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Nitrogen cycling microbes, including nitrite-oxidizing bacteria (NOB), perform critical ecosystem functions that help mitigate anthropogenic stresses and maintain ecosystem health. Activity of these beneficial nitrogen cycling microbes is dictated in part by the microorganisms’ response to physicochemical conditions, such as temperature, pH, and nutrient availability. NOB from the newly described Candidatus Nitrotoga genus have been detected in a wide range of habitats across the globe, yet only a few organisms within the genus have been physiologically characterized. For freshwater systems where NOB are critical for supporting aquatic life, Ca. Nitrotoga have been previously detected but little is known about the physiological potential of these organisms or their response to changing environmental conditions. Here, we determined functional response to environmental change for a representative freshwater species of Ca. Nitrotoga (Ca. Nitrotoga sp. CP45, enriched from a Colorado river). The physiological findings demonstrated that CP45 maintained nitrite oxidation at pH levels of 5–8, at temperatures from 4 to 28°C, and when incubated in the dark. Light exposure and elevated temperature (30°C) completely halted nitrite oxidation. Ca. Nitrotoga sp. CP45 maintained nitrite oxidation upon exposure to four different antibiotics, and potential rates of nitrite oxidation by river sediment communities were also resilient to antibiotic stress. We explored the Ca. Nitrotoga sp. CP45 genome to make predictions about adaptations to enable survival under specific conditions. Overall, these results contribute to our understanding of the versatility of a representative freshwater Ca. Nitrotoga sp. Identifying the specific environmental conditions that maximize NOB metabolic rates may ultimately direct future management decisions aimed at restoring impacted systems.

Study hypothesis Maternal ageing and ovarian stimulation result in the accumulation of mitochondrial DNA (mtDNA) deletions and heteroplasmy in individual oocytes from a novel bovine model for human assisted reproductive technology (ART). Study finding The levels of mtDNA deletions detected in oocytes increased with ovarian ageing. Low levels of mtDNA heteroplasmy were apparent across oocytes and no relationship was identified with respect to ovarian ageing or ovarian stimulation. What is known already Oocyte quality decreases with ovarian ageing and it is postulated that the mtDNA may have a role in this decline. The impact of ovarian stimulation on oocyte quality is poorly understood. Human studies investigating these effects are often limited by the use of low quality oocytes and embryos, variation in age and ovarian stimulation regimens within the patients studied, as well as genetic and environmental variability. Further, no study has investigated mtDNA heteroplasmy in individual oocytes using next-generation sequencing (NGS), and little is known about whether the oocyte accumulates heteroplasmic mtDNA mutations following ageing or ovarian stimulation. Study design, samples/materials, methods A novel bovine model for the effect of stimulation and age in human ART was undertaken using cows generated by somatic cell nuclear transfer (SCNT) from one founder, to produce a homogeneous population with reduced genetic and environmental variability. Oocytes and somatic tissues were collected from young (3 years of age; n = 4 females) and old (10 years of age; n = 5 females) cow clones following multiple natural ovarian cycles, as well as oocytes following multiple mild (FSH only) and standard (based on human a long GnRH agonist protocol) ovarian stimulation cycles. In addition, oocytes were recovered in a natural cycle from naturally conceived cows aged 4-13.5 years (n = 10) to provide a heterogeneous cohort for mtDNA deletion studies. The presence or absence of mtDNA deletions were investigated using long-range PCR in individual oocytes (n = 62). To determine the detection threshold for mtDNA heteroplasmy levels in individual oocytes, a novel NGS methodology was validated; artificial mixtures of the Bos taurus and Bos indicus mitochondrial genome were generated at 1, 2, 5, 15 and 50% ratios to experimentally mimic different levels of heteroplasmy. This NGS methodology was then employed to determine mtDNA heteroplasmy levels in single oocytes (n = 24). Oocyte mtDNA deletion and heteroplasmy data were analysed by binary logistic regression with respect to the effects of ovarian ageing and ovarian stimulation regimens. Main results and the role of chance Ovarian ageing, but not ovarian stimulation, increased the number of oocytes exhibiting mtDNA deletions (P = 0.04). A minimum mtDNA heteroplasmy level of 2% was validated as a sensitive (97-100%) threshold for variant detection in individual oocytes using NGS. Few mtDNA heteroplasmies were detected across the individual oocytes, with only 15 oocyte-specific variants confined to two of the 24 oocytes studied. There was no relationship (P > 0.05) evident between ovarian ageing or ovarian stimulation and the presence of mtDNA heteroplasmies. Limitations, reason for caution The low number of oocytes collected from the natural ovarian cycles limited the analysis. Fertilization and developmental potential of the oocytes was not assessed as the oocytes were destroyed for mtDNA deletion and heteroplasmy analysis. Wider implications of the findings If the findings of this model apply to the human, this study suggests that the incidence of mtDNA deletions increases with age, but not with degree of ovarian stimulation, while the frequency of mtDNA heteroplasmies may be low regardless of ovarian ageing or level of ovarian stimulation. Study funding and competing interests Funding was provided by Fertility Associates, the Nurture Foundation for Reproductive Research, the Fertility Society of Australia, and the Auckland Medical Research Foundation. J.C.P. is a shareholder of Fertility Associates and M.P.G. received a fellowship from Fertility Associates. The other authors of this manuscript declare no conflict of interest that could be perceived as prejudicing the impartiality of the reported research.

