Effect of centrifugation on early embryonic development and parthenogenetic activation of bovine oocytes matured in vitro

2001 ◽  
Vol 13 (6) ◽  
pp. 383 ◽  
Author(s):  
Jin-Tae Chung ◽  
Bruce R. Downey ◽  
Robert F. Casper ◽  
Ri-Cheng Chian
Keyword(s):  
Embryonic Development ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Early Embryonic Development ◽  
Ionophore A23187 ◽  
Vitro Fertilization ◽  
Parthenogenetic Activation ◽  
Treatment Groups ◽  
Bovine Oocytes

This study examined the fertilization, early developmental competence and capacity for parthenogenetic activation of bovine oocytes matured in vitro after centrifugation. Immature oocytes were cultured in tissue culture medium 199 supplemented with 10% fetal bovine serum and 75 mIU mL–1 FSH + LH at 5% CO2 to facilitate maturation. After culture for 24 or 30 h, the metaphase-II stage oocytes were centrifuged at 3000, 5000, 7000 or 10000g for 5 min before in vitro fertilization or parthenogenetic activation. Frozen–thawed bull semen was used for in vitro fertilization. For parthenogenetic activation, the oocytes were exposed to 20 M calcium ionophore A23187 for 5 min at room temperature. Fertilization rates were not different between control and treatment groups (87.7% v. 74.6%, 73.4%, 75.9% and 76.4% respectively). Also, there were no differences in early embryonic development between control and treatment groups (rates of blastocyst formation were 21.1% v. 20.2%, 28.8%, 31.2% and 24.1% respectively). When the oocytes were centrifuged at various speeds alone, the activation rate of oocytes was significantly higher (P<0.05) in the 10 000g treatment group compared with control (10.8% v. 0.0%). There were no differences in the activation rates of oocytes between control and treatment groups at speeds up to 7000g (70.9% v. 71.9%, 78.3% and 77.2% respectively) after centrifugation and stimulation with Ca2+-ionophore. However, the activation rate of oocytes was significantly higher (P<0.05) in the 10 000g treatment group compared with control (70.9% v. 83.1%). In addition, the percentage of activated oocytes with diploid formation was significantly higher in the oocytes after centrifugation at 10 000g and stimulation with calcium ionophore A23187 than in the control (18.4% v. 7.1%). These results indicate that centrifugation of oocytes matured in vitro has no detrimental effect on fertilization and subsequent early embryonic development. They also indicate that the oocytes might be parthenogenetically activated after centrifugation and that high-speed centrifugation may induce activation of some oocytes. The results suggest that the optimal speed for centrifugation of bovine oocytes might be ≤7000g to enhance the visibility of nuclear elements for further micromanipulation.

205 CAPACITATION OF STALLION SPERMATOZOA EVALUATED BY FERTILIZATION OF BOVINE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 181
Author(s):  
M. R. Hudson ◽  
G. E. Seidel Jr ◽  
E. L. Squires ◽  
B. E. Spizzirri ◽  
D. J. Walker ◽  
...  
Keyword(s):  
Calcium Ionophore ◽  
Cumulus Cells ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Cell Divisions ◽  
Bovine Oocytes

In vitro fertilization in the horse does not work reliably. Several methods of capacitating sperm in other species fail in the horse. The goal of this experiment was to develop a method to capacitate equine spermatozoa using calcium ionophore A23187 or phosphatidylcholine 12 (PC12). We also studied effects of maturing bovine oocytes for 24 or 28 h on fertilizability by capacitated equine sperm, hypothesizing that longer maturation would yield oocytes more easily fertilized by equine spermatozoa. Two sets of bovine oocytes were aspirated from 3 to 8 mm follicles of abattoir ovaries 4 h apart, but fertilized at the same time. On the day of fertilization, semen from 1 of 3 stallions was collected, evaluated, and centrifuged through 33% Percoll to remove seminal plasma. The resultant pellet was extended to 5 × 107 cells mL–1 in M199 containing 0.6% BSA, 2 mm caffeine, and 5 mm CaCl2. Sperm were treated with A23187 (1 or 3 μm) or PC12 (40 or 70 μm) or both A23187 and PC12 (1 μm/40 μm) in 500- μL aliquots. Sperm were incubated at 39°C for 10 min (for A23187 and combination treatments) or 15 min (for PC12 treatments), and then diluted 1:20 for fertilization. Oocytes from each maturation time were fertilized using the same semen preparation for each treatment. Oocytes and sperm were incubated together for 18 h in FCDM in 5% CO2 at 39°C (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596). Presumptive zygotes were cultured for 30 h in CDM-1, vortexed to remove cumulus cells, and evaluated for cleavage. Oocytes were also co-incubated with killed sperm to determine the level of parthenogenesis. Cleaved embryos were stained with orcein to ensure that each cell had a nucleus. Number of cell divisions were recorded as 0 for a 1-cell, 1 for a 2-cell, 1.5 for a 3-cell, etc. More oocytes cleaved after 28 h (18%) than 24 h (14%) maturation (P < 0.01). Sperm of Stallion 1 resulted in higher overall cleavage (24%) than Stallions 2 or 3 (11 and 12%; P < 0.01). Highest cleavage was seen with 28 h maturation and 70 μm PC12 and 3 μm A23187 (27 and 24%, respectively). The most cell divisions were seen with 28 h maturation and 70 μm PC12 (0.48); 28 of the 49 cleaved in this treatment reached ≥4-cell stage. In conclusion, both A23187 and PC12 were able to capacitate equine sperm in a dose-dependent manner as determined from cleavage of bovine oocytes matured for 28 h; maturation for the conventional 24 h was an inferior model for this purpose. Table 1. Mean responses of bovine oocytes fertilized by equine sperm

