163 IN VITRO CULTURE OF PORCINE OOCYTE-GRANULOSA CELL COMPLEXES

2011 ◽  
Vol 23 (1) ◽  
pp. 184
Author(s):  
Y. F. Diao ◽  
R. X. Han ◽  
H. R. Kim ◽  
C. S. Park ◽  
D. I. Jin
Keyword(s):  
Survival Rate ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Control Group ◽  
Oocyte Diameter ◽  
Cell Complexes ◽  
Treatment Groups ◽  
Significant Difference ◽  
Porcine Oocyte

The objective of this study was to investigate the effects of porcine follicular fluid (PFF) and insulin-like growth factor-1 (IGF-1) on the growth of porcine oocyte-granulosa cell complexes (OGC) in vitro, in an effort to improve meiotic and developmental competence in vitro. Porcine OGC were manually dissected from early antral follicles of diameter 400 to 700 μm, and intact oocytes with undamaged granulosa cells were selected for culture. Between 3 and 5 OGC were combined in 50-uL droplets and cultured for 12 days in M199 medium supplemented with PVP, oestradiol, FSH, transferrin, L-ascorbic acid, and insulin. The OGC were cultured at 38.5°C in a humidified atmosphere of 5% CO2 in air for 12 days, and the oocytes were matured for 44 h. Oocyte diameter, exclusive of the zona pellucida, was measured on day 0 and day 12 of culture. Control group was cultured in the absence of PFF or IGF-1. The experiment was divided into 2 parts. In part 1, treatment groups were cultured with 2.5, 5.0, or 7.5% (all v/v) PFF. Control OGC grew as spheres that were formed by granulosa cells. In treatment groups, the granulosa cells spread and grew on the bottom of dishes. When oocyte diameter was measured after 12 days of culture, no significant difference among groups was observed (104.07, 103.96, and 104.27 μm at the 3 PFF concentrations used; control: 104.03 μm). Similarly, the survival rate of oocytes did not differ significantly among groups. However, survival rate fell somewhat in the group treated with PFF (control: 65%; tests: 58.87, 58.33, and 50.83% at the 3 PFF concentrations used). The maturation rates of oocytes in the control was significantly higher than those of the treatment groups [25.83% (control) v. 14, 11.67, and 3.67% (the 3 treatment groups); P < 0.05]. Thus, the first conclusion is that supplementation of culture medium with PFF did not enhance the development of porcine OGC in vitro. In part 2 of the experiment, treatment groups were cultured with 10, 50, or 100 ng mL–1 IGF-1. The percentages of OGC showing antrum formation were 80, 80, 100, and 100% in groups treated with 0, 10, 50, or 100 ng mL–1 IGF-1, respectively. Average oocyte diameter was 94.16 to 94.58 μm just after OGC collection. However, the average diameter of oocytes cultured for 12 days with 50 or 100 ng mL–1 IGF-1 was significantly higher than that of the control or 10 ng mL–1 groups [108.88 and 108.31 μm (50 and 100 ng mL–1 IGF-1) v. 105.98 μm and 106.67 μm (0 and 10 ng mL–1 IGF-1); P < 0.05]. The maturation rate of oocytes grown with 10 and 50 ng mL–1 IGF-1 was higher than those of the other 2 groups [30 and 40.6% (10 and 50 ng mL–1 IGF-1, respectively) v. 25 and 26% (0 and 100 ng mL–1 IGF-1, respectively); P < 0.05]. Thus, the second conclusion is that 50 ng mL–1 IGF-1 improves the growth and maturation of porcine oocyte-granulosa cell complexes in vitro.

