70 VITRIFICATION OF IMMATURE OVINE OOCYTES WITH CRYOLOOP: EFFECT OF CYTOCHALASIN B PRETREATMENT ON SUBSEQUENT DEVELOPMENT

2009 ◽  
Vol 21 (1) ◽  
pp. 135
Author(s):  
A. R. Moawad ◽  
I. Choi ◽  
J. Zhu ◽  
K. H. S. Campbell
Keyword(s):  
Cytochalasin B ◽  
Mammalian Species ◽  
Subsequent Development ◽  
Total Cell ◽  
High Survival ◽  
Control Groups ◽  
Cell Numbers ◽  
Significant Difference ◽  
And Control

Oocyte cryopreservation is still a challenge in most mammalian species because of their extreme sensitivity to chilling injuries. Relaxation of the cytoskeleton during vitrification may improve post-thaw survival and subsequent development; however, a previous study in immature [germinal vesicle (GV) stage] oocytes from prepubertal lambs reported that pretreatment with cytochalasin B (CB) did not improve maturation (Silvestre MA et al. 2006 Anim. Reprod. Sci. 93, 176–182). We previously reported that GV oocytes from mature ewes can be vitrified using a cryoloop with high survival, maturation, and subsequent in vitro fertilization (Moawad AR and Campbell KHS 2008 Reprod. Fertil. Dev. 20, 122). The aim of this study was to evaluate the effects of CB pretreatment prior to vitrification of GV oocytes (from mature ewes) on subsequent development. Cumulus–oocyte complexes obtained at slaughter were randomly divided into 2 groups and incubated with or without 7.5 μg mL–1 CB for 60 min before vitrification. Oocytes from each group were vitrified or used as toxicity and controls. For vitrification, oocytes were equilibrated in 10% ethylene glycol (EG) and 0.25 m trehalose (T) in HEPES/TCM-199 plus 10% fetal bovine serum (BM) for 3 min. Oocytes were then exposed to vitrification solution (20% EG and 20% DMSO in BM), loaded into the cryoloop within 1 min, and immersed in liquid nitrogen. Oocytes were warmed by exposure to (1) 10% EG and 1 m T in BM, (2) 0.5 m T in BM, and (3) BM for 3 min in each solution at 39°C. Oocytes were then matured, fertilized, and cultured in vitro as previously described. The frequency of cleavage 24 and 48 h post-insemination (pi), development to morula (5 days pi), blastocyst (7 days pi), and total cell numbers of blastocyst-stage embryos were evaluated. Cleavage was significantly lower (P < 0.001) in vitrified and CB-vitrified groups at both 24 h pi (15.2 v. 14.7%) and 48 h pi (27.3 v. 23.5%) than in other groups. Development to morula stage was significantly lower (P < 0.001) in vitrified and CB-vitrified oocytes (12.1 v. 17.7%) than in toxicity, CB-control, and control groups (43.1, 42.6, and 52.8%, respectively); however, no significant difference (P > 0.05) was observed between CB-vitrified and CB-toxicity groups. There was a significant decrease (P < 0.01) in development to blastocyst in CB-vitrified (2.9%) compared with CB-control (22.9%) and control (24.5%), but this did not differ significantly (P > 0.05) from toxicity and CB-toxicity groups (9.8 v. 11.4%). No blastocysts developed from the vitrified group. Hatched blastocysts were observed only in CB-control and control groups (8.2 v. 5.7%). Total cell numbers were significantly greater (P < 0.05) in control blastocysts than in toxicity, CB-vitrified, and CB-control (143 v. 87.25, 73, and 82, respectively). However, this did not differ significantly from the CB-toxicity group (115). These results support our previous data and suggest that pretreatment of GV-stage ovine oocytes with CB prior to vitrification has a positive effect on subsequent development.