scholarly journals Coenzyme Q10 Supplementation Effects on In Vitro Maturation, Fertilization, and Early Embryonic Development in Pigs

2019 ◽  
Vol 119 (2) ◽  
pp. 2
Author(s):  
Caitlin Streacker ◽  
Brian D. Whitaker
Keyword(s):  
Embryonic Development ◽  
Oocyte Maturation ◽  
Coenzyme Q10 ◽  
Early Embryonic Development ◽  
Mitochondrial Activity ◽  
Mitochondrial Membranes ◽  
Vitro Fertilization ◽  
Treatment Groups ◽  
Development Rates

The objective of this study was to determine the reduction of polyspermic penetration, and increase of mitochondrial activity, in early pig embryonic development by supplementing different concentrations of coenzyme Q10 during oocyte maturation. Oocytes (n = 1,100) were supplemented during the last 24 h of maturation with 0 (control), 10, 50, or 100 μM of coenzyme Q10. After in vitro fertilization (IVF), embryos were evaluated for fertilization kinetics (penetration, polyspermic penetration, male pronuclear formation), and subsequent embryonic development and mitochondrial activity. Supplementation of 100 μM coenzyme Q10 was detrimental to the oocytes, as they had significantly lower (p < 0.05) fertilization kinetic and early embryonic development rates to the other treatment groups. There were no differences in fertilization kinetic and early embryonic development rates between the 0, 10 and 50 μM coenzyme Q10 treatment groups. Oocytes, matured in medium supplemented with 50 μM coenzyme Q10, ultimately developed into embryos with a significantly greater (p < 0.05) presence of intact mitochondrial membranes (observed at both 48 and 144 h post-IVF) compared to oocytes not supplemented with coenzyme Q10. In summary, supplementation of 100 μM coenzyme Q10 during oocyte maturation is detrimental, yet supplementation of 50 μM coenzyme Q10 leads to a higher occurrence of intact mitochondrial membranes in the in vitro produced pig embryos.

139 Effect of protein and calcium ionophore A23187 concentration on hyperactivated motility and acrosome status of stallion sperm

2019 ◽  
Vol 31 (1) ◽  
pp. 195
Author(s):  
I. Ortiz ◽  
H. Resende ◽  
M. Felix ◽  
C. Love ◽  
K. Hinrichs
Keyword(s):  
Repeated Measures ◽  
Calcium Influx ◽  
Semen Analysis ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Computer Assisted ◽  
Ionophore A23187 ◽  
Variable Effect ◽  
Vitro Fertilization

In vitro fertilization does not occur readily in the horse. Fertilization can be achieved using sperm treated with the calcium ionophore A23187 (CaI), but rates are low and variable. In order to fertilize, it is thought that the sperm must show hyperactivated motility and undergo the acrosome reaction. The presence of protein in the media is thought to suppress the effect of CaI, but protein is needed for maintenance of sperm motility. Therefore, the objective of this study was to assess the effect of CaI in the presence or absence of protein on the acrosome and on hyperactivated motility of equine sperm. For this purpose, sperm from 4 stallions were exposed for 10min at 37°C to vehicle or to 1 (C1), 5 (C5) or 10 (C10) μM CaI, with (BSA) or without (N) 7mg mL−1 BSA. The sperm were then washed and incubated at 37°C for 2h. Total and hyperactivated motility were measured by computer-assisted semen analysis. Sperm were considered hyperactivated if curvilinear velocity was &gt;180μm s−1, amplitude of lateral head displacement was &gt;12μm, linearity was &lt;30% and fractal dimension value was &gt;1.3. The percentage of live acrosome-reacted sperm was measured by flow cytometry after staining with propidium iodide and Pisum sativum agglutinin. Data were analysed by repeated-measures 2-way ANOVA. Results were expressed as mean±standard error. Total motility in C5 and C10 treatments was significantly decreased in relation to control (BSA-vehicle) starting at 30min of incubation (35.42±13.57 to 28.20±13.10% v. 71.72±9.21%, respectively; P&lt;0.05). Hyperactivated motility was significantly lower in C10, C5 and N-C1 than in control after 2h of incubation (1.46±0.64v. 3.10±0.58%, respectively). Live acrosome-reacted sperm were significantly higher (P&lt;0.05) for BSA-C5 (14.04±1.99%) and BSA-C10 (14.85±2.52%) than for control (7.50±1.62%) after 2h of incubation. The exposure to sperm of concentrations ≥5μM CaI was associated with loss of motility from 30min of incubation on. However, 2h of incubation after ≥5 μM CaI in the presence of BSA were needed to increase the percentage of live acrosome-reacted sperm. This mismatch between motility and acrosome response helps to clarify the reasons for the variable effect of sperm CaI treatment on equine IVF. Further studies measuring calcium influx and assessing the effect of sperm pre-incubation on CaI response are needed to explore mechanisms for equine in vitro sperm capacitation.

