In vitro developmental competence of domestic cat embryos after somatic cloning: a preliminary report

2002 ◽  
Vol 58 (8) ◽  
pp. 1615-1621 ◽  
Author(s):  
Maria Skrzyszowska ◽  
Lucyna Kątska ◽  
Bożenna Ryńska ◽  
Gabriela Kania ◽  
Zdzisław Smorąg ◽  
...  
Keyword(s):  
Preliminary Report ◽  
Domestic Cat ◽  
Developmental Competence

17 Effect of the zona-free aggregation on the developmental competence of kodkod (Leopardus guigna) embryos generated by interspecies somatic cell nuclear transfer

2019 ◽  
Vol 31 (1) ◽  
pp. 134
Author(s):  
D. Veraguas ◽  
C. Aguilera ◽  
D. Echeverry ◽  
D. Saez-Ruiz ◽  
F. O. Castro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Relative Expression ◽  
Standard Curve ◽  
Morula Stage ◽  
Leopardus Guigna

The kodkod is considered a vulnerable species by the International Union for Conservation of Nature. Phylogenetically, the kodkod is classified in the Leopardus genus, which has only 36 chromosome pairs compared with the domestic cat, which has 38. The proposed hypothesis was that domestic cat oocytes are capable of reprogramming somatic cells from kodkod after interspecies somatic cell NT (SCNT), allowing the in vitro embryo development up to blastocyst stage. Five experimental groups were made based on the technology and culture system: (1) cat embryos generated by IVF (IVF), (2) cat embryos generated by SCNT (Ca1x), (3) aggregated cat embryos generated by SCNT (Ca2x), (4) kodkod embryos generated by interspecies SCNT (K1x), and (5) aggregated kodkod embryos generated by interspecies SCNT (K2x). Interspecies SCNT was performed using a zona-free method. Reconstructed embryos were activated by 2 electrical pulses of 140 kV cm−1 for 40 µs and then incubated for 5h in 10μg mL−1 of cycloheximide and 5μg mL−1 of cytochalasin B. Embryos were cultured in SOF media using the well of the well system in a 5% O2, 5% CO2, and 90% N2 atmosphere at 38.5°C for 8 days. The morulae and blastocysts rates were estimated, and diameter of cloned blastocysts was measured. The relative expression of OCT4, SOX2, and NANOG was evaluated in blastocysts by RT-qPCR using the standard curve method; SDHA was used for normalization. The Kruskal-Wallis test was used to evaluate the developmental parameters and gene expression. The t-test was used to evaluate blastocyst diameter. Statistical differences were considered at P<0.05. The number of replicates was IVF=10, Ca1x=8, Ca2x=6, K1x=3, and K2x=8. The morulae rate was lower when clone embryos were cultured individually (IVF=97/153, 63.4%; Ca2x=28/51, 54.9%; K2x=63/110, 57.3%; Ca1x=48/126, 38.1%; K1x=22/87, 25.3%; P<0.05). In the domestic cat, blastocysts rate was higher in IVF (58/153, 37.9%) and Ca2x (28/51, 29.4%) groups than in the Ca1x group (21/126, 16.7%; P<0.05). No blastocysts were generated in the K1x group (0/87), whereas 5.5% of blastocysts were obtained from the K2x (6/110; 5.5%); this was not statistically different compared with the K1x group (P>0.05). No differences were found in blastocyst diameter between the Ca1x (220.4µm) and Ca2x (251.2µm) groups (P>0.05). However, the diameter of the blastocysts from the K2x group (172.8µm) tended to be lower than that of the blastocysts from the Ca2x group (P=0.05). Regarding gene expression, only 1 of the 6 kodkod blastocysts expressed OCT4, and none expressed SOX2 and NANOG. On the other hand, the relative expression of OCT4 tended to decrease in blastocysts from the Ca1x and Ca2x groups compared with the IVF group (P=0.09), but no differences were found in the expression of SOX2 and NANOG among groups (P>0.05). In conclusion, after interspecies SCNT, domestic cat oocytes support the development of kodkod embryos until the morula stage. However, the embryo aggregation did not significantly improve the blastocyst rate and gene expression.