150 IN VITRO AND IN VIVO DEVELOPMENT OF EMBRYO FOLLOWING BIOPSY WITH DIFFERENT TECHNIQUES IN MOUSE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 223
Author(s):  
A. C. Taskin ◽  
H. Bagis ◽  
H. Sagirkaya ◽  
T. Akkoc ◽  
S. Arat
Keyword(s):  
Fetal Development ◽  
Total Cell ◽  
Trophectoderm Biopsy ◽  
Control Groups ◽  
Cell Numbers ◽  
Development Rates ◽  
Significant Difference ◽  
Implantation Sites ◽  
And Control

In vitro development ratios, quality evaluation, in vivo implantation, and fetal development ratios were investigated following aspiration biopsy in 8-cell mouse embryos and trophectoderm biopsy in blastocyst developed from 8-cell stage embryos in vitro. Superovulated CB6F1 hybrid female mice (5–6 weeks) were sacrificed 68 to 72 hours after hCG administration. Eight-cell embryos were flushed from oviducts of the sacrificed mouse with HTF medium supplemented with HEPES and 3 mg mL–1 BSA. Embryos were randomly divided into two groups. In the first group, embryos at 8-cell stage were used for a single cell blastomer aspiration; in the second group, embryos were cultured in vitro until blastocyst stage. Trophectoderm cells (15% of trophoblastic cells) were biopsied from developing blastocysts. There were also control groups for both groups. Biopsy procedures for both groups were applied in 50 µL drops of Ca2+/Mg2+ free HTF medium containing HEPES+3 mg mL–1 BSA+5 µg mL–1 cytochalasine B. After biopsy, embryos were cultured in Quinn’s blastocyst medium supplemented with 4 mg mL–1 BSA and incubated in 5% CO2 and 5% O2 incubator at 37°C for 48 and 24 hours for blastomer aspiration and trophectoderm biopsy groups, respectively. While some developing blastocysts were used for determining total cell number, some of them were transferred to the recipients. Results were evaluated by independent t-test and ANOVA of SPSS 17.0 statistic program (SPSS Inc., Chicago, IL, USA). In blastomere biopsy and control groups, development rates were determined as 81.02% (121/152) and 96.37% (62/63), while the total cell numbers were determined as 50 and 50, respectively. There was no significant difference between groups in terms of development ratios and total cells. In blastomer biopsy and control groups, the implantation sites and fetal development rates were found as 25% (9/36) and 26% (8/30), and 19.44% (7/36) and 20% (6/30), respectively. No significant difference was observed between groups in terms of implantation sites and fetal development rates. In trophectoderm biopsy and control groups, while the development rates were found as 86.96% (69/79) and 93.33% (23/28), the total cell numbers were 26.66 and 55.33, respectively. Although there was not any significant difference between groups in terms of development rates, there was a significant difference between groups in terms of total cell numbers (P < 0.05). In trophectoderm biopsy and control groups, the implantation sites and fetal development rates were determined as 21.88% (7/32) and 59.09% (13/22), and 0% (0/32) and 18.18% (4/22), respectively. Although there was not any significant difference between groups in terms of implantation sites, there was a significant difference between groups in terms of fetal development rates (P < 0.05). Therefore, it was concluded that biopsy applied at early stage of embryonic development does not affect embryo development negatively and biopsy procedures applied at early developmental stages have more advantages especially in embryos developing faster with low total cell numbers such as mouse species. Supported by TUBITAK KAMAG-107G027).

