351 POLARIZED LIGHT MICROSCOPY: DETECTION OF MICROTUBULES AND ITS EFFECTS ON THE VIABILITY OF IN VITRO-MATURED PORCINE OOCYTES

2010 ◽  
Vol 22 (1) ◽  
pp. 332 ◽  
Author(s):  
I. Molina ◽  
M. Muñoz ◽  
C. Díez ◽  
E. Gómez ◽  
E. A. Martínez ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Polarized Light ◽  
Polarized Light Microscopy ◽  
Developmental Competence ◽  
Meiotic Spindle ◽  
Cleavage Rate ◽  
Cell Numbers ◽  
Oocyte Developmental Competence ◽  
Blastocyst Rate

The meiotic spindle in the oocyte is composed of microtubules and plays an important role during chromosome alignment and separation at meiosis. Polarized light microscopy (PLM) is used as a tool in human and, recently, in farm animals assisted reproductive technologies. PLM could be useful for a non-invasive evaluation of the meiotic spindle. The objectives of the present study were to assess the efficiency of PLM to detect microtubule-polymerized protein within in vitro-matured porcine oocytes and to examine the effects of PLM on the oocyte developmental competence. Cumulus-oocyte complexes from slaughterhouse ovaries were matured in vitro for 42 h as described by Gil et al. (2004 Theriogenology 62, 544-552). In the first experiment, a total of 97 oocytes from 6 replicates were placed individually in 10-μL drops of TCM-199-Hepes-FCS in a glass Petri dish. PLM was used to detect the presence of polymerized protein which could be forming a meiotic spindle. The presence of polymerized protein and a meiotic spindle was confirmed in individual oocytes by inmunostaining and chromatin detection as described by Morató et al. (2008 Mol. Reprod. Dev. 75, 191-201). In the second experiment, a total of 160 oocytes from 4 replicates were exposed or not (controls) to PLM for 10 minutes. Thereafter, the oocytes were parthenogenetically activated and cultured in vitro. Cleavage rate, total blastocyst rate, expanded blastocyst rate on Day 7 and total cell numbers in expanded blastocysts were assessed. Data were analyzed by GLM procedure of SAS. There was a positive correlation (r = 1; P < 0.0001) between the signal obtained by PLM and the presence of microtubule-polymerized protein as confirmed by inmunostaining. A positive PLM signal was detected in 98.9% of the oocytes. A barrel-shape spindle was observed in 94.8% of the individual samples by inmunostaining and all of these oocytes were positive to PLM. Moreover, oocytes exposed to PLM did not differ significantly from controls on cleavage rate (83.7 ± 1.5 v. 84.4 ± 1.5), total blastocyst rate (36.9 ± 3.6 v. 41.2 ± 3.6) and expanded blastocyst rate on Day 7 (21.9 ± 1.7 v. 26.2 ± 1.7), respectively. There were also no differences in total cell numbers counted in expanded blastocysts (32.8 ± 2.6 v. 35.6 ± 2.5). These results indicate that polarized light microscopy did not exert detrimental effects on porcine oocyte developmental competence and it seems an efficient system to detect polymerized protein in in vitro-matured porcine oocytes. Grant support: INIA: RZ2007-00013-00-00. I. Molina, M. Muñoz, B. Trigal and D. Martín are sponsored by INIA, RYC08-03454, Cajastur and PTA2007-0268-I, respectively.

18 DEVELOPMENTAL COMPETENCE OF CLONED PORCINE EMBRYOS PRODUCED WITH DIFFERENT CLONING PROCEDURES

2014 ◽  
Vol 26 (1) ◽  
pp. 123
Author(s):  
Y. Liu ◽  
A. Lucas-Hahn ◽  
B. Petersen ◽  
R. Li ◽  
P. Hassel ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Numbers ◽  
Blastocyst Rate ◽  
Handmade Cloning

