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.

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The objective of this study was to determine the effects of genetic manipulation, cell type, and culture conditions on developmental potential of bovine nuclear transfer (NT) embryos. Ovum pickup (OPU) technology was developed to obtain the oocytes for NT. A total 4044 cumulus-oocyte complexes (COCs) were obtained during 492 OPU sessions, with an average of 8.2 COCs recovered each session. Cultured granulosa cells (CGC), bovine fetal (150 days) oviduct epidermic cells (FOEC), and adult ear skin fibroblasts (ASFC) were used as donor cells for NT and were transfected with the expression vector including human FIX coding sequence directed by goat β-casein promoter and neomycin gene. The cells were screened under 800 µg mL −1 G418 for 10-14 days until the apperance of a "mono-colony" of cells which were then picked. Each cell population was expanded by consecutive passage culture under 300 µg mL −1 G418 until used for NT, ensuring that the majority of cells were transgenic. Oocytes were enucleated at 20 h post-maturation and a single donor cell was transferred into the perivitelline space of a recipient oocyte. After fusion and activation, the reconstructed embryos were co-cultured with vero cells in B2 medium for 7 days. NT efficiency between primary granulosa cells (PGC) without in vitro culture and CGC, as well as among CGC, FOEC and ASFC that were transfected with exogenous DNA (named TCGC, TFOEC, TASFC, respectively), were compared (Table 1). Differences between groups were verified by chi-square test using SAS 6.12 (SAS Institute, Inc., Cary, NC, USA) program. CGCs presented a higher fusion rate (P < 0.01) for reconstructed embryos and higher development to the blastocyst stage for NT embryos than did PGC (67% vs. 54% and 41% vs. 21%, respectively). There were no significant differences (P > 0.05) in cleavage rate (65%, 71%, and 69%, respectively) and development to the blastocyst stage for NT embryos (36%, 30% and 40%, respectively) for TCGC, TFOEC, and TASFC. A total of 86 blastocysts were selected for transfer into uteri of 86 cows, resulting in 26 pregnancies (30%) at 60 days by ultrasound scanning. Among these, 12 cows remain pregnant and 14 have aborted. The results indicated that oocytes recovered from OPU can be successfully used for NT with development to the blasocyst stage. PGC, CGC, FOEC, and ASFC can all be used for generating transgenic cattle by NT, although this needs to be verified by the birth of live calves. The cryopreservation of sperm has contributed greatly to animal breeding and reproduction. This study was designed to examine the effect of raffinose, sucrose, and trehalose as cryoprotectants for freezing of mouse sperm. The cryoprotectant solution (CPA) consisting of 3% skim milk (Skim Milk dehydrated, Bacto, Difco, Seoul, Korea) as buffer or extender was prepared and supplemented with 0.3 M raffinose (D[+]raffinose pentahydrate, Sigma) or sucrose or trehalose as non-permeating cryoprotectants. Sperm samples for cryopreservation were collected from caudae epididymides and vas deferens ...

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 500μL of TCM199+10% FCS (Life Technologies, Auckland, NZ), 10μgmL−1 FSH and LH (ICPBio, Auckland, NZ), 1μgmL−1 estradiol (Sigma, Auckland, NZ), 100μM cysteamine (Sigma) for 24h 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 500μL 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 0.25mL 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 48h 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.

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, 0.1μgkg−1 body weight). Blood samples were collected at −30, 0, 30, 60 and 90min (0min=time of injection) for cortisol measurements. There was no difference in BWT for MOET or IVP calves (40.9±4.7 v. 35.6±4.8kg, 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±0.6cm 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 (0min, 13.8±5.2; 30min, 46.6±9.8; 60min, 42.8±9.9; 90min, 28.1±8.9ngmL−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.