133 EFFECTS OF FROZEN STORED CULTURE MEDIA ON PRE-IMPLANTATION DEVELOPMENT OF PARTHENOTES AND CLONED EMBRYOS IN PIGS

2009 ◽  
Vol 21 (1) ◽  
pp. 166
Author(s):  
Y. H. Zhang ◽  
H. T. Xi ◽  
Y. Liu ◽  
J. Li ◽  
A. Pederson ◽  
...  
Keyword(s):  
Culture Media ◽  
Polar Body ◽  
Frozen Storage ◽  
Test Group ◽  
Total Cell ◽  
Control Group ◽  
Cell Numbers ◽  
Significant Difference ◽  
And Control

The present study was designed to examine if frozen storage of porcine zygote medium (PZM3) plus 3 mg mL–1 BSA (Yoshioka et al. 2002 Bio. Reprod. 66, 112–119) is feasible to culture pig embryos produced by parthenogenetic activation and somatic nuclear transfer. Slaughterhouse-derived sow cumulus–oocyte complexes (COCs) were matured in TCM199 supplemented with 10% porcine follicle fluid, 5% cattle serum, 10 IU mL–1 eCG, 5 IU mL–1 hCG, 0.8 mm L-glutamine and 0.05 mg mL–1 gentamicin at 38.5°C, 100% humidity and 5% CO2 in air. For activation, cumulus cells were removed after 42 to 44 h of maturation, and the denuded oocytes with 1st polar body were activated with a double 160 V mm–1, 100 μs direct pulse followed by culture in PZM3. Each experiment was replicated at least three times. Data were expressed as mean ± SEM and analyzed by using chi-square module in SPSS 11.0, with P < 0.05 denoting significant difference. In Experiment 1, after preparation, liquid PZM3 was aliquoted to 50 mL falcon centrifuge tubes. Randomly, half of the tubes with PZM3 were put into –80°C freezers, and the rest were placed into 4°C refrigerator. Within one week after storage, a tube of frozen PZM3, while that stored at 4°C served as control, was warmed at 38.5°C in CO2 incubator, and more than three 4-well culture dishes were then made with 400 μL PZM3 in each well and balanced for at least 4 h in the incubator before experiment. The results showed that both cleavage (78/93, 83.9 ± 1.2% v. 87/103, 84.5 ± 1.8%, P > 0.05) and blastocyst (60/93, 65.2 ± 2.1% v. 65/103, 63.1 ± 3.8%, P > 0.05) rates were similar between frozen-warmed PZM3 and control, as was total cell numbers per blastocyst (50 ± 7 v. 47 ± 5, P > 0.05) between groups. In Experiment 2, we used somatic cloned embryos to investigate the effect of frozen-warmed PZM3 on pre-implantation development of such embryos. Our results indicated that no significant difference in rates of cleavage (68/95, 71.5 ± 5.1% v. 78/100, 78.1 ± 1.9%, P > 0.05), blastocyst formation (33/95, 34.6 ± 7.6% v. 78/100, 38.2 ± 3.5%, P > 0.05) and total cell numbers per blastocyst (40 ± 11 v. 48 ± 9, P > 0.05) was found between the test and control groups, designed the same as in Experiment 1. In Experiment 3, we tested whether PZM3 in frozen storage for 5 months was able to support in vitro development of parthenotes comparable to freshly-made ones. PZM3 after frozen storage for 5 months was warmed using the same method as Experiment 1, and the newly made PZM3 within 1 week of storage at 4°C acted as control. The results showed that although the cleavage (135/138, 97.8 ± 2.7% v. 117/129, 90.7 ± 3.1%, P > 0.05) and blastocyst (104/138, 75.4 ± 1.6% v. 84/129, 65.1 ± 2.3, P > 0.05) rates in control group were both slightly higher than that in the test group, no statistical differences was observed. We also found no significant difference in total cell numbers per blastocyst (48 ± 7 v. 46 ± 6, P > 0.05) between groups. Taken together, our results imply that frozen storage of PZM3 is feasible, and of practical value for culture pig embryos.

