ORCID Profile
0000-0001-7625-5542
Current Organisation
University of Adelaide
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Publisher: CSIRO Publishing
Date: 1990
DOI: 10.1071/RD9900189
Abstract: Three groups of 40 parous Merino ewes were supplemented with 500 g of lupin grain/ewe/day for 7 days starting on either Day 3, 7 or 11 of the oestrous cycle and induced to ovulate by injecting cloprostenol on the sixth day of feeding. Supplementation with lupin grain significantly increased ovulation rate in all groups compared with corresponding controls by increasing the proportion of ewes with twin ovulations. Increases in ovulation rate did not depend on the stage of the cycle at which supplementation began or when during the luteal phase luteolysis was induced. It is concluded that the ovulatory response to lupin grain is initiated near the time of luteolysis.
Publisher: Wiley
Date: 05-2009
Publisher: Mary Ann Liebert Inc
Date: 12-2009
Abstract: Somatic cell nuclear transfer (SCNT) is a useful technique for the production of transgenic pigs that can be used for biomedical research. However, the efficiency of SCNT in pigs is low. In this study, we examined the effect of two postactivation treatments, cytochalasin B (CB) and trichostatin A (TSA), on the in vitro development of porcine SCNT embryos. Treating porcine parthenotes with 7.5 microg/mL CB for 3 h after electrical activation was effective in preventing the extrusion of the second polar body in 65% of the oocytes compared to 17% in the control group. Treating SCNT embryos with CB for 3 h after electrical activation significantly increased the average blastocyst cell number compared to the control group (CB treatment 51, Control 34, p < 0.05). Treatment of porcine SCNT embryos with CB for 3 h and 50 nM TSA for 24 h after electrical activation resulted in a threefold increase in blastocyst rate (CB + TSA 64%, CB 20%, p < 0.05) and an increase in the average blastocyst cell number (CB + TSA 63, CB 46, p < 0.05), compared to CB treatment alone. These results show that treatment with TSA and CB significantly improves the in vitro morphological development and quality of porcine SCNT embryos.
Publisher: CSIRO Publishing
Date: 1999
DOI: 10.1071/RD00033
Abstract: The current protocols used to activate pig nuclear transfer embryos are less efficient than those used for other species. To address this problem, the effect of multiple sets of electrical pulses on the parthenogenetic development of in vivo- and in vitro-derived porcine oocytes was examined. Each set of pulses consisted of two 1.5 kV cm–1 DC pulses of 60 s duration each, administered 1 s apart. For in vivo-derived oocytes, application of a second set of pulses 30 min after the first set increased the proportion of oocytes that developed to the blastocyst stage compared with a single treatment (51 v. 34%). Application of a third set of pulses 30 min after the second set reduced the rate of blastocyst formation compared with two sets of pulses. In contrast, the rate of blastocyst formation was greater with one set of pulses compared with two sets for in vitro matured oocytes (31 v. 16%). Additional sets of electrical pulses did not affect the number of cells in blastocysts obtained from either group of oocytes compared with a single treatment. In summary, the study demonstrates that the application of a second set of activating pulses 30 min after the first set is beneficial to in vivo-derived oocytes, but detrimental to in vitro matured oocytes, in terms of their ability to develop parthenogenetically to the blastocyst stage.
Publisher: No publisher found
Date: 2010
Abstract: We have developed a new method for the isolation of porcine embryonic stem cells (ESCs) from in vivo-derived and in vitro-produced embryos. Here we describe the isolation and characterization of several ESC lines established using this method. Cells from these lines were passaged up to 14 times, during which they were repeatedly cryopreserved. During this time, ESCs maintained their morphology and continued to express Oct 4, Nanog, and SSEA1. These cells formed embryoid bodies in suspension culture, and could be directed to differentiate into various lineages representative of all three germ layers in vitro. When injected into blastocysts these cells localized in the inner cell mass of blastocysts. To examine their pluripotency further, cells were injected into host blastocysts and transferred to recipient animals. Of the six transfers undertaken, one recipient became pregnant and gave birth to a litter of one male and three female piglets. Microsatellite analysis of DNA extracted from the tail tissue of these piglets indicated that two female piglets were chimaeric.
Publisher: Mary Ann Liebert Inc
Date: 09-2011
Abstract: For most of the derived human embryonic stem cell (ESC) lines thus far, the majority of human embryos used have been frozen in liquid nitrogen at or prior to the compacting stage for up to 10 years before human ESC derivation. As such they were grown in media that were relatively simple in their formulation compared with those used today. Here we report that culture of mouse embryos in media similar to these produces blastocysts in which both the inner cell mass cell number and the number of ESC progenitor cells (epiblast cells) in the inner cell mass are reduced compared with blastocysts cultured in a purpose-designed sequential (G1/G2) system commonly used today. Embryos cultured in a simple medium were less likely to attach and generate outgrowths. Further, these outgrowths had increased metabolic activity, which has been linked to differentiation, and altered gene expression. Culture of embryos in a simple medium to the compacting stage followed by culture in G2 to the blastocyst stage reduced some of these effects. However, none were improved to the level seen for culture in G1/G2. These results highlight the influence of embryo culture on embryo quality and pluripotency, which is a key factor in determining ESC isolation efficiencies.
Publisher: Wiley
Date: 11-1996
DOI: 10.1002/(SICI)1098-2795(199611)45:3<359::AID-MRD13>3.0.CO;2-U
Abstract: To examine the association between (GWG) and epithelial ovarian cancer (EOC). We compared GWG between 670 incident EOC cases and 1,551 community controls from a population-based, case-control study conducted in Pennsylvania, Ohio, and New York from 2003 to 2008. Multivariable unconditional logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) associated with GWG adjusting for potential confounders. To explore the potential effect of maternal long-term weight retention after childbearing, we restricted analyses to women who began their childbearing years as normal/underweight and examined differences in EOC risk between those who were normal/underweight versus those who were overweight/obese at study baseline reference date. Average GWG per full-term pregnancy did not differ between cases and controls. Among women who were normal/underweight at study baseline, greater average GWG was not associated with EOC (OR = 0.9, 0.8, 0.7 for quartiles 2, 3 and 4 of GWG gain, respectively, compared to quartile 1). In contrast, among women who were overweight/obese at study baseline, greater average GWG was positively associated with EOC (OR = 1.4, 1.8, 1.2, for quartiles 2, 3, and 4 compared to quartile 1 interaction p = 0.04). We posit that maternal post-partum weight retention and not gestational weight gain itself among normal/underweight women may impact subsequent risk of EOC. If our hypothesis is supported in other studies designed to assess this question directly, then counseling women on the importance of healthy weight management after a pregnancy could provide another means to help women reduce their risk of this often-fatal malignancy.
Publisher: CSIRO Publishing
Date: 2009
DOI: 10.1071/RD08165
Abstract: The lower ability of oocytes from prepubertal pigs to yield viable embryos than those from adult pigs appears, in part, a consequence of their reduced ability to accumulate cAMP during IVM. The present study examined the cAMP content of oocytes and cumulus–oocyte complexes (COCs), cumulus expansion and gap junctional communication (GJC) in COCs from 3- and 5–8-mm follicles during IVM. The effect of 1 mm dibutyryl cAMP (db-cAMP) treatment for the first 22 h of IVM was also examined for both follicle size classes. The cAMP concentration of oocytes from 5–8-mm follicles was threefold greater than that in oocytes from 3-mm follicles following 11 h of IVM (11.9 ± 5.9 v. 3.6 ± 1.8 fmol, respectively P 0.05). In the presence of db-cAMP, the cAMP content of oocytes from 3- and 5–8-mm follicles was no longer significantly different at 11 h IVM. The cAMP concentration of intact COCs from 5–8-mm follicles was significantly higher than that in COCs from 3-mm follicles at 11 h (1110.6 ± 318.0 v. 116.9 ± 55.7 fmol, respectively P 0.05). Despite maturation with db-cAMP, the cAMP content in COCs from 3- and 5–8-mm follicles at 11 h of IVM remained significantly different (15.1 ± 4.9 v. 196.2 ± 33.3 fmol, respectively P 0.05). The COCs from 3-mm follicles displayed lower cumulus expansion than did COCs from 5–8-mm follicles at both 11 h (cumulus expansion index (CEI) 1.0 ± 0.1 v. 1.8 ± 0.1, respectively P 0.01) and 22 h (CEI 1.9 ± 0.3 v. 2.9 ± 0.2, respectively P 0.05) of IVM. The level of cumulus cell–oocyte GJC decreased during IVM, with the number of GJC significantly greater in COCs from 3-mm compared with 5–8-mm follicles at both 6 h (613 ± 55 v. 304 ± 44 fluorescence intensity (FI), respectively P 0.05) and 11 h (644 ± 99 v. 337 ± 38 FI, respectively P 0.05) of IVM. By 22 h of IVM, the GJC of COCs from 3-mm follicles had decreased (227 ± 18 FI) and was no longer significantly different to that of COCs from 5–8-mm follicles (139 ± 15 FI P 0.05). Dibutyryl cAMP had no effect on the cAMP content, cumulus expansion or GJC of the whole COC. In conclusion, the results of the present study demonstrate that COCs from 3-mm follicles accumulate less intraoocyte and inter-COC cAMP, display lower cumulus expansion and maintain their cumulus cell–oocyte GJC for longer during IVM than do COCs from 5–8-mm follicles.
Publisher: Wiley
Date: 08-07-2014
DOI: 10.1111/XEN.12107
Abstract: Human corneal allografting is an established procedure to cure corneal blindness. However, a shortage of human donor corneas as well as compounding economic, cultural, and organizational reasons in many countries limit its widespread use. Artificial corneas as well as porcine corneal xenografts have been considered as possible alternatives. To date, all preclinical studies using de-cellularized pig corneas have shown encouraging graft survival results however, relatively few studies have been conducted in pig to non-human primate (NHP) models, and particularly using genetically engineered donors. In this study, we assessed the potential benefit of using either hCTLA4-Ig transgenic or α1,3-Galactosyl Transferase (GT) Knock-Out (KO) plus transgenic hCD39/hCD55/hCD59/fucosyl-transferase pig lines in an anterior lamellar keratoplasty pig to NHP model. Corneas from transgenic animals expressing hCTLA4-Ig under the transcriptional control of a neuron-specific enolase promoter showed transgene expression in corneal keratocytes of the stroma and expression was maintained after transplantation. Although a first acute rejection episode occurred in all animals during the second week post-keratoplasty, the median final rejection time was 70 days in the hCTLA4-Ig group vs. 21 days in the wild-type (WT) control group. In contrast, no benefit for corneal xenograft survival from the GTKO/transgenic pig line was found. At rejection, cell infiltration in hCTLA4Ig transgenic grafts was mainly composed of macrophages with fewer CD3+ CD4+ and CD79+ cells than in other types of grafts. Anti-donor xenoantibodies increased dramatically between days 9 and 14 post-surgery in all animals. Local expression of the hCTLA4-Ig transgene d ens rejection of xenogeneic corneal grafts in this pig-to-NHP lamellar keratoplasty model. The hCTLA4-Ig transgene seems to target T-cell responses without impacting humoral responses, the control of which would presumably require additional peripheral immunosuppression.
