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.

298 THE INFLUENCE OF DISTINCT CENTRAL BULL STATIONS ON THE IN VITRO EMBRYONIC DEVELOPMENT OF NELORE BULLS

2010 ◽  
Vol 22 (1) ◽  
pp. 305
Author(s):  
A. Renzi ◽  
F. P. Elias ◽  
R. A. Vila ◽  
R. B. Lôbo
Keyword(s):  
Calf Serum ◽  
Cumulus Cells ◽  
In Vitro Production ◽  
Freezing And Thawing ◽  
Chi Square ◽  
Frozen Semen ◽  
Chi Square Test ◽  
Vitro Production ◽  
In Vitro Embryonic Development

Reproductive biotechnology is growing worldwide as one of the most important tools of cattle breeding because it accelerates the process of genetic improvement. Most of the embryos produced are obtained using frozen semen from different AI centers. During freezing and thawing of semen, the sperm can be damaged by the rapid and dramatic changes in the physicochemical conditions that occur during cooling and ice formation. It has previously been described that bad management of frozen semen can result in reduced fertilization. This study investigated the influence of different central bull stations on the development of in vitro-produced bovine embryos. We compared semen of 154 Nelore bulls, used for IVF, from 8 different central bull stations (all of which used the same cryopreservation protocol) in the development of blastocysts. The in vitro production of embryos was performed as described: oocytes were collected from the slaughterhouse and matured in TCM-199 + 10% fetal calf serum (FCS) +0.5 μg mL-1 FSH + 50 μg mL-1 LH+ 1 μg mL-1 estradiol, for 24 hat 38.5°C in 5%CO2 in atmospheric air. Viable spermatozoids were obtained by centrifugation in Percoll gradient (45 and 90%), and used for IVF in a concentration of 2 million spermatozoa per mL in TALP + 10 μg mL-1 of heparin medium. After 12 h, the presumptive zygotes were transferred to a CR2+ 10% FCS medium and co-cultured with cumulus cells. After 168 h of IVF, we evaluated the number and stage of cleaved embryos produced with the semen of each bull. Statistical analyses were performed by using the chi-square test. Our results suggest that there are differences among distinct central bull stations in the proportion of embryos that developed into blastocysts and the different stages they hatched. FAPESP, CNPq, PROEX, FAEPA.

232 ADDITION OF VERY LOW AMOUNTS OF SERUM (ESTRUS COW SERUM) IMPROVES IN VITRO EMBRYO PRODUCTION IN DAIRY CATTLE

2015 ◽  
Vol 27 (1) ◽  
pp. 205 ◽  
Author(s):  
E. Mullaart ◽  
F. Dotinga ◽  
C. Ponsart ◽  
H. Knijn ◽  
J. Schouten
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Calf Serum ◽  
High Serum ◽  
In Vitro Production ◽  
Embryo Production ◽  
Chi Square ◽  
Embryo Yield ◽  
Vitro Production

Improving the efficiency of the in vitro production (IVP) process is very important because it results in more embryos to be used in breeding programs or as commercial service. At CRV, a culture medium consisting of SOF with amino acids and BSA is used. In the past, richer culture media were used with 10% fetal calf serum combined with BRL cell co-culture. Although the efficiency of the IVP process of these media was good, these rather high serum concentrations were quite often related to large offspring syndrome (LOS). The switch to a culture system without serum resulted in a significant reduction in LOS but also in a reduction of embryo yield. The aim of the present study was to investigate the effect of adding low amounts of serum to the culture medium on efficiency of embryo production. Immature cumulus-oocyte complexes (COC) were recovered from ovaries 6 to 8 h upon slaughter. The COC were matured in vitro in TCM199/FCS/LH/FSH supplemented with cysteamine (0.1 mM). Subsequently, matured oocytes were fertilised with frozen-thawed gradient-separated semen and further cultured for 7 days in SOFaaBSA. The SOF medium contained either 0 (control), 0.1, 0.5, or 1.0% oestrus cow serum (ECS). Embryos development was scored at Day 7. Three replicates were performed and results were analysed by chi-square analyses. The results clearly show that adding ECS significantly improved embryo production (Table 1). Interestingly, already very low amounts (0.1%) of serum gave a significant increase in embryo percentage. In conclusion, addition of very low amounts of ECS (0.1%) is beneficial for embryo production, resulting in significantly higher embryo production (from 19 to 27%). In a subsequent field trial with OPU-derived embryos, the effect of addition of 0.1% ECS on birth weight (LOS) of the calves has to be investigated. Table 1.Percentage of blastocysts at Day 7 after culture in SOF medium with different amounts of serum

