Efficiency of sperm-mediated gene transfer in the ovine by laparoscopic insemination, in vitro fertilization and ICSI

188-196Transgenesis constitutes an important tool for pharmacological protein production and livestock improvement. We evaluated the potential of laparoscopic insemination (LI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to produce egfp-expressing ovine embryos, using spermatozoa previously exposed to pCX-EGFP plasmid in two different sperm/DNA incubation treatments: "Long Incubation" (2 h at 17 C) and "Short Incubation" (5 min at 5 C). For LI, Merino sheep were superovulated and inseminated with treated fresh semen from Merino rams. The embryos were recovered by flushing the uterine horns. For IVF and ICSI, slaughterhouse oocytes were fertilized with DNA-treated frozen/thawed sperm. All recovered embryos were exposed to blue light (488 nm) to determine green fluorescent morulae and blastocysts rates. High cleavage and morulae/blastocysts rates accompanied the LI and IVF procedures, but no egfp-expressing embryos resulted. In contrast, regardless of the sperm/ plasmid incubation treatment, egfp-expressing morulae and blastocysts were always obtained by ICSI, and the highest transgenesis rate (91.6 percent) was achieved with Short Incubation. In addition, following the incubation of labeled plasmid DNA, after Long or Short exposure treatments, with fresh or frozen/thawed spermatozoa, only non-motile fresh spermatozoa could maintain an attached plasmid after washing procedures. No amplification product could be detected following PCR treatment of LI embryos whose zonae pellucidae (ZP) had been removed. In order to establish conditions for transgenic ICSI in the ovine, we compared three different activation treatments, and over 60 percent of the obtained blastocysts expressed the transgene. For ICSI embryos, FISH analysis found possible signals compatible with integration events. In conclusion, our results show that in the ovine, under the conditions studied, ICSI is the only method capable of producing exogenous gene-expressing embryos using spermatozoa as vectors

Plant Breeding Perspectives for Alfalfa (\u3cem\u3eMedicago sativa\u3c/em\u3e L.) Success in Warm Climates

Climate change can have major impacts on adaptation of forage species to agroecosystems around the world. The ability of breeders to select for traits that impart adaptability to climate resilience will be critical for the future of grasslands. Alfalfa (Medicago sativa L.) is the most important perennial forage legume in the world because of its relatively high yield and nutritional value. In Florida, nondormant cultivars were developed for improved adaptation to the state’s subtropical agroecosystem (‘Florida 66’, ‘Florida 77’, and ‘Florida 99’); however, these cultivars are not commercially available. Breeding efforts are underway to develop new nondormant alfalfa adapted to subtropical conditions. The main goal of the alfalfa breeding program at the University of Florida (UF) is to combine germplasm screening, genomics, enviromics, and phenomics to improve yield and persistence. The integration of multi-omics data can result in greater genetic gain by reducing the length of the breeding cycle and by increasing the size of breeding populations. The development of nondormant, persistent, and high yielding cultivars would be a big step towards establishing alfalfa systems in warmer climates

Simple gene transfer technique based on I-SceI meganuclease and cytoplasmic injection in IVF bovine embryos

Although transgenic methods in mammals are inefficient, an easy and highly efficient transgenesis systemusing I-SceI meganuclease (intron-encoded endonuclease fromS. cerevisiae)was recently described in Xenopus. The method consisted of injection into fertilized eggs of an I-SceI reaction mixture with a plasmid DNA carrying the transgene, flanked by the meganuclease recognition sites (pIS). In the present study, the effects of I-SceI on gene transfer were tested apparently for the first time in mammals, in particular, in cattle. Various conditions were evaluated, including three concentrations of the plasmid pIS Pax6egfp, carrying I-SceI recognition sites flanking egfp under Pax6 promoter and two injection times (before IVM and after IVF) of pIS CAGegfp, carrying I-SceI sites fanking egfp under CAG promoter. In addition, the quantity of transgenewasmeasured using quantitative polymerase chain reaction, and presence of transgene signals was evaluated using fluorescence in situ hybridization analysis. Transgene expression rateswere higher (P< 0.05) for groups treated after IVF (79.1%, 91/115 and 63.0%, 75/ 119) than before IVM (32.6%, 31/95 and 34.7%, 33/95), with and without I-SceI, respectively. Interestingly, injectionwith pIS plus I-SceI after IVF increased frequency (P<0.05) of nonmosaic transgene-expressing embryos (58.3%, 42/72 vs. 29.7%, 25/84) for pIS plus I-SceI and pIS alone. Based on fluorescence in situ hybridization analysis, injectionwith I-SceI increased (P<0.05) the proportion of embryos with transgene signals in all blastomeres compared with pIS alone (44.0%,11/25 vs. 6.9%, 2/29) for pIS plus I-SceI and pIS alone. In addition, transgene copy number was numerically higher for the group treated with pIS plus I-SceI compared with pIS alone. In conclusion, I-SceI gene transfer increased transgene signals in bovine embryos.Fil: Bevacqua, Romina Jimena. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Canel, Natalia Gabriela. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Hiriart, María Inés. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Sipowicz, P.. Universidad Nacional de San Martín. Laboratorio de Neuro y Citogenética Molecular; ArgentinaFil: Rozenblum, G. T.. Universidad Maimónides. Area de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; ArgentinaFil: Vitullo, Alfredo Daniel. Universidad Maimónides. Area de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Radrizzani Helguera, Martin. Universidad Nacional de San Martín. Laboratorio de Neuro y Citogenética Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Fernández y Martín, Rafael. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentin

6 EFFICIENT TRANSGENESIS IN BOVINE EMBRYOS BY FERTILIZATION WITH ANDROGENETIC TRANSGENIC BLASTOMERES

Pronuclear microinjection and intracytoplasmic sperm injection-mediated gene transfer (ICSI-mgt) are useful techniques to obtain transgenic animals. Nevertheless, a high frequency of mosaic expression is observed in embryos and offspring produced by these techniques. A possible explanation is that the transgene integrates in the embryo genome after the first cell division. Our main objective was to develop a new technique to generate transgenic bovine embryos without mosaic expression and with high efficiency. We hypothesize that fertilizing metaphase II (MII) oocytes with transgenic androgenetic haploidblastomeres (AHB) (from mosaic embryos) would result in non-mosaic transgenic embryos. To this aim, in the first experiment we generated AHB by enucleating IVM MII oocytes, before or after injecting with a single spermatozoon incubated with pCX-EGFP (enhanced green fluorescent protein) plasmid. These treatments were analyzed by Fisher test (P < 0.05). The rate of cleavage of the androgenetic transgenic embryos enucleated before and after ICSI-mgt was 35.1% (34/97) and 61.2% (71/116), respectively (P < 0.05). These embryos showed expression of EGFPof 11.8% (4/34) and 42.3% (30/71) (P < 0.05) with 0% (0/34) and 9.9% (7/71) of non-mosaic expression. The haploid condition of the androgenetic embryos was confirmed by karyotype analysis. After this first approach, we chose the procedure of enucleation after ICSI for successive experiments. In the second experiment, the haploid androgenetic embryos (4 to 16 cells) were disaggregated, and the AHB obtained were used to fertilize MII oocytes. Fertilization was carried out by fusing a single AHB to a zona-free MII oocyte, followed by chemical activation. Presumptive zygotes were cultured in SOF medium in the well of the well (WOW) system. To confirm fertilization, single AHB produced with sexed Y spermatozoa and embryos generated with them were checked by PCR using Y- and X-specific sequence primers. PCR analysis confirmed Y-specific sequences in all the AHB and XY-specific sequences in each of the analyzed embryos. FISH analysis on blastocysts was performed with a specific probe for a Y chromosome sequence, confirming the sexed sperm genome in all blastocyst cells. Additionally, the expression pattern of Oct-4 (pluripotent marker gene) was examined in the blastocysts by inmunocytochemistry with a confocal microscope. Blastocysts displayed a pattern of Oct-4 expression similar to that of IVF embryos, indicating efficient nuclear reprogramming. Finally, we fertilized MII oocytes with EGFP-AHB to produce transgenic bovine embryos without mosaic expression. The development reached 85.1% of cleavage and 9.0% of blastocysts (n = 84). One hundred percent of the embryos showed EGFP expression, with 90.1% non-mosaic expression. In conclusion, our results proved that it is possible to use AHB for fertilization of MII oocyte, and that fertilization with transgenic AHB is a highly efficient technique for the generation of transgenic non-mosaic bovine embryos.Fil: Vichera, Gabriel Damian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Pereyra Bonnet, Federico Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Olivera, Ramiro. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Sipowicz, Pablo. Universidad Nacional de San Martín; ArgentinaFil: Radrizzani Helguera, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín; ArgentinaFil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Perceptions of Supervisors and Performance: A Thematic Analysis

Observation is a common occurrence within the workplace, and can often manifest as either peer-to-peer monitoring or supervisor-to-peer monitoring. To date, there is a limited body of research that describes changes in performance due to either a positive or negative relationship between supervisors and employees. The present study reports qualitative data on supervisor-to-employee relationships and how the quality of the relationship can alter human performance. The results indicated that relationship with a supervisor was related to perceived performance under direct observation. Women were more likely to report a positive relationship with their supervisor, yet also indicated a negative emotion toward being monitored. These results are important in understanding how supervisor presence influences individual performance when completing job-specific tasks. The implications for future research are discussed