Two nuclear transfer (NT) techniques are routinely used to produce cloned animals, traditional cloning (TC) and handmade cloning (HMC). The TC embryos keep their zona and can be transferred at early stages, whereas HMC embryos are zona-free and must be cultured to the morula/blastocyst stage before transfer. Some studies have shown that in vitro culture reduces embryo development and quality, but it is not known whether embryos produced by TC or HMC differ because of the NT method or the in vitro culture. Therefore, we investigated the developmental competence and histone acetylation (H3K18ac) of porcine NT embryos produced by TC and HMC with (Day 5 and 6) or without (Day 0) in vitro culture. Nuclear transfer experiments were performed on same day (Day 0), using same batch of porcine oocytes and donor cells and same in vitro culture conditions. Cloning procedures were previously described (TC : Cloning Stem Cells 10 : 355; HMC : Zygote 20 : 61). Parthenogenetically activated embryos (PA) were used as control of activation and culture conditions. Embryos from all groups were collected for immunostaining of H3K18ac on Days 0, 5, and 6. The normalized H3K18ac level was calculated as previously described (Epigenetics 6 : 177). Cell numbers per blastocyst in each group were counted on Days 5 and 6. The cleavage rate (Day 2) and blastocyst rates (Days 5 and 6) between groups were analysed by Chi-squared test, whereas cell number per blastocysts and H3K18ac level between groups and days were analysed by ANOVA (SAS version 9.2; SAS Institute Inc., Cary, NC, USA). Cleavage rate of HMC embryos was lower than that of TC embryos, but blastocyst rate and cell number per blastocyst were higher in the HMC group compared with TC (Table 1). Differences of H3K18ac level between HMC, TC, and PA groups were only observed on Day 6 but not on Day 0 or Day 5. Within HMC and TC groups, there was no difference in H3K18ac level between Day 0 and Day 5, but the level was lower on Day 6 compared with Day 5 in the HMC group, whereas the TC group displayed the opposite pattern. In conclusion, NT embryos produced by HMC show higher blastocyst rate and cell number per blastocyst compared with TC embryos. Both in vitro culture and the NT method result in differences of the normalized H3K18ac levels. Further study is needed to investigate putative differences between NT embryos produced by HMC and TC compared to in vivo embryos also after transfer to recipients. Table 1.Cleavage and blastocyst rate, cell numbers, and normalized H3K18ac level for handmade cloning (HMC), traditional cloning (TC), and parthenogenetically activated (PA) embryos1

262 MEIOTIC SPINDLE CONFORMATION ASSESSMENT BY POLARIZED LIGHT MICROSCOPY IN SHEEP AND GOAT OOCYTES

2013 ◽  
Vol 25 (1) ◽  
pp. 279
Author(s):  
J. N. Caamaño ◽  
F. Cuadrado ◽  
C. Díez ◽  
M. Muñoz ◽  
D. Martín ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Linear Models ◽  
Polarized Light ◽  
Polarized Light Microscopy ◽  
Meiotic Spindle ◽  
System Research ◽  
Grant Support ◽  
Meiotic Spindles ◽  
The Mean

Polarized light microscopy (PLM) allows detection of microtubule-polymerized protein in in vitro-matured sheep and goat oocytes (Caamaño et al. 2011 Reprod. Fertil. Dev. 23, 226–227). Spindle birefringence, measured as the mean retardance value, has been proposed as a marker of meiotic spindle conformation in humans and mice. Because the conformation of the meiotic spindle cannot be readily assessed by PLM, in this study we aimed to measure the mean retardance value of normal and abnormal meiotic spindles from in vitro-matured prepubertal sheep and goat oocytes. Oocytes were matured in vitro for 27 h and were then individually assessed by PLM (Oosight System, Research Instruments Ltd., Falmouth, Cornwall, UK), and the mean retardance value was analysed in meiotic spindles using specific software (Oosight). Meiotic spindle conformation was determined in individual oocytes by immunostaining and chromatin detection, as described by Morató et al. (2008 Mol. Reprod. Dev. 75, 191–201). A barrel-shaped spindle was considered a normal spindle configuration. Only oocytes with meiotic spindles identified by both PLM and immunostaining were used in this study. The experiment was replicated four times. Data were analysed by the general linear models procedure of SAS (SAS Institute Inc., Cary, NC, USA). A normal barrel-shaped spindle was observed in 80.8% of the sheep oocytes (n = 47) and in 83.9% of the goat oocytes (n = 62). In sheep, the mean retardance value in oocytes with a normal meiotic spindle conformation did not differ from that in oocytes with abnormal spindles (4.42 ± 0.26 nm v. 3.92 ± 0.54 nm). Similar results were obtained with goat oocytes with normal (2.94 ± 0.20 nm) v. abnormal spindle conformation (2.77 ± 0.08 nm). These results indicate that the mean retardance value does not distinguish between oocytes with normal and abnormal meiotic spindle conformation, as assessed by subsequent immunostaining. Grant support from INIA-RZ2007-00013-00-00. M. Muñoz was sponsored by RYC08-03454.

