scholarly journals DNA damage in cumulus cells generated after the vitrification of in vitro matured porcine oocytes and its impact on fertilization and embryo development

2021 ◽  
Author(s):  
Alma López ◽  
Miguel Betancourt ◽  
Yvonne Ducolomb ◽  
Juan José Rodríguez ◽  
Eduardo Casas ◽  
...  
Keyword(s):  
Dna Damage ◽  
Embryo Development ◽  
Mammalian Species ◽  
Tail Length ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Limiting Factor ◽  
Oocyte Vitrification ◽  
Development Rates

Abstract Oocyte vitrification has become an important tool for the improvement of assisted reproduction in humans and other mammalian species. The toxicity and use of high cryoprotectants concentrations have been a limiting factor for cryopreservation success. The evaluation of the DNA damage generated in cumulus cells after mature cumulus-oocyte complexes vitrification can be considered as an indicator of oocyte quality since these cells play important roles in oocyte developmental competence. Alterations produced in these cells could compromise oocyte maturation, fertilization, and embryo development. Therefore, the aim of this study was to determine if matured cumulus-oocyte complexes exposure to cryoprotectants or vitrification affects both oocytes and cumulus cells viability, but also if DNA damage is generated in cumulus cells, affecting fertilization and embryo development. The DNA damage in cumulus cells was measured using the alkaline comet assay and expressed as Comet Tail Length and Olive Tail Moment. Results demonstrate that oocyte exposure to cryoprotectants or vitrification reduced oocyte and cumulus cells viability compared to control. Also, significantly higher DNA damage was generated in the cumulus cells after exposure to cryoprotectants and vitrification compared to control. In addition, fertilization and embryo development rates also decreased after exposure to cryoprotectants and vitrification. It was also found that fertilization and embryo development rates in granulose-intact oocytes were significantly higher compared to denuded oocytes in the control groups. However, a decline in oocyte fertilization and embryo development to the blastocyst stage was observed after cryoprotectants exposure or vitrification. This could be attributed to the reduction in both cell types viability, and the generation of DNA damage in the cumulus cells. These findings will allow to understand some of the mechanisms of oocyte damage after vitrification, and the search for new vitrification strategies to increase fertilization and embryo development rates.

scholarly journals DNA damage in cumulus cells generated after the vitrification of in vitro matured porcine oocytes and its impact on fertilization and embryo development

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Alma López ◽  
Miguel Betancourt ◽  
Yvonne Ducolomb ◽  
Juan José Rodríguez ◽  
Eduardo Casas ◽  
...  
Keyword(s):  
Dna Damage ◽  
Embryo Development ◽  
Tail Length ◽  
Cumulus Cells ◽  
Cell Types ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Development Rates

Abstract Background The evaluation of the DNA damage generated in cumulus cells after mature cumulus-oocyte complexes vitrification can be considered as an indicator of oocyte quality since these cells play important roles in oocyte developmental competence. Therefore, the aim of this study was to determine if matured cumulus-oocyte complexes exposure to cryoprotectants (CPAs) or vitrification affects oocytes and cumulus cells viability, but also if DNA damage is generated in cumulus cells, affecting fertilization and embryo development. Results The DNA damage in cumulus cells was measured using the alkaline comet assay and expressed as Comet Tail Length (CTL) and Olive Tail Moment (OTM). Results demonstrate that oocyte exposure to CPAs or vitrification reduced oocyte (75.5 ± 3.69%, Toxicity; 66.7 ± 4.57%, Vitrification) and cumulus cells viability (32.7 ± 5.85%, Toxicity; 7.7 ± 2.21%, Vitrification) compared to control (95.5 ± 4.04%, oocytes; 89 ± 4.24%, cumulus cells). Also, significantly higher DNA damage expressed as OTM was generated in the cumulus cells after exposure to CPAs and vitrification (39 ± 17.41, 33.6 ± 16.69, respectively) compared to control (7.4 ± 4.22). In addition, fertilization and embryo development rates also decreased after exposure to CPAs (35.3 ± 16.65%, 22.6 ± 3.05%, respectively) and vitrification (32.3 ± 9.29%, 20 ± 1%, respectively). It was also found that fertilization and embryo development rates in granulose-intact oocytes were significantly higher compared to denuded oocytes in the control groups. However, a decline in embryo development to the blastocyst stage was observed after CPAs exposure (1.66 ± 0.57%) or vitrification (2 ± 1%) compared to control (22.3 ± 2.51%). This could be attributed to the reduction in both cell types viability, and the generation of DNA damage in the cumulus cells. Conclusion This study demonstrates that oocyte exposure to CPAs or vitrification reduced viability in oocytes and cumulus cells, and generated DNA damage in the cumulus cells, affecting fertilization and embryo development rates. These findings will allow to understand some of the mechanisms of oocyte damage after vitrification that compromise their developmental capacity, as well as the search for new vitrification strategies to increase fertilization and embryo development rates by preserving the integrity of the cumulus cells.

