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

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.

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Effects of gonadotrophins, growth hormone and prolactin on developmental competence of domestic cat oocytes matured in vitro

Reproduction Fertility and Development ◽

10.1071/rd9951061 ◽

1995 ◽

Vol 7 (5) ◽

pp. 1061 ◽

Cited By ~ 23

Author(s):

RD Schramm ◽

BD Bavister

Keyword(s):

Growth Hormone ◽

Calf Serum ◽

Cell Number ◽

Blastocyst Stage ◽

Domestic Cat ◽

Developmental Competence ◽

Developmental Potential ◽

Nuclear Maturation ◽

Morula Stage

Specific aims were to (1) examine the developmental capacity of felid oocytes matured in vitro and (2) determine the effects of gonadotrophins, growth hormone and prolactin on nuclear and cytoplasmic maturation oocytes in vitro. Oocytes were obtained from excised ovaries of 21 cats, and were matured for 45-46 h in modified CMRL-1066 culture medium (1 mM glutamine, 1 mM pyruvate and 20% bovine calf serum), with one of the following: (1) gonadotrophins (1.0 micrograms mL-1 hFSH+10 micrograms mL-1 hLH), (2) gonadotrophins+10 micrograms mL-1 growth hormone, (3) gonadotrophins+10 micrograms mL-1 prolactin, or (4) no hormones. Oocytes were inseminated with ejaculated cat sperm capacitated in TALP medium. Embryos were cultured in modified CMRL-1066 medium until developmental arrest, then stained with Hoechst 33342 to assess nuclear status or cell number. Gonadotrophins enhanced (P < or = 0.05) the incidence of nuclear maturation, but neither gonadotrophins, growth hormone nor prolactin improved fertilization or developmental potential of oocytes matured in vitro. Mean percentages of mature oocytes that were fertilized and cleaved to or beyond the 2, 4, 8 and 16-cell stages were 80, 77, 66, 42 and 24%, respectively. Three embryos progressed to 40-60 cells, but none developed a blastocoel. Thus, although gonadotrophins enhance nuclear maturation of oocytes in vitro, and mature oocytes are capable of fertilization and development to the morula stage, culture with growth hormone, prolactin or gonadotrophins during maturation in vitro does not enhance developmental competence or overcome the morula-to-blastocyst-stage block in development of domestic-cat oocytes matured in vitro.

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Oocyte Competence Biomarkers Associated With Oocyte Maturation: A Review

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.710292 ◽

2021 ◽

Vol 9 ◽

Author(s):

Batara Sirait ◽

Budi Wiweko ◽

Ahmad Aulia Jusuf ◽

Dein Iftitah ◽

R. Muharam

Keyword(s):

Oocyte Maturation ◽

Blastocyst Stage ◽

Current Approach ◽

Developmental Competence ◽

Matrix Remodeling ◽

Oocyte Competence ◽

Nuclear Maturation ◽

Oocyte Developmental Competence ◽

Cumulus Oocyte Complex

Oocyte developmental competence is one of the determining factors that influence the outcomes of an IVF cycle regarding the ability of a female gamete to reach maturation, be fertilized, and uphold an embryonic development up until the blastocyst stage. The current approach of assessing the competency of an oocyte is confined to an ambiguous and subjective oocyte morphological evaluation. Over the years, a myriad of biomarkers in the cumulus-oocyte-complex has been identified that could potentially function as molecular predictors for IVF program prognosis. This review aims to describe the predictive significance of several cumulus-oocyte complex (COC) biomarkers in evaluating oocyte developmental competence. A total of eight acclaimed cumulus biomarkers are examined in the study. RT-PCR and microarray analysis were extensively used to assess the significance of these biomarkers in foreseeing oocyte developmental competence. Notably, these biomarkers regulate vital processes associated with oocyte maturation and were found to be differentially expressed in COC encapsulating oocytes of different maturity. The biomarkers were reviewed according to the respective oocyte maturation events namely: nuclear maturation, apoptosis, and extracellular matrix remodeling, and steroid metabolism. Although substantial in vitro evidence was presented to justify the potential use of cumulus biomarkers in predicting oocyte competency and IVF outcomes, the feasibility of assessing these biomarkers as an add-on prognostic procedure in IVF is still restricted due to study challenges.

