45 SPINDLE CONFIGURATION AND DNA FRAGMENTATION OF VITRIFIED BOVINE OOCYTES AFTER IN VITRO MATURATION WITH L-CARNITINE AND/OR RESVERATROL

2015 ◽  
Vol 27 (1) ◽  
pp. 115 ◽  
Author(s):  
J. F. W. Spricigo ◽  
N. Arcarons ◽  
T. Mogas ◽  
M. A. N. Dode ◽  
R. Morato
Keyword(s):  
Dna Fragmentation ◽  
In Vitro Maturation ◽  
Apoptotic Cell ◽  
Cumulus Cell ◽  
Meiotic Spindle ◽  
Significance Level ◽  
Chromosome Configuration ◽  
Chi Squared ◽  
Cell Layers

After cryopreservation, oocytes may suffer morphological and functional damage, due to the high cytoplasm lipid content and to the reactive oxygen species formation. The aim of this work was to evaluate the spindle configuration and the DNA fragmentation of vitrified/warmed oocytes after in vitro maturation (IVM) in a media supplemented with l-carnitine and/or resveratrol, a lipolytic and antioxidant agent, respectively. The IVM viable COC with at least 3 cumulus cell layers and homogenous cytoplasm were randomly distributed into 4 groups: (1) control: conventional IVM media with TCM-199, epidermal growth factor, and 10% FCS; (2) L-CAR: control media supplemented with 0.6 mg mL–1 of l-carnitine; (3) RES: control media supplemented with 1 μM mL–1 of resveratrol; and 4) L+R: control media supplemented with 0.6 mg mL–1 of l-carnitine and 1 μM mL–1 of resveratrol. After 22 h of IVM, half of the COC from each group were vitrified and warmed, using the cryotop methodology. After warming, the oocytes were allowed to recover in their respective media for 2 additional hours. After 24 h of IVM, oocytes from all treatments were completely denuded and fixed and stained using specific fluorescent probes. The microtubule/chromosome configuration and the DNA fragmentation were analysed by immunocytochemistry under a fluorescent microscope (A.40FL, Carl Zeiss, Oberkochen, Germany). All statistical analyses were conducted with IBM SPSS 19 (IBM; Chicago, IL, USA). ANOVA was performed to analyse differences in meiotic spindle configuration, and the Chi-squared test was used for DNA fragmentation. The significance level was 5%. Although vitrification may cause severe oocyte damage, IVM with l-carnitine alone or in association with resveratrol was able to reduce the percentage of abnormal spindle configurations (Table 1), whereas the addition of resveratrol alone or its association with l-carnitine reduced DNA fragmentation of IVM oocytes after a vitrification/warming process. These results indicate the IVM supplementation with RES and/or L-CAR could modify oocyte composition, increasing its cryotolerance. However further studies are required to confirm the beneficial effect of these molecular interactions. Table 1.Evaluation of spindle configuration (Experiment 1) and apoptotic cell status (Experiment 2) of fresh or vitrified/warmed oocytes matured with RES and/or L-CAR

188 IMPROVEMENT OF IN VITRO CANINE OOCYTE MATURATION BY OVIDUCTAL SECRETOME

2017 ◽  
Vol 29 (1) ◽  
pp. 202 ◽  
Author(s):  
A. Lange-Consiglio ◽  
C. Perrini ◽  
P. Esposti ◽  
F. Cremonesi
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Follicular Development ◽  
Cumulus Cells ◽  
Chi Square ◽  
Cell Layers ◽  
Hoechst Staining