Publisher: Japanese Society of Animal Reproduction
Date: 2013
DOI: 10.1262/JRD.2012-103
Publisher: Oxford University Press (OUP)
Date: 09-2011
Abstract: Supplementing diets with n-3 fatty acids from fish oil has been shown to improve reproductive performance in dairy cattle and sheep, but there is little published literature on its effects in sows. The aim of this study was to evaluate the reproductive performance of sows fed fish oil as a source of n-3 PUFA prefarrowing and during lactation. From d 107.7 ± 0.1 of pregnancy, 328 sows ranging in parity from 0 to 7 (parity 1.95 ± 0.09, mean ± SE) were fed either a diet containing tallow (control) or an isocaloric diet containing 3 g of fish oil/kg of diet (n-3). Diets were formulated to contain the same amount of DE (13.9 MJ/kg), crude fat (54 g/kg), and CP (174 g/kg). Sows were fed their treatment diet at 3 kg daily for 8 d before farrowing and continued on treatment diets ad libitum until weaning at 18.7 ± 0.1 d of lactation. After weaning, all sows were fed a gestation diet without fish oil until their subsequent farrowing. There was no effect (P > 0.310) of feeding n-3 diets prefarrowing on piglet birth weight, preweaning growth rate, piglet weaning weight, or sow feed intake. However, n-3 sows had a larger subsequent litter size (10.7 ± 0.3 vs. 9.7 ± 0.3 total born 10.2 ± 0.3 vs. 9.3 ± 0.3 born live P < 0.05). In conclusion, this is the first study to demonstrate that feeding sows a diet containing n-3 PUFA from fish oil fed before farrowing and during lactation increased litter size in the subsequent parity independent of energy intake.
Publisher: Elsevier BV
Date: 05-2011
DOI: 10.1016/J.THERIOGENOLOGY.2010.11.043
Abstract: The objective was to develop a simple successful porcine cryopreservation protocol that prevented contact between embryos and liquid nitrogen, avoiding potential contamination risks. In vivo-derived blastocysts were collected surgically from donor pigs, and two porcine embryo vitrification protocols (one used centrifugation to polarize intracytoplasmic lipids, whereas the other did not) were compared using the Cryologic Vitrification Method (CVM), which used solid surface vitrification. The CVM allowed embryos to be vitrified, without any contact between embryos and liquid nitrogen. Both protocols resulted in similar in vitro survival rates (90% and 94%) and cell number (89 ± 5 and 99 ± 5) after 48 h in vitro culture of vitrified and warmed blastocysts. The protocol that did not use centrifugation was selected for continued use. To protect vitrified embryos from contact with liquid nitrogen and potential contamination during storage, a sealed outer container was developed. Use of this sealed outer container did not affect in vitro survival of cryopreserved blastocysts. In vivo blastocysts (n = 151) were collected, vitrified, and stored using the selected protocol and sealed container. These embryos were subsequently warmed and transferred to six recipients five became pregnant and farrowed a total of 26 piglets. This embryo vitrification method allowed porcine embryos to be successfully vitrified and stored without any contact with liquid nitrogen.
Publisher: Bioscientifica
Date: 11-1988
Abstract: Parous Merino ewes were maintained outdoors in feedlots during the beginning of the spontaneous breeding season and fed a maintenance ration of wheaten hay. For 14 days, ewes in each of 4 groups (N = 40/group) were given supplements of lupin grain or formaldehyde-treated casein and/or wheat starch. These were calculated to supply equivalent amounts of protein post-ruminally and/or digestible energy. Supplementation with lupin grain significantly increased ovulation rate by 37% by increasing the proportion of ewes with two ovulations. Similar increases in ovulation rate were achieved by increasing the supply of digestible protein post-ruminally in the casein and casein + starch-supplement groups. Increasing the intake of digestible energy separately in the starch-supplement group did not increase ovulation rate. It is concluded that increases in ovulation rate in ewes fed a lupin supplement are the result of significant increases in the amount of protein digested post-ruminally.
Publisher: Wiley
Date: 08-05-2014
DOI: 10.1111/XEN.12100
Publisher: Wiley
Date: 08-1998
DOI: 10.1111/J.1399-3089.1998.TB00026.X
Abstract: High-level endothelial expression of the human complement regulatory factor CD59 has been shown to protect transgenic mouse hearts from human complement-mediated injury in an ex vivo perfusion model. In this study we examine whether co-expression of CD55 provides additional protection. CD55/CD59 double-transgenic mice were generated by co-injection of CD55 and CD59 expression constructs driven by the human intercellular adhesion molecule 2 (ICAM-2) promoter. A line was established from one mouse that exhibited strong expression of CD55 and CD59 on vascular endothelium in the heart and other transplantable organs. An ex vivo perfusion model was used to compare hearts from these CD55/CD59 mice with hearts from a previously established line, which expressed CD59 at a similar level to the double transgenic line. CD59 hearts displayed prolonged survival compared to wild-type hearts during perfusion with 40% human plasma and maintained approximately 20% maximum work after 60 min. CD55/CD59 hearts were further protected, with work maintained at 35% of the maximum level after 60 min. The data demonstrate that high-level endothelial co-expression of CD55 and CD59 provides greater protection from human complement-mediated injury in this model than expression of CD59 alone.
Publisher: Japanese Society of Animal Reproduction
Date: 2002
DOI: 10.1262/JRD.48.313
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 06-2000
DOI: 10.1097/00007890-200006270-00008
Abstract: The genetic modification of pigs is a powerful strategy that may ultimately enable successful xenotransplantation of porcine organs into humans. Transgenic pigs were produced by microinjection of gene constructs for human complement regulatory proteins CD55 and CD59 and the enzyme alpha1,2-fucosyltransferase (H-transferase, HT), which reduces expression of the major xenoepitope galactose-alpha1,3-galactose (alphaGal). Kidneys from CD55/HT and CD55/CD59/HT transgenic pigs were transplanted into nephrectomised, nonimmunosuppressed adult baboons. In several lines of transgenic pigs, CD55 and CD59 were expressed strongly in all tissues examined, whereas HT expression was relatively weak and did not significantly reduce alphaGal. Control nontransgenic kidneys (n=4) grafted into baboons were hyperacutely rejected within 1 hr. In contrast, kidneys from CD55/HT pigs (n=2) were rejected after 30 hr, although kidneys from CD55/CD59/HT pigs (n=6) maintained function for up to 5 days. In the latter grafts, infiltration by macrophages, T cells, and B cells was observed at days 3 and 5 posttransplantation. The recipients developed thrombocytopenia and abnormalities in coagulation, manifested in increased clotting times and an elevation in the plasma level of the fibrin degradation product D-dimer, within 2 days of transplantation. Treatment with low molecular weight heparin prevented profound thrombocytopenia but not the other aspects of coagulopathy. Strong expression of CD55 and CD59 completely protected porcine kidneys from hyperacute rejection and allowed a detailed analysis of xenograft rejection in the absence of immunosuppression. Coagulopathy appears to be a common feature of pig-to-baboon renal transplantation and represents yet another major barrier to its clinical application.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 02-1996
DOI: 10.1097/00007890-199602270-00012
Abstract: Transgenic mice expressing human CD55 were generated by microinjection of a CD55-minigene under the control of the mouse H2K(b) (MHC class I) promoter. Offspring were tested for transgene integration by PCR analysis, and for CD55 expression on peripheral blood leukocytes (PBLs) by flow cytometry. Expression levels of 15 founders ranged from 30 to 80% of that on human neutrophils. Immunohistochemical analysis of kidney, heart, liver, and lung tissue demonstrated staining for CD55 on endothelial surfaces as well as general diffuse staining throughout the tissues. The capacity of the transgenically expressed CD55 to prevent human C3 deposition on the surface of mouse splenocytes was assessed by flow cytometry. Cells from hemizygous mice incubated with 10% fresh human serum as a source of natural antibody and complement bound approximately 65% less C3 than control littermates. No further protection was seen using cells from homozygous littermates, and the protective effect was abrogated by prior incubation with an OFFi-CD55 monoclonal antibody. Similarly, transgenic mice were afforded significant protection from human serum-mediated lysis, determined using an LDH release assay. Hearts perfused with human plasma showed no increase in survival time in a modified Langendorff perfusion system, however deposition of human C3c was greatly reduced in transgenic hearts.
Publisher: Elsevier BV
Date: 07-1997
DOI: 10.1016/S0378-4320(97)00025-0
Abstract: A nutritional strategy for increasing ovulation rate in Merino ewes mated in late spring-early summer was evaluated on two commercial farms. The strategy used the 'ram effect' to induce oestrus in seasonally anoestrus ewes and supplementary feeding of lupin grain six days prior to oestrus to increase ovulation rate. Ewes that had been isolated from rams for 6 weeks were exposed to vasectomised rams for 2 weeks and then mated to fertile rams for 6 weeks. Feeding 500 g lupins/head/day for 14 days commencing 12 days after the introduction of vasectomised rams, increased the number of ovulations from 126 to 146 per 100 ewes exposed to rams (P < 0.05). This increase was reflected in an improvement in fecundity (lambs born per ewe lambing P < 0.05) but not fertility (ewes lambing per ewe mated to rams). Net reproductive performance (the product of fertility, fecundity and lamb survival) was increased by 11 lambs weaned per 100 ewes exposed to rams due to lupin supplementation at mating.
