Diet explains interpopulation variation of plasma carotenoids and skin pigmentation in nestling white storks

Carotenoids have a dietary origin in birds, but mechanisms by which they are absorbed in the gut, transported in the blood, metabolized at various sites, and deposited in the integument remain poorly understood. Variation in both plasma carotenoid levels and external color may reflect different access to dietary carotenoids or individual physiological differences in the uptake and deposition of carotenoids. We compared total plasma carotenoid concentration in nestling white storks (Ciconia ciconia) from 11 Spanish colonies in two consecutive years. The main food item in one of the colonies was the red swamp crayfish (Procambarus clarkii), a recently introduced species. Storks in the remaining colonies ate a variety of foods but no crayfish. Total plasma carotenoid levels in the colony where crayfish were consumed were about five times higher than in any other colony. These differences were maintained after controlling for the significant interyear variability, as well as for sex, age, and body mass of birds. Skin pigmentation also differed, being intensely orange in storks that consumed crayfish but white (unpigmented) in the remaining individuals. With thin-layer chromatography (TLC) and electronic absorption spectroscopy, astaxanthin was confirmed as the major carotenoid in crayfish as well as in the plasma, skin, and body fat of crayfish-eating storks, whereas lutein was the main carotenoid in plasma samples from the other colonies. These results indicate that a newly available carotenoid in the environment, astaxanthin, can be absorbed in large quantities from the gut and be transported in the blood before deposition in different tissues.     

On the relative efficiency of two- vs. one-stage production of astaxanthin by the green alga Haematococcus pluvialis

Haematococcus pluvialis under stress conditions overproduces the valuable red ketocarotenoid astaxanthin. Two proposed strategies for commercial production are under current analysis. One separates in time the production of biomass (optimal growth, green stage) and pigment (permanent stress, red stage), while the other uses an approach based on continuous culture under limiting stress at steady state. The productivities, efficiencies and yields for the pigment accumulation in each case have been compared and analyzed in terms of the algal basic physiology. The two-stage system indoors yields a richer astaxanthin product (4% of dry biomass) with a final astaxanthin productivity of 11.5 mg L(-1) day(-1), is more readily upscalable and amenable to outdoors production. Furthermore, each stage can be optimized for green biomass growth and red pigment accumulation by adjusting independently the respective ratio of effective irradiance to cell density. We conclude that the two-stage system performs better (by a factor of 2.5-5) than the one-stage system, and the former is best fit in an efficient mass production setup.     

Seasonal variation in energy expenditure and body composition in captive White Storks (Ciconia ciconia)

North Western European populations of White Storks (Ciconia ciconia) appear to have been saved from extinction by settling, i.e. stopping migration. Settled storks exposed to winter conditions must cope with periods of potentially high energy demands that would otherwise be avoided by the migration process. Doubly labeled water (DLW) was therefore used to examine the seasonal variation (summer vs winter) in daily energy expenditure (DEE) and the body composition of adult and immature storks of both sexes. Male White Storks showed a higher DEE over the winter period than in summer compared with females; in particular, immature males exhibited greater energy expenditure in winter than adult males. Thus, the DEE did not significantly differ between summer and winter (except for immature males), reflecting an absence of thermoregulation cost in winter. For both age classes, total body mass increased in winter, which was mainly due to an increase in fat mass. Adult storks were 5% heavier than immature storks. The sexes differed in body mass, with males weighing significantly more than females by 11%. Mean LBM (lean body mass) was 8.5% higher in adults than in immatures, and was 11.5% higher in males compared with females. Between their first and second summers, immatures accumulated a lean body mass to finally reach the same values as adults, indicating a phase of muscle development. The mean fat mass of the storks did not differ between age classes or between sexes. Based on physiological parameters, this study shows that settled White Storks are able to cope with mild winter periods when they are artificially provided with food. In a view to preserve favourable habitats for this species, it is therefore necessary to decide on a plan of action for breeding areas.     

