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.

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 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.

scholarly journals 136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Direct Role ◽  
Treatment Groups ◽  
And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

62 EXPOSURE TO ETHYLENE GLYCOL AND DIMETHYL SULFOXIDE CAUSES ACTIVATION AND SPINDLE ANOMALIES IN BUFFALO (BUBALUS BUBALIS) OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 131 ◽  
Author(s):  
M. De Blasi ◽  
E. Mariotti ◽  
M. Rubessa ◽  
S. Di Francesco ◽  
G. Campanile ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Embryo Development ◽  
Sucrose Solution ◽  
Bubalus Bubalis ◽  
Meiotic Spindle ◽  
Blastocyst Development ◽  
Chi Square ◽  
Chi Square Test ◽  
Simple Exposure

Despite the increasing interest, buffalo oocyte cryopreservation is still inefficient, especially in terms of blastocyst development after IVF. The aim of this work was to evaluate chromatin and spindle organization of buffalo in vitro-matured oocytes after vitrification/warming by cryotop and after their simple exposure to cryoprotectants (CP). An overall amount of 251 COC was selected and matured in vitro. In the vitrification group, COC were first exposed to 10% ethylene glycol (EG) + 10% DMSO for 3 min, and then to 20% EG + 20% of DMSO and 0.5 m sucrose, loaded on cryotops, and plunged into liquid nitrogen within 25 s. Oocytes were warmed into a 1.25 m sucrose solution for 1 min and then to decreasing concentrations of sucrose (0.625 m, 0.42 m, and 0.31 m) for 30s each. In order to test CP toxicity, COC were simply exposed to the vitrification and warming solutions. Two hours after warming, oocytes were fixed and immunostained for microtubules using a method previously described (Messinger SM and Albertini DF 1991 J. Cell Sci. 100, 289–298), stained for nuclei with Hoechst, and examined by fluorescence microscopy. Fresh in vitro-matured oocytes were fixed and stained as controls. Data were analyzed by chi-square test; results are shown in Table 1. The percentages of MII oocytes in the control and vitrification groups were greater than in the toxicity group, in which a greater percentage of telophase II stage oocytes were found compared with both the control and vitrification groups, indicating occurrence of activation. Of the MII oocytes, both exposure to CP and vitrification procedures gave greater percentages of oocytes with abnormal spindle and abnormal chromatin configuration compared with the control. An unexpected datum was the evidence of a significant percentage of spontaneously activated oocytes in the toxicity group. We speculate that the lack of activation in the vitrification group may be related to the slowing down of metabolic activity subsequent to thermal shock, and hence, that activation after vitrification may occur later than 2 h post-warming. In conclusion, the simple exposure to CP causes activation of the COC and damage to the cytoskeleton similar to that induced by the whole vitrification protocol. The damages to the meiotic spindle and DNA fragmentation may lead to aneuploidy incompatible with subsequent embryo development and account for the poor embryo development currently recorded in buffalo. Table 1.Chromatin and spindle organization in oocytes vitrified and exposed to cryoprotectants

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.

1 ARGININE SUPPLEMENTATION IN VITRO INCREASES PORCINE EMBRYO DEVELOPMENT AND AFFECTSmRNA TRANSCRIPT EXPRESSION

2011 ◽  
Vol 23 (1) ◽  
pp. 107 ◽  
Author(s):  
B. K. Bauer ◽  
L. D. Spate ◽  
C. N. Murphy ◽  
R. S. Prather
Keyword(s):  
Treatment Group ◽  
Mrna Expression ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Sequencing Analysis ◽  
Treatment Groups ◽  
Porcine Embryo ◽  
Arginine Supplementation

