Affect Animal Survival Research Articles

Persistence of social structuring in an arid zone mammal following reintroduction

Animal social relationships affect animal survival, reproduction, and resource exploitation, and are important to translocation success, but little is known on how they are impacted in reintroduced populations. Here, we investigate the social genetic structure in a reintroduced population of boodies (Bettongia lesueur), one of few social burrowing marsupial species in Australia. Pairwise relatedness of male and female individuals located within seven warrens was determined using 12 microsatellite markers, while mitochondrial DNA was used to identify maternal ancestral lineage. Females were significantly more related within warrens than between warrens, while the difference among males was not significant. We also found evidence of fine-scale genetic structure up to distances of 2 km in females consistent with patterns found in natural populations. Although mean relatedness values were low, our results support the hypothesis that boodies exhibit a female social structure. Multiple maternal lineages were present within all warrens with one exception, suggesting boodies appear to be non-selective when living with individuals from different ancestral backgrounds. We found a significant positive relationship between female sex bias and the number of active entrances in each warren. Together, these results suggest that social structuring should be a key consideration in future reintroductions of boodies.

Safe and Efficacious Near Superhydrophobic Hemostat for Reduced Blood Loss and Easy Detachment in Traumatic Wounds.

Hemorrhage is the leading cause of trauma death, and innovation in hemostatic technology is important. The strongly hydrophobic carbon nanofiber (CNF) coating has previously been shown to have excellent hemostatic properties. However, the understanding of how CNF coating guides the coagulation cascade and the biosafety of CNF as hemostatic agents has yet to be explored. Here, our thrombin generation assay investigation showed that CNF induced fast blood coagulation via factor (F) XII activation of the intrinsic pathway. We further performed studies of a rat vein injury and demonstrated that the CNF gauze enabled a substantial reduction of blood loss compared to both the plain gauze and kaolin-imbued gauze (QuikClot). Analysis of blood samples from the model revealed no acute toxicity from the CNF gauze, with no detectable CNF deposition in any organ, suggesting that the immobilization of CNF on our gauze prevented the infiltration of CNF into the bloodstream. Direct injection of CNF into the rat vein was also investigated and found not to elicit overt acute toxicity or affect animal survival or behavior. Finally, toxicity assays with primary keratinocytes revealed minimal toxicity responses to CNF. Our studies thus supported the safety and efficacy of the CNF hemostatic gauze, highlighting its potential as a promising approach in the field of hemostatic control.

Large Peritoneal Macrophages and Transitional Premonocytes Promote Survival during Abdominal Sepsis.

Monocytes and macrophages are early sentinels of infection. The peritoneum contains two resident populations: large and small peritoneal macrophages (LPMs and SPMs). While LPMs self-renew, circulating monocytes enter the peritoneum and differentiate into SPMs. We lack information on the dynamics of monocyte-macrophage trafficking during abdominal sepsis, reflecting an important knowledge gap. In this study, we characterize the presence of LPMs, SPMs, and monocytes in the peritoneum of mice following cecal ligation and puncture (CLP)-induced sepsis and sham surgery. LPMs rapidly disappeared from the peritoneum and were scarce at 18-66 h after CLP or sham surgery. By 14 d, LPMs returned for sham mice, but they remained scarce in CLP mice. Depletion of LPMs from the peritoneum of CD11b-DTR mice greatly increased animal mortality. These data imply that LPMs are critical for sepsis survival. Monocytes rapidly infiltrated the peritoneum and were abundant at 18-66 h after CLP or sham surgery. Surprisingly, SPMs only increased at 14 d post-CLP. Therefore, monocytes may defend hosts from acute sepsis mortality without generating SPMs. More monocytes were present in mice predicted to survive sepsis versus mice predicted to die. However, altering monocyte numbers via CCR2 deficiency or adoptive transfer did not significantly affect animal survival. We reasoned that animals destined to survive sepsis may exhibit a different monocyte phenotype, rather than merely enhanced numbers. Indeed, mice predicted to survive possessed more CD31+, CXCR4hi transitional premonocytes in their abdomen. Inhibition of CXCL12-CXCR4 signaling via AMD3100 exacerbated sepsis. These data imply that recruitment of transitional premonocytes to the abdomen promotes sepsis survival.