Requirements for carotenoids in fish diets

Carotenoids are among the most widespread and important pigment classes in living organisms, and in marine animals, astaxanthin is the most commonly occurring red carotenoid. Carotenoids are vitamin A precursors and are fundamental in photosynthesis and light protection in plants. Increasing attention has been drawn to a possible light protection, cancer prevention and immune enhancement by carotenoids in mammals. Reported functions in fish range from a general enhancement of performance to specific functions in reproduction and metabolism. In this paper, we show that astaxanthin is essential for growth and survival of fish and crawfish, and discuss this fact in relation to the use of purified and semipurified diets in nutritional studies. The similarity in action of astaxanthin and canthaxanthin compared to α-tocopherol (vitamin E) and retinol (vitamin A) suggests that these two carotenoids should be listed among the fat soluble vitamins.     

A new method for sexing Oriental White Storks

ABSTRACT.   For species where males and females are monomorphic, or nearly so, determining the sex of individual birds generally requires either capturing birds or collecting samples, such as feathers, for DNA analysis. We developed a new method, involving the use of photographs, to determine the sex of endangered Oriental White Storks ( Ciconia boyciana ). Using photographs, we analyzed the lateral features of the heads of 25 captive storks of known sex (12 males and 13 females) and found differences between males and females in the distance from the bill tip to the nape and the distance from the bill tip to the commissural point. These differences were used to generate a discriminant function that was then tested on 22 captive storks at Hyogo Homeland Park (Toyooka, Japan), and we correctly determined the sex of 18 individuals (82%). In addition, the sex of two wild storks was correctly assigned. Our results suggest that good-quality photographs can be useful for determining the sex of both captive and wild Oriental White Storks and, further, that similar methods may prove useful for determining sex in other species of birds.     

A factorial approach for a sugarcane juice-based low cost culture medium: increasing the astaxanthin production by the red yeast Phaffia rhodozyma

A sugarcane juice-based low cost culture medium was previously explored to produce the carotenoid pigment astaxanthin in liquid culture by the red yeast Phaffia rhodozyma (1300?μg astaxanthin/g of dry yeast and 6500?μg/l whole culture medium). Two peculiar limitations in Phaffia are growth temperature (<26?°C) and lack of sugar osmotolerance. Two advantages are the wide biochemical ability for the assimilation and metabolization of disaccharides and the prompt utilization of simple nitrogen sources. For instance, the sucrolytic/ureolytic enzymatic activities deserves exploration. In order to improve the culture medium composition and the conditions of fermentation for highly oxygenated carotenoids (e.g., astaxanthin) a study was carried out with a factorial design in two steps. As a first step, the production of astaxanthin was studied as a function of the nutrient concentration levels and their interactions. The production increase (μg/l) obtained was 23.0% but at the expense of 16.0% pigment content decrease (μg/g). In the second step, the variables pH and agitation level (OTR, oxygen transfer rate) were optimized and then, both goals were attained: the increase of pigment content (418?μg astaxanthin/g of yeast) as well as the absolute pigment production enhancement (1987?μg/l).     

Effects of dietary xanthophylls/astaxanthin ratios on the growth and skin pigmentation of large yellow croaker Larimichthys crocea (Richardson, 1846)

