46 EFFECT OF MANIPULATION MEDIUM ON THE DEVELOPMENT OF RECONSTRUCTED DOMESTIC CAT EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 141
Author(s):  
S. Imsoonthornruksa ◽  
C. Lorthongpanich ◽  
K. Srirattana ◽  
N. Sripunya ◽  
C. Laowtammathron ◽  
...  
Keyword(s):  
Fusion Rate ◽  
Domestic Cat ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Parthenogenetic Activation ◽  
Significant Difference ◽  
Reproductive Techniques ◽  
Vitro Development ◽  
Felid Species

Domestic cat can serve as a valuable model for assisted reproductive techniques studies of endangered felid species. Therefore, this study was conducted to examine the effect of different manipulation medium on in vitro development of reconstructed domestic cat oocytes. The oocytes were recovered by slicing the ovaries of the cats that had been superstimulated with 200 IU eCG (Intervet, Boxmeer, The Netherlands). The procedures for SCNT were described previously (Lorthongpanich et al. 2004 Reprod. Fertil. Dev. 16, 149 abst). The manipulation medium for SCNT procedures was evaluated between HEPES-buffered TCM-199 (Sigma-Aldrich Corp., St Louis, MO, USA) + 10% FBS (199H) and Emcare embryo holding solution (ICPbio, Ltd., Auckland, New Zealand) (Emcare) during the denuding, enucleation, injection, activation, and holding steps. Parthenogenetic activation (PA) embryos were used as a control for both media. There was no significant difference in fusion rate when either 199H (63%) or Emcare (76%) was used. The cleavage, 8-cell, and morulae development rates of SCNT and PA were not significantly different when using either 199H or Emcare (Table 1). However, the blastocyst formation rates of SCNT and PA in Emcare (44% and 29%, respectively) were significantly greater than those of 199H (14% and 18%, respectively; P < 0.05). These results indicated that the manipulation medium is important for SCNT blastocyst development. Table 1.In vitro development of cloned domestic cat and parthenogenetic activation of embryos from different manipulation media

scholarly journals 160EFFECTS OF EXTRACELLULAR ION CONCENTRATIONS ON IN VITRO DEVELOPMENT OF DOMESTIC CAT IVF EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 202 ◽  
Author(s):  
W.F. Swanson ◽  
A.L. Manharth ◽  
J.B. Bond ◽  
H.L. Bateman ◽  
R.L. Krisher ◽  
...  
Keyword(s):  
Culture Media ◽  
Ionic Composition ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Vitro Development