154 THE EFFECT OF OXYGEN TENSION ON IN VITRO DEVELOPMENT OF BOVINE EMBRYOS IN POLYDIMETHYLSILOXANE-BASED WELL OF THE WELL DISHES PREPARED UNDER ATMOSPHERIC OR REDUCED AIR PRESSURE

2010 ◽  
Vol 22 (1) ◽  
pp. 235
Author(s):  
T. Somfai ◽  
Y. Inaba ◽  
Y. Aikawa ◽  
M. Ohtake ◽  
S. Kobayashi ◽  
...  
Keyword(s):  
Oxygen Tension ◽  
Culture Medium ◽  
Production Method ◽  
Total Cell ◽  
Air Pressure ◽  
Bovine Embryos ◽  
Cell Numbers ◽  
Significant Difference ◽  
And Control

Polydimethylsiloxane (PDMS) is a non-toxic silicon compound. Its excellent optical characteristics and easy preparation make it a good candidate material for the molding of custom-shaped dishes for embryo culture. We investigated the feasibility of PDMS-based well of the well (WOW) dishes for in vitro culture of bovine embryos under different oxygen tensions. The WOW dishes with 25 micro-wells (each of 175 μm depth and 250 μm width in diameter arranged in 5 columns and 5 rows) were molded from PDMS prepared either under atmospheric (Experiment 1) or reduced (0.1 MPa) (Experiment 2) air pressure to remove air bubbles. Presumptive zygotes obtained by the in vitro maturation and fertilization of follicular oocytes were placed and cultured for 7 days in traditional micro-drops of culture medium (Control) or in the micro-wells of PDMS-based WOW dishes (PDMS-WOW), both covered by paraffin oil. The culture medium was CR1aa supplemented with 5% calf serum. The culture drop size was 125 μL (5 μL/oocyte) in both groups. Embryo development and blastocyst cell numbers between Control and PDMS-WOW groups were compared either under 20% or 5% O2 tensions. There was no statistical difference in cleavage and blastocyst rates (ranging between 82.3-86.4% and 34.0-45.8%, respectively) between Control and PDMS-WOW embryos irrespective of oxygen tension and dish production method. In Experiment 1, the mean total cell numbers in blastocysts were lower in the PDMS-WOW group than that in Control under 20% O2 (105.0 ± 5.5 and 130.4 ± 9.9, respectively) (P < 0.05, ANOVA); however, the application of 5% O2 significantly improved the cell numbers and eliminated the difference between the PDMS-WOW and Control groups (135.4 ± 6.2 and 148.0 ± 9.0, respectively). In Experiment 2, there was no significant difference in mean total cell numbers in blastocysts between the PDMS-WOW and Control either under 20% O2 (97.2 ± 5.7 and 103.9 ± 8.9, respectively) or 5% O2 (147.5 ± 12.1 and 157.3 ± 3.9, respectively). The numbers and rates of inner cell mass and trophectoderm cells did not differ between the Control and PDMS-WOW groups, irrespective of O2 tension and production method. Our results demonstrate that bovine embryos can develop to the blastocyst stage in PDMS-based WOW dishes; however, it may express detrimental effects on embryonic cell numbers, which can be neutralized by the application of low O2 tension during culture or reduced air pressure during the PDMS preparation. This work was supported by the Research and Development Program for New Bio-Industry Initiatives.

50 PRE-MATURATION OF BOVINE OOCYTES SUBMITTED TO NUCLEAR TRANSFER: EFFECTS ON IN VIVO DEVELOPMENT

2010 ◽  
Vol 22 (1) ◽  
pp. 183
Author(s):  
T. H. C. De Bem ◽  
P. R. Adona ◽  
R. Rochetti ◽  
F. F. Bressan ◽  
M. S. Miranda ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Total Cell ◽  
Control Group ◽  
Control Groups ◽  
Cell Numbers ◽  
Fragmented Dna ◽  
And Control ◽  
Bovine Oocytes