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219EFFECTS OF GROWTH HORMONE AND GNRH ANTAGONISTS ON FOLLICULAR AND OOCYTE DEVELOPMENT IN SHEEP

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab219 ◽

2004 ◽

Vol 16 (2) ◽

pp. 231

Author(s):

P. Gonzalez-Añover ◽

T. Encinas ◽

R.M. Garcia-Garcia ◽

A. Veiga-Lopez ◽

J. Santiago-Moreno ◽

...

Keyword(s):

Growth Hormone ◽

Breeding Season ◽

Developmental Competence ◽

Control Group ◽

Gh Treatment ◽

Gnrh Antagonists ◽

Nuclear Maturation ◽

Final Development ◽

Sigma Chemical ◽

Experimental Group

Embryo output in sheep is increased when superovulatory FSH treatments are started in the presence of a high number of small follicles (2–3mm in size) and in absence of large follicles (>6mm, Gonzalez-Bulnes et al., 2002. Theriogenology, 57, 1263–1272). Administration of GnRH antagonists (GnRHa) suppresses large follicles (Cognie et al., 2003. Theriogenology, 59, 171–188), whereas the use of growth hormone (GH) would increase the number of small follicles (Campbell et al., 1995. J Reprod Fertil Suppl, 49, 335–350). Our aim was to evaluate the usefulness of pre-treatments with GH or GH plus GnRH antagonists for sheep embryo production. First, we studied the effects on follicular population by serial ultrasonographies. Thereafter, we determined whether such treatments can affect oocyte developmental competence. In a first trial, a total of 18 Manchega ewes were treated with intravaginal FGA sponges (Chronogest®, Intervet Int., H) during breeding season (beginning of April). Six animals received daily i.m. doses of 15mg of ovine GH (Tuenre, GA) for 6 days, while six females received GH plus two s.c. doses of 1.5mg of GnRHa (Antarelix™, Zentaris, G) on Days 0 and 3 of GH treatment, and six ewes acted as controls receiving saline. Number of follicles >2mm, determined by daily transrectal ultrasonography, increased to reach significant differences on Day 4 in sheep treated with GH/GnRHa (22.7±0.8 v. 16.7±0.5, P<0.001) and on Day 5 in ewes injected with GH (20.3±0.4 v. 17.0±0.6, P<0.05). The second trial involved 18 Manchega ewes treated with progestagen sponges on Day 0 and distributed in three groups at the beginning of breeding season (end of July). In the first group (n=7), sheep were treated with two doses of GnRHa on Days 0 and 3 after sponge insertion and with three doses of 15mg of GH on Days 3, 4, and 5. Thereafter, ewes from this group and from a second experimental group (n=7) were treated with 3 doses of 1.5mL of FSH (Ovagen™, ICP, NZ) every 12h, starting on the afternoon of Day 5. A third group of sheep (n=4) did not receive GH/GnRHa or FSH, acting as controls. On Day 7, follicles were aspirated and the cumulus-oocyte complexes (COC) were cultured for 24h at 38.5°C, 5% CO2, in TCM-199 supplemented with ovine FSH (Ovagen), LH, FCS, 17-βoestradiol, cysteamine, and sodium pyruvate (Sigma Chemical Co., MO, USA). Nuclear maturation was measured by Hoechst 33342 fluorescence. Mean number of COC was higher in GH/GnRHa+FSH group (8.7±0.9 v. 6.8±1.3 in FSH group, NS and 4.5±0.8 in control, P<0.05) due to higher number of follicles with similar recovery rates (45.0±4.5, 40.3±1.4, and 39.1±7.1%, respectively). There were no significant differences on the ability of COC to resume meiosis, although this was higher in FSH group (63.1±9.5% for GH/GnRHa+FSH, 79.5±6.3% for FSH and 60.0±8.8% for control group), which can indicate the necessity of a higher FSH supply to induce final development in follicles/oocytes from ewes treated with GH and GnRHa. In conclusion, the use of GH and GnRHa would help to increase the number of gonadotrophin-responsive follicles prior to gonadotrophin injections;; also, adjustment of FSH treatments improved embryo yields in superovulatory protocols.