168 INHIBITION OF BOVINE OOCYTE CUMULUS CELL PROGESTERONE SYNTHESIS DURING IN VITRO MATURATION AFFECTS CHROMOSOME ALIGNMENT AND SPINDLE INTEGRITY IN FERTILIZED EGGS AND EARLY EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 198
Author(s):  
E. Daly ◽  
A. G. Fahey ◽  
M. M. Herlihy ◽  
T. Fair
Keyword(s):  
Embryo Development ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
Spindle Formation ◽  
Vitro Fertilization ◽  
Time Treatment

We have previously demonstrated the importance of progesterone (P4) synthesis by cumulus cells during oocyte maturation in vitro (IVM) for bovine oocyte acquisition of developmental competence and subsequent embryo development (Aparicio et al. 2011 Biol. Reprod. 84). The aim of this study was to identify key processes that may be deregulated by the inhibition of P4 signalling in the cumulus–oocyte complex (COC) during IVM. To this end, good quality immature COC were placed in IVM medium [TCM-199 supplemented with 10% (vol/vol) FCS and 10 ng mL–1 epidermal growth factor] and cultured at 39°C for 22 h in a humidified atmosphere containing 5% CO2, in the presence or absence of 10 μM trilostane (which blocks P4 synthesis by inhibiting 3 β-hydroxysteroid dehydrogenase; Stegram Pharmaceuticals Ltd., Surrey, UK). Matured COC were washed and placed in 250 μL of fertilization medium (25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6 mg mL–1 fatty acid-free BSA, and 10 mg mL–1 heparin). In vitro fertilization (IVF) was performed with 250 μL of frozen–thawed semen at a final concentration of 1 × 106 spermatozoa mL–1 at 39°C under 5% CO2 during 20 h. Presumptive zygotes were denuded, washed, and transferred to 25-μL culture droplets (SOF + 5% FCS) at 39°C under 5% CO2, 90% of N2, and 5% O2 atmosphere with maximum humidity. Subsets of presumptive fertilized eggs and developing embryos were recovered at 6, 72, 120, and 192 h postinsemination (hpi) and processed for confocal whole-mount immunocytochemistry. The meiotic and mitotic spindles and chromosomes were visualised by immunofluorescent labelling of α-tubulin and 4′,6-diamindino-2-phenylindole (DAPI), respectively, and classified as normal if the chromosomes were correctly aligned or appropriately segregated, or abnormal if lagging chromosomes or abnormal chromosome segregation were observed. Samples were collected from 5 replicates (n = 50 zygotes/embryos per treatment, per timepoint) and a total of 157 spindles were observed. Logistic regression analysis was conducted to determine the probability of abnormal spindle formation. The incidence of spindle abnormality was regressed on time, treatment, and treatment by time. For all time points, there was significant reduction in the odds of abnormal spindle formation in control samples versus trilostane-treated samples (P < 0.001). In conclusion, our data imply a role for P4 signalling in maintaining spindle integrity during oocyte meiotic maturation and progression through the initial mitotic divisions of early embryo development in cattle.

Glutathione content and embryo development after in vitro fertilisation of pig oocytes matured in the presence of a thiol compound and various concentrations of cysteine

Zygote ◽  
1999 ◽  
Vol 7 (3) ◽  
pp. 203-210 ◽  
Author(s):  
Lalantha R. Abeydeera ◽  
Wei-Hua Wang ◽  
Thomas C. Cantley ◽  
Randall S. Prather ◽  
Billy N. Day
Keyword(s):  
Embryo Development ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
In Vitro Fertilisation ◽  
Thiol Compound ◽  
Nuclear Maturation ◽  
Mean Differences ◽  
Pronuclear Formation

