34 COMPARISON OF PROLIFERATION AND TELOMERASE ACTIVITY IN FIBROBLASTS DERIVED FROM GYEONG-JU DONGGYEONG DOGS ACCORDING TO THEIR AGE

2015 ◽  
Vol 27 (1) ◽  
pp. 110
Author(s):  
Y. B. Choi ◽  
G. A. Kim ◽  
H. J. Oh ◽  
M. J. Kim ◽  
B. C. Lee

Donggyeong dog is a breed considered a natural monument in Korea since 2012. Nevertheless, this breed is only found in Gyeong-ju and is classified as endangered. The use of assisted reproductive technologies (ART) could be applied to preserve these endangered dogs. Among various ART, somatic cell nuclear transfer (SCNT) may be a good technique for achieving propagation of genetically identical individuals. For this reason, we investigated the effect of age of the cell donor on several characteristics, including growth pattern, doubling time of cell populations, cell size, viability, and telomerase activity in Gyeong-ju Donggyeong dog fibroblast cultures before SCNT. Primary fibroblast cell cultures were performed using ear biopsies from 1-year-old (D-1yr) and 7-year-old (D-7yr) Donggyeong dogs and cells at passage 2 to 6 from in this study. Cells were plated at 1 × 105 cells well–1 in a 6-well plate. Cells were harvested every 24 h for 6 days, and cell number was determined to measure growth pattern and doubling time. The harvested cells were stained with trypan blue, their size and viability were analysed using a Countess Automated Cell Counter (Life Technologies, Carlsbad, CA, USA). Telomerase activity was measured by telomeric repeat amplification protocol using TeloTAGGG Telomerase PCR ELISAPLUS. All experiments were replicated at least 3 times. Statistical analysis was performed using Graphpad Prism (GraphPad Software Inc., San Diego, CA, USA), and t-test (P < 0.05) was used to compare the growth pattern, doubling time, cell size, viability, and telomerase activity between D-1yr and D-7yr groups. Growth curves in both groups showed the typical “S” shape and no significant differences between D-1yr and D-7yr groups. However, doubling times from 2nd to 5th passages of D-1yr (20.0 ± 0.3 h, 38.0 ± 0.5 h, 38.7 ± 0.5 h, and 53.4 ± 1.4 h) were significantly shorter than those of D-7yr (37.0 ± 1.2 h, 54.0 ± 3.6 h, 63.3 ± 1.8 h, and 100.9 ± 4.4 h). Cells from 6th passage of D-7yr and 7th passage of D-1yr did not reach confluence. Cell size of D-1yr (13.6 ± 0.2 μm) was significantly smaller than that of D-7yr (14.9 ± 0.3 μm; P < 0.001). There was no significant difference between D-1yr (89.4 ± 1.4%) and D-7yr (88.9 ± 1.2%) in cell viability but relative telomerase activity was significantly higher in D-1yr (37.8 ± 7.5) compared with D-7yr (19.0 ± 6.2, P < 0.001). In conclusion, these results suggest that fibroblasts derived from young Donggyeong dogs are more suitable for SCNT than those from old donors. For further study, dog cloning using D-1yr and D-7yr should be performed to evaluate the effect of donor age in canine SCNT efficiency.

2016 ◽  
Vol 28 (2) ◽  
pp. 206
Author(s):  
Y. B. Choi ◽  
G. A. Kim ◽  
H. J. Oh ◽  
M. J. Kim ◽  
Y. K. Jo ◽  
...  

