Birth of Kittens After the Transfer of Frozen–Thawed Embryos Produced by Intracytoplasmic Sperm Injection with Spermatozoa Collected from Cryopreserved Testicular Tissue
Author's address (for correspondence): T Tharasanit, Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand.
The aim of this study is to produce live kittens from oocytes fertilized by intracytoplasmic sperm injection (ICSI) with frozen/thawed testicular spermatozoa. Spermatozoa were collected from thawed testicular tissue and subsequently injected into in vitro matured cat oocytes. At 24 h post-ICSI, presumptive zygotes/cleaved embryos were treated with 10 μm forskolin for 24 h to reduce intracellular lipid content of embryos (delipidation). At 48 h after oocyte injection, cleaved embryos (2- to 8-cell stage) were frozen in 10% (v/v) ethylene glycol-based medium by a slow controlled rate method and stored in liquid nitrogen. To evaluate in vitro and in vivo developmental competence, frozen embryos were thawed and then cultured for 6 days (n = 155) or cultured for 2 h before transferred (n = 209) to hormonal (equine chorionic gonadotropin/hCG)-treated cat recipients. Cleavage frequency at day 2 after ICSI with frozen/thawed testicular spermatozoa was ~30%. The percentages of frozen/thawed embryos that developed to morula and blastocyst stage (on day 3 and day 6 of in vitro culture, respectively) were significantly lower than that of fresh ICSI embryos (22.6 vs 45.2% and 21.3 vs 38.7%, respectively; p < 0.05). However, no difference was found in the number of blastomeres between frozen/thawed (242.5 ± 43.1) and fresh (320.2 ± 28.1) blastocysts. Three of seven cat recipients were pregnant and one pregnant cat delivered two healthy kittens. This is the first report of the birth of kittens after the transfer of frozen–thawed embryos produced by ICSI with frozen/thawed testicular sperm.
Assisted reproductive techniques, in particular, cryopreservation of gametes and embryos can be highly beneficial for genetic preservation and management of several endangered species (Pukazhenthi et al. 2006). Testicular tissue cryopreservation would increase the sperm source for valuable animals that died unexpectedly or are castrated for medical reason, while embryo cryopreservation is a potential technique for infinite storage of the complete genetic complement of both parents. The domestic cat is an important model to develop assisted reproductive technologies (ARTs) before their application to endangered wild felids. Cat embryos produced from testicular spermatozoa by intracytoplasmic sperm injection (ICSI) developed in vitro to the blastocyst stage (Comizzoli et al. 2006a; Buarpung et al. 2012). However, in vivo development of embryos produced by ICSI with cryopreserved testicular spermatozoa in cats has not been described. Embryos at early stages of development (2- to 8-cells) represent a valuable source for freezing because cleaved embryos produced by ICSI with testicular spermatozoa show poor development to the morula and blastocyst stage (S. Buarpung, unpublished). The aim of the present study is to produce kittens from frozen/thawed 2- to 8-cell embryos generated by ICSI with frozen–thawed testicular spermatozoa.
Materials and Methods
Testes and ovaries from adult mixed-breed domestic cats submitted for routine castration and ovariohysterectomy (OVH) at the Veterinary Public Health Division of The Bangkok Metropolitan Administration, Bangkok, Thailand, were used in this study.
Testicular tissues were cut into equally sized small pieces (approximately 2 × 3 × 5 mm in size) and cryopreserved in freezing medium containing 5% (v/v) glycerol by a conventional slow freezing technique. The tissue samples were loaded into 0.5-ml ministraws, exposed to nitrogen vapour (4 cm above the surface of liquid nitrogen) for 10 min, then plunged and stored in liquid nitrogen (−196°C). Tissue samples were thawed in air for 10 s and then placed in a warm water bath at 37°C for 30 s. Testicular spermatozoa were collected by mincing the tissues with sharp-ended scissors in Hepes-buffered synthetic oviductal fluid (Hepes-SOF) and maintained at 37°C approximately 30 min prior to ICSI. Cumulus oocyte complexes (COCs) were collected by mincing the ovaries in holding medium (HM), consisting of Hepes-buffered M199, 1 mm sodium pyruvate, 2 mm l-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin and 4 mg/ml bovine serum albumin (BSA, embryo tested). COCs were matured in vitro as described earlier by Sananmuang et al. (2010). Briefly, a group of 30–40 COCs were cultured in 500 μl of maturation medium (M199, NaHCO3 buffered) supplemented with 1 mm sodium pyruvate, 2 mm l-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, 4 mg/ml BSA and 0.1 IU/ml recombinant human follicle stimulating hormone (rhFSH; Organon, Bangkok, Thailand) at 38.5°C in a humidified atmosphere of 5% CO2 in air for 18–24 h. After maturation, oocytes were denuded of cumulus and corona cells, and oocytes with a first polar body extruded (MII stage) were fertilized by ICSI as described previously by Buarpung et al. (2012); however, no chemical activation was used after ICSI in this study. Sperm injected oocytes were cultured in SOF medium containing 4 mg/ml fatty acid-free BSA and 100 IU/ml penicillin (SOF-BSA) in a humidified condition of 5% CO2 in air at 38.5°C for 24 h before delipidation by culture in the culture medium supplemented with 10 μm forskolin for another 24 h. At 48 h post-injection, the number of cleaved embryos was recorded and 2- to 8-cell stage embryos were selected for freezing. Embryos were exposed to the cryoprotectant in four equilibration steps of Hepes-SOF containing 20% (v/v) foetal calf serum and 0.125 m Trehalose with 0%, 2.5%, 5% and 10% ethylene glycol 5 min/step, before loading into 250-μl ministraws and frozen in a programmable CL863 freezer (Cryologic PL, Mulgrave, Victoria, Australia) at a cooling rate of 2°C/min from 24°C to −6°C. After 5 min holding, each straw was seeded manually by touching with forceps pre-cooled in liquid nitrogen and held at −6°C for an additional 5 min. Cooling was resumed at 0.3°C/min to −33°C, where the straws were held for 10 min before being plunged into liquid nitrogen.