Publisher: Wiley
Date: 14-02-2013
DOI: 10.1111/XEN.12024
Abstract: Activation of the clotting cascade is central in acute xenograft rejection (AHXR) that occurs when pig organs are transplanted into primates. The coagulopathy reported in this model is a very complex process that involves simultaneously coagulation factors, platelets and phospholipid-bearing cells (i.e., leukocytes, red blood cells, and endothelial cells). Choosing whole blood for coagulation analysis theoretically appears more favorable compared with plasma. Whole blood rotation thromboelastometry (ROTEM(®) ) is a point-of-care global coagulation analyzer able to evaluate the characteristics of clot formation and lysis by dynamic monitoring. The aim of this study was to record thromboelastographic profiles, performed by ROTEM(®) , in a series of immunosuppressed nephrectomized primates that received a life-supporting kidney. Of the eight primates, n = 4 received a pig kidney transgenic for human decay-accelerating factor (hDAF/Gal+) n = 2, an α 1,3-galactosyltransferase gene-knockout (GT-KO) pig kidney transgenic for human CD39, CD55, CD59 and fucosyltransferase (HTF) and n = 2, a GT-KO pig kidney transgenic for hDAF. Blood s les were collected before and at least once per week after transplantation till euthanasia. Intrinsic (INTEM) and extrinsic (EXTEM) coagulation pathways and the function of fibrinogen (FIBTEM) were evaluated. Thromboelastographic parameters considered were clotting time (CT, seconds) and clot formation time (CFT, seconds) in INTEM and EXTEM and maximum clot firmness (MCF, mm) in FIBTEM. The correlations between CT in INTEM and activated partial thromboplastin time (aPTT), CT in EXTEM and PT, CFT in INTEM and EXTEM, and platelet counts and MCF in FIBTEM and fibrinogen plasma levels were also considered. In all animals, thromboelastographic profiles showed progressive prolongation of CT (activation of coagulative cascade) in INTEM. A close correspondence was observed between (i) the prolongation of the CFT values (propagation of clot formation), both in INTEM and EXTEM, and the decrease in platelet counts (ii) the reduction in MCF values (clot firmness) in FIBTEM and the decrease in fibrinogen plasma levels. No concordance between CT in INTEM and aPTT and between CT in EXTEM and PT was observed. Our study demonstrated that ROTEM(®) analyzer could be a useful and complementary tool to study the consumptive coagulopathy, either "compensated" or "non-compensated," that takes place when transgenic pig kidneys are transplanted into primates. Larger and prospective studies are needed to confirm our results and to evaluate the role of ROTEM(®) to guide the management of consumptive coagulopathy in order to prolong the survival of the transplanted organ.
Publisher: CSIRO Publishing
Date: 1997
DOI: 10.1071/R96087
Abstract: The progression of meiosis in porcine oocytes maturedin vitro was examined and the effect of maturation interval on the incidence of polyspermic fertilization and embryonic development in vitro was investigated. In Experiment 1, it was found that oocytes selected for in vitro maturation varied considerably in terms of their meiotic progression. Approximately half of the oocytes were already undergoing germinal vesicle breakdown at the start of maturation while the remainder were at the intact germinal vesicle stage. These two populations of oocytes developed to the metaphase-II stage by 24 h and 36 h of maturation respectively. Maturation for 40 h and 44 h did not increase the percentage of oocytes at metaphase-II stage observed at 36 h. In Experiment 2, reducing the maturation interval from 44 h to 36 h did not affect the percentage of oocytes penetrated but did decrease the rate of polyspermic fertilization (5% v. 34% P 0 ·05). In Experiment 3, development of the embryos produced in vitro was assessed after culture for seven days. Reducing the maturation interval from 44 h to 36 h decreased the percentage of embryos developing to the 8-cell (9% v. 18% P 0 · 05), morula (3% v. 10% P 0 · 01), and blastocyst (1% v. 8% P 0· 001) stages. These results suggest that maturation for 44 h gives rise to a population of ‘aged’ oocytes which is susceptible to polyspermic fertilization.
Publisher: Oxford University Press (OUP)
Date: 10-1994
DOI: 10.1095/BIOLREPROD51.4.618
Abstract: The lipid content of porcine 1-cell stage embryos was reduced (delipated) through the use of micromanipulation to remove the lipid layer formed after centrifugation. Of 94 delipated embryos chilled to 4 degrees C for 1 h at the 1-cell or 2- to 4-cell stage, 60 (64%) cleaved in culture with development to the morula-blastocyst stage, whereas all of the control embryos lysed within 24 h. Significantly more embryos developed beyond the 8-cell stage when they were chilled at the 2- to 4-cell stage compared with chilling at the 1-cell stage (44%, 20 of 45 vs. 18%, 9 of 49). Fewer embryos developed after chilling if they were only partially rather than fully delipated. Developmental rates of partially delipated embryos to the 8-cell and blastocyst stages were 33% (13 of 40) and 8% (3 of 40), rates significantly (p or = blastocyst: 40%, 19 of 48 vs. 57%, 17 of 30). These data demonstrate that the sensitivity of porcine embryos to chilling is related to their high lipid content and that they can become tolerant to chilling if their lipid content is reduced.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 1996
DOI: 10.1097/00007890-199601150-00004
Abstract: Organ xenografts in discordant combinations such as pig-to-man undergo hyperacute rejection due to the presence of naturally occurring human anti-pig xenoantibodies. The galactose alpha(1,3)-galactose epitope on glycolipids and glycoproteins is the major porcine xenoantigen recognized by these xenoantibodies. This epitope is formed by alpha(1,3)-galactosyltransferase, which is present in all mammals except man, apes, and Old World monkeys. We have generated mice lacking this major xenoantigen by inactivating the alpha(1,3)-galactosyltransferase gene. These mice are viable and have normal organs but develop cataracts. Substantially less xenoantibody from human serum binds to cells and tissues of these mice compared with normal mice. Similarly, there is less activation of human complement on cells from mice lacking the galactose alpha(1,3)-galactose epitope. These mice confirm the importance of the galactose alpha(1,3)-galactose epitope in human xenoreactivity and the logic of continuing efforts to generate pigs that lack this epitope as a source of donor organs.
Publisher: Mary Ann Liebert Inc
Date: 12-2007
Abstract: Two media used to mature adult porcine oocytes for somatic cell nuclear transfer were compared. In the first experiment, parthenogenetic embryos were produced using a maturation medium used by us previously to clone pigs (OMM199) and that described by Kühholzer et al. (2001) to transport oocytes overnight (BOMED). There was no difference in maturation rates between the two different media. However, BOMED medium increased the percentage of parthenogenetic embryos that developed to the blastocyst stage compared with OMM199 (49% vs. 29%, respectively). In a second experiment, BOMED medium increased the percentage of SCNT embryos that developed to the blastocyst stage compared with OMM199 (22% vs. 8%, respectively). The efficiency of our cloning protocol using adult oocytes matured in BOMED medium was then determined by transferring SCNT embryos reconstructed using adult fibroblasts to synchronized recipients. Primary cultures of adult fibroblasts were obtained from two adult male pigs and used for SCNT (passages 2-4). Between 82 and 146 fused couplets were transferred to seven recipients synchronized 1 day behind the embryos. Five recipients (71% pregnancy rate) subsequently farrowed a total of 23 piglets (4.4 average litter size). Overall efficiencies (liveborn/embryos transferred) were 3.2% for all transfers and 4.3% for animals that gave birth.
Publisher: Wiley
Date: 21-05-2002
DOI: 10.1002/MRD.10126
Abstract: The effect of the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), on the maturation promoting factor (MPF) activity, pronuclear formation, and parthenogenetic development of electrically activated in vitro matured (IVM) porcine oocytes was investigated. Oocytes were activated by exposure to two DC pulses, each of 1.5 kV/cm field strength and 60 microsec duration, applied 1 sec apart. In the first experiment, subsequent incubation with 2 or 5 mM 6-DMAP for 3 hr increased the incidence of blastocyst formation compared with no treatment, whereas incubation with 2 or 5 mM 6-DMAP for 5 hr did not. In the proceeding experiments, oocytes exposed to 6-DMAP were incubated with 2 mM of the reagent for 3 hr. Assaying histone H1 kinase activity in the second experiment revealed that the levels of active MPF in electrically activated oocytes treated with 6-DMAP were depleted more rapidly and remained depleted for longer compared with electrical activation alone. The kinetics of MPF activity following 6-DMAP treatment were similar to that found in inseminated oocytes in the third experiment. The effect of 6-DMAP was correlated with an increased incidence of parthenogenetic blastocyst formation. A fourth experiment was undertaken to examine the diploidizing effect of 6-DMAP. Electrically activated oocytes treated with 6-DMAP and cytochalasin B, either alone or in combination, displayed a higher incidence of second polar body retention compared with those that were untreated or treated with cycloheximide alone. After 6 days of culture in vitro, parthenotes exposed to 6-DMAP, either alone or in combination with cytochalasin B, formed blastocysts at a greater rate compared with those exposed to cytochalasin B alone, cycloheximide alone or no treatment. The combined 6-DMAP and cytochalasin B treatment induced the highest rate of blastocyst formation (47%), but the numbers of trophectoderm and total cells in these blastocysts were lower compared with those obtained following exposure to 6-DMAP alone. These results suggest that the increased developmental potential of 6-DMAP-treated parthenotes may be attributable to the MPF-inactivating effect of 6-DMAP, rather than the diploidizing effect of 6-DMAP.
Publisher: Japanese Society of Animal Reproduction
Date: 1995
DOI: 10.1262/JRD.41.165
Publisher: Wiley
Date: 24-09-2013
DOI: 10.1111/XEN.12063
Abstract: Immunological and histopathological features in pig-to-primate renal xenotransplantation are widely studied. Only limited data have been reported about clinicopathological findings in primate recipients of life-supporting renal xenografts. In human medicine, proteinuria represents a common complication in kidney transplantation and is associated with impaired graft survival. The detection of low molecular weight proteins of tubular origin is considered an early method for predicting potential graft rejection. In this study, the presence and the significance of quantitative and qualitative proteinuria were evaluated in xenotransplanted non-human primates in which kidney function was supported only by the transplanted organ. Eight bilaterally nephrectomized cynomolgus monkeys (Macaca fascicularis) were transplanted with a single kidney from α1,3-galactosyltransferase gene-knockout (GTKO) pigs transgenic for human CD39, CD55, CD59, and α1,2-fucosyltransferase. In addition to hematological and biochemical analyses, quantitative and qualitative analysis of proteinuria was evaluated by urinary protein-to-creatinine ratio (UPC ratio) and sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE), respectively. The main hematological and biochemical changes recorded after transplantation were a progressive anemia and a severe and progressive decrease in total proteins. In urine s les, the UPC ratio was low before transplantation and increased after transplantation. Similarly, SDS-AGE was negative before transplantation, but bands consistent with mixed (i.e., tubular and glomerular) proteinuria were observed in all s les collected post-transplantation. The study of clinicopathological changes in cynomolgus monkey renal xenograft recipients provides a valid help in monitoring the health conditions in the post-transplant period. Moreover, the evaluation of UPC ratio and the use of SDS-AGE technique in urine s les of cynomolgus monkey renal xenograft recipients may be considered a valid, inexpensive, and less time-consuming method than more sophisticated techniques in monitoring proteinuria. Proteinuria and presence of low molecular weight (LMW) proteins were consistently found in urine after transplantation, independent of fluctuations in renal function.
