Ursids evolved early and continuously to be low-protein macronutrient omnivores.

The eight species of bears world-wide consume a wide variety of diets. Some are specialists with extensive anatomical and physiological adaptations necessary to exploit specific foods or environments [e.g., polar bears (Ursus maritimus), giant pandas (Ailuropoda melanoleuca), and sloth bears (Melursus ursinus)], while the rest are generalists. Even though ursids evolved from a high-protein carnivore, we hypothesized that all have become low-protein macronutrient omnivores. While this dietary strategy has already been described for polar bears and brown bears (Ursus arctos), a recent study on giant pandas suggested their macronutrient selection was that of the ancestral high-protein carnivore. Consumption of diets with inappropriate macronutrient profiles has been associated with increased energy expenditure, ill health, failed reproduction, and premature death. Consequently, we conducted feeding and preference trials with giant pandas and sloth bears, a termite and ant-feeding specialist. Both giant pandas and sloth bears branched off from the ursid lineage a million or more years before polar bears and brown bears. We found that giant pandas are low-protein, high-carbohydrate omnivores, whereas sloth bears are low-protein, high-fat omnivores. The preference for low protein diets apparently occurred early in the evolution of ursids and may have been critical to their world-wide spread.

Microcystin accumulation in Sportfish from an agricultural reservoir differs among feeding guild, tissue type, and time of sampling.

Cyanobacterial blooms sometimes create secondary metabolites that can be transferred between trophic levels and accumulate in fish, but little is known about what time of year fish are most susceptible. Here, we examine microcystin in the muscle, liver, and kidney of bluegill and largemouth bass from an agricultural reservoir over 12 months. We identify which fish characteristics and water parameters best explain microcystin accumulation in fish tissues. Microcystin in bluegill was significantly higher than largemouth bass. In both species, microcystin was highest in livers (bluegill mean = 57.6 ng g - 1, largemouth bass mean = 71.8 ng g - 1 wet weight [ww]), then kidneys (bluegill mean = 27.1, largemouth bass mean = 22.7 ng g - 1 ww), followed by muscles (bluegill mean = 7.6, largemouth bass mean = 5.7 ng g - 1 ww). Adult bluegill feed on benthic macroinvertebrates and zooplankton, which may explain their higher microcystin concentrations compared to largemouth bass, which are primarily piscivorous. Harvest date emerged as the best predictor of microcystin in muscles and kidneys, with the highest concentrations occurring in April. Microcystin in water also emerged as a significant predictor, albeit much lower than harvest date, suggesting that low but persistent microcystin concentrations in water may result in accumulation of this cyanotoxin in fish. This study is the first to examine microcystin in fish from the North American Great Plains and one of only 5 studies that investigate microcystin in bluegill and largemouth bass. Additional investigation into the relationship between cyanobacteria and fish health is warranted, especially during spring when fish microcystin concentrations were highest.

Mercury, selenium, and fatty acids in the axial muscle of largemouth bass: evaluating the influence of seasonal and sexual changes in fish condition and reproductive status.

Largemouth bass (LMB, 265-475 mm) were collected to document whether changes in fish condition and reproductive status influenced the concentration of total mercury (Hg) and selenium (Se) in axial muscle by season and sex. The fatty acid (FA) composition of fish was also examined to describe seasonal and sexual differences and identify whether arachidonic acid (ARA) could be used as a biomarker of Hg toxicity. There was a trend for females to have lower (p < 0.062) Se concentrations than males. The concentration of Se for females during spring (mean ± SD, 686 ± 51 ng/g dw) was 15% lower than males (806 ± 67 ng/g dw). Lower Se concentrations in females than males continued through summer and fall. Concentration of Hg for females during spring (152 ± 39 ng/g ww) was also 59% lower than males (373 ± 303 ng/g ww), but the difference was not significant (p > 0.2). The percent of lipids was greatest in fall and winter (3%) and comprised primarily of omega-3 fatty acids (35 g/100 g lipid). Fish condition as measured by percent lipids and relative weight was negatively (p < 0.02) related to Hg concentration for females and males. Lipid content for both sexes was also positively (p < 0.05) related to the Se:Hg ratio. Relative weight was positively related to the Se:Hg ratio for females during all seasons (p = 0.014), but only during spring and summer for males (p < 0.007). A low Se:Hg value was associated with an elevation in ARA for both sexes and a reduced hepatosomatic index in males. Data suggested that females transferred muscle stores of Se and Hg to developing oocytes during spring. This study generates hypotheses regarding the physiological drivers of seasonal and sexual variability in Hg, Se, and FA in LMB that may be applicable to other species and have implications for fisheries health and management.

