114 Effect of in vivo heat stress on DNA methylation and DNA hydroxymethylation of bovine oocytes

2019 ◽  
Vol 31 (1) ◽  
pp. 183
Author(s):  
F. A. Diaz ◽  
E. J. Gutierrez ◽  
B. A. Foster ◽  
P. T. Hardin ◽  
K. R. Bondioli
Keyword(s):  
Dna Methylation ◽  
Heat Stress ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Dna Hydroxymethylation ◽  
Oocyte Collection ◽  
Grade 3 ◽  
Bovine Oocytes

Cattle under the effect of heat stress have reduced fertility, with negative effects on the oocyte observed at the morphological, biochemical, transcriptional and developmental levels. There are no studies evaluating the effect of heat stress on the epigenetic profile of bovine oocytes, which plays a fundamental role in the regulation of gamete development. The objective of this study was to evaluate the effect of in vivo heat stress during the spring to summer transition on DNA methylation and DNA hydroxymethylation of bovine oocytes at the germinal vesicle (GV) and metaphase II (MII) stages. Ten Bos taurus crossbred nonlactating beef cows located at Saint Gabriel, Louisiana, USA (30°16′11.1″ N, 91°06′12.1″ W), were used for oocyte collection once monthly from April to August. Dominant follicle removal was performed 5-7 days before oocyte collection. Cumulus-oocyte complexes were collected through ovum pick-up from follicles >2mm. Germinal vesicle (GV)-stage oocytes (50% of total obtained per cow) were subjected to a standard bovine in vitro maturation protocol to obtain metaphase II (MII) stage oocytes. The DNA methylation and DNA hydroxymethylation of GV and MII oocytes was assessed by fluorescence immunohistochemistry utilising primary antibodies against 5′-methylcytosine and 5′-hydromethylcytosine. Secondary antibodies utilised were Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 546 donkey anti-rabbit IgG. Oocytes were visualised utilising a fluorescence deconvolution microscope and immunofluorescence data were expressed as corrected relative fluorescence per nucleus. The polar body was not included for fluorescence quantification when evaluating MII stage oocytes. Results (least squares means±standard error) were evaluated as cold months (April and May) and hot months (June, July, and August). Results were analysed by the type III test of fixed effects and Tukey media separation utilising Proc Glimmix of SAS 9.4 (P<0.05; SAS Institute Inc., Cary, NC, USA). Maturation rates and percent of grade 1, grade 2, and grade 3 oocytes were square root arcsine transformed for statistical analysis. The number of total oocytes obtained per cow was higher in cold compared to hot months (21.88±2.34 and 14.23±2.17, respectively). Percent of grade-1 oocytes was higher in cold compared to hot months (38.25±3.69 and 27.59±3.09, respectively). There was no difference in percent of grade-2 oocytes between cold and hot months (21.80±2.44 and 22.60±2.20, respectively). There was a lower percent of grade-3 oocytes in cold compared to hot months (39.82±4.54 and 55.87±3.98, respectively). Maturation rate (in vitro maturation) was not different between cold and hot months (81.92±4.04 and 91.11±3.36, respectively). There was no difference between cold and hot months in DNA methylation (417,218.90±71,793.86 and 313,819.88±55,528.01, respectively) and DNA hydroxymethylation (444,931.10±67,920.78 and 352,254.68±56,425.96, respectively) of GV-stage oocytes. There was no difference between cold and hot months in DNA methylation (87,122.36±14,449.47 and 89,807.26±11,303.72 AU, respectively) and DNA hydroxymethylation (102,933.83±15,517.70 and 137,622.45±11,826.86 AU, respectively) of MII-stage oocytes.