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Factors affecting developmental competence of equine oocytes after intracytoplasmic sperm injection

Reproduction ◽

10.1530/rep.1.00087 ◽

2004 ◽

Vol 127 (2) ◽

pp. 187-194 ◽

Cited By ~ 26

Author(s):

Y H Choi ◽

L B Love ◽

D D Varner ◽

K Hinrichs

Keyword(s):

Embryo Culture ◽

Intracytoplasmic Sperm Injection ◽

Room Temperature ◽

Culture Media ◽

Developmental Competence ◽

Glucose Medium ◽

Factors Affecting ◽

Nuclear Maturation ◽

High Glucose Medium

This study was conducted to evaluate the effect of initial cumulus morphology (expanded or compact) and duration of in vitro maturation (24, 30 or 42 h) on the developmental competence of equine oocytes after intracytoplasmic sperm injection (ICSI). The effect of manipulation temperature (room temperature vs 37 °C) at the time of ICSI and concentration of glucose (0.55 vs 5.5 mM) during embryo culture was also investigated. The nuclear maturation rates of expanded (Ex) oocytes were significantly (P < 0.001) higher than those of compact (Cp) oocytes at all maturation times (61–72 vs 23–25% respectively). Forty-eight hours after ICSI of mature Ex oocytes, the rate of cleavage with normal nuclei was significantly (P < 0.05) higher for oocytes matured for 24 h than for those matured for 30 or 42 h (73 vs 57–59% respectively). For Cp oocytes, the morphologic cleavage rates for oocytes matured for 30 h were significantly higher (P < 0.05) than for those matured for 24 or 42 h (86 vs 55–61% respectively). The overall proportion of embryos having more than four normal nuclei at 48 h culture was significantly higher (P < 0.05) for Cp than for Ex oocytes. Manipulation temperature did not affect development of embryos from Ex or Cp oocytes at 96 h after ICSI. Culture in high-glucose medium significantly increased morphologic cleavage of Cp, but not Ex, oocytes (P < 0.05). Embryos from Cp oocytes had a significantly higher average nucleus number after 96-h culture than did embryos from Ex oocytes. These data indicate that developmental competence differs between Ex and Cp equine oocytes, and is differentially affected by the duration of maturation and by composition of embryo culture media.

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Effect of Hyaluronan on Developmental Competence and Quality of Oocytes and Obtained Blastocysts fromIn VitroMaturation of Bovine Oocytes

BioMed Research International ◽

10.1155/2014/519189 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 4

Author(s):

Jolanta Opiela ◽

Joanna Romanek ◽

Daniel Lipiński ◽

Zdzisław Smorąg

Keyword(s):

Dna Fragmentation ◽

Oocyte Maturation ◽

Developmental Competence ◽

Meiotic Maturation ◽

Nuclear Maturation ◽

Significant Difference ◽

The Mean ◽

Bovine Oocytes

The objective of the present study was to evaluate the effect of hyaluronan (HA) during IVM on meiotic maturation, embryonic development, and the quality of oocytes, granulosa cells (GC), and obtained blastocysts. COCs were maturedin vitroin control medium and medium with additional 0.035% or 0.07% of exogenous HA. The meiotic maturity did not differ between the analysed groups. The best rate and the highest quality of obtained blastocysts were observed when 0.07% HA was used. A highly significant difference (P<0.001) was noted in the mean number of apoptotic nuclei per blastocyst and in the DCI between the 0.07% HA and the control blastocysts (P<0.01). Our results suggest that addition of 0.035% HA and 0.07% HA to oocyte maturation media does not affect oocyte nuclear maturation and DNA fragmentation. However, the addition of 0.07% HA during IVM decreases the level of blastocysts DNA fragmentation. Finally, our results suggest that it may be risky to increase the HA concentration during IVM above 0.07% as we found significantly higherBaxmRNA expression levels in GC cultured with 0.07% HA. The final concentration of HA being supplemented to oocyte maturation media is critical for the success of the IVP procedure.

