Effect of Post-IVF Developmental Kinetics on In Vitro Survival of Vitrified-warmed Domestic Cat Blastocysts
Contents
A limited number of reports is available on cryopreservation of in vitro fertilization (IVF)-derived cat blastocysts. In the present study, IVF-derived domestic cat embryos which reached the blastocyst stage either on day 6 or day 7 were cryopreserved by vitrification using Cryotop as a cryodevice. Fresh control and post-warm surviving blastocysts were examined by differential cell staining with Hoechst 33342 and propidium iodide to determine total cell number and inner cell mass (ICM) ratio, and the post-warm survival rate was determined by re-expansion of the blastocoel during 24 h of in vitro culture. In fresh control, the mean number of total cells of day 7 blastocysts (61.4 cells) tended to be smaller than that of day 6 blastocysts (81.9 cells, p = 0.096). The post-warm survival rates of day 6 and day 7 blastocysts were not statistically different (73.8%; 31 of 42 vs 66.7%; 18 of 27). There were no significant differences in the total cell number and ICM ratio between fresh control and vitrified blastocysts, although the ICM ratio of surviving day 7 blastocysts was significantly smaller than that of fresh controls (stained at day 8, 18.9% vs 28.9%, p < 0.05). These results indicate that IVF-derived domestic cat embryos that reached the blastocyst stage earlier can survive the Cryotop vitrification without a reduction in the parameters studied.
Introduction
Cryopreservation of gametes and embryos from domestic cats provides a suitable model for conservation of genetically valuable endangered feline species. Cat embryos can be successfully produced by in vitro fertilization (IVF) of in vivo-matured (Pope et al. 1993; Roth et al. 1994) and in vitro-matured (Pope et al. 1997; Karja et al. 2002) oocytes, or by somatic cell nuclear transplantation (Shin et al. 2002; Gomez et al. 2004). However, only a limited number of reports is available for either conventional freezing or vitrification of cat embryos since the first kitten from transfer of frozen morulae and blastocysts produced in vivo had been reported by Dresser et al. (1988).
The development of in vitro-derived 2- to 8-cell stage (days 1–2) cat embryos into morulae and blastocysts was not affected by freezing and thawing and transfer of frozen-thawed embryos resulted in the birth of live kittens (Pope et al. 1994). Pope et al. (1997) reported that in vitro-derived cat embryos at the 16- to 30-cell stage (day 3, further development rate 69%) were more sensitive to conventional freezing than 2- to 8-cell stage embryos (days 1–2, 77–85%), while Gomez et al. (2003) reported that similar proportions of cat embryos developed into blastocysts regardless of the developmental stage at freezing (days 2, 4 and 5; 59%, 53% and 63%, respectively). Recently, it has been reported that 61% of IVF-derived day 7 cat blastocysts survived freezing and thawing (Karja et al. 2006).
To the best of our knowledge, there is no report on vitrification of cat embryos, except the IVF-derived Siberian tiger embryos at 2- to 4-cell stage (Crichton et al. 2003). Crichton et al. (2003) reported that IVF-derived 2- to 4-cell stage Siberian tiger embryos showed cleavage after vitrification in open-pulled straw system, but not after conventional two-step freezing. Cryotop device has been originally developed for vitrification of human oocytes and embryos (Kuwayama and Kato 2000) and later successfully used for pronuclear-stage rabbit zygotes (Hochi et al. 2004), pre-hatching-stage porcine embryos (Esaki et al. 2004), in vitro-matured bovine oocytes (Chian et al. 2004), germinal vesicle-stage whale oocytes (Iwayama et al. 2004), nuclear-transferred bovine and buffalo blastocysts (Laowtammathron et al. 2005) and in vitro-matured buffalo oocytes and cytoplasts (Muenthaisong et al. 2007).
The objective of the present study was to determine the effect of post-IVF developmental kinetics on in vitro survival of vitrified-warmed domestic cat blastocysts. Because the total number of cells and/or ratio of inner cell mass (ICM) of blastocysts are often used as a qualitative parameter, blastocysts surviving the Cryotop vitrification procedure were analysed by differential cell staining to determine total cell number and ICM ratio.