Evolutionary dynamics in the Anthropocene: Life history and intensity of human contact shape antipredator responses.

Humans profoundly impact landscapes, ecosystems, and animal behavior. In many cases, animals living near humans become tolerant of them and reduce antipredator responses. Yet, we still lack an understanding of the underlying evolutionary dynamics behind these shifts in traits that affect animal survival. Here, we used a phylogenetic meta-analysis to determine how the mean and variability in antipredator responses change as a function of the number of generations spent in contact with humans under 3 different contexts: urbanization, captivity, and domestication. We found that any contact with humans leads to a rapid reduction in mean antipredator responses as expected. Notably, the variance among individuals over time observed a short-term increase followed by a gradual decrease, significant for domesticated animals. This implies that intense human contact immediately releases animals from predation pressure and then imposes strong anthropogenic selection on traits. In addition, our results reveal that the loss of antipredator traits due to urbanization is similar to that of domestication but occurs 3 times more slowly. Furthermore, the rapid disappearance of antipredator traits was associated with 2 main life-history traits: foraging guild and whether the species was solitary or gregarious (i.e., group-living). For domesticated animals, this decrease in antipredator behavior was stronger for herbivores than for omnivores or carnivores and for solitary than for gregarious species. By contrast, the decrease in antipredator traits was stronger for gregarious, urbanized species, although this result is based mostly on birds. Our study offers 2 major insights on evolution in the Anthropocene: (1) changes in traits occur rapidly even under unintentional human "interventions" (i.e., urbanization) and (2) there are similarities between the selection pressures exerted by domestication and by urbanization. In all, such changes could affect animal survival in a predator-rich world, but through understanding evolutionary dynamics, we can better predict when and how exposure to humans modify these fitness-related traits.

Learning skills in Tropidurus lizards are associated with territory harshness

AbstractLearning increases the ability to cope with challenges experienced in natural environments and likely affects animal survival, especially in harsh environments. Most evidence for associations between environmental characteristics and learning ability derives from comparisons among closely related species of mammals or birds, although the contribution of such behavioral trait for variation in habitat usage may be established among individuals at the population level. Here, we used individuals of Tropidurus catalanensis lizards from the same population to test for associations between individual problem‐solving capacity and the type of territory used. Our prediction was that individuals living in harsher territories would exhibit enhanced capability for problem solving than lizards from more permissive (mild) territories. Lizards were tested during eight non‐consecutive days using a customized feeder and visual cues. After confirming that lizards learn to solve the proposed task, we corroborated the hypothesis that environmental parameters affecting territory harshness are associated with individual learning capacity. Specifically, individuals living in harsh territories presented greater enhancement of their performance for problem solving with exposure to the task. We also identified that some of these lizards shifted their behavior along the 2 weeks of trials, adopting an approach that apparently minimized attempts prior to success. Challenges imposed by structurally different territories may be overcome by individuals exhibiting enhanced learning, a skill that may initially allow colonization of harsh territories by a given set of individuals but might also be cryptic in the population and then become expressed in response to specific environmental settings.

The role of steroid hormones and individual traits in food intake in the wood mouse (Apodemus sylvaticus).

