Search

Background & aims: Clinical pig islet transplantation for the treatment of type I diabetes is still hindered by obvious immunological considerations and less investigated physiological incompatibilities. Cellular encapsulation, donor genetic engineering and host immunomodulation can help improve islet survival in a xenotransplantation context but adequate insulin output from transplanted islets remains the ultimate goal to be achieved for islet transplantation to be clinically efficient. We have previously shown that adenovirus-driven expression of a cassette carrying a dipeptidyl peptidase-resistant form of glucagon-like-peptide-1 (GLP1) and a constitutively activated form of type 3 muscarinic receptor (M3R) in isolated neonatal and adult pig islets significantly increases their secretory response to in vitro glucose stimulation that is otherwise 4-10 times lower than human islets. Our aim in the current study was to replicate our previous results in a transgenic pig model with beta-cell specific expression of our GLP1M3R cassette, to characterize in vivo and in vitro islet function of these pigs and to verify whether their offspring exhibit the same improved insulin secretion as founder animals. Material & methods: Cloned transgenic pigs were produced using Talens technology and validated by TLA sequencing analyses. Pancreata were collected from 14-day old piglets. Insulin secretion in response to different stimuli was evaluated during dynamic islet perifusion experiments. After reaching adult age, selected animals were subjected to IVGTT to study in vivo islet function. Transgenic animals were then bred and islet function of their offspring was studied as for founder animals. Results: Transgenic cloned pig founders (male and female) expressing the GLP1M3R cassette at the beta cell level were successfully obtained. Islets isolated from these piglets showed a 5.5 to 7.5-fold increase of their insulin output upon stimulation with 15 mM glucose. They maintained regulated insulin secretion as basal unstimulated secretion was not significantly increased: stimulation indices of 3.7 to 7.5 compared to 2.6 for control wild type islets. In vivo islet function during IVGTT was evaluated in two animals that both showed a 2 to 3-fold increase of insulin secretion compared to controls. Finally, we examined secretory function of islets isolated from piglets born from transgenic cloned parents. In vitro perifusion experiments showed increased stimulated insulin secretion from transgenic piglets compared to their wild type siblings. Conclusion & perspectives: We obtained the first lines of genetically modified pigs exhibiting enhanced function of their pancreatic islets. Specific expression of modified GLP1 and activated M3R at the beta-cell level enhanced insulin secretion both in vivo and in vitro without affecting viability or fertility and was transmitted to the descendants of cloned animals while maintaining its positive effect on beta-cell function.

The cloning of equines by somatic cell nuclear trans- fer (SCNT) was first reported in 2003 (Galli et al., 2003; Woods et al., 2003), 6 years after the birth of Dolly the sheep (Wilmut et al., 1997). Amongst the mammalian spe- cies that have been cloned (Campbell et al., 2007), equines are among the last. In general, the application of assisted reproduction technologies in equines has lagged behind that of other livestock species (Galli et al., 2007) in the poor interest expressed by the industry (still today, for example, the thoroughbred does not allow artificial insemi- nation) and also the limited resources devoted to this kind of research and the few laboratories working in this area. In fact the first equine clone, a mule (Woods et al., 2003), was obtained from in vivo matured oocytes that were transferred back to the oviduct of a synchronized recipi- ent mare immediately after nuclear transfer and activation. This approach was successful because it limited to a mini- mum the time exposure to the in vitro environment. This approach of using in vivo matured oocytes was, however, not sustainable from a practical point of view. In contrast, the first cloned horses (Galli et al., 2003; Lagutina et al., 2005) and those that followed later (Hinrichs et al., 2006, 2007) were all derived from abattoir-recovered and in vitro matured oocytes that, following enucleation and embryo reconstruction, were cultured in vitro to the blastocyst stage prior to non-surgical embryo transfer to a recipient mare. This method could be applied only after the develop- ment of the procedures for in vitro maturation of oocytes, fertilization by ICSI, and in vitro culture of embryos capable of establishing normal pregnancies and generat- ing offspring (Galli et al., 2007; Hinrichs, 2010). In addi- tion to the refinement of the in vitro protocols for oocyte maturation and embryo culture, SCNT protocols had to be adapted and optimized for the equine, which, because of its anatomical and physiological features, provides a lim- ited number of oocytes per ovary when compared to cat- tle or pigs. The protocols currently applied for nuclear transfer are based on a zona-free system (Lagutina et al., 2007) to enhance cell fusion or a piezo-electric device (Westhusin et al., 2003) for enucleation and microinjec- tion of the nucleus directly into the oocyte. Reconstructed embryos also require a specific activation protocol (Hinrichs et al., 2006; Lazzari et al., 2002).