321 OOCYTE DIAMETER INFLUENCES THE MEIOTIC RESUMPTION AND PROGRESSION INDUCED BY OKADAIC ACID IN DOG

2006 ◽  
Vol 18 (2) ◽  
pp. 268
Author(s):  
F. Ariu ◽  
L. Bogliolo ◽  
I. Rosati ◽  
M. T. Zedda ◽  
S. Pau ◽  
...  
Keyword(s):  
Okadaic Acid ◽  
In Vitro Maturation ◽  
Mammalian Species ◽  
Cumulus Cells ◽  
Meiotic Maturation ◽  
Control Group ◽  
Oocyte Diameter ◽  
Treatment Groups ◽  
Different Diameters

The acquisition of meiotic competence, in the bitch as in many other mammalian species, is related to the oocyte diameter. This study was designed to determine the effect of okadaic acid (OA), a potent inhibitor of seronine/threonine 1 and 2A phosphatases, on meiotic resumption and progression in canine oocytes with different diameters. In two experiments, healthy cumulus-oocytes complexes were collected from ovaries of bitches at various stages of the estrous cycle and divided, by diameters, into three treatment groups for in vitro maturation: <110 �m, 110-120 �m, and >120 �m. In Experiment 1, oocytes were pre-incubated for 1 h in TCM-199 + 20% estrous canine serum (SCE) + cysteamine + OA (0.5 �M). Then, oocytes were cultured for 48 h in the same medium without OA at 38.5�C, 5% CO2 in air. As a control group, oocytes were matured in vitro under the same conditions but without pre-incubation with OA. In Experiment 2, to determine if the effect of OA is mediated by cumulus cells, >120 �m oocytes were denuded from cumulus cells, incubated with or without OA, and cultured in vitro as previously described. At 48 h, all oocytes were stained and fixed with glycerol-Hoechst 33342 to assess the stage of meiotic maturation. In Experiment 1, OA induced a significantly higher incidence of meiotic resumption in oocytes <110 �m (16/108, 14.8%; P < 0.05) and 110-120 �m (70/130, 53.8%; P < 0.01) as compared to that of oocytes in the <110 �m and 110-120 �m control groups (2/58, 3.4%; 24/82, 29.3%). The percentage of oocytes in the 110-120 �m OA group that underwent in vitro maturation to metaphase II (MII) was significantly higher than in the 110-120 �m control group (18/130, 13.8% vs. 4/82, 4.9%, respectively; P < 0.05). In contrast, smaller oocytes (<110 �m) did not develop to MII with or whitout OA. Meiotic resumption rate of >120 �m OA group (64/78, 82.0%) was similar to the >120 �m control group (56/72, 77.8%), but a significantly higher proportion of the oocytes pre-incubated with OA progressed to MII than did the control oocytes (40/78, 51.3% vs. 12/72, 16.7%, respectively; P < 0.01). Low rates of meiotic resumption were observed in denuded >120-�m oocytes with (7/63, 11.1%) or without OA (7/55, 12.7%) and none of them progressed to MII. In conclusion, the results of the present study indicate that treatment of fully grown (>120 �m) oocytes with okadaic acid at the onset of in vitro maturation can result in a higher frequency of meiotic maturation than previously reported. Also, we determined that the beneficial effect of okadaic acid was mediated by cumulus cells.

47 CRYOPRESERVATION OF BOVINE GERM CELL USING ANTIFREEZE POLYAMINO-ACID (CARBOXYLATED POLY-L-LYSINE)

2017 ◽  
Vol 29 (1) ◽  
pp. 131
Author(s):  
T. Fujikawa ◽  
C. Kubota ◽  
T. Ando ◽  
S. Imamura ◽  
M. Tokumaru ◽  
...  
Keyword(s):  
Survival Rate ◽  
Germ Cell ◽  
Embryo Transfer ◽  
Conception Rate ◽  
Control Group ◽  
Vitro Fertilization ◽  
Polyamino Acid ◽  
Significant Difference

Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound, and it is obtained by converting 65% amino groups to carboxyl groups after synthesising ε-poly-l-lysine aqueous solution and succinic anhydride. CPLL has cryoprotective property similar to antifreeze protein, and addition of CPLL into cryopreservation medium improves the post-thaw survival rate of cells and embryos. In this research, we examined the effectiveness of CPLL as a bovine germ cell cryoprotective material. In experiment 1 (in sperm), the conventional cryopreservation medium used for control group was consisted of 6.5% (vol/vol) glycerin, and the cryopreservation medium used for CPLL group was consisted of 3.25% (vol/vol) glycerin and 0.5% CPLL (wt/vol). The post-thaw survival and motility were assessed by using Sperm Motility Analysis System (DITECT Corp., Tokyo, Japan). There was no significant difference for post-thaw survival rate and motility (control v. CPLL; 98.8% v. 96.6% and 69.7% v. 62.2%, respectively). Artificial insemination was carried out in 65 cows (control v. CPLL; 34 v. 31), and the conception rate of the CPLL group was higher than that of the control group (80.6% v. 67.6%; P = 0.23). In experiment 2 (embryos), the conventional cryopreservation medium used for control group was consisted of 5% (vol/vol) ethylene glycol and 6% (vol/vol) propylene glycol in PBS. In the CPLL group, 7% (wt/vol) CPLL was added to the conventional medium. In vitro fertilization embryos were cryopreserved at Day 7 and Day 8. There was no significant difference in survival rate at 0, 24, and 48 h and hatched rate until 72 h after thawing (control v. CPLL: 93.6% v. 93.2%, 69.0% v. 64.7%, 56.1% v. 56.3%, 12.9% v. 10.2%, respectively). Embryos obtained by superovulation treatment and in vivo fertilization at Day 7 were cryopreserved using above 2 media, and transferred non-surgically into synchronized recipient cows (1 embryo per animal). Embryo transfer (ET) was carried out in 81 cows (control v. CPLL: 31 v. 50), and recipients were diagnosed for pregnancy ultrasonically 50 days after embryo transfer. Conception rate of CPLL group was higher than control group (50.0% v. 29.0%; P = 0.063). In both experiments, the significant differences between control group and CPLL group were determined by chi-squared test. The effectiveness of CPLL in cells and embryos has been reported; however, there is no report using CPLL in bovine germ cells. In this research, CPLL improved the conception rate of AI and ET, probably due to its low toxicity and protection of the cell membrane. These results suggest that CPLL is available as a new cryoprotective material for bovine sperm and embryo in slow freezing methods.

Effect of estradiol during culture of bovine oocyte–granulosa cell complexes on the mitochondrial DNA copies of oocytes and telomere length of granulosa cells

Zygote ◽  
2012 ◽  
Vol 22 (4) ◽  
pp. 431-439 ◽  
Author(s):  
M. Endo ◽  
K. Kimura ◽  
T. Kuwayama ◽  
Y. Monji ◽  
H. Iwata
Keyword(s):  
Mitochondrial Dna ◽  
Telomere Length ◽  
Granulosa Cells ◽  
Culture Medium ◽  
Mt Dna ◽  
Cell Complexes ◽  
Antral Follicles ◽  
Mitochondrial Dna Copy Number ◽  
Significant Difference

SummaryDuring the development of oocytes from early antral follicles (EAFs) to antral follicles (AFs), the mitochondrial DNA copy number (Mt DNA number) increases, and granulosa cells markedly proliferate. This study examined the effect of supplementation of culture medium with estradiol-17β (E2) on the in vitro growth of oocytes, and increases in the Mt DNA number, and telomere length during the in vitro culture of oocytes derived from EAFs (0.4–0.7 mm in diameter). The E2 supplementation improved antrum formation and the ratio of oocytes reaching the metaphase II (MII) stage, and there was a significant difference in these values between addition E2 concentrations of 10 μg/ml and 0.1 μg/ml. When the oocytes were cultured in the medium containing 10 μg/ml E2, the Mt DNA number determined by real-time polymerase chain reaction (PCR) significantly increased, and the ratio of the Mt DNA number at the end of culture to the Mt DNA number at the beginning of the culture was greatly different among cows, and could be predicted by the degree of the difference between the Mt DNA number of oocytes derived from EAFs and that of oocytes derived from AFs (3–6 mm in diameter). When oocytes were cultured for 16 days in a medium containing 10 μg/ml E2 or 0.1 μg/ml E2, the Mt DNA number of oocytes grown in vitro did not differ, but the telomere length of the granulosa cells was significantly greater in the 10 μg/ml E2 group than in the 0.1 μg/ml group. In conclusion, E2 supplementation in culture medium improved the growth of oocytes derived from EAFs, and a high E2 concentration increased the telomere length of the granulosa cells.