scholarly journals Developmental Competence of Domestic Cat Vitrified Oocytes in 3D Enriched Culture Conditions

Animals ◽  
2019 ◽  
Vol 9 (6) ◽  
pp. 329 ◽  
Author(s):  
Martina Colombo ◽  
Maria Giorgia Morselli ◽  
Mariana Riboli Tavares ◽  
Maricy Apparicio ◽  
Gaia Cecilia Luvoni
Keyword(s):  
Developmental Stages ◽  
Culture Conditions ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Control Group ◽  
Vitro Fertilization ◽  
In Vitro Embryo Production ◽  
Physical And Chemical

Cryoinjuries severely affect the competence of vitrified oocytes (VOs) to develop into embryos after warming. The use of culture conditions that provide physical and chemical support and resemble the in vivo microenvironment in which oocytes develop, such as 3D scaffolds and coculture systems, might be useful to improve VOs outcomes. In this study, an enriched culture system of 3D barium alginate microcapsules was employed for the in vitro embryo production of domestic cat VOs. Cryotop vitrified-warmed oocytes were in vitro matured for 24 h in the 3D system with or without fresh cumulus-oocyte complexes (COCs) in coculture, whereas a control group of VOs was cultured in traditional 2D microdrops of medium. After in vitro fertilization, presumptive embryos were cultured in 3D or 2D systems according to the maturation conditions. Vitrified oocytes were able to mature and develop into embryos in 3D microcapsules (17.42 ± 11.83%) as well as in 2D microdrops (14.96 ± 8.80%), but the coculture with companion COCs in 3D resulted in similar proportions of VOs embryo development (18.39 ± 16.67%; p = 1.00), although COCs presence allowed for blastocyst formation (0.95 ± 2.52%). In conclusion, embryos until late developmental stages were obtained from cat VOs, and 3D microcapsules were comparable to 2D microdrops, but improvements in post-warming conditions are still needed.

scholarly journals Overcoming poor in vitro nuclear maturation and developmental competence of domestic cat oocytes during the non-breeding season

Reproduction ◽  
2003 ◽  
pp. 809-816 ◽  
Author(s):  
P Comizzoli ◽  
DE Wildt ◽  
BS Pukazhenthi
Keyword(s):  
Ascorbic Acid ◽  
In Vitro Culture ◽  
Breeding Season ◽  
Oocyte Quality ◽  
Fertilization Success ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Ovarian Activity ◽  
Nuclear Maturation

The domestic cat experiences circannual variations in ovarian activity and intrafollicular oocyte quality. One result is poor nuclear and cytoplasmic maturation during in vitro maturation (IVM) conducted during the annual non-breeding season (July through November). In an attempt to overcome this seasonal phenomenon immature oocytes were collected from July through November and cultured in a conventional IVM medium (IVM1) or in IVM1 supplemented with different FSH concentrations and antioxidant (ascorbic acid or cysteine). Nuclear status of oocytes was assessed after IVM or IVF. Embryo stage and blastocyst quality were evaluated after 7 days of in vitro culture. Although the addition of antioxidant alone had no effect, the presence of 10 microg FSH ml(-1) improved nuclear maturation (75.4+/-4.1% versus 48.7+/-8.8% in IVM1; P<0.05) and fertilization success (47.9+/-3.2% versus 35.0+/-5.1% in IVM1; P<0.05). Furthermore, developmental competence of fertilized oocytes was enhanced (P<0.05) only in the presence of ascorbic acid (30.6+/-6.7%) or cysteine (33.6+/-5.1%) compared with IVM1 (8.1+/-8.8%). Consequently, blastocyst yield (17% of total oocytes cultured) was highest when oocytes were matured in medium containing higher FSH concentration and antioxidants. The results of this study demonstrate that meiotic and developmental competences are inherent to the immature cat oocyte collected during the non-breeding season. However, appropriate mechanisms (perhaps seasonal variation in FSH receptors or lack of antioxidant capacity of the cumulus-oocyte complex) are inadequate during this period of gonadal quiescence. Regardless, this compromised oocyte function during the non-breeding season can be overridden by altering in vitro culture conditions to include supplemental FSH and antioxidant.