The in vitro maturation of canine oocyte is problematic because it is difficult to reproduce the oviducal microenvironment where the in vivo maturation occurs. Because cells are able to communicate with each other by paracrine action, oviducal cells could be in vitro cultivated to obtain the conditioned medium (CM) consisting of soluble factors and microvesicles (MV), which represent a carrier for nonsoluble molecules including microRNA. The aim of the present work was to investigate the effect of the addition of CM or MV, secreted by oviducal cells, to the canine in vitro maturation medium. To generate CM, cells from oviducts of 3 animals in late oestrus were cultured for 5 days at 38.5°C in a humidified atmosphere of 5% CO2. Supernatants were collected, pooled, centrifuged at 2500 × g, and stored at −80°C. Microvesicles were obtained by ultracentrifugation of CM at 100,000 × g for 1 h at 4°C and measured for concentration and size by a Nanosight instrument. Ovaries were obtained from 50 healthy domestic bitches (1–4 years old) of different breeds that underwent ovariectomy regardless of the oestrous cycle. Cumulus-oocyte complexes were released by slicing the ovarian cortex with a scalpel blade, and only Grade 1 cumulus-oocyte complexes (darkly granulated cytoplasm and surrounded by 3 or more compact cumulus cell layers) 110 to 120 µm in diameter were selected for culture. Maturation was performed at 38.5°C in a humidified atmosphere of 5% CO2 and 5% of O2 in bi-phasic systems: 24 h in SOF with 5.0 μg mL−1 of LH followed by 48 h in SOF supplemented with 10% of oestrous bitch serum and 10% CM or 50, 75, 100, or 150 × 106 MV mL−1 labelled with PKH-26. Control was the same medium without CM or MV. Oocytes were observed under a fluorescent microscope to detect metaphase II (MII), by Hoechst staining, and the incorporation of MV. Statistical analysis was performed by chi-square test. Results show that canine oviducal cells secreted MV of 234 ± 23 nm in size, underling that these MV fall within the shedding vesicles category. The incorporation of labelled MV occurred at first in cumulus cells, at 48 h of maturation, and then, at 72 h, in oocyte cytoplasm. These MV had a positive effect on maturation rate (MII) at the concentration of 75 and 100 × 106 MV mL−1 compared with CM and control (20.34 and 21.82 v. 9.09 and 3.95%, respectively). The concentration of 150 × 106 MV mL−1 provided only 9.26% of MII. To understand the role of MV, we assessed the expression of 3 microRNA (miRNA-30b, miR-375, and miR-503) that are involved in some key pathways (WNT, MAPK, ERbB, and TGFβ) regulating follicular development and meiotic resumption. The lower rate of MII with the higher concentration of MV is possibly due to the high level of miR-375, which recent literature shows to suppress the TGFβ pathway, leading to impaired oocyte maturation. In conclusion, the oviducal MV, or specific microRNA, are involved in cellular trafficking during oocyte maturation, and their possible use in vitro could facilitate the exploitation of canine reproductive biotechnologies.

116 EFFECTS OF l-GLUTAMINE ON CRYOPRESERVATION OF IMMATURE BOVINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 167
Author(s):  
C. Yamada ◽  
M. D. Goissis ◽  
H. V. A. Caetano ◽  
A. R. S. Coutinho ◽  
M. E. O. A. Assumpção ◽  
...  
Keyword(s):  
Amino Acids ◽  
In Vitro Maturation ◽  
Complex Structure ◽  
Cumulus Cell ◽  
Epifluorescence Microscopy ◽  
Control Group ◽  
Maturation Medium ◽  
Cell Layers ◽  
Bovine Oocytes