The present study examined the effect of different concentrations of cysteine in the presence of a thiol compound, β-mercaptoethanol (BME), during in vitro maturation (IVM) of pig oocytes on cumulus expansion, nuclear maturation, intracellular glutathione (GSH) level and subsequent embryonic development after in vitro fertilisation (IVF). In experiment 1, oocytes were matured in NCSU 23 medium containing 10% porcine follicular fluid, 25 μM BME, 0.5 μg/ml LH, 0.5 μg/ml FSH and 0, 0.1, 0.2 or 0.4 mg/ml cysteine for 20–22 h and then without hormonal supplements for an additional 20–22 h. After culture, cumulus cells were removed and a proportion of oocytes fixed to examine the rate of nuclear maturation. The remaining oocytes were co-incubated with spermatozoa for 5–6 h and putative zygotes were transferred to NCSU 23 medium containing 0.4% bovine serum albumin for 144 h. A proportion of putative zygotes were fixed 12 h after insemination to examine fertilisation parameters. In experiment 2, oocytes were matured as in experiment 1 and the GSH content was measured by a DTNB-GSSG reductase recycling assay. No mean differences among treatments were observed in nuclear maturation (78–89%). The mean differences in penetration rate (69–77%), polyspermy rate (31–40%), male pronuclear formation rate (93–96%) or mean number of sperm per oocyte (1.5-1.8) were not affected by the presence or absence of cysteine during oocyte maturation. Also no difference was observed in cleavage rates 48 h after insemination. However, compared with no addition (19%), the presence of 0.1-0.4 mg/ml cysteine during IVM increased (p < 0.001) the proportion of blastocysts (32–39%) at 144 h. In comparison with controls (5.6 pmol/oocyte), the GSH content of oocytes matured in the presence of cysteine was significantly (p < 0.001) higher (13–15 pmol/oocyte) with no mean differences among different cysteine concentrations. The results indicate that in the presence of a thiol compound, supplementation of IVM medium with cysteine can increase the GSH level and improve the developmental competence of pig oocytes following fertilisation. Further, no effect on either GSH level or embryo development was observed by increasing the levels of cysteine supplementation from 0.1 to 0.4 mg/ml.

scholarly journals cAMP Modulators before In Vitro Maturation Decrease DNA Damage and Boost Developmental Potential of Sheep Oocytes

Animals ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 2512
Author(s):  
Daniela-Alejandra Medina-Chávez ◽  
Irene Sánchez-Ajofrín ◽  
Patricia Peris-Frau ◽  
Carolina Maside ◽  
Vidal Montoro ◽  
...  
Keyword(s):  
Dna Damage ◽  
In Vitro Maturation ◽  
Phosphodiesterase Inhibitor ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Assisted Reproduction Technologies ◽  
Underlying Mechanisms

To date, the underlying mechanisms by which cAMP modulators act during in vitro maturation to improve oocyte developmental competence are poorly understood. Here, we sought to fill this knowledge gap by evaluating the use of phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and adenylyl cyclase activator forskolin during a culture period of 2 h before in vitro maturation (pre-IVM) on the nuclear and cytoplasmic maturation features in essential organelles, cumulus cells activity, and in vitro developmental potential of sheep oocytes. Results showed that pre-IVM treatment significantly decreased (p < 0.05) the DNA damage of mature oocytes (pre-IVM = 2.08% ± 3.51% vs. control = 20.58% ± 3.51%) and increased (p ≤ 0.05) expanded blastocyst rates compared to the control (from the total of oocytes: pre-IVM = 23.89% ± 1.47% vs. control = 18.22% ± 1.47%, and from the cleaved embryos: pre-IVM = 45.16% ± 1.73% vs. control = 32.88% ± 1.73%). Considering that oocytes are highly vulnerable to the accumulation of DNA damage because of exposure to in vitro culture conditions, our results suggest that the modulation of intra-oocyte cAMP levels with forskolin and IBMX before IVM might afford oocytes a more effective DNA repair mechanism to overcome damage obstacles and ultimately improve developmental competence. This previously unappreciated action of cAMP modulators could help to develop improved methods for assisted reproduction technologies in animal and clinical research.

scholarly journals Antioxidant Nobiletin Enhances Oocyte Maturation and Subsequent Embryo Development and Quality

2020 ◽  
Vol 21 (15) ◽  
pp. 5340
Author(s):  
Yulia N. Cajas ◽  
Karina Cañón-Beltrán ◽  
Magdalena Ladrón de Guevara ◽  
María G. Millán de la Blanca ◽  
Priscila Ramos-Ibeas ◽  
...  
Keyword(s):  
Embryo Development ◽  
Biological Effects ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Cortical Granules ◽  
Cleavage Rate ◽  
Cytoplasmic Maturation