In vitro embryo production by somatic cell nuclear transfer (SCNT) still presents low efficiency and blastocyst production rates are around 20%. Pre-maturation with cell cycle inhibitors is one alternative that has been studied to improve oocyte competence for use in in vitro production systems. The neurotrophin brain-derived neurotrophic factor (BDNF) has been reported to improve oocyte maturation. The aim of this work was to optimize the in vitro pre-maturation culture of bovine oocytes and its use in SCNT. Oocytes that were submitted to meiosis block before IVM (BL group) were cultured in TCM-199 medium supplemented with 10 ng mL-1 BDNF and 10 μM butyrolactone I for 24 h and then matured in IVM medium (TCM-199, 10% FCS, 0.5 μg mL-1 FSH, 5.0 μg mL-1 LH, 2.0 mM pyruvate, and 50 μg mL-1 gentamicin). Control oocytes (control group) were matured in IVM medium. After 19 h of IVM, oocytes from both groups were denuded with 0.2% hyarulonidase, enucleated, and reconstructed. Reconstructed embryos were chemically activated with ionomycin (5 min) and 6-DMAP (3h) and cultured in vitro in SOF medium for 7 or 8 days. Statistical analyses were performed by using BIOSTATS v.4.0 software. In vitro development variables [1st polar body (PB), fusion, cleavage, and blastocyst rates on Days 7 and 8] and in vivo development rates on Days 35, 60, 90, and 120 of pregnancy were analyzed by chi-square test. Total cell numbers and cells with fragmented DNA were analyzed by ANOVA. A level of 5% significance was considered. Extrusion of 1st PB (BL: n = 693; 69.3% and control: n = 639; 63.5%) and fusion rates (BL: n = 397; 79.2% and control: n = 345; 72.9%) were higher (P < 0.05) in the BL group. There were no differences between treatments for cleavage rates (BL: n = 268; 67.5% and control: n = 228; 66.1%) or blastocyst rates on Day 7 (BL: n = 77; 19.4% and control: n = 69; 20.0%) and Day 8 (BL: n = 81; 20.4% and control: n = 73; 21.2%). Cloned blastocysts from both groups were submitted to TUNEL reaction (Day 8 blastocysts, n = 15 for BL and control groups) for DNA fragmentation analysis or were transferred to synchronized recipients (Day 7 blastocysts, n = 26 and n = 28 for BL and control groups, respectively) for in vivo development analysis. No differences were observed (P > 0.05) between BL and control groups for total cell numbers (n = 127 and n = 138, respectively) and cells with fragmented DNA (0.0209 and 0.0188, respectively). Pregnancy rates at 35 (BL: n = 5; 19.2% and control: n = 9; 32.1%), 60 (BL: n = 3; 11.5 and control: n = 3; 10.7), 90 (BL: n = 3; 11.5 and control; n = 3; 10.7), and 120 days (BL: n = 3; 11.5 and control: n = 3; 10.7) also did not differ (P > 0.05) between treatments. In conclusion, pre-maturation enhanced 1st PB extrusion and fusion rates of oocytes submitted to SCNT, and moreover, it was able to establish pregnancies until 120 days, similarly to the control group. Financial support: FAPESP and CNPQ, Brazil.

Inhibition by saccharin of glucose-6-phosphatase: effects of alloxan in vivo and deoxycholate in vitro

1976 ◽  
Vol 54 (6) ◽  
pp. 587-590 ◽  
Author(s):  
David G. Lygre
Keyword(s):  
Kinetic Parameters ◽  
Rat Liver ◽  
Control Groups ◽  
Major Significance ◽  
Significant Difference ◽  
Hepatic Glucose ◽  
And Control

Inhibition by saccharin of rat liver glucose-6-phosphatase (EC 3.1.3.9) generally decreased as the pH increased in the range pH 4–8. This pattern was exhibited by homogenates from control and alloxan-treated animals assayed each in the absence and presence of 0.2% (w/v) deoxycholate. Saccharin inhibited in competitive fashion with respect to glucose-6-phosphate (glucose-6-P). There was a small increase in Km (glucose-6-P) but not Ki (saccharin) values in alloxan-treated rats when assays were conducted in the absence of deoxycholate. In the presence of this detergent there was no significant difference in these kinetic parameters between the alloxan-treated and control groups. Deoxycholate decreased Km (glucose-6-P) and increased Ki (saccharin) values. Calculations using these kinetic parameters indicate that, under usual hepatic glucose-6-P concentrations and relatively high levels of saccharin in liver, the inhibition by saccharin of glucose-6-phosphatase is unlikely to be of major significance in vivo.