In pig, cattle and mouse cloning, the age of the cell donor can influence cloning efficiency. However, little is known regarding the effects of the cell donor’s age related to cell proliferation and senescence and its importance for canine cloning. Therefore, the aim of this study was to investigate the effect of cell donor age on growth pattern and doubling time of cell populations, cell size, viability, and senescence-related gene expression (p53, p16, p21, Rb1, and E2F1) in canine fibroblasts. Cells were derived from 1-year-old and 7-year-old dogs. To obtain the fibroblasts, primary culture was performed using ear biopsies from three 1-year-old dogs (1yr) and three 7-year-old dogs (7yr), and cells at passage 2 to 6 were used for somatic cell cloning. Cells were plated at 1 × 105 cells well–1 and harvested every 24 h for 6 days. Cell number was determined for measurement of growth pattern and doubling time. The harvested cells were stained with trypan blue and their size and viability were analysed using a Countess automated cell counter (ThermoFisher Scientific, Waltham, MA, USA). Gene expression was measured by RT-qPCR. Cell proliferation and gene expression experiments were technically replicated 3 and 4 times, respectively. Statistical analysis was performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA) and t-test (P < 0.05) was used. Growth curves in both groups showed typical S-shape and at Day 6, 1yr samples had significantly higher cell numbers compared with 7yr samples. For doubling times, passages 2 to 5 of 1yr (29.8 ± 4.4 h, 41.0 ± 1.6 h, 49.5 ± 4.8 h, and 71.3 ± 8.4 h) were significantly shorter than those of 7yr (47.0 ± 4.5 h, 57.1 ± 2.3 h, 65.8 ± 1.8 h, and 126.4 ± 12.4 h). Cells from passage 6 of 7yr and passage 7 of 1yr did not reach confluence at Day 6. Cell size of 1yr (14.1 ± 0.2 µm) was significantly smaller than that of 7yr (15.3 ± 0.2 µm). Additionally, there was no significant difference in cell viability between 1yr (90.7 ± 0.8%) and 7yr (88.7 ± 0.7%). However, mRNA expression of p53, p21, and Rb1 was significantly lower in 1yr cells compared with 7yr cells. The expression level of E2F1 was significantly higher in 1yr cells. There was no significant difference between 1yr and 7yr cells for p16 expression. In conclusion, proliferation analysis indicated that somatic cells derived from old donor dogs reach a state of senescence more rapidly compared with those of young donor dogs. In addition, analysis of gene expression demonstrated that cells from old donor dogs have significantly increased p53, p21, and Rb1 expression levels, which indicate senescence. Further studies are needed to produce dogs using 1yr and 7yr cells to evaluate the effect of donor aging in canine somatic cell nuclear transfer efficiency. This study was supported by RAD (#PJ0109282015), IPET (#311062-04-3-SB010), Nature Cell, Research Institute for Veterinary Science, (Natural Balance Korea or Nestle Purina Korea) and the BK21 plus program. We thank Barry D. Bavister for his valuable editing of the manuscript.


Zygote ◽  
2020 ◽  
Vol 28 (2) ◽  
pp. 154-159
Author(s):  
Juliana I. Candelaria ◽  
Anna C. Denicol

SummaryPreantral follicles are a potential reservoir of oocytes to be used in assisted reproductive technologies. With the increasing interest in developing techniques to grow preantral follicles in vitro, and as the bovine emerges as an appropriate model species to understand human folliculogenesis, the establishment of an accurate classification of developmental stages is needed. Classification of bovine preantral follicles has been mostly based on histological analysis and estimation models, which may not translate well to correctly characterize preantral follicles isolated from the ovary. In this study, we classified bovine preantral follicles by morphology upon isolation, determined diameter and number of granulosa cells by direct counting, and compared our results with previous studies reporting bovine preantral follicle classification. Follicles were isolated via homogenization of ovary tissue and classified into primary, early secondary and secondary stage based on morphology and number of layers of granulosa cells. Diameter was individually measured and Hoechst 33342 was used as a nuclear stain to count granulosa cells. We found that follicles classified by morphology into primary, early secondary, and secondary had different mean diameter and cell number (P < 0.01); cell number and diameter were positively correlated, as were cell density and cell number in each developmental stage (P < 0.01). Results obtained here were mostly in agreement with previous classifications based on histological sections and on isolated follicles, with some discrepancies. The present data add accuracy to classification of bovine preantral follicles that is critical to optimize culture conditions to produce developmentally competent oocytes.


eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Sebastian Canovas ◽  
Elena Ivanova ◽  
Raquel Romar ◽  
Soledad García-Martínez ◽  
Cristina Soriano-Úbeda ◽  
...  

The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first whole-genome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT.


2007 ◽  
Vol 19 (1) ◽  
pp. 166
Author(s):  
X. J. Yin ◽  
H. S. Lee ◽  
E. G. Choi ◽  
X. F. Yu ◽  
B. H. Choi ◽  
...  

