126 EFFECT OF ASSISTED HATCHING ON THE SURVIVAL AND THE DEVELOPMENT OF BOVINE EMBRYOS PRODUCED IN VITRO AFTER CRYOPRESERVATION

2008 ◽  
Vol 20 (1) ◽  
pp. 143
Author(s):  
J. Fukuhara ◽  
T. Takuma ◽  
S. Kasa ◽  
K. Imai
Keyword(s):  
Survival Rates ◽  
Calf Serum ◽  
Assisted Hatching ◽  
Chi Square ◽  
Conventional Procedure ◽  
Custom Made ◽  
Frozen Embryos ◽  
Chi Square Test ◽  
Functional Peptides

The aim of this work is to investigate the effect of assisted hatching (AH) by partial zona pellucida (ZP) dissection on the survival and the development of bovine IVP embryos after ultra-rapid vitrification and slow freezing. COC obtained from abattoir bovine ovaries were matured and fertilized in vitro, and then cultured in IVD101 (Research Institute for the Functional Peptides, Yamagata, Japan) at 38.5�C in 5% CO2, 5% O2, 90% N2. The treatment of AH was done on compacted morulae by partially dissecting ZP with a micromanipulator. As a control, non-treated embryos with intact ZP were used. For vitrification, the blastocysts at days 7 and 8 were placed into a vitrification solution (Dulbecco's PBS (D-PBS) supplemented with 20% glycerol, 20% ethylene glycol (EG), 0.3 m sucrose (SUC), 0.3 m xylose, and 3% polyethylene glycol) for 30 s after two-step equilibration. Then, they were immediately placed on a custom-made vitrification tool made of nylon fishing line with a small piece of iron attached to one end (V-tool), and immersed into liquid nitrogen (LN2). After cooling, the embryos on the V-tool were placed into frozen 0.25 mL straws filled with a diluting solution (D-PBS supplemented with 0.5 m SUC and 20% new born calf serum) using a magnet, and then they were preserved in LN2. For warming, the straws were immersed into 25�C water. The V-tool was then introduced into the column of diluting solution using a magnet. For freezing, the blastocysts at days 7 and 8 were frozen by the conventional procedure with 10% EG. For thawing, the straws were immersed into 30�C water. In this study, 120 embryos were vitrified and 128 embryos were frozen. Warmed and thawed embryos were washed more than two times, and cultured in TCM199 supplemented with 20% fetal bovine serum and 0.1 mm β-mercaptoethanol for 72 h for assessment of survivability and developmental capacity of post-thaw embryos. Data were analyzed with the chi-square test. The survival rates of vitrified embryos were the same with or without AH (81.1 and 82.0%, P > 0.05). The survival rates of frozen embryos were also the same with or without AH (76.3 and 66.7%, P > 0.05). The survival rates of vitrified embryos without AH was significantly higher than that of frozen embryos without AH (82.0 v. 66.7%, P < 0.05). The hatched rates of frozen embryos without AH were significantly lower than that of frozen embryos with AH and those of vitrified embryos with and without AH (43.5 v. 64.4%, 67.9 and 68.9%, P < 0.05). These results indicated that AH enhanced the development of frozen bovine IVP embryos and that our vitrification method using a V-tool did not require AH for development of embryos.

scholarly journals Comparison of bacterial microleakage of three bioactive endodontic sealers in simulated underwater diving and aviation conditions

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Mehdi Dastorani ◽  
Behnam Malekpour ◽  
Mohsen AminSobhani ◽  
Mohammadsadegh Alemrajabi ◽  
Arezoo Mahdian ◽  
...  
Keyword(s):  
Atmospheric Pressure ◽  
Mineral Trioxide Aggregate ◽  
In Vitro Study ◽  
Chi Square ◽  
Custom Made ◽  
Single Cone ◽  
Registration Number ◽  
Chi Square Test ◽  
Pressure Changes

