Effect of hexoses and gonadotrophin supplementation on bovine oocyte nuclear maturation during in vitro maturation in a synthetic follicle fluid medium

2005 ◽  
Vol 17 (4) ◽  
pp. 407 ◽  
Author(s):  
Melanie L. Sutton-McDowall ◽  
Robert B. Gilchrist ◽  
Jeremy G. Thompson
Keyword(s):  
Polar Body ◽  
Culture Conditions ◽  
Fluid Medium ◽  
Nuclear Maturation ◽  
Meiotic Progression ◽  
Developmental Capacity ◽  
Polar Body Formation ◽  
Bovine Oocytes

In vitro oocyte maturation (IVM) culture conditions have been relatively unchanged over the past few decades and remain suboptimal. In contrast, studies of the in vivo environment have led to significant improvements to in vitro embryo culture technologies. The aim of the present study was to determine the effect of maturing bovine cumulus–oocyte complexes (COCs) in medium based on the composition of bovine follicular fluid (Bovine VitroMat; Cook Australia, Eight Mile Plain, Qld, Australia). In particular, the effect of different glucose concentrations and glucosamine supplementation on meiotic maturation was determined. Culturing COCs in the presence of gonadotrophins in Bovine VitroMat, containing either physiological glucose concentrations (2.3 mm) or 5.6 mm (equivalent to levels in Tissue Culture Medium 199 (TCM199)) supplemented with glucosamine resulted in comparable cumulus expansion to COCs cultured in TCM199 plus gonadotrophins. However, nuclear maturation was 1.3-fold lower in Bovine VitroMat cultures containing 2.3 mm glucose compared with 5.6 mm glucose and this effect was independent of glucosamine supplementation. Investigations into the effects of different glucose concentrations and gonadotrophin supplementation during the initial 6 h of maturation demonstrated that COCs cultured in Bovine VitroMat with 5.6 mm glucose without gonadotrophins had a twofold acceleration of the rate of meiotic resumption, yet the rate of polar body formation was decreased by approximately 20% compared with cultures in 2.3 mm glucose and TCM199. However, this effect was not seen when COCs were cultured for the initial 16 h in Bovine VitroMat + 5.6 mm minus gonadotrophins or in Bovine VitroMat + 2.3 mm glucose ± gonadotrophins. These data demonstrate that glucose concentrations and the timing of the introduction of gonadotrophin during IVM have variable effects on nuclear maturation. Manipulation of glucose concentrations may be a mechanism to influence oocyte meiotic progression and may lead to the development of improved IVM systems, allowing for an increased developmental capacity of bovine oocytes.

scholarly journals The presence of acylated ghrelin duringin vitromaturation of bovine oocytes induces cumulus cell DNA damage and apoptosis, and impairs early embryo development

Zygote ◽  
2017 ◽  
Vol 25 (5) ◽  
pp. 601-611 ◽  
Author(s):  
Matias A. Sirini ◽  
Juan Mateo Anchordoquy ◽  
Juan Patricio Anchordoquy ◽  
Ana M. Pascua ◽  
Noelia Nikoloff ◽  
...  
Keyword(s):  
Dna Damage ◽  
Embryo Quality ◽  
Cumulus Cell ◽  
Early Embryo ◽  
Acylated Ghrelin ◽  
Cumulus Expansion ◽  
Nuclear Maturation ◽  
Developmental Capacity ◽  
Bovine Oocytes

SummaryThe aim of this study was to investigate the effects of acylated ghrelin supplementation duringin vitromaturation (IVM) of bovine oocytes. IVM medium was supplemented with 20, 40 or 60 pM acylated ghrelin concentrations. Cumulus expansion area and oocyte nuclear maturation were studied as maturation parameters. Cumulus–oocyte complexes (COC) were assessed with the comet, apoptosis and viability assays. Thein vitroeffects of acylated ghrelin on embryo developmental capacity and embryo quality were also evaluated. Results demonstrated that acylated ghrelin did not affect oocyte nuclear maturation and cumulus expansion area. However, it induced cumulus cell (CC) death, apoptosis and DNA damage. The damage increased as a function of the concentration employed. Additionally, the percentages of blastocyst yield, hatching and embryo quality decreased with all acylated ghrelin concentrations tested. Our study highlights the importance of acylated ghrelin in bovine reproduction, suggesting that this metabolic hormone could function as a signal that prevents the progress to reproductive processes.