scholarly journals Evaluation of equine oocyte developmental competence using polarized light microscopy

Reproduction ◽  
2017 ◽  
Vol 153 (6) ◽  
pp. 775-784 ◽  
Author(s):  
A Bertero ◽  
F Ritrovato ◽  
F Evangelista ◽  
V Stabile ◽  
R Fortina ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Zona Pellucida ◽  
Light Microscope ◽  
Polarized Light ◽  
Developmental Competence ◽  
Immature Oocytes ◽  
Oocyte Developmental Competence ◽  
Morphological Evaluation ◽  
Meiotic Competence

The purpose of this study was to observe in vitro-matured equine oocytes with an objective computerized technique that involves the use of a polarized light microscope (PLM) in addition to the subjective morphological evaluation obtained using a classic light microscope (LM). Equine cumulus-oocyte complexes (COCs, n = 922) were subjected to different in vitro maturation times (24, 36 or 45 h), however, only 36-h matured oocytes were analyzed using CLM. The 36-h matured oocytes that reached maturity were parthenogenetically activated to evaluate the quality and meiotic competence. Average maturation percentages per session in groups 1, 2 and 3 (24-, 36- and 45-h matured oocytes respectively) were 29.31 ± 13.85, 47.01 ± 9.90 and 36.62 ± 5.28%, whereas the average percentages of immature oocytes per session were 28.78 ± 20.17, 7.83 ± 5.51 and 22.36 ± 8.39% respectively. The zona pellucida (ZP) birefringent properties were estimated and correlated with activation outcome. ZP thickness and retardance of the inner layer of the zona pellucida (IL-ZP) were significantly increased in immature oocytes compared with mature oocytes (P < 0.001 and P < 0.01 respectively). The comparison between parthenogenetically activated and non-activated oocytes showed a significant increase in the area and thickness of the IL-ZP in parthenogenetically activated oocytes (P < 0.01). These results show that the 36-h in vitro maturation (IVM) protocol allowed equine oocytes to reach maturity, and PLM observation of ZP can be used to distinguish mature and immature oocytes as well as activated and non-activated oocytes.

257 DETECTION OF MICROTUBULES BY POLARIZED LIGHT MICROSCOPY IN SHEEP AND GOAT OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 226 ◽  
Author(s):  
J. N. Caamaño ◽  
M. Catalá ◽  
R. Romaguera ◽  
C. Diez ◽  
M. Muñoz ◽  
...  
Keyword(s):  
Light Microscopy ◽  
In Vitro Maturation ◽  
Polarized Light ◽  
Petri Dish ◽  
Polarized Light Microscopy ◽  
Farm Animals ◽  
Meiotic Spindle ◽  
Efficient System ◽  
Positive Correlation