Availability of food resources affects animal survival and reproduction. Thus, coping with changes in food availability is one of the most crucial behavioural and physiological processes in wildlife. Food intake is a key concept in animal ecology that is directly conditioned by food quality and abundance or diet choice, but may also vary according to individual-related factors (e.g. foraging behaviours, social rank or energy-demanding periods) and the influence of the endocrine system on energy metabolism. Here, we studied food intake in relation to individual characteristics (sex, breeding condition and age) and whether steroid hormones (testosterone and corticosterone metabolites) mediate food intake in wild wood mouse (Apodemus sylvaticus). Field work was carried out in February-March 2014 in Monte de Valdelatas (Madrid, Spain). Wood mice were live-trapped for 10 consecutive days in four independent plots. Traps were baited with 4g of toasted corn and food intake was calculated by subtracting the remaining bait found inside traps. Fresh faecal samples from 130 different wood mice were collected and faecal testosterone and corticosterone metabolites (FTM and FCM, respectively) were analysed by enzyme immunoassays. Food intake was higher in females than males, probably due to greater energy requirements. Non-breeders and young individuals also showed a higher food intake. These individuals usually hold a lower social rank which is associated to limited food resources because of dominants; thus, increased food intake may be a result of freely exploit food bait inside traps while avoiding risky competition. In addition, food intake negatively correlated with FTM levels and positively with FCM levels indicating that both hormones have an active role mediating food intake in the wood mouse. Our data suggest that food intake is a function of both individual traits and the endocrine system that accordingly respond throughout different energy-demanding periods.

Diet and ambient temperature interact to shape plasma fatty acid composition, basal metabolic rate, and oxidative stress in great tits

Diet and ambient temperature affect animal physiology, survival and reproductive success. However, knowledge of how these environmental factors interact to shape physiological processes and life-history traits of birds and other animals is largely lacking. By exposing adult great tits (Parus major) to two contrasting diets (saturated or unsaturated fatty acids; SFAs and UFAs, respectively) and ambient temperatures (3°C versus 20°C) that the birds encounter in nature, we investigated the effects of these two factors on several physiological parameters. Our results show that diet and ambient temperature interact to affect the composition of plasma fatty acids, basal metabolic rate (BMR) and oxidative stress, which are thought to affect the life-history and survival of individuals. Specifically, birds provided the SFA-rich diet had higher mass-specific BMR and oxidative stress (levels of lipid peroxidation) after exposure to low compared with high ambient temperature, whereas the opposite pattern was evident for birds with a UFA-rich diet. Surprisingly, birds on the SFA diet had higher relative levels of monounsaturated fatty acids compared with the UFA-fed birds at low ambient temperature, whereas the opposite, and expected, pattern was found at the high temperature. Although the present study focuses on the physiological implications of the diet×temperature interaction, our results might also be important for the leading theories of ageing, which currently do not take interactions between environmental factors into account. In addition, the present results are important for wildlife management, especially with regards to anthropogenic feeding of wild animals across variable and changing climatic conditions.

Aquaporin-1 inhibition reduces metastatic formation in a mouse model of melanoma.

Aquaporin‐1 (AQP1) is a proangiogenic water channel protein promoting endothelial cell migration. We previously reported that AQP1 silencing by RNA interference reduces angiogenesis‐dependent primary tumour growth in a mouse model of melanoma. In this study, we tested the hypothesis that AQP1 inhibition also affects animal survival and lung nodule formation. Melanoma was induced by injecting B16F10 cells into the back of C57BL6J mice. Intratumoural injection of AQP1 siRNA and CTRL siRNA was performed 10 days after tumour cell implantation. Lung nodule formation was analysed after the death of the mice. Western blot was used to quantify HIF‐1α, caspase‐3 (CASP3) and metalloproteinase‐2 (MMP2) protein levels. We found that AQP1 knock‐down (KD) strongly inhibited metastatic lung nodule formation. Moreover, AQP1 siRNA‐treated mice showed a twofold survival advantage compared to mice receiving CTRL siRNAs. The reduced AQP1‐dependent tumour angiogenesis caused a hypoxic condition, evaluated by HIF‐1α significant increase, in turn causing an increased level of apoptosis in AQP1 KD tumours, assessed by CASP3 quantification and DNA fragmentation. Importantly, a decreased level of MMP2 after AQP1 KD indicated a decreased activity against extracellular matrix associated with reduced vascularization and metastatic formation. In conclusion, these findings highlight an additional role for AQP1 as an important determinant of tumour dissemination by facilitating tumour cell extravasation and metastatic formation. This study adds knowledge on the role played by AQP1 in tumour biology and supports the view of AQP1 as a potential drug target for cancer therapy.