Nobiletin is a polymethoxylated flavonoid isolated from citrus fruits with wide biological effects, including inhibition of reactive oxygen species (ROS) production and cell cycle regulation, important factors for oocyte in vitro maturation (IVM). Therefore, the objective of the present study was to evaluate the antioxidant activity of nobiletin during IVM on matured bovine oocyte quality (nuclear and cytoplasmic maturation; oocyte mitochondrial activity; intracellular ROS and glutathione (GSH) levels) and their developmental competence, steroidogenesis of granulosa cells after maturation, as well as quantitative changes of gene expression in matured oocytes, their cumulus cells, and resulting blastocysts. Bovine cumulus-oocyte complexes were in vitro matured in TCM-199 +10% fetal calf serum (FCS) and 10 ng/mL epidermal growth factor (EGF) (Control) supplemented with 10, 25, 50, or 100 μM of nobiletin (Nob10, Nob25, Nob50, and Nob100, respectively) or 0.1% dimethyl sulfoxide (CDMSO: vehicle for nobiletin dilution). A significantly higher percentage of matured oocytes in metaphase II was observed in Nob25 and Nob50 compared to other groups. Similarly, cleavage rate and cumulative blastocyst yield on Days 7 and 8 were significantly higher for Nob25 and Nob50 groups. Oocytes matured with 25 and 50 μM nobiletin showed a higher rate of migration of cortical granules and mitochondrial activity and a reduction in the ROS and GSH content in comparison with all other groups. This was linked to a modulation in the expression of genes related to metabolism (CYP51A1), communication (GJA1), apoptosis (BCL2), maturation (BMP15 and MAPK1), and oxidative stress (SOD2 and CLIC1). In conclusion, nobiletin offers a novel alternative for counteracting the effects of the increase in the production of ROS during IVM, improves oocyte nuclear and cytoplasmic maturation, and subsequent embryo development and quality in cattle.

Equine maternal aging affects oocyte lipid content, metabolic function and developmental potential

Reproduction ◽  
2021 ◽  
Vol 161 (4) ◽  
pp. 399-409
Author(s):  
Giovana D Catandi ◽  
Yusra M Obeidat ◽  
Corey D Broeckling ◽  
Thomas W Chen ◽  
Adam J Chicco ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Mammalian Species ◽  
Age Groups ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Metabolic Function ◽  
Mtdna Copy Number ◽  
Developmental Potential ◽  
Maternal Aging

Advanced maternal age is associated with a decline in fertility and oocyte quality. We used novel metabolic microsensors to assess effects of mare age on single oocyte and embryo metabolic function, which has not yet been similarly investigated in mammalian species. We hypothesized that equine maternal aging affects the metabolic function of oocytes and in vitro-produced early embryos, oocyte mitochondrial DNA (mtDNA) copy number, and relative abundance of metabolites involved in energy metabolism in oocytes and cumulus cells. Samples were collected from preovulatory follicles from young (≤14 years) and old (≥20 years) mares. Relative abundance of metabolites in metaphase II oocytes (MII) and their respective cumulus cells, detected by liquid and gas chromatography coupled to mass spectrometry, revealed that free fatty acids were less abundant in oocytes and more abundant in cumulus cells from old vs young mares. Quantification of aerobic and anaerobic metabolism, respectively measured as oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in a microchamber containing oxygen and pH microsensors, demonstrated reduced metabolic function and capacity in oocytes and day-2 embryos originating from oocytes of old when compared to young mares. In mature oocytes, mtDNA was quantified by real-time PCR and was not different between the age groups and not indicative of mitochondrial function. Significantly more sperm-injected oocytes from young than old mares resulted in blastocysts. Our results demonstrate a decline in oocyte and embryo metabolic activity that potentially contributes to the impaired developmental competence and fertility in aged females.