Domestic cats are a useful research model to develop assisted reproductive technologies for the conservation of endangered felids. Previously, we produced cloned offspring derived from somatic cell nuclear transfer of ear skin fibroblasts obtained from a deaf, odd-eyed, male Turkish Angora. The aim of this study was to assess the cloning efficiency of the fibroblasts derived from a cloned cat. Fibroblast cell lines were established from 6-mm skin biopsies taken from a deaf, odd-eyed, male Turkish Angora and his clone. The protocol for nuclear transfer was described previously (Yin et al. 2005 Reproduction 129, 245–249). Briefly, cumulus cells were removed from the ova by gently pipetting them into TCM-199 supplemented with 0.1% hyaluronidase. The denuded oocytes were then cultured in TCM-199 supplemented with 0.2 �g mL-1 demecolcine for 1 h and placed into TCM-199 containing 5 �g mL-1 cytochalasin B and 0.2 �g mL-1 demecolcine. The first polar body and protruded chromatin plate were removed with a beveled micropipette. Micromanipulation was used to place a single donor cell nucleus into the perivitelline space of enucleated ova. The ovum-cell couplets were fused and pulse activated. The activated couplets were cultured in 500 �L of CRI medium supplemented with 0.3% BSA for 2 days. The cleaved embryos were cultured in CRII medium supplemented with 10% FBS for 5 days. The cleavage and blastocyst development rates were 38.5% and 3.5% for second generation cloned embryos. A total of 310 second generation cloned embryos were transplanted to 9 surrogates, and 2 pregnancies at 30 days were determined by ultrasonography. One pregnancy was aborted at 40 days of gestation; the second pregnancy continued. These results indicate that the serial cloning of a cat can be generated efficiently up until pregnancy. This work was supported by KOSEF (grant #M10525010001-05N2501-00110).


2017 ◽  
Vol 29 (1) ◽  
pp. 190
Author(s):  
A. M. Raseona ◽  
O. A. Ajao ◽  
L. D. Nethengwe ◽  
L. R. Madzhie ◽  
T. L. Nedambale ◽  
...  

Preservation of semen is an important process to ensure that semen quality is sufficient for assisted reproductive technologies. The aim of this study was to evaluate the viability of bull semen collected by electro-ejaculation using commercial semen extender and 2 modified culture media stored at controlled RT (24°C) for 72 h. Two Nguni bulls were used for semen collection; after collection, the semen was evaluated macroscopically for volume, pH, and colour, and microscopically for sperm motility, viability, and morphology. Uncontaminated semen samples with progressive motility >70% and morphological defects <20% were pooled after collection before being aliquoted into 3 extenders, namely Triladyl, modified Ham’s F10, and TCM-199 culture media, at a dilution ratio of 1:4 and then stored at controlled RT (24°C). Sperm motility rate was analysed using the computer-aided sperm analyser after 0, 24, 48, and 72 h of storage. Sperm morphology and viability was performed after staining the sperm cells with spermac and nigrosine-eosin stain, respectively. The study was replicated 4 times and data were analysed using ANOVA. Triladyl had a higher sperm viability rate (41.3%) and total motility rate (96.3%) for 72 h (P < 0.01) compared with the 2 modified culture media, Ham’s F10 (26.5 and 86.8%) and TCM-199 (25.0 and 86.7%), respectively. However, Ham’s F10 had higher progressive motility rate (37.8%) as compared with the other extenders, TCM-199 (31.7%) and Triladyl (23.4). There was no significant difference (P > 0.05), in viability rate between Ham’s F10 (26.5%) and TCM-199 (25.0%). No significant difference (P > 0.05) in straight line velocity was observed for the three extenders. Furthermore, no significant difference was observed in total sperm abnormalities, except for reacted acrosomes and absent tails (P > 0.05), between the 2 Nguni bulls. Nguni semen can be preserved in Triladyl or modified Ham’s F10 and TCM-199 culture media stored at 24°C and stay viable for 72 h. Triladyl proved to be the best suitable extender of the 3 extenders, showing higher sperm viability and total motility rate as compared with Ham’s F10 and TCM-199 modified culture media.


2021 ◽  
pp. 26-31
Author(s):  
V.O. Beniuk ◽  
L.M. Vygivska ◽  
I.V. Maidannyk ◽  
T.V. Kovaliuk ◽  
O.O. Chorna ◽  
...  

