336 DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS PRODUCED USING SPERM CYTOSOLIC FACTOR (SCF) AT FUSION-ACTIVATION

2007 ◽  
Vol 19 (1) ◽  
pp. 283
Author(s):  
J. H. Shim ◽  
I. S. Hwang ◽  
H. J. Moon ◽  
M. R. Park ◽  
D. H. Kim ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Developmental Rate ◽  
Blastocyst Stage ◽  
Fetal Fibroblast ◽  
Statistical Analysis System ◽  
Donor Cells ◽  
Analysis System ◽  
Cytosolic Factor

At fertilization, the sperm activates the developmental program of the oocyte by inducing an elevation in the intracellular free Ca2+ concentration ([Ca2+]i). One possible explanation is that at sperm–oocyte fusion the fertilizing spermatozoon introduces a factor into the cytoplasm of the oocyte which opens the Ca2+ release channels from the intracellular stores through a yet unidentified mechanism. This study investigated the development of porcine nuclear transfer embryos fused and activated in the presence of sperm cytosolic factor (SCF) isolated from porcine sperm. Ovaries were collected at a local slaughterhouse and transported to the laboratory within 2 h at 35–39�C, and rinsed in 0.9% NaCl. Fetal fibroblast cells were prepared from a 35-day-old porcine fetus for use as donor cells. For parthenogenesis, matured oocytes were activated using 2 DC pulses of 1.2 kV cm-1 for 30 �s in fusion medium (0.1 mM CaCl2) supplemented with 100, 200, or 300 �g mL-1 SCF. For NT, matured oocytes were enucleated, reconstructed, and fused. Reconstructed embryos were divided into 2 groups. The embryos in one group were fused with fusion medium (1.0 mM CaCl2), and the embryos in the other group were fused with fusion medium (0.1 mM CaCl2) supplemented with 100 �g mL-1 SCF. After fusion, the embryos were cultured in PZM-3 under 5% CO2 in air at 38.5�C. A TUNEL assay was used to assess the presence of apoptotic cells (In Situ Cell Death Detection Kit, TMR red; Roche, Mannheim, Germany). Data were subjected to a generalized linear model procedure (PROC-GLM) of the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). Oocytes activated parthenogenetically in the presence of SCF showed significantly higher developmental rate to the blastocyst stage compared to that of controls (21.3–27.6% vs. 12.5%; P < 0.05). For NT, there was no difference between treatments in developmental rate to the blastocyst stage (18.2% vs. 17.1%). However, the apoptosis rate was slightly lower in blastocysts produced in the presence of SCF than that in controls. These results indicate that the presence of SCF in fusion medium can support a higher quality of porcine nuclear transfer embryos.

scholarly journals Dnmt3l-knockout donor cells improve somatic cell nuclear transfer reprogramming efficiency

Reproduction ◽  
2015 ◽  
Vol 150 (4) ◽  
pp. 245-256 ◽  
Author(s):  
Hung-Fu Liao ◽  
Chu-Fan Mo ◽  
Shinn-Chih Wu ◽  
Dai-Han Cheng ◽  
Chih-Yun Yu ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Dna Methyltransferase ◽  
Inner Cell Mass ◽  
Trichostatin A ◽  
Cell Mass ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Oct4 Expression ◽  
Donor Cells

Nuclear transfer (NT) is a technique used to investigate the development and reprogramming potential of a single cell. DNA methyltransferase-3-like, which has been characterized as a repressive transcriptional regulator, is expressed in naturally fertilized egg and morula/blastocyst at pre-implantation stages. In this study, we demonstrate that the use of Dnmt3l-knockout (Dnmt3l-KO) donor cells in combination with Trichostatin A treatment improved the developmental efficiency and quality of the cloned embryos. Compared with the WT group, Dnmt3l-KO donor cell-derived cloned embryos exhibited increased cell numbers as well as restricted OCT4 expression in the inner cell mass (ICM) and silencing of transposable elements at the blastocyst stage. In addition, our results indicate that zygotic Dnmt3l is dispensable for cloned embryo development at pre-implantation stages. In Dnmt3l-KO mouse embryonic fibroblasts, we observed reduced nuclear localization of HDAC1, increased levels of the active histone mark H3K27ac and decreased accumulation of the repressive histone marks H3K27me3 and H3K9me3, suggesting that Dnmt3l-KO donor cells may offer a more permissive epigenetic state that is beneficial for NT reprogramming.