In vitro culture systems are suboptimal as compared to in vivo. Previous next-generation sequencing analysis of in vivo fertilized and in vitro cultured (IVC) or in vivo cultured (IVV) porcine blastocyst stage embryos identified an arginine transporter (SLC7A1) expressed 63 fold higher in IVC compared to IVV blastocysts (Bauer et al. 2010 Biol. Reprod. Epub ahead of print). Arginine catabolism may play important roles in placental and conceptus growth and development as it is a substrate for synthesis of nitric oxide synthase and polyamines. The objective of this study was to determine the effects arginine had on both embryo development and mRNA expression in in vitro fertilized embryos. Cumulus–oocyte complexes were matured for 44 h in M199 supplemented with EGF, FSH, and LH. Oocytes with a visible polar body (metaphase II) were selected and fertilized in modified Tris Buffered Medium for 5 h and then placed into one of 5 treatment groups (Porcine Zygote Medium 3 (PZM3) with 0 mM, 0.12 mM (current concentration of arginine in PZM3), 0.36 mM, 0.72 mM, or 1.69 mM arginine). Twenty-eight hours post-fertilization, cleaved embryos were selected and moved into 25 μL drops of respective culture media and cultured to day 6 in 5% CO2, 5% O2, 90% N2 at 38.5°C. To determine the effect arginine had on development the percent of embryos that made it to the blastocyst stage for each treatment group were analysed using PROC GLM in SAS (SAS Institute, Cary, NC). A least significant difference post test comparison was completed to determine if significant differences existed between treatment groups (a,b,cP < 0.05). The percentage of cleaved embryos on Day 6 that developed to blastocyst was 57.2%b,c, 50.2%c, 67.3%a,b, 67.3%a,b, 70.4%a (N = 147, 163, 150, 120, and 134) in 0 mM, 0.12 mM, 0.36 mM, 0.72 mM, and 1.69 mM arginine, respectively. Real-time PCR was then completed to assess the affect arginine supplementation had on SLC7A1 mRNA expression. Three biological replicates, each containing 10 blastocyst pools to ensure enough starting material, were collected for each treatment group. RNA was isolated from each sample and 5 μL was linearly amplified (NuGEN Ovation Pico WTA System) so multiple genes could be compared and then purified using Bio-Rad MicroSpin Columns. Expression levels were calculated relative to the reference sample and the housekeeping gene, YWHAG. The ΔΔCT values were log-transformed and analysed using PROC GLM in SAS. The expression of SLC7A1 mRNA was decreased (P = 0.0006) compared to PZM3 in the 1.69 mM arginine group. These results illustrate the positive effects that additional arginine may be having on porcine embryo development during culture from the 2-cell to the blastocyst stage. Supplementing arginine to a final concentration of 1.69 mM during culture increases development of porcine embryos to blastocyst compared to PZM3 and also decreases the expression of SLC7A1. Evaluation of the transcriptional profile appears to be a good method of letting the embryo tell us what it needs for development, and in this case arginine. Funded by F21C.

14 EQUINE CLONING AND EMBRYO AGGREGATION: EFFECT OF BOVINE, PORCINE, FELINE AND EQUINE OOPLAST

2012 ◽  
Vol 24 (1) ◽  
pp. 118
Author(s):  
A. Gambini ◽  
J. Jarazo ◽  
A. De Stefano ◽  
F. Karlanian ◽  
D. Salamone
Keyword(s):  
Embryo Development ◽  
Metaphase Plate ◽  
Donor Cell ◽  
Development Stage ◽  
Blastocyst Stage ◽  
Chi Square ◽  
Cell Stage ◽  
Aggregation Effect ◽  
Group I