The focus of this study was to investigate the effect of subclinical endometritis (scEndo) on ovarian follicular steroid concentrations in early postpartum pasture-fed dairy cows. Mixed-age lactating dairy cows (n = 169) were examined to ascertain uterine health status on d 21 postpartum (±3 d). From this herd, a cohort of scEndo and uninfected cows (n = 47) were selected using uterine cytology to determine scEndo. To ensure cows with scEndo were selected for the study, a conservative threshold [>18% polymorphonuclear (PMN) cells among uterine nucleated cells] was chosen as a selection threshold. Ovarian follicular dynamics were assessed by ultrasonography on d 21, 42, and 63 postpartum. On the latter 2 d, all follicles >4 mm in diameter were ablated, and 4 d later, the largest (F1) and second largest (F2) follicles were measured and their follicular fluid aspirated. Hematological variables and plasma metabolites were measured also on these days to further characterize scEndo cows. On d 21, the prevalence of scEndo was approximately 9% in this herd; by d 42 infections had self-resolved in the majority (81%) of those cows classified as having scEndo on d 21. The scEndo cows had a delayed return to cyclicity; however, no effect was evident on ovarian follicle size or growth rate. Weeks after scEndo had self-resolved and cyclicity was restored, decreased (P = 0.07) testosterone and increased (P = 0.07) cortisol concentrations were evident in F1 follicles of scEndo compared with uninfected cows. Progesterone concentrations of F1 increased (P < 0.05) in 11- to 16-mm diameter follicles of scEndo cows, whereas estradiol, androstendione, and dehydroepiandrosterone concentrations were decreased (P < 0.05) in F1 8- to 10-mm diameter follicles of scEndo cows. These 3 steroids also differed (P < 0.05) between F1 follicle size categories of scEndo but not uninfected cows. On d 21, mean plasma albumin concentration was decreased (P = 0.02) in scEndo cows. In summary, early postpartum scEndo had surprisingly long-term influences on the steroid concentrations of ovarian follicles long after infections had self-resolved. This is likely to affect oocyte quality and may partially explain the reduced conception rates and longer interval between calving and conception that are often associated with scEndo, although more detailed investigations are required to substantiate this theory.

Somatic cell nuclear transfer (NT)-specific effects on postblastocyst early cattle embryogenesis were investigated by comparison to in vitro-produced (IVP) embryos grown under identical conditions to embryonic days (E) 14 and 15. Recipient effects were excluded by transferring mixed batches of NT and IVP embryos into each cow. Embryo recovery rates, proportions with an epiblast and embryo, as well as epiblast dimensions did not differ between NT and IVP embryos. A developmental expression profile was determined for nine trophoblast markers, two inner cell mass (ICM)/epiblast markers, and E-cadherin at nine time points between E7 and E26, providing a molecular gene signature assay for developmental progression. Gene expression levels for these genes (Cdx2, Elf5, Mash2, Ifn-tau, Furin, Kunitz1, Pag11, Gata3, Oct4 and Ifitm3) were equal in NT and IVP embryos of equivalent length. Furthermore, the average residual deviation of all 10 genes did not differ significantly suggesting an overall "normality" in gene expression of E14/15 NT embryos. The absence of NT-specific defects during the second, highly selective, week of cattle embryogenesis is interpreted as supportive for the view that NT-associated defects are predominantly of an epigenetic nature.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are essential for ovarian follicular growth in sheep, whereas only GDF9 is essential in mice suggesting that the roles of these oocyte-derived growth factors differ among species. At present, however, there is only limited information on the action of BMP15 and GDF9 in other species. Thus, the aim of this experiment was to determine the effect of neutralizing GDF9 and/or BMP15in vivoon ovarian follicular development and ovulation rate in cattle through active immunization using the mature regions of the proteins or peptides from the N-terminal area of mature regions. Immunization with the BMP15 peptide, with or without GDF9 peptide, significantly altered (increased or decreased) ovulation rate. In some animals, there were no functional corpora lutea (CL), whereas in others up to four CL were observed. From morphometric examination of the ovaries, immunization with GDF9 and/or BMP15 reduced the level of ovarian follicular development as assessed by a reduced proportion of the ovarian section occupied by antral follicles. In addition, immunization against GDF9 and/or BMP15 peptides reduced follicular size to

Study question Does maternal ageing and ovarian stimulation alter mitochondrial DNA (mtDNA) copy number and gene expression of oocytes and cumulus cells from a novel bovine model for human IVF? Summary answer Oocytes collected from females with identical nuclear genetics show decreased mtDNA copy number and increased expression of an endoplasmic reticulum (ER) stress gene with repect to ovarian stimulation, whilst differences in the expression of genes involved in mitochondrial function, antioxidant protection and apoptosis were evident in relation to maternal ageing and the degree of ovarian stimulation in cumulus cells. What is known already Oocyte quality declines with advancing maternal age; however, the underlying mechanism, as well as the effects of ovarian stimulation are poorly understood. Human studies investigating these effects are often limited by differences in age and ovarian stimulation regimens within a patient cohort, as well as genetic and environmental variability. Study design, size, duration A novel bovine cross-sectional maternal age model for human IVF was undertaken. Follicles were aspirated from young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian clones following multiple unstimulated, mild and standard ovarian stimulation cycles. These bovine cloned females were generated by the process of somatic cell nuclear transfer (SCNT) from the same founder and represent a homogeneous population with reduced genetic and environmental variability. Maternal age and ovarian stimulation effects were investigated in relation to mtDNA copy number, and the expression of 19 genes involved in mitochondrial function, antioxidant protection, oocyte-cumulus cell signalling and follicle development in both oocytes and cumulus cells. Materials, setting, methods Young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian bovine clones were maintained as one herd. Stimulation cycles were based on the long GnRH agonist down-regulation regimen used in human fertility clinics. Follicle growth rates, numbers and diameters were monitored by ultrasonography and aspirated when the lead follicles were >14 mm in diameter. Follicle characteristics were analysed using a mixed model procedure. Quantitative PCR (qPCR) was used to determine mtDNA copy number and reverse transcriptase-qPCR (RT-qPCR) was used to measure gene expression in oocytes and cumulus cells. Main results and the role of chance Method of ovarian stimulation (P = 0.04), but not maternal age (P > 0.1), was associated with a lower mtDNA copy number in oocytes. Neither factor affected mtDNA copy number in cumulus cells. In oocytes, maternal age had no effect on gene expression; however, ovarian stimulation in older females increased the expression of GRP78 (P = 0.02), a gene involved in ER stress. In cumulus cells, increasing maternal age was associated with the higher expression of genes involved in mitochondrial maintenance (TXN2 P = 0.008 and TFAM P = 0.03), whereas ovarian stimulation decreased the expression of genes involved in mitochondrial oxidative stress and apoptosis (TXN2 P = 0.002, PRDX3 P = 0.03 and BAX P = 0.03). Limitations, reason for caution The low number of oocyte and cumulus cell samples collected from the unstimulated cycles limited the analysis. Fertilization and developmental potential of the oocytes was not assessed because these were used for mtDNA and gene expression quantification. Wider implications of the findings Delineation of the independent effects of maternal age and ovarian stimulation regimen on mtDNA copy number gene expression in oocytes and cumulus cells was enabled by the removal of genetic and environmental variability in this bovine model for human IVF. Therefore, these extend upon previous knowledge and findings provide relevant insights that are applicable for improving human ovarian stimulation regimens. Study funding/competing interests Funding was provided by Fertility Associates and the University of Auckland. J.C.P. is a shareholder of Fertility Associates and M.P.G. received a fellowship from Fertility Associates. The other authors of this manuscript declare no conflict of interest that could be perceived as prejudicing the impartiality of the reported research.