Domestic cat embryos typically are cultured in media formulated for somatic cells or embryos from rodents or livestock species. Under these conditions, blastocyst development has been inconsistent and delayed relative to embryos grown in vivo, and embryo viability following transfer has been low. Our goal is to systematically define the culture requirements of the feline embryo to improve embryo development and viability. The objective of this study was to determine the ionic (NaCl, KCl, KH2PO4, and CaCl2:MgSO4) preferences of domestic cat IVF embryos. Anestral female cats were injected (i.m.) with 150IU eCG followed 84h later by 100IUhCG. Oocytes were recovered via laparoscopic follicular aspiration approximately 24h post-hCG injection (Day 0). Semen was collected from one of two males by means of an artificial vagina and washed once in HEPES-buffered IVF medium. Mature cumulus-oocyte complexes were co-incubated with 2.5–5×105 motile sperm mL−1 in IVF medium (100mM NaCl, 4.0mM KCl, 1.0mM KH2 PO4, 2.0mM CaCl2, 1.0mM MgSO4-7H2O, 25.0mM NaHCO3, 3.0mM glucose, 0.1mM pyruvate, 6.0mM L-lactate, 1.0mM glutamine, 0.1mM taurine, 1×MEM nonessential amino acids, 50μgmL−1 gentamicin, and 4.0mgmL−1 BSA) for 19 to 22h in 6% CO2 in air (38.7°C). Cumulus cells were removed and embryos cultured (8–11 embryos/50μL drop; 6% CO2, 5% O2, 89% N2, 38.7°C) in media containing 100.0 or 120.0mM NaCl, 4.0 or 8.0mM KCl, 0.25 or 1.0mM KH2PO4, and 1.0mM:2.0mM or 2.0mM:1.0mM CaCl2:MgSO4 (2×2×2×2 factorial design). The remaining components of the culture medium were identical to the IVF medium (but w/o gentamicin). Development to the blastocyst stage by Day 6, metabolism (glycolysis and pyruvate) of each blastocyst, and final cell number (Hoechst 33342 staining) of all embryos were evaluated. Final cell number of cleaved embryos and development to the blastocyst stage were analyzed using analysis of variance in the GLIMMIX macro of SAS. A total of 236 oocytes were inseminated, yielding 128 cleaved embryos (54%), including 6 blastocysts (4.7% of cleaved embryos). Cell number was not (P>0.05) affected by NaCl, KCl, or KH2PO4 concentrations, but tended (P=0.057) to be higher after culture in 2.0mM:1.0mM CaCl2:MgSO4. Treatments did not significantly affect (P>0.05) development to the blastocyst stage, but numerically more blastocysts were produced in 100.0mM NaCl (4/6), 8.0mM KCl (5/6), or 1.0mM KH2PO4 (5/6). Both CaCl2:MgSO4 ratios resulted in 3 blastocysts. Blastocysts contained 61.08±5.1 (mean±SEM, n=6) cells and actively metabolized glucose (glycolysis, 3.7±0.8pmol/embryo/3h or 0.06±0.01pmol/cell/3h) and pyruvate (0.75±0.27pmol/embryo/3h or 0.013±0.005pmol/cell/3h). These results suggest that the ionic composition of culture media influences the in vitro development of cat IVF embryos. (Supported by NIH grant RR15388.)

74 MECHANICAL DELIPIDATION IMPROVES CRYOSURVIVAL AND IN VITRO DEVELOPMENT OF VITRIFIED CAT OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 142
Author(s):  
J. Galiguis ◽  
M. C. Gómez ◽  
C. E. Pope ◽  
B. L. Dresser ◽  
S. P. Leibo
Keyword(s):  
Lipid Content ◽  
High Speed ◽  
Domestic Cat ◽  
Blastocyst Development ◽  
Chi Square ◽  
In Vitro Development ◽  
Intracellular Lipids ◽  
Fetal Fibroblasts ◽  
Vitro Development

Although considerable progress has been made in the development of successful methods for cryopreservation of embryos, oocytes are much less cryotolerant. There appears to be an inverse relationship between cryosurvival and intracellular lipid levels. For example, cat oocytes, which appear microscopically as coffee-coloured, nearly opaque spheres due to their high lipid content, are extremely sensitive to cryopreservation. Oocyte delipidation thus represents a potential approach to improving cryosurvival. The objectives of the present study were to examine 1) the effects of calcium (Ca2+, 0 v. 10 nM), FBS (0 v. 10%), and cytochalasin B (CB, 7.5 v. 20.0 μg mL–1) during mechanical delipidation by high-speed centrifugation on in vitro development of IVM cat oocytes, and 2) the influence of centrifugation, degree of lipid polarization (partial v. full), and co-culture with cat fetal fibroblasts (CFF) on in vitro development of vitrified IVM cat oocytes. In Experiment 1, oocytes were randomly allocated to each centrifugation medium and centrifuged at 12 000 × g for 20 min. Oocytes were then fertilized with epididymal sperm (motile sperm mL–1) and cultured until Day 8 (Pope et al. 2006 Theriogenology 66, 59–71). In Experiment 2, oocytes were centrifuged with the optimal centrifugation medium obtained in experiment 1, allocated to each treatment and vitrified in a solution of 15% DMSO, 15% ethylene glycol, and 18% sucrose (2008 Reprod. Fertil. Dev. 20, 188). Liquified oocytes were fertilized and cultured until Day 8. In both experiments, cleavage and degeneration rates were determined on Day 2 and blastocyst development on Day 8. Data were analysed by 2-way ANOVA and chi-square tests. In Experiment 1, of 939 oocytes that were centrifuged and fertilized, 40% of those treated in 0 nM Ca2+ cleaved and 22% developed into blastocysts, v. 33 and 6%, respectively, in 10 nM Ca2+ (P < 0.05). The respective cleavage and degeneration frequencies for oocytes treated in 10% FBS were 43 and 19% v. 19 and 3% in 0% FBS (P < 0.05). Cleavage and blastocyst development after treatment with 7.5 and 20.0 μg mL–1 CB were 36 and 15% v. 42 and 22%, respectively. In Experiment 2, 493 oocytes were vitrified/liquified and fertilized. The degeneration, cleavage, and blastocyst rates of non-centrifuged oocytes were 49, 21, and 0% v. 31 (P < 0.05), 38 (P < 0.05), and 7%, respectively, of centrifuged oocytes. Of centrifuged oocytes with partially extruded lipids, 34% degenerated, 34% cleaved, and 4% developed into blastocysts v. 29, 42, and 10%, respectively, of oocytes with fully extruded lipids. Degeneration, cleavage and blastocyst rates of co-cultured v. control oocytes were 18, 36, and 10%, v. 26 (P < 0.05), 34, and 3%, respectively. In summary, cryotolerance of domestic cat oocytes to vitrification was 1) affected by their lipid content, and 2) improved by mechanical reduction of intracellular lipids. When oocytes were fully delipidated in Ca2+-free medium containing 10% FBS and 20.0 μg mL–1 CB before vitrification and co-cultured after IVF with CFF, blastocyst development was similar to that of control, non-vitrified oocytes.