To examine in vitro development, frozen embryos (n = 163) were thawed by holding in air for 10 s and then immersed in a water bath at 37°C for 20 s. Cryoprotectant was removed from the embryos by a four-step rinse into Hepes-SOF medium containing 20% (v/v) FCS and 0.25, 0.125, 0.062, 0.031 m trehalose (3 min/step). Thawed embryos were then cultured in SOF medium containing 10% (v/v) FCS (SOF-FCS) for 6 days. Development to morula and blastocyst stage was evaluated on Day 3 and Day 6, respectively, of in vitro culture (IVC). After 6 days of IVC, thawed embryos were fixed in 4% (w/v) paraformaldehyde and stained by fluorescent DNA labelling (DAPI; 4′6′ diamidino-2-phenylindole dihydrochloride) to determine the blastocyst quality by counting the total cell numbers. Non-frozen embryos, generated by ICSI with cryopreserved testicular spermatozoa (n = 93), served as control group and were cultured for 8 days in parallel to that of frozen/thawed ICSI embryos (day of ICSI = Day 0).
To examine in vivo developmental competence of frozen embryos, seven recipient cats were injected intramuscularly with 150 IU equine chorionic gonadotropin (eCG, Folligon; Intervet-Schering Plough, Boxmeer, the Netherlands) to induce oestrus. Ovulation was induced by intramuscular injection of 100 IU human chorionic gonadotropin (hCG) after 96 h of eCG injection. For embryo transfer, the recipients were anaesthetized with a combination of 0.04 mg/kg atropine sulphate (A.N.B. Laboratories, Bangkok, Thailand), 3 mg/kg xylazine (Laboratorios calier, Barcelona, Spain) and 10 mg/kg ketamine hydrochloride (Gedeon Richter, Budapest, Hungary). The 209 embryos were thawed (as previously described) and cultured in SOF-FCS for 2 h, then transferred by surgery into the oviducts of seven recipient cats (mean = 29.8 ± 3.6) at day 3 post-hCG.
Statistical analysis was performed using the Statistical Analysis Systems software package (Version 9.0; SAS Institute Inc., 1996, NC, USA). The frequency of morula and blastocyst development between frozen/thawed and fresh ICSI embryos was analysed by chi-square test. Differences were considered significant with a p < 0.05.
As showed in Table 1, cleavage frequencies at day 2 after ICSI with cryopreserved testicular spermatozoa for freezing and culture as a control were 34.9% (186/533) and 32.4% (93/287), respectively. Some of these cleaved embryos may be the result of parthenogenesis, according to our previous study (unpublished) that 5.1% cleavage could be achieved after sham injection without external activation. The percentage of frozen/thawed embryos that developed to morula (35/155; 22.6%) and blastocyst (33/155; 21.3%) stage was significantly lower than that of ICSI fresh embryos (42/93; 45.2%, 36/93; 38.7%, respectively, p < 0.05). However, the number of blastomeres per blastocyst was comparable between frozen/thawed (242.5 ± 43.1; n = 33) and fresh (320.2± 28.1; n = 36) embryos.