scholarly journals PERBANDINGAN EFEKTIVITAS BUAH STROBERI (Fragaria x ananassa) DENGAN BUAH JERUK NIPIS (Citrus aurantifolia) SEBAGAI BAHAN ALAMI PEMUTIH GIGI SECARA IN VITRO

2017 ◽  
Vol 5 (2) ◽  
pp. 129-135
Author(s):  
Nia Nurhaeni ◽  
Denas Symond ◽  
Bambang Ristiono
Keyword(s):  
Fragaria X Ananassa ◽  
Control Group ◽  
Carbamide Peroxide ◽  
Tooth Whitening ◽  
Citrus Aurantifolia ◽  
Treatment Groups ◽  
Color Changes ◽  
Significant Difference ◽  
Lime Concentration

One of the aesthetic problem which had bother and become a complaint was teeth discoloration that can be overcome by dental bleaching procedures. The use of tooth whitening ingredient can cause side effects such as tooth sensitivity and mucous irritation. Therefore, many researchers have been looking for a safer alternatives materials  to be used as tooth whitening ingredients including Strawberry (Fragaria x ananassa) contain of elegat acid and malic acid and Lime (Citrus aurantifolia) contain of citric acid which have potential to whiten the teeth. The purpose of this research is to determine differences strawberry and lime fruit as a natural ingredient of tooth whitening. This research used in vitro laboratory experiment method by using 30 post-extraction premolar on teeth divided into three groups, namely the treatment of strawberry concentration of 100% (K1), the treatment group lime concentration of  2.5% (K2 ) and the treatment control group carbamide peroxide 10% (K3). Color changes measurements was observed pretest and posttest by 15 observers using Shade Guide VITAPAN classical. The research showed that strawberry concentration of 100% have an average difference in the color of teeth is 6.40, lime concentration of 2.5 at 6.20 and carbamide peroxide 10% as the control group amounted to 3.20. Based on the Kruskal Wallis test there are differences in the average value of the observation color of the teeth was significant (P <0.05) and continued with different test further the Post Hoc Test Mann Whitney gained significant difference (p> 0.05) between treatment groups strawberry 100% with carbamide peroxide 10%, lime and 2.5% carbamide peroxide 10%, while among the treatment groups strawberries 100% with 2.5% lime fruit is not significantly different because it has a value of p> 0.05. Strawberry concentration of 100% more effective to whiten teeth than lime concentration of 2.5%, but there is no significant difference in teeth whitening, while lime is more effective than carbamide peroxide 10%. Keywords:Strawberry, lime, carbamide peroxide 10%, tooth whitening.

190 EFFECT OF TRANS-ε-VINIFERIN ON IN VITRO PORCINE OOCYTE MATURATION AND SUBSEQUENT DEVELOPMENTAL COMPETENCE IN PRE-IMPLANTATION EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 207
Author(s):  
Y. Jeon ◽  
S.-S. Kwak ◽  
S.-A. Jeong ◽  
R. Salehi ◽  
Y. H. Seong ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Vitis Amurensis ◽  
Total Cell Number ◽  
Treatment Groups ◽  
Nuclear Maturation ◽  
Porcine Oocyte