Neuroprotective effects of p-tyrosol after the global cerebral ischemia in rats

BackgroundSalidroside is a biologically active compound derived from Rhodiola rosea L. Studies showed that salidroside after i.v. injection is extensively metabolized to p-tyrosol and only trace amounts of salidroside are found in the brain tissue. ObjectiveThe aim of the study was to investigate the neuroprotective effects of p-tyrosol in the global cerebral ischemia-reperfusion (GCI) model. Study designA total of 103 Wistar rats were assigned to groups of sham-operated (n=10), control (n=42), p-tyrosol-treated (n=36), and pentoxifylline-treated (n=15) animals. The rats of control, p-tyrosol-treated, and pentoxifylline-treated groups received intravenously 0.9% NaCl solution, 2% solution of p-tyrosol in doses of 5mg/kg, 10mg/kg, and 20mg/kg, and pentoxifylline in a dose of 100mg/kg, respectively, daily for 5 days. Rats were examined at days 1, 3, and 5 after GCI. After evaluation of neurological deficit, animals were euthanized for morphological and biochemical characterization. MethodsRats of control, p-tyrosol-treated, and pentoxifylline-treated groups were exposed to three-vessel model of GCI. Neurological deficit, numeric density of neurons in hippocampal CA1 region, and percentage of neurons with focal and total chromatolysis were studied. Biochemical study assessed contents of conjugated dienes and fluorescent products in brain homogenate. ResultsIn control group, only 50.0% of rats survived by day 5 after the GCI; 38.1% of survived animals had severe neurologic deficit. In brain tissue of PTX-treated rats, the levels of diene conjugates and fluorescent products were 79% and 73%, respectivley, at day 5 compared with control. Differences in diene conjugates were statistically significant compared with control. The survival rate of animals treated with 20mg/kg p-tyrosol was 82.3% at day 5 after GCI. In p-tyrosol-treated GCI rats, the numeric density of neurons in the hippocampal CA1 region was higher by 31% compared with control. The percentage of neurons with focal and total chromatolysis decreased by 27% and 43%, respectively. At day 5 after GCI, the levels of conjugated dienes and fluorescent products were significantly lower (by 37% and 45%, respectively) in group of animals treated with 20mg/kg p-tyrosol compared with control. Moderate neuroprotective effects of 5mg/kg p-tyrosol administration were documented only at day 5 after GCI. In case of 10mg/kg p-tyrosol administration, neuroprotection was documented sooner: at day 1 or 3 after GCI. However, administration of 5 and 10mg/kg p-tyrosol did not affect animal survival. ConclusionCourse administration of intravenous p-tyrosol in a dose of 20mg/kg increased survival, reduced neurological deficit after GCI, attenuated neuronal damage in the hippocampus, and attenuated lipid peroxidation in brain tissue in animals subject to GCI with reperfusion.

Alpha-Linolenic Acid-Induced Increase in Neurogenesis is a Key Factor in the Improvement in the Passive Avoidance Task After Soman Exposure.

Exposure to organophosphorous (OP) nerve agents such as soman inhibits the critical enzyme acetylcholinesterase (AChE) leading to excessive acetylcholine accumulation in synapses, resulting in cholinergic crisis, status epilepticus and brain damage in survivors. The hippocampus is profoundly damaged after soman exposure leading to long-term memory deficits. We have previously shown that treatment with three sequential doses of alpha-linolenic acid, an essential omega-3 polyunsaturated fatty acid, increases brain plasticity in naïve animals. However, the effects of this dosing schedule administered after a brain insult and the underlying molecular mechanisms in the hippocampus are unknown. We now show that injection of three sequential doses of alpha-linolenic acid after soman exposure increases the endogenous expression of mature BDNF, activates Akt and the mammalian target of rapamycin complex 1 (mTORC1), increases neurogenesis in the subgranular zone of the dentate gyrus, increases retention latency in the passive avoidance task and increases animal survival. In sharp contrast, while soman exposure also increases mature BDNF, this increase did not activate downstream signaling pathways or neurogenesis. Administration of the inhibitor of mTORC1, rapamycin, blocked the alpha-linolenic acid-induced neurogenesis and the enhanced retention latency but did not affect animal survival. Our results suggest that alpha-linolenic acid induces a long-lasting neurorestorative effect that involves activation of mTORC1 possibly via a BDNF-TrkB-mediated mechanism.