The meiotic spindle in the oocyte is composed of microtubules and plays a key role in normal chromosome alignment and segregation during meiosis. In oocytes from farm animals, the meiotic spindle cannot be detected by conventional light microscopy due to the characteristic of their cytoplasm. Conventional methods to image the meiotic spindle rely on fixation of the oocytes. Polarized light microscopy (PLM) allows noninvasive evaluation of the meiotic spindle of metaphase oocytes. The aim of this study was to assess the efficiency of polarized light microscopy to detect microtubule-polymerized protein within in vitro matured prepubertal sheep and goat oocytes. We carried out 2 studies. In the first one, cumulus–oocyte complexes from slaughterhouse sheep ovaries were matured in vitro for 27 h. After in vitro maturation, oocytes (n = 77) were denuded of cumulus cells and placed individually in 10-μL drops of TCM-199-HEPES-BSA in a glass Petri dish. Polarized light microscopy was used to detect the presence of polymerized protein, which could be associated with the forming of a meiotic spindle. To confirm the presence of the polymerized protein and the meiotic spindle, each individual oocyte was subjected to immunostaining and chromatin detection as described by (Morató et al. 2008 Mol. Reprod. Dev. 75, 191–201). The experiment was replicated 4 times. The correlation analysis was performed using the Proc Corr procedure of SAS. There was a positive correlation (r = 0.87; P < 0.001) between the signal obtained by PLM and the presence of microtubule-polymerized protein as confirmed by immunostaining. A positive PLM signal was detected in 87.0% of the oocytes, and 69.0% of the oocytes reached the metaphase II (MII) stage after in vitro maturation. A barrel-shaped spindle was observed in 77.3% of the MII oocytes. In the second study, we performed a similar experiment but used goat oocytes. A total of 78 oocytes were used, and PLM and immunostaining were performed in each individual oocyte as it was described with sheep oocytes. There was also a positive correlation (r = 1; P < 0.001) between the signal obtained by PLM and the presence of microtubule-polymerized protein. A positive PLM signal was detected in 98.7% of the oocytes, and 80.7% of the oocytes reached the MII stage after in vitro maturation. A barrel-shaped spindle was observed in 92.0% of the MII oocytes. These results indicate that PLM is an efficient system to detect polymerized protein in in vitro matured sheep and goat oocytes. This work was supported by the following grant: INIA: RZ2007-00013-00-00. M. Muñoz and D. Martín are sponsored by RYC08-03454 and PTA2007-0268-I, respectively.

242 DETECTION OF MICROTUBULES BY POLARIZED LIGHT MICROSCOPY IN BOVINE OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 219 ◽  
Author(s):  
J. N. Caamaño ◽  
C. Díez ◽  
M. Muñoz ◽  
D. Martin ◽  
R. Morató ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Calf Serum ◽  
Polarized Light ◽  
Reproductive Technologies ◽  
Polarized Light Microscopy ◽  
Farm Animals ◽  
Meiotic Spindle ◽  
Efficient System ◽  
Bovine Oocytes

Polarized light microscopy (PLM) is being used as a tool in assisted reproductive technologies in humans and, more recently, in farm animals. Polarized light microscopy may allow for a quick localization or removal of nuclear structures, or both in the oocyte without fluorochrome staining and ultraviolet exposure, and may reduce nuclear damage when performing intra-cytoplasmic sperm injection. The aim of this study was to assess the efficiency of PLM to detect microtubule-polymerized protein within in vitro-matured bovine oocytes. Cumulus–oocyte complexes from slaughterhouse ovaries were matured in vitro for 23 h in bicarbonate-buffered TCM-199, FSHp (1 μg mL–1), LH (5 μg mL–1), 17β-estradiol (1 μg mL–1), and 10% fetal calf serum at 38.7°C in 5% CO2 in air with high humidity. After in vitro maturation, oocytes (n = 98) were denuded with 0.5% hyaluronidase and were placed individually in 10-μL drops of TCM-199-HEPES-BSA in a glass Petri dish. Polarized light microscopy was used to detect the presence of polymerized protein that could be forming a meiotic spindle. To confirm the presence of the polymerized protein and the meiotic spindle, each individual oocyte was subjected to immunostaining and chromatin detection as described by Morató et al. 2008 Mol. Reprod. Dev. 75, 191–201. The experiment was replicated 6 times. There was an absolute positive correlation (r = 1; P < 0.0001) between the signal obtained by PLM and the presence of microtubule-polymerized protein as confirmed by immunostaining. A barrel-shaped spindle was observed in 40% of the individual samples, whereas disorganized microtubule structures were obtained in 55% of the samples. The biological implication of these findings needs to be explored. However, PLM seems to be an efficient system to detect polymerized protein in in vitro-matured bovine oocytes.