The cryopreservation of bovine oocytes remains a challenge despite significant reported progress. Immature bovine oocytes have a complex structure and the conventional cryoprotectants (penetrating cryoprotectants, sugars, and macromolecules) appear to be not sufficient to preserve them efficiently during freezing. Studies on semen and fibroblast cryopreservation indicate that amino acids, particularly l-glutamine, protect enzymes during freezing and increase the post-thaw viability. Therefore, the amino acids may optimize oocyte cryopreservation when associated with conventional cryoprotectants. This work evaluated the effect of l-glutamine on cryopreservation of immature bovine oocytes after in vitro maturation. Oocytes with homogeneous cytoplasm and several cumulus cell layers from slaughterhouse ovaries were distributed randomly in three groups: non-vitrified control, vitrified control, and vitrified with l-glutamine. Oocytes from vitrified groups were exposed for 10 min to PBS + 10% FCS + 10% ethylene glycol (EG) + 0.25 m trehalose (T), and for 30 s to PBS + 10% FCS + 25% EG + 25% dimethylsulfoxide + 0.5 m T at room temperature, adding 80 mm l-glutamine for the third group. Oocytes were loaded into OPS and plunged in liquid nitrogen. For thawing, OPS were immersed in PBS + 10% FCS + 10% EG + 1 m T for three min. Oocytes werethen placed in PBS + 10% FCS + 0.5 m T and in PBS + 10% FCS, remaining three min in each solution. For in vitro maturation, oocytes were washed three times on holding medium (TCM-HEPES + FCS + pyruvate + gentamycin), washed three times in maturation medium (TCM-bicarbonate + FCS + pyruvate + gentamycin + hCG + FSH + estradiol), and cultured in microdrops (90 μL) of maturation medium covered with mineral oil at 38.5°C under 5% CO2 in air and high humidity for 24 h. Oocytes were denuded, fixed in paraformaldehyde and triton, stained with Hoechst 33342, and evaluated under epifluorescence microscopy. Oocytes at metaphase II were considered matured. The group vitrified with l-glutamine had a significantly higher maturation rate than the group vitrified without l-glutamine; however, both had significantly lower maturation rates than the non-vitrified control group. In conclusion, l-glutamine improved the viability of vitrified oocytes. Table 1. Oocyte maturation rates of non-vitrified control, vitrified control, and vitrified with glutamine groups This work was supported by FAPESP 03/08543-1.

scholarly journals Apoptosis in cumulus cells during in vitro maturation of bovine cumulus-enclosed oocytes

Reproduction ◽  
2003 ◽  
pp. 369-376 ◽  
Author(s):  
S Ikeda ◽  
H Imai ◽  
M Yamada
Keyword(s):  
Dna Fragmentation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Treated Group ◽  
Developmental Competence ◽  
Pcr Analysis ◽  
Control Group ◽  
Dependent Manner

The aim of this study was to investigate whether apoptosis occurs in cumulus cells during in vitro maturation (IVM) of bovine cumulus-enclosed oocytes (CEOs). The bovine CEOs obtained from ovaries from an abattoir were cultured for 24 h in IVM medium in the presence or absence of 10% (v/v) fetal bovine serum. The developmental competence of enclosed oocytes, as assessed by the development of the blastocyst after IVF, was significantly higher in the serum-treated group than in the control group. The morphological features of apoptosis that were analysed by orcein staining were hardly detectable in the cumulus cells at the start (0 h) of IVM, but were evident at the end (24 h) of IVM both in the control and serum-treated groups. Genomic DNA was extracted from CEOs at 0, 6, 12, 18 and 24 h of IVM and subjected to ligation-mediated PCR (LM-PCR) to detect apoptotic internucleosomal DNA fragmentation. DNA fragmentation was hardly detectable at the start of IVM, but increased in a time-dependent manner as the IVM culture proceeded. DNA fragmentation was not observed in the oocytes, indicating that fragmentation occurs in cumulus cells. The degree of fragmentation was lower in the serum-treated group compared with the control group. The LM-PCR analysis of DNA extracted from CEOs at 24 h of IVM, in which the DNA had been pretreated with Klenow enzyme or T4 DNA polymerase, revealed that the characteristic forms of the DNA ends generated during cumulus cell apoptosis were mainly 3'-overhangs and blunt ends. In conclusion, the results of the present study demonstrate that cumulus cells in bovine CEOs spontaneously undergo apoptosis during IVM. The degree of apoptosis may be correlated with the developmental competence of the enclosed oocytes.

scholarly journals Effect of D-Glucuronic Acid and N-acetyl-D-Glucosamine Treatment during In Vitro Maturation on Embryonic Development after Parthenogenesis and Somatic Cell Nuclear Transfer in Pigs

Animals ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 1034
Author(s):  
Joohyeong Lee ◽  
Eunhye Kim ◽  
Seon-Ung Hwang ◽  
Lian Cai ◽  
Mirae Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
In Vitro Maturation ◽  
Glucuronic Acid ◽  
Cumulus Cell ◽  
Cortical Granule ◽  
Oocyte Diameter