Simultaneous choice bioassays accompanied by physiological changes identify civetone and decanoic acid as pheromone candidates for giant pandas.

Chemicals present in urine are thought to play an important role in mate identification in the solitary giant panda (Ailuropoda melanoleuca). During the breeding season, females will deposit chemical signals to advertise sexual receptivity to potential mates. The goal of this study was to determine if specific volatile compounds found in female urine could be considered as pheromones that elicit behavioral and physiological responses in males. Experimental simultaneous choice trials were conducted with captive male giant pandas (n = 3) housed at Memphis Zoo, San Diego Zoo, and Zoo Atlanta. Octanoic acid, 1H-pyrrole-2-carboxaldehyde, decanoic acid, and civetone were selected as stimuli because previous studies reported their elevation in urine during the breeding season. Male interest was determined by a behavioral preference toward these volatile compounds diluted in synthetic urine compared with nontreated synthetic urine. Male urine samples were collected 1 week prior, during, and 1 week after the experimental period to assess changes in urinary semiochemical composition and urinary androgen concentrations. No significant differences in investigation response (p = .395) or flehmen response (p = .600) were found when stimuli were compared; however, decanoic acid and civetone elicited a behavioral preference over the control (response ratio > 0.5). The relative abundance of 16 compounds identified in male urine was significantly elevated (p < .05) above baseline values after the males were exposed to the stimuli. Androgen levels were significantly elevated (p < .05) in one male after exposure to 1H-pyrrole-2-carboxaldehyde, decanoic acid, and civetone. These data suggested that civetone and decanoic acid in female urine may motivate sexual responses in males.

Physical, Chemical, and Biological Factors that Contribute to the Variability of Mercury Concentrations in Largemouth Bass Micropterus salmoides from Missouri Reservoirs.

Large-bodied predatory sportfish from Missouri reservoirs can contain elevated methylmercury concentrations that are of concern to the health of consumers. The concentration of total mercury (tHg) in the muscle (which > 95% is in the methylated-Hg form) of harvestable-sized largemouth bass (Micropterus salmoides; LMB) was examined to determine which factors contributed to the variability of tHg concentration in sportfish populations among Missouri reservoirs. Mean tHg concentrations in LMB from each reservoir were compared to physical and chemical characteristics of the reservoir and to biological attributes of each LMB population. Low concentrations of tHg (70-170 ng/g wet weight) in LMB from large reservoirs (surface area ≥ 35,680 acres) were likely related to the dilution of chemical Hg forms with water volume and depth. The highest tHg concentrations in LMB (268-542 ng/g) were from reservoirs with low particulate inorganic material (< 1.5 mg/L) and chlorophyll a concentrations (< 14.6 µg/L), and from LMB populations with a low proportion of large fish (proportional size distribution of LMB > 12 inches was < 33%). These relationships suggest that resource competition among LMB likely contributed to tHg bioaccumulation in reservoirs < 930 acres. Small reservoirs located in northern Missouri also may have greater methylation potential due to warmer water temperatures and anoxic conditions, but more data are needed to confirm these interactions. Fish consumption advisories for reservoirs with large surface area and volume could be reduced from one fish meal per month to one per week. To improve Missouri fisheries and protect consumers, management strategies to limit methylation and improve fish growth should be considered to reduce methylmercury bioaccumulation in small- and medium-sized reservoirs.

Implementing solid phase microextraction (SPME) as a tool to detect volatile compounds produced by giant pandas in the environment.

