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Biomedical subjects

M B Nottle

Publications and source records attributed to M B Nottle.

At least 19 recordsLinked to original sources
PubMed2002

Piglets born from centrifuged and vitrified early and peri-hatching blastocysts.

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.

Animals↗
PubMed2001

Effect of DNA concentration on transgenesis rates in mice and pigs.

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.

Animals↗
PubMed2000

Renal xenografts from triple-transgenic pigs are not hyperacutely rejected but cause coagulopathy in non-immunosuppressed baboons.

BACKGROUND: The genetic modification of pigs is a powerful strategy that may ultimately enable successful xenotransplantation of porcine organs into humans. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Animals↗
PubMed2000

In vitro development of porcine nuclear transfer embryos constructed using fetal fibroblasts.

The in vitro development of porcine nuclear transfer embryos constructed using primary cultures from day 25 fetal fibroblasts which were either rapidly dividing (cycling) or had their cell-cycle synchronized in G0/G1 using serum starvation (serum-starved) was examined. Oocyte-karyoplast complexes were fused and activated simultaneously and then cultured in vitro for seven days to assess development. Fusion rates were not different for either cell population. The proportion of reconstructed embryos that cleaved was higher in the cycling group compared to the serum-starved group (79 vs. 56% respectively; P < 0.05). Development to the 4-cell stage was not different using either population. Both treatments supported similar rates of development to the morula (1.5 vs. 7%, cycling vs. serum-starved) and blastocyst stage (1.5 vs. 3%, cycling vs. serum-starved). The blastocyst produced using cycling cells had a total cell number of 10. Total cell numbers for the three blastocysts produced serum-starved cells were 22, 24, and 33. These blastocysts had inner cell mass numbers of 0, 15, and 4, respectively. Six hundred and thirty-five nuclear transfer embryos reconstructed using serum-starved cells were transferred to 15 temporarily mated recipients for 3-4 days. Of these, 486 were recovered (77% recovery rate) of which 106 (22%) had developed to the 4-cell stage or later. These were transferred to a total of 15 recipients which were either unmated or mated. Seven recipients farrowed a total of 51 piglets. Microsatellite analysis revealed that none of these were derived from the nuclear transfer embryos transferred.

Animals↗
PubMed1999

Activation of in vivo- and in vitro-derived porcine oocytes by using multiple electrical pulses.

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 micros 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.

Animals↗
PubMed1998

Development of a nutritional strategy for increasing lamb survival in Merino ewes mated in late spring/early summer.

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.

Animal Feed↗
PubMed1998

High-level co-expression of complement regulators on vascular endothelium in transgenic mice: CD55 and CD59 provide greater protection from human complement-mediated injury than CD59 alone.

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.

Animals↗
PubMed1997

Nuclear transfer of porcine embryos using cryopreserved delipated blastomeres as donor nuclei.

Nuclear transfer protocol for the pig using cryopreserved delipated four- to eight-cell and morula stage embryos as nucleus donors was developed. Donor embryos, which had been delipated by micromanipulation following centrifugation for polarizing cytoplasmic lipid droplets, were cryopreserved with 1.5 M 1,2-propanediol and 0.1 M sucrose. Recipient cytoplasts were prepared from ovulated oocytes. Activation of oocytes could be induced more efficiently when electric stimulation was given 53 hr after the hCG injection or later (66-83%), compared with 52 hr or earlier (11-16%, P < 0.05), suggesting that aging after ovulation may be required for in vivo matured porcine oocytes to be activated by electric stimuli. Membrane fusion rates between donor blastomeres and enucleated oocytes were 88% (127/144) and 97% (56/58, P > 0.05) for the four- to eight-cell and morula stage embryos, respectively. In vitro developmental rates to the two-cell (53/100 vs. 35/65), four-cell (34/100 vs. 26/65), and morula stage (17/100 vs. 18/65) were the same between the nuclear transfer embryos with four- to eight-cell and morula nuclei. However, more embryos reconstituted with morula nuclei developed to blastocysts (15% vs. 6%, P < 0.05). These data demonstrated that blastomeres of cryopreserved, delipated porcine embryos can be used as donor nuclei for nuclear transfer. Frozen-thawed, delipated blastomeres can be efficiently isolated and fused, and therefore provide a useful source of donor nuclei.