75 EPIGENETIC EFFECTS OF VITRIFICATION ON PRONUCLEAR DOMESTIC CAT EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 131 ◽  
Author(s):  
J. H. Galiguis ◽  
C. E. Pope ◽  
M. N. Biancardi ◽  
C. Dumas ◽  
G. Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Genetic Material ◽  
Domestic Cat ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Epigenetic Effects ◽  
Donor Cells ◽  
Vitro Development

Vitrification remains a promising technique in the preservation of valuable genetic material; however, in the cat, success has varied. Live kittens have been produced from embryos vitrified at early cleavage stages, but phenotypic abnormalities in some kittens suggest possible epigenetic effects of the vitrification process. It has been reported that cryopreservation alters epigenetic events in somatic donor cells, which indirectly influences physical status of cloned offspring. However, extending post-warming in vitro culture of donor cells corrects these epigenetic modifications, resulting in normal embryos/clones. Accordingly, in the present study, vitrification was performed at the pronuclear stage to lengthen pretransfer culture time, and vitrified cat zygotes were assessed by analysing (1) histone acetylation/methylation, (2) global DNA methylation, (3) pluripotent gene expression, (4) in vitro development, and () in vivo viability. In vivo matured/IVF oocytes were vitrified in 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose at 16 h post-insemination (PI). After warming in 1.0 M sucrose at 38°C, embryos were fixed at 18 h or 40 h PI, and the nuclear intensity of either acetyl/dimethyl-H3K9 or 5-methylcytosine was determined by immunofluorescence. Results showed that at 18 h PI, mean H3K9ac intensity of vitrified embryos (11.8; n = 6) was higher than that of corresponding nonvitrified (fresh) controls (4.5; n = 6) and the fresh (3.2; n = 11) and vitrified (0.6; n = 7) 40-h groups (2-way ANOVA; P < 0.05). H3K9me2 in the fresh (36.9) and vitrified (32.5) 18-h embryos was similar but increased relative to both fresh (10.7) and vitrified (9.2) 40-h groups (P < 0.05). Mean DNA methylation (5MeC) in the fresh (31.6; n = 1) and vitrified (24.7; n = 3) 18-h groups was similar to that of the fresh 40-h group (19.8; n = 4) but higher than that of the vitrified 40-h group (15.0; n = 5; P < 0.05). To assess expression of POU5F1 and Nanog, qRT-PCR was performed on Day 8 blastocysts. Relative to controls (n = 9), mean POU5F1 and Nanog levels in vitrified blastocysts (n = 24) were 1.38- and 1.98-fold higher, respectively (one-way ANOVA; P > 0.05). In terms of in vitro development, Day 2 cleavage of vitrified zygotes (59%; n = 508) was similar to that of controls (66%; n = 340), but Day 8 blastocyst formation was reduced (9 v. 31%; t-test; P < 0.05). In vivo viability was assessed by oviducal transfer of 41 Day 1 embryos into 2 recipients. One pregnancy was established (50%), with 3 live kittens weighing 70, 79, and 131 g delivered without assistance on Day 65 of gestation. The 2 smaller kittens died within a few hours of birth, with the smallest exhibiting an umbilical hernia and organ exteriorization. The third kitten developed into a normal, healthy adult. In summary, mean H3K9me2, 5MeC, and POU5F1/Nanog expression of vitrified zygotes was similar to corresponding controls. H3K9ac increased at 18h PI as a result of vitrification, but was reduced after culture to 40 h PI. Although vitrified zygotes cleaved in vitro at rates similar to controls, blastocyst development was reduced. In vivo viability was demonstrated; however, postnatal survival of kittens produced was low.