Chemical cues are thought to play an important role in mate identification in the solitary giant panda (Ailuropoda melanoleuca). The goal of this study was to detect and identify volatile compounds present in the enclosure air of captive giant pandas. We hypothesized that a subset of compounds produced from breeding animals would be detected in environmental samples because highly volatile chemicals are likely to facilitate mate detection. Samples were collected from the enclosures of 8 giant pandas (n = 4 male, n = 4 female) during the Mar-June breeding season and the Aug-Jan non-breeding period from 2012-2015. Volatile compounds were captured by securing a solid phase micro extraction fiber approximately 3 meters above the ground within a panda enclosure for 6-12 hours. Compounds adsorbed onto the SPME fibers were analyzed by gas chromatography mass spectrometry. Thirty-three compounds were detected in at least 10% of all samples within individual and season and across all subjects within each season. Aromatic compounds made up 27.3% of the enclosure volatile profile, while 21.2% was made of cyclic aliphatic compounds and 51.5% of the enclosure profile was comprised of acyclic aliphatic compounds. Three compounds were likely to be present in male enclosures regardless of season, while Undecane, 4-methyl had a significant (p<0.05) predicted probability of being present in female enclosures. 3,3'-(1,1-Ethanediyl)bis(1H-indole) had a significant (p<0.05) probability of occurrence in male enclosures during the breeding season. Given the prevalence of these compounds, we suspect that these chemicals are important in giant panda communication. This novel sampling technique can detect volatile compounds produced by captive species and also may be a useful tool for detecting pheromones in free-ranging individuals.

Behavioral, semiochemical and androgen responses by male giant pandas to the olfactory sexual receptivity cues of females.

Male giant pandas identify female sexual receptivity through the detection of olfactory cues in estrous urine. However, it is yet unknown which specific days of the female estrous cycle may provoke male sexual-social responses and a physiological readiness to mate. We hypothesized that female urine from specific days of the estrous cycle will be positively associated with specific changes in male behaviors, urinary semiochemical production, and steroidogenic activity. Experimental simultaneous choice trials were conducted in captivity with four male giant pandas during the spring breeding season and during fall. Male interest was determined by a behavioral preference toward peri-estrual urine collected from a specific day of the estrous cycle encompassing proestrus (Day -13, Day -6, Day -3, Day -2), estrus (Day -1 and Day 0), and metestrus (Day four and Day nine) over that of anestrous urine. Provocation of male sexual motivation was examined by changes in urinary semiochemical composition and urinary androgen concentrations. During the spring, male investigative behaviors indicated a preference for Day -13, Day -3 and Day 0 urine over anestrous urine, while no significant preferences for estrous urine could be detected during fall. The relative abundance of only three compounds in male urine were significantly higher above baseline values after males were exposed to peri-estrual urine during spring; whereas 34 compounds significantly increased in the fall. Similarly, androgen concentrations increased above baseline in only two out of four males during spring, while all males had elevated androgen concentrations after exposure to Day -3 urine during the fall. Our results suggest that peri-estrual urine from Day -13, Day -3, and Day 0 elicited the greatest duration of male investigation, changes in the semiochemical profile, and elevations in androgen levels. These data suggest that managers should incorporate a combination of behavioral, semiochemical, and endocrinological assessment of males in the reproductive management of giant pandas to determine impending ovulation and pinpoint the best time for male-female introductions and artificial inseminations.

Changes in urinary androgen concentration indicate that male giant pandas (Ailuropoda melanoleuca) respond to impending female oestrus during and outside the typical spring breeding season.