A Hepatic Protein, Fetuin-A, Occupies a Protective Role in Lethal Systemic Inflammation

BackgroundA liver-derived protein, fetuin-A, was first purified from calf fetal serum in 1944, but its potential role in lethal systemic inflammation was previously unknown. This study aims to delineate the molecular mechanisms underlying the regulation of hepatic fetuin-A expression during lethal systemic inflammation (LSI), and investigated whether alterations of fetuin-A levels affect animal survival, and influence systemic accumulation of a late mediator, HMGB1.Methods and FindingsLSI was induced by endotoxemia or cecal ligation and puncture (CLP) in fetuin-A knock-out or wild-type mice, and animal survival rates were compared. Murine peritoneal macrophages were challenged with exogenous (endotoxin) or endogenous (IFN-γ) stimuli in the absence or presence of fetuin-A, and HMGB1 expression and release was assessed. Circulating fetuin-A levels were decreased in a time-dependent manner, starting between 26 h, reaching a nadir around 24–48 h, and returning towards base-line approximately 72 h post onset of endotoxemia or sepsis. These dynamic changes were mirrored by an early cytokine IFN-γ-mediated inhibition (up to 50–70%) of hepatic fetuin-A expression. Disruption of fetuin-A expression rendered animals more susceptible to LSI, whereas supplementation of fetuin-A (20–100 mg/kg) dose-dependently increased animal survival rates. The protection was associated with a significant reduction in systemic HMGB1 accumulation in vivo, and parallel inhibition of IFN-γ- or LPS-induced HMGB1 release in vitro.ConclusionsThese experimental data suggest that fetuin-A is protective against lethal systemic inflammation partly by inhibiting active HMGB1 release.

Cytotoxicity from sulfide exposure in a sulfide-tolerant marine invertebrate

It is unknown whether sulfide-tolerant marine invertebrates suffer cytotoxicity from sulfide exposure in vivo at environmentally-relevant concentrations. We tested this with the mudflat polychaete Glycera dibranchiata. Exposure of live animals to sulfide up to 2.4 mmol l − 1 for 24 h did not affect animal survival, and animal condition (gross appearance and activity) was unaffected at sulfide concentrations up to 0.25 mmol l − 1 . However, animal condition was decreased at higher sulfide concentrations (P < 0.0001, n = 33). Coelomic fluid obtained from the animals showed decreased erythrocyte count (P = 0.0035, n = 14), indicating cell loss, and increased propidium iodide and Hoechst 33324 staining (P < 0.0001 and P = 0.0003, respectively, n = 14), indicating loss of plasma membrane integrity, even at sulfide concentrations that produced no change in animal condition. When G. dibranchiata were allowed a 72 h recovery period following 24 h sulfide exposure, there was no overall improvement in condition (P ≥ 0.12, n = 7–8), and worms that had been exposed to 1 mmol l − 1 sulfide still had an erythrocyte count that was less than half that of control animals (P = 0.028, n = 7). The inability to completely recover the cell count was at least partially due to impaired production of new erythrocytes, since analysis of BrdU incorporation indicated that erythrocyte proliferation was reduced from 2% per day in control animals to 0.12% per day in animals exposed to 1 mmol l − 1 sulfide (P = 0.010, n = 21). Together, these findings indicate that at least some sulfide-tolerant marine invertebrates experience significant cellular injury and impaired tissue proliferation when exposed to environmentally relevant sulfide concentrations, even when the appearance and behavior of the animal appear unaffected.