Abstract Background Bacterial microleakage is an important cause of apical periodontitis and endodontic treatment failure. This study aimed to assess the bacterial microleakage of nano-mineral trioxide aggregate (nano-MTA) as a sealer, Endoseal MTA, and GuttaFlow Bioseal sealers in atmospheric pressure, and simulated underwater diving and aviation conditions. Methods In this in vitro, experimental study, 180 extracted single-rooted teeth were cleaned and shaped, and were then randomly divided into three groups for single-cone obturation using Endoseal MTA, GuttaFlow Bioseal, or nano-MTA as a sealer. Each group was then randomly divided into three subgroups, and subjected to ambient atmospheric pressure, 2 atm pressure (to simulate underwater diving), and 0.5 atm pressure (to simulate aviation) using a custom-made pressure chamber. The teeth then underwent microbial leakage test using Streptococcus mutans (S. mutans), and the percentage of samples showing microleakage was recorded for up to 1 month, and analyzed using the Chi-square test. Results The three sealer groups were significantly different regarding bacterial microleakage (P < 0.05). The nano-MTA group showed significantly higher microleakage after 15 days than the other two groups (P = 0.006). The effect of pressure on bacterial microleakage was not significant in any sealer group (P > 0.05). Conclusion Within the limitations of this in vitro study, it may be concluded that single-cone obturation technique using nano-MTA as a sealer results in lower resistance to bacterial microleakage compared with the use of GuttaFlow Bioseal, and Endoseal MTA. Pressure changes in simulated underwater diving and aviation conditions had no significant effect on bacterial microleakage. Trial Registration Number This is not a human subject research.

242 PROMISING TWO-STEP EVALUATION SYSTEM FOR SELECTING HIGH DEVELOPMENTAL COMPETENCE IN VITRO FERTILIZED EMBRYOS IN CATTLE USING WELL-OF-THE-WELL DISH

2015 ◽  
Vol 27 (1) ◽  
pp. 210
Author(s):  
M. Taniai ◽  
M. Takayama ◽  
O. Dochi ◽  
K. Imai
Keyword(s):  
Evaluation System ◽  
Evaluation Method ◽  
Calf Serum ◽  
Time Lapse ◽  
Developmental Competence ◽  
Bovine Embryo ◽  
Chi Square ◽  
Cell Stage ◽  
Chi Square Test

Bovine IVF embryos are evaluated morphologically using light microscopy just before transfer. However, this evaluation method is subjective, and an objective method with more certainty is needed. Sugimura et al. (PLoS ONE 2012 7, e36627) reported a promising system for selecting healthy IVF bovine embryo by using time-lapse cinematography and 5 prognostic factors. This study was to investigate the efficacy of a 2-step evaluation system of IVF embryos using microscopy for selecting high developmental competence IVF embryos. Cumulus-oocyte complexes (COC) were collected by ovarian follicular aspiration (2 to 5 mm diameter) obtained from a local abattoir. The COC (n = 488) were matured in TCM-199 medium supplemented with 5% calf serum (CS) and 0.02 IU mL–1 of FSH at 38.5°C for 20 h in an atmosphere of 5% CO2 (20 COC 100 µL–1 droplets). After 10 h of gametes co-culture (5.0 × 106 sperm cells mL–1), the presumptive zygotes were cultured in 125 µL of CR1 aa medium supplemented with 5% CS in well of-the-well culture dishes (AS ONE, Japan; 25 zygotes well–1) at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 for 9 days. Two-step evaluations of embryos were done at 27 and 55 h post-IVF (hpi). In the first step of evaluation, cleavage patterns at 27 hpi were categorized as mono-cell, 2-cell with even blastomeres and without fragments (normal cleavage), 2-cell with uneven blastomeres, and ≥3 blastomeres. During the second step of evaluation, embryos were classified by their number of blastomeres (2 to 5 cells, 6 to 8 cells, and >8 cells) and the absence or presence of multiple fragments (<20 or >20%) at 55 hpi. The data were analysed by chi-square test. The blastocyst rate (BL%) of embryos cleaved before 27 hpi (56.6%, n = 106) was higher (P < 0.01) than those of embryos cleaved after 27 hpi (37.0%, n = 235). A greater percentage (P < 0.05) of 2-cell embryos with normal cleavage (68.0%, n = 50) developed to blastocysts than from with =3 blastomeres at 27 hpi (40.6%, n = 32). Superior BL% (P < 0.01) was obtained from embryos categorized as 6- to 8-cell stage (58.6%, n = 140) and >8 cell stage (70.6%, n = 25) compared with those embryos at the 2- to 5-cell stage at 55 hpi (26.1%, n = 176). Embryos with no fragments (58.0%, n = 467) had higher BL% (P < 0.01) compared with those with <20% fragments (30.7%, n = 127) and having with >20% fragments (17.5%, n = 25) at 55 hpi. The highest of BL% was observed in embryos showing a normal cleavage to 2-cells with at 27 hpi and having >6 cells with no fragments at 55 hpi (95.2%, n = 21, P < 0.01). These results demonstrate that the 2-step evaluation system at 27 and 55 hpi using microscopy is an effective method for selecting IVF embryos with high developmental competence.