Giant pandas have been described as mono-oestrus spring breeders, yet males exposed to aseasonal oestrous females in the autumn or winter exhibit breeding behaviours and interest in mating. In the present study, urinary androgens and sperm parameters were quantified for males exposed to females expressing oestrus during spring, autumn or winter to examine plasticity of reproductive seasonality in giant pandas. Monthly average androgen concentrations for two males exposed to females in either seasonal or aseasonal oestrus were greater (P<0.001) than baseline concentrations. Evaluation of daily androgen concentrations revealed a peak that was three- to fivefold greater than baseline, occurring an average of 5 days before ovulation for both seasonal and aseasonal cycles. There were no significant differences in testes volume, sperm motility, forward progression or sperm concentration in males between female seasonal and aseasonal cycle years. Male gonadal activity was more variable without a clear pattern in years when the female was anovulatory than when she was ovulatory (seasonal or aseasonal). These data show the flexible reproductive capacity of male giant pandas as demonstrated by a rapid physiological readiness to mate in response to female oestrous cues within or outside the normal breeding season and may suggest a facultative seasonal reproduction with a 'female-induced rut'.

Phenological changes in bamboo carbohydrates explain the preference for culm over leaves by giant pandas (Ailuropoda melanoleuca) during spring.

Seasonal changes in the foodscape force herbivores to select different plant species or plant parts to meet nutritional requirements. We examined whether the search for calorie-rich carbohydrates explained giant panda's selection for bamboo culm over leaves during spring. Leaves and culms were collected from four Phyllostachys bamboos (P. aurea, P. aureosulcata, P. glauca, and P. nuda) once per month over 18-27 months. Monthly changes in annual plant part nutrients were examined, and compared to seasonal foraging behaviors of captive giant pandas. Although total fiber was greater (p<0.0001) in culm (85.6 ± 0.5%) than leaves (55.3 ± 0.4%) throughout the year, culm fiber was at its lowest in spring (79-85%) when culm selection by giant pandas exceeded 70% of their overall diet. Culm starch also was greatest (p = 0.044) during spring (5.5 ± 1.1%) and 2.5-fold the percentage of starch in leaves (2.2 ± 0.6%). The free sugars in spring culm consisted of a high proportion of glucose (35%) and fructose (47%), whereas sucrose made up 42% of the total free sugar content of spring leaves. Bound sugars in culm consisted of 60% glucose and 38% xylose likely representative of hemicellulose. The concentrations of bound sugars (hemicelluloses) in spring culms (543.7 ± 13.0 mg/g) was greater (p<0.001) than in leaves (373.0 ± 14.8 mg/g). These data help explain a long-standing question in giant panda foraging ecology: why consume the plant part with the lowest protein and fat during the energetically intensive spring breeding season? Giant pandas likely prefer spring culm that contains abundant mono- and polysaccharides made more bioavailable as a result of reduced fiber content. These data suggest that phenological changes in bamboo plant part nutrition drive foraging decisions by giant pandas.

Urinary profiles of progestin and androgen metabolites in female polar bears during parturient and non-parturient cycles.

Due to the environmental and anthropogenic impacts that continue to threaten the reproductive success of polar bears, a more detailed understanding of their reproductive cycle is needed. Captive populations of polar bears provide an excellent opportunity to learn more about the reproductive physiology of the species. Progestin (P4) and androgen (T) metabolites in urine, and their ratio (P4:T), were examined during 11 reproductive cycles of captive female polar bears (n = 4) to characterize the steroid hormone profile during pregnancy and determine possible variations related to reproductive failure. The concentration of hormone metabolites in urine were determined through enzyme immunoassay. Reproductive cycles were classified as pregnant (n = 3), anovulatory (n = 4) and ovulatory-non-parturient (n = 4) based on the changes in urinary hormone metabolite values and cub production. In the absence of a lactational suppression of estrus, elevated androgen concentrations suggested resumption of follicular development within 3 weeks of parturition. Breeding behaviours were most often observed when androgen values were at their highest or in decline. Ovulation was identified by a return to basal androgen concentration and elevation of progestins within 1-4 weeks after breeding. As a result, urinary concentrations of progestins were greater than androgens (P4:T ratio ≥ 1.0) during ovulatory cycles whereas the P4:T ratio was <1.0 when females were anovulatory. Progestins and the P4:T ratio of parturient cycles were greatest beginning in June/July (17-20 weeks after breeding) and reached a peak at 24-37 weeks (mid-October/mid-November, 4-9 weeks before birth of cubs). Non-invasive monitoring of hormone metabolites in urine provided a rapid determination of endocrine function for improved husbandry and reproductive management of polar bears in captivity. Further research is warranted to understand the reproductive endocrinology of polar bears and its impact on conservation and management of this species in captivity and the wild.