Publisher: Wiley
Date: 14-04-2003
DOI: 10.1034/J.1399-3089.2003.01140.X
Abstract: It is highly likely that successful pig-to-human xenotransplantation of vascularized organs will require genetic modification of the donor pig, and in particular of donor vascular endothelium. Promoters are generally tested in transgenic mice before generating transgenic pigs. Several promoters have been used to drive endothelial cell-specific expression in mice but none have yet been tested in pigs. We compared the promoters of three human genes that are predominantly expressed in vascular endothelium: intercellular adhesion molecule 2 (ICAM-2), platelet endothelial cell adhesion molecule 1 (PECAM-1) and endoglin. Expression of human complement regulatory proteins (hCRPs), directed by each of the promoters in mice, was largely restricted to vascular endothelium and leukocyte subpopulations. However, expression from the PECAM-1 promoter was weak in liver and non-uniform in the small vessels of heart, kidney, and lung. Conversely, expression from the endoglin promoter was consistently strong in the small vessels of these organs but was absent in larger vessels. The ICAM-2 promoter, which produced strong and uniform endothelial expression in all organs examined, was therefore used to generate hCRP transgenic pigs. Leukocytes from 57 pigs containing at least one intact transgene were tested for transgene expression by flow cytometry. Forty-seven of these transgenic pigs were further analyzed by immunohistochemical staining of liver biopsies, and 18 by staining of heart and kidney sections. Only two of the pigs showed expression, which appeared to be restricted to vascular endothelium in heart and kidney but was markedly weaker than in transgenic mice produced with the same batch of DNA. Thus, in this case, promoter performance in mice and pigs was not equivalent. The weak expression driven by the human ICAM-2 promoter in pigs relative to mice suggests the need for additional regulatory elements to achieve species-specific gene expression in pigs.
Publisher: Mary Ann Liebert Inc
Date: 12-2004
Abstract: We have reported relatively efficient methods for somatic cell nuclear transfer and for knocking out the alpha(1,3)-galactosyltransferase (alpha1,3-GT) gene in porcine fetal fibroblasts using a nonisogenic promoterless construct approach. Here we report the production of alpha1,3-GT gene knockout pigs using these procedures. Seven alpha1,3-GT gene knockout cell clones were identified by long-range PCR from 108 neomycin resistant (neo(R)) colonies, giving a 6.5% targeting efficiency. Three cell clones were used for nuclear transfer. Nuclear transfer was performed using a fusion before activation protocol using in vitro-matured adult oocytes. Between 51 and 110 fused couplets were transferred to 10 recipients synchronized 1 day behind the embryos. Parturition was induced on day 115, and piglets were delivered by caesarean section. Four recipients gave birth to a total of 18 live piglets. All pigs were female, and all three clones resulted in the birth of live pigs. alpha1,3-GT gene knockout pigs were identified by long-range PCR and confirmed by Southern blot analysis. The efficiency (embryos transferred iglets born) of our cloning protocol was 1.9% for all transfers and 4.6% for animals that gave birth.
Publisher: Mary Ann Liebert Inc
Date: 06-2007
Abstract: As the pig becomes increasingly used for biomedical research, an effective and efficient in vitro culture system is essential. This study aimed to improve the commonly used porcine embryo culture medium, NCSU23, by altering the energy substrates and adding amino acids, using electrically activated diploid parthenotes from oocytes obtained from the ovaries of prepubertal and adult animals. Morphological development to day 6 and blastocyst cell number were examined. Glucose (5.56 mM) was replaced by pyruvate and lactate (0.2 mM and 5.7 mM, respectively) for either the entire culture period or for the first 48 h only. Blastocyst rates were not different between any of the treatments, and were similar for prepubertal and adult oocytes. When the embryos were cultured with pyruvate and lactate for the first 48 h and then glucose, there was a significant increase in blastocyst cell number compared to glucose only. Blastocysts produced using pyruvate and lactate for the entire time tended to have more cells than those exposed to glucose only and less than those who were cultured in pyruvate and lactate for the first 48 h and then glucose. Nonessential amino acids added for the first 48 h and nonessential and essential amino acids added for the remaining time significantly increased blastocyst cell number only when the embryos were grown in pyruvate and lactate followed by glucose. Blastocyst rates were not different between any of the treatments, and this result was the same when using sow or gilt oocytes. The modified medium was then tested using in vitro matured and fertilized embryos from sow oocytes. Blastocyst rates and cell number were significantly increased in the modified medium compared to those grown in unmodified NCSU23. This shows that altering energy substrates and adding amino acids can increase the quantity and cell number of IVP blastocysts compared with NCSU23.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 04-2006
Publisher: CSIRO Publishing
Date: 2003
DOI: 10.1071/RD02086
Abstract: The developmental competence of oocytes recovered from the ovaries of slaughtered prepubertal and adult pigs was evaluated after in vitro maturation, parthenogenetic activation and culture in vitro. In addition, the effect of prepubertal and adult follicular fluid (FF) on the developmental competence of prepubertal and adult oocytes was investigated. When matured in adult FF, the rates of cleavage (92 v. 73% P 0.01) and blastocyst formation (57 v. 38% P 0.05) were greater for adult oocytes than for prepubertal oocytes. Blastocysts derived from adult oocytes had more trophectoderm cells (43 v. 30 P 0.05) and total cells (51 v. 36 P 0.05) than blastocysts derived from prepubertal oocytes. The developmental competence of prepubertal oocytes was not affected by the FF donor age, whereas the developmental competence of adult oocytes was. Blastocysts derived from adult oocytes matured in adult FF had more trophectoderm cells (38 v. 24 P 0.005), inner cell mass cells (7 v. 3 P 0.01) and total cells (45 v. 27 P 0.001) than blastocysts derived from adult oocytes matured in prepubertal FF. Characterization of the steroid content of the FF used to supplement the maturation medium revealed that adult FF contained more progesterone (42 v. 23 ng mL−1 P 0.005) and androstenedione (70 v. 16 ng mL−1 P 0.05) than prepubertal FF. In addition, the molar ratios of progesterone to androstenedione, androstenedione to 17β-oestradiol and androstenedione to testosterone differed (P 0.05) between prepubertal and adult FF. The results support the hypothesis that a greater proportion of adult oocytes than of prepubertal oocytes has completed ‘oocyte capacitation’. The differences in FF steroid content are indicative of the different follicular environments from which the prepubertal and adult oocytes were isolated, and may be attributed to the observed effects on oocyte developmental competence.
Publisher: CSIRO Publishing
Date: 2011
DOI: 10.1071/RD10333
Abstract: This study employed a unilateral ovariectomy model to investigate the relevance of the local supply of progesterone (ovary) compared with the systemic supply of progesterone, in terms of embryo survival in the ipsilateral uterine horn as opposed to the contralateral uterine horn. Thirty gilts were unilaterally ovariectomised (ULO) during the luteal stage of their first oestrous cycle. Half of the ULO gilts were fed at 1.2 maintenance requirement (M), while the other half were fed at 2.4 M. Across ULO gilts 0.8 more embryos survived in the ipsilateral horn compared with the contralateral horn at Day 35 of gestation (P 0.05). In ULO gilts on the 2.4 M feed level the difference (+1.3 P 0.05) between the ipsi- and contralateral horn was more pronounced than on the 1.2 M feed level (+0.4 NS). The higher feed level reduced circulating levels of systemic progesterone on Day 5 of pregnancy but not embryo survival at Day 35. However, post-implantation embryo survival was lower on the low feed level. In conclusion, these data indicate that local progesterone supply from the ovaries to the uterus contributes to the probability of embryo survival.
Publisher: Japanese Society of Animal Reproduction
Date: 2010
DOI: 10.1262/JRD.10-060T
Abstract: Litter size and progeny birth weights are lower in gilts than in sows. Somatotropin (ST) is an important regulator of ovulation, fetal growth and survival. We therefore investigated effects of pST treatment of gilts for two to four weeks before mating on ovulation rate, behavioural estrus, fetal growth and survival, litter size and birth weights. In Experiment One, gilts were injected with 0, 30, 60 or 90 µg pST/kg/day for 14 days commencing 7 days after first estrus. Reproductive tracts were collected and corpora lutea and follicle numbers counted 5.5 days after second estrus. Ovulation rate (P=0.031) and number of medium-sized follicles (P=0.059) correlated positively with pST dose. In Experiment Two, gilts were injected with 0, 12.5, 25 or 50 µg pST/kg/day for 21 days from first estrus, and mated at second estrus. Numbers of corpora lutea, follicles and fetuses were counted at day 31 of pregnancy. Numbers of medium follicles and ovary weights were positively related to pST dose. In Experiment Three, 31 week old (1(st) replicate) or 27 week old (2(nd) replicate) gilts were injected daily with 0 or 12.5 µg pST/kg/day until mating 25.9 ± 0.6 days later, and delivered at term. Pre-mating pST increased total litter size in younger gilts in the 2(nd) replicate only (P<0.05). In conclusion, injecting gilts with pST before mating does not consistently alter ovulation rate, increases the number of medium follicles available for recruitment at the second mating after treatment and increases subsequent litter size in younger gilts.
Publisher: Public Library of Science (PLoS)
Date: 14-03-2018
Publisher: Springer Science and Business Media LLC
Date: 03-1995
DOI: 10.1038/374416A0
Publisher: CSIRO Publishing
Date: 1997
DOI: 10.1071/R96115
Abstract: The effects of epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) on the in vitro maturation of porcine oocytes were examined. Oocytes obtained from the ovaries of slaughtered prepubertal gilts were matured in modified Medium 199 supplemented with 25% porcine follicular ßuid and gonadotropins, and fertilized in vitro. Oocytes were either xed 16 h later to assess fertilization or cultured for 7 days to assess embryonic development. In Experiment 1, the addition of EGF to maturation medium increased the percentage of meiotically mature oocytes (88% v. 70% P 0· 001) but did not affect the proportion of fertilized or cleaved oocytes. Blastocysts derived from oocytes matured in medium supplemented with 10 ng mL-1 EGF had a greater number of cells compared with those of control blastocysts (51·1 ± 5· 1 v. 36·0 ± 3·1 P 0· 02). In Experiment 2, the addition of IGF-I to maturation medium had no effect on meiotic maturation, fertilization or embryonic development. Our ndings demonstrate that EGF plays an important role in both the meiotic and cytoplasmic maturation of porcine oocytes in vitro.