Trans-ε-viniferin is a naturally occurring polyphenol belonging to the stilbenoids family. Trans-ε-viniferin is isolated from Vitis amurensis, 1 of the most common wild grapes in Korea, Japan and China. We investigated the effects of trans-ε-viniferin on in vitro maturation (IVM) and developmental competence after IVF or parthenogenesis (PA). At the laboratory of Veterinary Pharmacology, College of Veterinary Medicine, Chungbuk National University, trans-ε-viniferin was purified from the leaves and stems of Vitis amurensis. Data were analyzed with SPSS 17.0 using Duncan's multiple range test. First, in total, 594 cumulus–oocyte complexes (COC) were used for the evaluation of nuclear maturation. The COC were matured in TCM-199 medium supplemented with various concentrations of trans-ε-viniferin (0, 0.1, 0.5, 1.0 and 5.0 μM) with 10% porcine follicular fluid, 10 IU mL–1 of eCG and 10 IU mL–1 of hCG. After 22 h in maturation culture, the COC were cultured in hormone-free medium supplemented with various concentrations of trans-ε-viniferin for an additional 22 h and then nuclear maturation was evaluated. Second, in total, 300 matured oocytes were used to examine the effects of different trans-ε-viniferin concentrations (0, 0.5 and 5.0 μM) on porcine oocyte intracellular glutathione (GSH) and reactive oxygen species (ROS) levels. Lastly, the developmental competence of oocytes matured with different concentrations of trans-ε-viniferin (0, 0.5 and 5.0 μM) was evaluated after IVF or PA. In total, 711 embryos were evaluated. As results, we observed that trans-ε-viniferin treatment during IVM did not improve the nuclear maturation of oocytes in any group (84.2, 86.6, 85.5, 83.3 and 79.2%, respectively), but significantly increased (P < 0.05) intracellular GSH levels in the 0.5 μM group (0 μM vs 0.5 μM; 14.6 vs 16.8 pmol oocyte–1) and reduced ROS levels (0 μM vs 0.5 μM and 50 μM; 174.6 vs 25.7 and 23.8 pixel oocyte–1). Oocytes treated with trans-ε-viniferin during IVM did not have significantly different cleavage rates or blastocyst formation rates after IVF, but total cell numbers were significantly higher (P < 0.05) in the 0.5 and 5.0 μM treatment groups (53.6 ± 4.0 and 47.9 ± 3.1) compared to the control group (36.4 ± 2.2). The PA embryos showed similar results; there were no significant differences in cleavage rates and blastocyst formation rates, but the total cell number significantly increased in the 0.5 and 5.0 μM treatment groups (59.6 ± 4.2 and 60.8 ± 4.6) compared to the control group (43.1 ± 2.1). In conclusion, these results indicate that trans-ε-viniferin treatment during porcine IVM increased total cell number of blastocysts, possibly through increasing intracellular GSH synthesis and reducing ROS levels. This work was supported by a grant from the Korea institute of Planning & Evaluation for Technology in Food, Agriculture, Forestry & Fisheries, Republic of Korea.

186 EFFECT OF POLYVINYLPYRROLIDONE SUPPLEMENTATION IN CULTURE MEDIUM ON THE GROWTH OF MOUSE OOCYTES

2013 ◽  
Vol 25 (1) ◽  
pp. 242
Author(s):  
S. Mizumachi ◽  
K. Sasaki ◽  
K. Matsubara ◽  
Y. Hirao
Keyword(s):  
Granulosa Cell ◽  
Culture Medium ◽  
Polar Body ◽  
Cumulus Cell ◽  
Mouse Oocyte ◽  
Oocyte Diameter ◽  
Oocyte Growth ◽  
Cell Complexes ◽  
Bovine Oocytes