Using Domestic and Free-Ranging Arctic Canid Models for Environmental Molecular Toxicology Research.

The use of sentinel species for population and ecosystem health assessments has been advocated as part of a One Health perspective. The Arctic is experiencing rapid change, including climate and environmental shifts, as well as increased resource development, which will alter exposure of biota to environmental agents of disease. Arctic canid species have wide geographic ranges and feeding ecologies and are often exposed to high concentrations of both terrestrial and marine-based contaminants. The domestic dog (Canis lupus familiaris) has been used in biomedical research for a number of years and has been advocated as a sentinel for human health due to its proximity to humans and, in some instances, similar diet. Exploiting the potential of molecular tools for describing the toxicogenomics of Arctic canids is critical for their development as biomedical models as well as environmental sentinels. Here, we present three approaches analyzing toxicogenomics of Arctic contaminants in both domestic and free-ranging canids (Arctic fox, Vulpes lagopus). We describe a number of confounding variables that must be addressed when conducting toxicogenomics studies in canid and other mammalian models. The ability for canids to act as models for Arctic molecular toxicology research is unique and significant for advancing our understanding and expanding the tool box for assessing the changing landscape of environmental agents of disease in the Arctic.

Fecal estrogen, progestagen and glucocorticoid metabolites during the estrous cycle and pregnancy in the giant anteater (Myrmecophaga tridactyla): evidence for delayed implantation.

BACKGROUND: Declining numbers of wild giant anteaters highlight the importance of sustainable captive populations. Unfortunately, captive reproductive management is limited by the lack of external physical indicators of female reproductive status and the aggressive behavior of males. We examined the endocrinology of the estrous cycle and pregnancy, and whether delayed implantation is a gestational strategy for giant anteaters as described for other xenarthrans. METHODS: Feces were collected from seven captive females 3-5 times weekly and mating was recorded. Concentrations of estrogen (estrone-glucuronide, E1, and estradiol-17ß, E2), progestagen (20-oxo-progestagens, P4), and glucocorticoid (GC) metabolites were examined in fecal extracts by enzyme immunoassay. RESULTS: Estrous cycles for nulliparous females (6 cycles, n = 2) compared to the multiparous female (6 cycles, n = 1) were shorter (47.3 +/- 4.3 days versus 62.5 +/- 2.6 days) with relatively lower luteal phase concentrations of P4 (49.4 +/- 2.9 ng/g versus 136.8 +/- 1.8 ng/g). The four remaining females had unclear ovarian activity: two females exhibited apparent luteal activity but unclear fluctuations in estrogens, while the other two females had parallel fecal P4 and estrogens concentrations. Pregnancy ranged 171-183 days with females returning to estrus post-partum as early as 60 days (n = 3, 1.8-4 years of age at mating). Delayed implantation was indicated by a biphasic elevation in fecal P4 metabolites: the initial 4-fold increase occurred for 81-105 days and was followed by a 26-fold secondary rise in P4 metabolites lasting 66-94 days prior to parturition. Fecal GC was correlated with fecal estrogens and greatest during estrus, late pregnancy, and six days prior to parturition (estrous cycle GC, 14.4-62.8 ng/g; pregnancy GC, 13.6-232.7 ng/g). CONCLUSIONS: Estrous cycles of giant anteaters occurred year-round, but were shorter and more intermittent in younger nulliparous animals compared to a multiparous female. A pronounced elevation in fecal P4, estrogen, and GC occurred during late gestation after an initial post-mating delay providing evidence for delayed implantation. Adrenocorticoid activity indicated impending parturition. Differences in estrous cycle characteristics with age and the protracted but variable gestation length must be considered to improve reproductive success and neonatal survival in giant anteaters.