scholarly journals 38BIRTH OF AFRICAN WILD CAT CLONED KITTENS

2004 ◽  
Vol 16 (2) ◽  
pp. 141 ◽  
Author(s):  
M.C. Gomez ◽  
C.E. Pope ◽  
A.M. Giraldo ◽  
L. Lyons ◽  
R.F. Harris ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Nuclear Transfer ◽  
Pcr Amplification ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Felis Silvestris ◽  
Cell Nuclei ◽  
Oocyte Aspiration

The African wild cat (AWC, Felis silvestris lybica; 2n=38) is one of the smallest wildcats, and it’s future is threatened by hybridization with domestic cats (Felis silvestris catus; 2n=38). Nuclear transfer (NT) is a potentially valuable tool for retaining genetic variability, and could assist in the continuation of species with few remaining individuals. Inter-species nuclear transfer into domestic cat (DSH) supports development of somatic cell nuclei from AWC (Gomez et al., 2003, Biol Reprod 69, 1032–1041). Therefore, the purpose of the present study was to evaluate the in vivo developmental competence of nuclear transfer embryos derived by fusion of African wildcat fibroblasts with domestic cat cytoplasts, after transfer into domestic cat recipients. In vivo- and in vitro-matured domestic cat oocytes were mechanically enucleated in modified Tyrodes salt solution supplemented with 20μgmL−1 of cytochalasin B (CCB) and 2mgmL−1 of sucrose, and reconstructed with AWC fibroblast cells derived from an adult male; cultured and passaged 1 to 3 times before serum-starved with DMEM +0.5% FBS and cultured for 5 additional days before use. Fusion took place in fusion medium (0.3M mannitol and 0.1mMMg+2), and membrane fusion was induced by applying a 3s AC pre-pulse of 20V, 1MHz; followed by two 30μs DC pulses of 240V/mm at intervals of 0.5s. Fused couplets were activated 2–3h after fusion by placing the couplets between two electrodes in a fusion chamber containing 3mL of fusion medium and exposing them to two 60μs DC pulses of 120V/mm. Then, couplets were incubated in 30μL drops of Tyrodes solution containing 1% MEM nonessential amino acids, 3mgmL−1 BSA (IVC-1 medium), and supplemented with 10μgmL−1 cycloheximide and 5μgmL−1 CCB at 38°C in 5% CO2 for 4h. After activation, cloned embryos were cultured in 500μL of IVC-1 medium until the day of the transfer. Derived AWC NT embryos were transferred into the oviducts (Day 1) or uteri (Days 5, 6, 7) of 36 gonadotrophin-treated DSH recipients on Day 1 after ovulation or on Days 5, 6, or 7 after oocyte aspiration, respectively. Pregnancy was assessed by ultrasonography on Days 21 to 23. One domestic cat was still pregnant and ongoing on Day 60. Kittens were delivered by Cesarean section in each of the seven pregnant recipients on days 61 to 67 of gestation. The kittens weighed an average of 86.2g (50.0 to 103g) and died within 36h after delivery. The post-mortem pathology reports revealed that most of them had an immature respiratory system. The clonal status of the kittens was assessed by multiplex PCR amplification of 20 microsatellite markers, including seven markers that are known to be on the X chromosome. Results from these assays confirmed that the AWC kittens had originated from the AWC donor somatic cell line and were not related to the DSH recipient cats. In summary, these results indicate that AWC cloned kittens can be produced by ET of embryos derived from AWC cells into DSH cytoplasts. Research was funded partially by the John &amp; Shirley Davies Foundation. Table 1

scholarly journals hCG Priming Before Ovary Collection Increasing The Oocyte Quality In The Domestic Cat