44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS

2011 ◽  
Vol 23 (1) ◽  
pp. 128
Author(s):  
J. Lee ◽  
J. Park ◽  
Y. Chun ◽  
W. Lee ◽  
K. Song
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Skin Fibroblasts ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.

228 microRNA REGULATION OF GENES IN BOVINE OOCYTES AND EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 272
Author(s):  
J. P. Barfield ◽  
G. J. Bouma ◽  
G. E. Seidel Jr
Keyword(s):  
Mirna Expression ◽  
Polar Body ◽  
Prostaglandin F2α ◽  
Defined Medium ◽  
Tight Junction Formation ◽  
First Polar Body ◽  
Lysis Buffer ◽  
Bovine Oocytes

Little is known about expression of microRNA (miRNA) in bovine oocytes and pre-implantation embryos. These molecules likely have an important role in regulating development. For example, differences in quality of oocytes matured in vivo v. in vitro might be due, in part, to altered miRNA expression. In Experiment 1, in vivo-matured COC were collected by transvaginal aspiration of 7 superstimulated cows 21 to 23 h after GnRH injection, given 48 h after prostaglandin F2α and the last of 6 FSH injections given b.i.d. Oocytes aspirated from abattoir ovaries were matured in vitro for 23 h in a chemically defined medium. After vortexing, maturation of both groups of oocytes was confirmed by visualization of the first polar body, and oocytes were snap frozen in mirVana lysis buffer (Applied Biosciences, Foster City, CA, USA). In Experiment 2, in vitro-matured oocytes were generated as described. Subsets were fertilized in vitro or activated parthenogenetically by incubation in 5-μM ionomycin for 5 min followed by 10 μg mL-1 cycloheximide plus 5 μg mL-1 cytochalasin B for 5 h. After 18 h and 12 h, respectively, fertilized and activated oocytes were centrifuged at 10 000 × g for 10 min to enable visualization of pronuclei. Zygotes with 2 polar bodies and 2 pronuclei and parthenotes with 2 pronuclei were snap frozen in mirVana lysis buffer. Total RNA was extracted from 30 pooled oocytes for each replicate using the mirVana MiRNA Isolation Kit (Ambion, Inc., Austin, TX, USA). Reverse transcription of RNA was performed using the QuantiMir RT kit (System Biosciences, Mountain View, CA, USA), and miRNA expression was evaluated by real-time PCR using the Mouse miRNome Profiler plate, which contains primers for 384 miRNA (System Biosciences). Three plates were analyzed for each group (30 oocytes per plate). Changes in relative expression levels were analyzed with a t-test of values normalized to miR-181a, which was consistently expressed in all samples. In Experiment 1, compared with in vitro-matured oocytes, in vivo-matured oocytes had 11-fold higher (P = 0.02) expression of miR-375, which targets numerous genes involved in electron transport chain and oxidative phosphorylation pathways according to the bioinformatic database mirGator. MiR-291a-5p, miR-494, miR-539, and miR-547 were expressed in in vivo-matured oocytes only; the converse was found for miR-575-5p. Results from Experiment 2 are in the table. Major pathways associated with potential targets of the detected miRNA include TGF-beta signaling, Wnt signaling, tight junction formation, DNA replication reactome, steroid biosynthesis, mRNA processing binding reactome, and glutamate metabolism. Several of these candidate miRNA might be important for regulation of bovine oocyte maturation and embryo development. Table 1.Experiment 2: Fold change expression of miRNA