Human Immunoglobulin G2 (IgG2) and IgG4, but Not IgG1 or IgG3, Protect Mice againstCryptococcus neoformansInfection

The encapsulated yeast Cryptococcus neoformans is a significant cause of meningitis and death in patients with AIDS. Some murine monoclonal antibodies (MAbs) against the glucuronoxylomannan (GXM) component of the C. neoformans capsular polysaccharide can prolong the lives of infected mice, while others have no effect or can even shorten survival. To date, no one has systematically compared the efficacies of antibodies with the same variable regions and different human constant regions with their unique combination of effector functions in providing protection against murine C. neoformans infection. In the present study, we examined the efficacies of anti-GXM MAbs of the four human immunoglobulin G (IgG) subclasses, which have identical variable regions but differ in their capacities to bind the three types of Fc receptors for IgG (FcgammaR), their abilities to activate complement, and their half-lives. IgG2 and IgG4 anti-GXM prolonged the lives of infected BALB/c mice, IgG3 anti-GXM did not affect animal survival, while mice treated with IgG1 anti-GXM died earlier than mice treated with phosphate-buffered saline or irrelevant isotype-matched MAbs. All MAbs decreased serum GXM in infected animals. Effector pathways traditionally believed to be important in defense against microbes, such as opsonophagocytosis and complement binding, negatively correlated with antibody efficacy. It is generally accepted that human IgG1 has the most favorable combination of effector functions for therapeutic use against infections. Therefore, our findings have significant implications for humanization of the mouse IgG1 currently in clinical trials for cryptococcal meningitis and for the design of antibody therapeutics to treat other infectious diseases as well.

Superior T Cell and Humoral Immunity Generated Against B Cell Lymphomas Using a Sulfhydryl-Based Idiotype Carrier Protein Conjugation Method.

Therapeutic vaccination of lymphoma patients with tumor-specific immunoglobulin (idiotype, Id) chemically-coupled to the highly immunogenic foreign carrier protein keyhole limpet hemocyanin (Id-KLH) has shown promising results in phase I/II clinical trials, and phase III trials are underway. However, many patients fail to mount anti-Id immune responses. Traditionally Id is coupled to KLH using glutaraldehyde (glut), which crosslinks via lysine, cysteine, tyrosine and histidine residues. Maleimide (mal) crosslinks only via cysteine sulfhydryl groups, thus limiting the potential for epitope destruction, and potentially allowing more efficient lysosomal processing in antigen presenting cells. The murine B cell lymphoma A20 was reported to be unresponsive to vaccination with glut/Id-KLH. We hypothesized that Id epitopes might be damaged by glut, but preserved using the more specificmal conjugation method. Id proteins were partially reduced to generate free sulfhydryl groups, and reacted with mal-activated KLH. Mice with 4-day established A20 lymphoma were vaccinated with 3 weekly glut/Id-KLH or mal/Id-KLH plus GM-CSF. As previously reported, tumor eradication was uncommon in glut/Id-KLH-treated mice (8–25%), with no statistical advantage over control-treated mice. In contrast, tumor was eradicated in 33–66% of mice vaccinated with mal/Id-KLH, with tumor free-survival being highly significant compared to control mice (p=0.0001) and glut/Id-KLH-vaccinated mice (p=0.0084). Combined vaccination with glut/Id-KLH+mal/Id-KLH did not improve survival over mal/Id-KLH alone (p=0.99). KLH conjugated to an irrelevant antibody did not result in any long-term survivors when challenged with A20. The length of time Id and KLH were allowed to conjugate with glut showed a statistically significant time dependence that affected survival (highest 58%) and anti-Id titers, while mal conjugation showed no such time dependence, and gave superior protection (83%). Mice vaccinated with A20 mal/Id-KLH generated 14-fold higher anti-Id antibody titers compared to glut/Id-KLH, and had superior survival after lethal tumor challenge (58% vs. 25%, respectively). A20 immune sera stained A20 cells via FACS analysis and the dominant isotype was shown to be IgG1 by ELISA. Adoptive transfer of high titer A20 anti-Id immune sera resulted in no protective advantage compared to naïve sera indicating antibodies are not the dominant protective mechanism against A20. Depletion of CD8+ T cells demonstrated their critical role in A20 tumor eradication (p<0.0001). In the prototypical 38C13 model, mice vaccinated with mal/Id-KLH developed 3-fold higher anti-Id antibody titers compared to glut/Id-KLH treated animals, and had superior survival after tumor challenge (85% vs. 47%, respectively). Depletion of T cell subsets did not affect animal survival when challenged with 38C13. Here protection was dependent on antibodies rather than T cells. In a third model (BCL-1), 50% of mal/Id-KLH mice survived compared to 25% for glut/Id-KLH, with 3-fold higher titers. Human IgG could be efficiently bound to mal-activated KLH, demonstrating the feasibility of this method for human applications. These results show that the method of Id conjugation to carrier protein can substantially affect the level of tumor protection elicited by Id-KLH vaccination for B cell lymphoma, and may explain why some patients fail to respond to traditional glut/Id-KLH vaccinations. These findings have important implications for the ongoing development of vaccines targeting tumor Id.

