28 DOUBLING OOCYTE CYTOPLASM VOLUME INCREASES BLASTOCYST QUALITY FOLLOWING INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER IN ARGALI SHEEP (OVIS AMMON)

2016 ◽  
Vol 28 (2) ◽  
pp. 144
Author(s):  
A. L. Green ◽  
F. C. Oback ◽  
J. E. Oliver ◽  
L. Popovic ◽  
L. T. McGowan ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Single Cells ◽  
Statistical Significance ◽  
Ovis Aries ◽  
Domestic Sheep ◽  
Custom Made ◽  
Blastocyst Quality ◽  
Ovis Ammon

Interspecies somatic cell NT (SCNT) can be used in the conservation of endangered animals but only when there is an abundant source of compatible oocytes and recipients. The objective of this study was to compare the efficiency of intra- and interspecies SCNT in sheep using zona-free embryo reconstruction methods. Skin fibroblasts from either an argali (Ovis ammon) or control (Ovis aries) ram were used as donor cells for SCNT between passages 2 to 5 and following culture in medium containing 0.5% FCS for 4 to 6 days. Single cells were electrically fused to cytoplasts prepared following enucleation of in vitro-matured zona-free metaphase II-arrested oocytes obtained from domestic ewes. In an additional experiment with argali, a double cytoplast (DC-SCNT) procedure was used whereby a second cytoplast was fused to the first reconstruct within 1 h. Reconstructs were artificially activated ~25 h after the start of maturation using ionomycin and 6-DMAP. Zona-free parthenogenote (PG) control oocytes were activated around the same time. In each treatment, 10 to 12 zona-free embryos where cultured in microwells formed in 20-μL drops of modified synthetic oviduct fluid under oil. Half the medium was replaced on Day 3, and developing embryos were transferred to individual 5-μL drops on Day 6. Development on Day 7 was expressed as a percentage of cleaved embryos. Statistical significance was determined using Fisher’s exact test for embryo development and two-tailed t-test for embryo cell numbers. Total embryo development on Day 7 was significantly greater with intraspecies sheep SCNT compared with interspecies argali SCNT (34/157 = 21.7% v. 34/363 = 9.4%, respectively; P < 0.001). Moreover, SCNT embryo development was significantly less than PG controls (122/360 = 33.9%; P < 0.001). Although argali DC-SCNT had no effect on total embryo development compared with SCNT (9/69 = 13.0% v. 7/56 = 12.5%, respectively), doubling cytoplasm volume increased the proportion of grade 1 and 2 embryos on Day 7 (8.7 v. 0%; P < 0.05). Consequently, DC-SCNT blastocysts had greater numbers of nuclei compared with SCNT (108 ± 47 v. 65 ± 9; n = 6, n = 6, respectively; P = 0.054). In comparison, PG blastocysts possessed on average 122 ± 27 nuclei (n = 7). A sample of 14 argali cloned blastocysts were all confirmed to have been derived from the respective ram after genotyping ~6000 ovine single nucleotide polymorphisms on a custom-made chip (Illumina, San Diego, CA, USA). Chromosome spreads of argali embryos revealed a modal number of 4 bi-armed autosomes as opposed to 6 in Ovis aries. In conclusion, in vitro development following interspecies SCNT in argali was about half that compared with domestic sheep. Blastocyst quality was improved by increasing oocyte cytoplasmic volume facilitated by zona-free cloning. Alternative sources of cytoplasm may further improve development. Confirmation that Ovis aries cytoplasm can fully reprogram a differentiated argali nucleus remains to be determined. This research was supported by AgResearch Core Funding.

scholarly journals Comparison of ovine herpesvirus 2 genomes isolated from domestic sheep (Ovis aries) and a clinically affected cow (Bos bovis)

