Mastitis or other infectious diseases have been related to reduced fertility in cattle. Inflammatory cytokines such as tumor necrosis factor α (TNFα) are released in response to infection and may have negative effects on embryo development. In the current study the effect of exposure to TNFα on the development of in vitro fertilized bovine embryos was examined. Indomethacin, a prostaglandin synthesis inhibitor, was used to determine if blockade of prostaglandin synthesis would alter the effects of TNFα. Ovaries were obtained from a local abattoir and immature COC were isolated from 2-10 mm follicles, in vitro matured and fertilized. After fertilization, groups of presumptive zygotes were randomly placed into either control development medium, medium containing 25 ng/mL TNFα or medium containing 25 ng/mL TNFα plus 1 μg/mL indomethacin. The proportion of blastocysts formed was assessed at day 7 of culture. Fewer embryos exposed to TNFα alone reached the blastocyst stage (17.5 ± 2.4%, P < 0.01) compared with controls (30.5 ± 2.4%) or embryos developed in TNFα plus indomethacin (25.8 ± 2.8%). There was no difference between control embryos and embryos developed in TNFα plus indomethacin. These results indicate that TNFα is inhibitory to the in vitro development of bovine embryos and that this inhibition may be mediated by prostaglandins because it can be blocked by indomethacin.

The male germ line in mammals is composed of self-renewing cells, spermatogonia, the meiotic spermatocytes and spermiogenic spermatids. Identification of these cell stages in vitro has been problematic. Transgenic animals expressing a marker gene with a promoter specific to certain cell stages in the testis would be a useful approach to identifying these cells in a viable state. Towards this end, we have produced transgenic pigs expressing mitochondrial localized enhanced yellow fluorescent protein (EYFP-mito) under control of the germ cell specific Stimulated by Retinoic Acid 8 (Stra8) promoter. Stra8 has been shown to be expressed in pre-meiotic germ cells of mice. Twelve clones harboring the Stra8-EYFP-mito transgene were produced. Analysis by Western blot indicated that expression of the transgene was limited to testicular tissue in the transgenic pigs. Single cells and seminiferous tubules were cultured in vitro and subsequently examined with epifluorescent microscopy. Expression of EYFP was noted in cells cultured for up to 5 days. Both EYFP-mito and STRA8 antibodies were shown to bind and co-localize in seminiferous tubule cells in whole mounts and in histological sections. EYFP-mito in the transgenic pigs co-localized with the endogenous stem cell marker, NANOG. Expression of the Stra8-EYFP transgene in spermatogenic cells indicates that these pigs will be useful by providing labelled cells for use in such technologies such as germ cell transplantation and in vitro spermatogenic studies.

Polymer and polymer/ceramic composite coatings were produced by ball-milling 60 lm Nylon-11 together with nominal 10 vol.% of nano and multiscale ceramic reinforcements and by HVOF spraying these composite feedstocks onto steel substrates to produce semicrystalline micron and nanoscale reinforced polymer matrix composites. Room temperature dry sliding wear performance of pure Nylon-11, Nylon-11 reinforced with 7 nm silica, and multiscale Nylon-11/silica composite coatings incorporating 7-40 nm and 10 lm ceramic particles were characterized using a pin-on-disk tribometer. Coefficient of friction and wear rate were determined as a function of applied load and coating composition. Surface profilometry and scanning electron microscopy were used to characterize and analyze the coatings and wear scars. The pure Nylon-11 coating experienced less wear than the composites due to the occurrence of two additional wear mechanisms: abrasive and fatigue wear.

Stimulated by retinoic acid 8 (STRA8) is a protein that is required for meiotic initiation in both male and female gametes in vertebrates. It is also expressed in embryonic germ cells and neonatal male germ cells of mice. The utility of using the Stra8 promoter to recognise and isolate pre-meiotic male germ cells has been reported by others in the mouse. In order to mark germ cells in male pigs, we cloned 1.6 kb of the mouse Stra8 promoter and used it to develop a reporter plasmid using mitochondrial-localised enhanced yellow fluorescent protein (mEYFP). The Stra8-mEYFP transgenic male pigs were produced using somatic cell nuclear transfer. The mEYFP reporter was expressed and easily detectable in the live germ cells of the mature animals and could be observed during tissue culture. The mitochondrial-localised expression of the EYFP reporter was helpful in observing the size and stage of the germ cell. The mEYPF protein was found to be expressed only in the testis of the transgenic pigs using Western blot analysis, whereas endogenous STRA8 protein was also detected in the lung and brain. Fluorescent immunohistochemistry of testicular sections of the transgenic pigs indicated a similar expression pattern to that of the endogenous STRA8 protein. There was an overlap in the expression of the mEYFP and the endogenous STRA8 protein; however, it was observed that the mEYFP protein was present at an earlier stage of spermatogenesis than the STRA8 protein. Immunocytochemistry performed on plated tubules similarly showed varying intensity in expression between the mEYFP transgene and the endogenous STRA8. The difference in the timing of protein expression may be due to the model created or the use of the mouse Stra8 promoter for the expression of mEYFP. Alternatively, the lag in expression between that of the endogenous STRA8 and mEYFP protein may be due to attenuated translation of the Stra8 mRNA. This transgenic model should be useful for the study of reproduction, development, transplantation, biotechnology, and culture of the pig male germ line. Supported by North Carolina Agricultural Research Service 02234.