188 EFFECT OF TRANSFER OF FROZEN-THAWED IVF EMBRYOS ON PREGNANCY IN REPEAT-BREEDER INSEMINATED OR NON-INSEMINATED HOLSTEIN CATTLE

2006 ◽  
Vol 18 (2) ◽  
pp. 202 ◽  
Author(s):  
O. Dochi ◽  
M. Tanisawa ◽  
S. Goda ◽  
H. Koyama
Keyword(s):  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Calf Serum ◽  
Pregnancy Rates ◽  
Holstein Cattle ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Chi Square Test ◽  
Repeat Breeder

Repeat-breeding is one of the important factors that affect dairy management. The objective of this study was to investigate the effect of transfer of frozen–thawed IVF embryos on pregnancy in repeat-breeder Holstein cattle. Cumulus–oocyte complexes (COCs) were collected by aspiration of 2–1-mm follicles from ovaries obtained at a local abattoir. COCs were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 mg/mL of FSH at 38.5°C under a 5% CO2 atmosphere in air. Matured oocytes were inseminated with spermatozoa of 5 × 106/mL in BO solution (Brackett and Oliphant 1975 Biol. Reprod. 12, 260–274) containing 10 mM hypotaurine and 4 units/mL heparin. After 18 h of gamete co-culture, presumptive zygotes were cultured in CR1aa (Rosenkrans et al. 1991 Theriogenology 35, 266) supplemented with 5% CS for 8 days at 38.5°C under 5% CO2, 5% O2, 90% N2 atmosphere in air. After in vitro fertilization, Day 7 and Day 8 blastocysts were frozen in 1.5 M ethylene glycol (EG) in Dulbecco's PBS (DPBS) supplemented with 0.1 M sucrose and 20% CS. Embryos were transferred into a freezing medium, loaded into 0.25-mL straws, and allowed to stand for 15–20 min for equilibration. The straws were then plunged into a −7°C methanol bath of a programmable freezer for 1 min, seeded at −7°C, maintained at −7°C for 15 min, cooled to −30°C at the rate of −0.3°C/min, and then plunged into liquid nitrogen. Recipient animals (43 heifers, 131 cows) included those that did not conceive after being artificially inseminated (AI) 3 to 15 times. The frozen–thawed IVF embryos were directly transferred to the recipient animals 7 days after estrus or AI. Pregnancy rates were analyzed by chi-square test. The results are presented in Table 1. There were no significant differences in the pregnancy rates between treatments. However, a slightly higher pregnancy rate was achieved by embryo transfer after AI. These results suggest that embryo transfer may increase the pregnancy rate in repeat-breeder Holstein cattle. Table 1. Pregnancy rates after transfer of IVF frozen–thawed embryos in repeat-breeder Holstein cattle

137 EFFECT OF THE ADDITION OF FOLIC ACID TO MATURATION AND CULTURE MEDIA ON DEVELOPMENT OF THE BOVINE BLASTOCYST AND ITS SURVIVAL RATE AFTER FREEZE-THAWING

2017 ◽  
Vol 29 (1) ◽  
pp. 177
Author(s):  
S. Sato ◽  
O. Dochi ◽  
K. Imai
Keyword(s):  
Folic Acid ◽  
Survival Rate ◽  
Culture Media ◽  
Cell Damage ◽  
Calf Serum ◽  
Cleavage Rate ◽  
Chi Square ◽  
Exact Test ◽  
Chi Square Test