37 Effect of invivo and invitro heat stress on DNA methylation and DNA hydroxymethylation of bovine oocytes and embryos

2021 ◽  
Vol 33 (2) ◽  
pp. 126
Author(s):  
F. A. Diaz ◽  
E. J. Gutierrez ◽  
B. A. Foster ◽  
P. T. Hardin ◽  
K. R. Bondioli
Keyword(s):  
Dna Methylation ◽  
Heat Stress ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Dna Hydroxymethylation ◽  
Early Embryos ◽  
Oocyte Collection ◽  
Maturation Rate ◽  
Bovine Oocytes ◽  
Fluorescence Immunohistochemistry

Reduced reproductive performance is one of the main effects caused by heat stress in cattle. Its negative effects have been observed at the transcriptional, biochemical, morphological, and developmental levels on the oocyte and embryo. There are no studies evaluating the effect of heat stress on the epigenetic profile of bovine oocytes and early embryos. The objective of this study was to evaluate the effect of invivo and invitro heat stress on DNA methylation and DNA hydroxymethylation in bovine MII oocytes, pronuclear, and 2- to 4-cell stage embryos. Seven Bos taurus crossbred nonpregnant, non-lactating beef cows located in Saint Gabriel, Louisiana (30.269746, −91.103357) were used for oocyte collection. Dominant follicle removal was performed 5 days before oocyte collection. Cumulus–oocyte complexes were collected by ovum pickup from follicles >2mm. Samples were collected during the summer (August) and winter (February) (5 collections each). Three treatments were utilised: invivo heat stress (August samples), invitro heat stress (February samples subjected to 41°C during the first 12h of IVM and then to 38.5°C during the next 12h of IVM), and control (February samples IVM at 38.5°C). All oocytes collected per treatment were assigned to 3 developmental stages: MII oocytes, pronuclear, and 2- to 4-cell stage embryos. Embryos were obtained through standard IVF. DNA methylation and DNA hydroxymethylation was assessed by fluorescence immunohistochemistry utilising primary antibodies against 5′-methylcytosine and 5′-hydromethylcytosine and secondary antibodies Alexa Fluor 488 and Alexa Fluor 546, respectively. Samples were visualised with a fluorescence deconvolution microscope, and immunofluorescence data were expressed as corrected relative fluorescence per nucleus. Results were analysed by the Type III test of fixed effects and Tukey media separation utilising the Proc Glimmix of SAS 9.4 (P<0.05). Maturation rate, 2 pronuclei (2PN) rate, cleavage rate, and 2- to 4-cell rate were analysed by Chi-square. There was no difference in maturation rate (88.19±7.57, 82.91±5.18, 94.51±5.04; P=0.2516), 2PN rate (79.34±10.23, 93.75±7.21, 81.74±12.53; P=0.1757), cleavage rate (79.26±2.69, 70.65±7.22, 81.85±16.65; P=0.2388) and 2- to 4-cell rate (69.38±7.83, 81.25±10.34, 61.11±11.69; P=0.4392) between invivo and invitro heat stress compared with control, respectively. No difference was found in DNA methylation (P=0.0537) or DNA hydroxymethylation (P=0.4632) between treatments in MII oocytes. When evaluating the paternal and maternal pronuclei, there was no difference in DNA methylation (P=0.9766; P=0.1954, respectively) or DNA hydroxymethylation (P=0.6440; P=0.1932, respectively) between invivo and invitro heat stress compared with control. Similarly, there was no difference in DNA methylation (P=0.0903) or DNA hydroxymethylation (P=0.2452) between treatments when evaluating the 2- to 4-cell embryos. In conclusion, we detected no effect of invivo or invitro heat stress on MII oocytes and early embryos when evaluating global DNA methylation and hydroxymethylation through fluorescence immunohistochemistry.

scholarly journals Evaluation of Seasonal Heat Stress on Transcriptomic Profiles and Global DNA Methylation of Bovine Oocytes

2021 ◽  
Vol 12 ◽  
Author(s):  
Fabian A Diaz ◽  
Emilio J Gutierrez-Castillo ◽  
Brittany A Foster ◽  
Paige T Hardin ◽  
Kenneth R Bondioli ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Heat Stress ◽  
Molecular Mechanisms ◽  
Developmental Competence ◽  
Beta Catenin ◽  
Hippo Signaling ◽  
Sequencing Analysis ◽  
Dna Hydroxymethylation ◽  
Bovine Oocytes