74 EFFECT OF BETA-MERCAPTOETHANOL ON THE VITRIFICATION CRYOTOLERANCE OF BOVINE IN VITRO-PRODUCED EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 137
Author(s):  
E. S. Ribeiro ◽  
M. C. Gonçalves ◽  
M. C. Pedrotti ◽  
L. T. Martins ◽  
R. P. C. Gerger ◽  
...  
Keyword(s):  
Cell Number ◽  
Blastocyst Stage ◽  
High Humidity ◽  
In Vitro Production ◽  
Chi Square ◽  
Embryo Survival ◽  
Chi Square Test ◽  
Vitro Production ◽  
Hatching Rates

The control of oxidative processes in in vitro production (IVP) systems by the use of additives may be an alternative approach to improve embryo cryotolerance. The aim of this study was to verify the effect of β-mercaptoethanol (βME) on the cryotolerance of bovine IVP embryos. In 7 replications, and following IVM-IVF, presumptive zygotes (n = 3735) were in vitro-cultured in SOF medium supplemented or not with 100 μm βME (IVC treatment), at 38.5°C and high humidity. The initial 24 h of IVC was performed in 5% CO2 in air, with the remaining 6 days of IVC carried out in 5% CO2, 5% O2, and 90% N2. On Day 7, resulting blastocysts and expanded blastocysts were vitrified in glass micropipettes in a solution with 20% ethylene glycol + 20% propylene glycol. After warming, embryos were randomly allocated to 1 of 2 sub-groups for an additional 72 h of IVC to the hatching blastocyst (HBL) stage, in fresh SOF medium supplemented or not with 100 μm βME (PVC treatment), at 38.5°C, high humidity and 5% CO2. Experimental groups were as follows: G1 (βME-free medium during IVC and PVC); G2 (βME only during PVC); G3 (βME only during IVC); and G4 (βME during IVC and PVC). Cleavage (Day 2) and blastocyst (Day 7) rates in the IVC treatment and hatching rates (Days 7 to 9) for the PVC treatment were analyzed by the chi-square test, for P < 0.05. Total cell number (TCN) estimated by fluorescence staining in HBL derived from vitrified and nonvitrified embryos was analyzed by ANOVA. The use of βME during IVC did not affect cleavage rates (βME-free, 1491/1858, 80.2% v. βME, 1522/1877, 81.1%), but negatively affected development to the blastocyst stage (βME-free, 813/1858, 43.8% v. βME, 525/1877, 28.0%). Following vitrification, however, βME supplementation during PVC improved hatching rates (G2, 58.1% and G4, 63.8%) compared with groups without the additive (G1, 36.6% and G3, 42.0%). In addition, the presence of βME either during IVC or PVC, or during both culture periods, increased TCN in HBL from vitrified embryos (Table 1). The use of βME during IVC, irrespective of the presence of βME during the PCV period, caused an increase in TCN in HBL in G3 + G4, with no effects on hatching rates (Table 1b), whereas the addition of βME during PVC, irrespective of the presence of βME during the IVC period, resulted in greater hatching rates and TCN in HBL in G2 + G4 than in G1 + G3 (Table 1). In conclusion, the addition of βME during the IVC period did not affect cleavage, but reduced blastocyst yield. Despite that, βME supplementation during the IVC period appeared to have increased the cryotolerance of the resulting blastocysts, expressed by greater TCN in HBL, whereas βME supplementation during the PVC period also improved embryo survival to the vitrification process, manifested by greater hatching rates and TCN in HBL. Table 1.Effect of βME on the cryotolerance of bovine IVP embryos This study was supported by a grant from CNPq/Brazil.