40 TRICHOSTATIN A TREATMENT EFFECTS ON IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER CAT EMBRYOS DEPEND ON RECIPIENT CYTOPLASM SPECIES

2015 ◽  
Vol 27 (1) ◽  
pp. 113
Author(s):  
L. T. K. Do ◽  
Y. Sato ◽  
M. Taniguchi ◽  
T. Otoi
Keyword(s):  
Nuclear Transfer ◽  
Treatment Effects ◽  
Trichostatin A ◽  
Polar Body ◽  
Blastocyst Stage ◽  
Control Group ◽  
Fluid Medium ◽  
First Polar Body ◽  
Bovine Oocytes

The developmental ability of interspecies somatic cell nuclear transfer (iSCNT) embryos decreases as the taxonomic distance between the donor and recipient species increases. Treatment of cat iSCNT embryos using bovine oocytes with 50 nM of trichostatin A (TSA) improves in vitro embryonic development (Wittayarat et al. 2013 Cell. Reprogram. 15, 301–308). This study investigated whether the TSA treatment effects differ between the development of cat iSCNT embryos reconstructed with porcine and bovine oocytes. Porcine and bovine cumulus-oocyte complexes were in vitro matured for 44 h and 24 h, respectively. After cumulus cell removal, enucleation was performed by aspiration of the metaphase II plate and the first polar body using a piezo-driven pipette. A cat fibroblast cell was then injected into cytoplasm of successfully enucleated oocyte. Reconstructed cybrids were electrically activated by a single 1.5 kV cm–1 pulse for 100 µs (pig-cat embryos), or a 2.3 kV cm–1 pulse for 30 µs (cow-cat embryos). Pig-cat and cow-cat embryos were cultured in porcine zygote medium (PZM)-5 and modified synthetic oviducal fluid medium (mSOF), respectively. After electrical activation, pig-cat and cow-cat embryos were cultured in medium supplemented with 5 µg mL–1 cytochalasin B + 50 nM TSA (TSA group) or without TSA (control group), and the cow-cat embryo medium was also supplemented with 10 µg mL–1 cycloheximide. After 2 h, TSA-treated pig-cat and cow-cat embryos were incubated in medium supplemented with TSA for 22 h, followed by 48 h incubation without TSA. Pig-cat and cow-cat control embryos were cultured in medium without TSA for 70 h after activation. Then, all pig-cat and cow-cat embryos were cultured in porcine blastocyst medium (PBM) or mSOF medium supplemented with 5% fetal bovine serum, respectively, for 5 additional days. Four to seven replicates were performed for each experiment. Data were analysed using Student's t-test. For pig-cat embryos, no difference was observed in cleavage rates between both groups, but development to the blastocyst stage was higher in the pig control group (n = 147, 8.0%) than that of pig TSA group (n = 131, 0.7%; P < 0.05). In contrast, development to the blastocyst stage in cow-cat embryos was not observed in the cow control group (n = 125, 0%), but it was observed in cow TSA group (n = 136, 3.7%). These results indicate that TSA treatment effects are species-specific, but those effects remain to be clarified.

174 Effects of Melatonin on In Vitro Maturation of Bovine Oocytes and Gene Expression in Cumulus Cells: Preliminary Results

2018 ◽  
Vol 30 (1) ◽  
pp. 226
Author(s):  
F. C. Castro ◽  
L. Schefer ◽  
K. L. Schwarz ◽  
H. Fernandes ◽  
R. C. Botigelli ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Reverse Transcription ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Nuclear Maturation ◽  
Maturation Rate ◽  
Bovine Oocytes