Previous work suggests that a number of factors such as follicle size, day of estrous cycle, and level of atresia influence the developmental potential of bovine oocytes in vitro. To understand better the interactions of these factors, 1299 follicles > or =3 mm in diameter were dissected from ovaries of synchronized dairy cows on four days (d2, d7, d10, or d15) during the estrous cycle. The oocyte from each follicle was collected and matured, fertilized, and cultured singly to d8 (d0 of culture = IVF). Control follicles (302) were similarly dissected and processed from an ovary pair randomly collected from the abattoir on each slaughter day. Results showed that development to blastocyst was greater in oocytes collected during phases of follicular growth (d2 and d10) than those collected during phases of follicular dominance (d7 and d15; 44.8% vs. 36.0%, respectively: P or =13 mm). Oocyte competence tended to increase with increasing follicle size (P < 0.1). Follicular cells from follicles containing an oocyte that developed to morula or greater by d8 (484 samples) were analyzed by flow cytometry to measure the level of apoptosis. Results showed an increase in mean percent apoptotic cells in subordinate follicles (18.65 +/- 0.86 over all size categories), particularly those of medium size (25.55 +/- 2.2 for 6-8 mm size follicles; P < 0.001), during the dominance phase compared to growth phase (9.25 +/- 0.95 over all sizes; P < 0.05). These results show a significant affect of the stage of estrous cycle on both oocyte competence and levels of follicular atresia.

The uterus provides the nurturing environment that supports the growth of the early preimplantation bovine conceptus. To determine critical time points of uterine influence, in vitro-produced Day 7 blastocysts were transferred into synchronous (Day 7) uteri and asynchronous uteri (Days 5 or 9). Embryo growth was evaluated 7 and 15 days after transfer and compared with that of embryos generated by AI. Conceptuses recovered from asynchronous Day 9 transfers were fourfold larger than synchronous transfer or gestational Day 14 AI conceptuses; by 15 days after transfer, differences were less marked. Two-dimensional gel electrophoresis was used to compare the histotroph protein composition of uterine luminal flushings (ULF) on Days 5 and 9 after oestrous to determine any protein differences that would promote embryo growth. The ULF were collected by serially flushing the uteri of the same heifers and mature cows at different times of the cycle. Ten proteins that differed in abundance between Day 5 and 9 were identified by mass spectrometry. Three, namely phosphoserine aminotransferase 1, purine nucleoside phosphorylase and aldose reductase, were verified by western blot analysis as more abundant on Day 9 (P

The focus of this study was to investigate the effect of subclinical endometritis (scEndo) on ovarian follicular steroid concentrations in early postpartum pasture-fed dairy cows. Mixed-age lactating dairy cows (n = 169) were examined to ascertain uterine health status on d 21 postpartum (±3 d). From this herd, a cohort of scEndo and uninfected cows (n = 47) were selected using uterine cytology to determine scEndo. To ensure cows with scEndo were selected for the study, a conservative threshold [18% polymorphonuclear (PMN) cells among uterine nucleated cells] was chosen as a selection threshold. Ovarian follicular dynamics were assessed by ultrasonography on d 21, 42, and 63 postpartum. On the latter 2 d, all follicles4 mm in diameter were ablated, and 4 d later, the largest (F1) and second largest (F2) follicles were measured and their follicular fluid aspirated. Hematological variables and plasma metabolites were measured also on these days to further characterize scEndo cows. On d 21, the prevalence of scEndo was approximately 9% in this herd; by d 42 infections had self-resolved in the majority (81%) of those cows classified as having scEndo on d 21. The scEndo cows had a delayed return to cyclicity; however, no effect was evident on ovarian follicle size or growth rate. Weeks after scEndo had self-resolved and cyclicity was restored, decreased (P = 0.07) testosterone and increased (P = 0.07) cortisol concentrations were evident in F1 follicles of scEndo compared with uninfected cows. Progesterone concentrations of F1 increased (P0.05) in 11- to 16-mm diameter follicles of scEndo cows, whereas estradiol, androstendione, and dehydroepiandrosterone concentrations were decreased (P0.05) in F1 8- to 10-mm diameter follicles of scEndo cows. These 3 steroids also differed (P0.05) between F1 follicle size categories of scEndo but not uninfected cows. On d 21, mean plasma albumin concentration was decreased (P = 0.02) in scEndo cows. In summary, early postpartum scEndo had surprisingly long-term influences on the steroid concentrations of ovarian follicles long after infections had self-resolved. This is likely to affect oocyte quality and may partially explain the reduced conception rates and longer interval between calving and conception that are often associated with scEndo, although more detailed investigations are required to substantiate this theory.