2021 ◽  
pp. 123-126
Author(s):  
Karisma Mardatillah ◽  
Rini Widyastuti ◽  
Diah Nugrahani Pristihadi ◽  
Wahyudin ◽  
Sigit Prastowo ◽  
...  
Keyword(s):  
Oocyte Quality ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Control Group ◽  
Oocyte Competence ◽  
Slicing Method ◽  
Determining Factor

Oocyte competence is a determining factor that influences the embryo development. Embryos produced in vitro have a reduced developmental competence than embryos produced in vivo. Therefore, human Chorionic Gonadotropin (hCG) injection was carried out to improve the quality of the oocytes. The objective of this study was to evaluate the effect of ovarian stimulation with hCG before ovary collection on oocyte quality in the domestic cat. Oocyte donors were either 1) treated with a single dose of 200 IU hCG four days before ovary collection (hCG group), or, 2) no treatment before ovary collection (control group). The oocytes were collected by the slicing method. Immature cumulus oophorus complexes (COCs) from both groups were pooled and matured in vitro for 24-26 hours. Then mature oocytes were fertilized with epididymal sperm and cultured in vitro for seven days. The results study showed that the number of the dominant follicle (DF) and the number of COCs in the hCG group was higher than the control group in right and left ovaries (p<0.05). The morulae and blastocyst rates from cleavage embryos were 88% and 75%, respectively. These results demonstrate that hCG priming of oocytes donors before ovary collection improve oocyte quality.

163 DOMESTIC CAT (FELIS CATUS) EMBRYO CRYOPRESERVATION: SLOW-COOLING VERSUS VITRIFICATION

2009 ◽  
Vol 21 (1) ◽  
pp. 180 ◽  
Author(s):  
M. J. Pedersen ◽  
C. A. Watson ◽  
B. A. Blevins ◽  
N. M. Loskutoff
Keyword(s):  
Domestic Cat ◽  
Developmental Competence ◽  
Embryo Cryopreservation ◽  
Control Group ◽  
Panthera Tigris ◽  
Slow Cooling ◽  
Vitro Fertilization ◽  
Chi Square Analysis ◽  
Felid Species

A variety of small domestic or nondomestic felid species have been produced from cryopreserved embryos using slow-cooling (controlled rate) cryopreservation methods (Pope et al. 2006 Theriogenology 66, 59–71; 1518–1524). However, in our laboratory these methods have not been as successful as vitrification in freezing in vitro-derived embryos of larger felid species such as tigers, Panthera tigris spp. (Crichton et al. 2000 Theriogenology 53, 238). The objective of this study was to examine the effects of slow-cooling cryopreservation v. vitrification methods on the developmental competence of in vitro-produced 2- to 4-celled domestic cat embryos as compared to non-frozen controls. Mature tomcat testicles and queen ovaries were collected from local veterinary clinics. Epididymal sperm were processed using one of three methods according to availability and time of use: 1) freshly washed if used the same day for IVF, 2) stored in cooled, non-electrolyte medium (Pope et al. 2000 ICAR Proc. 2, 191) if used within one week, or 3) frozen according to Tebet et al. (2006 Theriogenology 66, 1629–1632). Batches of oocytes were inseminated with the same pool and type of processed sperm per replicate. All in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC) procedures followed those previously reported (Pope et al. 2000 Theriogenology 53, 163–174). After 48 h in IVC, 2- to 4-celled embryos were equally divided into the three groups: 1) control, non-frozen; 2) slow-cooling cryopreservation using propanediol and sucrose; and 3) vitrification using DMSO, ethylene glycol and sucrose. The cryopreservation/thawing procedure followed that of Gomez et al. (2003) and the vitrification/warming procedure followed that of Vajta et al. (2000 Anim. Reprod. Sci. 60, 357–364). Results were recorded as the percentages of blastocyst formations at Day 8 of IVC and were compared between groups using chi-square analysis. In March 2000 to 2001, 806 feline oocytes were recovered from 101 ovaries and 286 blastocysts were obtained on Day 8 IVC in seven replicates. There were no significant differences in the numbers of degenerated embryos (2- to 8-celled) or morulae in all three groups (P > 0.05). There were more blastocysts that developed in the non-frozen control group (45/97; 46.4%), but this was not statistically different (P > 0.05) from the cryopreserved group (37/91; 40.7%). However, there were significantly more blastocysts (P < 0.01) produced in the non-frozen control group than the vitrified group (28/98, 28.6%). There were no differences between the cryopreserved and vitrified groups (P > 0.05). In conclusion, this study verifies previous reports that slow, or controlled-rate cryopreservation is effective for domestic cat embryos. Vitrification is less effective, as compared to non-frozen control embryos, but this may be improved with modified protocols.