Melatonin mediates several processes in animal reproduction and has drawn attention for its potent antioxidant, anti-apoptotic, anti-inflammatory action and, more recently, for its benefits on oocyte maturation and embryo development in vitro. The aim of this study was to assess the effect of melatonin during the in vitro maturation (IVM) on nuclear maturation of bovine oocytes and gene expression in their corresponding cumulus cells (CC). Bovine cumulus–oocyte complexes (COC) were obtained by aspiration of follicles (2-6 mm) from slaughterhouse ovaries, selected (grades I and II) and transferred to 4 well plates (25-30 COC/well) containing IVM medium [TCM-199 supplemented with sodium bicarbonate (26 mM), sodium pyruvate (0.25 mM), FSH (0.5 µg mL−1), LH (5.0 µg mL−1), 0.3% BSA, and gentamicin (50 µg mL−1)] with 0, 10−5, 10−7, 10−9 or 10−11 M melatonin and cultured for 24 h at 38.5°C and 5% CO2. At the end of IVM, oocytes were stained with Hoechst 33342 (10 μg mL−1) and evaluated for nuclear maturation rate. The CC were evaluated for the expression of antioxidant (SOD1, SOD2, GPX4), pro-apoptotic (P53, BAX) and expansion-related genes (PTX3, HAS1, HAS2). For transcript detection in CC, RNA isolation was performed with TRIzol®Reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcription with High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Relative quantification of transcripts was performed by RT-qPCR using 3 endogenous controls (β-actin, GAPDH, PPIA). Nuclear maturation rate and gene expression were tested by ANOVA and means were compared by Tukey’s test (6 replicates). In CC, the different concentrations of melatonin did not significantly alter expression of the investigated genes (P > 0.05), although all concentrations provided a numerical increase in the expression of the antioxidant SOD1 and of the expansion-related genes PTX3 and HAS2. Regarding the pro-apoptotic genes, concentrations of 10−11 and 10−9 M were able to reduce only numerically the expression of BAX and P53, respectively. In oocytes, the rate of nuclear maturation was not different among the tested treatments (P > 0.05), but it was numerically higher in the 10−7 M melatonin treated group compared with the control (69.71 ± 13.76% v. 88.1 ± 12.54%). In conclusion, under the studied conditions, melatonin was unable to improve maturation rate or to affect the expression of antioxidant, pro-apoptotic, and expansion-related genes in CC. Melatonin during IVM has shown variable results in different studies and appears to show different effects depending on culture conditions and parameters studied. In order to take advantage of the possible positive antioxidant effects of melatonin, other culture conditions and parameters should be investigated. In a next step, melatonin will be included during in vitro culture of embryos to evaluate its possible cytoprotective role, because such embryos are more exposed to oxidative stress during in vitro culture, and to investigate its benefits on developmental competence in vitro. This reaesrch was funded by FAPESP (2015/20379-0; 2014/17181-0).

Development in vitro and in vivo of aggregated parthenogenetic bovine embryos

1995 ◽  
Vol 7 (5) ◽  
pp. 1073 ◽  
Author(s):  
A Boediono ◽  
S Saha ◽  
C Sumantri ◽  
T Suzuki
Keyword(s):  
Cytochalasin B ◽  
Polar Body ◽  
Cleavage Rate ◽  
Development In Vitro ◽  
Second Polar Body ◽  
Bovine Oocytes ◽  
Longitudinal Diameter ◽  
Parthenogenetic Embryos

