Oocyte cleavage at 48 hpi was reduced for short (36/57; 63.2%) compared with long (49/55; 89.1%) co-incubation, although blastocyst formation (% cultured oocytes; Day 7) did not differ between groups (22/57; 38.6% and 23/55; 41.8%, respectively). These results demonstrate that increasing the FSH concentration above normal levels during IVM of prepubertal lamb oocytes improves development in vitro. Gamete co-incubation length did not influence the proportion of oocytes progressing to the blastocyst stage. (1) Morton et al. (2003) Proc. Soc. Reprod. Fert. P18.

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Impact of gonadotropins on oocyte maturation, fertilisation and developmental competence in vitro

Reproduction Fertility and Development ◽

10.1071/rd13024 ◽

2014 ◽

Vol 26 (5) ◽

pp. 752 ◽

Cited By ~ 5

Author(s):

Xuemei Wang ◽

Tony Tsai ◽

Jie Qiao ◽

Zhan Zhang ◽

Huai L. Feng

Keyword(s):

Oocyte Maturation ◽

Early Embryo ◽

Developmental Competence ◽

Dependent Manner ◽

Success Rates ◽

Maturation In Vitro ◽

High Concentrations ◽

Development In Vitro ◽

Dose Dependent

The aim of the present study was to evaluate the dose-dependent effects of gonadotropins, either singly (Bravelle (B), Luveris (L), Menupur (M), Repronex (R), Gonal-F (G), Follism (F) and Norvarel (N)) or in combination (Menupur + Bravelle; Repronext + Bravelle; and Bravelle + Norvarel), on rates of oocyte maturation, fertilisation and early embryo development in vitro in an animal model. Bovine cumulus–oocyte complexes (COCs) were purchased commercially and cultured in TCM-199 with 10% fetal bovine serum supplemented with varying concentrations of gonadotropin (0, 5, 10, 20, 40 IU or United States Pharmacopoeia (USP) mL–1) for 24 and 48 h according to current IVF clinical stimulation protocols. All gonadotropins enhanced oocyte maturation in vitro in a dose-dependent manner. Individually, Gonal-F (Merck KGaA, Darmstadt, Germany), Follism (Merck Co, Whitehouse Station, NJ, USA) and Repronext (Ferring, Parsippany, NJ, USA) promoted oocyte maturation; in combination, they effectively enhanced COC expansion and increased the maturation competence of MII oocytes. However, high concentrations of gonadotropins may result in maturation arrest. Specific combinations of gonadotropins may change the rate of early embryonic development (8–16-cells) and morula–blastocyst formation. These data provide support for the responsiveness of bovine oocytes to gonadotropins in vitro and the need to consider variations in the relative concentrations and ratio of combinations (FSH/LH or human chorionic gonadotropin) for optimisation of oocyte developmental competence. The results of the present study could be applied to therapeutic clinical stimulation protocols and help improve IVF success rates.

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94MEIOSIS INHIBITION OF BOVINE OOCYTES: EFFECTS ON SURVIVAL AFTER VITRIFICATION BY OPEN PULLED-STRAW METHOD

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab94 ◽

2004 ◽

Vol 16 (2) ◽

pp. 168

Author(s):

C. Diez ◽

M. Carbajo ◽

L. Fernandez ◽

C.O. Hidalgo ◽

S. de la Varga ◽

...

Keyword(s):

