Effect of GnRH injection timing in the production of pronuclear-stage zygotes used for DNA microinjection

Zygote ◽  
2004 ◽  
Vol 12 (3) ◽  
pp. 257-261 ◽  
Author(s):  
Hernan Baldassarre ◽  
Bin Wang ◽  
Melanie Gauthier ◽  
Nathalie Neveu ◽  
Anthoula Lazaris ◽  
...  
Keyword(s):  
Medroxyprogesterone Acetate ◽  
Treatment Protocol ◽  
Hormonal Treatment ◽  
Injection Timing ◽  
Chi Square ◽  
Cell Stage ◽  
Embryo Recovery ◽  
Stage Of Development ◽  
Production Programme ◽  
Dna Microinjection

This study was aimed at developing a hormonal treatment protocol in order to optimize the proportion of pronuclear-stage embryos to be used for DNA microinjection in a goat transgenic founder production programme. A total of 46 adult BELE® and 47 adult standard goats (1–5 years old) were used as donors and recipients, respectively. They were heat-synchronized using intravaginal sponges containing 60 mg medroxyprogesterone acetate for 10 days with an injection of 125 μg cloprostenol on the morning of the eighth day. Recipients were injected with 400 IU eCG at the time of sponge removal while donors received a total of 133 mg NIH-FSH-P1 (Folltropin-V) given twice daily in decreasing doses over 3 days starting 48 h before sponge removal. Ovulation was induced in donors by injecting 100 μg of GnRH at 24 h (GnRH24) or 36 h (GnRH36) after sponge removal. Embryo recovery was performed by oviduct flushing following a standard mid-ventral laparotomy procedure. The proportion of embryos in the pronuclear stage of development was higher in the GnRH36 group (90% vs 34%, p<0.01). Embryos were microinjected with a DNA expression cassette followed by transfer to the oviduct of synchronized recipients. A higher, yet not statistically significant, pregnancy rate was found in the recipients transferred with pronuclear-stage embryos compared with those transferred with 2-cell-stage embryos (64% vs 37%, chi-square p=0.06). One transgenic female founder was produced from the group of recipients transferred with pronuclear-stage microinjected embryos.

scholarly journals Manipulating the onset of lambing season in communal ewes through hormonal intervention

2020 ◽  
Vol 50 (2) ◽  
pp. 207-214
Author(s):  
J.M. Rust ◽  
S. Mthi ◽  
T. Rust
Keyword(s):  
Medroxyprogesterone Acetate ◽  
Phase 1 ◽  
Treatment Protocol ◽  
Hormonal Treatment ◽  
Residual Effects ◽  
Seasonal Effect ◽  
Second Phase ◽  
Effective Prevention ◽  
Pre Treatment ◽  
Long Acting

The study was conducted to evaluate the effectiveness of using long-acting medroxyprogesterone acetate (MPA) to delay the lambing season in communal wool sheep ewes. The study was conducted in three phases. In phase 1, a random assessment was made to determine whether the hormone had any effect on delaying the onset of the lambing season. In the second phase, the administration of the hormone at different times during the perceived mating season was assessed. In the third phase, it was investigated whether the use of the hormone had residual effects in the subsequent lambing season after the treatment was discontinued. From the results it is evident that the administration of 150 mg MPA before conception could delay lambing between two and three months. November seems to be the optimum month for hormone administration. However, administration of the hormone did not guarantee effective prevention of conception in all treated ewes and discontinuation of treatment resulted in ewes reverting to pre-treatment lambing patterns. In conclusion, long-acting MPA can be used selectively as an effective method to delay the lambing season in communal ewes and to manipulate it towards more favourable environmental conditions for ewes and lambs. There can be a seasonal effect on time of conception in communal ewes and this should be considered when timing a hormonal treatment protocol. Keywords: communal sheep farming, lambing season, manipulation, medroxyprogesterone acetate

162 EVALUATION OF THE DEMI-EMBRYOS AGGREGATION TECHNIQUE EFFICACY ON THE PRODUCTION OF CHIMERAS WITH IN VIVO PRODUCED MURINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 239
Author(s):  
M. P. M. Mancini ◽  
B. C. S. Campanha ◽  
D. M. Souza ◽  
C. P. Godoi ◽  
F. Frei ◽  
...  
Keyword(s):  
Advanced Stage ◽  
Chi Square ◽  
Cell Stage ◽  
Exact Test ◽  
Stage Of Development ◽  
Embryo Cells ◽  
Genotype Composition ◽  
23 Gauge ◽  
Aggregation Technique