Publisher: Oxford University Press (OUP)
Date: 07-1995
DOI: 10.1095/BIOLREPROD53.1.173
Abstract: Most porcine oocytes matured and fertilized in vitro fail to develop normally due to abnormal fertilization. The aim of this study was to determine the effect of cysteamine on pronuclear formation and developmental competence in pig embryos produced in vitro. Follicular oocytes were matured in vitro with or without cysteamine in Medium 199 supplemented with sodium pyruvate, FSH, LH, 17 beta-estradiol, antibiotics, and 25% porcine follicular fluid. Matured oocytes were then inseminated for 10 h. In the first experiment, pronuclear formation was assessed immediately after the insemination period. The addition of 50 microM or 500 microM cysteamine to the maturation medium increased male pronuclear development, resulting in a higher proportion of oocytes with synchronously formed male and female pronuclei. The frequency of synchronous pronuclear formation was increased in monospermic oocytes from 10% in the control group to 43% (p < 0.001) and 45% (p < 0.001), respectively, and in polyspermic oocytes from 43% in the control group to 68% (p < 0.05) and 75% (p < 0.001), respectively. In the second experiment, development of the in-vitro produced embryos was assessed after 7 days of culture in vitro. The addition 500 microM cysteamine to the maturation medium increased the percentage of cleaving embryos developing to the 8-cell (37% vs. 16% p < 0.001), morula (19% vs. 6% p < 0.001), and blastocyst (12% vs. 1% p < 0.001) stages. These results demonstrate that the addition of cysteamine to maturation medium increases synchronous pronuclear formation and normal embryonic development in porcine oocytes matured and fertilized in vitro.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 29-06-2018
Abstract: National laws fearing biopiracy squelch taxonomy studies
Publisher: Bioscientifica
Date: 03-08-2009
DOI: 10.1677/JOE-09-0131
Abstract: Fetal growth is restricted in primiparous pigs (gilts) compared with dams who have had previous pregnancies (sows), as in other species. In gilts, daily maternal porcine GH (pGH) injections from day 25 to 50 of pregnancy (term ∼115 day) increase fetal growth and progeny muscularity, and responses in sows are unknown. Whether feeding the β 2 -adrenergic agonist ractopamine during this period increases progeny growth rates in either parity and fetal responses in gilts, have not been investigated. We hypothesised that fetal and placental growth and fetal muscle development would be increased more by maternal pGH and/or ractopamine during early–mid pregnancy in gilts than sows, since fetal growth is restricted in gilts causing lower birth weights. Large White×Landrace gilts and sows were injected daily with water (controls) or pGH (∼15 μg/kg per day), or were fed 20 ppm ractopamine, between day 25 and 50 of pregnancy. Maternal pGH increased litter average fetal weight (11%, P =0.007) and length (3%, P =0.022), but not placental weight, at day 50 of pregnancy, irrespective of parity, and had the greatest effects in the heaviest fetuses of each litter. Maternal ractopamine increased average fetal weight (9%, P =0.018), but not length. Muscle fiber diameter was increased by pGH in heavy littermates and by ractopamine in median littermates. Similar fetal growth responses to pGH and ractopamine in gilts and sows suggest that these hormones increase fetal nutrient availability similarly in both parities. We therefore predict that sustained pGH treatment will increase progeny birth weight, postnatal growth and survival, in both sows and gilts.
Publisher: Mary Ann Liebert Inc
Date: 09-2006
Abstract: Mesenchymal stem cells (MSCs) isolated from bone marrow were used to examine the hypothesis that a less differentiated cell type could increase adult somatic cell nuclear transfer (SCNT) efficiencies in the pig. SCNT embryos were produced using a fusion before activation protocol described previously and the rate at which these developed to the blastocyst stage compared with that using fibroblasts obtained from ear tissue from the same animal. The use of bone marrow MSCs did not increase cleavage rates compared with adult fibroblasts. However, the percentage of embryos that developed to the blastocyst stage was almost doubled, providing support for the hypothesis that a less differentiated cell can increase cloning efficiencies. As MSCs are relatively difficult to isolate from the bone marrow of live animals, a second experiment was undertaken to determine whether MSCs could be isolated from the peripheral circulation and used for SCNT. Blood MSCs were successfully isolated from four of the five pigs s led. These cells had a similar differentiation capacity and marker profile to those isolated from bone marrow but did not result in increased rates of development. This is the first study to our knowledge, to report that MSCs can be derived from peripheral blood and used for SCNT for any species. These cells can be readily obtained under relatively sterile conditions compared with adult fibroblasts and as such, may provide an alternative cell type for cloning live animals.
Publisher: Wiley
Date: 20-03-2014
DOI: 10.1111/XEN.12091
Publisher: Wiley
Date: 07-2007
DOI: 10.1111/J.1399-3089.2007.00417.X
Abstract: We report here our experience regarding the production of double or homozygous Gal knockout (Gal KO) pigs by breeding and somatic cell nuclear transfer (SCNT). Large White x Landrace female heterozygous Gal KO founders produced using SCNT were mated with H shire or Duroc males to produce a F1 generation. F1 heterozygous pigs were then bred to half-sibs to produce a F2 generation which contained Gal KO pigs. To determine the viability of mating Gal KO pigs with each other, one female F2 Gal KO pig was bred to a half-sib and subsequently a full-sib Gal KO. F1 and F2 heterozygous females were also mated to F2 Gal KO males. All three types of matings produced Gal KO pigs. To produce Gal KO pigs by SCNT, heterozygous F1s were bred together and F2 fetuses were harvested to establish primary cultures of Gal KO fetal fibroblasts. Gal KO embryos were transferred to five recipients, one of which became pregnant and had a litter of four piglets. Together our results demonstrate that Gal KO pigs can be produced by breeding with each other and by SCNT using Gal KO fetal fibroblasts.
Publisher: Elsevier BV
Date: 09-1998
DOI: 10.1016/S0378-4320(98)00102-X
Abstract: A nutritional strategy for increasing lamb survival in Merino ewes mated in late spring/early summer was evaluated in a commercial flock over two consecutive years (Year 1, n = 680 Year 2, n = 325). The strategy combined the 'ram effect' to synchronise oestrus and hence parturition, plus supplementary feeding of lupin grain for 14 days in the expected early post-parturient period. Supplementary lupin feeding commenced 12 days after the expected start of lambing. Lambing was highly synchronised over a 14-day period commencing 17-19 days after the expected start of lambing, in both years. Supplementary feeding did not affect lamb birthweight in either year but subsequent increases in weight were observed at weaning in Year 1 (1.4 kg P = 0.06) and tail docking in Year 2 (1.3 kg P < 0.05). Lamb survival was increased by 7 lambs per 100 ewes exposed to rams in both years. (Year 1 at weaning, NS Year 2 at tail docking, P < 0.001). It was concluded that the strategy improved both lamb survival and lamb performance possibly due to an effect of lupin supplementation on colostrum and subsequent milk production.
Publisher: Springer Science and Business Media LLC
Date: 2001
Abstract: A retrospective analysis of transgenesis rates obtained in seven pronuclear microinjection programs was undertaken to determine if a relationship existed between the amount of DNA injected and transgenesis rates in the pig. Logistic regression analysis showed that as the concentration of DNA injected increased from 1 to 10 ng/microl, the number of transgenics when expressed as a proportion of the number liveborn (integration rate) increased from 4% to an average of 26%. A similar relationship was found when the number of molecules of DNA injected per picolitre was analysed. No evidence was obtained to suggest either parameter influenced integration rate in mice when the same constructs were injected. The number of transgenics liveborn when expressed as a proportion of ova injected (efficiency rate), increased as DNA concentration increased up to 7.5 ng/microl and then decreased at 10 ng/microl for both species suggesting that at this concentration DNA (or possible contaminants) may have influenced embryo survival. The relationship between efficiency and the number of molecules injected per picolitre was complex suggesting that the concentration at which DNA was injected was a better determinant of integration and efficiency rates. In conclusion, the present study suggests that transgenes need to be injected at concentrations of between 5 and 10 ng/microl to maximise integration and efficiency rates in pigs.
Publisher: Humana Press
Date: 2013
DOI: 10.1007/978-1-62703-628-3_7
Abstract: Despite their agricultural and biomedical importance, embryonic stem cells (ESCs) are yet to be isolated for the pig or the domestic ungulates in general. This suggests that methods which have been used successfully in mice may not be applicable to these. In this chapter we describe a new method for the isolation of porcine ESCs. This method differs from those described previously in that it produces homogeneous outgrowths from undifferentiated inner cell mass cells when embryos are plated onto inactivated mouse embryonic feeder layers.
Publisher: Hindawi Limited
Date: 2013
DOI: 10.1155/2013/150901
Abstract: Human embryos donated for embryonic stem cell (ESC) derivation have often been cryopreserved for 5–10 years. As a consequence, many of these embryos have been cultured in media now known to affect embryo viability and the number of ESC progenitor epiblast cells. Historically, these conditions supported only low levels of blastocyst development necessitating their transfer or cryopreservation at the 4–8-cell stage. As such, these embryos are donated at the cleavage stage and require further culture to the blastocyst stage before hESC derivation can be attempted. These are generally of poor quality, and, consequently, the efficiency of hESC derivation is low. Recent work using a mouse model has shown that the culture of embryos from the cleavage stage with insulin to day 6 increases the blastocyst epiblast cell number, which in turn increases the number of pluripotent cells in outgrowths following plating, and results in an increased capacity to give rise to ESCs. These findings suggest that culture with insulin may provide a strategy to improve the efficiency with which hESCs are derived from embryos donated at the cleavage stage.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 02-2019
Publisher: Japanese Society of Animal Reproduction
Date: 2009
DOI: 10.1262/JRD.20176
Abstract: The aims of this study were to investigate improvements to the pig preimplantation embryo culture system using in vitro produced embryos. For experiment 1, the optimum time to change the medium from NCSU23 containing 0.6 mM glucose, 0.2 mM pyruvate, 5.7 mM lactate and nonessential amino acids to NCSU23 containing 5.6 mM glucose and both essential and nonessential amino acids was examined. There were no statistically significant differences in blastocyst rates or cell number when the medium was changed at 48, 72 or 96 h, although there was a consistent trend for the 96 h treatment to produce fewer blastocysts with fewer cells. For experiment 2, the addition of essential amino acids at either a 1:50 or a 1:100 dilution of the purchased stock solution for day 1 to 6 or for days 3 to 6 only was investigated. Adding essential amino acids at a 1:50 dilution for day 3 to 6 significantly reduced the blastocyst rate and adding them at a 1:50 dilution from day 1 to 6 significantly reduced both the blastocyst rate and blastocyst cell number compared to when it was added at a 1:100 dilution. Embryos were produced by IVF, cultured for 6 days and good quality blastocysts were transferred into 6 synchronized pseudopregnant recipients (24 to 35 blastocysts per recipient) resulting in 4 pregnancies and 21 live birth piglets. These results show that adding essential amino acids at a 1:100 dilution provided the best culture conditions and the blastocysts produced were able to attain full term development after transfer.