80 EFFECT OF MEDIA, SYNCHRONIZATION OF FIBROBLAST CELLS, CULTURE TIME, OXYGEN CONCENTRATION, AND ACTIVATION ON NUCLEAR-TRANSFERRED PORCINE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 157
Author(s):  
J. H. Quan ◽  
H. B. Seok ◽  
S. K. Kim
Keyword(s):  
Nuclear Transfer ◽  
Culture Medium ◽  
Atmospheric Condition ◽  
Blastocyst Stage ◽  
Fibroblast Cells ◽  
Fetal Fibroblast ◽  
Donor Cells ◽  
The Impact ◽  
Cells Cultured

The purpose of this study was to investigate the impact of culture medium, culture duration, and atmospheric condition on the fusion and in vitro development rates of nuclear transfer porcine embryos constructed by the microinjection of fetal fibroblast cells into in vitro-matured oocytes. Single fetal donor cells were deposited into the perivitelline space of enucleated oocytes, followed by electrical fusion and activation. Activated embryos were cultured in NCSU-23 medium supplemented with 5% FBS, at 38.5�C for 6 to 8 days in 5% CO2 and air. In Experiment 1, the fusion rates of nuclear transfer embryos did not differ from those of fetal fibroblast cells incubated in 5% FBS + NCSU-23 or 5% FBS + TL-HEPES medium, nor did fusion rates of donor cells differ among 1–8-h incubation durations. Fusion rates for the 4 treatment subclasses ranged from 72.1% to 78.0%. In Experiment 2, pre-synchronization in medium containing 0.1 �g mL-1 Hoechst(H) 33342 increased during the period from 0 and 8 h of culture up to 15 h, the end of the synchronization period, at which time there was a significantly increased percentage of porcine fibroblast cells at the G2/M stage (12.4%, 17.5%, and 47.6%; P < 0.01). Neither an increase in the concentration of H 33342 (0.2–1.6 �g mL-1) nor a longer exposure time (12 h, 18 h, and 24 h) increased the proportion of porcine G2/M fibroblasts. In Experiment 3, fusion rates did not differ significantly between nuclear transfer embryos constructed using donor cells cultured in 5% FBS + NCSU-23 medium for 1–2, 6–8, or 12–14 days (60.0%, 73.3%, and 62.5%, respectively). The cleavage rate for nuclear transplant embryos using fetal fibroblast cells cultured for 1–2 days was 44.0%, which was significantly less than the 56.7% and 50.0% for 6–8 or 12–14 days of culture, respectively (P < 0.05). In Experiment 4, the proportions of nuclear transfer embryos that developed to the e2 cell and to the blastocyst stage were not affected significantly by culture medium (5% FBS + NCSU-23 or 5% FBS + TL-HEPES) or by O2 concentration during culture (5% vs. 10%). The developmental rates to the e2 cell stage ranged from 65.9% to 70.1%, and those to the blastocyst stage ranged from 9.8% to 12.5%, for the 4 treatment subclasses. Blastocyst rate was highest for embryos cultured in 5% FBS + NCSU-23 under a gas atmosphere of 5% O2 in air.

scholarly journals 73EFFECT OF CALF RECLONING ON EMBRYO AND FETAL SURVIVAL

2004 ◽  
Vol 16 (2) ◽  
pp. 158
Author(s):  
D.F. Salamone ◽  
C.B. Santos ◽  
J.L. Barañao ◽  
J.L. Bussmann ◽  
J. Artuso ◽  
...  
Keyword(s):  
Cell Line ◽  
Umbilical Cord ◽  
Nuclear Transfer ◽  
Transgenic Animals ◽  
Blastocyst Stage ◽  
Fetal Cell ◽  
Fetal Fibroblast ◽  
Fetal Survival ◽  
Donor Cells ◽  
Different Sources