A high volume of polyvinylpyrrolidone (PVP) supplementation in culture medium has a significant impact on the growth of bovine oocytes. The objective of the present study was to determine whether or not PVP affects oocyte growth in the mouse. Oocyte–granulosa cell complexes were isolated from 11- or 12-day-old mice (ICR) by mechanical isolation of follicles, followed by a collagenase treatment (0.1%; 10 min). Twenty complexes were placed on each insert fit in the 24-well culture plate and cultured for 10 days in an atmosphere of 5% CO2 in air at 37°C. The culture medium was a modified α-MEM supplemented with 5% fetal bovine serum and 1 ng mL–1 FSH. The concentration of PVP (molecular weight of 360 000) was 0%, 1%, 2%, or 3% (w/v). During the first 2 days, only medium with 0% PVP was used. The oocytes recovered on Day 10 were subjected to in vitro maturation, IVF, and embryo culture. In 12 replications, the total numbers of oocytes cultured in medium with 0%, 1%, 2%, and 3% PVP were 235, 233, 233, and 231, respectively. In some additional experiments, oocytes were fixed on Day 10 and processed for transmission electron microscopy (TEM). The oocytes in medium with 0% PVP became located within an enlarged dome-like structure. In medium with 2% PVP and 3% PVP, no such domes were formed, and the oocytes within several granulosa cell layers were exposed to medium; however, the cumulus cell mass specifically became larger than that in medium with 0% PVP. The viabilities of oocytes recovered from medium with 0%, 1%, 2%, and 3% PVP were 83%, 81%, 91%, and 93%, respectively. The survival rate was significantly higher in medium with 3% PVP than in medium with 0% PVP or 1% PVP (P < 0.05). The mean oocyte diameter increased from 59 µm (Day 0) to 72, 71, 71, and 72 µm in medium with 0, 1, 2, and 3% PVP, respectively, but they continued to be smaller than in vivo grown oocytes (81.0 µm; P < 0.01). When maturation was induced, cumulus cell mucification occurred irrespective of PVP concentration during the growth. No significant differences were found between the groups in the percentage of polar body extrusion (ranging from 78 to 88%). Developmental outcomes based on oocytes used for in vitro fertilization were the following: cleavage rates were 67, 78, 74, and 76%; and blastocyst rates were 37, 44, 47, and 36% of oocytes that had been grown in medium with 0, 1, 2, and 3% PVP, respectively. The numbers of oocytes included were 60, 59, 68, and 66, respectively. The TEM observation suggests that more intimate contacts were maintained between the oocyte and cumulus cells in medium with 2% PVP than in medium with 0% PVP. Taken together, PVP supplementation in medium has a considerable influence on the morphology of mouse oocyte–granulosa cell complexes and close contacts within the complexes in the long-term culture, as having been observed with bovine oocytes.

scholarly journals Growing Porcine Oocyte-Granulosa Cell Complexes Acquired Meiotic Competence During In Vitro Culture

2007 ◽  
Vol 53 (2) ◽  
pp. 379-384 ◽  
Author(s):  
Shu HASHIMOTO ◽  
Kanako OHSUMI ◽  
Yoko TSUJI ◽  
Naoko HARAUMA ◽  
Yuko MIYATA ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Granulosa Cell ◽  
Cell Complexes ◽  
Porcine Oocyte ◽  
Meiotic Competence

scholarly journals 117EFFECTS OF PREVIOUS PRESSURE TREATMENT ON THE SURVIVAL AND DEVELOPMENTAL SPEED OF EXPANDED MOUSE BLASTOCYSTS FROZEN RAPIDLY (PILOT STUDY)

2004 ◽  
Vol 16 (2) ◽  
pp. 181
Author(s):  
C. Pribenszky ◽  
M. Molnar ◽  
S. Cseh ◽  
L. Solti
Keyword(s):  
Survival Rate ◽  
Applied Pressure ◽  
Control Group ◽  
Pressure Treatment ◽  
Embryo Viability ◽  
Group I ◽  
Significant Difference ◽  
Vitrification Solution ◽  
Group Ii

