Nitrite to nitrate molar ratio is inversely proportional to oxidative cell damages and granulocytic apoptosis at the wound site following cutaneous injury in rats

Nitric oxide (NO) metabolism in response to the inflammatory cell infiltration and their apoptosis at the wound site, using a model of subcutaneously implanted sponges in Albino Oxford rats, were examined. The injured animals were sacrificed at days 1, 2 and 3 after the injury. Nitrites, nitrates (final products of NO metabolism), malondialdehyde (an indicator of oxidative cell damages), urea (product of arginase activity) and other parameters were measured both in plasma and wound fluid samples. Nitrite to nitrate molar ratio and sum of nitrites and nitrates (NO_x) were calculated. The total cell numbers were at similar level throughout the examined period, but a gradual decrease of viable granulocytes, mainly due to the increased apoptosis, and the increase of monocyte-macrophage number occurred after the second day. A gradual increase of wound fluid nitrates, NO_x and malondialdehyde suggested the increases of both NO and free oxygen radicals production. Interestingly, wound fluid nitrites peaked at the first day decreasing to the corresponding plasma levels thereafter. Wound fluid nitrite to nitrate molar ratio gradually decreased and negatively correlated both with the number of apoptotic cells (r = −0.752, p < 0.05) and malondialdehyde (r = −0.694, p < 0.05) levels. In conclusion, the inversely proportional relation between nitrite to nitrate molar ratio and both malondialdehyde and apoptotic cell number indicated a mutual relationship between NO metabolism, oxidative cell damages and cell apoptosis at the wound site early after the cutaneous wound. Moreover, the obtained findings suggest that measurement of both nitrites and nitrates contribute to better insight into overall wound NO metabolism.     

Growth hormone inhibits apoptosis in in vitro produced bovine embryos.

Growth hormone (GH) has recently been shown to exert distinct effects on the differentiation and metabolism of early embryos. Up to now, however, it is not clear whether GH is able to modulate apoptosis during early embryogenesis. Differential cell staining of 8-day-old bovine embryos cultured with 100 ng bovine recombinant GH (rbGH) per ml medium (synthetic oviduct fluid-polyvinylalcohol) demonstrated that GH significantly increased the number of inner cell mass (ICM) and trophectoderm cells in bovine expanded blastocysts. As shown by terminal deoxynucleotidyl transferase mediated dUTP labeling (TUNEL) supplementation of bGH decreased the percentage of 8-day-old embryos showing at least one apoptotic cell from 58 to 21%. The percentage of apoptotic cells in one blastocyst was significantly (P < 0.01) reduced from 4.6 to 1.1% by GH treatment. Incubation of the embryos with 150 mM vanillylnonanamide induced apoptosis in all embryos. Whereas in control embryos 14% of the embryonic cells were TUNEL-positive, the percentage of apoptotic cells declined to 2.7% in the GH treated embryos. Expression of immunoreactive bcl-2 in blastocysts was not affected by GH treatment. Synthesis of the bax protein which is known to promote apoptosis was reduced in embryos cultured with GH. Our results suggest that GH acts as survival factor during in vitro culture and reduces apoptosis by altering the bax to bcl-2 ratio during early embryogenesis.     

Comparison of Animal Infectivity and Nucleic Acid Staining for Assessment of Cryptosporidium parvum Viability in Water

Cryptosporidium parvum oocysts were stained with the fluorogenic dyes SYTO-9 and SYTO-59 and sorted by flow cytometry in order to determine whether the fluorescent staining intensity correlated with the ability of oocysts to infect neonatal CD-1 mice. Oocysts that did not fluoresce or that displayed weak fluorescent intensity when stained with SYTO-9 or SYTO-59 readily established infections in mice, whereas those oocysts that fluoresced brightly did not. Although fluorescent staining profiles varied among different batches of oocysts, a relative cutoff in fluorescent staining intensity that correlated with animal infectivity was observed for all batches.     

Bovine somatic cell nuclear transfer using recipient oocytes recovered by ovum pick-up: effect of maternal lineage of oocyte donors.

The efficiency of bovine nuclear transfer using recipient oocytes recovered by ultrasound-guided follicle aspiration (ovum pick-up [OPU]) was investigated. Oocyte donors were selected from 2 distinct maternal lineages (A and B) differing in 11 nucleotide positions of the mitochondrial DNA control region. A total of 1342 cumulus-oocyte complexes (COCs) were recovered. The numbers of total COCs and class I/II COCs recovered from donors of lineage A were higher (P < 0.001) than those obtained from lineage B. Follicle aspiration once per week yielded a higher (P < 0.001) total number of COCs per session than aspiration twice per week, whereas the reproduction status of donors (heifer vs. cow) had no effect on OPU results. Of the 1342 oocytes recovered, 733 (55%) were successfully matured in vitro and used for nuclear transfer. Fusion was achieved in 550 (75%) karyoplast-cytoplast complexes (KCCs), resulting in 277 (50%) cleaved embryos on Day 3. On Day 7 of culture, 84 transferable embryos (15% based on fused KCCs) were obtained. After 38 transfers (10 single, 22 double, and 6 triple transfers), 9 recipients (8 double and 1 triple transfer) were diagnosed as pregnant on Day 28, corresponding to a pregnancy rate of 24%. The proportion of transferable embryos on Day 7 was significantly (P < 0.05) influenced by maternal lineage of oocyte donors and by the frequency of follicle aspiration. Our study demonstrates the feasibility of generating nuclear transfer embryos with defined cytoplasmic background. These will be valuable tools to experimentally dissect the effects of nuclear and cytoplasmic components on embryonic, fetal, and postnatal development.