Reactive oxygen species (ROS) are the main causes of cell damage in bovine embryos in vitro. Folic acid (FA) is an antioxidant that protects cells from ROS. We studied the effect of the addition of FA to maturation and culture media on development of bovine blastocysts and their survival rate after freeze-thawing. Cell-oocyte complexes (COC) were allowed to mature in HEPES (25 mM)-buffered TCM199 (TCM199) supplemented with 5% calf serum (CS), 0.02 AU mL−1 of FSH, and FA (0, 2.5, 25, and 50 µM) for 20 hours (20–25 COC/100-µL droplet of the medium). After 6 hours of gamete co-culture (5 × 106 sperm/mL), presumptive zygotes were cultured in CR1aa medium supplemented with 5% CS and FA (0, 2.5, 25, and 50 μM) for 9 days (day of fertilization = Day 0). Expanded blastocysts that developed from Day 7 to 9 were frozen for further study. Each embryo was frozen in Dulbecco’s PBS (D-PBS) supplemented with 20% CS, 1.5 M ethylene glycol (EG), and 0.1 M sucrose (SUC). Embryos were equilibrated with their freezing medium for 15 min and loaded individually into a 0.25-mL straw. These straws were put into the cooling chamber of a programmable freezer precooled at −7°C. After 2 min, straws were seeded and held for 13 min at −7°C. Next, straws were cooled to −30°C at −0.3°C/min before being plunged into liquid nitrogen. Frozen embryos were thawed by allowing straws to stand in air for 7 s and warming them in a 30°C water bath for 20 s. Thawed embryos were washed twice with D-PBS supplemented with 20% fetal calf serum (FCS), which was warmed to 38°C. They were immersed into the same medium at 38°C for 10 min, and each embryo was cultured in a 20-μL droplet of TCM199 supplemented with 10% FCS and 0.1 mM β-mercaptoethanol (TCM-199-βME) for 72 h. Embryo cleavage rate was observed at 55 h post-insemination. Blastocyst rates were analysed at 9 days post-insemination. Rates of embryos developing into reexpanded, hatching, and hatched blastocyst stages were determined after 72 h of thawing. All data were analysed by the chi-square test and Fisher’s exact test. Cleavage and blastocyst rates after insemination at 55 hours and 9 days, respectively, were not significantly different among media containing 0 μM (n = 278; 74.1% and 39.9%), 2.5 μM (n = 260; 74.2% and 45.8%), 25 μM (n = 258; 75.6% and 45.7%), and 50 μM (n = 253; 76.3% and 42.7%) FA. Survival and hatching rates of frozen and thawed expanded blastocysts after 72 h in culture were 62.5% and 56.3%, respectively, in 0 μM FA (n = 16); 85.2% and 74.1% in 2.5 μM FA (n = 27); 66.7% and 62.5% in 25 μM FA (n = 24); and 68.0% and 64.0% in 50 μM FA (n = 25). Blastocysts cultured in media containing 2.5 μM FA tended to have a higher survival rate than those cultured in media containing 0 μM FA, although this difference was not significant (P = 0.09). Inclusion of FA did not appear to influence development or post-thaw survival of bovine blastocysts produced in vitro.

scholarly journals 282MATURATION MEDIUM EXCHANGE IS EFFECTIVE ON BOVINE EMBRYO DEVELOPMENT IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 261
Author(s):  
Y.S. Park ◽  
S.H. Choi ◽  
H.D. Park ◽  
M.D. Byun
Keyword(s):  
Embryo Development ◽  
Total Protein ◽  
Harmful Effect ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Chi Square ◽  
Medium Exchange ◽  
Chi Square Test