Heat stress affects oocyte developmental competence and is a major cause of reduced fertility in heat stressed cattle. Negative effects of heat stress on the oocyte have been observed at morphological, biochemical and developmental levels. However, the mechanisms by which heat stress affects the oocyte at the transcriptional and epigenetic levels remain to be further elucidated. Here we aimed to investigate the effect of heat stress on oocyte quality, transcriptomic profiles and DNA methylation of oocytes collected through the transition from spring to summer under Louisiana conditions. Summer season resulted in a lower number of high quality oocytes obtained compared to the spring season. There was no difference in in vitro maturation rates of oocytes collected during spring as compared to summer. RNA sequencing analysis showed that a total of 211 and 92 genes were differentially expressed as a result of heat stress in GV and MII oocytes, respectively. Five common genes (E2F8, GATAD2B, BHLHE41, FBXO44, and RAB39B) were significantly affected by heat in both GV and MII oocytes. A number of pathways were also influenced by heat stress including glucocorticoid biosynthesis, apoptosis signaling, and HIPPO signaling in GV oocytes, and Oct4 pluripotency, Wnt/beta-catenin signaling, and melatonin degradation I in MII oocytes. In addition, fluorescent immunocytochemistry analysis showed no difference in global levels of DNA methylation and DNA hydroxymethylation at either the GV or MII stage between spring and summer oocytes. The results of this study contribute to a better understanding of the effect of heat stress on the molecular mechanisms altered in bovine oocytes.

261 INFLUENCE OF IN VITRO MATURATION ON EPIGENETIC MARKS AND GENE EXPRESSION IN BOVINE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 228
Author(s):  
J. Heinzmann ◽  
T. Hansmann ◽  
C. Wrenzycki ◽  
U. Zechner ◽  
T. Haaf ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Embryonic Development ◽  
Quantitative Pcr ◽  
Assisted Reproduction ◽  
In Vitro Maturation ◽  
Imprinted Genes ◽  
Assisted Reproduction Technology ◽  
Bovine Oocytes

In cattle, in vitro maturation (IVM) of oocytes is an integral part of assisted reproduction technology. However, only 30% of in vitro matured bovine oocytes develop to the blastocyst stage after fertilization (compared with 60% for in vivo matured oocytes), indicating critical involvement of maturation conditions in the developmental competence of oocytes. Oocytes for IVF and intracytoplasmic sperm injection in humans are typically allowed to mature in vivo after superovulation because IVM is not considered to be a safe medical procedure. Several studies have shown that assisted reproduction technology involving prolonged in vitro culture of human and ruminant embryos can be associated with increased risk of fetal or placental abnormalities due to aberrant DNA methylation of imprinted and non-imprinted genes. Similarities between the bovine large offspring syndrome and imprinting-related human Beckwith–Wiedemann syndrome and the general similarity of bovine and human pre-implantation development make bovine oocyte maturation and embryonic development an increasingly accepted model of human development. Differentially methylated regions and imprinting control regions for the bovine paternally imprinted gene H19 and the maternally imprinted genes PEG3 and SNRPN were identified and characterised in this study. The DNA methylation profiles of bovine oocytes could be determined by bisulfite treatment of DNA from pools of 10 oocytes, but no significant differences were observed between IVM in TCM medium with 20% O2, in SOF medium with 5% O2, or after in vivo maturation. In contrast, quantitative PCR analysis of single oocyte preparations (n ≥ 8) revealed significant differences between these groups in the expression of the 3 genes. We then investigated the expression of genes involved in other critical processes in the developing oocyte and early embryo by quantitative PCR, including SLC2A8 (glucose transport), GDF9 (growth factor), PRDX1 (antioxidant and intercellular communication), DNMT1a/b (maintenance of methylation), and DNMT3a/b (de novo methylation). We also studied IGF2R, an imprinted gene implicated in large offspring syndrome. We observed significant differences in the expression of several of these genes. Interestingly, the expression of DNMT3a and DNMT3b was significantly higher in in vitro matured oocytes than in in vivo matured oocytes and could result in the above-mentioned aberrant methylation patterns established later in embryonic development. This work was funded by DFG (FOR1041).

