Oocytes vitribanking in the domestic cat model: Developmental competence in 3d culture

The leopard cat (Prionailurus bengalensis), a member of the felidae family, is currently listed as threatened by the Ministry of Environment in South Korea. In exotic or endangered species, the lack of oocytes and recipients precludes the use of traditional somatic cell nuclear transfer (NT), and an approach such as intragenus NT may be the only alternative for producing embryos and offspring. In the present study, we used the leopard cat (LC) as a somatic cell donor to evaluate the in vivo developmental competence, after transfer into domestic cat recipients, of cloned embryos produced by the fusion of LC fibroblast cell nuclei with domestic cat cytoplasts. A total of 412 enucleated domestic cat oocytes were reconstructed with either male (Treatment A) or female (Treatment B) adult LC fibroblasts. There was no significant difference in fusion rate (60.4 vs. 56.9%) between Treatment A and B. Of the fused couplets, the cleavage and blastocyst developmental rate in Treatment A were greater than those in Treatment B (69.5 vs. 60.9%; 8.3 vs. 7.8%; P < 0.05). In treatment A, in vivo developmental studies at 30-45 days postimplantation demonstrated 4.8% (21/435) of reconstructed embryos (n = 435) had entered into the uterine lining of recipients, but only 1.4% (6/435) formed fetuses. However, all of the reconstructed embryos failed to develop to term (65 days). Microsatellite analyses confirmed that the nuclear genome of the cloned fetuses were LC in origin.

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56 EFFECTS OF PHYLOGENIC GENERA OF RECIPIENT CYTOPLASTS ON DEVELOPMENT AND VIABILITY OF CANADA LYNX (LYNX CANADENSIS) CLONED EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab56 ◽

2010 ◽

Vol 22 (1) ◽

pp. 186 ◽

Cited By ~ 1

Author(s):

M. C. Gómez ◽

J. I. Lyons ◽

C. E. Pope ◽

M. Biancardi ◽

C. Dumas ◽

...

Keyword(s):

Somatic Cell ◽

Donor Cell ◽

Domestic Cat ◽

Developmental Competence ◽

Lynx Lynx ◽

Phylogenetic Distance ◽

Lynx Canadensis ◽

Gestational Length ◽

Canada Lynx ◽

Oocyte Donor

Canada lynx (Lynx canadensis; CL) once occupied 16 states in the Unites States of America, but small populations remain in only 3 states. Interspecies-somatic cell nuclear transfer (Is-SCNT) offers the possibility of preventing their extinction; however, developmental constraints on Is-SCNT embryos are proportional to the phylogenetic distance between the donor cell and the recipient oocyte. Mitochondrial DNA (mtDNA) heteroplasmy may be involved in nuclear-cytoplasmic incompatibilities, thus inhibiting development of cloned embryos at the time of genomic activation. Minimizing the phylogenetic distance between the donor cell and recipient oocyte may enhance development of clone embryos. Caracal (Caracal caracal) may be suitable as an oocyte donor for SCNT and a recipient of CL cloned embryos because caracals hybridize with other felid species and share physical characteristics with the lynx family, marked by being previously classified in the lynx genera and having similar gestational length. To ensure compatibilities between the donor nuclei of the CL and the mitochondria of recipient oocytes, we (1) compared in vitro development of CL cloned embryos reconstructed with domestic cat (Felis catus; DSH) or caracal cytoplasts, (2) examined the mtDNA genotypes in CL cloned embryos, and (3) evaluated in vivo developmental competence of CL cloned embryos after transfer into caracal recipients. A total of 160 and 217 preovulatory oocytes were collected by laparoscopy from gonadotropin-treated caracals (n = 8) and DSH (n = 10) and used as recipient cytoplasts for reconstructing CL embryos. Results indicated that the phylogenetic genera of recipient cytoplasts did not affect embryo cleavage at Day 2 (caracal 50/55, 91% v. DSH 63/65, 97%), but development of CL cloned embryos to the blastocyst stage was higher when caracal oocytes were used as recipient cytoplasts (15/50; 30%) than with DSH cytoplasts (9/63, 14%; P < 0.05). The extent of mtDNA homoplasmy or heteroplasmy in CL cloned embryos was calculated by the number of single nucleotide polymorphisms (SNP) derived from the DSH or caracal oocyte donors and from the somatic cell donor CL. DNA was isolated from 25 and 35 CL cloned embryos reconstructed with caracal or DSH cytoplasts, respectively. All amplified products after PCR were sequenced and SNP analyzed. All CL embryos reconstructed with DSH cytoplasts were homoplasmic, carrying mtDNA only from the DSH oocyte donor (n = 35; SNP DSH = 2-6). Embryos reconstructed with caracal cytoplasts were homoplasmic for CL mtDNA (n = 9; SNPCL = 10-12) or heteroplasmic (caracal × CL, n = 17; SNPCL = 7-9; SNP caracal = 2-3). A total of 69 (mean = 34.5 ± 4.9 per caracal) and 70 (mean = 35.0 ± 9.8 per caracal) CL cloned embryos reconstructed with caracal and DSH cytoplasts, respectively, were transferred into 4 caracal recipients; however, no pregnancies were established. In summary, Is-SCNT between 2 phylogenetically closer species favors retention of the donor’s mitochondria, which might lead to a better nucleo-cytoplasmic interaction for reprogramming of donor nucleus.