In vitro embryo development is strongly influenced by IVM conditions. Increased duration of IVM may cause aging of the oocytes, which has a harmful effect on the embryo development. Oocyte maturation depends upon the synthesis of several proteins that may play important roles in the cytoplasmic maturation. These experiments were conducted to determine the effect of IVM duration(18-h or 24-h) and medium exchange (at 18h) on embryo development, and to investigate the protein quantities in IVM medium. Korean Native Cow (KNC) ovaries were obtained from a local slaughterhouse, and cumulus-oocyte complexes (COCs) were aspirated from 2- to 8-mm follicles. Groups of 15 COCs were matured in 50-μL drops of TCM-199 supplemented with 10% fetal calf serum (FBS), 1μgmL−1 MFSH, 10μgmLLH and 1μgmL−1 Estradiol-17β for 18h or 24h. In vitro-matured oocytes were fertilized using frozen-thawed percoll separated spermatozoa (Day 0) in fer-TALP medium for 20h and cultured in CR1aa medium supplemented with 0.3% BSA (before Day 3) or 10% FBS (After Day 3). All types of cultures were carried out in an incubator at 39°C, 5% CO2 in air. The total protein quantity in IVM medium at 18h or 24h were compared by 2-dimensional gel electrophoresis using a 10–15% polyacrylamide gradient gels. Data from three replicates were analyzed by chi-square test. The proportions of oocytes reaching the blastocyst stage was significantly higher in 18h IVM group than 24h IVM group (Table 1). However, there was no difference detected in blastocyst rate between 18h IVM group and 18h medium exchange group. Total protein quantity was reduced between 18h and 24h in IVM medium. There were 299 protein spots identified in IVM medium;; there was an increase at 10 spots in the IVM medium analyzed at 18h and a decrease of 20 spots at 24h. This study suggests that duration of IVM affects subsequent embryo development. The total protein quantity was decreased between 18h and 24h in IVM medium. These proteins may be absorbed into the oocytes and reduce development to the blastocyst stage. However, this may be overcome by IVM medium exchange. Table 1 Effects of duration of IVM and medium exchange on embryo development of KNC oocytes

scholarly journals 292 MATURATION IN A STRAW IS EFFECTIVE ON THE DEVELOPMENT OF BOVINE OOCYTES IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 296
Author(s):  
Y.M. Park ◽  
S.S. Kim ◽  
J.H. Lee ◽  
Y.S. Park ◽  
H.D. Park
Keyword(s):  
Embryo Development ◽  
Calf Serum ◽  
Blastocyst Stage ◽  
Development Rate ◽  
Culture Dish ◽  
Chi Square ◽  
Treatment Groups ◽  
Chi Square Test ◽  
Bovine Oocytes

In vitro embryo development is strongly influenced by oocyte maturation environments. Maturation of bovine oocytes is processed in a culture dish. However, the development rate to the transferable blastocyst stage was 10 to 30%. This experiment was to examine the effect of the size of straw and the medium exchange on the development of Korean Native Cow (KNC) oocytes. Ovaries of KNC were obtained from a local slaughterhouse and cumulus oocyte complexes (COCs) were aspirated from 2- to 8-mm follicles. Groups of 15 COCs were matured in TCM-199 supplemented with 10% fetal calf serum (FCS), 1 μg/mL FSH, 10 μg/mL LH, and 1 μg/mL estradiol-17β for 18 h. In vitro-matured oocytes were fertilized using frozen-thawed percoll-separated spermatozoa (Day 0) in fer-TALP medium for 20 h and cultured in CR1aa medium supplemented with 0.3% BSA (before Day 3) or 10% FBS (after Day 3). All cultures were maintained in an incubator at 39°C, 5% CO2 in air with maximum humidity. Data from three replicates were analyzed by chi-square test. In Experiment 1, we examined the effect of the instrument of maturation (dish or 0.25-mL and 0.5-mL straws) on embryo development. There were no difference in the cleavage (2-cell) among treatment groups. However, the development rate to the 8-cell and blastocyst stage was significantly higher in the 0.5-mL straw (38.5 and 17.0%) than in the 0.25 mL-straw (26.6 and 7.4%, all respectively). In Experiment 2, the KNC oocytes were matured in 0.5-mL straws based on the results of Experiment 1, and we examined the effect of the conditions such as circulation and exchange of maturation medium at 9 h after the start of IVM on embryo development. The development rates to the 2-cell, 8-cell, and blastocyst stage were significantly higher in the circulation group (83.3, 58.0 and 31.3%) than in the control (72.0, 44.7 and 19.3%) and exchange groups (71.3, 40.0, and 18.0%, all respectively). The results of this study suggest that the maturation of KNC oocytes in 0.5-mL straws accompanied by circulation of medium at 9 h is effective in the development to the blastocyst stage.