74 Treatment with Melatonin During In Vitro Culture Enhances Porcine Parthenogenetically Activated Embryo Development

2018 ◽  
Vol 30 (1) ◽  
pp. 175
Author(s):  
G. A. Kim ◽  
J.-X. Jin ◽  
S. Lee ◽  
A. Taweechaipaisankul ◽  
B. C. Lee
Keyword(s):  
Oxidative Stress ◽  
Embryo Development ◽  
Developmental Competence ◽  
Free Radical Scavengers ◽  
Control Group ◽  
Cleavage Rate ◽  
Cell Numbers ◽  
Significant Difference ◽  
Blastocyst Rate

Melatonin and its metabolites are powerful antioxidants and free radical scavengers. Because porcine embryos are vulnerable to oxidative stress in vitro, the addition of various protective chemicals to the culture medium, including melatonin, has been explored. The aim of this study was to investigate the effect of melatonin on in vitro developmental competence of porcine parthenogenetically activated (PA) embryos. Immature cumulus–oocyte complexes (COC) were collected and cultured in medium comprising TCM-199 supplemented with 10 ng mL−1 epidermal growth factor, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 5 μL mL−1 insulin, transferrin selenium solution 100×, 10% porcine follicular fluid, 10 IU mL−1 eCG, and 10 IU mL−1 hCG for 44 h. Then, COC were denuded and PA with electrical stimulation, and PA embryos were cultured in porcine zygote medium 5 (PZM-5) supplemented with melatonin at increased concentrations (10−9, 10−7, 10−5 M) at 39°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 for 7 days. Subsequent embryo development, including cleavage rate, blastocyst rate, and blastocyst cell numbers, was compared between groups (mean no. of embryos; control, 27.14; 10−9 M, 28.86; 10−7 M, 27.71; 10−5 M, 26.43). The experiments were repeated 7 times for each treatment group. Statistical analyses of all data were performed using one-way ANOVA with Dunn’s multiple comparison test. Results are expressed as the mean ± SEM and all differences were considered significant at P < 0.05. No apparent effect on cleavage rate of melatonin treatment of various concentrations was noted. Blastocyst cell number did not show any significant difference between groups. However, the potential of PA oocytes to develop into blastocysts was significantly higher in the group supplemented with 10−9 M melatonin compared with the control group (35.44 ± 3.84 v. 24.71 ± 1.59) and other melatonin treated groups (10−5 M, 21.35 ± 2.82; 10−7 M, 24.01 ± 2.31; P < 0.05). These indicated that treatment with 10−9 M melatonin in embryo culture might reduce the oxidative stress properly compared with other concentrations, which results in improvement of blastocyst rate formation. In conclusion, treatment with 10−9 M melatonin positively promoted the blastocyst formation rate of porcine PA embryos with no beneficial effects on their blastocyst cell numbers or cleavage rate. This study was supported by the National Research Foundation (#2015R1C1A2A01054373; 2016M3A9B6903410), Research Institute for Veterinary Science and the BK21 PLUS Program.

292 INHIBITION OF HSP90 AGGRAVATES THE EFFECTS OF HEAT SHOCK ON DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 235
Author(s):  
E. D. Souza ◽  
N. C. Rabelo ◽  
T. D. Araujo ◽  
C. M. Assunção ◽  
C. C. R. Quintão ◽  
...  
Keyword(s):  
Heat Shock ◽  
In Vitro Maturation ◽  
Calf Serum ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Oocyte Developmental Competence ◽  
Significant Difference ◽  
Blastocyst Rate ◽  
Bovine Oocytes