scholarly journals 64COMPARISON OF DEVELOPMENTAL POTENTIAL OF IN VIVO AND IN VITRO RECIPIENT OOCYTES AFTER NUCLEAR TRANSFER IN GOAT

2004 ◽  
Vol 16 (2) ◽  
pp. 154
Author(s):  
H.S. Park ◽  
M.Y. Lee ◽  
S.P. Hong ◽  
J.I. Jin ◽  
J.K. Park ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Serum Starvation ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

Recent techniques in somatic cell nuclear transfer (SCNT) have been widely used for animal research. In addition, SCNT techniques may allow for the rescue of endangered species. Despite efforts for wildlife preservation, however, some threatened or endangered wild animal species will likely become extinct. As a preliminary experiment of a series in wildlife research, we tried to identify an improved method for the production of more transferable NT embryos in goats. Mature donor animals of Korean native goats (20–25kg) were synchronized with a CIDR (type G; InterAg, New Zealand) vaginal implant for 10 days followed by a total of 8 twice daily injections of 70mg of FSH (Folltropine, London, Ontario, Canada) and 400IU of hCG (Chorulon, Intervet, Moxmeer, The Netherlands). Oocytes were then collected surgically by retograde oviduct flush or direct aspiration from ovarian follicles in vivo at 29–34h after hCG. Oocytes collected from follicles were matured in TCM-199 containing 10% FBS and hormones. Prepared ear skin cells from the goat were cultured in TCM-199 containing 10% FBS at 39°C, 5% CO2 in air, and confluent monolayers were obtained. Oocytes were enucleated and donor cells from serum starvation (0.5%) culture were fused through a single electric pulse (DC 2.36kvcm−1, 17μs), and then activated by a single electric pulse (AC 5vmm−1, 5s+DC 1.56kvcm−1, 30μs) or chemical treatment (5μgmL−1 ionomycin 5min−1, 1.9mM 6-DMAP/4h). Reconstructed oocytes were cultured in M16 medium with 10% goat serum (GS) for 6–7 days. Data were analyzed by chi-square test. In in vitro development, significantly (P&lt;0.05) more oocytes were cleaved (24/30, 80.0%) and developed (7/24, 29.2%) to morula or blastocyst stage, respectively, in NT oocytes activated by Iono + DMAP compared to electric stimulated oocytes (2/21, 40.0%; 0/2, 0%). There was a significant difference in in vitro development of NT embryos by the method of oocyte collection. Cleavage rate was higher (P&lt;0.05) in NT embryos from in vivo oocytes (23/28, 82.1%) than in in vitro matured oocytes (19/35, 54.3%), and further development to morula or blastocyst was also significantly (P&lt;0.05%) higher in NT embryos from in vivo oocytes (7/23, 30.4%) than in NT embryos from in vitro matured oocytes (0/19, 0%). When we compared NT embryos to parthenotes, developmental rate was not significantly different between NT embryos and parthenotes. These results strongly suggest that the in vivo oocytes will have superior developmental potential to oocytes matured in vitro. Table 1 Effect of different oocyte source on in vitro development following caprine SCNT