The low number of horse slaughterhouses is one of the reasons for the limited availability of horse oocytes for research in cloning. The aim of our study was to assess the capability of equine, bovine, porcine, or feline ooplast to produce cloned embryos when equine cells are used as donor nuclei and to evaluate if embryo aggregation improves their development. Oocytes from mentioned species were collected from ovaries derived from slaughterhouses, except for cat ovaries that were obtained from ovariectomized queens. Oocytes were matured in TCM199 supplemented following standard protocols for each species. After maturation, cumulus and zona pellucida were removed. Enucleation was performed by aspiration of the metaphase plate under ultraviolet light. Donor cell and ooplast were attached by phytohemagglutinin treatment and then electrofused. Activation protocols were ionomycin for 4 min, except for porcine, which were electrically activated, followed by culture in 1.9 mM 6-DMAP for bovine, feline and porcine, except for equine: 1 mM 6-DMAP with 5 mg mL–1 of cycloheximide. Reconstructed embryos (RE) were cultured in SOF in the well of well system in 2 different groups: only one RE per well (1X) and three RE per well (3X, aggregated embryos, AE). Blastocysts derived from homospecific clones were transferred to synchronized mares. Cleavage and maximum development stage achieved of all experimental groups were assessed. In vitro development was compared using the chi-square test. In group 1X, a total of 64, 49, 38 and 145 RE were performed for porcine, bovine, feline and equine, respectively and in group 3X, 88, 48, 48 and 195 RE. Cleavage of cloned embryos ranged from 67 to 87%. Aggregated of homospecific equine clones showed the highest blastocyst rates (1X: 5.5%, 3X: 34%) and after embryo transfer (4 recipients for each group), an ongoing pregnancy (day 300, at the time of submission) was only achieved with aggregated embryo confirming the positive effect of embryo aggregation in these clones. The stages with higher developmental arrest of heterospecific nonaggregated embryos were 2 to 4 cells for porcine ooplast (23/64, 36%) and 4 to 8 cells for bovine and feline ooplast (37/49, 75% and 18/38, 47%, respectively). Blastocyst stage was only reached using feline ooplast (group I: 2/38, 5.26% and group II: 2/16, 12.5%). Heterospecific aggregated clones were able to achieve 16-cell stage, showing statistic differences compared with group 1X. As we reported previously, embryo aggregation shows benefits for homospecific equine clones, although more studies are needed to clarify if aggregation of heterospecific clones has the same effect. All heterospecific ooplasm was able to support embryo development. The stage of major developmental arrests was similar to embryonic genomic activation stage. Our results suggest that cat oocyte seems to be the best receptor to support equine cloned embryo development.

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 266IN VITRO-DERIVED EMBRYO PRODUCTION WITH SEXED AND UNSEXED SEMEN FROM DIFFERENT BULLS

2004 ◽  
Vol 16 (2) ◽  
pp. 253 ◽  
Author(s):  
L. Ferré ◽  
C. Ohlrichs ◽  
D. Faber
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Calf Serum ◽  
Sperm Concentration ◽  
Reproductive Technologies ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Sexed Semen ◽  
Chi Square Analysis

The production of pre-sex-selected calves by in vitro fertilization (IVF), using sexed semen, does show some benefits due to the small quantity of sperms needed for the process as compared to other reproductive technologies. The objective of this study was to determine differences among bulls and sperm concentrations in embryo development with sexed and unsexed semen. Follicles ranging from 2 to 6mm in diameter were aspirated from slaughterhouse ovaries. COC were selected and matured in groups of maximum of 30 in 1.8mL of TCM-199, supplemented with 10% fetal calf serum, 0.01UmL−1 bFSH, 0.01UmL−1 bLH and 10μLmL−1 penicillin-streptomycin for 24h at 38.5°C. Fertilization (Day 0) was carried out in micro-drops (50μL) with TALP-FERT medium containing PHE (3μgmL−1 penicillamine, 11μgmL−1 hypotaurine and 0.18μgmL−1 epinephrine), 10μLmL−1 non-essential amino acid and 2μgmL−1 heparin. Frozen/thawed sexed (female) and non-sexed sperms from five bulls were selected in a discontinuous percoll gradient. Sperm concentration was 1×106 for non-sexed semen and 1×106 or 2×106 for sexed semen. After 18–20h, presumptive zygotes were denuded and cultured in groups of 10 in 50-μL micro-drops of SOF citrate with 5% FCS (Holm P et al., 1999 Theriogenology 52, 683–700) under paraffin oil in a 5% O2, 5% CO2, 90% N2 atmosphere with high humidity. On Day 7, blastocysts (BL) were morphologically evaluated and recorded. Results are shown in Table 1. Data was compared by chi-square analysis. Sexed frozen bovine sperm can be used successfully in IVF systems. More research needs to be done to optimize and standardize bovine in vitro fertilization with sexed semen. Table 1 Results of comparisons between bulls, sperm concentrations, cleavage and embryo development