Mixing embryo cells coming from different fertilizations (i.e. embryonic chimera) have been used as a tool to understand embryogenesis, organo- genesis, and pluripotency, as well as a source to obtain transgenic mammals. The objectives of this work were to evaluate the potential of mice demi-embryos, in advanced stage of the development (morulae and blastocysts) to aggregate in chimeras; to compare the chimerism rate of those embryos with the rate of whole 8- to 16-cell-stage embryos; and to measure the genotype composition of the resultant chimera. One-month-old transgenic (C57/BL6/EGFP strain, GFP) or non-transgenic (Swiss Webster strain, SW) mice weighing approximately 35 g were superstimulated with 5 or 10IU of eCG (for GFP or SW mice, respectively) followed with hCG injection of 5 or 10IU (GFP or SW mice, respectively) 48 h later. Embryos were harvested at different stages of development and allocated in 3 groups for aggregation technique. Blastocysts and morulae were bisected (microblade mounted on TransferMan NK-2, Eppendorf), whereas 8- to 16-cell-stage embryos had their zona pellucida mechanically removed (23-gauge needle). Embryos were manipulated in M2 culture medium at room temperature, and aggregation groups consisted of G1 (2 demi-blastocysts, n = 28), G2 (demi-blastocyst and demi-morula, n = 20), and G3 (2 whole 8- to 16-cell-stage embryos, n = 25). All embryos were placed in wells (Embryo GPS dish, SunIVF) containing KSOMaa medium (EmbryoMax, Millipore) under oil (Sigma, St. Louis, MO, USA) and were incubated at 37°C, 5% CO2 in air saturated with humidity. After 24 h of incubation, the presence of chimera was verified, and the percentage of area (square pixel) occupied by each embryonic type (GFP or SW) from both G2 (n = 3) and G3 (n = 3) were measured by the ImageJ program (v. 1.42i, USA). General results of the chimerism rate were 3.6%, 15.0%, and 60.0% (G1, G2, and G3, respectively; P < 0.001, chi-square). The G3 group differed from others (G1, P < 0.001 and G2, P = 0.003), which appeared similar (P = 0.294; Fisher’s exact test). The mean percentage (±SD) of GFP cells in the resultant chimera were 51.3 ± 4.1% and 50.6 ± 10.0% (for G2 and G3, respectively; P = 0.91, t-test). Moreover, the percentages of GFP cells within the same group of G2 or G3 at 0 v. 24 h of culture were not statistically different (data not shown). It was concluded that in our conditions, the embryonic chimerism by aggregation of murine demi-embryos is a feasible procedure, even for embryos in an advanced stage of development (morulae and blastocysts). Nevertheless, the chimerism rate with whole pre-compaction embryos (G3) was higher than that of G1 and G2 groups. Furthermore, the phenotype of embryonic chimera was equally composed, with no effect of strain (GFP or SW cells) or culture (0 or 24 h) on its composition. Supported by FAPESP, Brazil: 2006/06491-2 and 2007/07705-9 (MFGN) and 2007/04291-9 (MPMM).

9 SINGLE FIXED-TIME LAPAROSCOPIC INTRAUTERINE INSEMINATION IN PIGS TO PRODUCE LOW-DIVERSE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 152
Author(s):  
K.-P. Brüssow ◽  
H. Torner ◽  
J. Rátky
Keyword(s):  
Intrauterine Insemination ◽  
Fixed Time ◽  
Uterine Wall ◽  
Sperm Cells ◽  
Chi Square ◽  
Cell Stage ◽  
Stage Of Development ◽  
Sperm Migration ◽  
Chi Square Test