Study objective: to determine the role and effectiveness of the proposed therapeutic and preventive complex and psychoemotional correction of hormonal disorders in the pregnancy dynamics after assisted reproductive technologies (ART) to improve the antenatal observation and prevention of obstetric and perinatal complications.Materials and methods. The study included 299 pregnant women: the main group included 249 women whose pregnancy occurred as an ART result; the control group included 50 pregnant women with spontaneous pregnancy. Therapeutic and prophylactic complex for pregnant women after ART included: micronized progesterone, magnesium oxide, folic acid, L-arginine aspartate, ω3-polyunsaturated fatty acids and long-term psychological correction on the eve of the ART program, at 8–10, 16–18 and 28–30 weeks of pregnancy. Results. There was a significant increase in the β-chorionic gonadotropin (β-hCG) level in women of the study groups in the first trimester of pregnancy against the background of the proposed treatment. Mean β-HCG value at 7–8 weeks of gestation in the subgroup IA exceeded the subgroup IB by 37% (p <0.05), in subgroup IIA it exceeded the subgroup IIB by 33% (p <0.05). The mean β-hCG value in subgroups IIIA and IIIB did not have a significant difference in the dynamics of the first trimester compared with the control group and among themselves (p >0.05).Mean progesterone value at 7–8 weeks of gestation in subgroup IA increased by 38% in comparison with pregnant women who received the conventional treatment complex (p <0.05), in subgroup IIA it was 73% higher than in subgroup IIB (p <0.05). There was no significant difference in the progesterone level in subgroups IIIA and IIIB in the dynamics of the first trimester.The average cortisol value at 23–24 weeks of pregnancy in subgroup IA decreased by 42% (p <0.05), in pregnant women with endocrine infertility against the background of the proposed treatment complex it was 62% less than in subgroup IIB (p <0.05). The average cortisol level in women with a male factor of infertility was 63% lower than in subgroup IIIB against the background of the proposed complex (p <0.05).Conclusion. Advanced therapy with micronized progesterone in combination with magnesium saturation, L-arginine aspartate, folic acid, ω-3 polyunsaturated fatty acids, as well as long-term psychoemotional correction is appropriate and effective compared to conventional therapy for pregnant women.


2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Y Kida ◽  
M Tokoro ◽  
H Kitasaka ◽  
T Yoshimura ◽  
N Fukunaga ◽  
...  

Abstract Study question Do ACA have an effect on pregnancy and miscarriage rates of human embryos? Summary answer The present results suggest that in ACA-positive cases, the pregnancy rate per transfer was significantly lower, although the miscarriage rate was not affected. What is known already We have previously shown that patients with high levels of anti-centromere antibody (ACA), (one of the anti-nuclear antibodies (ANA)), frequently have dispersal of the female chromosomes in the cytoplasm. Additionally, we reported that the clinical outcome was characterized by a low oocyte maturation rate following ovum pick up and high multiple pronuclear formation rate after fertilization. However, the post-implantation course of embryos with ACA-positive cases has not yet been reported. Therefore, in this study, we analyzed the pregnancy and miscarriage rates in ACA-positive patients treated with Assisted Reproductive Technologies (ART). Study design, size, duration 6581 patients who underwent embryo transfer after antinuclear antibody testing between January 2014 and February 2020 were included in the analysis. Participants/materials, setting, methods The subjects were classified into three groups: ANA-negative (without ACA or any other ANA), ACA-positive (with only ACA) and ANA-positive (with ANA but not ACA). The cycle in which the gestational sac was confirmed was considered a positive pregnancy. The pregnancy and miscarriage rates were compared among the groups using “Ryan Test” for statistical analysis. Main results and the role of chance Of the 6581 eligible cases, the incidence of antinuclear antibody were 71.3% (4695/6581; ANA-negative), 0.9% (61/6581; ACA-positive) and 27.7% (1825/6581; ANA-positive). The pregnancy rates based on the total number of embryo transfer cycles for each were ANA-negative: 31.5% (5283/16792), ACA-positive: 17.6% (41/233), and ANA-positive: 32.4% (1891/5833). The pregnancy rates were significantly lower in the ACA-positive group than in the other groups. The miscarriage rate was 29.4% (1553/5283) in ANA-negative, 31.7% (13/41) in ACA-positive, and 28.0% (529/1891) in ANA-positive, with no significant difference between the three groups. Limitations, reasons for caution Retrospective analysis Wider implications of the findings: ACA-positive patients may benefit from a treatment strategy to increase the absolute number of oocytes by obtained in order to increase the chances of normal fertilization and attainment of implantation. Trial registration number none


2018 ◽  
Vol 19 (2) ◽  
pp. 82-87 ◽  
Author(s):  
S. I. Gamidov ◽  
R. I. Ovchinnikov ◽  
A. Yu. Popova ◽  
V. V. Polozov ◽  
N. P. Naumov ◽  
...  