Dose-dependent effects of gonadotropin on oocyte developmental competence and apoptosis

2011 ◽  
Vol 23 (8) ◽  
pp. 990 ◽  
Author(s):  
Shan Liu ◽  
Huai L. Feng ◽  
Dennis Marchesi ◽  
Zi-Jiang Chen ◽  
Avner Hershlag
Keyword(s):  
Embryo Development ◽  
Assisted Reproductive Technologies ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Beneficial Effects ◽  
Nuclear Maturation ◽  
High Concentrations ◽  
Vitro Development

The aim of the present study was to evaluate the effect of gonadotropins (Gn) on oocyte maturation, developmental competence and apoptosis in an animal model. Bovine cumulus–oocyte complexes (COCs) were matured for 24 h in media supplemented with varying concentrations of Bravelle (B), B + Menopur (B + M) or B + Repronex (B + R) (Ferring Pharmaceuticals, Parsiappany, NJ, USA). Then, nuclear maturation, embryo development, and apoptosis in cumulus cells and oocytes were evaluated. Low to moderate Gn concentrations (75–7500 mIU mL–1) effectively improved nuclear maturation and in vitro development. Higher concentrations of Gn (75 000 mIU mL–1) did not have any added beneficial effects and nuclear maturation and blastocyst rates in the presence of these concentrations were comparable to control (P > 0.05). Most COCs showed slight apoptosis when exposed to 75, 750 and 7500 mIU mL–1 Gn; however, when the concentration was increased to 75 000 mIU mL–1, the proportion of moderately apoptotic COCs increased. In conclusion, extremely high concentrations of Gn have detrimental effects on oocyte nuclear maturation and embryo development and increase apoptosis in cumulus cells, suggesting the importance of judicious use of Gn in assisted reproductive technologies (ART).

scholarly journals Lipids and oocyte developmental competence: the role of fatty acids and β-oxidation

Reproduction ◽  
2014 ◽  
Vol 148 (1) ◽  
pp. R15-R27 ◽  
Author(s):  
Kylie R Dunning ◽  
Darryl L Russell ◽  
Rebecca L Robker
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Unsaturated Fatty Acids ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Oocyte Developmental Competence

Metabolism and ATP levels within the oocyte and adjacent cumulus cells are associated with quality of oocyte and optimal development of a healthy embryo. Lipid metabolism provides a potent source of energy and its importance during oocyte maturation is being increasingly recognised. The triglyceride and fatty acid composition of ovarian follicular fluid has been characterised for many species and is influenced by nutritional status (i.e. dietary fat, fasting, obesity and season) as well as lactation in cows. Lipid in oocytes is a primarily triglyceride of specific fatty acids which differ by species, stored in distinct droplet organelles that re-localise during oocyte maturation. The presence of lipids, particularly saturated vs unsaturated fatty acids, in in vitro maturation systems affects oocyte lipid content as well as developmental competence. Triglycerides are metabolised by lipases that have been localised to cumulus cells as well as oocytes. Fatty acids generated by lipolysis are further metabolised by β-oxidation in mitochondria for the production of ATP. β-oxidation is induced in cumulus–oocyte complexes (COCs) by the LH surge, and pharmacological inhibition of β-oxidation impairs oocyte maturation and embryo development. Promoting β-oxidation with l-carnitine improves embryo development in many species. Thus, fatty acid metabolism in the mammalian COC is regulated by maternal physiological and in vitro environmental conditions; and is important for oocyte developmental competence.

127 L-Carnitine Supplementation During In Vitro Maturation Improves Developmental Competence of Canine Oocytes

2018 ◽  
Vol 30 (1) ◽  
pp. 203 ◽  
Author(s):  
A. Salama ◽  
M. Fathi ◽  
M. R. Badr ◽  
A. R. Moawad
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Assisted Reproductive Technologies ◽  
Mammalian Species ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Carnitine Supplementation ◽  
Cell Stage ◽  
Nuclear Maturation