Embryo loss between embryonic Days 7 and 16 (Day 0=day of IVF) in nonlactating cattle, Bos taurus, was analyzed using transfer of 2449 (in groups of 3 to 30) in vitro-produced (IVP) blastocysts. In 152 transfers, pregnancy losses attributable solely to recipient failings amounted to between 6% (beef heifers) and 16% (parous dairy cows), of which 3% were caused by uterine infections. Neither season, year, nor the age of the embryos on retrieval affected pregnancy rates. The latter observation indicated that the reason that a recipient failed to retain embryos was already present at the time of transfer. Notably, the proportion of embryos recovered decreased (P=0.03) as more embryos were transferred, particularly at later stages (Day 14, P

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.

In cattle, oocytes obtained from follicles smaller than 3 mm in diameter can undergo maturation in vitro, progressing to MII and undergoing fertilization, but are developmentally incompetent. Cytoplasts were prepared from in vitro matured oocytes aspirated from small (1-3 mm) or large (6-12 mm) follicles and fused to serum starved mural granulosa cells. Following activation, reconstructed embryos were cultured for 7 days and classified G1 to G4, before being processed for nuclei counting or transferred to synchronized recipients. Oocytes from small follicles had lower rates of polar body extrusion (59.6 vs. 69%; 731/1230 vs. 608/857) and fusion (71.4 vs. 78.8%; 360/497 vs. 364/465; P < 0.06). There were no differences in total rate of blastocysts development (60 vs. 59.8%; small vs. large), or any grade classification. A significant interaction was detected between follicle size and embryo grade with G3 embryos from small follicles having a greater cell number. Developmental competence of G1 and G2 embryos did not differ at day 27 (48 vs. 46%; 16/33 vs. 17/37; small vs. large). Although there were no differences in fetal size between the two groups, differences in allantois length (53 vs. 86 mm; small vs. large; P < 0.002) and allantois width (9.5 vs. 13 mm; small vs. large; P < 0.06) were seen. No differences in survival to term (2/13 in each group) were observed. These results indicate that cytoplasts from follicles of 1-3 and 6-12 mm in diameter are equally developmentally competent when used in a nuclear transfer procedure.

Dairy animals provide an attractive production platform for biosimilar antibodies due to the high protein production capacity of the mammary gland and easy access to milk. Goats are well suited for this approach as they offer a relatively short gestation time and good milk yield and are fully validated for the production of recombinant therapeutics. To generate transgenic goats capable of producing a biosimilar version of cetuximab, a monoclonal antibody for epidermal growth factor receptor and approved for the treatment of specific cancers, we co-transfected primary female fetal fibroblasts with expression constructs for cetuximab’s heavy (HC) and light (LC) chains under the control of the goat β-casein regulatory sequences. Beta-globin insulators were added to both transgenes to minimize position effects, and an antibiotic selection marker was placed downstream of the HC transgene sequences to allow for the isolation of stable transgenic cell clones. Selected cell clones were screened by PCR for the presence of both transgenes. Positive cell clones were analysed by Southern blot with a β-casein-specific probe. This allowed for the simultaneous detection of both transgenes, and the endogenous β-casein gene served as a standard to determine transgene copy numbers. The cell clones showed a broad range of copy numbers, from single copy insertions to >100 copies for the HC and LC transgenes. Interestingly, most of the cell clones had more LC than HC transgene copies. Ten cell clones were selected to generate transgenic founders using somatic cell nuclear transfer. We were able to produce 43 live kids from 9 cell lines following transfer of between 26 and 153 one- and two-cell embryos per line into recipients (range of 4 to 15 embryos per recipient). The one cell clone that we used unsuccessfully had the lowest number of transferred embryos (11). The efficiency for the production of live kids per transferred embryos was, on average, 5.1% (range of 1.0 to 9.7%). Kids from 5 lines were hormonally induced into lactation at the age of 10 weeks. Two lines with high copy numbers (≥30) produced either no or only a few drops of milk, whereas the lines with ≤25 transgene copies gave up to several milliliters of milk per day. Western analyses confirmed cetuximab production levels of 15 g L–1 in 2 of the lines with ≤25 transgene copies and ~45 g L–1 in a high copy number line; one low copy number line showed good HC but very low LC expression. Our data demonstrate that cetuximab can be produced in significant quantities in transgenic goats. Future work is aimed at determining production levels under natural lactation conditions and characterising glycosylation patterns to fully understand the pharmacodynamic properties of the antibody. Supported by GTC, the NZ Ministry of Science and Innovation and AgResearch.

The effects of activation method and timing between fusion and activation in goat somatic cell nuclear transfer (NT) were investigated. In vivo-ovulated oocytes were surgically flushed from donors 54 to 62 h after CIDR withdrawal in the breeding season and enucleated after brief ultraviolet exposure. Transfected fibroblasts and epithelial cells from 4 clonal strains were serum-starved for 4 days before NT. Two direct current electric pulses (2 kV cm–1 each for 10 μs) were used to induce fusion and simultaneous activation. Forty-five minutes after successful fusion, reconstructs received a second activation stimulus delivered either electrically as above (group 1) or by exposure to 2.5 μM ionomycin for 1 min (group 2). Non-fused couplets received another electrical stimulus in a second fusion attempt (group 3). Fused reconstructs from all three groups were cultured in 5 μg mL–1 of cycloheximide and 5 μg mL–1 of cytochalasin B for 3 h before culture overnight in AgResearch SOF media. Embryos at the 1- and 2-cell stages were transferred to the oviducts of synchronized recipients 2 days after oestrus. Each recipient received on average 10 to 12 embryos. Pregnancy and fetal development was monitored regularly by ultrasound. Parturition was induced up to 5 days before expected full term. Kids were reared on the recipients until weaning, with supplemental feeding as required. Embryo survival data were analysed by Fisher's exact test. There were no significant differences between groups 1 and 2 in terms of pregnancy and embryo survival rates throughout development. In group 1, 110 embryos were transferred to 11 recipients. Four does (36%) were diagnosed pregnant on Day 30 of gestation, carrying a total of 8 fetuses (7.3%). All 8 were delivered at term; however, one died at birth and another before weaning. In group 2, 202 embryos were transferred to 20 recipients. Thirteen does (65%) were pregnant on Day 30 of gestation, with a total of 23 fetuses (11.4%). One pregnancy was lost by Day 50 and another by Day 100. The remaining 11 pregnancies (55%) were maintained to term, with 18 kids delivered (8.9%). Four died within 1 day of birth, with the other 14 surviving to weaning. In group 3, a total of 63 embryos were transferred to five recipients. However, no fetuses were detected at Day 30; significantly less than for either group 1 (P