An 8‐week feeding trial was conducted to evaluate the effects of dietary xanthophylls/ astaxanthin ratio on the growth and skin color of large yellow croaker, Larimichthys crocea. Five pigment‐supplemented diets were formulated to contain 75/0, 50/25, 37.5/37.5, 25/50 and 0/75 mg kg^?1 of xanthophylls/astaxanthin. The xanthophylls contain 89.31% lutein and 6.12% zeaxanthin. A diet without pigment supplementation was used as the control. The large yellow croaker juveniles (13.80 ± 0.03 g) were randomly distributed in 18 sea cages (1.0 × 1.0 × 1.5 m) at a density of 45 fish per cage. Water temperature ranged from 21 to 31°C during the feeding trial. To obtain results, the survival rate, specific growth rate, feed conversion ratio, skin redness, skin yellowness, skin lightness, skin carotenoid content and skin melanin content were measured. The results showed that the survival rate, specific growth rate and feed conversion ratio were not significantly affected by dietary treatments (P > 0.05). The ventral skin lightness was also not affected by dietary treatments (P > 0.05); however, the dorsal skin lightness of fish fed with the control diet was significantly lower than those fed with pigment‐supplemented diets (P < 0.05). The lowest values of yellowness and carotenoid content both in the ventral skin and dorsal skin were found in the control group. Yellowness and carotenoid content increased with an increasing proportion of dietary xanthophylls in both the ventral and dorsal skin. Higher redness values were found in the compound pigment groups, either in the dorsal skin or ventral skin. Fish fed with the control diet showed a higher melanin content in the dorsal skin than those fed with pigment‐supplemented diets, although differences were not significant (P > 0.05). Lightness and yellowness were linearly related to skin carotenoid content. Meanwhile, skin yellowness and carotenoid content were linearly related to the proportion of xanthophylls in dietary pigments.     

Microbial Community Structure, Pigment Composition, and Nitrogen Source of Red Snow in Antarctica

“Red snow” refers to red-colored snow, caused by bloom of cold-adapted phototrophs, so-called snow algae. The red snow found in Langhovde, Antarctica, was investigated from several viewpoints. Various sizes of rounded red cells were observed in the red snow samples under microscopy. Pigment analysis demonstrated accumulation of astaxanthin in the red snow. Community structure of microorganisms was analyzed by culture-independent methods. In the analyses of small subunit rRNA genes, several species of green algae, fungus, and various phylotypes of bacteria were detected. The detected bacteria were closely related to psychrophilic or psychrotolerant heterotrophic strains, or sequences detected from low-temperature environments. As predominant lineage of bacteria, members of the genus Hymenobacter were consistently detected from samples obtained in two different years. Nitrogen isotopic compositions analysis indicated that the red snow was significantly ¹⁵N-enriched. Based on an estimation of trophic level, it was suggested that primary nitrogen sources of the red snow were supplied from fecal pellet of seabirds including a marine top predator of Antarctica.     

ASTAXANTHIN ACCUMULATION IN HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE) AS AN ACTIVE PHOTOPROTECTIVE PROCESS UNDER HIGH IRRADIANCE1

Green cells of Haematococcus pluvialis Flotow accumulate the ketocarotenoid astaxanthin under stress conditions, such as high irradiance, nutrient deficiency, high salinity, and high temperature. Though some photoprotective mechanisms have been suggested, the function of astaxanthin in red cysts is still questioned. We studied the role of astaxanthin in photoprotection by inducing its formation in logarithmically growing cultures by high irradiance, thus avoiding unrelated processes that can occur in H. pluvialis when carotenogenesis is induced by other stresses. On exposure to high irradiance, the green Haematococcus culture turned red as lipid globules loaded with astaxanthin esters were formed and concentrated at the periphery of the cell. During this phase of induction, the photosynthesis rates remained high, but the amount of the D1 protein of PSII was significantly reduced. The decline in D1 protein content stopped after 1 day; the level then increased, returning to normal after 5 days. The response of the D1 protein was indicative of a transitional phase in the acclimation of Haematococcus to high light. The formation and deposition of astaxanthin seemed to prevent further reduction in D1 protein level, thus enabling the cell to maintain PSII function and structural integrity. This result seems to be a clear indication of the light screening by astaxanthin, which absorbs light in the blue region, thus protecting the photosynthetic apparatus. When the cells recovered from the high light stress, the astaxanthin globules concentrated around the nucleus, indicating that the pigment also serves as a physicochemical barrier, protecting the replicating DNA from oxidation as the cells divide.     