This study aimed to examine the effects of treatment with glucuronic acid (GA) and N-acetyl-D-glucosamine (AG), which are components of hyaluronic acid (HA), during porcine oocyte in vitro maturation (IVM). We measured the diameter of the oocyte, the thickness of the perivitelline space (PVS), the reactive oxygen species (ROS) level, and the expression of cumulus cell expansion and ROS-related genes and examined the cortical granule (CG) reaction of oocytes. The addition of 0.05 mM GA and 0.05 mM AG during the first 22 h of oocyte IVM significantly increased oocyte diameter and PVS size compared with the control (non-treatment). The addition of GA and AG reduced the intra-oocyte ROS content and improved the CG of the oocyte. GA and AG treatment increased the expression of CD44 and CX43 in cumulus cells and PRDX1 and TXN2 in oocytes. In both the chemically defined and the complex medium (Medium-199 + porcine follicular fluid), oocytes derived from the GA and AG treatments presented significantly higher blastocyst rates than the control after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). In conclusion, the addition of GA and AG during IVM in pig oocytes has beneficial effects on oocyte IVM and early embryonic development after PA and SCNT.

scholarly journals Effects of BMP15 and GDF9 on Ovine Oocytes in an In Vitro Maturation System

2021 ◽  
Author(s):  
◽  
Aanchal Singh
Keyword(s):  
Cell Death ◽  
Litter Size ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Oocyte Quality ◽  
Mature Oocyte ◽  
High Ratio ◽  
High Fecundity ◽  
Species Specific

<p>Oocyte developmental competency is the intrinsic measure of oocyte quality and the capacity for a mature oocyte to support the early stages of embryo development and implantation. Oocyte-secreted factors (OSFs), such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), play a pivotal role in regulating the synchrony of various complex maturation events within the cumulus-oocyte complex (COC) through the induction of paracrine and endocrine signalling. These proteins act synergistically to influence the proliferation and differentiation of granulosa cells (GCs), cumulus cell (CC) expansion, promote survival, ovulation, the attainment of developmental competency and fertility. Species-specific ratios suggest that poly-ovulatory mammals have increased fecundity due to high ratios of GDF9:BMP15, which is directly reflected in their large litter size. Interestingly, it has also been found that higher ratios of GDF9:BMP15 also increased blastocyst rate in sheep implying that these embryos develop from oocytes that are more developmentally competent.  In this study, I investigated the hypothesis that supplementing a commercial in vitro maturation (IVM) system with a high ratio of GDF9:BMP15 would increase the developmental competency sheep oocytes; a species with low-moderate litter size. To test this hypothesis, ovine oocytes were matured in a biphasic IVM system containing GDF9 and BMP15 at three divergent ratios (1:6, 1:1, 6:1). The results herein show that the 6:1 ratio resulted in higher levels of reagent transfer to the ovine oocyte through gap junctions (GJs) after 24 hours of incubation. Similarly, it was also observed that at the higher ratio, glutathione (GSH) levels were higher at 7.5 hours of incubation. The high GDF9:BMP15 ratio also facilitated the increased consumption of pyruvate by the COC consistently throughout the culture period. Importantly, the high GDF9:BMP15 ratio showed higher expression of the gene that encodes GJ (CX43) at 24 hours relative to the control. It was also demonstrated through decreased apoptotic factor (BAX:BCL2) ratios, that the addition of OSFs, regardless of ratio, protected against cell death. In summary, this study provides novel results that support the notion that a high GDF9:BMP15 ratio improves oocyte quality by delaying the timing of meiotic resumption. This subsequently improves the transport of key metabolites and antioxidants to protect against oxidative stress and cell death and aid in the completion of maturation, ultimately resulting in the increased developmental competency observed in high fecundity poly-ovulatory species.</p>

scholarly journals Prediction of maturational competence of feline oocytes using supravital staining of cumulus cells by propidium iodide