Materials and Methods
Experimental design
Domestic cat embryos were produced by in vitro maturation (IVM), IVF and in vitro culture (IVC) of follicular oocytes. All embryos reaching the blastocyst stage at day 6 (day 0 was defined as the day of IVF) were randomly allocated to either the vitrification or control group. Fresh control blastocysts were either subjected immediately to differential cell staining or after a 24-h culture period. Blastocysts that survived vitrification/warming (re-expansion during 24 h of culture) were also stained. Additional embryos reaching the blastocyst stage initially at day 7 were harvested and allocated to either the vitrification or fresh group, and were treated as well.
In vitro maturation of cat oocytes
Queen ovaries were supplied from the Clinic for Veterinary Gynecology and Obstetrics, Ludwig-Maximilians-University and local veterinary clinics in Muenchen, Germany, after routine ovariohysterectomy. The ovaries were kept at room temperature (22–26°C) for 1–8 h in phosphate-buffered saline (PBS; Biochrom, Berlin, Germany) supplemented with 10 IU/ml penicillin and streptomycin (Sigma, St Louis, MO, USA) until oocyte recovery. Ovaries were sliced with a scalpel blade and washed repeatedly to release cumulus-oocyte-complexes (COCs). The COCs were collected into TCM-199 (Sigma) supplemented with 3 mg/ml NaHCO3 (Sigma), 0.05 mg/ml gentamycin (Sigma), 0.275 mg/ml sodium pyruvate (Sigma), 0.6 mg/ml calcium lactate (Sigma), 0.1 mg/ml cystein (Sigma) and 3 mg/ml BSA (Sigma). Only COCs with homogenous dark cytoplasm and several layers of cumulus cells were selected and rinsed three times in the TCM-199. Then, they were cultured in 400 μl of the TCM-199 containing 1 mg/ml estradiol-17β (E2; Sigma), 0.1 IU/ml FSH (Sioux Biochemical, Sioux City, IA, USA) and 0.0063 IU/ml LH (Sioux Biochemical) in 4-well dish (Nunc, Roskilde, Denmark) for 24 h at 38.5°C in 5% CO2 in air.
Preparation of cat spermatozoa
Testes and epididymis from adult male cats following elective castration were kept at room temperature for 1–8 h in PBS supplemented with penicillin and streptomycin before sperm collection. The epididymidis was removed from the testes and sliced repeatedly with a scalpel blade to release spermatozoa in a canine extender consisting of Tris buffer and glycerol (Triladyl canine; Minitube, Tiefenbach, Germany). Only semen samples with >50% progressively motile spermatozoa were used for cryopreservation as follows: the semen sample was diluted at room temperature with an equal volume of the Triladyl extender containing 10% egg yolk, and the mixture was immediately loaded into 0.25-ml straws. The straws were cooled to 4°C for 1 h and placed in vapour of liquid nitrogen (LN2) for 10 min before being stored in liquid nitrogen. On the day of IVF, the straw was submerged into a 37°C water bath for 10 s. Post-thaw semen samples were placed at the bottom of TALP medium (Freistedt et al. 2001a) supplemented with 0.22 mg/ml sodium pyruvate and 6 mg/ml BSA allow to swim-up of the spermatozoa. After washing the supernatant by centrifugation at 600 × g for 10 min, the sperm pellet was resuspended with 500 μl of the TALP medium.