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Improvement of the developmental competence of canine oocyte using caffeine supplementation during IVM at different maturation time

Zygote ◽

10.1017/s0967199418000059 ◽

2018 ◽

Vol 26 (2) ◽

pp. 162-167 ◽

Cited By ~ 1

Author(s):

Mohamed Fathi ◽

A. Salama ◽

Magdy R. Badr

Keyword(s):

Developmental Competence ◽

Control Group ◽

Free Medium ◽

Maturation Time ◽

Fluid Medium ◽

Developmental Potential ◽

Nuclear Maturation ◽

Maturation Medium ◽

Oviductal Fluid

SummaryThe aim of the current study was to investigate the effect of caffeine supplementation during in vitro maturation (IVM) for different maturation times on the developmental potential of canine oocytes recovered from ovariohysterectomized bitches. The recovered cumulus–oocytes complexes were in vitro matured for 72 h. Here, 10 mM caffeine was added to the maturation medium for different incubation times (caffeine from 0–72 h maturation, caffeine for the first 24 h of maturation only, caffeine addition from 24 to 48 h maturation time, caffeine addition from 48 to 72 h maturation or in caffeine-free medium, control group). The matured oocytes were in vitro fertilized using frozen–thawed spermatozoa. The presumptive zygotes were in vitro cultured in synthetic oviductal fluid medium for 5 days. The results showed that both maturation and fertilization rates were significantly higher (P ˂ 0.05) using caffeine-treated medium for the first 24 h of maturation compared with the control and other two groups of caffeine treatment (from 24 to 48 h and from 48 to 72 h), whereas use of caffeine-treated medium for a 0–72 h incubation time did not affect these rates (P > 0.05). Interestingly, the matured oocytes in caffeine-supplemented medium for the first 24 h or from 0–72 h showed a significant (P ˂ 0.05) increase in the total number of cleaved embryos compared with the control group. In conclusion, supplementation of the maturation medium with 10 mM caffeine for the first 24 h of maturation or during the whole maturation time (0–72 h) improved nuclear maturation and subsequent embryo development preimplantation following in vitro fertilization.

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44 PRODUCTION OF LIVE PIGLETS AFTER CRYOPRESERVATION OF IMMATURE PORCINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab44 ◽

2014 ◽

Vol 26 (1) ◽

pp. 136

Author(s):

T. Somfai ◽

K. Kikuchi ◽

K. Yoshioka ◽

F. Tanihara ◽

H. Kaneko ◽

...

Keyword(s):

Polar Body ◽

Petri Dish ◽

Developmental Competence ◽

Basic Medium ◽

High Survival ◽

Blastocyst Development ◽

Nuclear Maturation ◽

First Polar Body ◽

Vitrification Solution

Development to term of vitrified porcine follicular oocytes is reported in the present study. Immature cumulus-oocyte complexes (COC) were collected from slaughtered prepubertal gilts and were vitrified according to our method published recently (Somfai et al. 2013 J. Reprod. Dev., in press). Briefly, after pretreatment with 7.5 μg mL–1 of cytochalasin B (CB) for 30 min in modified NCSU-37 (a basic medium, BM) at 38.5°C, groups of 88 to 121 COC were equilibrated in a mixture of 2% ethylene glycol (EG), 2% propylene glycol (PG), and 7.5 μg mL–1 CB for 13 to 15 min. Then, COC were washed in vitrification solution (17.5% EG, 17.5% PG, 5% polyvinyl pyrrolidone, and 0.3 M trehalose in BM) and then dropped with 2 μL of vitrification solution onto the surface of aluminum foil floating on liquid nitrogen (LN2). Microdroplets (each containing 10–25 COC) were transferred into cryotubes. After storage in LN2 for 2 to 4 weeks, the oocytes were warmed by dropping the microdroplets directly into 2.5 mL of warming solution (0.4 M trehalose in BM) kept in a 35-mm Petri dish on a 42°C hotplate for less than 1 min. Then, the warming dish was placed on a 38°C hotplate and COC were consecutively transferred for 1-min periods into BM containing 0.2, 0.1, or 0.05 M trehalose at 38°C. The COC were matured in vitro for 44 h using porcine oocyte medium (POM) supplemented with 10% follicular fluid (Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213). Then, oocytes were denuded, and their live/dead status and nuclear maturation were determined by their morphology and the presence of the first polar body, respectively. To assess their developmental competence, vitrified and non-vitrified (control) oocytes were in vitro fertilized (IVF; Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) and then in vitro cultured in porcine zygote medium-5 (PZM-5; Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213). Blastocyst rates were recorded on Days 5, 6, and 7 of culture (Day 0 = the day of IVF). The experiment was replicated 4 times. Data were analysed with 1-way ANOVA and the Tukey test. The results revealed that 86.4% (364/424) of oocytes survived after vitrification, which was significantly lower (P < 0.05) than that of controls [100% (326/326)]. Live oocytes in vitrified and control groups did not differ statistically in terms of nuclear maturation (63.9 v. 65.3%). Blastocyst rates of surviving vitrified oocytes were significantly lower compared with controls on Days 5 (2.4 v. 12.7%), 6 (4.8 v. 17.6%), and 7 (5.6 v. 18.4%). To test their ability to develop to term, 16 and 27 blastocysts on Day 5 developing from vitrified COC were transferred into 2 recipients. Both recipients became pregnant and farrowed a total of 10 live piglets (4 and 6 piglets, respectively). These data demonstrate that large groups of immature porcine oocytes could be cryopreserved by this method showing high survival and maturation rates. Furthermore, despite a low rate of blastocyst development, transfer of Day-5 blastocysts generated from vitrified oocytes resulted in piglet production for the first time in the world. Partially supported by JSPS and HAS under the Japan-Hungary Research Cooperative Program.