scholarly journals 271 ASSESSMENT OF NUCLEAR STATUS OF ACTIVATED BOVINE OOCYTES MATURED IN DIFFERENT MATURATION CONDITIONS IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 285
Author(s):  
J.I. Park ◽  
Y. Jang
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Calf Serum ◽  
Chi Square ◽  
Cell Stage ◽  
Maturation Medium ◽  
Second Polar Body ◽  
Humidified Air ◽  
Bovine Oocytes

This study was carried out to assess the nuclear status after parthenogenetic activation in in vitro matured oocytes under different conditions. Bovine ovaries were collected from slaughtered cows at a local abattoir. Oocytes were aspirated from follicles of 3–8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, 100 mg/mL l-cysteine, 20 mg/mL sodium pyruvate, gonadotropins (each 250 IU of eCG and hCG/mL), and 10 mg/mL epidermal growth factor, with or without 5 mM hypotaurine and taurine. Oocytes were cultured at 38.9°C in 5% CO2 in humidified air. After 24 h of culture, oocytes with polar body were selected and submitted to activation treatments. Oocytes were exposed to calcium ionomycin (5 μM for 5 min) followed by incubation with 6-DMAP (2 mM), roscovitine (50 μM), or 6-DMAP + roscovitine for 3.5 h. After activation, oocytes were cultured in mSOF medium containing 0.8% BSA at 38.9°C in 5% CO2, 5% O2 in humidified air for 16 h and stained with Hoechst 33342 or aceto-orcein for assessment of nuclear status. Nuclear status was recorded as follows: 1PB (polar body) + 1PN (pronucleus), 2PB + 1PN and others. Data were analyzed using chi-square test. The maturation rate of bovine oocytes cultured in maturation medium containing hypotaurine/taurine (89.3%, n = 84) was higher (P < 0.05) than those cultured without hypotaurine/taurine (72%, n = 93). In the oocytes matured with hypotaurine/taurine, the rates of diploid activation (1PB + 1PN) were 84% (n = 50) in oocytes treated with 6-DMAP + roscovitine, 78.6% (n = 56) with 6-DMAP, and 52% (n = 50) with roscovitine. In the oocytes matured without hypotaurine/taurine, the rates of diploid activation were 80% (n = 60) in oocytes treated with 6-DMAP + roscovitine, 72% (n = 50) with 6-DMAP, and 54% (n = 50) with roscovitine. The rates of diploid activation were not different in oocytes matured with or without hypotaurine/taurine and among activation treatments. The oocytes treated with roscovitine showed a lower rate (P < 0.05) of diploid activation and higher rate (39.3–40%) of second polar body extrusion (1PN + 2PB) than the other activation groups in both maturation conditions. Cleavage rates to 2-cell stage were 40–45% in all groups. Development rate of blastocysts were 7–10% in all the groups treated with 6-DMAP and 6-DMAP + roscovitine and no blastocysts were obtained from the groups treated with roscovitine alone. Hypotaurine/taurine are known to be stable and potent antioxidants, and have shown the properties of supporting oocyte maturation and further embryonic development (Guerin and Menezo 1995 Zygote 3, 333–43; Mizushima and Fukui 2001 Theriogenology 55, 1432–45). In this study, although the effectiveness of hypotaurine/taurine on promoting oocyte maturation was observed, there were no significant improvements in the rate of diploid activation in oocytes matured with hypotaurine/taurine. These results suggest that the nuclear status of activated oocytes may not have a direct relationship with the enhanced maturation condition. This work was supported by BioGreen 21 Program(#1000520030100000-1), Republic of Korea.

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