214 EFFECT OF LONG-DISTANCE TRANSPORTATION OF OVARIES ON THE DEVELOPMENT OF IN VITRO-MATURED - IN VITRO-FERTILIZED - IN VITRO-CULTURED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 206
Author(s):  
M. Kanae ◽  
K. Hisaichi ◽  
S. Jaswant ◽  
D. Osamu
Keyword(s):  
Calf Serum ◽  
Control Group ◽  
Long Distance ◽  
Chi Square ◽  
Maturation Rate ◽  
Long Time ◽  
Chi Square Test ◽  
Thermos Flask ◽  
Vitro Development

Keeping ovaries for a long time before oocyte recovery affects the maturation rate and blastocyst rate (BL rate), but a change in the temperature of the ovary at the time of transportation does not affect the BL rate (Ribeiro et al. 2008 Anim. Reprod. Sci. 108, 171–179). The objective of this study was to investigate the effect of long-distance transportation of ovaries on the in vitro development of bovine oocytes into blastocysts. Ovaries in the transportation group (Group T) were collected from a slaughterhouse, placed inside a Thermos flask, and transported to the laboratory within 17 to 21 h. The Thermos flask was covered with a freezer pack in a foam polystyrene box (Nakatate et al. 2006 Reprod. Fertil. Dev. 18, 193). The temperature of the Thermos flask was measured with a temperature recorder. Cumulus–oocyte complexes (COC) were collected by aspirating 2- to 6-mm follicles from the ovaries. The COC were matured in TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 of FSH. Groups of 20 COC were incubated in 100-μL drops of IVM media at 38.5°C under an atmosphere of 5% CO2 in air for 20 h. After 18 h of gamete co-culture (3 × 106 sperm mL–1), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum for 9 days at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2. Embryonic development was evaluated at 48 h after IVF (total cleavage rates, CL rate) and on Days 7 to 9 (BL rate). The ovaries in the control group (Group C) were collected from a slaughterhouse different from that of the Group T ovaries and transported to the laboratory in saline (23°C) in a Thermos flask within 3 h. These ovaries were then used for the collection of COC in the same way as for Group T. Data were analysed by chi-square test. A total of 25 experiments were performed using the ovaries from Groups C and T (n = 3945 v. n = 7218). The CL rate and the BL rate for Group T were significantly lower than those for Group C (41.8 v. 62.0% and 16.1 v. 29.7%; P < 0.01). In Group T, the duration of ovary transportation did not affect the CL rate (within 19 h, between 19 and 20 h, and more than 20 h), but the BL rate was significantly decreased with an increase in transport time (18.7 v. 16.1 v. 14.8%). The temperature change in the Thermos flask during transportation ranged from 12.0 to 25.1°C; the temperature was maintained virtually constant or decreased. The CL rates of the groups that underwent a temperature (t) change (t ≤ 5.0 and 5.0 < t ≤ 10.0) were not significantly different, but the BL rate in the group with 5.0 < t ≤ 10.0 was significantly lower than that of the other group (16.7 v. 13.5%; P < 0.01). These results suggest that the long-distance transportation of ovaries affects in vitro embryo development. Moreover, a decrease in temperature during transportation may have an adverse effect on blastocyst formation.