Cloning cattle by somatic cell nuclear transfer (NT) results in low survival and high frequencies of abnormal placentation and fetal development. We postulate that such anomalies may be overcome by complementing NT embryos with in vitro fertilised (IVF) embryos to form chimaeras. The gender and germline composition of chimaeras can be experimentally manipulated. Using embryological methods, we aim to produce chimaeric fetuses that are functionally male and produce sperm derived from the somatic NT embryo. Provided sufficient contribution from the IVF embryo, such chimeras should develop more normally than clones. At the 12- to 16-cell stage, individual male NT embryos were aggregated with female IVF embryos derived from X-sorted sperm. Following aggregation, there were no significant difference in blastocyst development between NT/IVF aggregates and disaggregated and re-aggregated IVF and NT controls (86/183 = 47% v. 77/233 = 33% v. 47/109 = 43%, respectively). Suitable quality embryos were transferred individually into synchronised recipient animals. Pregnancy establishment at Day (D) 35 was not significantly different between aggregate, IVF and NT groups (18/57 = 32% v. 11/45 = 24% v. 6/31 = 19%, respectively). Whilst there was no difference in survival between aggregates and IVF controls to ~D100, aggregates survived significantly better than NT controls (16% v. 18% v. 0%; respectively; P < 0.05). In the aggregate group, 7/8 fetuses recovered were phenotypically male. Using RT-PCR, expression of the female-specific mRNA for Xist was detected in 4/5 liver samples, indicating chimaerism. Despite improved survival to ~D100 compared to NT, 3/7 fetuses in the aggregate group still displayed evidence of abnormalities, such as fetal overgrowth. Further studies will explore alternative aggregation strategies and germline transmission of the NT-derived genome in chimaeras.

Previous studies in the mouse have shown treatment of somatic cell nuclear transfer (SCNT) embryos with histone deacetylase inhibitors (HDACi) to significantly increase cloning efficiency (Kishigami S et al. 2006 BBRC 340, 183–189; van Thuan N 2007 Asian Reproductive Biology Society 4, 9 abst). Increasing histone acetylation may open donor chromatin allowing better access for oocyte cytoplasmic factors to facilitate reprogramming. Here, we determined the effect of two HDACi, Trichostatin A (TSA), and scriptaid (Sigma-Aldrich, Castle Hill, NSW, Australia), on bovine cloning efficiency. Zona-free SCNT was performed with serum starved fibroblasts fused to enucleated MII-arrested IVM oocytes. After 4 h, reconstructs were activated with 5 μm ionomycin and 2 mm 6-dimethylaminopurine (DMAP) and cultured individually in 5 μL drops of AgResearch synthetic oviduct fluid (SOF) medium. Treatment with HDACi commenced concomitant with the 4 h DMAP incubation and continued in SOF for the remainder of the treatment period; totalling either 18 or 48 h post activation (hpa). TSA concentrations examined were: 0, 5, 50, and 500 nm, with all treatments containing 0.5% DMSO (n = 1121). Following TSA treatment, increased histone (H) acetylation at lysine (K) of H4K5 was confirmed by semi-quantitative immunofluorescence at the eight-cell stage. Scriptaid concentrations examined were: 0, 5, 50, 250, and 1000 nm, with all treatments containing 0.5% DMSO during DMAP and 0.1% DMSO during IVC (n = 1059). In vitro development on Day 7 was expressed in terms of transferable quality embryos as a percentage of reconstructs cultured. Data were analyzed using a generalized linear model with binomial variation and logit link. Embryos from selected treatments were transferred singularly to recipient cows on Day 7 with pregnancy data analyzed using Fisher’s exact test. Day 7 in vitro development was significantly greater with 5 nm TSA treatment for 18 hpa compared to controls (47.1% v. 34.5%; P < 0.02). Treatment of embryos with TSA for 48 hpa had no effect at any concentration tested. In contrast, scriptaid treatment for 18 hpa had no effect in vitro, while exposure for 48 hpa at 1000 nm significantly increased the development of transferable quality embryos compared to 0 nm (44.0% v. 32.4%; P < 0.005). There was no significant difference in embryo survival rates at D150 of gestation between embryos treated with 0 or 5 nm TSA for 18 hpa (8/48 v. 10/48; 16.7% v. 20.8%). However, in vivo development at Day 150 of gestation following treatment of embryos with 1000 nm scriptaid for 48 hpa was significantly lower compared to controls (1/37 v. 6/31; 2.7% v. 19.4%; P < 0.05). Contrary to the mouse, TSA or scriptaid treatment as used in this study did not increase cloning efficiency in cattle. The use of various HDACi either alone or in combination with DNA demethylating agents may still prove beneficial for reprogramming following nuclear transfer. Supported by FRST C10X0303.