Publisher: Wiley
Date: 03-2011
Publisher: Wiley
Date: 29-08-2002
Publisher: Wiley
Date: 2006
DOI: 10.1002/MRD.20555
Abstract: Pre-pubertal pig oocytes display reduced developmental competence compared with adult oocytes following in vitro maturation (IVM). Exposure to dibutyryl cyclic adenosine monophosphate (dbcAMP) for the first 20 hr IVM improves development of pre-pubertal oocytes, suggesting that their cAMP content may be inadequate. This study examined the effect of 1 mM dbcAMP treatment for the first 22 hr of IVM on the cAMP content, meiotic progression, and embryo development of pre-pubertal and adult oocytes. In control groups, a two-fold increase in cAMP was observed in adult oocytes after 22 hr IVM, with no change in pre-pubertal oocyte cAMP content. At 22 hr IVM, dbcAMP treatment resulted in two- and five-fold increases in pre-pubertal and adult oocyte cAMP, respectively. After 22 hr control IVM, a greater proportion of pre-pubertal oocytes occupied metaphase I (MI) compared with adult oocytes (69% vs. 49%). dbcAMP treatment reduced the proportion of pre-pubertal and adult oocytes in MI stage at 22 hr. Despite dbcAMP treatment, the proportion of pre-pubertal oocytes in the MI stage at 22 hr remained higher than that of adult oocytes. In control groups, adult oocytes displayed a greater ability to form blastocysts compared with pre-pubertal oocytes following either parthenogenetic activation (59% vs. 25%) or in vitro fertilization (IVF) (47% vs. 19%). dbcAMP treatment increased subsequent blastocyst formation rates of pre-pubertal oocytes, whereas blastocyst formation rates of adult oocytes remained unchanged. Our results suggest that the reduced developmental capacity of pre-pubertal oocytes may be a consequence of their reduced ability to accumulate cAMP during IVM.
Publisher: Elsevier BV
Date: 2011
DOI: 10.1016/J.TRRE.2010.10.001
Abstract: Xenotransplantation of solid organs will only ever become a clinical reality with genetic modification of the pig, which is now widely accepted as the most likely donor species for humans. The understanding of the barriers to xenotransplantation has required advances in genetic technologies to resolve these problems. Hyperacute rejection has been overcome by overexpression of complement regulatory proteins or targeted disruption of the enzyme associated with the major carbohydrate xenoantigen. The subsequent barriers of disordered coagulation, induced antibody, and cell-mediated rejection remain challenging. The mechanisms for these incompatibilities are being deciphered, and multiple genetic manipulations to resolve these issues are currently in progress. Moreover, new technologies offer help to producing sizeable numbers of modified pigs in a timely manner. This article retraces the basis and foreshadows progress of the genetically modified pig for xenotransplantation as it advances toward the clinic.
Publisher: Oxford University Press (OUP)
Date: 04-2010
Abstract: Piglet neonatal survival and postnatal growth and efficiency are positively related to birth weight. In gilts, daily maternal porcine ST (pST) injections from d 25 to 100 (term approximately 115 d), but not d 25 to 50, of pregnancy increase progeny birth weight. Daily maternal pST injections from d 25 to 50 increase fetal weight at d 50 in gilts and sows. We therefore hypothesized that daily pST injections from d 25 to 100, but not d 25 to 50, of pregnancy would increase birth weight similarly in both parities. Landrace x Large White gilts and sows were uninjected (controls) or were injected daily with pST (gilts: 2.5 mg/d sows: 4.0 mg/d, each approximately 15 microg of pST/kg per day) from d 25 to 50 or 100 of pregnancy. Litter size and BW were recorded at birth, midlactation, and weaning. Dams were followed through the subsequent mating and pregnancy. Maternal pST injections from d 25 to 100, but not d 25 to 50, increased mean piglet birth weight by 11.6% in sows (P <or= 0.001) and by 5.6% in gilts (P = 0.008). Both pST treatments decreased litter size by approximately 0.6 live-born piglets (each P 0.1) the weaning-remating interval, conception rate, or subsequent litter size. Greater pST-induced birth weight increases in sows than in gilts may mean that underlying metabolic or placental mechanisms for pST action are constrained by maternal competition for nutrients in rapidly growing gilts.
Publisher: Wiley
Date: 11-2010
Publisher: Elsevier BV
Date: 06-2014
DOI: 10.1111/AJT.12722
Publisher: CSIRO Publishing
Date: 2007
DOI: 10.1071/RD07018
Abstract: The present study compared the distribution and steroid composition of 3-, 4- and 5–8-mm follicles on the surface of prepubertal and adult ovaries, and determined the relationship between follicle size and developmental competence of oocytes following parthenogenetic activation. The effect of 1 mm dibutyryl cAMP (dbcAMP) for the first 22 h of in vitro maturation (IVM) on the embryo development of prepubertal oocytes from the three follicle size cohorts was also determined. Compared with adult, prepubertal ovaries contained a higher proportion of 3-mm follicles (46 v. 72%, respectively), but a lower proportion of 4-mm (33 v. 22%, respectively) and 5–8-mm follicles (21 v. 6%, respectively). Adult follicular fluid (FF) contained 11-fold higher levels of progesterone (P4) than prepubertal FF, with similar levels observed between all adult follicle sizes. In prepubertal FF, the P4 concentration increased with follicle size from 3 to 4 to 5–8 mm. Rates of blastocyst development following parthenogenetic activation of adult oocytes from all three follicles sizes were similar (approximately 55%), whereas rates from prepubertal oocytes increased with increasing follicle size from 3 (17%) to 4 (36%) to 5–8 mm (55%). Treatment with dbcAMP for the first 22 h of IVM led to a 1.5-fold increase in the rate of blastocyst development for prepubertal oocytes from 3-mm follicles, but had no effect on prepubertal oocytes from the 4 and 5–8 mm classes. Mean blastocyst cell number increased with follicle size in prepubertal ovaries and was similar for all follicle sizes in adult ovaries. The present study demonstrates that the low efficiency of in vitro embryo production observed using prepubertal compared with adult pig oocytes is due to a greater proportion of 3-mm follicles on prepubertal ovaries, which contain oocytes of inferior developmental competence.
Publisher: Oxford University Press (OUP)
Date: 03-2004
Publisher: Elsevier BV
Date: 09-1997
DOI: 10.1016/S0378-4320(97)00066-3
Abstract: The effect of undernutrition on the ovulation rate of Merino ewes supplemented with lupins was examined in two experiments using a 2 x 2 factorial (low vs high nutritional plane x none vs supplemented) design. In both experiments, ewes were assigned at random to two equal-sized groups and differentially grazed for 8 weeks (low and high). In Experiment 1, flocks were recombined and managed as one group for 18 weeks and then ided into their original nutritional treatments 17 days prior to ovulation. Each of these groups was ided at random into equal-sized subgroups and one subgroup fed lupins for 10 days prior to ovulation. Restricting nutrition 6 months prior to ovulation resulted in a difference in mean liveweight between the low and high groups of 9.3 kg at the end of the 8-week period (P < 0.001). Ovulation rates per ewe were 1.06 +/- 0.07 (low, no supplement), 1.63 +/- 0.09 (low, lupin-supplemented), 1.28 +/- 0.09 (high, no supplement) and 1.57 +/- 0.08 (high, lupin-supplemented). The increase of 0.22 ovulations per ewe for the low vs high plane of nutrition without supplement was significant (P < 0.05). There was a significant interaction (P < 0.05) between previous nutrition imposed 6 months prior to ovulation and lupin supplementation, indicating that the ovulatory response to lupins was greater at the low compared with the high plane of nutrition (0.57 vs 0.29 extra ovulations per 100 ewes). In Experiment 2, the previous nutritional treatments were imposed for 8 weeks immediately before ovulation. Restricting feed intake in the low group resulted in a difference in mean liveweights between the two groups of 6.2 kg (P < 0.001) 6 weeks after the start of the nutritional treatments. Ovulation rates were 1.22 +/- 0.06 (low, no lupin supplement), 1.38 +/- 0.09 (low, lupin-supplemented), 1.67 +/- 0.08 (high, no lupin supplement) and 1.64 +/- 0.09 (high, lupin-supplemented). The effect of previous nutrition on ovulation rate was significant (P < 0.001) with 0.35 extra ovulations per ewe fed the high plane. Ewes in the low group responded to lupin supplementation with 0.16 extra ovulations per ewe (P = 0.06), whereas ewes previously fed on a high plane did not respond to the lupin treatment.
Publisher: Wiley
Date: 07-2013
DOI: 10.1111/XEN.12044
Abstract: Glutaraldehyde fixation does not guarantee complete tissue biocompatibility in current clinical bioprosthetic heart valves (BHVs). Particularly, circulating anti-αGal human antibodies increase significantly from just 10 days after a BHV implantation. The inactivation of such epitope should be mandatory to meet the requirements for a perspectively safe clinical application nevertheless, its quantitative assessment in commercially available BHVs has never been carried out. In this investigation, seven different models of BHVs were tested. The number of epitopes was determined with reference to a standard αGal source by an ELISA test. The presence of xenoantigen was subsequently confirmed by immunofluorescence analysis. Porcine tissue, knockout for the αGal epitopes, was used as negative control. Epic™ valve was the only model among those tested, in which the αGal antigen appeared to be completely shielded. Composite Trifecta™ valve exhibited conflicting results: cusps of bovine pericardial tissue were devoid of reactive αGal epitopes, while the stent cover strip of porcine pericardium still maintained 30% of active antigens originally present in native tissue. All other tested BHVs express an αGal amount not significantly different from that exhibited by porcine Mosaic(®) valve (5.2 ± 0.6 × 10(10) each 10 mg of tissue). For the first time, the quantitative evaluation of the αGal epitope in heart valve bioprostheses, already in clinical practice for about 40 yrs, was finally determined. Such quantification might provide indications of biocompatibility relevant for the selection of bioprosthetic devices and an increase in the confidence of the patient. It might become a major quality control tool in the production and redirection of future investigation in the quest for αGal-free long-lasting substitutes.
Publisher: Wiley
Date: 11-1997
DOI: 10.1002/(SICI)1098-2795(199711)48:3<339::AID-MRD6>3.0.CO;2-S
Publisher: Bioscientifica
Date: 03-2017
DOI: 10.1530/REP-16-0517
Abstract: The preimplantation embryo in vivo is exposed to numerous growth factors in the female reproductive tract, which are not recapitulated in embryo culture media in vitro . The IGF2 and plasminogen activator systems facilitate blastocyst development. We hypothesized that the addition of IGF2 in combination with urokinase plasminogen activator (uPA) and plasminogen could improve rates of blastocyst hatching and implantation in mice. B6BcF1 and CBAB6F2 mouse embryos were ided into one of four supplemented culture media treatment groups: (1) control (media only) (2) 12.5 nM IGF2 (3) 10 µg/mL uPA and 5 µg/mL plasminogen or (4) a combination of IGF2, uPA and plasminogen treatments. Embryo development to blastocyst stage and hatching were assessed before transfer to pseudopregnant recipient females and implantation, pregnancy rates and postnatal growth were assessed. After 90.5 h of culture, IGF2 + U + P treatment increased the percentage of B6BcF1 embryos that were hatching/hatched and percentage developing to blastocyst stage compared with controls ( P 0.02). Following B6BcF1 embryo transfer, IGF2 + U + P treatment increased implantation sites at day 8 of pregnancy compared with controls ( P 0.05). Replication in the CBAB6F2 mouse strain showed significant improvements in pregnancy rates at days 8 and 18 but not in blastocyst development. No adverse effects were seen on gestational age, litter size or birthweight, or the reproductive capacity of offspring of IGF2 + U + P treated embryos. For embryos susceptible to detrimental effects of in vitro culture, IGF2, uPA and plasminogen supplementation of culture media can improve pregnancy success, but the effect of treatment is dependent on the mouse strain.