ACCUMULATION OF ASTAXANTHIN IN HAEMATOCOCCUS LACUSTRIS (CHLOROPHYTA)1

In N-limited continuous chemostat cultures of the green alga Haematococcus lacustris (Gir.) Rostaf. (UTEX 16), the steady-state astaxanthin content of the cells was determined by the specific growth rate of the cultures. The highest, pigment content was obtained at the lowest dilution rate. The specific rate of astaxanthin accumulation was, however, a function of the photon flux density measured at the illuminated culture surface. In nongrowing Haematococcus cultures, the specific rate of astaxanthin accumulation was determined by the growth rate of the culture during growth phase. The highest possible cellular astaxanthin content of all cultures was comparable and independent of the culture parameters.     

Intraperitoneal and dietary administration of astaxanthin in rainbow trout (Oncorhynchus mykiss)--plasma uptake and tissue distribution of geometrical E/Z isomers

Astaxanthin enters circulation in salmonid fishes upon intraperitoneal injection (IP) of small doses. Blood uptake and tissue distribution of geometrical E/Z astaxanthin isomers were determined in tissues and plasma of duplicated groups of rainbow trout (Oncorhynchus mykiss, initial weight 550 g) some of which were administered high doses of astaxanthin by IP in a trial lasting for 8 weeks. Doses of 10 (IP10), 50 (IP50) or 100 mg (IP100) astaxanthin (Lucantin Pink, BASF, Germany), respectively, dispersed in phosphate buffered saline were tested in comparison with diets containing 10 (Control) or 60 (Fed 60) mg astaxanthin kg(-1). Astaxanthin concentrations in all examined tissues and plasma were significantly higher in IP50 and IP100 than in controls and Fed 60 (p<0.05). In IP50, 11 mg astaxanthin kg(-1) muscle was detected after 4 weeks, compared to 4 mg kg(-1) in rainbow trout fed 60 mg kg(-1). Concentrations up to 80 and 100 mg astaxanthin kg(-1) were detected in liver and kidney after IP, respectively, whereas fish only fed astaxanthin contained about 2 mg astaxanthin kg(-1). No increase in muscle astaxanthin concentration was found between 4 and 8 weeks in fish given IP, and the muscle astaxanthin concentration in IP50 and IP100 were similar. Muscle concentration and injected dose were curvilinearly correlated and the proportion of ingested dose retained by the muscle was negatively correlated with the amount of injected astaxanthin. Plasma and muscle concentrations of astaxanthin were highly correlated (p<0.0001). Astaxanthin Z-isomers accumulated selectively in the various tissues after IP, whereas all-E-astaxanthin was preferably absorbed into plasma when administered via the diet. There was a selective uptake of all-E-astaxanthin in the muscle of all fish. Mortality was not affected by treatment, but a dose-dependent reduction in SGR was evident after IP. In conclusion, a more rapid and higher uptake of astaxanthin in plasma, muscle, kidney and liver of rainbow trout takes place after IP compared to when astaxanthin is fed via the diet.     

小龙虾肠道细菌群落多样性及产蛋白酶细菌的筛选

【背景】养殖动物对饲料的消化利用往往与肠道菌群密切相关。【目的】揭示小龙虾肠道细菌群落组成,并从小龙虾肠道筛选产蛋白酶细菌。【方法】在Illumina MiSeq PE300平台对细菌16S rRNA基因V3-V4区进行高通量测序,分析小龙虾肠道细菌群落多样性;通过酪蛋白平板法筛选产蛋白酶细菌并进行分子生物学鉴定。【结果】小龙虾肠道细菌优势门包括变形菌门、软壁菌门、厚壁菌门、拟杆菌门等4个门,累计占比为98.53%;优势属包括柠檬酸杆菌属、支原体科Candidatus_Bacilloplasma暂定属、哈夫尼菌属、肠球菌属、拟杆菌属、梭菌科未分类属、希瓦氏菌属等7个属,累计占比为91.67%。通过酪蛋白平板法筛选的产蛋白酶细菌来自哈夫尼菌属、柠檬酸杆菌属、克雷伯氏菌属和芽孢杆菌属。【结论】小龙虾肠道细菌核心类群在蛋白质消化利用中发挥了重要作用。     