Zygote ◽  
2011 ◽  
Vol 20 (4) ◽  
pp. 333-337 ◽  
Author(s):  
Kenzo Uchikura ◽  
Masashi Nagano ◽  
Mitsugu Hishinuma
Keyword(s):  
Propidium Iodide ◽  
Cumulus Cell ◽  
Membrane Integrity ◽  
Cumulus Cells ◽  
Nuclear Maturation ◽  
Non Invasive ◽  
Maturation Rate ◽  
Cell Layers ◽  
The Relationship

SummaryWe examined the relationship between integrity of cumulus cells and nuclear maturation rate after in vitro culture to determine a non-invasive prediction of the maturational competence of feline oocytes. Feline cumulus–oocyte complexes (COCs) were collected from either small (400–800 μm) or large (≥800 μm) follicles. Immediately after collection, cumulus cells were evaluated morphologically (thickness of cumulus cell layers) and stained with propidium iodide (PI), which penetrates only non-viable cells. Cumulus cells without PI staining were judged as having good membrane integrity. After evaluation, COCs were cultured for 30 h and their nuclear maturation rate was determined. The nuclear maturation rate of oocytes derived from large follicles (89.8%) was higher (p < 0.05) than that from small follicles (60.8%). There was no difference in the maturation rate of oocytes from follicles with the same size regardless of cumulus morphology. In contrast, oocytes that had cumulus cells with good membrane integrity showed a higher maturation rate (93.8%) than oocytes with poor cumulus integrity (76.9%) in large follicles (p < 0.05). We conclude that evaluation of membrane integrity of cumulus cells by propidium iodide staining can be used to predict the maturational competence of oocytes.

scholarly journals α-Tocopherol modifies the expression of genes related to oxidative stress and apoptosis during in vitro maturation and enhances the developmental competence of rabbit oocytes

2018 ◽  
Vol 30 (12) ◽  
pp. 1728 ◽  
Author(s):  
M. Arias-Álvarez ◽  
R. M. García-García ◽  
J. López-Tello ◽  
P. G. Rebollar ◽  
A. Gutiérrez-Adán ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Cycle ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Rabbit Model ◽  
Developmental Competence ◽  
Antioxidant Agents ◽  
Junction Protein

The developmental competence of in vitro maturation (IVM) oocytes can be enhanced by antioxidant agents. The present study investigated, for the first time in the rabbit model, the effect of adding α-tocopherol (0, 100, 200 and 400 µM) during IVM on putative transcripts involved in antioxidant defence (superoxide dismutase 2, mitochondrial (SOD2), glutathione peroxidase 1 (GPX1), catalase (CAT)), cell cycle regulation and apoptosis cascade (apoptosis tumour protein 53 (TP53), caspase 3, apoptosis-related cysteine protease (CASP3)), cell cycle progression (cellular cycle V-Akt murine thymoma viral oncogene homologue 1 (AKT1)), cumulus expansion (gap junction protein, alpha 1, 43 kDa (GJA1) and prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclo-oxygenase) (PTGS2)) and metabolism (glucose-6-phosphate dehydrogenase (G6PD)). Meiotic progression, mitochondrial reallocation, cumulus cell apoptosis and the developmental competence of oocytes after IVF were also assessed. Expression of SOD2, CAT, TP53, CASP3 and GJA1 was downregulated in cumulus–oocyte complexes (COCs) after IVM with 100 μM α-tocopherol compared with the group without the antioxidant. The apoptotic rate and the percentage of a non-migrated mitochondrial pattern were lower in COCs cultured with 100 μM α-tocopherol, consistent with better-quality oocytes. In fact, early embryo development was improved when 100 μM α-tocopherol was included in the IVM medium, but remained low compared with in vivo-matured oocytes. In conclusion, the addition of 100 μM α-tocopherol to the maturation medium is a suitable approach to manage oxidative stress and apoptosis, as well as for increasing the in vitro developmental competence of rabbit oocytes.

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

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

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

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Germinal Vesicle Breakdown ◽  
Significant Difference ◽  
Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

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