Publisher: Oxford University Press (OUP)
Date: 05-2002
DOI: 10.1095/BIOLREPROD66.5.1283
Abstract: Somatic cell nuclear transfer was used to produce live piglets from cultured fetal fibroblast cells. This was achieved by exposing donor cell nuclei to oocyte cytoplasm for approximately 3 h before activation by chemical means. Initially, an experiment was performed to optimize a cell fusion system that prevented concurrent activation in the majority of recipient cytoplasts. Cultured fibroblast cells were fused in medium with or without calcium into enucleated oocytes flushed from superovulated gilts. Cybrids fused in the presence of calcium cleaved at a significantly (P < 0.05) greater rate (69%, 37 out of 54) after 2 days of culture compared with those fused without calcium (10%, 7 out of 73), suggesting that calcium-free conditions are needed to avoid activation in the majority of recipient cytoplasts during fusion. In the second experiment, cybrids fused in calcium-free medium were activated approximately 3 h later with ionomycin, followed by incubation in 6-dimethylaminopurine to determine development in vitro. Following 2 days of culture, cleavage rates of chemically activated and unactivated cybrids (fusion without activation control) were 93% (100 out of 108) and 7% (2 out of 27), respectively. After an additional 5 days of culture, activated cloned embryos formed blastocysts at a rate of 23% (25 out of 108) with an average inner cell mass and trophectoderm cell number of 10 (range, 3 to 38) and 31 (range, 16 to 58), respectively. In the third experiment, activated nuclear transfer embryos were transferred to the uteri of synchronized recipients after 3 days of culture to assess their development in vivo. Of 10 recipients receiving an average of 80 cleaved embryos (range, 40 to 107), 5 became pregnant (50%) as determined by ultrasound between Day 25 and Day 35 of gestation. Of the five pregnant recipients, two subsequently farrowed one piglet per litter originating from two different cell culture lines. In this study, efficient reprogramming of porcine donor nuclei by fusing cells in the absence of calcium followed by chemical activation of recipient cytoplasts was reflected in high rates of development to blastocyst and pregnancy initiation leading to full term development.
Publisher: Springer Science and Business Media LLC
Date: 16-08-2017
DOI: 10.1038/S41598-017-09030-6
Abstract: Xenotransplantation from pigs has been advocated as a solution to the perennial shortage of donated human organs and tissues. CRISPR/Cas9 has facilitated the silencing of genes in donor pigs that contribute to xenograft rejection. However, the generation of modified pigs using second-generation nucleases with much lower off-target mutation rates than Cas9, such as Fok I-dCas9, has not been reported. Furthermore, there have been no reports on the use of CRISPR to knock protective transgenes into detrimental porcine genes. In this study, we used Fok I-dCas9 with two guide RNAs to integrate a 7.1 kilobase pair transgene into exon 9 of the GGTA1 gene in porcine fetal fibroblasts. The modified cells lacked expression of the αGal xenoantigen, and secreted an anti-CD2 monoclonal antibody encoded by the transgene. PCR and sequencing revealed precise integration of the transgene into one allele of GGTA1 , and a small deletion in the second allele. The cells were used for somatic cell nuclear transfer to generate healthy male knock-in piglets, which did not express αGal and which contained anti-CD2 in their serum. We have therefore developed a versatile high-fidelity system for knocking transgenes into the pig genome for xenotransplantation purposes.
Publisher: CSIRO Publishing
Date: 2004
DOI: 10.1071/EA03237
Abstract: The limitations of existing transgenic technology, the potential of cloning technology to overcome these, as well as technologies which may be available in the future for inserting new genetic material are discussed. Currently, transgenic livestock are produced by injecting hundreds to thousands of copies of a particular transgene into the pronucleus of a fertilised egg. This method suffers from a number of inherent limitations that prevent the full potential of this technology from being explored. Most of these limitations stem from the fact that it is impossible to control the site at which the transgene becomes inserted. Transgenic technology holds considerable promise for the livestock industries as well as having important biomedical applications. However, before any of these possibilities can be realised, technology is required whereby a single copy of a particular transgene can be inserted or ‘knocked in’ at a site that does not interfere with expression, as well as having the capacity to ‘knockout’ existing genes. This is possible in mice using a combination of homologous recombination and embryonic stem cell technologies. However, despite considerable effort worldwide, embryonic stem cells are yet to be isolated from any of the livestock species. The ability to clone these now means that somatic cells most notably fetal fibroblasts, can used for gene targeting purposes instead of embryonic stem cells. However, this method is not without its limitations and it is possible that more efficient methods will be developed in the future. In particular, the use of mammalian artificial chromosomes will extend this technology to allow combinations of transgenes as well as chromosomal segments to be incorporated, allowing us to explore the full potential of transgenic technology for agricultural as well as biomedical applications.
Publisher: Oxford University Press (OUP)
Date: 05-2012
Abstract: Birth weight positively predicts postnatal growth and performance in pigs and can be increased by sustained maternal porcine ST (pST) treatment from d 25 to 100 of pregnancy (term ∼115 d). The objective of this study was to test whether a shorter period of maternal pST treatment in late pregnancy (d 75 to 100) could also increase birth and weaning weights of progeny under commercial conditions. Gilts (parity 0) and sows (parities 2 and 3) were not injected (controls) or injected daily with pST (gilts: 2.5 mg•d(-1), sows: 4.0 mg•d(-1), both ∼13 to 14 μg•kg(-1)•d(-1)) from d 75 to 100 of pregnancy. Litter size and BW were recorded at birth and weaning, and dams were followed through the subsequent mating and pregnancy. Maternal pST injections from d 75 to 100 increased litter average progeny weight at birth (+96 g, P = 0.034) and weaning (+430 g, P = 0.038) in sows, but had no effect on progeny weight in gilts (each P > 0.5). Maternal pST treatment did not affect numbers of live-born piglets and increased numbers of stillborn piglets in sows only (+0.4 pigs/litter, P = 0.034). Maternal pST treatment did not affect subsequent reproduction of dams. Together with our previous data, these results suggest that sustained increases in maternal pST are required to increase fetal and postnatal growth in gilt progeny, but that increasing maternal pST in late pregnancy may only be an effective strategy to increase fetal and possibly postnatal growth in sow progeny.
Publisher: Elsevier BV
Date: 05-2012
Publisher: Wiley
Date: 03-2000
Publisher: Japanese Society of Animal Reproduction
Date: 2010
DOI: 10.1262/JRD.09-197A
Abstract: In the present study we examined the effect of culture media and protein source on the formation of pluripotent primary outgrowths from in vitro produced and in vivo derived porcine embryos as the first step towards the isolation of embryonic stem cells (ESCs). To do this we compared high glucose Dulbeccos Modified Eagles Medium (DMEM) with Minimal Essential Alpha Medium (αMEM) both supplemented with fetal bovine serum (FBS) or serum replacement (SR) in a 2 × 2 factorial design. Culture in DMEM or αMEM supplemented with 10% SR resulted in the establishment of homogenous populations of cells which expressed Oct 4 and Nanog. In contrast culture in either media with FBS resulted in the formation of embryonal outgrowths composed entirely of differentiated cells or a mixture of differentiated cells and putative ESCs which grew poorly and could not be passaged. Using αMEM medium containing 10% SR and culturing in 5% oxygen, putative ESC lines were isolated from in vitro and in vivo derived embryos at efficiencies of 2 and 10% respectively.
Publisher: Wiley
Date: 2000
Publisher: Springer Science and Business Media LLC
Date: 2002
Abstract: Pigs are currently considered the most likely source of organs for human xenotransplantation because of anatomical and physiological similarities to humans, and the relative ease with which they can be bred in large numbers. A severe form of rejection known as hyperacute rejection has been the major barrier to the use of xenografts. Generating transgenic pigs for organ transplantation is likely to involve precise genetic manipulation to ablate the alpha(1,3) galactosyltransferase (galT) gene. In contrast to the mouse, homologous recombination in livestock species to ablate genes is h ered by the inability to isolate functional embryonic stem cells. However, nuclear transfer using genetically targeted cultured somatic cells provides an alternative means to producing pigs deficient for galT. In this study we successfully produced galT+/- somatic porcine fetal fibroblasts using two approaches positive negative selection (PNS) using an isogenic targeting construct, and with a promoterless vector using non-isogenic DNA.
Publisher: Japanese Society of Animal Reproduction
Date: 1999
DOI: 10.1262/JRD.45.167
Publisher: Elsevier BV
Date: 11-1986
DOI: 10.1016/0093-691X(86)90169-X
Abstract: The effect of melatonin treatment on intervals from calving to first postpartum estrus and ovulation was determined in Shorthorn cows which calved May 8 to June 14. Melatonin (500 mg in beef tallow) was injected subcutaneously (s.c.) into 20 cows on June 15 (4 to 38 d postpartum). Ovulation was determined from progesterone concentrations in jugular venous blood collected weekly from June to August. Mean intervals to first estrus and first ovulation were significantly longer in primiparous than in multiparous cows (85 +/- 4 vs 55 +/- 3 d and 83 +/- 4 vs 52 +/-3 d). Melatonin treatment caused a significant increase in the intervals to first postpartum estrus (68 +/- 4 vs 58 +/- 5d) and ovulation (68 +/- 4 vs 55 +/- 5 d). Mean plasma melatonin concentrations during the daytime were significantly higher in treated than in control cows one and two weeks after melatonin injection and were within the lower range of nighttime values reported previously for cows. Thus melatonin treatment raised daytime plasma concentrations of melatonin and delayed the onset of estrus and ovulation. These results support the possibility of a role of photoperiod through melatonin secretion in the onset of postpartum ovarian activity in cattle.