133 EFFECT OF PROGESTERONE SUPPLEMENTATION OF MATURATION MEDIUM ON THE DEVELOPMENT OF IN VITRO-MATURED-IN VITRO-FERTILIZED-IN VITRO-CULTURED BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 180 ◽  
Author(s):  
K. Syoji ◽  
K. Imai ◽  
H. Koyama ◽  
O. Dochi
Keyword(s):  
Cell Mass ◽  
Calf Serum ◽  
Total Cell ◽  
Differential Staining ◽  
Control Group ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Maturation Medium ◽  
Significant Difference

The objective of this study was to investigate whether progesterone (P4) supplementation to in vitro maturation (IVM) medium could affect the competence of bovine oocyte to develop into blastocysts in vitro. Cumulus–oocyte complexes (COC) were collected by aspiration of ovarian follicles (2 to 6 mm in diameter) obtained from a local abattoir. The COC were matured for 20 h in TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 FSH at 38.5° in an atmosphere of 5% CO2 in air. After 18 h of gamete co-culture (5 × 106 sperms mL–1), presumptive zygotes were cultured in CRlaa containing 5% calf serum at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 for 9 days (fertilization = Day 0). Progesterone was added to the IVM medium 10 h after the start of the culture (1 μg group = 1 μg mL–1 of P4; 5 μg group = 5 μg mL–1 of P4; control group = no P4). The maturation (MII) rates were investigated after 20 h of starting the IVM culture. After maturation, the COC were denuded mechanically, and a part of the oocytes were mounted on slides, fixed with aceto-alcohol (1 : 3) solution for 48 h, stained with aceto-orcein, and observed under a phase-contrast microscope to determine their nuclear status (1 μg group: n = 32; 5 μg group: n = 28; control group: n = 31). The remaining COC were used for IVF. The cleavage rates were investigated on day 2, and the blastocyst formation rates were investigated on Days 7 to 9, respectively (1 μg group: n = 264; 5 μg group: n = 274; control group: n = 277). The blastocysts from Day 7 were used for differential staining of the inner cell mass (ICM) and trophectoderm cells (TE). The total cell numbers, ICM, and TE in the blastocysts were counted (1 μg group: n = 28; 5 μg group: n = 24; control group: n = 24). The rates of MII, cleavage, and blastocyst formation were expressed and analysed by the chi-squared test. Each set of cell numbers (mean ± standard error) was analysed by the unpaired t-test. The MII rate in the control group (76.7%) was significantly lower (P < 0.05) than that in the 1 μg group (93.8%). The cleavage rate in the 1 μg group (85.6%) was significantly higher (P < 0.05) than those in the control group (74.7%) and 5 μg group (77.4%). Further, the blastocyst formation rate in the 1 μg group (47.7%) and 5 μg group (43.4%) were significantly higher (P < 0.05) than that in the control group (35.0%). The ICM numbers (mean ± s.e.) were 39.5 ± 13.8 to 36.2 ± 8.9, the TE numbers were 74.4 ± 22.4 to 66.2 ± 12.9, the total cell numbers of blastocysts were 110.6 ± 28.2 to 103.0 ± 13.8. There was no significant difference in cell numbers among the groups. These results indicate that the cleavage and blastocyst formation rates can be improved by the addition of 1 μg mL–1 of P4 to the maturation medium.