Table 1. Developmental competence in vitro of frozen–thawed embryos produced by ICSI with cryopreserved testicular spermatozoa
|FT||533||10||186 (34.9)a||163||155*||35 (22.6)a||33 (21.3)a||242.5 ± 43.1a|
|NF||287||5||93 (32.4)a||0||93||42 (45.2)b||35 (38.7)b||320.2 ± 28.1a|
Three of seven cat recipients of frozen/thawed embryos were diagnosed as pregnant by radiographic imaging at day 49 after embryo transfer because of one of recipient queen aborted a foetus in that day. However, the other two recipients carried their pregnancies to term. One queen delivered two healthy kittens on the 64th day of gestation, while a kitten from the third pregnant cat died in utero at 65 days of gestation.
Our results demonstrated for the first time that live kittens can be produced after the transfer of frozen/thawed embryos fertilized by ICSI with frozen testicular spermatozoa. However, frozen–thawed cleaved embryos developed to the morula and blastocyst stage in vitro at lower proportions than that of non-frozen embryos.
Percentage of cleavage after ICSI with frozen–thawed testicular spermatozoa in this study was comparable to our previous results (32.7% cleavage; unpublished data). However, these results were less than the report of Comizzoli et al. (2006a) that ICSI with non-preserved testicular spermatozoa and ejaculated spermatozoa yielded the similar percentage of cleavage at approximately 60%. It may be due to lack of external stimulation of ICSI oocytes in this study or due to sperm immaturity. It is known that immaturity of cat testicular sperm centrosome influences embryo cleavage after ICSI (Comizzoli et al. 2006b). Alternatively, the lower cleavage rate may have been due to deleterious effects of cryopreservation on testicular spermatozoa. However, it has been demonstrated in bovine that the function of the microtubule-organizing centre (MTOC; referred to sperm centrosome), which originate the microtubule network and sperm aster formation, is not impaired during the freezing procedure (Hara et al. 2011). Although we did not include an ICSI control group fertilized with non-frozen testicular spermatozoa or analyse the cat testicular sperm centrosome, we suggest that the lower cleavage rate of cat ICSI embryos fertilized with frozen/thawed testicular spermatozoa may be a consequence of immature centrosome in testicular spermatozoa, and not because of deleterious effects of cryopreservation on the spermatozoa. This analysis supported by our previous data (unpublished) showed that the number of cleaved embryos after ICSI with cryopreserved testicular spermatozoa was comparable to that of non-cryopreserved testicular spermatozoa.
Although it was not clear that why development of frozen cleaved-ICSI-embryos to the morula and blastocyst stage was lower than that of fresh embryos, there was a possibility that embryonic cytoskeleton, an essential structure for embryonic development, was affected by freezing and thawing process as report in horse (Tharasanit et al. 2005) and mouse (Sathananthan et al. 1988). However, these results contradicted the finding of Gómez et al. (2003), who reported that there was no difference between development to morula and blastocyst stage of frozen–thawed and fresh cat IVM/IVF embryos. Moreover, that study also reported lower cell numbers at the blastocyst stage in frozen groups compared to fresh control.
In this present study, we treated cat ICSI embryos with forskolin to reduce intracellular lipid content before freezing because of there were several reports mentioned that high lipid content in embryos produced in vitro can increase sensitivity to freezing and thawing (Nagashima et al. 1994; Abe et al. 2002). Moreover, our previous study demonstrated that frozen cat embryos prior treated with forskolin increased cryoresistance and improved in vitro development to the morula and blastocyst stage (Tharasanit and Techakumphu 2011).
Our study supported the results of a previous report in which cryopreserved testicular spermatozoa are able to fertilize oocytes after ICSI and result in embryonic development, and establish pregnancies after transfer of such embryos (Fukunaga et al. 2001). However, spontaneous abortion and foetal death following transfer of frozen–thawed embryos may be due to embryonic damage during the freezing and thawing processes (Edgar et al. 2000).
In summary, our study showed that cryopreserved testicular spermatozoa of domestic cat retained their ability to fertilize oocytes after ICSI. We confirmed that embryos can be frozen with 10% (v/v) ethylene glycol by controlled slow freezing. Frozen–thawed ICSI cleaved embryos maintained the potential to develop to the blastocyst stage in vitro and to full-term kittens in vivo. This study demonstrated that cryopreservation program using cryopreserved testicular tissue as a source of spermatozoa combined with embryo freezing and transfer of embryos into cat recipients is feasible. However, improvement of all these techniques is a prerequisite for preserving the genetic potential of endangered species.
This study was supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Programme (Grant No. PHD/0189/2550) and the National Research University Project of CHE, the Ratchadaphiseksomphot Endowment fund (HR1166IB-55). Ovaries and testes were kindly provided by The Veterinary Public Health Division of The Bangkok Metropolitan Administration, Bangkok, Thailand. We would like to thank Chanapiwat P. for helping with statistical analysis.
Conflicts of interest
None of the authors have any conflicts of interest to declare.
All authors contributed extensively to the work presented in this paper.