scholarly journals Fetal calf serum enhances in vitro production of Bos taurus indicus embryos

2011 ◽  
Vol 75 (3) ◽  
pp. 429-433 ◽  
Author(s):  
F.G. Leivas ◽  
D.S. Brum ◽  
S.S. Fialho ◽  
W.P. Saliba ◽  
M.T.T. Alvim ◽  
...  
Keyword(s):  
Fetal Calf Serum ◽  
Bos Taurus ◽  
Calf Serum ◽  
In Vitro Production ◽  
Bos Taurus Indicus ◽  
Vitro Production

221 EFFECT OF STAGE OF CORPUS LUTEUM DEVELOPMENT ON THE IN VITRO PRODUCTION OF BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 209
Author(s):  
S. Miyashita ◽  
K. Miyata ◽  
C. Tachibana ◽  
Y. Inaba ◽  
H. Koyama ◽  
...  
Keyword(s):  
Corpus Luteum ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Large Mass ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Stage Of Development ◽  
The Mean ◽  
Vitro Production

The objective of this study was to investigate the effect of stage of corpus luteum (CL) development on the in vitro production of bovine embryos. Ovaries were classified according to the expected day of the oestrous cycle based on the morphology of the ovaries. Ovaries with a corpus hemorrhagicum and the remnant of the follicular lumen filled with blood were considered the early luteal stage (Days 2 to 4; Day 0 = day of ovulation, n = 46). Ovaries with a large mass of orange tissue in the CL were classified as the midluteal stage (Days 7 to 10, n = 42). Cumulus–oocyte complexes (COC) were collected by aspiration of 2- to 6-mm follicles. The COC were classified into the following grades: COC with >3 compact layers of cumulus cells and evenly granulated cytoplasm were classified into Grade 1; COC with >3 layers cumulus cells and evenly granulated cytoplasm were classified into Grade 2; COC with partially remaining cumulus cells and abnormal cytoplasm were classified into Grade 3; COC without cumulus cells or those with expanded cumulus cells were classified into Grades 4 and 5, respectively. Grades 1 and 2 COC were in vitro matured for 20 h in TCM-199 supplemented with 5% calf serum and 0.02 mg mL–1 of FSH at 38.5°C under an atmosphere of 5% CO2 in air. Matured COC were inseminated with 5 × 106 sperm for 18 h. Presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum at 38.5°C under an atmosphere of 5% O2, 5% CO2, and 90% N2 for 9 days (fertilization = Day 0). The mean number of COC and the proportion of COC classified as Grades 1 and 2 were analysed by ANOVA. Cleavage rates on Day 3 and blastocyst rates on Days 7 to 9 were analysed by a chi-square test. The mean number of recovered oocytes in the early luteal stage (18.7 ± 9.5) was significantly higher (P < 0.05) than the number in the midluteal stage (12.2 ± 5.7). The proportion of Grades 1 and 2 oocytes in the early luteal stage [66.7% (531/789)] was significantly higher (P < 0.01) than that in the midluteal stage [51.6% (252/484)]. The cleavage and blastocyst rates in the early luteal stage [60.9% (181/297) and 32.7% (97/297), respectively] were significantly higher (P < 0.05) than those in the midluteal stage [50.7% (76/150) and 20.7% (31/150) respectively].The present study suggests that the stage of development of the CL in bovine ovaries influences the number of recovered oocytes per ovary and the development of in vitro production of bovine embryos.

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.

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.

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.

In vitro production of large osteoclasts by calf serum

1970 ◽  
Vol 62 (2-3) ◽  
pp. 483-486 ◽  
Author(s):  
Maria Liskova ◽  
P. Jean
Keyword(s):  
Calf Serum ◽  
In Vitro Production ◽  
Vitro Production
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