The hematological characterization of clones derived by somatic cell nuclear transfer (SCNT) has not been extensively reported. Studies show that, generally, hematological parameters are within normal ranges, although distinct divergence between specific cohorts of clones and contemporaries exist. The aim of this study was to identify similarities and differences between cohorts of bovine clones and control animals and analyze the variations over time as the collective cohorts mature. Hematological profiles of 47 clones derived from 4 cell lines and 23 of their age- and sex-matched contemporary controls were compared. These donor cell lines were from 2 beef (male, n = 30) and 2 dairy (1 male, n = 9 and 1 female, n = 8) breeds and derived from myogenic cells, skin fibroblasts, and granulosa cells. Matched contemporaries, analyzed as one group, were produced via natural mating (n = 5) and AI (n = 14), with an additional in vitro-produced (IVP) group (n = 4) in the female cohort. All animals were subjected to similar management, nutrition, and environmental conditions. Serial samples were collected from birth until 15 months. Samples were assessed for the standard hematological parameters and cell morphology by a commercial clinical lab. Parameters were analyzed by one-way or as repeated measures ANOVA. The mean values for erythroid, myeloid, and lymphoid parameters were within normal ranges for both SCNT and controls, indicative of normal physiology. Red blood cells (RBC) from SCNT and control calves showed anisocytosis, poikilocytosis, cell fragmentation, and stippling, with a greater prevalence found in SCNT than in the controls. These abnormal morphologies were still evident in SCNT animals at 15 months of age, suggestive of delayed or incomplete erythroid maturation. Numbers of RBC, mean corpuscular volume (MCV), and hemoglobin (MCH) were different (P < 0.0001) between the collective SCNT cohorts and control animals over time, irrespective of genetics, sex, or breed. Taken together, these data suggest that erythropoiesis is generally perturbed in SCNT animals. In beef SCNT lines, platelet numbers were consistently different (P < 0.0001) from controls. White blood cell counts (WBC) were greater (P < 0.05) collectively in SCNT, although within the normal range, and the differential WBC changed with age (P < 0.05). Lymphocyte counts were greater (P < 0.05) in the collective SCNT cohorts. Further differences were seen in myeloid counts between specific SCNT and control cohorts. The greater variance evident in the myeloid parameters of SCNT animals was presumably because of an increased incidence of transient infections or inflammation in these animals. In summary, although most parameters were within the normal ranges over time, SCNT animals commonly display altered RBC, MCV, MCH, WBC, and lymphocyte parameters, which may be linked to cloning per se. This could partially explain the greater susceptibility of SCNT animals to external stressors. Supported by FRST contract C10X0311 and NRCGD.

Somatic cell nuclear transfer (NT) is associated with an increased incidence of abnormal placental and fetal development. Postnatal survival of NT offspring to weaning and even into adult life is lower when compared to contemporary offspring produced by artificial insemination (AI). Neonatal NT calves are hypoglycemic and lethargic initially but show increased frequency of feeding later on. These and other symptoms suggest that NT animals have impaired homeostatic mechanisms. Despite this, NTs that survive beyond weaning appear normal, but underlying health problems can become apparent when NTs are subjected to physiological stressors. The health and physiology of predominantly (75%) Jersey NT bull calves (n = 9), derived from a fetal myogenic cell line, and control Jersey AI bull calves (n = 5) were investigated. At 6 months of age, animals were subjected to a series of physiological intravenous hormonal challenges to test the responses of individual organs. Pancreatic insulin secretion response was assessed via a glucose tolerance test (GTT) and direct adrenal resistance via ACTH administration. The response of the hypothalamic-pituitary-adrenal (HPA) axis was assessed via a glucagon challenge. Except for glucagon, the doses given were adjusted for body weight at the time of challenge and all animals were fasted overnight. Plasma glucose, insulin, glucagon, and cortisol concentrations were measured using commercial assays. All animals were euthanized at approximately 7 months of age and a full postmortem (PM) was undertaken. Calf and organ weights were recorded. Liver glycogen content was also determined. Parameters and challenge data were analyzed by one-way or as repeated measures ANOVA. NT and AI calves appeared healthy at the time of the physiological challenges, and at PM, no major gross or histological organ abnormalities were recorded. There were no differences in the relative mean liver and adrenal weights between NT and AI. Basal plasma glucose concentrations were similar between NT and AI controls but the post-fasting decrease in glucose concentration was greater (P < 0.05) in controls than in NTs. There was no difference in response between the groups to the GTT. The NTs showed a slower response to ACTH than did AI controls. Glucose and insulin secretion were significantly higher (P < 0.05) whereas initial cortisol release was significantly lower (P < 0.05) after glucagon administration. Plasma glucagon levels and liver glycogen content did not differ between NT and controls. The current study indicates that surviving NT calves appear healthy, but when physiologically challenged, demonstrate reduced adrenal sensitivity and altered HPA axis response. These response deviations are indicative of underlying physiological differences and could explain the increased susceptibility of NT animals to physiological stressors. Ultimately, the cloning procedures may affect the long-term health of cloned offspring.

Bovine somatic cell nuclear transfer (NT) is associated with increased incidence of abnormal placental and fetal development. NT fetuses often show an overgrowth phenotype involving the liver, kidney, heart, adrenals, and thyroids. About 10% of NT embryos will result in a live calf and only 67% of these will survive to weaning at 3 months of age. Those that survive beyond weaning appear normal but underlying health problems may not be revealed until the animals are stressed, physiologically challenged, or subjected to postmortem (PM) examination. The health and physiology of NT predominantly (75%) Jersey bull calves (n = 11) and Jersey bull control calves (n = 5) derived from artificial insemination (AI) were investigated. Routine blood analyses were carried out at 3 weeks and then at 3 and 6 months of age. The animals were euthanized at approximately 7 months of age and a full PM undertaken, including histological examination of organ tissues. Calf and organ weights were recorded. The bone mineral density (BMD, g cm2) of the left femur was determined by dual energy x-ray absorptiometry (DEXA). The mean values for the various parameters were compared between treatments using one-way ANOVA. One of the calves died at 40 days from acute hemorrhaging in the abomasum, and a second, at 50 days from respiratory infection. The remaining 9 NT and 5 AI calves appeared healthy at the time of euthanasia, and PM results showed no major gross or histological organ abnormalities. Plasma electrolytes of all animals were within the normal range. Mean NT body weight was significantly higher than that of AI calves (169 vs. 141 kg, respectively; P < 0.0001) but this may have been due in part to the minor beef genetic contribution. Comparison of organ weights relative to the total body weight identified the mean relative NT brain (P < 0.0001) and lung (P < 0.01) weights to be lighter than those of controls, whereas the mean relative weight of the chest thymus (P < 0.05), thyroid glands (P < 0.001), and left testis (P < 0.05) were heavier in NT than in controls. Interestingly, mean femur weight (P < 0.01) and mean overall BMD (P < 0.05) were higher in NT than in AI calves. The mean bone shaft BMD of NT calves was greater (P < 0.05) than that of the controls, although no difference in BMD was evident at the growth plate. The current study indicates that surviving NT calves are apparently healthy but still manifest certain organ abnormalities frequently seen in NT fetuses of failing pregnancies. Identification of osteopetrosis of the long bones suggests that osteoclast differentiation or function is altered in NT calves. Modulation of hematopoietic progenitor differentiation by the NT process may explain the osteopetrosis and the reported compromised immune function and response of NT animals subjected to mild stressors since osteoclasts, monocytes, and macrophages are derived from the same progenitor cells. However, further studies are required to test this hypothesis. This work was supported by NRCGD and FRST contract C10X0311.