The Kathmandu Valley in the Himalayan foothills, considered as one of the global "hot spots" for what concerns air pollution, is currently facing severe air quality problems due to rapid urbanization processes, dramatic land use changes, socioeconomic transformation and high population growth. In this work, we present the first full year (February 2013 - February 2014) analysis of simultaneous measurements of two short-lived climate forcers/pollutants (SLCF/P), i.e. ozone (O3) and equivalent black carbon (BC), and aerosol number concentration at Paknajol (27°43'4'' N, 85°18'32'' E, 1380 m a.s.l.), in the city center of Kathmandu. These observations were carried out in the framework of the SusKat-ABC (A Sustainable Atmosphere for the Kathmandu Valley - Atmospheric Brown Cloud) campaign in Nepal. The diurnal behavior of BC and aerosol number concentration indicated that local pollution sources represent the major contribution to air pollution in this city. In addition to photochemistry, the planetary boundary layer (PBL) dynamic plays an important role in determining O3 variability, as suggested by the analysis of seasonal changes of the diurnal cycles and the correlation with meteorological parameters and aerosol properties. Especially during pre-monsoon, high values of O3 were observed during the afternoon/evening. This could be related to mixing and entrainment processes between upper residual layers and the PBL. During this season, the high O3 appeared well related to the impact of major open vegetation fires occurring in Nepal. On a synoptic-scale perspective, westerly and regional atmospheric circulations appeared to be especially conducive for the occurrence of the high BC and O3 values. The very high values of the SLCF/P, detected during the whole measurement period, indicated persisting adverse air quality conditions, dangerous for the health of over 3 million residents of the Kathmandu Valley, and the environment. Consequently, all of this information may be useful for implementing control measures to mitigate the occurrence of acute pollution levels in the Kathmandu Valley and the surrounding area.

Equine Assisted Reproduction and Embryo Technologies

International audience

Tropospheric ozone (O3) is an atmospheric key compound. It is recognised as a powerful short-lived greenhouse gas, it influences the oxidative capacity of the troposphere, and it affects population health as well as the ecosystem integrity and crop yields. Levels of tropospheric O3 at regional scale are strongly affected by anthropogenic emissions but production from lightning and Stratosphere Troposphere Exchange (STE) still represent not-negligible natural sources. South Asia and the Himalayas are hot-spot regions in terms of current surface O3 levels. For this reason, since March 2006, continuous surface O3 observations are carried out at the Nepal Climate Observatory - Pyramid (NCO-P; 27.95 N, 86.82 E), located at 5079 m a.s.l. in the Nepali Himalayas. As reported in previous investigations dealing with NCO-P observations, surface O3 variability in the Himalayas is strongly affected by upward transport of air-masses from the Indo-Gangetic Plain and Himalayan foothills, as well as stratospheric intrusion events. In this work, episodes of stratospheric air-mass intrusions at NCO-P are studied over the period 2006 - 2013, when continuous O3 measurements are available at the measurement site. The aim is to assess the influence of STE on the surface O3 variability. The analysis will be based on the global STE climatology produced at ETH Zurich for the ERA-Interim reanalysis data set from the European Centre for Medium-Range Weather Forecasts (ECMWF). This dataset, relying on a Lagrangian methodology to diagnose STE, will be used to understand the interannual O3 variations at NCO-P in relation to the corresponding STE variability.

Mt. Cimone (44.18N, 10.70E, 2165 m a.s.l.) is the highest peak of the Italian northern Apennines and is characterised by a 360° free horizon. As being located South of the Alps and of the polluted Po basin, the observations of atmospheric composition conducted at this GAW-WMO station, provide useful information for evaluating the role of regional and long-range transport processes in modifying the tropospheric background conditions in the Mediterranean basin/Southern Europe. Moreover, this long-term monitoring activity helps in quantifying the existing trend of climate-altering substances and atmospheric pollutants (e.g. Maione et al., 2010).