The heat shock protein 90kDa (HSP90) is a chaperone involved in protein homeostasis under normal and stress conditions. Its inhibition by 17-(allylamino)-17-demethoxygeldanamycin (17AAG, Sigma, St. Louis, MO, USA) for 12 or 24 h during in vitro maturation reduces the oocyte's ability to develop after in vitro fertilization (Souza et al. 2014 Reprod. Fert. Dev. 26, 197). This study aimed to evaluate the effect of treatment with 17AAG during the heat shock on oocyte developmental competence. Immature bovine COC were randomly allocated in 4 treatments during IVM: control = no heat shock or 17AAG; HS = heat shock (41.5°C) for the first 12 h of IVM; 17AAG = 2 µM 17AAG for the first 12 h of IVM; and 17AAG + HS = 2 µM 17AAG plus heat shock for the first 12 h of IVM. In vitro maturation was performed in Nunc plate containing 400 µL of TCM199 medium (Invitrogen, Carlsbad, CA, USA) supplemented with porcine FSH (Hertape Calier, Juatuba, Brazil) and 10% oestrus cow serum under 5% CO2 in air, 95% humidity, and 38.5°C for 24 h. Semen was processed by Percoll gradient (Nutricell, Campinas, Brazil) and oocytes were in vitro fertilized for 20 h with 2 × 106 spermatozoa mL–1 under the same IVM atmospheric conditions. Presumptive zygotes were completely denuded in a PBS solution with 0.1% hyaluronidase and then cultured in wells with 500 µL of modified CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell) in an incubator at 38.5°C under 5% CO2, 5% O2, 90% N2, and saturated humidity. Cleavage rate was evaluated 72 h postfertilization and blastocyst rate was evaluated at Day 7 (D7) and 8 (D8). Data from 7 replicates were submitted to analysis of variance and means were compared by Student Newman Keul's test. There was no difference (P > 0.05) on cleavage rate among treatments. Heat shock or treatment with 17AAG, both for 12 h of IVM, decreased (P < 0.05) the blastocyst rate at D7 and D8 when compared to control but no significant difference between HS and 17AAG treatments was found (Table 1). However, the lowest (P < 0.05) blastocyst rate at D7 and D8 was achieved when oocytes were submitted simultaneously to 17AAG and heat shock for 12 h of IVM (17AAG + HS treatment, Table 1). In conclusion, the treatment with 17AAG during IVM worsens the deleterious effect of heat shock on oocyte developmental competence and suggests that HSP90 may also play role on cellular protection during heat shock in bovine oocytes. Table 1.Cleavage and blastocyst (Bl) rates at D7 and D8 for control, 17AAG, Heat Shock (HS), and 17AAG plus HS treatments Financial support comes from CNPq, FAPEMIG, and FAPES.

An in vitro Evaluation of Artificial Caries-like Lesions on Restored Overdenture Abutments

1988 ◽  
Vol 67 (3) ◽  
pp. 582-584 ◽  
Author(s):  
P.P. Kambhu ◽  
R.L. Ettinger ◽  
J.S. Wefel
Keyword(s):  
Light Microscopy ◽  
Polarized Light ◽  
Polarized Light Microscopy ◽  
The Other ◽  
Type Ii ◽  
Restorative Materials ◽  
Abutment Teeth ◽  
The Mean ◽  
Materials Used

An acidified dialyzed gelatin gel system was used to determine the caries resistance of a variety of restorative materials used to obturate the canal orifice of overdenture abutment teeth. The restorative materials used were Tytin, Tytin + Copalite, P30 + Scotchbond, Fuji Ionomer-Type II, and Miracle Mix. Polarized light microscopy and microradiography were used to examine the caries-like lesions adjacent to the restorations. The lesions formed in the Fuji Ionomer-Type II and Miracle Mix groups appeared arrested at the wall adjacent to the restoration, and did not penetrate apically down the wall as did those associated with the other restorative materials. The mean depths of lesions adjacent to Fuji Ionomer-Type II and Miracle Mix restorations were significantly less than those of Tytin, Tytin + Copalite, or P30 + Scotchbond.

286 INFLUENCE OF REVERSIBLE MEIOSIS INHIBITION ON SWINE EMBRYOS PRODUCED BY IN VITRO FERTILIZATION AND PATHENOGENETIC ACTIVATION

2006 ◽  
Vol 18 (2) ◽  
pp. 250
Author(s):  
M. G. Marques ◽  
A. B. Nascimento ◽  
V. P. Oliveira ◽  
A. R. S. Coutinho ◽  
M. E. O. A. Assumpção ◽  
...  
Keyword(s):  
Culture Medium ◽  
Cell Number ◽  
Control Group ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Electric Pulses ◽  
Cell Numbers ◽  
Blastocyst Rate ◽  
And Control