Analysis of Variance↗
PubMed1997

Evaluation of a nutritional strategy to increase ovulation rate in merino ewes mated in late spring-early summer.

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.

Animal Nutritional Physiological Phenomena↗
PubMed1997

Effect of previous undernutrition on the ovulation rate of Merino ewes supplemented with lupin grain.

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 divided into their original nutritional treatments 17 days prior to ovulation. Each of these groups was divided 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.

Animal Nutritional Physiological Phenomena↗
PubMed1997

Asynchronous meiotic progression in porcine oocytes matured in vitro: a cause of polyspermic fertilization?

The progression of meiosis in porcine oocytes matured in 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.

Animals↗
PubMed1997

Role of epidermal growth factor and insulin-like growth factor-I on porcine oocyte maturation and embryonic development in vitro.

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 fluid and gonadotropins, and fertilized in vitro. Oocytes were either fixed 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 findings demonstrate that EGF plays an important role in both the meiotic and cytoplasmic maturation of porcine oocytes in vitro.

Animals↗
PubMed1997

Developments in transgenic techniques in pigs.

Manipulation of the pig genome is currently restricted to the random insertion of new DNA using pronuclear microinjection. This method suffers from a number of inherent limitations, the majority of which result from the inability to control the site at which the transgene becomes integrated. These drawbacks, together with the need to be able to target existing genes, will result in the replacement of pronuclear injection by new methods that have the capability to direct insertion to a particular genomic site that does not influence expression. Currently, it is possible to control the site of insertion in mice using embryonic stem (ES) cell and homologous recombination technologies. However, pluripotent ES cells have yet to be isolated in pigs. The possibility of using nuclear transfer to reprogramme early differentiated embryonic cells as well as somatic cells from adult animals may provide an alternative method for generating precise genetic modifications. Methods that allow these changes to be carried out in situ are also likely to be developed in the future.

Animals↗
PubMed1996

Expression of functional decay-accelerating factor (CD55) in transgenic mice protects against human complement-mediated attack.

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.

Animals↗
PubMed1996

The alpha-1,3-galactosyltransferase knockout mouse. Implications for xenotransplantation.

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.

Animals↗
PubMed1996

Developmental competence of in vivo and in vitro matured porcine oocytes after subzonal sperm injection.

In vivo and in vitro matured porcine oocytes were fertilized by subzonal sperm injection (SUZI), and their subsequent development in vitro was examined to determine whether ooplasmic incompetence is the major cause of limited developmental ability of in vitro matured/ fertilized porcine oocytes (Experiment 1). There was no significant difference in rates of fertilization (61% vs. 70%), monospermy (37% vs. 45%), and male pronuclear formation (77% vs. 61%) between in vivo and in vitro matured oocytes. Blastocyst formation rate was significantly lower for in vitro matured oocytes (11% vs. 42%; P < 0.001). Forty-six percent of in vivo matured oocytes cleaved to the 2-4 cell stage by 24 hr in culture after SUZI, compared with 3% of in vitro matured oocytes (P < 0.01). In experiment 2, in vitro development of in vitro matured oocytes with evenly and unevenly granulated cytoplasm were compared after SUZI to examine whether developmentally competent in vitro matured oocytes can be identified on the basis of morphological appearance. Most of the blastocysts obtained developed from oocytes with unevenly granulated cytoplasm (7/56 vs. 1/45; P > 0.05). Experiment 3 revealed that the proportion of oocytes with evenly granulated cytoplasm was originally low (11%) in the population of oocytes used for in vitro maturation, and it increased approximately 3-fold (36%; P < 0.001) after maturation. These results suggest that ooplasmic incompetence in porcine in vitro matured oocytes is the major cause of their limited developmental competence. Cytoplasmic maturation measured by male pronucleus formation does not directly reflect developmental competence of the oocytes. It was also shown that evenness of granulation of the cytoplasm is not a useful morphological indicator of developmental competence.

Animals↗