146 DETRIMENTAL EFFECTS OF PROSTAGLANDIN F2α ON IN VITRO EMBRYO DEVELOPMENT IN BOVINE ARE INHIBITED BY A RECEPTOR ANTAGONIST

2008 ◽  
Vol 20 (1) ◽  
pp. 153 ◽  
Author(s):  
F. N. Scenna ◽  
J. L. Edwards ◽  
F. N. Schrick
Keyword(s):  
Prostaglandin F2α ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Negative Effects ◽  
In Vitro Development ◽  
Ann Arbor ◽  
Reproductive Techniques ◽  
Vitro Development ◽  
Cayman Chemical

Numerous studies have demonstrated negative effects of prostaglandin F2α (PGF2α) on bovine reproduction. Discovery of a PGF2α receptor (FPr) in bovine embryos (Scenna et al. 2006 Reprod. Fertil. Dev. 18, 180) allows for development of new therapeutic strategies to improve success of embryo transfer. Therefore, two experiments were performed to investigate the occurrence of any toxic effect of AL-8810 (Cayman Chemical Inc., Ann Arbor, MI, USA), an FPr antagonist, on in vitro development of bovine embryos. In Exp. 1, pre-compacted embryos were cultured in (1) 100 AL (100 nm AL-8810 in potassium simplex optimized medium with polyvinyl alcohol (KSOM-PVA); n = 94); (2) 50 AL (50 nm AL-8810 in KSOM-PVA; n = 94); (3) 25 AL (25 nm AL-8810 in KSOM-PVA; n = 94); and (4) CON (control: KSOM-PVA; n = 95). In Exp. 2, pre-compacted embryos were cultured in (1) 1000 AL (1000 nm AL-8810 in KSOM-PVA; n = 282); (2) 500 AL (500 nm AL-8810 in KSOM-PVA; n = 274); (3) 250 AL (250 nm AL-8810 in KSOM-PVA; n = 274); and (4) CON (control: KSOM-PVA; n = 278). Embryos remained in treatments until blastocyst assessment. Next, two experiments were performed to determine the efficiency of AL-8810 on preventing detrimental effects of PGF2α on pre-compacted embryos. In Exp. 3, pre-compacted embryos were cultured in (1) 100 AL (100 nm AL-8810 in KSOM-PVA; n = 121); (2) 10 PGF (10 ng mL–1 of PGF2α (Cayman Chemical Inc.) in KSOM-PVA; n = 91); (3) AL100+PGF (100 nm AL-8810 and 10 ng mL–1 of PGF2� in KSOM-PVA; n = 116); (4) CON (control: KSOM-PVA; n = 96). In Exp. 4, embryos were cultured in (1) 1000 AL (1000 nm AL-8810 in KSOM-PVA; n = 87); (2) 10 PGF (10 ng mL–1 of PGF2α in KSOM-PVA; n = 87); (3) AL1000+PGF (1000 nm AL-8810 and 10 ng mL–1 of PGF2α in KSOM-PVA; n = 84); (4) CON (control: KSOM-PVA; n = 84). In Exp. 3 and 4, embryos remained in treatments for 48 h when development to morula was assessed. Data for all experiments were analyzed using the GLIMMIX procedure of SAS (SAS Institute, Inc., Cary, NC, USA). For Exp. 1, results indicated that addition of 100, 50, and 25 nm did not compromise embryonic development to the blastocyst stage compared to controls (60.2%, 55.8%, 55.4%, and 49.9%, respectively). In addition, orthogonal contrasts indicated that 100 nm AL-8810 improved development to the blastocyst stage (100 AL = 61% v. CON = 50.6%, P = 0.01). Similarly for Exp. 2, 1000, 500, and 250 nm AL-8810 did not affect in vitro development to the blastocyst stage compared to controls (40%, 39%, 34.8%, and 37.7%, respectively). In Exp. 3 and 4, addition of 1000 nm AL-8810, but not 100 nm, to culture medium of pre-compacted embryos exposed to PGF2α increased the ability of embryos to undergo compaction 48 h later (1000 AL+PGF = 51% v. PGF = 40%; P = 0.05). In conclusion, AL-8810 at a concentration of 1000 nm inhibits detrimental effects of PGF2α on the development of pre-compacted bovine embryos and may prove beneficial for other assisted reproductive techniques in cattle. Funding was provided by Ultimate Genetics and the Tennessee Agricultural Experiment Station for completion of these studies.

