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As the demand for cloned embryos and offspring increases, the need arises for the development of nuclear transfer procedures that are improved in both efficiency and ease of operation. Here, we describe a novel zona-free cloning method that doubles the throughput in cloned bovine embryo production over current procedures and generates viable offspring with the same efficiency. Elements of the procedure include zona-free enucleation without a holding pipette, automated fusion of 5-10 oocyte-donor cell pairs and microdrop in vitro culture. Using this system, zona-free embryos were reconstructed from five independent primary cell lines and cultured either singularly (single-IVC) or as aggregates of three (triple-IVC). Blastocysts of transferable quality were obtained at similar rates from zona-free single-IVC, triple-IVC, and control zona-intact embryos (33%, 25%, and 29%, respectively). In a direct comparison, there was no significant difference in development to live calves at term between single-IVC, triple-IVC, and zona-intact embryos derived from the same adult fibroblast line (10%, 13%, and 15%, respectively). This zona-free cloning method could be straightforward for users of conventional cloning procedures to adopt and may prove a simple, fast, and efficient alternative for nuclear cloning of other species as well.

Several studies have shown that both quiescent and proliferating somatic donor cells can be fully reprogrammed after nuclear transfer (NT) and result in viable offspring. So far, however, no comparative study has conclusively demonstrated the relative importance of donor cell cycle stage on nuclear cloning efficiency. Here, we compare two different types of bovine fetal fibroblasts (BFFs) that were synchronized in G 0 , G 1 , and different phases within G 1 . We show that for non-transgenic (non-TG) fibroblasts, serum starvation into G 0 results in a significantly higher percentage of viable calves at term than synchronization in early G 1 or late G 1 . For transgenic fibroblasts, however, cells selected in G 1 show significantly higher development to calves at term and higher post-natal survival to weaning than cells in G 0 . This suggests that it may be necessary to coordinate donor cell type and cell cycle stage to maximize overall cloning efficiency.

Vitrification has become the method of choice for the preservation of in vitro derived embryos of a number of species, and several methods of vitrification have been developed. One such method, the cryoLogic vitrification method (CVM) yields high survival rates of warmed embryos (Lindemans W et al. 2004 Reprod. Fertil. Dev. 16, 174 abst). In this study, the post-warm viability of bovine IVP embryos following either vitrification using CVM or slow freezing using ethylene glycol (EG) was compared. In addition, the survival of embryos following triple transfer to synchronized recipients was measured and the embryo (“e”) and recipient (“r”) contributions to embryo survival was determined using the “er” model for embryo survival (McMillan WH et al. 1998 Theriogenology 50, 1053–1070). Bovine IVP methods were those of van Wagtendonk et al. 2004 Reprod. Fertil. Dev. 16, 214 (abst). On day 7 of culture (Day 0 = IVF), Grade 1 and 2 embryos that had reached at least the late morula stage were selected for vitrification (20% DMSO, 20% ethylene glycol) or freezing in 1.5 M ethylene glycol + 0.1 M sucrose (0.5°C/min to −35°C). Following storage in LN2 for at least 24 h the embryos were thawed, the cryoprotectant removed, and the embryos cultured for 72 h in mSOF medium under 5% CO2, 7% O2, 88% N2. The number of hatching embryos was recorded at 24-h intervals. In addition, blastocyst and expanded blastocyst embryos were thawed and immediately transferred nonsurgically to recipients (three embryos of the same grade to each recipient) on Day 7 of a synchronized cycle (Day 0 = heat). The recipients were ultrasound-scanned for the presence of, and number of, fetuses on Days 35 and 62, respectively. The invitro assessment of 148 CVM and 230 EG frozen embryos indicated that more vitrified than EG embryos hatched by 72 h (73% vs. 62%; CVM vs. EG, χ2 = 4.5, P < 0.05). Overall, more Grade 1 embryos hatched than Grade 2 (74% vs. 60%, χ2 = 7.2, P < 0.01). CVM embryos (105) were triple-transferred to 35 recipients, and EG embryos (30) were triple-transferred to 10 recipients. Recipient pregnancy rates at Day 62 were 80% and 70%, respectively. Overall embryo survival was 38.5% (41% for CVM and 30% for EG). The overall calculated “e” and “r” values were 0.39 and 1.0 (“e”: 0.42 and 1.0, and “r”: 0.31 and 1.0, respectively, CVM and EG groups). Survival rates of CVM embryos to Day 62 (41%) were slightly lower than that previously obtained for fresh embryos produced using an identical IVP procedure (44% – van Wagtendonk AM 2004).