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

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab17 ◽

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.

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78 Radio electric asymmetric conveyer treatment during prolonged cold storage of ovaries enhances developmental competence of oocytes in the domestic cat model

Reproduction Fertility and Development ◽

10.1071/rdv32n2ab78 ◽

2020 ◽

Vol 32 (2) ◽

pp. 165

Author(s):

F. Ariu ◽

E. Sanna Passino ◽

A. Piras ◽

V. Melosu ◽

M. Maioli ◽

...

Keyword(s):

Fertility Preservation ◽

Cold Storage ◽

Ovarian Tissue ◽

Morphological Characteristics ◽

Domestic Cat ◽

Developmental Competence ◽

Cleavage Rate ◽

Long Distance ◽

Functional Changes ◽

Phosphate Buffered Saline

Hypothermic storage (4°C) of ovaries for long-distance transport holds great potential to expand access to fertility preservation in animals and humans (Duncan et al. 2016 Reproduction 152, 201-210). However, storage for prolonged periods (over 24h) leads to structural/functional changes in the ovarian tissue with a critical reduction of follicular viability and oocyte quality (Piras et al. 2018 Reprod. Biol. Endocrinol. 10, 16-76; Isachenko et al. 2009 Fertil. Steril. 91, 1556-1559). The radio electric asymmetric conveyer (REAC) is a novel technology platform for neuro and bio-modulation that optimize the ion fluxes and the mechanisms driving cellular asymmetry and polarization in biological structures (Maioli et al. 2016 Sci. Rep. 6, 28682). The REAC has already proved to be effective in promoting cell differentiation and reprogramming and to counteract the biological mechanisms linked to aging and the degenerative process (Berlinguer et al. 2017 Reprod. Biol. Endocrinol. 15, 11; Maioli et al. 2014 Age 36, 9-20). In the present study, the domestic cat was used as an experimental model to evaluate the effect of REAC treatment during ovary storage at 4°C for 48h on invitro developmental ability of oocytes retrieved from antral follicles. Ovaries harvested from healthy domestic queens during ovariectomy were randomly assigned to the REAC-treated (R: n=13) and untreated (C: n=13) groups. In detail, ovaries were maintained in 4mL of phosphate-buffered saline at 4°C for 48h. The REAC device was set at 2.4 GHz, and its conveyer electrodes were immersed into the phosphate-buffered saline. After 48h, ovaries were sliced to release cumulus-oocyte complexes, which were selected according to their morphological characteristics (Johnston et al. 1991 Biol. Reprod. 45, 898-906) for IVM (R: n=130; C: n=133). Matured oocytes were fertilised (IVF) with frozen-thawed epididymal spermatozoa and presumptive zygote were invitro cultured (IVC) for 7 days. On Day 2 and Day 7 of IVC, respectively, the number of embryos cleaved and developing to the blastocyst stage was determined. The IVM, IVF, and IVC were performed according to the procedure of Piras et al. (2018 Reprod. Biol. End. 16, 76). Data were analysed by chi-square test with STATA\IC 11.0. Maturation rate of oocytes did not differ between groups (R: n=59/130, 45.4%; C: n=66/133, 49.6%). Cleavage rate was higher (P<0.05) in the R group (n=34/59, 57.6%) compared with the C group (n=25/66, 37.9%). The percentages of blastocyst formation relative to the number of cleaved embryos (R: n=12/34, 35.3%; C: n=3/25 12.0%) and to the total number of MII oocytes (R: n=12/59, 20.4%; C: n=3/66, 4.5%) increased (P<0.05) after REAC treatment compared with the untreated counterpart. In conclusion, REAC treatment during cold storage of cat ovaries for 48h positively affected the quality of oocytes as assessed by invitro embryo production outcome. The REAC technology could provide a useful approach for the optimization of ovarian tissue transport conditions for fertility preservation especially for endangered species and patients with fertility-threatening conditions.

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Proteomic analysis of germinal vesicles in the domestic cat model reveals candidate nuclear proteins involved in oocyte competence acquisition

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gax059 ◽

2017 ◽

Vol 24 (1) ◽

pp. 14-26 ◽

Cited By ~ 7

Author(s):

P -C Lee ◽

D E Wildt ◽

P Comizzoli

Keyword(s):

Proteomic Analysis ◽

Domestic Cat ◽

Nuclear Proteins ◽

Oocyte Competence ◽

Germinal Vesicles ◽

Competence Acquisition ◽

Cat Model

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24 Asynchronic tetraploid complementation and embryo quality in domestic cat and Leopardus geoffroyi hybrid embryos

Reproduction Fertility and Development ◽

10.1071/rdv32n2ab24 ◽

2020 ◽

Vol 32 (2) ◽

pp. 138

Author(s):

M. Duque Rodriguez ◽

A. Gambini ◽

C. Gutnisky ◽

L. Ratner ◽

S. Rulli ◽

...