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172 DETERMINING THE REQUIREMENTS FOR LH AND FSH DURING SHEEP IN VITRO OOCYTE MATURATION

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab172 ◽

2014 ◽

Vol 26 (1) ◽

pp. 200 ◽

Cited By ~ 5

Author(s):

C. de Frutos ◽

R. Vicente-Perez ◽

P. J. Ross

Keyword(s):

Oocyte Maturation ◽

In Vitro Maturation ◽

Mixed Logit ◽

Cumulus Cells ◽

Pituitary Hormones ◽

Developmental Competence ◽

Cumulus Expansion ◽

Nuclear Maturation ◽

Grade 3

In vitro maturation (IVM) of oocytes in domestic animals is a widespread practice of research and commercial relevance. Gonadotropic hormones are typically supplemented to the IVM medium to stimulate resumption of meiosis, progression to metaphase II (MII), and oocyte developmental competence. The common use of pituitary-derived products presents 2 problems: contamination from other pituitary hormones and inconsistences from batch-to-batch variation. Recombinant hormones can help circumvent these issues and identify specific gonadotropin requirements for in vitro maturation. The aim of the present study was to determine the effect of supplementing recombinant bovine LH and/or FSH (AspenBio) to the maturation of ovine oocytes in terms of cumulus expansion and progression to the MII stage. Abattoir-derived sheep cumulus–oocyte complexes (COC) were obtained from 1- to 5-mm-diameter antral follicles by ovary slicing. Oocytes with a homogeneous cytoplasm surrounded by at least 3 layers of cumulus cells were selected and cultured in serum-free IVM medium (Cotterill et al. 2012 Reproduction 144, 195–207) at 38.5°C and 5% CO2. The COC obtained from 8 replicates were allocated into 4 experimental groups: (1) no hormones; (2) 1.5 μg mL–1 recombinant bovine LH (rbLH); (3) 1.5 μg mL–1 recombinant bovine FSH (rbFSH); and (4) rbLH and rbFSH. The expansion of cumulus cells was recorded in each group after 24 h of IVM and COC classified as (1) very poor or no cumulus expansion (grade 1); (2) limited cumulus expansion (grade 2); and (3) full cumulus expansion (grade 3). Nuclear maturation in the 4 treatments was evaluated by assessing progression to the MII stage via DNA staining with Hoechst 33342 and fluorescence imaging. The effect of treatment on the observed proportion of MII oocytes was evaluated using a mixed logit model including treatment and replicate as fixed and random effects, respectively. Culture in IVM medium in the absence of gonadotropins or in the presence of rbLH resulted in poor cumulus expansion (grade 1). The supplementation of IVM medium with rbFSH (with or without rbLH) yielded a high degree of cumulus expansion (grades 2–3). Likewise, addition of rbFSH enhanced progression of oocytes to the MII stage, whereas use of rbLH, although it had an effect on progression to MII, did not augment the effect of rbFSH (Table 1). These results indicate that rbFSH is necessary and sufficient to induce sheep oocyte maturation in a high proportion of oocytes. Table 1.Cumulus expansion and oocyte nuclear stage after IVM

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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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