2007 ◽  
Vol 88 (1) ◽  
pp. 40-45 ◽  
Author(s):  
Naomi S. Taus ◽  
David R. Herndon ◽  
Donald L. Traul ◽  
James P. Stewart ◽  
Mathias Ackermann ◽  
...  
Keyword(s):  
Cell Line ◽  
Amino Acid Identity ◽  
Ovis Aries ◽  
Open Reading Frames ◽  
Domestic Sheep ◽  
Sequence Information ◽  
Virus Shedding ◽  
Worldwide Distribution ◽  
Nasal Secretions

The rhadinovirus Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever. OvHV-2 primarily affects ruminants and has a worldwide distribution. In this study, a composite sequence of OvHV-2 genomic DNA isolated from nasal secretions of sheep experiencing virus-shedding episodes was determined and compared with the sequence of OvHV-2 DNA isolated from a lymphoblastoid cell line derived from a clinically affected cow. The study confirmed the OvHV-2 sequence information determined for the cell line-isolated DNA and showed no apparently significant changes in the OvHV-2 genome during passage through a clinically susceptible species with subsequent maintenance in vitro. Amino acid identity between the predicted open reading frames (ORFs) of the two genomes was 94–100 %, except for ORF73, which had an identity of 83 %. Polymorphism in ORF73 was due primarily to variability in the G/E-rich repetitive central region of the ORF.

27 SOMATIC CELL NUCLEAR TRANSFER CLONING AND EMBRYO AGGREGATION IN PIGS

2014 ◽  
Vol 26 (1) ◽  
pp. 128
Author(s):  
C. P. Buemo ◽  
A. Gambini ◽  
I. Hiriart ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Electric Pulse ◽  
Blastocyst Stage ◽  
Control Group

Somatic cell nuclear transfer (SCNT) derived blastocysts have lower cell number than IVF-derived blastocysts and their in vivo counterparts. The aim of this study was to improve the blastocyst rates and quality of SCNT blastocysts by the aggregation of genetically identical free zona pellucida (ZP) porcine clones. Cumulus–oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the ZP using a protease and then enucleated by micromanipulation; staining was performed with Hoechst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80 V for 30 μs). All reconstituted embryos (RE) were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% PVA) by a DC pulse of 1.2 kV cm–1 for 80 μs. Then, the oocytes were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of free ZP embryos was achieved in a system of well of wells in 100 μL of medium, placing 3 activated oocytes per microwell (aggregation embryo), whereas the control group was cultivated with equal drops without microwells. Embryos were cultivated at 39°C in 5% O2, 5% CO2 for 7 days in SOF medium with a supplement of 10% fetal bovine serum on the fifth day. The RE were placed in microwells. Two experimental groups were used, control group (not added 1X) and 3 RE per microwell (3X). At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality and evaluate if the embryo aggregation improves it. Results demonstrated that aggregation improves in vitro embryo development rates until blastocyst stage and indicated that blastocysts rates calculated over total number of oocytes do not differ between groups (Table 1). Embryo aggregation improves cleavage per oocyte and cleavage per microwell rates, presenting statistical significant differences and increasing the probabilities of higher embryo development generation until the blastocyst stage with better quality and higher diameter. Table 1.Somatic cell nuclear transfer cloning and embryo aggregation

Identification of novel variants of interferon-tau gene in Bighorn sheep (Ovis canadensis mexicana), Pelibuey sheep (Ovis aries) and its expression in hybrid blastocysts (Ovis canadensis × Ovis aries)

2012 ◽  
Vol 92 (3) ◽  
pp. 275-283 ◽  
Author(s):  
M. Palma-Irizarry ◽  
P. Damian-Matsumura ◽  
M. Betancourt ◽  
S. Romo ◽  
G. Perales ◽  
...  
Keyword(s):  
Bighorn Sheep ◽  
Ovis Canadensis ◽  
Ovis Aries ◽  
Domestic Sheep ◽  
Interferon Tau ◽  
Pelibuey Sheep ◽  
Novel Variants ◽  
Ovis Canadensis Mexicana