Intracellular pH increase accompanies parthenogenetic activation of porcine, bovine and murine oocytes

2000 ◽  
Vol 12 (4) ◽  
pp. 201 ◽  
Author(s):  
Nancy T. Ruddock ◽  
Zoltán Macháty ◽  
Randall S. Prather
Keyword(s):  
Intracellular Ph ◽  
Prolonged Exposure ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Oocyte Activation ◽  
Parthenogenetic Activation ◽  
Sea Urchin Egg ◽  
Porcine Oocyte ◽  
Bovine Oocytes

Although an intracellular pH (pH i ) increase at the time of fertilization is necessary for activation of the sea urchin egg, recent reports in the mouse and rat have indicated that there is not a pHi increase during fertilization or during 7% ethanol activation in the mouse. It has been suggested that mammals may have lost the need for a pH i increase at the time of fertilization and the present study reports significant pH i changes during parthenogenetic activation of porcine IVM oocytes, as well as pH i responses to activation in bovine and murine oocytes. Transient intracellular pH changes were found during porcine oocyte activation when using 7% ethanol and with 50 or 100 M calcium ionophore (A23187). Treatment with 200 M thimerosal resulted in an increase in pH i after a delay of approximately 12 min. Murine oocytes showed a significant increase during activation with 7% ethanol and A23187 as well as during prolonged exposure to thimerosal. Bovine oocytes exhibited an increase in pH i only when activated with 50 or 100 M A23187. The final set of experiments aimed to determine whether the porcine oocyte has mechanisms to alleviate induced acidic and alkaline challenges. Both acidic (~20 mM acetic acid) and alkaline (~30 mM ammonium chloride) challenges caused significant changes in pH i that porcine IVM oocytes were capable of recovering from within 35 min. Future studies will focus on determining which of the mechanisms is producing the pH i increase at the time of parthenogenetic activation in the porcine oocyte.

Inhibition of Platelet Aggregation by Ethanol in Vitro Shows Specificity for Aggregating Agent Used and Is Influenced by Platelet Lipid Composition

1982 ◽  
Vol 48 (01) ◽  
pp. 049-053 ◽  
Author(s):  
C G Fenn ◽  
J M Littleton
Keyword(s):  
Platelet Aggregation ◽  
Lipid Composition ◽  
Saturated Fatty Acids ◽  
Calcium Ionophore ◽  
Cholesterol Content ◽  
Membrane Lipid ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Increased Sensitivity

SummaryEthanol at physiologically tolerable concentrations inhibited platelet aggregation in vitro in a relatively specific way, which may be influenced by platelet membrane lipid composition. Aggregation to collagen, calcium ionophore A23187 and thrombin (low doses) were often markedly inhibited by ethanol, adrenaline and ADP responses were little affected, and aggregation to exogenous arachidonic acid was actually potentiated by ethanol. Aggregation to collagen, thrombin and A23187 was inhibited more by ethanol in platelets enriched with saturated fatty acids than in those enriched with unsaturated fats. Platelets enriched with cholesterol showed increased sensitivity to ADP, arachidonate and adrenaline but this increase in cholesterol content did not appear to influence the inhibition by ethanol of platelet responses. The results suggest that ethanol may inhibit aggregation by an effect on membrane fluidity and/or calcium mobilization resulting in decreased activity of a membrane-bound phospholipase.

scholarly journals Decreased miR-149 expression in sperm is correlated with the quality of early embryonic development in conventional in vitro fertilization

2021 ◽  
Vol 101 ◽  
pp. 28-32
Author(s):  
Hongping Li ◽  
Lejun Li ◽  
Chuanping Lin ◽  
Minhao Hu ◽  
Xiaozhen Liu ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Embryonic Development ◽  
Early Embryonic Development ◽  
Vitro Fertilization
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