Mature bovine oocytes were activated with 7% ethanol followed by cytochalasin B or D treatment. Most oocytes extruded a second polar body and formed one pronucleus when treated with 7% ethanol alone [35/43 (81%)]. With ethanol followed by cytochalasin B or D, overall activation frequency was 70% (309/441), with activated oocytes containing two pronuclei. The cleavage rate was not significantly different between treatment with ethanol alone and ethanol followed by 5 micrograms mL-1 cytochalasin B, but it was significantly lower than in fertilized oocytes (P < 0.01). However, the blastocyst production rate was significantly different (P < 0.01) among the treatments. The incidence of parthenogenetic embryos with normal (diploid) complements and with chromosome anomalies (2N/4N) was 68% (17/25) and 32% (8/25) respectively, and this was not affected by cryopreservation treatment. The longitudinal diameter of aggregated-four embryos cultured in vitro was greater (P < 0.01) than aggregated-two or single embryos. One of the aggregated-four parthenogenetic embryos was further cultured in vitro and developed up to Day 27 after activation, with a diameter of 2980 microns. The aggregated-four parthenogenetic embryos were transferred to five recipients. The oestrus was prolonged in three recipients and they returned to oestrus on Day 57, 62 and 67 after the previous oestrus. These results indicate that aggregating parthenogenetic embryos can prolong their survival in vitro and in vivo.

scholarly journals Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

Reproduction ◽  
2002 ◽  
pp. 455-465 ◽  
Author(s):  
YH Choi ◽  
CC Love ◽  
LB Love ◽  
DD Varner ◽  
S Brinsko ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
First Polar Body ◽  
Bovine Oocytes

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.

355 INFLUENCE OF NITRIC OXIDE AND CYCLIC GMP SIGNALING PATHWAY ON THE IN VITRO MATURATION OF BOVINE OOCYTES: PRELIMINARY RESULTS

2010 ◽  
Vol 22 (1) ◽  
pp. 334
Author(s):  
K. R. L. Schwarz ◽  
T. H. C. de Bem ◽  
P. R. L. Pires ◽  
L. G. Mesquita ◽  
L. Remy ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Signaling Pathway ◽  
Polar Body ◽  
Calf Serum ◽  
Guanosine Monophosphate ◽  
No Donor ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Bovine Oocytes

Nitric oxide (NO) is a chemical messenger generated by the activity of the nitric oxide synthase enzyme (NOS) and has been shown to be involved in oocyte maturation. NO is known to act through the guanylate cyclase (GC) signaling pathway, stimulating the production of cyclic guanosine monophosphate (cGMP), which in turn activates protein kinase G (PKG). The objective of the present study was to investigate the involvement of NO and GC/cGMP/PKG pathway on the IVM of bovine oocytes. Slaughterhouse ovaries were transported to the laboratory and oocytes were aspirated from 2 to 8 mm follicles. Oocytes were submitted to IVM (TCM-199+10% fetal calf serum + hormones) for 24 h (38.5°C and 5% CO2 in air) and were assessed for nuclear maturation by acetic-orcein (1%) staining. Maturation rates were analyzed by ANOVA. Five replicates were performed with 20 oocytes per group per replicate. When the oocytes were matured with the NO donor [(0, 10-9, 10-8 and 10-7M S-nitroso-N-acteyl-D,L- penicillamine (SNAP)] germinal vesicle break down (GVBD) rates after 7 h in IVM were 36, 31, 42, and 24%, respectively (P > 0.05). Maturation rates after 24 h IVM ranged from 80 to 85% (P > 0.05). The inhibition of GC [(0, 0.1, 10, and 100 μM 1, H-[1, 2, 4]oxadiazole[4, 3-a]quinoxalon-1-one (ODQ)] and PKG (0, 1, 10, and 100 μM KT5823) did not affect (P > 0.05) the ability of oocytes to form the first polar body (average of 83 and 88%, respectively). When the cGMP-analogue (0, 1, 2, and 4 mM 8-Bromo-cGMP) and the GC-stimulator (0, 5, 10, and 50 μM Protoporphyrin IX) were used during IVM, maturation rates were over 85% in all groups (P > 0.05). To confirm the lack of effect of the inhibitors, another evaluation with higher concentrations of inhibitors in semi-defined IVM medium (TCM-199 + 0.04% BSA) was carried out. Maturation rates were 70 to 75% (P > 0.05) with ODQ and 57 to 76% (P > 0.05) with KT5823. The evaluation with the GC stimulator and the cGMP analogue in semi-defined medium is currently underway. In conclusion, under the conditions studied, the GC/cGMP/PKG signaling pathway is not involved in the nuclear maturation of bovine oocytes. Supported by FAPESP, Brazil.