Palma-Irizarry, M., Damian-Matsumura, P., Betancourt, M., Romo, S., Perales, G., Lagunas-Martínez, A., Madrid-Marina, M. V., Mejía, O. and Ducolomb, Y. 2012. Identification of novel variants of interferon-tau gene in Bighorn sheep ( Ovis canadensis mexicana ), Pelibuey sheep ( Ovis aries ) and its expression in hybrid blastocysts ( Ovis canadensis×Ovis aries ). Can. J. Anim. Sci. 92: 275–283. The major signal for maternal recognition of pregnancy (MRP) in ruminants is interferon-tau (IFNT). Four novel sequences of the IFNT gene were isolated from genomic DNA obtained from blood samples of Bighorn, Pelibuey or the hybrid ewes. The deduced amino acid sequence of the hybrid ewe showed 100% identity with one Bighorn sheep IFNT sequence, suggesting that this sequence was inherited from Bighorn sheep. The high degree of identity between Pelibuey and Bighorn sheep sequences could be a possible explanation for the successful MRP in some Bighorn hybrid pregnancies. In vivo and in vitro domestic sheep (Ovis aries) and hybrid sheep (Ovis aries×Ovis canadensis) blastocysts were obtained, and IFNT expression was measured by semiquantitative RT-PCR. In vivo and in vitro hybrid blastocysts showed lower expression of IFNT when compared with domestic blastocysts. In conclusion this study showed that there exists an effect of hybridization on IFNT expression and this can affect the MRP.

P–233 The spatial arrangement of blastomeres and time of cavitation forming as predictors of blastocyst quality

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
N Sayme ◽  
T Krebs ◽  
M Kasoha ◽  
D H A Maas ◽  
E F Solomayer ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Statistical Significance ◽  
Spatial Arrangement ◽  
Time Frame ◽  
Classification Model ◽  
Time Duration ◽  
Vitro Fertilization ◽  
Morphokinetic Parameters ◽  
Blastocyst Quality

Abstract Study question Does the spatial arrangement of blastomeres and the start of blastulation affect blastocyst quality? Summary answer Better blastocyst quality is associated with the spatial arrangement of the embryo and the shorter time frame of blastulation (cavitation). What is known already The ability to select the human embryo with the highest implantation potential remains one of the greatest challenges in the management of In Vitro Fertilization patients. Several publications have proposed that additional morphological evaluations of blastomere arrangement and the dynamics of late-stage embryonic divisions might be a useful non-invasive way for embryo selection. In the last decade, the introduction of time-lapse technology enables continuous monitoring of embryo development, which leads to better outcomes than a selection based on the traditional morphology assessment. Study design, size, duration The spatial arrangement was defined as tetrahedrally if the cleavage planes were perpendicularly orientated, while embryos with rather parallelly orientated cleavage axes were considered as non-tetrahedral embryos. The injection time of ICSI was designated as “time zero” (t0), and EmbryoViewer software was used to calculate the time duration between injection and start of blastulation (cavitation). Obtained results were later correlated with the embryo’s capability to form a blastocyst as well as with blastocyst quality. Participants/materials, setting, methods A total of 195 oocytes from 40 patients undergoing the antagonist cycle for ICSI treatment were evaluated. All blastocysts were cultured in Embryoscope™ according to the manufacturer’s specifications (Vitrolife, Sweden). The Gardner and Schoolcraft scoring system was used to describe blastocyst quality. Statistical analyses were performed using IBM SPSS version 24. Data were reported as median and range. Differences between groups were tested using the Mann-Whitney U test. Statistical significance was defined as p &lt; 0.05. Main results and the role of chance Obtained data showed that 83.6% (61/73) of embryos with tetrahedral arrangement formed blastocysts compared to 42.4% (50/116) of embryos with the non-tetrahedral arrangement (p &lt; 0,001). Moreover, tetrahedral embryos more frequently formed good quality blastocyst compare to the non-tetrahedral [59% (36/61) vs 18 (9/50)% respectively; p &lt; 0,001]. In addition, we found that good quality blastocyst had a significantly shorter time frame between injection and blastulation start, compared with blastocysts which did not reach good quality [95.00h (84–118) vs 102h (77–121) respectively; p = 0,006]. Limitations, reasons for caution The limitation of the present study was that due to the double-embryo transfer correlation between those morphokinetic parameters and pregnancy rate can not be calculated. Further research should link these morphokinetic parameters with pregnancy rate and live birth rate as well. Wider implications of the findings: The potential of our findings is considerable, especially for countries with strict Embryo Law Regulation. Obtained results might be highly useful for selecting embryos with high implantation potential. In addition, the present work illustrates the possibility of additional information that can potentially be incorporated into an embryo classification model. Trial registration number Not applicable