Fertilization and culture in vitro
After 24 h of IVM culture, COCs were washed once with the TALP medium and transferred to 400 μl of the TALP medium. They were co-incubated with spermatozoa (final concentration of 2 × 106 cells/ml) for 22 h at 38.5°C in 5% CO2 in air. Cumulus cells were removed by frequent pipetting, and the presumptive zygotes were washed once with m-SOF medium (Freistedt et al. 2001a) supplemented with 0.36 mg/ml sodium pyruvate, 4 mg/ml BSA, 1% essential amino acid solution (BME; Sigma) and 0.5% non-essential amino acid solution (MEM; Sigma). The zygotes were cultured for 3 days in 400 μl of the m-SOF medium at 38.5°C in 5% CO2 in air. Then, only cleaved embryos were selected and further cultured for an additional 2–3 days in m-SOF supplemented with 0.36 mg/ml sodium pyruvate, 5.5 mm glucose (Sigma), 1% BME, 0.5% MEM and 5% autologous oestrous cow serum (ECS) at 38.5°C in 5% CO2 in air. Cleavage rate of the inseminated oocytes was recorded on day 3. In each experiment of IVM/IVF/IVC, all embryos at the blastocyst stage with a clearly visible ICM were harvested on day 6 and newly formed blastocysts among the remaining embryos were harvested on day 7.
Vitrification and warming
Vitrification was performed by the procedure previously reported by Hochi et al. (2004). Briefly, day 6 or day 7 blastocysts were first exposed to pre-treatment solution consisting of 7.5% ethylene glycol (EG; Sigma) + 7.5% DMSO (Sigma) + 20% fetal calf serum (FCS; Biochrom) in TCM-199 for 3 min at room temperature, and then transferred into a vitrification solution consisting of 15% EG + 15% DMSO + 0.5 m sucrose (Sigma) + 20% FCS in TCM-199 for 1 min at room temperature. Within this 1 min, up to five blastocysts were placed on a sheet of each Cryotop (Kitazato Supply, Tokyo, Japan) in a small volume of the vitrification solution (<1 μl) and the Cryotop device was plunged into LN2. After storage in LN2, the blastocysts were warmed by immersing the Cryotop into 1 ml of 0.5 m sucrose in the TCM-199 + 20% FCS for 30 s at 37°C, and then transferred to the TCM-199 + 20% FCS. The blastocysts were washed three times at 5-min intervals with the TCM-199 + 20% FCS at 37°C, and cultured in 400 μl of the m-SOF supplemented with sodium pyruvate, glucose, BME, MEM and ECS for 24 h at 38.5°C in 5% CO2 in air. Blastocysts with a re-expanded blastocoel cavity were considered as surviving.
Differential cell staining
Total cell number and ICM and trophectoderm (TE) cell ratio were determined using protocol described by Comizzoli et al. (2004). Briefly, the blastocysts were incubated for 30 s at room temperature in PBS containing 1% Triton-X (Sigma) and 100 μg/ml propidium iodide (PI; Sigma) to stain the TE cells. Further, the blastocysts were incubated for 3–5 h at 4°C in ethanol with 25 μg/ml Hoechst 33342 to fix and stain the ICM cells. Then, the blastocysts were mounted on slide glass with glycerol (Sigma) under a cover glass and sealed with nail polish. The ICM cells and TE cells were counted under a fluorescence microscope (Axiovert 25; Zeiss, Jena, Germany) at 320–350 nm, allowing the determination of the total number of cells of the blastocysts and the percentage of ICM cells based on the total number of blastocyst cells.
Statistical analysis
In vitro survival rate of vitrified blastocysts was analysed by Fisher's exact probability test. Mean total number of blastocyst cells and the ICM ratio between fresh control and vitrified groups were compared by Student's t-test. A p-value of <0.05 was chosen as an indication of significant difference.
Results
Nearly half of the inseminated oocytes (47.1%) cleaved (Table 1). On day 6 after insemination, 9.8% of all inseminated oocytes had developed to blastocysts. A similar proportion of blastocysts could be detected in the remaining embryos on day 7 (9.5%). Regardless of the developmental kinetics, a similar variation in the size and morphology of blastocysts was observed (Fig. 1a).