Introduction. There’re some pathological mechanisms of male fertility disorders that still don’t have proper diagnostic tests. This significantly decreases diagnostic value of a spermogram and makes the problem of evaluation of the characteristics of spermogram changes and their effects on the effectiveness of assisted reproductive technologies (ART) a pressing problem.The study objectiveis to identify the correlation between effectiveness of ART programs and the characteristics of spermogram changes, in particular sperm concentration, motility, and morphology Materials and methods. At the V.I. Kulakov Research Center for Obstetrics, Gynecology and Perinatology in the period from December of 2012 to December of 2016, 10,042 married couples who underwent treatment using ART (2221 – in vitro insemination (IVF), 7821 – IVF with intracytoplasmic sperm injection) were examined.Results. In patients after IVF, the frequency of live births significantly depended on sperm concentration: 28.6 % for concentration above 5 mil/ml and 51.5 % for concentration above 15 mil/ml (p <0.0001). No significant difference was observed for the dependence of the frequency of live births on the number of progressive-motile sperm (grade А): 38.2 % for <5 % and 57.7 % for >15 % (p = 0.11), or on the number of spermatozoa with normal morphology: 50 % for ≥4 % and 45.5 % for <4 % (p = 0,23). In patients after IVF with intracytoplasmic sperm injection, the concentration of spermatozoa, number of progressive-motile sperm (grade А), and number of spermatozoa with normal morphology didn’t affect the frequency of live births in a statistically significant way.Conclusion. Sperm concentration, motility, and morphology can affect the frequency of live births in the IVF program, but statistically significant correlation was observed only for sperm concentration. After IVF with intracytoplasmic sperm injection, only sperm morphology affects the frequency of live births, but not in a statistically significant way. 


Author(s):  
Inci Kahyaoglu ◽  
Cavidan Gülerman ◽  
Nafiye Yılmaz ◽  
Mehmet Ufuk Ceran ◽  
Ayşe Seval Ozgu-Erdinc ◽  
...  

Objectives: Data regarding whether the assisted reproductive technologies (ART) outcome of patients with decreased ovarian reserve (DOR) secondary to an endometrioma surgery differs from that of patients with DOR secondary to other etiologies is limited. The aim of this study was to compare the ART outcomes of DOR patients diagnosed by Bologna criteria secondary to a previous endometrioma surgery with DOR patients without a history of an endometrioma surgery. Methods: This retrospective cohort study was conducted in the assisted reproduction unit of a tertiary research and education hospital. Medical records of the patients with DOR were reviewed retrospectively. Group I included 23 cycles of DOR patients secondary to a previous endometrioma surgery and Group II consisted of 260 cycles of DOR patients without a history of endometrioma surgery. Results: No significant difference was demonstrated between groups regarding demographic features except for the age (32 (24–41) in Group I vs 35 (23–47) in Group II, p = 0.031). The data comparing the controlled ovarian stimulation parameters and ART outcomes showed similar results in both groups. No statistically significant difference was found between groups regarding pregnancy rates per transfer (23% vs 22.2 %) and per cycle (13% vs 15.4%) ( p > 0.05). Conclusion: Cycle outcomes of DOR patients secondary to an endometrioma surgery did not differ from that of DOR without an endometrioma surgery history.


2015 ◽  
Vol 143 (9-10) ◽  
pp. 632-638 ◽  
Author(s):  
Tamara Sljivancanin ◽  
Olivera Kontic-Vucinic

Introduction. Recent epidemiological studies showed significantly higher incidence of perinatal complications in newborns and women after the use of assisted reproductive technologies (ART). Multiple pregnancies are more frequent after the use of ART. Singleton pregnancies following ART are more prone to preterm birth, low and very low birth weight (LBW and VLBW), small for gestational age (SGA) and perinatal mortality. Objective. The aim of this study was to summarize the results of relevant articles and to evaluate whether the mode of conception is the determining factor for different pregnancy outcomes after assisted and natural conceptions. Methods. Eleven studies were included in this review. The following outcomes were observed: preterm and very preterm birth, SGA, LBW, VLBW, perinatal mortality, admission to neonatal intensive care unit (NICU), and Apgar score (As) ?7 at fifth minute. Qualitative analysis and quantitative assessment were performed. Results. For singletons, odds ratios were 1.794 (95% confidence interval 1.660-1.939) for preterm birth, 1.649 (1.301-2.089) for LBW, 1.265 (1.048-1.527) for SGA. Admission to NICU, As?7 at fifth minute and perinatal mortality showed significantly different frequency after assisted conception. Summary of results for twin gestations showed no significant difference between ART and spontaneous conception for preterm birth (32-36 weeks), very preterm birth (<32 weeks), LBW and VLBW. Conclusion. Analyzed studies showed that infants from ART have significantly worse perinatal outcome compared with natural conception. More observational studies should be conducted in order to establish the exact mechanism leading to more frequent perinatal morbidity and mortality after the use of ART.


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