83 IN LOW OXYGEN CULTURE, IS HYPOTAURINE NECESSARY FOR IN VITRO DEVELOPMENT OF PORCINE EMBRYOS?

2016 ◽  
Vol 28 (2) ◽  
pp. 171
Author(s):  
J. A. Benne ◽  
L. D. Spate ◽  
B. M. Elliott ◽  
R. S. Prather
Keyword(s):  
Embryo Culture ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Free Radical Scavengers ◽  
Total Cell Number ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

For decades it has been known that reactive oxidative species (ROS) form during in vitro embryo culture. A buildup of ROS can be detrimental to individual cells in the embryo and lead to a decrease in development and quality. To overcome oxidative stress in culture systems, additives, such as taurine and/or hypotaurine, have been used. In the pig, taurine or hypotaurine addition is deemed necessary for normal in vitro development. Another commonly used technique to reduce ROS is to culture embryos in a lowered oxygen environment (e.g. 5%). Porcine zygote medium 3 (PZM3) base culture medium is used in the following experiments and contains 5 mM hypotaurine, which is one of the most costly additives in the medium. The objective of this experiment was to determine if hypotaurine is still necessary if the embryos were cultured in 5% O2 from the zygote to the Day 6 blastocyst stage. In Experiment 1, oocytes were matured for 44 h and fertilized in vitro. After fertilization, presumptive zygotes were then transferred to 500 µL of MU-1 medium (PZM3 with 1.69 mM arginine) that either contained or did not contain hypotaurine for overnight culture at 20% O2. On Day 1, the same embryo culture plates were moved to 5% O2, 5% CO2, and 90% N2 and cultured to Day 6. The percent blastocyst stage was determined, and total cell number was counted in 3 of the 5 replicates in order to give us an indication of the embryo quality. The percent blastocyst in the controls (+hypotaurine) was 34.4% ± 2.8 and not different from the no hypotaurine (32.9% ± 2.2; N = 830; 5 replications; P > 0.10). Furthermore, total cell number was not different between the two groups (30.8 ± 1.5 v. 33.6 ± 1.8, respectively, N = 146; 3 replications; P > 0.10). In Experiment 2, the same experiment was repeated in somatic cell nuclear transfer derived embryos, which may be more sensitive to ROS due to the micromanipulation procedure. Wild type fetal fibroblast cells were used as donor cells. There was no significant difference in development to the blastocyst stage due to the presence or absence of hypotaurine (17.7% ± 2.5 v. 11.8% ± 2.3, respectively; N = 454; 4 replications; P = 0.07). All blastocyst data were analysed using the GENMOD procedure in SAS 9.4 (SAS Institute Inc., Cary, NC, USA), and cell number data were analysed using the PROC GLM also with SAS 9.4. These data show that porcine embryos can be efficiently cultured to the blastocyst stage without adding any oxygen free radical scavengers to the media when culturing in reduced oxygen atmosphere. Further studies include evaluating term development via embryo transfers and measuring ROS production of these embryos. Funding was provided by Food for the 21st Century and the National Institutes of Health (U42 OD011140).

Effects of electric field strengths on fusion and in vitro development of domestic cat embryos derived by somatic cell nuclear transfer

2006 ◽  
Vol 66 (5) ◽  
pp. 1237-1242 ◽  
Author(s):  
Ni Wayan Kurniani Karja ◽  
Takeshige Otoi ◽  
Pimprapar Wongsrikeao ◽  
Ryohei Shimizu ◽  
Masako Murakami ◽  
...  
Keyword(s):  
Electric Field ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Domestic Cat ◽  
In Vitro Development ◽  
Vitro Development ◽  
Field Strengths
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