The aim of the present study was to assess the in vivo competence of in vitro-produced sika deer (Cervus nippon nippon) embryos after freezing–thawing and transfer into red deer (Cervus elaphus hippelaphus) recipients. During the breeding season, 11 adult sika deer hinds were synchronized as oocyte donors with an intravaginal sponge (45 mg of fluorogestone acetate, FGA) inserted for 12 days and removed immediately after laparoscopic ovum pick-up (LOPU), and renewed after 3 days. Ovarian stimulation was induced with an i.m. injection of 75 µg of cloprostenol (Estrumate) given on Day 8, followed by 3 i.m. injections of 0.1, 0.1, and 0.05 IU of ovine FSH (Ovagen) on Days 10 and 11 at 12-h intervals. On Day 12, hinds were anesthetized and oocytes were collected by LOPU from follicles >2 mm using an 18 G needle under moderate vacuum. COC were recovered and morphologically evaluated for quality (graded from 1 to 5). COC were then submitted to in vitro maturation, fertilization, and culture (IVM, IVF, and IVC) as described previously (Locatelli Y et al. 2005 Theriogenology 64, 1729–1739). For IVC, embryos were co-cultured with a monolayer of ovine oviduct epithelial cells in synthetic oviduct fluid medium supplemented with 10% FCS. On Day 8 post-insemination, all sika deer embryos at the blastocyst stage were cryopreserved via a standard bovine slow-freezing protocol. Of 44 LOPU sessions performed during the 1-month study, an average of 7.5 � 0.38 follicles were aspirated (mean � SEM), allowing the recovery of 3.65 � 0.38 COC per hind and per session, of which 80.0% were suitable for IVM (grades 1 and 2). Of 142 oocytes recovered, 57 cleaved after IVF (40.1%), and 14 embryos (24.6% of cleaved) reached the blastocyst stage after 8 days. At the end of the breeding season, 7 adult red deer hinds were synchronized as embryo recipients by inserting 2 intravaginal sponges per female (90 mg of FGA), for 13 days. Injections (i.m.) of 400 IU of eCG and 125 µg of cloprostenol (Estrumate) were administered 72 h before sponge removal. At Day 8 after sponge removal, straws containing frozen embryos were thawed and cryoprotectant was removed as described previously (see Locatelli Y et al. 2005 Theriogenology 64, 1729–1739). Two sika deer embryos were surgically transferred into uterine horn (unilaterally) of each red deer recipient. One of 7 red deer recipients was diagnosed pregnant by ultrasonography on Day 56. A healthy male sika deer fawn was born unassisted after 224 days of gestation. No complications were observed in initial recognition of the sika deer fawn by the red deer surrogate mother, nor in subsequent interactions. To our knowledge, this is the first report of an interspecific pregnancy obtained after in vitro embryo production and embryo transfer in deer species. In conclusion, interspecific embryo transfer after IVP may represent a useful tool for the preservation and amplification of captive residual populations of endangered deer species. Further studies are required to increase the rate of cleavage after LOPU-IVF as well as viability of frozen–thawed IVP embryos.
Impact of interspecific relations between native red deer (Cervus elaphus) and introduced sika deer (Cervus nippon) on their rutting season in the Doupovské hory Mts.