In vivo-derived embryos at a defined stage of development are often a necessary requirement for ongoing biotechnological applications. Because double fixed-time insemination after ovulation induction is commonly used in pigs to produce embryos, variations in the time of ovulation and fertilization of the ovulated oocytes by spermatozoa mainly of 1 of the 2 inseminations can cause, however, diversities in embryo development. To moderate embryo diversity and to realize a uniform outcome of porcine embryo stages, single laparoscopic fixed-time insemination can be used to minimize embryo diversity. The potential of laparoscopic intrauterine insemination (LIUI) has been demonstrated in sperm-mediated gene transfer (Fantinati et al. 2005) and evaluation of sperm migration (Brüssow et al. 2006, 2011). The aim of the present study was to analyze the development and possible diversity of embryos after LIUI. Forty-eight puberal German Landrace gilts were included in the study. Estrus of gilts was synchronized by 15-day Regu-Mate® (Intervet, Millsboro, DE, USA) feeding and follicle development was stimulated with 850 IU of eCG 24 h after Regu-Mate® treatment. Ovulation was induced by 500 IU of hCG 80 h after eCG treatment. The LIUI was performed 31 h after hCG treatment. To that, ketamine/azaperone-anaesthetized gilts were fixed in a dorsal position, a pneumoperitoneum was produced and 3 trocar cannulas were inserted into the abdomen for optics and instruments. Laparoscopic handling was observed on a television monitor. Each uterine horn was carefully fixed with an atraumatic forceps 10 to 15 cm caudal from the utero-tubal junction and the uterine wall was punctured with a 2.5-mm diameter trocar. A 2.2-mm catheter connected to a syringe was inserted about 3 cm into the uterine lumen and 20 mL of extended, fresh boar semen (32.2 × 106 sperm cells mL–1; 65% motility) was deposited in the lumen. Embryos were surgically flushed from the genital tract on Day 2 and 3, respectively. Altogether, 778 oocytes were recovered (recovery rate 68 ± 17%); 45 of 48 gilts (93.8%) revealed fertilization and 76.1% of the recovered embryos (n = 592) were at the 2- and 4-cell stage. On Day 2 (n = 22 gilts), a higher percentage of gilts displayed only 2-cell embryos compared with both 2- and 4-cell, and only 4-cell embryos (72.2 v. 22.7 and 4.6%, P < 0.05; chi-square test). On Day 3 (n = 23 gilts), there was a shift regarding the embryo stage. The proportion of gilts with 2-cell, 2- and 4-cell, and only 4-cell embryos was 4.3, 0, and 95.7%, respectively (P < 0.05). Results of the present study demonstrate high rates of fertilization and of non-diverse developed embryos after single fixed-time LIUI in gilts. Additionally, these results were achieved after inseminating a 75% lower number of sperm cells per insemination dose. Laparoscopic intrauterine insemination can be suggested as an alternative for insemination of sex-sorted semen where the number of available sperm cells after the sorting procedure is restricted.

142 CHIMERISM BY AGGREGATION OF BISECTED IN VITRO PRODUCED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 175
Author(s):  
E. M. Razza ◽  
I. P. Emanuelli ◽  
C. M. Barros ◽  
M. F. G. Nogueira
Keyword(s):  
In Vitro Culture ◽  
Cell Aggregation ◽  
Bovine Embryos ◽  
Chi Square ◽  
Cell Stage ◽  
Exact Test ◽  
Aggregation Rate ◽  
Stage Of Development

Aggregation is one of the main techniques used to obtain embryonic chimeras. This procedure can be performed with whole or demi-embryos, in different stages of development and produced by in vivo or in vitro systems. However, aggregation efficiency tends to be reduced when using embryos in advanced stages (e.g. morulae and blastocysts). The aim of this work was to evaluate the effect of the agglutinating agent phytohemagglutinin-L (PHA) in the percentage of chimeras produced with in vitro-produced (IVP) bovine embryos. Cumulus–oocyte complexes (COC; 445; quality I and II) were matured in drops of 90 μL of TCM-199 bicarbonate supplemented with 10% of FCS and incubated for 22 to 24 h. Fertilization was performed in TALP-IVF medium for 18 h. Presumptive zygotes were transferred to SOF medium for in vitro culture. Incubation conditions were 38.5°C and 5% CO2 in air. To conduct the manual bisection, embryos were placed into 3-μL microdrops of protein-free HEPES-buffered SOF medium. The bisection was executed with a microblade (Ultra-Sharp Splitting Blade, Bioniche, Bogart, GA, USA) under stereomicroscope (35× magnification). Half-structures were joined and transferred to an embryo reconstruction plate, where they were kept for 3 min in drops containing 500 μg mL–1 phytohemagglutinin-L, before the approximated pairs were transferred to SOF medium in cell aggregation well-of-the-well (WOW) micro-wells to in vitro culture. The structures were randomly allocated and the aggregation was performed between 2 whole (zona free) 8- to 16-cell stage embryos to construct aggregated chimeras in the presence [group (G)1, n = 32] or absence of PHA (G2, n = 34) and between demi-morula and demi-blastocyst with PHA (G3, n = 28) or without (G4, n = 29). The aggregation of structures was evaluated after 24 h. Aggregation rates among the 4 experimental groups and the main effects were analysed by Chi-square or Fisher’s exact test and significance was considered when P < 0.05. Embryo aggregation was higher in group G1 than G2 (75.0 and 50.0%, respectively; P = 0.045). Aggregation rate of demi-embryos was similar either in the presence (G3, 39.3%) or in the absence of PHA (G4, 20.7%; P = 0.16). The presence of PHA significantly increased the aggregation rates of the whole pre-compaction embryos (G1) compared with G3 (75.0 and 39.3%, respectively; P < 0.01). The use of PHA resulted in higher aggregation rates (58.3%) than non-use (36.5%; P = 0.03), whereas the embryonic stage of pre-compaction development (G1+G2) produced a higher rate of aggregation (62.1%) than post-compaction demi-embryos (G3+G4, 29.8%; P < 0.001). We could infer a positive effect of PHA on the aggregation rate of bovine IVP embryos only to the 8- to 16-cell stage of development. Financial support: FAPESP, Brazil (06/06491-2, 07/07705-9, 09/10679-5, and 09/04888-0).