Water or ice?--the challenge for invertebrate cold survival.

The ecophysiology of cold tolerance in many terrestrial invertebrate animals is based on water and its activity at low temperatures, affecting cell, tissue and whole organism functions. The normal body water content of invertebrates varies from 40 to 90% of their live weight, which is influenced by water in their immediate environment, especially in species with a water vapour permeable cuticle. Water gain from, or loss to, the surrounding atmosphere may affect animal survival, but under sub-zero conditions body water status becomes more critical for overwinter survival in many species. Water content influences the supercooling capacity of many insects and other arthropods. Trehalose is known to maintain membrane integrity during desiccation stress in several taxa. Dehydration affects potential ice nucleators by reducing or masking their activity and a desiccation protection strategy has been detected in some species. When water crystallises to ice in an animal it greatly influences the physiology of nearby cells, even if the cells remain unfrozen. A proportion of body water remains unfrozen in many cold hardened invertebrates when they are frozen, which allows basal metabolism to continue at a low level and aids recovery to normal function when thawing occurs. About 22% of total body water remains unfrozen from calculations using differential scanning calorimetry (compared with ca 19% in food materials). The ratio of unfrozen to frozen water components in insects is 1:4 (1:6 for foods). Such unfrozen water may aid recovery of freezing tolerant species after a freezing exposure. Rapid changes in cold hardiness of some arthropods may be brought about by subtle shifts in body water management. It is recognised that cold tolerance strategies of many invertebrates are related to desiccation resistance, and possibly to mechanisms inherent in insect diapause, but the role of water is fundamental to them all. Detailed experimental studies are needed to provide information which will allow a more complete and coherent understanding of the behaviour of water in biological systems and aid the cryopreservation of a wide range of biological material.

Ovariectomy increases squamous metaplasia of the uterine horns and survival of SENCAR mice fed a vitamin A–deficient diet

Retinoic acid is necessary for the growth and differentiation of organisms and exerts its molecular actions by binding to specific nuclear receptors that belong to the thyroid-steroid hormone receptor superfamily. Steroids and retinoids control the differentiation of the female reproductive epithelia: estrogen maintains the squamous differentiation of vaginal and ectocervical epithelia, whereas retinoic acid maintains the simple columnar endocervical and uterine epithelia. These lining epithelia transform into a squamous metaplastic phenotype in vitamin A-deficient animals. Furthermore, mortality due to vitamin A deficiency is usually attributed to infection resulting in part from dysfunction of the protective epithelia. Our objective was to test the hypothesis that estrogen depletion might change the squamous metaplastic response to vitamin A deficiency and affect animal survival. We used female SENCAR mice maintained on a purified vitamin A-deficient diet containing either 0 or 3 microg retinoic acid/g diet. Mice were either ovariectomized or intact. Squamous cells arising in the normally simple columnar epithelium of the endocervix and uterine cavity were monitored by keratin 5 expression with immunohistochemistry. Ovariectomy did not change the time to onset of vitamin A deficiency. It increased the number of squamous metaplastic cells and prolonged survival in mice consuming a vitamin A-deficient diet by as much as 40%. Factors other than epithelial differentiation per se control survival outcome of vitamin A-deficient mice. The results also show a significant increase in longevity of vitamin A- deficient mice when ovariectomized.