In a large-scale cloning program destined to produce transgenic animals, it is very important to incorporate well-characterized transgene integration and gene expression. However, after non-homologous transfection, a wide variety of transgene copies are introduced, and these occur in different chromosome locations. Recloning a selected first-generation transgenic calf offers the opportunity to increase the homogeneity among transgenic animals. Calf recloning was performed in an experiment in which the survival rate was evaluated after a second round of cloning from transgenic umbilical cord and ear calf fibroblasts. The original genetically modified fetal cell line that produced the clones was used as control. Oocytes were aspirated from slaughterhouse ovaries and matured in TCM-199+5% FCS at 39°C for 24h. Matured oocytes were denuded by vortexing for 3min in TL HEPES with 1mgmL−1 bovine testis hyaluronidase. Metaphases were assessed and oocytes were enucleated by visualization with Hoechst 33342 (5μgmL−1) under UV light (<6s). A fetal fibroblast cell line was initially established from a 75-day old Jersey female fetus. Genetically modified cells were isolated after selection with geneticin for 10–15 days following liposome transfection with a DNA construct containing a selectable neomycin resistance gene. Following nuclear transfer with these transgenic cells, new cell lines were isolated from umbilical cord and ear fibroblasts obtained from one of the cloned-transgenic calves so-produced. Donor cells from all three sources were used for nuclear transfer at G0/G1 cell cycle stages and were fused to enucleated oocytes by an electrical pulse. After 3h, activation was induced by incubation in TL-HEPES with 5μM ionomycin for 4min and then 2mM 6-DMAP for 3h. The oocytes were then washed with TL- HEPES and cultured in SOF medium with an atmosphere of 5% CO2+5% O2+90% N2. Development to blastocyst stage (Days 7 to 9) was recorded. One or two blastocysts were transferred non-surgically per recipient cow, and pregnancies at 30 days or 60 days were determined by ultrasonography. All data were analyzed by chi-square test. Seven births were obtained from the original fetal cell line, one birth was obtained from recloned umbilical cord and four pregnancies are in the last third of gestation from recloned ear fibroblasts. Development to blastocyst stage was significantly different between transfected fetal fibroblast and both recloned treatment groups. Differences were observed in pregnancy rates between blastocysts generated by the different sources of donor cells. In spite of the lower blastocyst production, our results suggest that recloning provides an additional method to obtain transgenic animals, and that fibroblasts from umbilical cord could give better results for recloning than those obtained from young calf ear. Effect of different sources of donor cells for calf recloning on embryo and fetal survival

scholarly journals Manipulating the Mitochondrial Genome To Enhance Cattle Embryo Development

2017 ◽  
Vol 7 (7) ◽  
pp. 2065-2080 ◽  
Author(s):  
Kanokwan Srirattana ◽  
Justin C St. John
Keyword(s):  
Embryo Development ◽  
Nuclear Transfer ◽  
Bos Taurus ◽  
Trichostatin A ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Genetic Integrity ◽  
Rna Seq ◽  
Mtdna Copy Number ◽  
Donor Cells

Abstract The mixing of mitochondrial DNA (mtDNA) from the donor cell and the recipient oocyte in embryos and offspring derived from somatic cell nuclear transfer (SCNT) compromises genetic integrity and affects embryo development. We set out to generate SCNT embryos that inherited their mtDNA from the recipient oocyte only, as is the case following natural conception. While SCNT blastocysts produced from Holstein (Bos taurus) fibroblasts were depleted of their mtDNA, and oocytes derived from Angus (Bos taurus) cattle possessed oocyte mtDNA only, the coexistence of donor cell and oocyte mtDNA resulted in blastocysts derived from nondepleted cells. Moreover, the use of the reprogramming agent, Trichostatin A (TSA), further improved the development of embryos derived from depleted cells. RNA-seq analysis highlighted 35 differentially expressed genes from the comparison between blastocysts generated from nondepleted cells and blastocysts from depleted cells, both in the presence of TSA. The only differences between these two sets of embryos were the presence of donor cell mtDNA, and a significantly higher mtDNA copy number for embryos derived from nondepleted cells. Furthermore, the use of TSA on embryos derived from depleted cells positively modulated the expression of CLDN8, TMEM38A, and FREM1, which affect embryonic development. In conclusion, SCNT embryos produced by mtDNA depleted donor cells have the same potential to develop to the blastocyst stage without the presumed damaging effect resulting from the mixture of donor and recipient mtDNA.