The early development of haploid and aneuploid parthenogenetic embryos

Development ◽  
1975 ◽  
Vol 34 (3) ◽  
pp. 645-655
Author(s):  
Matthew H. Kaufman ◽  
Leo Sachs
Keyword(s):  
Early Development ◽  
Polar Body ◽  
Chromosome Counts ◽  
Cell Stage ◽  
Haploid Chromosome Number ◽  
Stage Of Development ◽  
Morula Stage ◽  
Second Polar Body ◽  
Similar Development ◽  
Parthenogenetic Embryos

The early development of parthenogenetically activated oocytes has been studied in C57BL × CBA-T6T6 (F1T6) translocation heterozygote mice and C57BL × CBA-LAC (F1LAC) mice. All F1T6 oocytes had either a quadrivalent or a univalent-trivalent configuration at meiosis I; no such chromosome configurations were observed in the F1LAC oocytes. At ovulation 36·5 % of the F1T6 oocytes had 19 or 21 chromosomes, whereas 97 % of the F1LAC had the normal haploid chromosome number of 20. After parthenogenetic activation, chromosome counts at metaphase of the first cleavage mitosis were made of the eggs with a single pronucleus following extrusion of the second polar body. These activated eggs had similar frequencies of 19, 20 and 21 chromosomes as had the oocytes at ovulation. The activated 1-cell eggs were transferred to the oviducts of pseudopregnant recipients and the embryos recovered 3 days later. At this stage of development, most of the F1T6 embryos with 19 chromosomes were no longer found, but the frequency of 21-chromosome embryos was similar to the frequency of 21-chromosome oocytes and activated eggs. There was a similar mean number of cells in the embryos with 20 and 21 chromosomes. The results indicate that nearly all the embryos with 19 chromosomes failed to develop, probably beyond the 2-cell stage, whereas oocytes with 21 chromosomes had a similar development to oocytes with 20 chromosomes up to the morula stage.

scholarly journals The Effect of Probiotics on Bacterial Pneumonia

2021 ◽  
Vol In Press (In Press) ◽  
Author(s):  
Ahmad Najafi ◽  
Alireza Sharif ◽  
Mohammadreza Sharif ◽  
Hamidreza Gilassi
Keyword(s):  
Bacterial Pneumonia ◽  
Treatment Protocol ◽  
Response To Treatment ◽  
Control Group ◽  
Case Group ◽  
Chi Square ◽  
Double Blind ◽  
Duration Of Symptoms ◽  
Mortality And Morbidity ◽  
Significant Difference