In Vitro Maturation ◽

Cumulus Cells ◽

Cell Types ◽

Blastocyst Stage ◽

Developmental Competence ◽

Bovine Oocyte ◽

17Β Estradiol ◽

Bovine Oocytes ◽

Range Test

Mammalian oocytes remain one of the most difficult cell types to successfully cryopreserve. The in vitro-maturation protocols (IVM) have a large impact on the oocyte maturation. Consequently, inhibition of meiosis has been used to improve developmental competence of oocytes without reducing blastocyst rates. Moreover, the meiotic stage influences the ability of oocytes to survive cryopreservation. This work analyzes the effect of the inhibition of meiosis (prematuration) on the freezability of the bovine oocyte. Cumulus-oocyte complexes (COCs) were recovered from slaughterhouse ovaries. Inmature oocytes (I) with compact cumulus and evenly granulated cytoplasm were selected. Prematuration (PM) was performed by incubating COCs for 22h in TCM199 NaHCO3 and roscovitine 25μM. IVM was accomplished in TCM199 NaHCO3, 10% FCS, FSH-LH and 17β-estradiol. Oocytes were subjected to 5 treatments prior the vitrification (see table). COCs were partially denuded from cumulus cells and vitrified/warmed using the OPS system (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58). Warmed oocytes were fertilized (Day 0) and presumptive zygotes having a normal morphological appearance were cultured in SOFaa+5% of FCS (Day 3); elements with degenerated appearance were discarded and recorded. Fresh oocytes submitted to IVM (c-M) or prematured and matured (c-PM+M) were fertilized and cultured as controls. Data were analyzed by ANOVA and Duncan’s multiple range test and expressed as LSM±SE. Developmental data are referred to the zygotes cultured. Only oocytes vitrified after IVM reached the blastocyst stage, but at lower rates than fresh controls. However, no differences were found between treatments at any developmental stage. Oocytes vitrified both as prematured+matured and immature oocytes showed increased proportions (P<0.01) of degenerated oocytes (37.3±5.9 and 49.9±5.9, respectively), as compared with oocytes matured before vitrification (17.6±5.9). These results show that effects induced by incubation with roscovitine (Lonergan et al. 2003 Mol. Reprod. Dev. 64, 369–378) in combination with cryodamage compromise the oocyte developmental ability. Supported by CICYT, AGL2001-379.

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207 DEVELOPMENT OF A ROUTINE IN VITRO EMBRYO PRODUCTION SYSTEM USING SINGLY IN VITRO-MATURED, FERTILIZED, AND CULTURED BOVINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab207 ◽

2009 ◽

Vol 21 (1) ◽

pp. 201

Author(s):

I. G. F. Goovaerts ◽

J. L. M. R. Leroy ◽

J. B. P. De Clercq ◽

S. Andries ◽

P. E. J. Bols

Keyword(s):

Production System ◽

Cumulus Cell ◽

Developmental Competence ◽

Control Individual ◽

Embryo Production ◽

Group Culture ◽

In Vitro Embryo Production ◽

Bovine Oocytes ◽

Hatching Rates

An in vitro embryo production system (IVP), in which a single oocyte can be tracked from the moment of retrieval up to the blastocyst stage, would be a valuable tool for studies linking developmental competence and embryo metabolism to oocyte quality and follicular environment. Unfortunately, to date, data on individual IVP are inconsistent, and in most cases show unsatisfactory blastocyst rates. Earlier studies revealed that individual culture on a cumulus cell (CC) monolayer resulted in comparable numbers of good-quality embryos as obtained after regular group culture (Goovaerts et al. 2008 Reprod. Dom. Anim. 43 (Suppl. 3), 190). Because, in the latter study, single culture was performed after group maturation and fertilization, the aim of this study was to develop and test an individual IVP system using bovine oocytes or zygotes obtained after single maturation and single fertilization. Therefore, 532 grade I COC from slaughterhouse ovaries (3 replicates) were randomly assigned to 1 of 2 treatments: a complete individual IVP protocol, or a routine group IVP as a control. Individual maturation (TCM-199 + 20% serum) and fertilization were performed in 20-μL droplets under oil in 24-well plates. Subsequently, each zygote was cultured in 20 μL of medium (SOF + 5% serum, 90% N2, 5% CO2, 5% O2) on a 6-day-old monolayer of matured CC (5% CO2 in air). Group maturation and fertilization were carried out per 100 COC in 500 μL, whereas group culture was performed per 25 zygotes in 50-μL droplets under oil. Cleavage, blastocyst, and hatching rates were assessed 2, 8, and 10 days postfertilization, respectively. Possible effects of individual and group culture were evaluated with binary logistic regression (SPSS 15.0). No interactions between replicate and treatment could be found (P > 0.05). Cleavage and blastocyst rates were significantly lower after individual IVP, compared with group IVP, whereas the blastocyst rates on cleaved zygotes and the hatching rates did not differ significantly (Table 1). In conclusion, acceptable blastocyst rates (25.1%) could be obtained after individual IVP. The lower blastocyst rates were associated with the lower cleavage rates, and no effect of the individual embryo culture system on embryo development could be found. Table 1.Cleavage, blastocyst, and hatching rates after individual and group in vitro embryo production (IVP)