Lactational transfer of mercury and polychlorinated biphenyls in polar bears.

We examined concentrations of total mercury (tHg, inorganic and methylated forms) and polychlorinated biphenyls (PCBs) in blood and milk from free-ranging Southern Beaufort-Chukchi Sea polar bears (Ursus maritimus) to assess maternal transfer of contaminants during lactation and the potential health risk to nursing young. Concentrations of contaminants in the blood of dependent and juvenile animals (ages 1-5 years) ranged from 35.9 to 52.2 µg kg(-1) ww for tHg and 13.9 to 52.2 µg kg(-1) ww (3255.81-11067.79 µg kg(-1) lw) for ΣPCB(7)s, similar to those of adult females, but greater than adult males. Contaminant concentrations in milk ranged from 5.7 to 71.8 µg tHg kg(-1)ww and 160 to 690 µg ΣPCB(11)s kg(-1) ww (547-5190 µg kg(-1) lw). The daily intake levels for tHg by milk consumption estimated for dependent young were below the tolerable daily intake level (TDIL) of tHg established for adult humans. Although the daily intake levels of PCBs through milk consumption for cubs of the year exceeded the TDIL thresholds, calculated dioxin equivalents for PCBs in milk were below adverse physiological thresholds for aquatic mammals. Relatively high concentrations of non-dioxin like PCBs in polar bear milk and blood could impact endocrine function of Southern Beaufort-Chukchi Sea polar bears, but this is uncertain. Transfer of contaminants during mid to late lactation likely limits bioaccumulation of dietary contaminants in female polar bears during spring. As polar bears respond to changes in their arctic sea ice habitat, the adverse health impacts associated with nutritional stress may be exacerbated by tHg and PCBs exposure, especially in ecologically and toxicologically sensitive polar bear cohorts such as reproductive females and young.

Histologic findings in free-ranging Sarasota Bay bottlenose dolphin (Tursiops truncatus) skin: mercury, selenium, and seasonal factors.

Full-thickness epidermal biopsy samples were collected from free-ranging common bottlenose dolphins (Tursiops truncatus) in Sarasota Bay, Florida, USA. Season (summer or winter) of collection, mercury (Hg) concentration, and selenium (Se) concentration were compared to histologic parameters. Epidermal Hg concentration was positively related to age (P<0.001) and negatively related to height of the stratum spinosum (P<0.05). The mitotic index and heights of the stratum externum and intermedium were lower in summer than in winter (P<0.01). Transmission electron microscopic examination revealed variation in the diameters (60-138 nm) and arrangements of collagen fibers, regardless of age or concentrations of Hg and Se. The significance of the variation in height of the stratum spinosum and the perivascular collagen degeneration to dolphin health need further investigation.

Blood-based biomarkers of selenium and thyroid status indicate possible adverse biological effects of mercury and polychlorinated biphenyls in Southern Beaufort Sea polar bears.

We examined biomarkers of selenium status (whole blood Se; serum Se; glutathione peroxidase activity) and thyroid status (concentrations and ratios of thyroxine, T4; tri-iodothyronine, T3; albumin) in polar bears to assess variations among cohorts, and relationships to circulating concentrations of contaminants. Concentrations of total mercury (Hg) in whole blood were similar among cohorts (prime aged males and females, older animals, ages≥16 years, and young animals, ages 1-5 years; 48.44±35. 81; p=0.253). Concentrations of sum of seven polychlorinated biphenyls (∑PCB7) in whole blood were greater in females (with and without cubs, 26.44±25.82 ng/g ww) and young (26.81±10.67 ng/g ww) compared to males (8.88±5.76 ng/g ww, p<0.001), and significantly related to reduced body condition scores (p<0.001). Concentrations of Se and albumin were significantly greater in males than females (whole blood Se, males, 42.34 pmol/g ww, females, 36.25±6.27 pmol/g ww, p=0.019; albumin, males, 4.34±0.34 g/dl, females, 4.10±0.29 g/dL, p=0.018). Glutathione peroxidase activity ranged from 109.1 to 207.8 mU/mg hemoglobin, but did not differ significantly by sex or age (p>0.08). Thyroid hormones were greater in females (solitary females and females with cubs) compared to males (p<0.001). Biomarkers of Se status and concentrations of T3 were significantly positively related to Hg in all prime aged polar bears (p<0.03). Albumin concentrations were significantly positively related to total TT4, and significantly negatively related to concentrations of ∑PCB7 (p<0.003). Total thyroxine (TT4) was significantly negatively associated with blood concentrations of ∑PCB7 in solitary females (p=0.045). These data suggest that female polar bears were more susceptible to changes in blood-based biomarkers of selenium and thyroid status than males. Further classifications of the physiologic states of polar bears and repeated measures of individuals over time are needed to accurately assess the biological impact of combined toxicant exposures.