169 Nuclear Maturation Kinetics and In Vitro Fecundation of Immature Bovine Oocytes Injected into Preovulatory Follicles

2018 ◽  
Vol 30 (1) ◽  
pp. 224
Author(s):  
L. M. S. Simoes ◽  
A. P. C. Santos ◽  
E. A. Lima ◽  
R. E. Orlandi ◽  
M. P. Bottino ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Causative Factor ◽  
Nuclear Maturation ◽  
Preovulatory Follicles ◽  
Fertilization Capacity ◽  
Bovine Oocytes ◽  
Metaphase I

The objective was to evaluate in vitro nuclear maturation and fecundation kinetics of oocytes injected into preovulatory follicles of synchronized cows using the intra-follicular oocyte injection (IFOI) technique. In experiment 1, 438 immature abattoir-bovine cumulus–oocyte complexes (COC) of grades I, II, and III were randomly allocated to 1 of 3 groups: Matvitro (n = 111), COC matured in vitro for 22 h; Matvivo20 (n = 172) and Matvivo30 (n = 155), 30 oocytes were injected into each preovulatory follicle of pre-synchronized recipients. In Matvivo20, oocytes were matured for 19.8 ± 0.1 h and in Matvivo30, for 28.3 ± 0.1 h. All cows received 12.5 mg of LH (Lutropin, Bioniche, Canada) at IFOI (Matvivo20) or 10 h after IFOI (Matvivo30). Oocytes from Matvivo20 and Matvivo30 were aspirated 20 h after LH injection for assessment of oocyte maturation and recovery rates. Oocytes were evaluated according to maturation kinetics as germinal vesicle, metaphase I, anaphase I, telophase I, metaphase II, parthenogenetically activated, and degenerated (chromosomal aberrations, presence of diffuse or indefinite chromatin). In experiment 2, immature abattoir-bovine COC (n = 202) of grades I, II, and III were randomly distributed into 2 groups: Matvitro (n = 103), COC were matured and fertilized in vitro; Matvivo (n = 99), same as Matvivo20 protocol, and COC fertilized in vitro. Presumptive zygotes were evaluated as fertilized, unfertilized, or polyspermic. Statistical analyses were performed by the GLIMMIX procedure of SAS (SAS Institute Inc., Cary, NC, USA). Recovery rate was lower (P < 0.001) in Matvivo20 (52.9%, 91/172) compared with Matvivo30 (72.9%, 113/155). Germinal vesicle (P = 0.94), metaphase I (P = 0.98), anaphase I (P = 0.99), and telophase I (P = 0.20) rates were similar. However, there were differences in metaphase II [Matvitro: 81.0% (90/111)a, Matvivo20: 74.5% (35/47)a, and Matvivo30: 41.6% (32/77)b; P = 0.001], degenerate [Matvitro: 5.4% (6/111)c, Matvivo20: 21.3% (10/47)b and Matvivo30: 48.1% (37/77); P = 0.001] and parthenogenetically activated [Matvitro: 0.0% (0/111)b, Matvivo20: 0.0% (0/47)b and Matvivo30: 9.1% (7/77)a; P = 0.001]. Polyspermic (P = 0.18) and abnormal (P = 0.98) rates were similar. However, there was a higher rate (P = 0.05) of fertilized oocytes in Matvivo (60.6%, 60/99) than in Matvitro (46.6%, 48/103). In conclusion, oocyte maturation in vivo after IFOI for 20 h does not alter maturation kinetics and increases in vitro oocyte fertilization capacity. However, the 10-h increase in intra-follicular oocyte permanence decreased the proportion of viable oocytes. Thus, the oocyte maturation phase is not the limiting causative factor for the low IFOI-embryo production rates.

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