Keyword(s):

Cell Number ◽

Felis Catus ◽

Domestic Cat ◽

Developmental Competence ◽

Terminal Deoxynucleotidyl Transferase ◽

Control Group ◽

Exact Test ◽

Domestic Species ◽

Healthy Pregnancy ◽

Tetraploid Complementation

Fusion of 2-cell embryos generates tetraploid (4n) blastomeres with an increased commitment to trophectoderm. Complementation of embryos from endangered species with 4n blastomeres derived from a phylogenetically related domestic species could improve healthy pregnancy establishment after embryo transfer in domestic females. However, generation, development, and quality of tetraploid complemented embryos in felids remain unstudied. Therefore, our objectives were (1) to evaluate tetraploidy of 2-cell fused embryos; (2) to analyse the blastocyst cell number, distribution after synchronic (S) or asynchronic (AS) complementation, OCT4+ cells, DNA-fragmentation levels and CDX2 gene expression of IVF complemented embryos; and (3) to evaluate the developmental rates of tetraploid complemented Felis catus-Leopardus geoffroyi hybrid embryos. After ovariectomy, Felis catus oocytes were IVM and subjected to IVF. For Experiment 1 (n=66), 2-cell embryos (2n) were exposed to two 30-ms DC pulses at 8 kV cm−1 electric field in fusion media. Fused (4n) and nonfused embryos were cultured invitro in 50-μL drops of modified Tyrode's medium. Karyotype analysis was performed at Day 4. For Experiment 2 (n=24), zona-free IVF embryos were aggregated S (4-cell-2n/4-cell-2n) or AS (4-cell-2n/2-cell-2n and 4-cell-2n/1-cell-4n). For Experiment 3 (n=36), blastocysts generated by AS complementation (4-cell-2n/2-cell-2n and 4-cell-2n/1-cell-4n) were either fixed with 4% paraformaldehyde for immunofluorescence and terminal deoxynucleotidyl transferase dUTP nick end labeling assay or saved in RNA-Later for RT-qPCR analysis. For this experiment, nonaggregated 2n and 4n blastocysts were used as a control. For Experiment 4 (n=60), IVM oocytes were co-incubated with Leopardus geoffroyi and Felis catus (control) spermatozoa and then 4-cell-2n heterologous embryos were complemented with 1-cell-4n homologous IVF embryos. Data were analysed by Fisher's exact test. Our results showed that 67% of the 2-cell fused embryos were 4n. Moreover, 82% of nonfused embryos were aneuploids compared with 78% of 2n embryos in the control group. The AS complemented blastocysts (4-cell-2n/1-cell-4n and 4-cell-2n/2-cell-2n) had significantly higher cell number compared with S complemented (4-cell-2n/4-cell-2n) or noncomplemented embryos. The AS complementation also increased the number of OCT4+ cells independently of the ploidy of the embryos. Interestingly, AS tetraploid complemented embryos had significantly lower number of cells with fragmented DNA. No differences were found in CDX2 expression among complemented embryos; however, noncomplemented 2n blastocysts showed a significantly lower expression compared with the others group. Finally, we observed that AS complementation of 2n hybrid embryos with 4n homologous embryos reached similar blastocyst rates, 70 and 88%, respectively. Our findings support the use of 2-cell fused embryos to generate 4n blastomeres and demonstrated that tetraploid complementation generates good quality embryos, providing evidences that encourage the use of this technology to improve the developmental competence of interspecific embryos after transfer.

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Domestic Cat Model for Predicting Human Nucleoside Analogue Pharmacokinetics in Blood and Seminal Plasma

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.7.2173-2176.2001 ◽

2001 ◽

Vol 45 (7) ◽

pp. 2173-2176 ◽

Cited By ~ 4

Author(s):

Holly L. Jordan ◽

Arlene S. Pereira ◽

Myron S. Cohen ◽

Angela D. M. Kashuba

Keyword(s):

Seminal Plasma ◽

Nucleoside Analogue ◽

Domestic Cat ◽

Pharmacokinetic Parameters ◽

Time Curve ◽

Concentration Time ◽

Feline Model ◽

Cat Model ◽

Male Genital

ABSTRACT To establish whether a feline model can predict nucleoside analogue behavior in human semen, zidovudine (ZDV) and lamivudine (3TC) pharmacokinetic parameters (PKs) were determined in the blood and seminal plasma of healthy cats. Our results show considerable similarity in ZDV and 3TC PKs between cats and humans. As in humans, ZDV and 3TC tend to accumulate in feline seminal plasma. Area under the blood plasma concentration-time curve was predictive of seminal plasma excretion. The felid model offers a unique in vivo experimental alternative for investigating the pharmacokinetics of nucleoside analogues in the male genital tract.

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