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86 IN VITRO-MATURED GILT OOCYTES CAN HAVE EQUAL OR BETTER DEVELOPMENTAL COMPETENCE THAN SOW OOCYTES WITH NEW MATURATION MEDIA

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab86 ◽

2017 ◽

Vol 29 (1) ◽

pp. 150 ◽

Cited By ~ 2

Author(s):

L. D. Spate ◽

S. L. Murphy ◽

J. A. Benne ◽

A. Giraldo ◽

D. Hylan ◽

...

Keyword(s):

Growth Factor ◽

Somatic Cell ◽

Culture Media ◽

Blastocyst Stage ◽

Developmental Competence ◽

Blastocyst Development ◽

Development In Vitro ◽

Humidified Air

It has long been thought that oocytes obtained from sows yielded a higher level of developmental competence compared with oocytes obtained from prepubertal gilts. Because gilt-derived oocytes are more readily available to our laboratory and they are less developmentally competent, we hypothesised that by making alterations to our maturation system we could improve the developmental competence of the gilt-derived oocytes to that of their sow-derived counterparts. We performed 2 experiments that evaluated the ability of each source of oocyte to develop to the blastocyst stage, using altered maturation media. The first experiment focused on the developmental ability of each source of oocytes, through IVF and culture. The second experiment again focused on the developmental competence of each oocyte source but through somatic cell NT. For both experiments, the sow-derived oocytes were obtained from Desoto Biosciences and the gilt ovaries were collected from Smithfield Inc. in Milan, Missouri. Both sets of oocytes were in vitro matured in M199 supplemented with 0.57 mM cysteine, 5 μg mL−1 LH and FSH, and 10 ng mL−1 epidermal growth factor; however, the gilt derived media was altered to contain 40 ng mL−1 fibroblast growth factor 2 and 20 ng mL−1 insulin-like growth factor and leukemia inhibitory factor. Additionally, the maturation media for the sow-derived oocytes contained the addition of 5 μg mL−1 insulin and 10% follicular fluid. In the first experiment we performed IVF on oocytes from the 2 sources as per our laboratory standard IVF procedure, co-incubating the oocytes with 0.25 × 106 porcine semen for 4 h, followed by washing and moving the oocytes to MU2 culture media at 38.50°C in 5% CO2, humidified air overnight. After overnight culture the presumptive zygotes were transferred to the same conditions with 5% CO2, 5% O2, and 90% N2. After an additional 5 days, blastocyst development was assessed. The gilt oocytes yielded 39.3a ± 7.2% blastocyst, and the sow oocytes had a blastocyst rate of 24.9b ± 6.9%, with an n of 389 and 313, respectfully. Statistical analysis was performed by using Genmod in SAS 9.4. In the second experiment, using standard laboratory protocol for somatic cell NT, we activated both sets of oocytes with 200 μM thimerosal for 10 min followed by 30-min incubation with 4 mM dithiothreitol. The embryos were co-incubated for 15 h with 500 nM Scriptaid in the MU2 culture media in 5% CO2, humidified air; then these embryos were also moved to 5% CO2, 5% O2, and 90% N2 and cultured to Day 6. The sow oocytes produced a blastocyst percentage of 38.6%, and the gilt oocyte group had a blastocyst percentage of 43.5%, with an n of 290 and 285, respectfully. There was no difference statistically between these treatments. Both gilt and the sow oocyte sources have yielded live piglets at this time. We concluded that the maturation system used for our gilt-derived oocytes resulted in equal or better development in vitro compared with the sow-derived oocytes. Follow-up experiments evaluating in vivo development are needed for a complete comparison. This work was funded by Food for the 21st Century University of MO, and the NIH U42OD011140.

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