81 Generation of presumptive domestic cat tetraploid embryos and its application for asynchronic complementation with diploid blastomeres

2019 ◽  
Vol 31 (1) ◽  
pp. 166
Author(s):  
M. D. Rodriguez ◽  
A. Gambini ◽  
A. Sestelo ◽  
O. Briski ◽  
R. Fernandez-Martin ◽  
...  
Keyword(s):  
Fusion Rate ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Electric Pulses ◽  
Tetraploid Complementation ◽  
Cell Embryo ◽  
Tetraploid Embryo ◽  
Electric Fusion

Tetraploid complementation has been extensively used to verify the pluripotency of stem cells and also for improving placenta formation when tetraploid embryos are aggregated synchronously or asynchronously with diploid (2n) embryos. Generation of tetraploid embryos can be achieved by the electric fusion of a 2-cell embryo. However, the optimal electric intensity pulse to generate tetraploid embryos has not been studied in the feline. The aims of this study were to (1) evaluate the optimal fusion conditions to achieve the highest fusion rate without affecting embryo developmental competence, (2) compare the in vitro development of synchronic and asynchronic aggregated domestic cat IVF embryos, and (3) assess pre-implantation development of embryos generated by asynchronic complementation of presumptive 1-cell tetraploid embryos with diploid blastomeres. Domestic cat cumulus-oocyte complexes were matured in vitro on 21% O2 in air at 38.5°C for 22h. The IVF embryos were generated by co-incubation of in vitro-matured oocytes with 2×106 motile spermatozoa mL−1 on 21% O2 in air at 38.5°C for 18 to 20h. After 24h of IVF, 2-cell embryos were selected. For Experiment 1, membrane fusion of 2-cell IVF embryos (n=164) was performed with two 30-ms DC pulses at different electric field (0.8, 2, 4, and 8 kV/cm) in fusion media (Mannitol, MgSO4, CaCl2, and polyvinyl alcohol). Presumptive fused embryos and nonfused were cultured in vitro in 50-µL drops of modified Tyrode’s medium on 6.5% CO2 in air at 38.5°C (Pope et al. 2006 Methods in Molecular Biology 254, 227-244). Cleavage was determined 24h after pulse. For Experiment 2, zona pellucida-free IVF embryos (n=110) were synchronically (two 4-cell embryos) or asynchronically (one 4-cell embryo and one 2-cell embryo) aggregated in 1 microwell. For Experiment 3, 1-cell presumptive tetraploid embryo (2-cell fused embryo) was asynchronically complemented with a 4-cell embryo (n=38). For all experiments, blastocyst stage was evaluated at Day 8, and embryos presenting more than one structure per microwell were considered non-aggregated. Data were analysed by Fisher’s exact test using GraphPad Prism 6.0 (GraphPad Inc., San Diego, CA, USA), and differences were considered significant at P&lt;0.05. The highest fusion rates (30 and 46%) with the best developmental competence (31 and 46%) were observed with 4 and 8 Kv/cm electric pulses, respectively. Electric fusion did not affect the embryo developmental competence. We observed that synchronic and asynchronic complementation reached similar blastocysts rates (54 and 65%, respectively), indicating that both techniques are suitable for tetraploid embryo complementation. Finally, when presumptive tetraploid embryos were asynchronically complemented with diploid blastomeres, the high blastocyst rate (90%) was obtained from embryos that form only one structure (aggregated embryos). Further experiments will be performed to track the distribution of cells using mitotrackers after complementation using tetraploid IVF and diploid somatic cell nuclear transfer embryos.

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