It has been demonstrated that embryos can survive exposure to a substantial amount of pressure. (Pribenszky et al., 2003 Theriogenology 59, 329, and 2002 Theriogenology 57, 506). Other studies report that, if a biological system is challenged by certain stresses, its ability to react and survive other stresses can be improved. The aim of our present study was to examine whether the survival rate of expanded mouse blastocysts could be improved by a certain pressure treatment before the freezing procedure. Morula stage mouse embryos were collected and cultured at 37°C with 5% CO2 and maximal humidity in air in G 2.2 medium (Vitrolife, Göteborg, Sweden) to the expanded blastocyst stage. Embryos were randomly allocated to three groups. Embryos in Group I were equilibrated for 5 minutes in a solution containing 1.5M ethylene glycol (EG) and 0.25M sucrose in M2 (Sigma, St. Louis, MO, USA), supplemented with 10% FCS (Sigma), and then transferred into a vitrification solution (7M EG, 0.5M sucrose in M2 with 10% FCS) pre-loaded in a 0.25-ml plastic straw (7–9 embryos/straw). After 1-min exposure to the vitrification solution, the straw was slowly immersed in liquid nitrogen. Embryos in Group II were loaded into 0.08-mL straws (7–9 embryos/straw) with M2. Straws were placed into the chamber, filled with M2, of a special laboratory-made device that is capable of generating and precisely detecting hydrostatic pressure up to 150MPa (1500atm), and were exposed to 60MPa pressure for 30min. After the pressure treatment, embryos were frozen as described above. Straws were thawed by transfer into 30°C water for 30s and then the embryos were recovered and placed in rehydration medium (0.5M sucrose in M2 supplemented with 10% FCS) for 5min. Embryos then were cultured in medium G2.2 as described above. A total of 27, 29 and 26 embryos were assigned to Group I, Group II and the untreated control group, respectively. Embryo viability and development were assessed at 6 and 20h after culture as determined by morphological appearance and hatching. At 6h, 16% (4/27) of the non-pressurized embryos were one-half expanded, at 20 hours 37% (10/27) were two-thirds and 30% (8/27) were one-half expanded; none of them were hatching. While at the pressure treated groups 89% (26/29) of the embryos were fully expanded at 6 hours, and 68% (20/29) were hatching at 20h (untreated: 25/26 fully expanded at 6h, 24/26 hatched at 20h). Data were analyzed by chi-square test. We considered embryos which were at least two-thirds expanded. After 6 hours Group I differed from Group II and the control (P&lt;0.01). There was no significant difference between Group II and the control (P&lt;0.01). After 20 hours the same relations were seen. In the case of hatching, Group I differed from Group II and the control (P&lt;0.01). There was no significant difference between Group II and the control (P&lt;0.05). According to our results, the applied pressure treatment improved the in vitro development of the embryos after freezing. The re-expansion was faster and the survival rate was higher for those embryos that received pressure treatment before cryopreservation. Further experiments are needed to confirm and explore the in vitro and in vivo effects and benefits of pressure treatment before freezing.

Promotion of glucose utilization by insulin enhances granulosa cell proliferation and developmental competence of porcine oocyte grown in vitro

Zygote ◽  
2016 ◽  
Vol 25 (1) ◽  
pp. 65-74 ◽  
Author(s):  
Nobuhiko Itami ◽  
Yasuhisa Munakata ◽  
Koumei Shirasuna ◽  
Takehito Kuwayama ◽  
Hisataka Iwata
Keyword(s):  
Granulosa Cell ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Oocyte Development ◽  
Energy Status ◽  
Cell Complexes ◽  
Porcine Oocyte ◽  
High Insulin

SummaryIn vitro culture of the oocyte granulosa cell complexes (OGCs) from early antral follicles (EAFs) shows granulosa cell (GC) proliferation, but to a lesser extent than that observed in vivo during follicle development. As the number of GCs closely relates to energy sufficiency of the oocytes, enhancement of GC proliferation influences oocyte development. GC proliferation depends on glycolysis and insulin-mediated AKT/mTOR signaling pathway; therefore, addition of culture medium containing insulin and glucose may potentially promote GC proliferation and hence improve oocyte development. In the present study, we assessed the effect of exogenous insulin and glucose concentration on GC proliferation and oocyte energy status as well as developmental abilities of porcine oocytes grown in vitro. In the presence of 5.5 mM of glucose (Low), a comparison of 10 versus 20 μg/ml insulin showed that high insulin enhanced GC proliferation but exhausted glucose from the medium, which resulted in low energy status including lipid and adenosine triphosphate of the oocyte. Whereas, in the presence of 20 μg/ml insulin, medium with 11 mM glucose (High) enhanced GC proliferation and oocyte energy status as well as developmental ability up to the blastocyst stage. Considering that there was no difference in OGCs development observed with medium (10 μg/ml insulin) containing 5.5 versus 11 mM glucose, we concluded that the combination of high insulin and glucose enhanced GC proliferation and energy status of oocytes as well as the developmental ability of the oocytes grown in vitro.

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