In Vitro Mesenchymal Stem Cell Culture Using Calcium Phosphate Glass Scaffold

2005 ◽  
Vol 284-286 ◽  
pp. 679-682
Author(s):  
J. Kim ◽  
J.K. Ryu ◽  
Min Chul Kim ◽  
Yeon Ung Kim ◽  
Seong Ho Choi ◽  
...  
Keyword(s):  
Calcium Phosphate ◽  
Phosphate Glass ◽  
Bone Marrow Cells ◽  
Extracellular Matrices ◽  
Control Group ◽  
Control Groups ◽  
Significant Difference ◽  
And Control ◽  
Experimental Group

The purpose of this study was to evaluate the cell affinity of calcium phosphate glass scaffold in the system of CaO-CaF2-P2O5-MgO-ZnO, which is already reported that promoted the bone-like tissue formation in vitro and formed new bone in Sprague-Dawley rats. We prepared calcium phosphate glass saffolds with three-dimensionally interconnected pores of 200~500 µm. Commercial HA scaffold was employed as a control in this study. Bone marrow cells were collected from the healthy human donors and cultured within the prepared scaffolds. After 2, 4, 6, and 8 weeks, hMSCs/scaffold were fixed and stained with hematoxylin and eosin. hMSCs were continuously proliferated both in the experimental and control groups at every incubation period. The number of cells was higher in the experimental group than that of the control group, however, there was no significant difference (p>0.05). Extracellular matrices could be observed at the 2nd and 4th days in the experimental and control groups, respectively. The extracellular matrices were more abundant in the experimental group at all periods. The prepared calcium phosphate glass scaffolds are expected effective in bone tissue engineering.

Blastocyst production by in vitro maturation and development of porcine oocytes in defined media following intracytoplasmic sperm injection

Zygote ◽  
2007 ◽  
Vol 15 (2) ◽  
pp. 93-102 ◽  
Author(s):  
M. Kobayashi ◽  
S. Asakuma ◽  
Y. Fukui
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Subsequent Development ◽  
Control Group ◽  
Control Medium ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Defined Media ◽  
Significant Difference ◽  
The Mean

SummaryThe present study was carried out to establish porcine defined IVP. In Experiments 1 and 2, we investigated the efficacy of additional 0.6 mM cystine and/or 100 µM cysteamine (Cys) to a defined TCM199 maturation medium with regard to the intracellular glutathione (GSH) concentration and the developmental competence of in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The control medium was a modified TCM199 containing 0.05% (w/v) polyvinyl alcohol (PVA). Cys and/or cystine were added to the control medium. The control group and immature oocytes (presumptive germinal vesicle oocytes; GV) were prepared for GSH assay. In Experiment 3, the efficacy of epidermal growth factor (EGF) addition to a modified porcine zygote medium (mPZM) for in vitro culture (IVC) medium was investigated on embryonic development and the mean cell number of blastocysts following ICSI. As a positive or negative control, 0.3% BSA (mPZM-3) or 0.3% PVA (mPZM-4), respectively, was added to the base medium. The defined IVC medium was supplemented with 5 or 10 ng/ml EGF. In Experiment 1, no significant difference was found in the rates of cleavage (31.4–64.3%) and blastocyst formation (6.5–22.9%) among the treatment and control groups. The mean cell numbers per blastocyst ranged from 30 to 48 among the groups without significant differences. However, in Experiment 2, the intracellular GSH concentrations in the oocytes cultured in the medium supplemented with 100 µM Cys (9.6 pmol/oocyte) or Cys + cystine (9.9 pmol/oocyte) were significantly (p < 0.05) higher than the control (2.5 pmol/oocyte) and 0.6 mM cystine (6.5 pmol/oocyte) groups, but not different from the GV group (9.0 pmol/oocyte). The GSH concentration in the cystine group was also significantly (p < 0.05) higher than that in the control group, but not different from the GV group. In Experiment 3, the rates of cleavage and blastocyst formation and the mean cell numbers of blastocysts were not significantly different among the groups. However, the addition of 5 ng/ml EGF into the mPZM-4 resulted in a significantly (p < 0.05) higher blastocyst rate per cleaved embryo than the other two defined groups (mPZM-4 + 5 ng/ml: 48.6%, mPZM-4 and mPZM-4 +10 ng/ml: 23.4% and 23.1%, respectively).The present results indicate that the addition of Cys to a defined medium for in vitro maturation (IVM) of porcine oocytes increases intracellular GSH concentration. Further addition of cystine into the IVM medium containing 100 µM Cys is not necessary and TCM199 plus Cys (100 µM) could be used as a defined IVM medium for porcine oocytes. The addition of 5 ng/ml EGF to a defined IVC medium has enhanced subsequent development after ICSI. This study shows that porcine blastocysts can be produced by defined media throughout the steps of IVP (IVM, ICSI and IVC).