Maximum advantage can be gained from gene discovery programs, by screening embryos carrying the desired genes(s) prior to immediate transfer. This requires an efficient and reliable genotyping system and a method for biopsy preparation that does not compromise subsequent embryo or fetal development. The present study examined the effect of removing a single-cell from the developing 8–16 cell embryo on its subsequent ability to continue development to at least the late morula stage in vitro and then survive following triple transfer to recipients. Abattoir-sourced ovaries were obtained and subjected to IVP as previously described (van Wagtendonk-De Leeuw AM et al. 2004 Reprod. Fert. Dev. 16, 214 abst). Briefly, oocytes were matured in TCM199 +10% FCS, 10 μg/mL FSH, 10 μg/mL LH, 1 μg/mL estradiol, and 100 μM cysteamine under 5% CO2 in air at 38.5°C for 24 h. Percoll-separated sperm (1 × 106/mL) were then co-incubated with the matured oocytes (Day 0) for 24 h with the presumptive zygotes further cultured in mSOF medium under 5%CO2, 7% O2, 88% N2. On Day 4 embryos with a minimum of 8 cells were selected and held at 38.5°C in HEPES-buffered SOF (HSOF) until biopsy at ambient temperature. Embryo biopsy was performed in HSOF medium + 5 μg/mL cytochalasin B. A single cell was removed using a 30 μm biopsy pipette. Both biopsied and control embryos were then further cultured in mSOF in individual wells prepared in a 1% agarose matrix (Peura TT 2003 Cloning Stem Cells 5, 13–24). Embryos were scored for grade and stage of development reached on Day 7, and Grades 1 and 2 blastocysts and expanded blastocysts were transferred to synchronized recipients (three embryos of the same stage and grade to each recipient; n = 50). Fetal number was determined on Day 35 and 62 of gestation. A model for embryo survival was fitted to the data (McMillan WH et al. 1998 Theriogenology 50, 1053–1070) in order to estimate embryo (“e”) and recipient (“r”) contributions to embryo survival. Values were then compared to those determined for control embryos, produced using identical IVP methods (van Wagtendonk-De Leeuw AM et al. 2004 Reprod. Fert. Dev. 16, 214 abst). A total of 358 control and 561 biopsied embryos were cultured. Removal of a single cell did not significantly affect in vitro development (60.1% vs. 56.0%; control vs. biopsy). Day 35 survival of biopsied embryos was 44.7% with calculated “e” and “r” values of 0.48 and 0.94, respectively, which did not differ from control values (44.1%; 0.50 and 0.89). However, by Day 62 fetal survival had significantly decreased with a concomitant drop in “e” but not “r” (30.0%; 0.32 and 0.94, respectively; control “e” and “r” were unchanged). In conclusion, single-cell biopsy of the 8–16-cell embryo does not affect in vitro development or embryo survival to Day 35. However, significant fetal loss occurs by Day 62 that may limit commercial application. Further work is required to elucidate the cause of and overcome fetal loss.

Heavy birth weight, increased calving difficulty, heart function defects, increased perinatal mortality and organ immaturity have been reported for calves produced from IVP embryos compared to those produced from MOET or AI (van Wagtendonk AM et al., 2000 Theriogenology 53, 575–597; Jacobsen H et al., 2002 Anim Reprod Sci 70, 1–11). In this study we examined birth weight (BWT), and blood chemistry at 1 day of age, gestation length and heart function at 7 days, and response to an ACTH challenge at 21 days of calves derived from IVP in a ‘semi-defined’ IVC system (Thompson JG et al., 2000 J. Reprod. Fertil. 118, 47–55) and of contemporary MOET or AI calves. Holstein Friesian (HF) 2- and 3-year-old recipients carrying single HF calves (101 × IVP and 21 × MOET) were monitored in this study. Within 1 day of birth the calves were weighed and a blood sample taken for analysis. At 7d, ultrasound measurement of the left ventricle diastolic diameter (LVEDd) and % ejection fraction (EF%) was determined. Each calf was then transported to a rearing unit. At 3 weeks of age, 30 IVP and 30 control AI calves of the same age were injected i.v. with Synacthen (synthetic ACTH, Ciba Corporation, 0.1 μg kg−1 body weight). Blood samples were collected at −30, 0, 30, 60 and 90 min (0 min = time of injection) for cortisol measurements. There was no difference in BWT for MOET or IVP calves (40.9 ± 4.7 v. 35.6 ± 4.8 kg, respectively). Moreover, gestation lengths (279 days v. 281 days) and calving assistance scores (1.3 v. 1.6) did not differ. Calf mortality at birth was higher for IVP calves (16%) than for MOET calves (5%). All but 7 surviving calves (6 × IVP and 1 × MOET) had high GGT levels at 1 day. Blood chemistry revealed no differences between the calf types, all measures being within normal ranges. For all calves, heart function analysis revealed no abnormalities with mean LVEDd = 4.1 ± 0.6 cm and mean EF% = 78.5 ± 8.4%. All calves exhibited elevated cortisol following ACTH challenge. There was no difference between control and IVP calves for mean cortisol concentration at any time point (0 min, 13.8 ± 5.2; 30 min, 46.6 ± 9.8; 60 min, 42.8 ± 9.9; 90 min, 28.1 ± 8.9 ng mL−1). These data suggest that, unlike calves produced in less defined culture systems, calves produced by IVP in a semi-defined culture system have birth weight and gestation lengths similar to those of MOET calves. Moreover, no abnormalities in organ (heart, adrenal) function were detected. However, of concern was the high number of unexplained deaths for IVP calves. This may be due to an overall lack of vigour in IVP calves that, in an unsupervised calving, results in calf death. More vigilence at calving may be needed to ensure calf survival. The authors thank Juliet Jensen, Waikato Hospital, for ultrasound measurements and David Stewart, Morrinsville Veterinary Services, for calf care. This study was funded by Vialactia Biosciences and FRST.