| Number of oocytes inseminated | Number (%) of oocytes cleaved | Number (%) of blastocysts initially appearing | Total number (%) of blastocysts | |
|---|---|---|---|---|
| Day 6 | Day 7 | |||
| 666 | 314 (47.1) | 65 (9.8) | 63 (9.5) | 128 (19.2) |
Morphology of domestic cat blastocysts before and after vitrification. (a) Fresh blastocysts with different sizes, harvested on day 6. A similar variation in size was observed for day 7 blastocysts. (b) Post-warm blastocysts immediately after removal of the cryoprotectants. (c) Re-expanding blastocyst (=surviving; top) and contracted blastocyst (=died; bottom) after 24-h culture
Disappearance of the blastocoel was observed during pre-treatment (7.5% EG + 7.5% DMSO) or equilibration (15% EG + 15% DMSO + 0.5 m sucrose) before cooling, as a result of osmotic shock and dehydration. After vitrification and warming, all blastocysts appeared to be normal but they were still contracted (Fig. 1b). Neither zona fracture nor cell lysis was observed in the post-warm blastocysts. Surviving blastocysts regained their blastocoel cavity during the subsequent 24-h culture, while non-surviving blastocysts arrested their development (Fig. 1c). A high proportion of post-warm day 6 and day 7 blastocysts survived the process of vitrification (73.8% and 66.7%, respectively; Table 2).
| Harvest of blastocysts | Number of blastocysts | % | |
|---|---|---|---|
| Vitrified-warmed | Re-expanded | ||
| Day 6 | 42 | 31 | 73.8 |
| Day 7 | 27 | 18 | 66.7 |
- Ten and six replicates for day 6 and day 7 blastocysts, respectively.
As shown in Table 3, differential cell staining of fresh control blastocysts indicated that the mean number of total cells in day 6 blastocysts tended to be higher than that in embryos developing to the blastocyst stage on day 7 (81.9 vs 61.4 cells, p = 0.096), that after 24-h culture of the harvested blastocysts, the total cell number between day 6 and day 7 blastocysts was significantly different (99.8 vs 71.9 cells), and that the ICM ratio of day 6 blastocysts was significantly lower after the 24-h culture (42.6–27.5%). Comparison of these parameters between post-warm surviving blastocysts and fresh control blastocysts indicated that both total cell number (post-warm 117.2 cells vs fresh 99.8 cells) and ICM ratio (post-warm 26.8% vs fresh 27.5%) of blastocysts harvested on day 6 were comparable, and that the ICM ratio of day 7 blastocysts was impaired by vitrification and warming (post-warm 18.9% vs fresh 28.9%; p < 0.05).
| Harvest of blastocysts | Groups (additional culture period) | Number of blastocysts evaluated | Mean number of total cells* | Percentage of ICM cells* |
|---|---|---|---|---|
| Day 6 | Fresh (0 h) | 12 | 81.9 ± 24.4bc | 42.6 ± 10.1a |
| Fresh (24 h) | 10 | 99.8 ± 27.4ab | 27.5 ± 10.8c | |
| Vitrified (24 h) | 28 | 117.2 ± 37.2a | 26.8 ± 15.0c | |
| Day 7 | Fresh (0 h) | 8 | 61.4 ± 27.5c | 36.8 ± 20.3ab |
| Fresh (24 h) | 8 | 71.9 ± 25.8c | 28.9 ± 9.6bc | |
| Vitrified (24 h) | 15 | 68.6 ± 30.6c | 18.9 ± 10.3d |
- *Values are expressed as mean ± SD.
- a-dValues with different superscripts in same columns differ significantly (p < 0.05).
Discussion
In the present study, we were able to demonstrate that embryos which reached the blastocyst stage slower were constituted from smaller number of cells than the faster developing blastocysts, as previously reported in bovine (Van Soom et al. 1997; Enright et al. 2000) and ovine (Leoni et al. 2006) species. A relatively large variation in cell number of IVF-derived day 8 cat blastocysts (104.4 ± 28.7 cells; differential cell staining) has been reported, with an ICM ratio of 27.8% (Comizzoli et al. 2004). The mean cell number of day 8 cat blastocysts ranged from 86.9 to 108.4 in different culture media (Murakami et al. 2002), and 65.3 in unexpanded blastocysts to 121.4 in expanding blastocysts (Freistedt et al. 2001b). A range of 129–169 cells had been found in expanding blastocysts and 160–269 cells in hatching blastocysts (Gomez et al. 2003).