Mercury concentrations in Southern Beaufort Sea polar bears: variation based on stable isotopes of carbon and nitrogen.

Total Hg concentration was measured in hair and whole blood of 52 adult Southern Beaufort Sea polar bears (Ursus maritimus) captured in the spring of 2005. Stable isotopic signatures (i.e., 13C/12C, delta13C; 15N/14N, delta15N) in hair and two blood compartments (packed blood cells/clot and serum) were determined to assess the variation of Hg concentrations among polar bears in relation to their feeding ecology and other biological factors. Concentrations of Hg in hair and blood (2.2-23.9 microg/g dry wt and 0.007-0.213 microg/g wet wt, respectively) were within the range of values previously reported for polar bears in Canada and East Greenland. Mercury concentration in hair from females was higher than that in hair from males, and concentration was related to interactions between delta13C, delta15N, and longitude of capture location. Mercury concentrations in hair were inversely correlated to delta13C in hair and blood, suggesting that polar bears with greater total Hg concentrations fed more on pelagic prey, such as ringed seals or beluga whale, than on benthic prey. Variability in Hg concentrations in polar bear hair and blood may be the result of intraspecific or regional variation in prey selection rather than strictly trophic level interactions.

Targeting angiogenesis for mammary cancer prevention: factors to consider in experimental design and analysis.

An experimental model developed to investigate premalignant stages of breast cancer was used to establish a rationale for designing experiments that target angiogenesis for cancer prevention. Blood vessels were identified via CD31 immunostaining, and all vessels that occurred in a 50 microm wide region circumscribing each pathology were counted using a digital imaging technique. The blood vessel density associated with terminal end buds was unaffected by carcinogen treatment, whereas vessel density was higher in intraductal proliferations and ductal carcinoma in situ than in terminal end buds (P < 0.001) and total vascularity increased with morphologic progression. In comparison with intraductal proliferation or ductal carcinoma in situ, mammary carcinomas had higher vascular density in the tissue surrounding the cancer with a marked increase in the number of blood vessels <25 microm(2). These data suggest that antiangiogenic chemopreventive agents would inhibit cancer occurrence if initiated at any premalignant stage of the carcinogenic process. Because increased vascular density observed during premalignancy could be explained by the size expansion of the lesion and its encroachment on a preexisting blood supply, by pathology-associated vessel expansion, and/or by angiogenesis, it remains to be determined if antiangiogenic agents will reduce the prevalence of premalignant lesions or cause their accumulation by blocking conversion to carcinomas. Failure to recognize the patterns of vascularization that accompany morphologic progression could limit the success of efforts to target angiogenesis for cancer prevention and lead to misinformation about how agents that affect blood vessel formation or growth inhibit the carcinogenic process.

Nutritional development of feeding strategies in arctic ruminants: digestive morphometry of reindeer, Rangifer tarandus, and muskoxen, Ovibos moschatus.