Overexpression of MicroRNA-29b Decreases Expression of DNA Methyltransferases and Improves Quality of the Blastocysts Derived from Somatic Cell Nuclear Transfer in Cattle

2018 ◽  
Vol 24 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Shuang Liang ◽  
Zheng-Wen Nie ◽  
Jing Guo ◽  
Ying-Jie Niu ◽  
Kyung-Tae Shin ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cellular Proliferation ◽  
Dna Methyltransferases ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Somatic Cell Reprogramming

AbstractMicroRNA (miR)-29b plays a crucial role during somatic cell reprogramming. The aim of the current study was to explore the effects of miR-29b on the developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos, as well as the underlying mechanisms of action. The expression level of miR-29b was lower in bovine SCNT embryos at the pronuclear, 8-cell, and blastocyst stages compared within vitrofertilized embryos. In addition, miR-29b regulates the expression of DNA methyltransferases (Dnmt3a/3bandDnmt1) in bovine SCNT embryos. We further investigated SCNT embryo developmental competence and found that miR-29b overexpression during bovine SCNT embryonic development does not improve developmental potency and downregulation inhibits developmental potency. Nevertheless, the quality of bovine SCNT embryos at the blastocyst stage improved significantly. The expression of pluripotency factors and cellular proliferation were significantly higher in blastocysts from the miR-29b overexpression group than the control and downregulation groups. In addition, outgrowth potential in blastocysts after miR-29b overexpression was also significantly greater in the miR-29b overexpression group than in the control and downregulation groups. Taken together, these results demonstrated that miR-29b plays an important role in bovine SCNT embryo development.

Sensory acceptability and quality of flavored yogurt enriched with Spinacia oleracea extract

2014 ◽  
Vol 44 (3) ◽  
pp. 182-192 ◽  
Author(s):  
Jafar Hayaty Nejad ◽  
Ali Mohamadi Sani ◽  
Mohammad Hojjatoleslamy
Keyword(s):  
Design Methodology ◽  
Spinacia Oleracea ◽  
Titratable Acidity ◽  
Organoleptic Properties ◽  
Content Type ◽  
Overall Acceptability ◽  
Spinach Extract ◽  
Statistical Analysis System ◽  
Analysis System

Purpose – The purpose of this paper was to determine the effects of the spinach extract and kiwi flavor on the physicochemical and organoleptic properties of yogurt. Design/methodology/approach – A total of 48 yogurt samples including yogurts flavored with kiwi flavor (1, 2 and 4 percent) and colored with spinach extract (1.25, 2.5 and 4 percent) and a control yogurt (no kiwi flavor or spinach extract) were evaluated for chemical, physical and sensory properties during 21 days of storage. Data were analyzed by ANOVA using statistical analysis system. Findings – Statistically significant differences (p<0.05) were found between the control and kiwi-spinach yogurts in terms of viscosity and syneresis. The addition of the spinach extract to yogurt resulted in an increase in the syneresis, and a decrease in the viscosity. During the storage, the values of the titratable acidity, viscosity and syneresis of yogurt samples increased, while pH decreased significantly (p<0.05). Yogurt enriched with 4 percent spinach extract and 4 percent kiwi flavor was more acceptable than the other samples, and high scored with respect to overall acceptability by panelists. Originality/value – No research had been done to formulate and compare the sensory and physicochemical properties of kiwi-spinach yogurt in Iran.

scholarly journals 43IMPROVED IN VITRO DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS WITH 6-DMAP FOLLOWING FUSION

2004 ◽  
Vol 16 (2) ◽  
pp. 144
Author(s):  
G.-S. IM ◽  
L. Lai ◽  
Z. Liu ◽  
Y. Hao ◽  
C.M. Murphy ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Developmental Rate ◽  
Cell Number ◽  
Blastocyst Stage ◽  
In Vitro Development ◽  
Electric Pulses ◽  
Developmental Rates ◽  
Vitro Development