In vitro embryo production (IVP) in the domestic bitch is important for conservation of endangered canids. Compared with various domestic animals, the development of assisted reproductive technologies (ART) in the dog has lagged behind, mainly due to the low percentage of oocytes that can reach metaphase II (MII) stage after in vitro maturation (IVM). Beneficial effects of l-carnitine (LC) on embryonic development in culture have been reported in many mammalian species; however, no studies have been conducted in dogs. The aim of the present study was to investigate the effect of LC supplementation during IVM of canine oocytes on nuclear maturation, fertilization status, and pre-implantation development following IVM/IVF. Cumulus-oocyte complexes (COC) were collected by slicing ovaries obtained from dogs (n = 20, 1 to 6 years of age) after ovariohysterectomy. The COC were subjected to IVM for 72 h in a medium (TCM-199) supplemented with LC at different concentrations (0.1, 0.3, 0.6, 1.0, or 2.0 mg mL−1) or without LC supplements (0 mg mL−1; control). Matured oocytes were fertilized in vitro with frozen–thawed spermatozoa, and presumptive zygotes were cultured in SOF medium for 7 days. Frequencies of nuclear maturation (72 h post-IVM), fertilization rates (18 h post-insemination), and embryo development (Days 2 to 5 post-insemination) were evaluated. Data were analysed by one-way ANOVA followed by Tukey’s multiple comparisons test. Supplementation of IVM medium with 0.3 or 0.6 mg mL−1 LC significantly improved (P ≤ 0.05) maturation (35.4% and 41.4%) and fertilization (21.3% and 25.8%) rates compared with the controls and with other LC-supplemented groups; values ranged from 20.1% to 25.0% for maturation and from 12.1% to 14.6% for fertilization. Cleavage (2- to 16-cell stages) was significantly higher (P ≤ 0.05) in the 0.6 mg mL−1 LC supplemented group than the 0.3 mg mL−1 supplemented group (16.3% v. 13.3%). These values were significantly higher (P ≤ 0.05) than those in other groups. Interestingly, 4.5% of IVM/IVF oocytes were developed to morula in 0.6 mg mL−1 LC supplemented group which was significantly higher (P ≤ 0.05) than those developed in the 0.3 mg mL−1 supplemented group (1.0%). No embryos developed beyond the 2- to 16-cell stage in the rest of the groups. In conclusion, l-carnitine supplementation during IVM is particularly efficient in improving nuclear maturation and pre-implantation embryo development of canine oocytes after IVF. These outcomes are important for the improvement of IVM conditions that can advance the efficiency of ART in dogs.

scholarly journals 248 EMBRYO DEVELOPMENT IN MICRODROPS AND MICROCHANNELS: COMPARISON OF NCSU23 SEQUENTIAL AND NON-SEQUENTIAL CULTURE MEDIUM

2005 ◽  
Vol 17 (2) ◽  
pp. 274
Author(s):  
A.S. Lima ◽  
C.E. Ferguson ◽  
M.B. Wheeler
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Culture Media ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
The Other ◽  
Cell Stage ◽  
Development Rates ◽  
Hatching Rates

The in vitro culture systems used to produce pig embryos generally result in few embryos developing to the blastocyst stage. The use of pyruvate (pyr) and lactate (lac) during the culture of zygotes to the 8-cell stage followed by glucose (glu) supplementation replacing pyr and lac appears to be beneficial for embryo development in the pig. The aim of this study was to compare the embryo development rates from pig oocytes fertilized with and without cumulus cells in 100-μL microdrops (MD) and cultured in 100-μL MD or microchannels (MC), using NCSU23 containing 8 mg/mL of BSA and supplemented with (1) glu or (2) pyr/lac or (3) pyr/lac for the first three days and then with just glu for the remainder of culture period (pyr/lac-glu). Sow oocytes were matured in TCM199 supplemented with gonadotropins for the first 22 h, and for an additional 22 h without hormones. After 44 h of maturation, oocytes were placed in MD of modified tris-buffered medium to be fertilized using 3 × 105 sperm/mL. Oocytes were divided into two groups for fertilization: with and without cumulus cells. Following 6 h of fertilization, all inseminated oocytes were washed, divided into groups of 15, allotted to the three culture media treatment groups as described above, and incubated in either MD or MC. With the exception of one treatment there were no significant differences in development rates among embryos cultured in MD or MC, hence data were pooled from these two culture devices. Only oocytes fertilized without cumulus cells and cultured in pyr/lac in MC appeared to have lower rates of blastocyst formation (11.67%) than those cultured in MD (26.67%) in the same culture medium. When the six treatments were compared, oocytes fertilized with cumulus cells and cultured in glu had significantly higher (P < 0.05) blastocyst rates and hatching rates compared with the other treatments, with the exception of those fertilized without cumulus cells and cultured in pyr/lac-glu. There were no significant differences among other treatments in Day 7 blastocyst or in Day 9 hatching rates. In conclusion, both culture devices can be used to reach similar blastocyst rates with different treatments. In this experiment, the removal of cumulus cells before fertilization appeared to enhance embryo development in vitro when sequential media are used. On the other hand, the presence of cumulus cells before fertilization seems to enhance embryo development when non-sequential glu medium is used. Table 1. Embryo development rates on Day 9 for three different culture treatments

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