Hematology and blood chemistry of chicks of white and black storks (Ciconia ciconia and Ciconia nigra)

1. The hematology and blood chemistry of 15-68-day-old chicks have been studied. 2. Red cell numbers (1,900,000/mm3), hemoglobin content (11.5 g/dl) and hematocrit (37.5) were similar in both species of storks. 3. Total numbers of leucocytes (63,000/mm3) were similar in both species, whilst differential leucocyte counts were not. 4. Higher levels of plasma protein, GOT and cholesterol in HDL were found in white storks when compared with black storks but not differences were detected in plasma levels of triglycerides, total cholesterol, urea, uric acid, GPT and alkaline phosphatase.     

Esterified astaxanthin levels in lobster epithelia correlate with shell colour intensity: potential role in crustacean shell colour formation

Carotenoids, particularly astaxanthin, are the primary pigment in crustacean shell colour. Sub-adults of the western rock lobster, Panulirus cygnus, moult from a deep red colour (termed the red phase) to a much paler colour (the white phase) at sexual maturation. We observe a 2.4-fold difference in the amount of total carotenoid present in the shell extracts of reds compared to whites, as might be expected. However, analysis of the underlying epithelium shows that there is no correlation with shell colour and the amount of free (unesterified) astaxanthin-the level of free astaxanthin in reds and whites is not significantly different. Instead, we observe a correlated two-fold difference in the amount of esterified astaxanthin present in the epithelium of red versus white individuals. These data suggest a role for esterified astaxanthin in regulating shell colour formation and suggest that esterification may promote secretion and eventual incorporation of unesterified astaxanthin into the exoskeleton.     

枫香秋季变色期叶色变化及其生理基础

为探究秋季枫叶呈色的关键生理因素,该文以转色期叶色为绿色、黄色和红色的枫香单株为试材,研究了L^*、a^*、b^*值变化与叶片色素、可溶性糖及可溶性蛋白质含量变化的相关性。结果表明:(1)在变色期,3种色彩枫香叶片叶绿素a、叶绿素b、总叶绿素和类胡萝卜素均大量降解,花色素苷不同程度积累。(2)绿色叶单株叶绿素和类胡萝卜素始终保持较高含量,花色素苷含量上升4.2倍,叶片内色素含量比值始终保持稳定; 黄色叶单株叶绿素和类胡萝卜素含量最低,花色素苷含量上升4.4倍,b^*值与叶绿素含量极显著负相关,与类胡萝卜素含量显著负相关,与花色素苷/类胡萝卜素含量比值极显著正相关; 红色叶单株叶绿素和类胡萝卜素含量略高于黄色叶单株,花色素苷含量上升27.2倍,a^*值与叶绿素含量、类胡萝卜素含量极显著负相关,与花色素苷含量显著正相关,与色素含量比值无显著相关性。(3)红色叶单株具有较高的可溶性糖含量和可溶性蛋白质含量。因此,在枫香叶片变色期,保持较高的叶绿素和类胡萝卜素含量,维持色素含量比值稳定使叶片呈现绿色; 叶绿素和类胡萝卜素的大量降解,以及花色素苷/类胡萝卜素含量比值的升高使叶片呈现黄色; 叶绿素的降解和花色素苷的大量合成使叶片呈现红色。     

An efficient method for the extraction of astaxanthin from the red yeast Xanthophyllomyces dendrorhous