Bovine blastocysts with a slower developmental kinetics exhibited a higher sensitivity to cryopreservation than the faster developing blastocysts (Han et al. 1994; Massip et al. 1995; Nedambale et al. 2004). In the present study, in vitro survival judged by blastocoel re-expansion of post-warm blastocysts was similar regardless of the day of blastocyst harvest (Table 2). Total cell number of slower developing day 7 cat blastocysts after vitrification and 24-h culture was comparable with that of non-vitrified control blastocysts, but the ICM cells were significantly affected by the vitrification procedure (Table 3). A lower number of ICM cells in frozen-thawed bovine blastocysts compared with that in non-frozen control embryos has also been reported by Takagi et al. (1996). As a sufficient number of ICM is necessary for development of post-implantation embryos, the vitrification procedure needs to be improved before it can be applied in practice. During the Cryotop vitrification procedure employed here, blastocysts were treated for 3 min with the permeable cryoprotective additives (CPAs) before they were exposed to a 1-min equilibration period with vitrification solution of high osmorality. The intracellular level of CPAs before cooling depends on time of equilibration, CPAs concentration and equilibration temperature. Longer pre-treatment with the CPAs (10 min) was effective for improving the post-warm survival in pronuclear-stage rabbit zygotes (Hochi et al. 2004).
The culture conditions of IVF-derived presumptive bovine zygotes affect the morphological feature of the blastocysts, resulting in the different sensitivity to cryopreservation (Massip 2001). Shamsuddin et al. (1994) and Abe et al. (2002) reported that the cryotolerance of bovine embryos produced in serum-free medium is higher than embryos produced in serum-containing medium. These results are attributed to the fact that culture in serum-free media can reduce intracellular lipid content (Sata et al. 1999; Abe et al. 2002). One distinct feature of cat oocytes and embryos is a uniformly dark appearance, indicating a high consent of intracellular lipids (Luvoni 2006). An invasive approach to reduce the volume of intracytoplasmic lipids resulted in a significant improvement of cryotolerance in porcine (Nagashima et al. 1995) and bovine (Ushijima et al. 1999) IVF-derived blastocysts. Karja et al. (2006) recently reported that mechanical aspiration of the lipid layer from centrifuged domestic cat zygotes had no effect on blastocyst production in vitro and resulted in a higher tendency in post-thaw survival of the blastocysts. The protocol for the blastocyst production employed in the present study includes IVM, IVF and IVC until day 4 in serum-free media (BSA was supplemented instead of serum), and an additional period of IVC until day 6 or day 7 in ECS-supplemented medium. Further investigations on the culture conditions as well as cryopreservation protocol are necessary to improve the cryosurvival of domestic cat blastocysts. In addition, optimization of post-warm embryo culture may improve post-warm embryo development as was shown by Kaidi et al. (1998).
Vitrified-warmed oocytes or embryos are usually exposed to sucrose solution for >3 min during the dilution process of permeable CPAs. Murakami et al. (2004) reported that development into blastocysts of vitrified-warmed cat oocytes was observed only when the exposure of post-warm oocytes to sucrose solution was restricted to 30 s. Therefore, the exposure period of post-warm cat blastocysts to sucrose solution in the present study was limited to 30 s. Further investigations are required to clarify the potential toxic effect of sucrose for the cat embryos. Moreover, the developmental ability of cat blastocysts after the Cryotop vitrification should be verified by transfer of post-warm embryos into recipient females.
In conclusion, IVF-derived domestic cat blastocysts can survive vitrification and warming without a reduction of embryo quality, when fast-developing blastocysts on day 6 are used. This is also applicable to slow-developing day 7 blastocysts regardless of a significant loss of ICM cells after vitrification.
Acknowledgement
The Cryotop device was kindly provided by Dr M. Kuwayama of Kato Ladies Clinic, Tokyo, Japan.