The importance of manganese in the cytoplasmic maturation of cattle oocytes: blastocyst production improvement regardless of cumulus cells presence duringin vitromaturation

Zygote ◽  
2015 ◽  
Vol 24 (1) ◽  
pp. 139-148 ◽  
Author(s):  
Juan Patricio Anchordoquy ◽  
Juan Mateo Anchordoquy ◽  
Matias Angel Sirini ◽  
Juan Alberto Testa ◽  
Pilar Peral-García ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Quality ◽  
Cumulus Cells ◽  
Sod Activity ◽  
Cumulus Expansion ◽  
Production Improvement ◽  
Blastocyst Quality ◽  
Cytoplasmic Maturation

SummaryAdequate dietary intake of manganese (Mn) is required for normal reproductive performance in cattle. This study was carried out to investigate the effect of Mn duringin vitromaturation of bovine cumulus–oocyte complexes (COC) on apoptosis of cumulus cells, cumulus expansion, and superoxide dismutase (SOD) activity in the COC. The role of cumulus cells on Mn transport and subsequent embryo development was also evaluated. Early apoptosis decreased in cumulus cells matured with Mn compared with medium alone. Cumulus expansion did not show differences in COC matured with or without Mn supplementation. SOD activity was higher in COC matured with 6 ng/ml Mn than with 0 ng/ml Mn. Cleavage rates were higher in COC and denuded oocytes co-cultured with cumulus cells, either with or without Mn added toin vitromaturation (IVM) medium. Regardless of the presence of cumulus cells during IVM, the blastocyst rates were higher when 6 ng/ml Mn was supplemented into IVM medium compared with growth in medium alone. Blastocyst quality was enhanced when COC were matured in medium with Mn supplementation. The results of the present study indicated that Mn supplementation to IVM medium enhanced the ‘health’ of COC, and improved subsequent embryo development and embryo quality.

scholarly journals Embryo development after successful somatic cell nuclear transfer to in vitro matured human germinal vesicle oocytes

2007 ◽  
Vol 22 (7) ◽  
pp. 1982-1990 ◽  
Author(s):  
B. Heindryckx ◽  
P. De Sutter ◽  
J. Gerris ◽  
M. Dhont ◽  
J. Van der Elst
Keyword(s):  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Germinal Vesicle

scholarly journals Comparison of neuroglobin expression in the retina of Tibetan sheep (Ovis ammon or argali ) and domestic sheep (Ovis aries )

2021 ◽  
Vol 77 (04) ◽  
pp. 6512-2021
Author(s):  
XIAOHUA DU ◽  
XIA LIU ◽  
JAMES BLACKAR MAWOLO
Keyword(s):  
Oxygen Transport ◽  
Inner Core ◽  
Morphological Characteristics ◽  
Oxygen Metabolism ◽  
Ovis Aries ◽  
Domestic Sheep ◽  
Positive Expression ◽  
Tibetan Sheep ◽  
Ovis Ammon ◽  
The Brain