Publisher: Elsevier BV
Date: 1994
Publisher: Elsevier BV
Date: 11-2001
Publisher: Elsevier BV
Date: 09-2016
DOI: 10.1016/J.THERIOGENOLOGY.2016.03.029
Abstract: The present study was undertaken to examine the effect of feed restriction on ovulation rate and in vivo blastocyst development in gilts and sows. In the first experiment, gilts were feed restricted (1 vs. 2.5 times maintenance requirement) during the luteal and follicular phases before ovulation. In the second experiment, primiparous sows were feed restricted (ad lib vs. 60% thereof) during the last week of lactation before weaning. Gilts and sows were slaughtered at 5 days after ovulation to determine ovulation rate and blastocyst development. Blastocysts were also differentially stained to determine the effect of feed restriction on total, trophectoderm, and inner cell mass cell numbers. In both experiments, feed restriction delayed ovulation and reduced the number of ovulations in gilts (14.8 ± 1.3 vs. 12.0 ± 0.2 P < 0.05) and in sows (19.9 ± 1.0 vs. 18.4 ± 0.7). The number of blastocysts recovered on Day 5 was similarly reduced in gilts (12.0 ± 1.7 vs. 9.1 ± 1.1 P < 0.10) and in sows (15.9 ± 1.5 vs. 14.7 ± 1.0). However, feed restriction did not affect total, trophectoderm, or inner cell mass cell numbers in gilts or sows. In conclusion, the present study reported that energy balance influences ovulation rate and blastocyst number rather than blastocyst viability as measured by cell number.
Publisher: Elsevier BV
Date: 06-2002
DOI: 10.1016/S0093-691X(01)00720-8
Abstract: Cryopreservation of zona-intact porcine embryos has been relatively unsuccessful to date, although some success has been obtained with lipid reduced morulae and early blastocysts. This study adapted some vitrification protocols used successfully with late blastocysts for use with early zona-intact blastocysts, using actin depolymerization, centrifugation, and open-pulled (OPS) straws. Initially, Day 6 peri-hatching blastocysts were collected, cultured for 40 min in 7.5 microg/ml cytochalasin B and vitrified in 6.5 M glycerol and 6% BSA (VS1) in either heat-sealed (HS) or open straws (OS). The post-thaw survival of those stored in HS was 15.4% after 24 and 48 h in vitro storage in OS significantly improved survival (58.8% for both 24 and 48 h). When similar stage blastocysts were cultured in cytochalasin B and vitrified with 8 M ethylene glycol and 7% polyvinylpyrrolidone (PVP VS2) in OS, survival was 44.4 and 33.3% for 24 and 48 h, respectively. Day 5 late morulae and early blastocysts were collected, cultured with cytochalasin B, and centrifuged or left intact (control), then vitrified with VS1 in HS or OS, or vitrified in VS2 in OS only. None of the intact control embryos survived thawing and 48 h culture in vitro. Centrifuged early blastocysts vitrified with VS1 showed good post-thaw survival in culture when stored in HS (62.8 and 60.5% for 24 and 48 h, respectively), or OS (75 and 63.6%). When vitrified with VS2 in OS, survival improved (80 and 76.7%). Peri-hatching blastocysts were vitrified in VS1, and early blastocysts were vitrified with VS1 and VS2. All blastocysts were stored in OS. The embryos were recovered and transferred to Day 4 and 5 pseudopregnant recipients (for Day 5 and 6 blastocysts, respectively). Of the five recipients receiving peri-hatching blastocysts, two became pregnant and delivered a total of eight piglets. All three recipients of early blastocysts vitrified in VS1 had a delayed return to estrus while of the four receiving embryos vitrified with VS2, two were delayed in returning to estrus, and one was confirmed pregnant after 45 days. A litter of five piglets, one male and four female, was produced at 116 days of gestation. To our knowledge, this is the first litter of piglets produced from early blastocysts vitrified without micromanipulation to remove polarized lipid droplets.
Publisher: Mary Ann Liebert Inc
Date: 06-2011
Abstract: We report here the establishment and characterization of putative porcine embryonic stem cell (ESC) lines derived from somatic cell nuclear transfer embryos (NT-ESCs). These cells had a similar morphology to that described previously by us for ESCs derived from in vitro produced embryos, namely, a polygonal shape, a relatively small (10-15 μm) diameter, a small cytoplasmic/nuclear ratio, a single nucleus with multiple nucleoli and multiple lipid inclusions in the cytoplasm. NT-ESCs could be passaged at least 15 times and vitrified repeatedly without changes in their morphology, karyotype, or Oct-4 and Nanog expression. These cells formed embryoid bodies and could be directed to differentiate in vitro to cell types representative of all three germ layers. Following their injection into blastocysts, these cells preferentially localized in the inner cell mass. In conclusion, we have isolated putative porcine ESCs from cloned embryos that have the potential to be used for a variety of applications including as a model for human therapeutic cloning.
Publisher: Mary Ann Liebert Inc
Date: 09-2012
Abstract: High-quality embryos give rise to embryonic stem cells (ESCs) at greater efficiencies than poor-quality embryos. However, most embryos available for human ESC derivation are of a reduced quality as a result of culture in relatively simple media up to 10 years earlier, before cryopreservation, or before compaction. In the present study, we used a mouse model to determine whether a culture with insulin from the 8-cell stage could increase the number of ESC progenitor epiblast cells in blastocysts, as well as endeavor to determine the molecular mechanism of the insulin's effect. Culture in media containing 1.7 ρM insulin increased epiblast cell number (determined by Oct4 and Nanog co-expression), and proportion in day 6 blastocysts. The inhibition of phosphoinositide 3 kinase (PI3K) (via LY294002), an early second messenger of the insulin receptor, blocked this effect. The inhibition of glycogen synthase kinase 3 (GSK3) or p53, 2 s messengers inactivated by insulin signaling (via CT99021 or pifithrin-α, respectively), increased epiblast cell numbers. When active, GSK3 and p53 block the transcription of Nanog, which is important for maintaining pluripotency. A simultaneous inhibition of GSK3 and p53 had no synergistic effects on epiblast cell number. The induced activation of GSK3 and p53, via the inhibition of proteins responsible for their inactivation (PKA via H-89 and SIRT-1 via nicotinamide, respectively), blocked the insulin's effect on the epiblast.From our findings, we conclude that insulin increases epiblast cell number via the activation of PI3K, which ultimately inactivates GSK3 and p53. Furthermore, we suggest that the inclusion of insulin in culture media could be used as a strategy for increasing the efficiency with which the ESC lines can be derived from cultured embryos.
Publisher: CSIRO Publishing
Date: 2013
DOI: 10.1071/AN12119
Abstract: The response in reproductive performance when pigs are fed diets supplemented with fats high in polyunsaturated fatty acids (PUFA) has not been widely studied. Improved fertility has been reported in sows and other species fed diets with added fish oil, a rich source of omega-3 PUFA, but results are inconsistent. The aim of the present study was to determine the effect of the duration and the level of supplementation of omega-3 PUFA from fish oil on the reproductive performance of gilts. In Experiment 1, 570 Large White and Landrace purebred gilts were fed ad libitum either an unsupplemented diet containing tallow (Control) or a diet containing 3 g fish oil/kg (Omega-3) as a partial replacement for tallow from 24 weeks (Omega-3 for 6 weeks) or 27 weeks of age (Omega-3 for 3 weeks) before mating. Liveweight and backfat gain between 24 weeks of age and mating were recorded. Gilts were then fed an unsupplemented diet during gestation and farrowing rate and first litter size were recorded. In Experiment 2, 356 Large White × Landrace F1 cross gilts were fed ad libitum diets containing either 0, 3 or 10 g fish oil/kg of diet as a partial replacement of tallow from 24 weeks of age and continued after mating at 2.2 kg/day until slaughter at 25 days of gestation. Pregnancy rate, ovulation and embryo survival were recorded. Data were analysed by general linear model ANOVA and Chi-square methods. In Experiment 1, there was no increase in farrowing rate or litter size born in gilts fed the omega-3-supplemented diet for either 3 or 6 weeks before mating compared with Control gilts. In Experiment 2, supplementation with omega-3 PUFA from 24 weeks of age through to mating and continued during early gestation did not increase ovulation rate but there was a trend (P 0.10) for an increase in embryo survival measured at Day 25 of gestation in gilts fed diets containing fish oil. Embryo survival was higher in gilts fed diets containing 3 g fish oil/kg of diet than in those fed the Control diet (P 0.05). Increasing the supplementation level to 10 g fish oil/kg did not increase embryo survival further. In both experiments, supplementation of omega-3 as fish oil did not affect the onset of oestrous, gilt removal and weight and backfat gain. In conclusion, supplementation of omega-3 PUFA before mating did not improve farrowing rate or litter size in gilts. It may be necessary to continue feeding diets with low concentrations of fish oil during early gestation to maximise the reproductive response to elevated omega-3 PUFA.
Publisher: Wiley
Date: 21-10-2015
DOI: 10.1111/XEN.12199
Publisher: Wiley
Date: 12-08-2020
DOI: 10.1111/XEN.12551
Abstract: Gene editing using clustered regularly interspaced short palindromic repeats/Cas9 has great potential for improving the compatibility of porcine organs with human recipients. However, the risk of detrimental off-target mutations in gene-edited pigs remains largely undefined. We have previously generated GGTA1 knock-in pigs for xenotransplantation using FokI-dCas9, a variant of Cas9 that is reported to reduce the frequency of off-target mutagenesis. In this study, we used whole genome sequencing (WGS) and optimized bioinformatic analysis to assess the fidelity of FokI-dCas9 editing in the generation of these pigs. Genomic DNA was isolated from porcine cells before and after gene editing and sequenced by WGS. The genomic sequences were analyzed using GRIDSS variant-calling software to detect putative structural variations (SVs), which were validated by PCR of DNA from knock-in and wild-type pigs. Platypus variant-calling software was used to detect single-nucleotide variations (SNVs) and small insertions/deletions (indels). GRIDSS analysis confirmed the precise integration of one copy of the knock-in construct in the gene-edited cells. Three additional SVs were detected by GRIDSS: deletions in intergenic regions in chromosome 6 and the X chromosome and a duplication of part of the CALD1 gene on chromosome 18. These mutations were not associated with plausible off-target sites, and were not detected in a second line of knock-in pigs generated using the same pair of guide RNAs, suggesting that they were the result of background mutation rather than off-target activity. Platypus identified 1375 SNVs/indels after quality filtering, but none of these were located in proximity to potential off-target sites, indicating that they were probably also spontaneous mutations. This is the first WGS analysis of pigs generated from FokI-dCas9-edited cells. Our results demonstrate that FokI-dCas9 is capable of high-fidelity gene editing with negligible off-target or undesired on-target mutagenesis.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 02-2007
Start Date: 2006
End Date: 2008
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2009
End Date: 2012
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2005
End Date: 2010
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2013
End Date: 2017
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2013
End Date: 2017
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2014
End Date: 2018
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2005
End Date: 2014
Funder: Juvenile Diabetes Research Foundation
View Funded ActivityStart Date: 2004
End Date: 2007
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2004
End Date: 2006
Funder: Australian Research Council
View Funded ActivityStart Date: 2008
End Date: 2010
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2007
End Date: 2013
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2001
End Date: 2006
Funder: National Health and Medical Research Council
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