Background: Pneumonia, as a fairly prevalent illness, is the main cause of hospital mortality. The major cause of mortality and morbidity of pneumonia is due to bacteria. The presence of multi-drug resistant pathogens and no response to treatment have aroused considerable interest in the use of probiotic components to prevent infections. Objectives: Given that few studies have evaluated the efficacy of probiotics in reducing bacterial pneumonia, the current aimed to evaluate the role of probiotics in decreasing pneumonia. Methods: This double-blind, randomized clinical trial study was conducted on 100 patients diagnosed with bacterial pneumonia in Shahid Beheshti Hospital, Kashan, Iran, during 2018. Patients were randomly classified into two groups (n = 50). One group (case) received two sachets of probiotic/daily for five days, and another group (control) received placebo. Moreover, patients in both groups received the same treatment protocol. All data were extracted from medical records. Chi-square test and independent t-test were used for analysis of data. P < 0.05 was considered statistically significant. Results: No significant difference was seen between case and control groups regarding age, gender, and duration of symptoms before hospitalization (P > 0.05), which implies a completely random classification of two groups. The mean duration of hospitalization, dyspnea, tachypnea, cough, fever, and crackles was significantly decreased in the case group compared to the control group (P < 0.05). Conclusion: The use of probiotics can be effective in reducing the duration of dyspnea, tachypnea, cough, fever, and length of hospitalization. Therefore, probiotics may be considered a promising treatment for the development of new anti-infectious therapy. In addition, the usage of probiotics along with antibiotics is suggested for decreasing pneumonia complications and improving the efficacy of therapy.

scholarly journals 452. Antibiotic Duration, but Not Size, Impacts Clinical Cure of Limited Skin and Soft-Tissue Infection After Incision and Drainage

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S222-S223
Author(s):  
Jason G Lake ◽  
Stephanie Fritz
Keyword(s):  
Antibiotic Therapy ◽  
Antibiotic Treatment ◽  
Clinical Cure ◽  
Day Treatment ◽  
Treatment Protocol ◽  
Chi Square ◽  
Incision And Drainage ◽  
Exact Test ◽  
Days Of Therapy ◽  
Antibiotic Duration

Abstract Background Incision and drainage (I&D) is the most common treatment for skin abscesses. A recent randomized clinical trial (RCT) of outpatients with limited (≤5 cm) skin abscesses demonstrated antibiotic therapy with clindamycin or trimethoprim-sulfamethoxazole (TMP-SMX) was superior to I&D alone. We performed a subgroup analysis to measure the effect of antibiotic duration and abscess size on clinical cure at 7–10 days after antibiotic completion. Methods Participants with complete data regarding adherence to the 10-day treatment were included. Demographic and baseline clinical features were compared using t-test, Pearson’s chi-square or Fisher’s exact test, or a non-parametric equivalent where appropriate. Largest abscess dimension (cm) was dichotomized by median size. The effect of antibiotic duration, abscess size (≤ median vs. >median) and covariates on clinical cure were measured using logistic regression. Breslow-Day Test for Homogeneity was used to assess the interaction between treatment and abscess size. Results Of 786 participants in the intention-to-treat analysis, complete adherence data were available for 680 (87%) participants. Of these, 463 (68%) received either antibiotic: 421 (91%) completed 10 days of therapy, 29 (6.3%) ≤7 days and 20 (4.3%) ≤5 days. Only antibiotic treatment duration was associated with clinical cure (table). Odds of clinical cure were 1.7 (95% CI: 1.5, 2.0) times higher for each additional day of treatment. Median abscess size was 2.5 cm (range: 0.2–5); 364 participants had abscesses ≤ median vs. 316 >median. Assessed continuously, abscess size was not associated with cure within antibiotic groups (table) or between placebo and treatment groups (OR 0.94, 95% CI: 0.58–1.5). Stratifying on size, no significant interaction was observed with antibiotic treatment (Breslow-Day P = 0.13). Conclusion Adherence to the treatment protocol was high. These data suggest that longer courses of antibiotic therapy in conjunction with I&D are associated with successful treatment of limited skin abscesses. Size was not associated with clinical cure. Prospective RCTs to determine the optimal length of treatment are needed. Disclosures All authors: No reported disclosures.

HPRT activity in embryos of a South American opossum Monodelphis domestica

1994 ◽  
Vol 6 (4) ◽  
pp. 529 ◽  
Author(s):  
PG Johnston ◽  
D Dean ◽  
JL VandeBerg ◽  
ES Robinson
Keyword(s):  
X Chromosome ◽  
Blastocyst Stage ◽  
Monodelphis Domestica ◽  
X Inactivation ◽  
Hypoxanthine Phosphoribosyl Transferase ◽  
South American ◽  
Cell Stage ◽  
Phosphoribosyl Transferase ◽  
Stage Of Development ◽  
American Opossum

Marsupial females show preferential paternal X-inactivation. However, the time at which X-inactivation occurs in early development has not yet been determined. A double microassay which measures the activities of X-linked hypoxanthine phosphoribosyl transferase (HPRT) and the autosomally-coded adenine phosphoribosyl transferase (APRT) from the same sample was performed on a collection of embryos from a South American opossum Monodelphis domestica. The embryos ranged in age from the 2-cell stage to the bilaminar blastocyst stage. The results indicate that their embryonic HPRT and APRT are not expressed until just before the unilaminar blastocyst stage in M. domestica. This is at a later stage of development than that in the mouse where embryonic HPRT and APRT expression first occurs at the 4-8-cell stage. It is concluded that HPRT is an uniformative enzyme for assessing X chromosome activity in cleaving embryos of M. domestica. The widespread distribution of HPRT:APRT ratios after the unilaminar blastocyst stage also makes it difficult to draw conclusions about the state of X chromosome activity in early marsupial development.