In cattle, a significant proportion of in vitro-produced (IVP) blastocysts do not result in viable pregnancies after transfer to recipient surrogates. Betteridge et al. (1980 J. Reprod. Fert. 59, 205–216) showed that it was possible after superovulation to recover elongated bovine embryos up to Day 17, transfer them into synchronized recipient cows, and have them develop further. We investigated the feasibility of recovering cattle embryos at Day 15, taking a sample of the trophoblast and transferring the embryos into recipients afterward for further development. The biopsied material could be used later to evaluate gene expression and correlate the profile retrospectively with developmental potential. With this approach, a larger amount of material is available for study and only embryos surviving to the elongation stage would be examined. In our experience, 30–40% of transferred blastocysts do not develop to the elongation stage. In three separate experiments, IVP embryos were generated using abattoir derived oocytes and cultured in SOF-aa supplemented with BSA (Thompson et al. 2000 J. Reprod. Fert. 118, 47–55). Six graded Day 7 (Day 0 = day of IVF) blastocysts were transferred into synchronized recipient heifers (n = 10 for each experiment). At Day 15 of gestation, concepti were flushed from the uteri after slaughter with EmCare Flush (ICPbio, Ltd., Suckland, New Zealand) containing 25 mg/mL kanamycin sulfate and then put into EmCare Hold. Conceptus lengths were measured and a proportion of those >30 mm long were cut off (5–15 mm) at one end and the trophoblast kept for future analysis. Pairs of cut or uncut (control) concepti were loaded into 0.25-mL embryo transfer straws. Each pair was transferred nonsurgically into recipients synchronized at Days 15 (Expt. 1, n = 17) or 13 (Expts. 2 and 3, n = 16 and 17, respectively) of the estrous cycle. The time between embryo flush and transfer to a recipient was noted. At Day 30, embryo survival was assessed at slaughter and compared using the Fisher's exact and chi-square. Day 15 conceptus lengths varied between 1 and 140 mm. The time between flush and transfer varied between 13 and 126 min and did not affect the ability of the concepti to subsequently establish pregnancies. Transfer to an earlier uterine environment did not significantly improve embryo survival. The proportion of embryos recovered at Day 30 was not affected by the biopsy. Up to 10 mm can be removed from 40–10 mm concepti without effect on subsequent survival. However, the overall survival post-Day 15 transfer is still too low for practical application. Table 1.

The cloning of cattle by somatic cell nuclear transfer (NT) is associated with a high incidence of abnormal placentation, excessive fluid accumulation in the fetal sacs (hydrops syndrome) and fetal overgrowth (Lee RSF et al. 2004 Biol. Reprod. 70, 1–11). Early embryonic loss in bovine NT pregnancies may also be due to immunological rejection (Hill JR et al. 2002 Biol. Reprod. 67, 55–63). As a means of overcoming placental abnormalities and improving pregnancy outcome in bovine NT, reconstructed blastocysts were produced by combining immunosurgically isolated inner cell masses (ICM) from Day 7 NT embryos with the trophectoderm (TE) of Day 7 IVF embryos. Oocytes for the production of NT and IVF embryos were obtained from abattoir-collected ovaries of dairy cows. The semen used for IVF was from the bull from which the cell line for NT was derived. The NT blastocysts were produced as described previously (Oback B et al. 2003 Cloning Stem Cells 5, 3–12) except that two one-cell embryos were aggregated together after NT (2NT). Blastocyst reconstruction was achieved using a modified procedure (Rorie RW et al. 1994 Vet. Record 135, 186–187). Embryos from four experimental groups were transferred individually to synchronized recipient heifers on Day 8 of culture: (1) ICM from 2NT embryos reconstructed with IVF TE (R-2NT, n = 15); (2) ICM from IVF embryos reconstructed with IVF TE (R-IVF, n = 15); (3) control 2NT (n = 10); and (4) control IVF (n = 10). Pregnancy rates were recorded and treatments compared using Fisher's exact test. After slaughter between Days 149 and 161 of gestation, morphometric measurements were determined for the fetuses, fetal organ weights, fluid volumes, and placentomes. Data were rank transformed; treatments were compared using Student's t-test with standard errors calculated from the pooled variation. Pregnancy rates on Day 35 were R-2NT (60%), R-IVF (47%), 2NT (90%), and IVF (10%). Pregnancy rates on Day 150 were R-2NT (40%), R-IVF (40%), 2NT (70%), and IVF (10%). The reason for the low IVF pregnancy rate was unknown. Previously, pregnancy rates using the same sire and cell line (but using Day 7 embryo transfer) on Day 35 were 63% (n = 40) and 69% (n = 42) for IVF and single, non-aggregated NT, respectively, and 50% and 33% for IVF and NT on Day 150. The single NT pregnancy rate was not significantly different from that for the 2NT embryos. There was no significant difference in pregnancy rates on Day 35 and Day 150 between R-2NT v. 2NT, R-2NT v. R-IVF, or 2NT v. R-IVF. The blastocyst reconstruction procedure did not have any impact on fetal development or influence pregnancy rates. All fetuses recovered were male. No significant differences were found between R-2NT and 2NT fetuses in terms of fetal weight, fluid volume, total placentome weight, and placentome numbers or in the relative and absolute weights of the brain, heart, liver, and kidneys. Thus, replacement of the TE in NT embryos with TE from IVF embryos did not overcome placental abnormalities or decrease fetal overgrowth prevalence.