159 EFFECT OF 3-ISOBUTYL-1-METHYLXANTHINE (IBMX) ON THE GERMINAL VESICLE STAGE OF PORCINE OOCYTES DURING OOCYTE COLLECTION IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 193
Author(s):  
R. Appeltant ◽  
J. Beek ◽  
D. Maes ◽  
A. Van Soom
Keyword(s):  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Guanosine Monophosphate ◽  
Developmental Competence ◽  
Meiotic Arrest ◽  
Oocyte Collection

When using modern maturation conditions for in vitro maturation, pig oocytes yield ~20% blastocysts only. One problem is that cumulus cells, which are normally connected with the immature oocyte by cellular projections penetrating through the zona pellucida and with the oolemma via gap junctions, are prematurely losing these connections after the cumulus–oocyte complex is removed from the follicle. The oocyte possesses a type 3 phosphodiesterase, which degrades 3′,5′-cyclic adenosine monophosphate (cAMP), and this activity is inhibited by supply of 3′,5′-cyclic guanosine monophosphate (cGMP) to the oocyte via the cumulus cells. Consequently, cAMP levels, which are typically high during early stages of oocyte maturation in vivo, decrease, leading to spontaneous nuclear maturation and oocytes of low developmental competence. Therefore, the maintenance of these cumulus-oocyte connections is important to keep cAMP high and the oocyte under meiotic arrest. One way to prevent this drop in cAMP is using N6, 2′-o-dibutyryladenosine 3′,5′-cyclic monophosphate sodium (dbcAMP) that causes an arrest at germinal vesicle (GV) stage II (Funahashi et al. 1997 Biol. Reprod. 57, 49–53). Another option is collecting the oocytes in a medium containing the phoshodiesterase inhibitor, IBMX. The present study investigated the influence of IBMX on the progression of the GV of the oocyte after collection, just before the start of the maturation procedure. The GV stage was defined according to Sun et al. (2004 Mol. Reprod. Dev. 69, 228–234). In parallel with the findings on dbcAMP, we hypothesised an arrest at GV II by the presence of IBMX during collection. One group of oocytes were collected in HEPES-buffered TALP without IBMX (n = 375) and another group in the same medium containing 0.5 mM IBMX (n = 586). An average incubation time of 140 min was applied in both groups, and 3 replicates were performed. The proportions of oocytes before or at GV II and beyond GV II were compared in both groups using logistic regression analysis. The proportion of oocytes was included as dependent variable and group (IBMX addition or not) as independent variable. Replicate was also included in the model. The proportion of oocytes before or at GV II was not statistically significant between the group without and the group with IBMX (59.2 v. 58.7% respectively; P > 0.05). In conclusion, the use of IBMX during oocyte collection did not influence the state of the germinal vesicle of the oocyte during collection, indicating that IBMX did not cause a meiotic arrest in the oocytes during collecting in vitro.

45 THE EFFECT OF MEIOTIC STAGE OF BOVINE OOCYTES ON THE SURVIVAL OF VITRIFIED CUMULUS–OOCYTE COMPLEXES

2012 ◽  
Vol 24 (1) ◽  
pp. 135 ◽  
Author(s):  
J. R. Prentice ◽  
J. Singh ◽  
M. Anzar
Keyword(s):  
Embryo Development ◽  
Culture System ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Early Embryo ◽  
Time Intervals ◽  
Early Embryo Development ◽  
In Vitro Culture System ◽  
Bovine Oocytes