Clinical application of Ovum Pick Up, Intracytoplasmic sperm injection and Embryo culture in equine reproduction

Offspring of pre-determined sex has been obtained in several species with the use of sex-sorted semen by flowcytometry followed by artificial insemination or assisted reproduction techniques. Nevertheless in some species such as pig, sheep and horse efficiency is highly reduced compared to conventional AI due to the high number of sperm required for insemination. To obtain offspring from semen available in very limited amounts and with poor motility, as always occurs with sex-sorted frozen stallion semen, the application of the in vitro assisted reproductive techniques, in particular intracytoplasmic sperm injection has become a real option. The scope of this work was to investigate the use of sex-sorted frozen stallion semen for the production of embryos in vitro following ICSI, in vitro culture, freezing and thawing, and their viability after transfer to recipients to produce offsprings. Ejaculates were collected from 2 Standardbred stallions of proven fertility (A, B). Only the ejaculates containing >60% motile spermatozoa after dilution were used for subsequent assessment. Briefly semen samples were diluted, stained with Hoechst 33342 and food dye and sorted with a MoFlo SX® flow cytometer/sperm sorter. Flow-cytometrically sorted spermatozoa were deflected into polypropylene tubes containing 500 μl of 2 % Tes-Tris-egg yolk buffer, than the collected semen was centrifuged, diluted in freezing medium and frozen. Sex-sorted and control non-sexed frozen semen (two stallions of in vitro proven fertility, C, D) was thawed, centrifuged on a Percoll gradient, washed and diluted 1:1 in PVP before ICSI. Oocytes were collected from ovaries of slaughtered mares at the beginning of the breeding season and matured in vitro. Metaphase II oocytes were injected with sperm, subsequently cultured up to the blastocyst stage and frozen in glycerol. Six embryos were subsequently thawed and non-surgically transferred in naturally cycling synchronous recipient 5 days after spontaneous ovulation. Overall 70, 58, 30 and 15 oocytes were injected with sex-sorted and control frozen semen from stallion A, B, C, D respectively. Mean cleavage rates were 20.31% for sorted sexed semen and 71.11% for control showing a significantly lower cleavage rate for sexed semen. This difference was reflected on the number of blastocyst obtained (4.18% vs 20.00%). Out of 6 frozen-thawed embryos transferred we obtained 4 pregnancies, one was lost at 21 days, a second was pharmacologically aborted at 21 days and two were maintained to go to term (one male and one female are currently in the 8th month of gestation). In conclusion, in this study we demonstrated that sex-sorted sperm can be used for producing blastocysts able to establish pregnancies at high rate when transplanted in recipient mares. However the efficiency of the system is low due to the very low cleavage rate obtained with such sex-sorted frozen thawed semen.

Ovum pick-up, intracytoplasmic sperm injection and embryo culture applied to subfertile mares and stallions are safe and reliable techniques for the production of offspring; often, they are the only options available. Embryos produced outside the breeding season can be frozen for later transfer, which makes these techniques very versatile.

In this study, we compared the developmental capacity of bovine haploid and diploid androgenetic and parthenogenetic embryos obtained by different methods. Androgenetic embryos were produced by piezo-intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF) of enucleated oocytes with or without subsequent pronuclear transfer from one haploid zygote to another. Parthenogenetic embryos were obtained by activation of matured oocytes by ionomycin combined with cycloheximide or 6-dimethylaminopurine (DMAP) treatment. Only few cleaved androgenetic haploid embryos were able to compact (2.7%) and to form blastocysts (1.8%), while significantly more haploid parthenogenotes underwent compaction (24-37%) and a minority developed to blastocysts at different rates, depending on the activation procedure (cycloheximide 3%, 6-DMAP 14.5%). By contrast, development to blastocyst of diploid androgenotes, cloned androgenetic embryos, and parthenogenotes (31%, 39%, and 43%, respectively) was similar to IVF control embryos (35%). Cell number on Day 7 was higher for IVF blastocysts and decreased in consecutive order in diploid androgenotes, diploid parthenogenotes, and haploid uniparental embryos. Following transfer of diploid androgenetic embryos, a pregnancy was established and maintained up to Day 28.