Reindeer have been classified as intermediate feeders and muskoxen as grazers based on differences in digestive morphology and consumption of fibrous plants. We hypothesized that the digestive morphology of young (< 2 months) reindeer and muskoxen anticipates transitions in diet and determines the feeding strategy of each species at adulthood. We compared structural morphology and rates of cell division in the rumen, abomasum, duodenum and liver of reindeer and muskoxen as neonates (1 day old), during the transition from milk to forage (30-60 days old) and in adults (> 7 yr). Development in utero provided the neonate with a functioning mucosa of the gastric abomasum and duodenal mucosa with high surface enlargement for digestion and absorption of concentrated milks. Transition to forage was preceded by changes in ruminal papillae structure that increased surface area and likely contributed to active fermentation by 60 days of age. The abomasum also increased in acid-secreting parietal cells during the transition to forage, which may enhance digestion of plant and microbial proteins. Rates of cell division also indicated a sustained differentiation of tissue structure during the transitional period. Young arctic ruminants expressed digestive structures that preceded full function, which indicated the ultimate feeding strategy of each species. For example, the rumen of young muskoxen had thick cornified epithelia and muscle layers that would provide ruminal mucosa with better protection from fibrous abrasion and enhance motility of bulky diets. Conversely, young reindeer had more complex papillary shapes in the rumen and more foliate villi in the duodenum, indicating a greater absorptive capacity of these structures than in muskoxen. Ontogenetic programs, therefore, play the primary role for digestive development of reindeer and muskoxen and determine the nutritional strategies of adults.

Vascular density profile of rat mammary carcinomas induced by 1-methyl-1-nitrosourea: implications for the investigation of angiogenesis.

Given the mounting interest in studying the effects of various cancer preventive regimes on the angiogenic process in autochthonously growing mammary tumors, the experiment reported here was conducted to identify factors that should be considered in the collection of data from experiments in which a primary endpoint is the measurement of blood vessel density as a reflection of angiogenic activity. Female Sprague-Dawley rats were injected i.p. with 50 mg 1-methyl-1-nitrosourea (MNU)/kg body weight at 21 days of age. Tumor-bearing rats were killed 35 days post-carcinogen. Immunohistochemical detection of blood vessels using antiserum directed against the CD31 epitope was performed on paraffin sections of 36 carcinomas representing four histological types. Census counting was used to detect all blood vessels that were subsequently divided into one of five vessel size categories. Vessels were counted in both the intra-tumoral region and in a 50 microm width band of mammary tissue circumscribing each tumor; the circumscribed area was referred to as the extra-tumoral region. Vascular density was reported in units of vessel counts or vessel area per unit of assessed area. Overall, vascular density was observed to be highly variable and was not affected by tumor histology. Vascular density in the extra-tumoral region was significantly higher than in the intra-tumoral region. Correlation analyses indicated that similar information was obtained when vascular density was reported as either vessel counts or vessel area; however, correlations across vessel size categories and between the intra-tumoral and extra-tumoral regions were low and generally not statistically significant. Collectively, the data provide a complete description of vascularization in autochthonously growing mammary tumors, and a reference for comparison in studies of various cancer preventive agents that modulate the angiogenic process.

Semi-automated method of quantifying vasculature of 1-methyl-1-nitrosourea-induced rat mammary carcinomas using immunohistochemical detection.

Studies of the vascularization of autochthonous rodent mammary tumors are limited in number, and the majority have used Factor VIII staining for blood vessel detection. Moreover, little effort has been directed at measuring the vascularization of tissue immediately adjacent to a tumor despite its central importance in the process of angiogenesis. Thirty-six chemically-induced mammary carcinomas and tissue immediately adjacent to these carcinomas were used to develop a census counting method for quantitative assessment of intra- and extra-tumor vascularization. Blood vessels were identified using antiserum directed against either CD31 or Factor VIII. Techniques used to create digitized images of all tumors and the semi-automated methods for circumscribing the extra-tumoral region are described. For Factor VIII, CD31 allowed greater discrimination of blood vessels with areas <25 microm(2) and demonstrated crisp staining of blood vessels, with minimal background and excellent preservation of tissue architecture. Census counting data support the use of CD31 for quantifying both intra- and extra-tumoral vascularization. This method provides a basis for standardizing the approach to evaluation of experimentally induced premalignant and malignant mammary lesions in rodent model systems used to investigate potential anti-angiogenic cancer preventive agents.