Vitrification is a rapid freezing method in which cells/tissues are frozen in a glass state without ice crystal formation. However, vitrification of bovine oocytes is challenging due to their complex structure and sensitivity to chilling. Oocytes at the germinal vesicle (GV) stage of maturation are thought to be less prone to chromosomal and microtubular damage during cryopreservation because no spindle is present and genetic material is contained within the nucleus. However, immature oocytes are thought to be more sensitive to osmotic stress and have lower cell membrane stability than mature, metaphase II (MII) stage oocytes. The present studies aimed to validate the in vitro culture system used in our laboratory and to evaluate the effect of vitrification of bovine cumulus-oocyte complexes (COC) at different meiotic stages on their in vitro maturation (IVM), cleavage and early embryo development. Analyses were conducted on each dataset with PROC GLIMMIX in SAS using binary distribution (for yes/no response variable) and considering replicate as a random factor. In Experiment 1, meiotic progression of oocytes was evaluated at different time intervals during IVM. The following COC stages were predominantly found at different IVM time intervals: GV (89%) at 0 h, GV (47%) and germinal vesicle breakdown (GVBD; 44%) at 6 h, metaphase I (MI; 90%) at 12 h and MII (84%) at 22 h (n > 62 oocytes at each time group). In Experiment 2, bovine COC at 0, 6, 12 and 22 h of IVM were exposed to vitrification solution (15% dimethyl sulfoxide + 15% ethylene glycol + 0.5 M sucrose + 20% CS in TCM-199), loaded onto a cryotop device and vitrified by plunging in liquid nitrogen. Following warming (1 min in 0.5 M sucrose + 20% CS in TCM-199), COC completed 22 h of IVM and the nuclear stage was evaluated with lamin A/C-4′6-diamidino-2-phenylindole staining. Upon completion of 22 h of IVM, 23, 23, 35 and 89% of oocytes from 0-, 6-, 12- and 22-h groups, respectively were detected at MII (P < 0.0001). In Experiment 3, cleavage and embryo development of oocytes vitrified at 0, 12 and 22 h of IVM were evaluated. The cleavage rate did not differ among vitrification groups (i.e. 14% at 0 h, 17% at 12 h and 14% at 22 h; P = 0.825). Cleavage and blastocyst rates were higher (P < 0.0001) in the non-vitrified (control) group than in vitrified groups (i.e. 73 vs 15% and 22 vs 0.3%, respectively). In conclusion, the maturation kinetics validated our in vitro culture system and vitrification adversely affected the ability of bovine oocytes to undergo in vitro maturation to the MII stage, in vitro fertilization and early embryo development. Vitrification of oocytes at GV, MI and MII stages of nuclear maturation did not differ in their subsequent survivability. This study was supported by the Canadian Animal Genetic Resources Program, Agriculture and Agri-Food Canada.

Dynamic changes in the global transcriptome of bovine germinal vesicle oocytes after vitrification followed by in vitro maturation

2018 ◽  
Vol 30 (10) ◽  
pp. 1298 ◽  
Author(s):  
Jianwei Huang ◽  
YongShun Ma ◽  
Shao Wei ◽  
Bo Pan ◽  
Yu Qi ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Oocyte Vitrification ◽  
Kegg Pathway Database ◽  
Global Transcriptome ◽  
Vitro Development ◽  
Bovine Oocytes ◽  
Regulation Of Actin Cytoskeleton ◽  
Mrna Transcriptome

This study was conducted to investigate the effect of vitrification on the dynamics of the global transcriptome in bovine germinal vesicle (GV) oocytes and their in vitro-derived metaphase II (MII) oocytes. The GV oocytes were vitrified using the open-pulled straw method. After warming, GV oocytes and the resulting MII-stage oocytes were cultured in vitro for 2 h and 24 h respectively and were then collected. The fresh GV oocytes and their in vitro-derived MII oocytes were used as controls. Then, each pool (fresh GV, n = 3; vitrified GV, n = 4; fresh MII, n = 1 and MII derived from vitrified GV, n = 2) from the different stages was used for mRNA transcriptome sequencing. The results showed that the in vitro maturation rates of GV oocytes were significantly decreased (32.36% vs 53.14%) after vitrification. Bovine GV oocyte vitrification leads to 12 significantly upregulated and 19 downregulated genes. After culturing in vitro, the vitrification-derived MII oocytes showed 47 significantly upregulated and six downregulated genes when compared with those from fresh GV oocytes. Based on molecular function–gene ontology terms analysis and the Kyoto encyclopaedia of genes (KEGG) pathway database, the differentially expressed genes were associated with the pathways of cell differentiation and mitosis, transcription regulation, regulation of actin cytoskeleton, apoptosis and so on, which potentially result in the lower in vitro development of GV bovine oocytes.