80 EFFECT OF PRE-EQUILIBRATION TIME ON THE SURVIVAL RATE OF IN VITRO-FERTILIZED BOVINE EMBRYOS AFTER VITRIFICATION

2008 ◽  
Vol 20 (1) ◽  
pp. 120 ◽  
Author(s):  
H. Koyama ◽  
A. H. Sugulle ◽  
O. Dochi
Keyword(s):  
Calf Serum ◽  
Equilibration Time ◽  
Bovine Embryos ◽  
Chi Square ◽  
Chi Square Test ◽  
Vitrification Solution ◽  
Short Time ◽  
Further Development ◽  
Hatching Rates

Successful cryopreservation of embryos depends on the pre-equilibration time. This study was designed to compare 2 pre-equilibration times – short (1 min) and long (5 min) – and to evaluate the re-expansion and hatching rates of different stages of embryos using the short pre-equilibration method. Cumulus–oocyte complexes (COCs) from ovaries obtained from a slaughterhouse were matured, fertilized, and cultured in vitro. In Experiment 1, expanded blastocysts between 7 and 9 days of culture were pre-equilibrated for the short time (1 min) in 100 μL of vitrification solution 1 (VS1: containing 7.5% ethylene glycol (EG), 7.5% dimethyl sulfoxide (DMSO), and 20% calf serum (CS) in TCM-199), followed by incubation in 100 μL of vitrification solution 2 (VS2: containing 15% EG, 15% DMSO, 20% CS, and 1 m sucrose (Suc) in TCM-199) for 30 s. Another group of blastocysts was subjected to the long pre-equilibration (5 min) in 100 μL VS1, followed by incubation in 100 μL of VS2 for 30 s. The embryos were placed into Cryotops® (Kitasato Supply Co., Tokyo, Japan) and immediately submerged in liquid nitrogen and kept there for 1 week. Blastocysts were warmed by plunging the Cryotops into 1 m Suc in TCM-199 for 1 min, placed in 0.5 m Suc in TCM-199 for 3 min, and finally placed in CR1aa alone for 5 min before being cultured. In Experiment 2, 8-cell embryos, morulae, and expanded blastocysts were vitrified by the previously described short equilibration method. The re-expansion and hatching rates of embryos were determined as the percentage of vitrified–warmed embryos undergoing further development in the in vitro culture. The data were analyzed by the chi-square test. Results are presented in Table 1. There was no difference between the short and long pre-equilibration times in terms of survival (94.0% v. 94.1%, respectively) and morphological appearance immediately after warming. However, re-expansion of blastocysts (ability to develop further) was slightly higher with the short pre-equilibration than with the long pre-equilibration (90.0% v. 85.9%, respectively). In Experiment 2, there were no differences in embryo re-expansion, but the hatching rates of 8-cell embryos were lower than those of morulae, blastocysts, and controls. In conclusion, the results of this study indicate that the length of pre-equilibration time prior to vitrification does not influence embryo re-expansion, and that bovine morulae and blastocysts can be vitrified with equal success. We also conclude that insufficient permeation of cryoprotectants may occur in 8-cell embryos. Table 1. Survival and hatching rates of vitrified bovine embryos after warming

scholarly journals Effect of insulin-like growth factor-1 during in vitro oocyte maturation and in vitro culture of bovine embryos

2001 ◽  
Vol 53 (2) ◽  
pp. 207-211 ◽  
Author(s):  
M.D. Quetglas ◽  
L.A. Coelho ◽  
J.M. Garcia ◽  
E.B. Oliveira Filho ◽  
C.R. Esper
Keyword(s):  
Growth Factor ◽  
In Vitro Culture ◽  
Embryo Development ◽  
Calf Serum ◽  
Culture Conditions ◽  
Insulin Like Growth Factor ◽  
Chi Square ◽  
Igf I ◽  
Chi Square Test