Neuroglobin (Ngb) is a member of the globin member that is mainly expressed in the brain and retina. The expression in the retina is greater than it is in the brain because of the high demand for oxygen by the retina. This protein reversibly binds oxygen and is involved in oxygen transport or protection against oxidative stress. The study aims to explore the pattern of Ngb expression in Tibetan sheep (Ovis ammon or argali ) living in a plateau region and domestic sheep (Ovis aries) dwelling on low land. The study also provides detailed insight into the morphological characteristics of Ngb distribution closely related to oxygen metabolism in the retina of Tibetan and domestic sheep. Immunohistochemical staining procedures were performed to detect the Ngb protein expression in the retinae of both sheep, while the Inter’s integrated performance primitives (IPP) semi-quantitative analysis was employed to compare the quantities of Ngb expressed in the retinae of the two types of sheep. The outer nuclear layer and the outer segment of the photoreceptor showed a negative Ngb expression, while a weak positive expression was observed in the inner core layer and ganglion cell layer, while a strong positive expression was found in the other layers. The independent T-test revealed that the expression intensity in the nerve fiber layer, inner and outer plexus layers, inner segment of the photoreceptor, and superior pigment cortex were significantly higher in the Tibetan sheep as compared to the domestic sheep. The overall Ngb expression in the Tibetan sheep was higher than the domestic sheep. The study emphasized that the different Ngb expression in the Tibetan and domestic sheep may influence their adaptation to the respective environments. The stronger Ngb expression in the retina of Tibetan sheep may indicate that Ngb is closely related to the oxygen metabolism of these animals and has a function during oxygen transport.

scholarly journals Analysis of parameters characterizing argyrophilic zones in intact lymphocytes of domestic sheep (Ovis aries L., 1758) and their hybrids with argali (Ovis ammon L., 1758)

2021 ◽  
Vol 344 (1) ◽  
pp. 52-56
Author(s):  
P. M. Klenovitsky ◽  
B. S. Iolchiev ◽  
A. N. Vetokh
Keyword(s):  
Peripheral Blood Lymphocytes ◽  
Ovis Aries ◽  
F1 Hybrids ◽  
Domestic Sheep ◽  
Functional Evaluation ◽  
Group 4 ◽  
Average Color ◽  
Color Density ◽  
Group 2 ◽  
Ovis Ammon

Relevance and research material. The study of the characteristics of the nucleolus in cells of various tissues allows us to assess the intensity of the processes of protein proliferation and biosynthesis in animals. In interphase cells, the nucleoli correspond to argyrophilic zones (AgNOR) stained with silver nitric acid. The purpose of this study is to determine the characteristics of AgNOR in sheep interphase lymphocytes of different genotypes based on computer analysis and to select parameters for functional evaluation of nucleoli. AgNOR status was studied in peripheral blood lymphocytes from hybrid sheep four genotypes: purebred Romanov sheep, and their F1 hybrids with argali (groups 1 and 2), hybrids, bearing 3/4 of Romanov sheep blood and 1/4 blood argali (group 3), and hybrids that had 7/8 of domestic sheep and 1/8 blood argali (group 4). The number of argyrophilic zones (AgNOR), their total area (SSNOR), their average color density (DNOR), and the average color density of the core (DN) and its AgNOR-free area (DF) were taken into account.Results. Image processing and analysis were performed using the Image Scope 1.0 program. The average AgNOR value in the studied groups of sheep ranges from 2.64 (group 1) to 3.50 in hybrids in groups 2 and 3. There were no significant differences in the value of SSNOR between the groups. In terms of DN and DF, all hybrids were significantly inferior to purebred sheep (р < 0.001). In terms of DNOR, the sheep of group 2 were significantly inferior to the animals of the other groups (р < 0.001). When evaluating the state of the nucleolus-forming system, it is advisable to take into account the AgNOR number, as well as the average optical densities DN and DF.

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