Although nuclear transfer (NT) has successfully produced cloned piglets, the development to blastocyst and to term is still low. Activation of the NT embryos is one of the key factors to improve the developmental ability of porcine NT embryos. Electric pulses as well as chemicals have been used to activate porcine NT embryos. This study was conducted to investigate the effect of continued activation following fusion pulses on in vitro development of porcine NT Embryos. Oocytes derived from a local abattoir were matured for 42 to 44h and enucleated. Ear skin cells were obtained from a 4-day-old transgenic pig transduced with eGFP recombinant retrovirus. Enucleated oocytes were reconstructed and cultured in PZM-3 in a gas atmosphere of 5% CO2 in air. Cleavage and blastocyst developmental rates were assessed under a stereomicroscope on Day 3 or 6. Blastocysts were stained with 5μg of Hoechst 33342 and total cell number was determined with an epifluorescent microscope. In Experiment 1, oocytes were activated with two 1.2kV/cm for 30μs (E) in 0.3M mannitol supplemented with either 0.1 or 1.0mM Ca2+. In each treatment, activated oocytes were divided into three groups. The first group was control (E). Other two groups were exposed to either ionomycin and 6-DMAP (E+I+D) or 6-DMAP (E+D) immediately after the electric pulses. In Experiment 2, fusion was conducted by using 1.0mM Ca2+ in the fusion medium. Fused NT embryos were divided into three treatments. NT embryos were fused and activated simultaneously with electric pulse as a control (C); the second group was treated with 6-DMAP immediately after fusion treatment (D0); and the third group was treated with 6-DMAP at 20min (D20) after fusion. In experiment 1, for 0.1mM Ca2+, developmental rates to the blastocyst stage for E, E+I+D or E+D were 12.5, 26.7 and 22.5%, respectively. For 1.0mM Ca2+, developmental rates to the blastocyst stage were 11.4, 28.3 and 35.6%, respectively. The activated oocytes treated with 6-DMAP following the electric pulses by using 1.0mM Ca2+ in fusion medium had higher (P&lt;0.05) developmental rates to the blastocyst stage. In Experiment 2, developmental rates to the blastocyst stage for C, D0 or D20 were 10.0, 12.3, and 19.9%, respectively. Developmental rate to the blastocyst stage was higher (P&lt;0.05) in D20. Fragmentation rates were 19.9, 10.8, and 9.0%, respectively. Regardless of Ca2+ concentration in fusion medium, continued treatments with chemicals following electric pulses supported more development of porcine activated oocytes. Treating NT embryos with 6-DMAP alone after fusion was completed by using 1.0mM Ca2+ in fusion medium improved the developmental rates to the blastocyst stage and prevented fragmentation accompanied by electric fusion. This study was supported by NIH NCRR 13438 and Food for the 21st Century.

scholarly journals 231EXPRESSION PATTERN OF CERTAIN DEVELOPMENTALLY IMPORTANT GENES IN BOVINE NUCLEAR TRANSFER EMBRYOS PRODUCED USING CELL LINES OF DIFFERENT EFFICIENCY

2004 ◽  
Vol 16 (2) ◽  
pp. 236 ◽  
Author(s):  
Z. Beyhan ◽  
N.L. First
Keyword(s):  
Gene Expression ◽  
Statistical Analysis ◽  
Cell Line ◽  
Cell Lines ◽  
Live Birth ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Gene Expression Patterns ◽  
Donor Cells

Developmental abnormalities associated with the cloning process suggest that reprogramming of donor nuclei into an embryonic state may not be fully completed in most of the cloned animals. One of the areas of interest in this respect is the analysis of gene expression patterns in nuclear transfer embryos to dissect the processes that failed and to develop means to overcome the limitations imposed by these factors. In this study, we investigated the expression patterns of histone deacetylase-1,-2,-3 (HDAC-1,-2,-3), DNA methyltransferase-3A (DNMT3A) and octamer binding protein-4 gene (POU5F1) in donor cells with different cloning efficiencies (low: no-pregnancy, medium: pregnancy but no live birth and high: live birth) and nuclear transfer embryos derived from these cell lines using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay with SYBR green chemistry. Employing standard protocols, we produced nuclear transfer embryos from three different cell lines categorized as having varying efficiencies in supporting development to term. Embryos were collected at morula, blastocyst and hatched blastocyst stages and total RNA was extracted from pools of 4–5 embryos using Absolutely RNA nanoprep kit (Stratagene, La Jolla, CA, USA). Relative level of expression at these stages was analyzed using ΔΔCT method with HH2A as the reference gene and in vitro-fertilized embryos as the control samples. Statistical analysis was performed on ranked expression data employing SAS statistical analysis software procedure ANOVA. Same set of genes were also analyzed on donor cells using standard curve method. All genes investigated were affected by nuclear transfer and followed somewhat altered expression patterns. In general, expression of HDAC genes was elevated especially at the compact morula stage but became comparable to control embryos at the hatched blastocyst stage. DNMT3A expression in NT embryos was lower than in IVF embryos at all stages. POU5F1 transcript levels were also reduced in nuclear transfer embryos at the compact morula and blastocyst stages. The difference, however, disappeared at the hatched blastocyst stage. There was a cell line effect on the expression patterns of all genes investigated. Cell lines efficient in producing offspring tended to resemble control embryos in gene expression patterns compared to inefficient cell lines. These results agree with several studies reporting altered gene expression patterns for certain genes in cloned embryos. Our data also suggest that cell line differences in developmental competency observed in cloning experiments might be related to physiological differences in transcriptional regulation and nuclear remodeling, DNA methylation, and lineage differentiation in embryos cloned from these cell lines.