The effects of insulin-like growth factor-I (IGF-I) on in vitro maturation (IVM) (experiment I) and on in vitro embryo development (experiment II) of bovine oocytes fertilized in vitro, were evaluated in terms of cleavage (CR), blastocyst (BR) and hatching (HR) rates. For IVM, immature cumulus-oocyte complexes were cultured in TCM-199 medium supplemented with Hepes, sodium bicarbonate, sodium pyruvate, additives, fetal calf serum (B-199 medium) and gonadotropins (14 U/ml PMSG and 7 U/ml hCG). For embryo development, the oocytes/zygotes were cultured in B-199 medium with bovine oviduct epithelial cells in suspension under silicon oil. Treatments for in vitro culture conditions for both experiments were: 1- B-199 + 200 ng/ml IGF-I; 2- B-199 + 100 ng/ml IGF-I; 3- B-199 + 50 ng/ml IGF-I; 4- B-199 + 10 ng/ml IGF-I; 5- B-199 + 0 ng/ml IGF-I. All cultures were performed at 38.5ºC in 5% CO2 in air and the data were analyzed by chi-square test. In experiment I, there were no differences (P>0.05) among treatments for CR, BR or HR. In experiment II, the addition of IGF-I to the embryo culture medium (ECM) resulted in a significant increase in CR while for BR and HR this effect was not observed. The addition of 200 ng/ml IGF-I to ECM increased CR (71.1%) when compared to 100 ng/ml IGF-I (57.6%) or control (56.7%) groups, however, there were no differences when compared to 50 (69.4%) or 10 ng/ml (73.1%) groups. There was no beneficial effect of the addition of IGF-I in the IVM or ECM media on the in vitro development of embryos produced from oocytes matured and fertilized in vitro.

290 THE FACTORS ON RATES OF ABNORMALITY, DISEASE AND MORTALITY OF CALVES DERIVED FROM IN VITRO EMBRYOS OF KOREAN NATIVE CATTLE

2006 ◽  
Vol 18 (2) ◽  
pp. 252
Author(s):  
Y.-S. Park ◽  
S.-S. Kim ◽  
M.-C. Park ◽  
H.-D. Park
Keyword(s):  
Calf Serum ◽  
Treatment Technique ◽  
In Vitro Production ◽  
Chi Square ◽  
Offspring Number ◽  
Chi Square Test ◽  
Native Cattle ◽  
Vitro Production ◽  
Delivery Type

In Korea, in vitro production and transfer of bovine embryos has advanced remarkably and applied commercially. However, in vitro-produced embryos result in lower pregnancy and higher abortion rates and in some cases increased rates of abnormality and mortality in calves. The present study was conducted to investigate the effects of various factors such as recipient parity, delivery season, offspring number, pregnancy period, delivery type, midwifery type, dystocia and vaccination, on the viability of calves derived from embryos produced in vitro. Korean Native Cow ovaries were obtained from local slaughterhouse and cumulus-oocyte complexes (COCs) were aspirated from 2 to 8 mm follicles. Selected COCs were matured in TCM-199 supplemented with 10% fetal calf serum (FBS), 1 �ML FSH, 10 �ML LH and 1 �ML Estradiol-17� for 20-22 h. In vitro-matured oocytes were fertilized using frozen-thawed percoll separated semen (Day 0) in fer-TALP medium for 20 h. The presumptive zygotes were cultured in CR1aa medium supplemented with 0.3% BSA (before Day 3) or 10%FBS (After Day 3). All types of cultures were made in an incubator at 38.5�, 5% CO2 in air. Statistical analysis was performed using the Chi-square test. Two blastocysts were transferred to the Holstein recipients (n = 1888). The parturition was occurred in total 755 recipients. There was no difference in the abnormality of calves among treatments. The incidence of disease was significantly higher in single calf than twin calves (18.4 vs. 6.7%), in multiparous than nulliparous group (40.0 vs. 9.9%), in eutocia than dystocia group (20.0 vs. 4.8%), in spring and winter groups than summer and autumn groups (20.3, 22.7 vs. 4.3, 0.0%), and in non-vaccinated than vaccinated group (22.7 vs. 1.6%), respectively (p < 0.05). The rate of mortality was significantly higher when transferred into nulliparous than multiparous (22.3 vs. 0.0%), when were dystocia than eutocia group (71.4 vs. 14.1%), when were non-midwifery than midwifery (45.0 vs. 13.6), when delayed midwifery than earlier midwifery (31.6 vs. 11.5%), and when were non-vaccinated than vaccinated group (28.0 vs. 9.8%), respectively (P < 0.05). The present study suggested that the viability of bovine calves derived from in vitro was affected by the recipient parity, parturition treatment technique and vaccination. This study was supported by the BIO-GREEN 21 PROGRAM.

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