scholarly journals Effect of heat stress during in vitro maturation on developmental competence of vitrified bovine oocytes

2017 ◽  
Vol 52 ◽  
pp. 48-51 ◽  
Author(s):  
M Vendrell-Flotats ◽  
N Arcarons ◽  
E Barau ◽  
M López-Béjar ◽  
T Mogas
Keyword(s):  
Heat Stress ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Bovine Oocytes

313 PRE-IN VITRO MATURATION WITH CYCLIC AMP AND CYCLIC GMP MODULATORS IMPROVES DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 245 ◽  
Author(s):  
N. W. Santiquet ◽  
A. F. Greene ◽  
W. B. Schoolcraft ◽  
R. L. Krisher
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Subsequent Development ◽  
Developmental Competence ◽  
Nuclear Maturation ◽  
Oocyte Developmental Competence ◽  
Oocyte Collection ◽  
Short Period

In vitro maturation (IVM) of cumulus-oocyte complexes (COC) results in oocytes with reduced quality and is still not as efficient as in vivo maturation in most species. One hypothesis that could explain the low developmental competence of oocytes following IVM is that the oocytes resume meiosis too quickly after being retrieved from the follicles. Studies in mice and bovine have shown that a short period of prematuration in the presence of cAMP modulators, before IVM, enhances oocyte developmental competence. Moreover, other studies have recently demonstrated that cGMP is also a crucial molecule involved in meiotic resumption. Here, our objective was to examine the effect of a cGMP modulator in combination with a cAMP modulator during a short period of prematuration on mouse oocyte nuclear maturation and subsequent embryo development following IVF. The COC were collected (6 replicates) from 2-month-old outbred CF1 mice 48 h after PMSG (5 IU) injection in the presence (pre-IVM) or absence (control) of cGMP and cAMP modulators. Pre-IVM COC (n = 184) were then placed in prematuration medium that also contained these cGMP and cAMP modulators. After 2 h, pre-IVM COC were washed and transferred to our in-house prepared, completely defined IVM medium (Paczkowski et al. 2014 Reprod.) for the remaining 16 h of culture; 10 oocytes per 50 µL drop under oil, at 37°C in 7.5% CO2 and 6.5% O2 due to the increased altitude at our location. Control COC (n = 161) were matured in the same IVM medium under identical conditions for 18 h, without prematuration. After IVM, oocytes were fixed for assessment of nuclear maturation, or fertilized and cultured in vitro and subsequent development (96 and 112 h) was recorded (Paczkowski et al. 2014 Reprod.). Results were analysed by ANOVA. A short 2-h prematuration period in the presence of cGMP and cAMP modulators had no impact on oocyte nuclear maturation to metaphase II after IVM or on embryo cleavage after IVF. However, pre-IVM treatment improved the developmental competence of the oocyte, as demonstrated by increased embryo development. More (P < 0.02) blastocysts (96 h of culture) and hatched blastocysts (112 h of culture) developed in the pre-IVM treatment compared to control (31.0 ± 3.4 v. 19.9 ± 3.2%; 31.5 ± 3.4 v. 19.9 ± 3.2%, respectively). In conclusion, a combination of cGMP and cAMP modulators during oocyte collection and a subsequent short pre-IVM improves oocyte developmental competence and could therefore be a potential tool to improve embryo yield following IVM.

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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