The present work evaluated the reversible meiosis inhibition effect on the development of swine embryos produced by in vitro fertilization (IVF) or parthenogenetic activation (PA). The efficiency of PZM3 and NCSU23 embryo culture media was also evaluated. Oocytes from ovaries collected at a slaughterhouse were subjected to IVM in two different groups: CHX (cycloheximide 5 µM for 10 h) and control, both with TCM-199 + 3.05 mM glucose + 0.91 mM sodium pyruvate + 10% porcine follicular fluid (pFF) + 0.57 mM cystein + 10 ng epidermal growth factor (EGF)/mL + 10 IU eCG/mL + 10 IU hCG/mL for the initial 22 h. In the remaining period (20 h for CHX and 22 h for control), medium without hormones was utilized. After IVM, oocytes were denuded and fertilized for 6 h (IFV) or the matured oocytes were submitted to activation by electric pulses (PA) (2 DC of 1.5 kV/cm for 30 µs), incubated for 1 h in culture medium with 10 μM of CHX, and again submitted to the same electric pulses for 60 µs. Embryo development was evaluated by cleavage rate on Day 3 and blastocyst rate and blastocyst cell number on Day 7 of culture. Cleavage and blastocyst rates were analyzed by the equality-of-two-ratios test and cell number by the Kruskal-Wallis and Mann-Whitney tests (P < 0.05). In relation to IVF, the PZM3 medium was more efficient than NCSU23 for cleavage rate in the CHX group (PZM3: 68.4%, NCSU23: 44.4%) and had a better blastocyst rate in the control group (PZM3: 13.4%, NCSU23: 5.6%). With reference to PA, NCSU23 presented better cleavage and blastocyst rates than PZM3 in the CHX group (NCSU23: 89.5%, PZM3: 78.5% and NCSU23: 20.4%, PZM3: 13.0%, respectively). In the control group, only the NCSU23 blastocyst rate was higher than that for PZM3 (NCSU23: 22.5%, PZM3: 10.8%). No culture medium effect on cell number mean of IVF and PA blastocysts was observed. Maturation block improved cleavage rates in IVF groups cultured with PZM3 (68.4% and 50.6%, respectively, for CHX and control) and in PA groups cultured with NCSU23 (89.5% and 80.3%, respectively, for CHX and control), but no improvement of blastocyst rates in both groups (IVF and PA) was verified. Table 1 below shows that maturation block decreased the IVF and increased the PA blastocyst cell numbers. As older oocytes are more effectively activated, oocytes blocked with CHX achieved the maturation stage faster than the control group, therefore resulting in high-quality PA blastocysts. In conclusion, PZM3 was more efficient for IVF embryo production in contrast to NCSU23, whereas NCSU23 can be indicated for PA embryo production. Moreover, maturation blockage with CHX influenced blastocyst cell number, decreasing in IVF embryos and increasing in PA embryos. Table 1. Mean (±SD) of blastocyst cell numbers for IVF or PA groups after in vitro maturation without (control) or with cycloheximide (CHX) and cultured in NCSU23 or PZM3 medium This work was supported by FAPESP 02/10747–1.

Polarized Light Microscopy for Analyzing Tissue Mechanics During In Vitro Acupuncture

2008 ◽  
Author(s):  
Lowell Taylor Edgar ◽  
Margaret Julias ◽  
David I. Shreiber ◽  
Helen M. Buettner
Keyword(s):  
Connective Tissue ◽  
Light Microscopy ◽  
Type I Collagen ◽  
Polarized Light ◽  
Tissue Mechanics ◽  
Polarized Light Microscopy ◽  
Tissue Response ◽  
Type I ◽  
Collagen Gels

Acupuncture is a traditional therapy originating in China almost 2000 years ago. Acupuncture has slowly been growing in popularity in the West, and clinical evidence has shown the potential for acupuncture as a low-cost ‘alternative’ therapy for an assortment of ailments [1]. The practice of acupuncture involves inserting fine needles into the skin followed by needle manipulation, usually by rotation. Recent studies by Langevin et al demonstrate that this rotation causes the subcutaneous connective tissue to couple to and wind around the needle [2–4], which suggests that mechanotransduction in the connective tissue might play a role in the therapeutic mechanisms that underlay acupuncture [2, 3]. To begin to decompose and quantify this complex mechanism at the tissue level in a controlled setting, we have simulated acupuncture in type I collagen gels in vitro, and have developed algorithms to quantify the tissue response following imaging with polarized light microscopy (PLM).

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