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

Lobe-specific increases in malondialdehyde DNA adduct formation in the livers of mice following infection with Helicobacter hepaticus

Helicobacter hepaticus infection is associated with chronic hepatitis and the development of liver tumours in mice. The underlying mechanism of this liver carcinogenesis is not clear but the oxidative stress associated with H. hepaticus infection may result in induction of lipid peroxidation and the generation of malondialdehyde. Malondialdehyde can react with deoxyguanosine in DNA resulting in the formation of the cyclic pyrimidopurinone N-1,N-2 malondialdehyde-deoxyguanosine (M(1)dG) adduct. This adduct has the potential to cause mutations that may ultimately lead to liver carcinogenesis. The objective of this study was to determine the control and infection-related levels of M(1)dG in the liver DNA of mice over time, using an immunoslot-blot procedure. The level of M(1)dG in control A/J mouse livers at 3, 6, 9 and 12 months averaged 37.5, 36.6, 24.8 and 30.1 adducts per 10(8) nucleotides, respectively. Higher levels of M(1)dG were detected in the liver DNA of H. hepaticus infected A/JCr mice, with levels averaging 40.7, 47.0, 42.5 and 52.5 adducts per 10(8) nucleotides at 3, 6, 9 and 12 months, respectively. There was a significant age dependent increase in the level of M(1)dG in the caudate and median lobes of the A/JCr mice relative to control mice. A lobe specific distribution of the M(1)dG adduct in both infected and control mice was noted, with the left lobe showing the lowest level of the adduct compared with the right and median lobes at all time points. In a separate series of mice experimentally infected with H. hepaticus, levels of 8-hydroxy-deoxyguanosine were significantly greater in the median compared with the left lobe at 12 weeks after treatment. In conclusion, these results suggest that M(1)dG occurs as a result of oxidative stress associated with H. hepaticus infection of mice, and may contribute to liver carcinogenesis in this model

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