scholarly journals Platelet Aggregability in Sleep-Related Stroke

1989 ◽  
Vol 16 (1) ◽  
pp. 71-77
Author(s):  
C.L. Voll ◽  
N. Chetty ◽  
P. Atkinson
Keyword(s):  
Stroke Onset ◽  
Control Groups ◽  
Nocturnal Sleep ◽  
Platelet Aggregability ◽  
Significant Difference ◽  
History Of ◽  
Circadian Fluctuation ◽  
The Mean ◽  
And Control

ABSTRACT:We examined platelet aggregability during nocturnal sleep and daytime wakefulness in patients with a history of sleep-related stroke onset (SOS) and compared it to that of matched awake-onset stroke (AOS) patients and controls without evidence of vascular disease. Aggregability was evaluated in-vitro at least seven weeks following stroke onset. Platelets were more aggregable to ADP, collagen and arachidonic acid (AA) during both sleep and wakefulness in patients with AOS (p<0.01). No significant difference in the mean aggregation thresholds during sleeping or waking periods were found between SOS and control groups. However, platelets were significantly more responsive to AA during sleep than during wakefulness in the SOS patients (p<0.01). This difference was confined to the subgroup of SOS patients who had experienced nocturnal as opposed to daytime sleep-related stroke onset, suggesting that the observed difference in platelet responsiveness to AA may be related to a circadian fluctuation in platelet aggregability rather than to a sleep-related fluctuation. Significant sleep-related changes in platelet aggregability were not identified in the other two groups.

Effects of Virgin Coconut Oil as Adjunct Therapy in the Treatment of Allergic Rhinitis

2016 ◽  
Vol 1 (1) ◽  
pp. 22
Author(s):  
Nazli Zainuddin ◽  
Nurul Azira Mohd Shah ◽  
Rosdan Salim
Keyword(s):  
Allergic Rhinitis ◽  
Side Effects ◽  
Coconut Oil ◽  
Test Group ◽  
Nasal Symptom ◽  
Control Group ◽  
Virgin Coconut Oil ◽  
Control Groups ◽  
Significant Difference ◽  
And Control

Introduction: The role of virgin coconut oil in the treatment of allergic rhinitis is controversial. Thus, the aim of the present study is to determine the effects of virgin coconut oil ingestion, in addition to standard medications, on allergic rhinitis. We also studied the side effects of consumption of virgin coconut oil. Methods: Fifty two subjects were equally divided into test and control groups. All subjects received a daily dose of 10mg of loratadine for 28 days. The test group was given 10ml of virgin coconut oil three times a day in addition to loratadine. The symptoms of allergic rhinitis were scored at the beginning and end of the study. Results:, the symptom score were divided into nasal and non-nasal symptom scores. Sneezing score showed a significant difference, however the score was more in control group than test group, indicating that improvement in symptom was more in control group. The rest of the nasal symptom and non-nasal symptom score showed no significant difference between test and control groups. Approximately 58% of the test subjects developed side effects from consumption of virgin coconut oil, mainly gastrointestinal side effects. Conclusion: In the present study, ingestion of virgin coconut oil does not improve the overall and individual symptoms of allergic rhinitis, furthermore it has side effects.

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