Debra K. Berg

The seasonal demand for farmed venison in New Zealand has necessitated the concentration of red deer breeding into the first month of the four-month breeding season. Because of this constraint it is difficult to obtain enough in vitro-produced blastocysts for transfer. Successful cryopreservation would enable embryos produced and stored throughout the breeding season to be available for transfer the following year. In vitro red deer calves have been successfully produced after trans-cervical transfers in a limited number of red deer (Berg DK et al. 2004 Reprod. Fert. Dev. 16, 201 abst). We determined the viability of frozen blastocysts following trans-cervical transfer to recipient hinds using the direct-thaw method. In two replications, abattoir derived red deer COCs were selected and matured in vitro (Berg DK et al. 2002 Ani. Reprod. Sci. 70, 85–98). Oocytes were randomly divided into two groups and fertilized with either red deer sperm using IVF-Deer SOF (DSOF), or wapiti sperm using IVF-SOF. All presumptive zygotes were cultured for 6 days in DSOF (Beaumont SE et al. 2004 Reprod. Fert. Dev. 16, 268 abst). Cleavage was recorded on Day 4 and embryos were evaluated on Day 7. Grade 1 and 2 blastocysts were selected and equilibrated in 1.5 M ethylene glycol with 0.1 M sucrose, frozen from -5 to -38°C at a rate of 0.3°C per min and plunged into liquid nitrogen. Twenty synchronized farmed deer hinds (13 red deer to receive red deer blastocysts, and 7 F1 wapiti/red hybrids to receive F1 blastocysts) were prepared for transfer (Berg DK et al. 2003 Theriogenology 59, 189–205). Only Grade 1 blastocysts were selected for transfer. Straws were thawed for 5 s in air, immersed in a 30°C water bath for 20 s, directly diluted, and loaded into cattle transfer pistolettes. Each embryo was deposited in the uterine horn. A modified pistolette, fitted with a Mariensee tip (Minitüb, 84184 Tiefenbach, Germany) was used to dilate difficult cervices (n = 4). Pregnancies were confirmed by ultrasonography on Day 35. Results were evaluated using chi-square analysis. Embryo cleavage rates ranged from 74 to 85% and were not different between the two sires. Blastocyst development rates (from cleaved zygotes) were similar for both sires; wapiti 15% (43/279) and red deer 14% (34/246). A total of 24 wapiti/red hybrid and 17 red deer blastocysts were frozen. Eighteen of 20 hinds (90%) received embryos, 11/13 red deer receiving red deer embryos and 7/7 F1 wapiti/red hybrids receiving F1 wapiti/red hybrid embryos. The cervices of two red deer hinds were impenetrable. Pregnancy rates were not different between the 2 groups of recipients, with 29% (2/7) of the wapiti hybrids and 45% (5/11) of red deer confirmed pregnant. These preliminary results demonstrate, for the first time, that farmed deer pregnancies can be established from frozen in vitro-produced embryos after direct-thaw and trans-cervical transfer to synchronized hinds.

Deer Synthetic Oviduct Fluid medium (DSOF;; Berg and Asher 2003 Theriogenology 59, 189–205), based upon the composition of red deer oviduct fluid, has been shown to support routine in vitro fertilization and blastocyst development (15%) of in vitro-matured red deer oocytes without the use of somatic cell co-culture or serum supplementation. However, pregnancy establishment and fetal survival remained to be determined. The objectives of this study were to transfer in vitro red deer blastocysts produced by the DSOF culture system, follow fetal survival through calving and investigate trans-cervical embryo transfer for red deer. Red deer hinds of mixed age were synchronized using a 12-day CIDR (Pharmacia & Upjohn, Auckland, NZ) synchrony program (Berg DK et al. 2002 Ani. Reprod. Sci. 70, 85–98). Onset of estrus was synchronized to the day of IVF and embryos were transferred 7 days later. in vitro red deer blastocysts were produced after aspirating oocytes from abattoir-sourced ovaries. Selected COCs were matured and fertilized and presumptive zygotes cultured in vitro (Berg and Asher 2003 Theriogenology 59, 189–205) with modified Ca2+ concentrations: 3.0 mM and 1.5 mM for early and late DSOF, respectively. in vitro blastocyst development was 14.7% (21/143) on Day 7 and 22.4% (32/143) on Day 8. Ten blastocysts (grade 1 and 2) were selected for transfer on Day 7 (post-IVF) and placed into Emcare embryo holding medium (ICPbio, Auckland, NZ). Blastocysts were loaded into 0.25-cc straws (n = 5) or tom cat catheters (n = 5) and transported to the Ruakura Deer Unit at 25°C. Hinds were restrained and sedated as described for OPU (Berg and Asher 2003 Theriogenology 59, 189–205) and an attempt was made to pass a cattle transfer pistolette through the cervix. If unsuccessful, the hind underwent laparoscopic uterine transfer. Serial serum progesterone values diagnosed pregnancies at Day 21 and fetal survival was determined using rectal ultrasonography on Day 35, 45, 60 and 90. Seven single-embryo transfers were completed;; 2 of 5 trans-cervical attempts and 5 using the laparoscopic method. Serum progesterone levels confirmed 57% (4/7) of the hinds were pregnant on Day 21;; 2/2 (100%) from trans-cervical and 2/5 (40%) from laparoscopic transfers. No pregnancy losses occurred after Day 21. Four calves, 1 male and 3 female, were born unassisted after 230 to 233 days of gestation. Birth weights ranged from 7.3 to 10 kg. Our results indicate that in vitro red deer blastocysts produced using the DSOF culture system can establish pregnancies after transfer and result in normal healthy calves.