Abnormal placentation, pregnancy failure, and hydroallantois are associated with somatic cell nuclear transfer (SCNT) in cattle. Identification of diagnostic markers for abnormal placentation in early gestation would permit therapeutic intervention. Ultrasonography and transvaginal sampling of amniotic and/or allantoic (fetal) fluid enables regular monitoring of fetal health. We report on the composition of serial samples of fetal fluid from individual cows between Days 70–130 of gestation and the potential of steroid and electrolyte composition as an early diagnostic marker for the subsequent occurrence of hydroallantois in SCNT pregnancies in cattle. On Day 70, pregnancy rates were 50% and 60% for cows or heifers implanted with single in vitro-fertilized (IVP, 20/40) or SCNT (25/42) embryos, respectively, and 67% for pregnancies generated by artificial insemination (AI, 12/18). Resulting fetuses were either clones (SCNT) or offspring (IVP/AI) of a donor Holstein bull. Fetal fluids, sampled using ultrasound-guided transvaginal puncture, were collected on Days 70, 100, and 130 of gestation (n = 12 and 139 for amniotic and allantoic samples, respectively). Placental and fetal morphological data were collected following slaughter between Days 135–163 of gestation (n = 14, 20, and 10 for SCNT, IVP, and AI groups, respectively). Fetal fluids were analyzed for progesterone, estrone sulphate, sodium, chloride, potassium, creatinine, urea, calcium, magnesium and phosphate. Pregnancy outcomes for the SCNT group were retrospectively classified as: Fail 100 (pregnancies failing between Days 70–99; n = 6); Fail 130 (failing between Days 100–129; n = 5); Hydrops (greater than 10 L combined amniotic and allantoic fluid at postmortem between Days 135–163; n = 8) and SCNT Pregnant 150 (pregnant between Days 135–163; n = 6). IVP and AI pregnancies were classified as IVP or AI Pregnant 150. Fluid composition was analyzed by ANOVA on log-transformed data. On Day 70, allantoic progesterone and estrone sulphate concentrations were significantly higher (P < 0.05) for the SCNT cows compared to the IVP/AI Pregnant 150 cows. On Day 70, allantoic potassium, chloride, creatinine, and urea concentrations were significantly higher (P < 0.05) for the SCNT Hydrops cows compared to the IVP/AI Pregnant 150 cows. In addition, Day 70 allantoic creatinine and urea concentrations were significantly higher (P < 0.05) for the SCNT Hydrops cows compared to other SCNT groups. By Day 100, allantoic chloride, creatinine, and urea concentrations in SCNT Hydrops cows were significantly lower (P < 0.05) than in IVP/AI Pregnant 150 groups. We conclude that elevated Day 70 allantoic urea and creatinine concentrations are potential early diagnostic markers predicting hydroallantois in recipient cattle carrying SCNT fetuses. Further investigation of these markers in other somatic donor cell lines used for nuclear transfer is warranted to determine their general utility.

Our previous work successfully analysed follicular fluid (45/61, 74% sampling success) from live cows using a portable clinical gas tension analyser (iSTAT Corp., Princeton, NJ). However, the oocyte recovery rate (7/24, 29%) from preovulatory follicles (POF) was disappointingly low, perhaps due to a need for the second puncture to aspirate the remaining follicular contents. This work describes a modified procedure that allowed a follicular fluid (FF) sample to be collected and stored in a gas-impermeable manner and the remaining follicular contents to be directly aspirated with or without follicular flushing from a single puncture. This modified dual port slip-luer hub, designed for use with a disposable needle ovum pick-up (OPU) system (PieMed, Netherlands), used a low negative pressure (30 mmHg), medium flow rate (25 mL/min) aspiration system with syringe flushing. FF samples for analysis were collected and stored in glass capillary tubes and the rest of the follicular contents/flushings were collected into tubes containing 10 mL collection medium and searched for oocytes. The hub's larger port, with a fitted Y connector, allows an initial flow of FF to be drawn into a capillary tube which has a polyvinylchloride (PVC) powder plugged end. When the PVC powder is permeated with FF it forms a seal effectively capping the tube. The remaining FF is diverted down the other leg of the Y connector using negative pressure. Flushing medium can be injected through the smaller port into the follicle while negative pressure is interrupted. A pilot trial examined gas tension, pH and ion concentrations from POF, dominant (D), and hormonally stimulated (S) follicles. Twenty synchronised Friesian cattle were scanned daily and follicles were tracked and mapped. The POF and D follicles were sampled on Day 20 or Day 10 respectively of the estrous cycle. The cows were then subjected to four weekly stimulations (used CIDR-B device inserted Day 2 and removed Day 5, 120 mg NIH-FSH-P1 administered twice daily on Day 5 and 6). Follicles were sampled and aspirated on Day 7. Successful FF analysis was achieved in 88% (110/125) of follicles attempted while oocyte recovery rates from POF, D, and S follicles were 56% (9/16), 54% (6/11) and 43% (36/83) respectively, showing it is possible to combine portable gas tension analysis with effective OPU. However, further work is needed to elucidate the correlation between the follicular environment and the oocyte developmental competence. Table 1. Follicular fluid sample analysis

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: 3.0mM and 1.5mM 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 10kg. 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.