Evaluation of the potential carcinogenicity of 60 Hz linear sinusoidal continuous-wave magnetic fields in Fischer F344 rats.

Electric and magnetic fields (EMFs) associated with the production, transmission, and use of electricity are ubiquitous in industrialized societies. These fields are predominantly of low frequency (50/60 Hz) and are generally of low intensity. Review of the epidemiological evidence shows that the association between exposure to EMFs and cancer is weak and inconsistent, and generally fails to show a dose-response relationship. Moreover, in view of the methodological problems of these epidemiological studies, animal and laboratory studies are urgently needed to determine whether EMFs could be initiators and/or promoters of cancers. The objective of the present study was to determine whether chronic exposure to 60 Hz linear (single axis) sinusoidal, continuous-wave magnetic fields (MFs) of different intensities might increase the risk of leukemia and solid tumor development in rodents born and raised under these fields. Five groups of 50 female F344 rats were exposed for 20 h/day to 60 Hz MFs at intensities of <0.02 (sham controls), 2, 20, 200, and 2000 microT. Full body exposure to the different fields was administered for 104 wk starting from the prenatal period (2 days before birth) and continuing during lactation and weaning until late adult life. Body weight, survival, and clinical observations were evaluated in all groups of animals during in-life exposure. Necropsy was performed on all exposed and control animals that died, were found moribund, or were killed at termination of the study. To preserve and demonstrate the absence of any experimental bias, all clinical observations and pathological evaluations were conducted under "blinded" conditions. Fifty organs and tissues were evaluated in each animal, with special attention to the incidence of mononuclear cell leukemia, brain tumors, and mammary tumors. The findings from this chronic carcinogenicity study demonstrate that, under our defined experimental conditions, exposure to 60 Hz linear (single axis) sinusoidal, continuous wave MFs did not affect animal survival, solid tumor, or mononuclear cell leukemia development in female F344 rats. No statistically significant, consistent, positive dose-related trends with the number of tumor-bearing animals per study group could be attributed to MF exposure.

Contraceptive steroids and hypertension: an experimental model.

An experimental model is described to determine the relationship of oral contraceptives and hypertension by examining the effect of gonadal steroids on a strain of rats genetically predisposed to develop hypertension from a chronic excess of NaCl. 52 female salt-sensitive rats divided into 4 groups were maintained on a .38% salt diet during Weeks 3-5 of life while control blood pressure measurements were taken. Group A was then maintained on its diet and administered contraceptive steroids subcutaneously daily. Group B maintained its diet without change. The diet of Group C was changed to a 4% salt food and the animals were administered the steroids while Group D received the 4% NaCl diet without the steroids. All treatments were continued for 38 weeks and blood pressure was maintained weekly. The rats receiving contraceptive steroids showed significantly lower body weight than those not receiving steroids (p=.0001). The animals on the high salt diet had significantly higher blood pressure than those rats on the low salt diet (p=.0001). Group C rats had significantly higher blood pressure than Group D (p=.0001) but Group A rats had significantly lower blood pressure than Group B (p=.006). The administration of contraceptive steroids enhanced the hypertension of the rats receiving the high salt diet but had no relationship to hypertension in the rats receiving the low salt diet. The steroids did not affect animal survival but the salt intake did. The mechanism of the hypertension enhancement described here is unknown but this experimental model should prove useful for further research.