Factors that affect the viability of in vitro-produced (IVP) embryos are usually evaluated by comparing pregnancy rates of a treatment and a control group. The ‘er’ model of embryo survival (McMillan WH et al., 1998 Theriogenology 50, 1053–1070) utilizes twin embryo transfer to estimate embryo (‘e’) and recipient (‘r’) contributions to embryo survival, and allows the comparison of treatment effects without using a control group, when treatment is the only change in operations. Application of the model to data of contemporaneous single and twin transfer indicates that ‘e’ and ‘r’ are independent of the number of embryos transferred. Thus, twin transfers enable the efficient use of costly recipients while providing meaningful estimates of single embryo survival rates. The objective of this study was to assess the embryo survival rates of fresh IVP embryos of a newly established IVP lab by applying the model to triple transfers and comparing the expected embryo survival rates with those achieved for single transfers. Cumulus-oocyte complexes (COCs) were aspirated from abattoir-derived ovaries of cows of unknown breeds or by ovum pick-up (OPU) from Holstein-Friesian 2- or 3-yr-old donor cows. COCs were matured in 500 μL of TCM199 + 10% FCS (Life Technologies, Auckland, NZ), 10 μg mL−1 FSH and LH (ICPBio, Auckland, NZ), 1 μg mL−1 estradiol (Sigma, Auckland, NZ), 100 μM cysteamine (Sigma) for 24 h under 5% CO2 and then fertilized with 1 × 106 percoll-separated sperm mL−1 from a single bull (Tervit HR and Pugh PA, 2000 14th ICAR 18, 37(abst)). Twenty-four h after insemination, presumptive zygotes were transferred into 500 μL mSOF (Pugh A et al., 2001 Theriogenology 55, 314 (abst)) and cultured for 4 days under humidified 5% CO2, 7% O2 and 88% N2. On Day 4, cleaved embryos were transferred into fresh culture medium and culture continued for a further 3 days under the same conditions. Embryo stage and grade were evaluated on Day 7 of culture. Grades 1, 2 and 3 (IETS manual, 2002) compact morulae and blastocysts produced from abattoir-derived COCs were transferred in triplets, while grades 1 and 2 compact morulae and blastocysts from OPU-derived COCs were transferred singly, in 0.25 mL insemination straws into synchronized Holstein-Friesian heifers. Recipients received a CIDR (CIDR Cattle Insert, Pharmacia, Auckland, NZ) at Day −12 followed by a prostaglandin (Estroplan, Parnell Laboratories, Auckland, NZ ) injection at Day −6. CIDRs were removed at Day −2, followed by estrus at Day 0 (= day of IVF). Embryos were transferred on Day 7 and recipients received a CIDR after transfer (ET). CIDRs were removed at Day 19 to synchronize any returns. Two experienced practitioners performed all the transfers. Pregnancies (single transfers) and number of live fetuses (triple transfers) were confirmed at Days 60 and 42, respectively. Pregnancies were terminated between Days 62 and 65 by two prostaglandin injections 48 h apart. A total of 76 single transfers resulted in 36 pregnancies (47.4%, binomial SD 5.7%). A total of 75 triple transfers (225 embryos) resulted in 98 viable fetuses (44%) and 58 pregnant recipients (77.3%). For triple transfers, the estimates for ‘e’ and ‘r’ were 0.50 and 0.89, respectively, with the product yielding an expected triple embryo survival rate of 44.1%. The actual distribution of 17, 23, 30 and 5 recipients carrying 0, 1, 2, or 3 fetuses, respectively, was not significantly different from the expected values of 16, 25, 25 and 8 estimated from the model (chi-square = 2.49, NS). Estimates for ‘e’ and ‘r’ were not significantly different when combined single and triple data were included in the model (‘e’ = 0.55 and ‘r’ = 0.90), indicating that embryo survival is independent of the number of embryos transferred. Results indicate that multiple transfers do increase pregnancy rate (from 47.4 to 77.3%), but not embryo survival posttransfer (44.1 v. 47.4%). Although single ET was done with OPU-derived embryos and triple with slaughterhouse-derived embryos and results are not strictly comparable, the similarity of estimates for ‘e’ suggests that using the same in vitro-embryo assessment criteria resulted in embryos of similar intrinsic viability from the two sources. In the near future, we will perform triple transfers of cryopreserved IVP embryos and use the model to estimate embryo and recipient contributions to embryo survival of frozen IVP embryos, without using a fresh control. We will continue to build a dataset based on triple and single transfers to further assess the effect on embryo survival rates of triple and single transfers.