221 SOMATIC CELL GOAT CLONING USING ALLOGENEIC OR SYNGENEIC TRANSGENIC CELL LINES

2014 ◽  
Vol 26 (1) ◽  
pp. 224
Author(s):  
L. T. Martins ◽  
L. H. Aguiar ◽  
C. E. M. Calderón ◽  
S. G. Neto ◽  
K. C. S. Tavares ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Donor Cell ◽  
Serum Starvation ◽  
Chi Square ◽  
Cell Stage ◽  
Primary Fibroblast ◽  
Skin Cell ◽  
Standard Procedures

The aim of this study was to compare the efficiency of goat cloning by using cell lineages from distinct transgenic backgrounds. Primary fibroblast skin cell cultures from 2 females (allogeneic), transgenic for the human lysozyme gene (hLZ), were established following standard procedures. Cells from one hLZ genotype were used for the establishment of 2 double transgenic syngeneic cell lines by cell transfection (Nucleofector®, Lonza, Germany) with transgene cassettes containing either the human glucocerebrosidase gene (hGC) and neomycin resistance gene, or the human lactoferrin gene (hLF) with no selection gene. The hGC-transfected hLZ cells were antibiotic-selected (G418, Sigma-Aldrich, St. Louis, MO, USA) until the isolation of positive cell colonies, whereas hLF-transfected hLZ cells were seeded onto 100-mm culture plates (100 cells/plate) to allow colony outgrowth from individual cells. Isolated colonies were screened by PCR using specific primers for each transgene (hGC or hLF) and for hLZ and GAPDH (controls). Positive cells from one hLZ-hGC and one hLZ-hLF colony were used for cloning at passage 9, whereas hLZ cells from the other genotype were at passage 4. Cells were synchronized by high confluence and 24 h of serum starvation. Goat cloning was performed according to standard procedures (Feltrin et al. 2012 Reprod. Fertil. Dev. 25, 163). Briefly, cumulus-oocyte complexes from abattoir ovaries were in vitro-matured for 20 h. Oocyte enucleation and hLZ, hLZ-hGC, or hLZ-hLF donor cell insertion were done by micromanipulation. Reconstructed structures were fused by two 1.2-KV cm–1 DC pulses for 20 μs. Cloned embryos were cultured for 1 h in cytochalasin B and then activated in ionomycin/6-DMAP. After 12 h of in vitro culture in G-1™ medium (Vitrolife, USA), 1-cell stage embryos were transferred into the oviduct of synchronous females (Keefer et al. 2002 Biol. Reprod. 66, 199-203). Pregnancy diagnosis was performed by ultrasonography on Day 30, with weekly monitoring afterwards. Preliminary data from 6 replicates were analysed by the chi-square test (P < 0.05). Maturation rate and survival after enucleation were 42.8% (610/1425) and 72.9% (291/399), respectively. A total of 271 structures were reconstructed using the 3 donor cell lines. Fusion rates did not differ between hLZ (59.5%), hLZ-hGC (47.5%), and hLZ-hLF (48.5%) groups. A total of 68 hLZ, 92 hLZ-hGC, and 39 hLZ-hLF-derived embryos were transferred to 5, 7, and 3 recipients, respectively. No pregnancies were detected with the use of hLZ and hLZ-hLF cells. However, 3 pregnancies (one nonviable) were detected on Day 30 with hLZ-hGC cells (42.9%), with both viable pregnancies lost on Days 40 and 130 of gestation. Molecular analyses confirmed both concepti as transgenic clones from the hLZ-hGC cell line. In summary, antibiotic selection of positive colonies was effective at maintaining cell viability, with a positive response when used for cloning. Replications are in progress to evaluate the effect of cell colony isolation from individual cells (e.g. hLZ-hLF cells) on cell viability over time and on cloning outcome.

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.

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