Yield of “Pipiana” squash (Cucurbita argyrosperma Huber) in Vertisol soil due to the effect of biostimulants

2020 ◽  
Vol 13 (10) ◽  
Author(s):  
Enriqueta García Peña ◽  
Víctor Manuel Interián-Ku ◽  
Benjamín Abraham Ayil-Gutiérrez ◽  
Esmeralda Cázares-Sánchez ◽  
Pablo Santiago Sánchez-Azcorra ◽  
...  
Keyword(s):  
Statistical Analysis ◽  
Experimental Design ◽  
Design Methodology ◽  
Scientific Literature ◽  
Fresh Fruit ◽  
Comparison Test ◽  
Statistical Analysis System ◽  
Analysis System

Objective: To evaluate the effect of liquid (supermagro) and solid (bocashi) biostimulants on the yield of Pipiana squash (Cucurbita argyrosperma Huber) in a Vertisol soil. Design/Methodology/Approach: The research was carried out at “Central Flores” farm, in May-August 2018. Treatments were: T1 = Control, T2 = 400 g of bocashi + supermagro 1:20 v/v, T3 = 400 g of bocashi + supermagro 1:30 v/v, T4= 500 g of bocashi + supermagro 1:20 v/v, T5 = 500 g of bocashi + supermagro 1:30 v/v, with four replicates (plots) each. For the statistical analysis, 11 variables were recorded in plants, flowers and fruits, and 12 variables in seed, the experimental design used was completely randomized. An ANOVA and a means comparison test (Tukey, a?0.05) were performed with the Statistical Analysis System version 9.1 (SAS, 2003). Results: The plants of treatment 4, showed the highest values in most of the variables evaluated, the yield of fresh fruit and dry seed was 4.48 t ha-1 and 1.76 t ha-1, respectively, higher results than those reported in the scientific literature. Limitations/Implications: No limitations were found in this study. Findings/Conclusions: The application of foliar and soil bio-stimulant increases quantity and quality of fruits and seed of Cucurbita argyrosperma Huber.  

Effects of the time interval between fusion and activation on in vitro rabbit nuclear transfer efficiency when nuclear donor cells are derived from older adults

Zygote ◽  
2004 ◽  
Vol 12 (2) ◽  
pp. 133-141 ◽  
Author(s):  
R.P. Cervera ◽  
F. Garcia-Ximénez
Keyword(s):  
Older Adult ◽  
Exposure Time ◽  
Blastocyst Stage ◽  
Adult Rabbit ◽  
Time Interval ◽  
Unfavourable Effect ◽  
Donor Cells ◽  
Hatching Rates

Cloning older adult rabbits can serve as a model in animal breeding, biodiversity preservation and in human therapeutic cloning. To establish the required exposure time of fibroblasts from these kind of animals to reprogramming factors, in the present study three different time intervals between fusion and activation were tested (30 min, 30-ADF group; 60 min, 60-ADF group; and 90 min, 90-ADF group). Vitrified epithelial fibroblasts derived from four older adult rabbit females (D1, D2, D3 and D4) and cultured from passages 0 to 4 were used as nuclear donors. Nuclear status of reconstructed embryos was not evaluated. No differences were observed in blastocyst rate (30-ADF 21% vs 60-ADF 19% vs 90-ADF 18%). Differences in hatching rates did not reach significance (30-ADF 11% vs 60-ADF 18% vs 90-ADF 18%). However, in the 60- and 90-ADF groups, embryos reached the blastocyst stage earlier than in the 30-ADF group (day 4: 40% and 50% vs 8%; p>0.05). Moreover, the quality of blastocysts (good vs poor) was lower in the 30-ADF group (good: 30-ADF 38% vs 60-ADF 90% vs 90-ADF 90%; p>0.05). Overall, these results suggest an unfavourable effect of the shortest exposure time tested (30 min). Differences between specimen origins were detected (blastocyst and hatching rates: D2 (26%; 25%) and D4 (25%; 27%) vs D1 (10%; 11%) and D3 (12%; 12%)), but significance were not reached. Effect of culture passage was not detected in any parameter studied.

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