This study investigated an efficient method for the extraction of astaxanthin from the red yeast Xanthophyllomyces dendrorhous. The extraction process comprised three steps: (1) cultivating the yeast; (2) treating the yeast culture suspension with microwaves to destroy the cell walls and microbodies; and (3) drying the yeast and extracting the astaxanthin pigment using ethanol, methanol, acetone, or a mixture of the three as the extraction solvent. Ultimately, various treatment tests were performed to determine the conditions for optimal pigment extraction, and the total carotenoid and astaxanthin contents were quantified. A frequency of 2,450 MHz, an output of 500 watts, and irradiation time of 60 s were the most optimum conditions for yeast cell wall destruction. Furthermore, optimal pigment extraction occurred when using a cell density of 10 g/l at 30 C over 24 h, with a 10% volume of ethanol.     

Apparent digestibility coefficients and accumulation of astaxanthin E/Z isomers in Atlantic salmon (Salmo salar L.) and Atlantic halibut (Hippoglossus hippoglossus L.)

Apparent astaxanthin (3,3'-dihydroxy-beta,beta-carotene-4,4'-dione) digestibility coefficients (ADC) and carotenoid compositions of the muscle, liver, whole kidney and plasma were compared in Atlantic salmon (Salmo salar) and Atlantic halibut (Hippoglossus hippoglossus) fed a diet supplemented with 66 mg astaxanthin kg(-1) dry matter for 112 days. The astaxanthin source consisted of 75% all-E-, 3% 9Z- and 22% 13Z-astaxanthin, of (3R,3'R)-, (3R,3'S; meso)-, and (3S,3'S)-astaxanthin in a 1:2:1 ratio. The ADC of astaxanthin was significantly higher in Atlantic halibut than in Atlantic salmon after 56 and 112 days of feeding (P < 0.05). The ADC of all-E-astaxanthin was significantly higher than ADC of 9Z-astaxanthin (P < 0.05). Considerably more carotenoids were present in all plasma and tissue samples of salmon than in halibut. Retention of astaxanthin in salmon muscle was 3.9% in salmon and 0 in halibut. All-E-astaxanthin accumulated selectively in the muscle of salmon, and in plasma of salmon and halibut compared with diet. 13Z-astaxanthin accumulated selectively in liver and whole kidney of salmon and halibut, when compared with plasma. A reductive pathway for astaxanthin metabolism in halibut similar to that of salmon was shown by the presence of 3',4'-cis and trans glycolic isomers of idoxanthin (3,3',4'-trihydroxy-beta,beta-carotene-4'-one) in plasma, liver and whole kidney. In conclusion, the higher ADC of astaxanthin in halibut than Atlantic salmon may be explained by lower feed intake in halibut, and the lower retention of astaxanthin by a higher capacity to transform astaxanthin metabolically.     

UV-B辐射对雨生红球藻光合特性和虾青素含量的影响及其响应

实验研究了不同强度的UV-B(280-320 nm)辐射对雨生红球藻(Haematococcus pluvialis)的光合活性、生物量、色素含量、活性氧(ROS)含量和抗氧化酶活性等的影响, 以探讨利用UV-B辐射诱导虾青素生物合成增强的可能性。结果发现, 经UV-B辐射处理后,雨生红球藻的光合活性降低、生物量增长被抑制。UV-B辐射对叶绿素影响不大, 但会改变细胞的类胡萝卜素和虾青素含量:0.1和0.3 W/m2强度的UV-B辐射使细胞中的这两种色素含量升高, 0.5 W/m2组的色素含量短暂升高后恢复到对照水平。中低强度的UV-B可以促进雨生红球藻单细胞虾青素含量的增加, 但由于其对细胞生长的抑制作用, 并不能使虾青素大量积累。随辐射时间延长, 细胞内ROS含量未明显增加,但抗氧化酶(过氧化氢酶和超氧化物歧化酶)活性下降, 雨生红球藻可能主要依靠虾青素来淬灭ROS。以上结果表明, UV-B辐射对雨生红球藻的主要生理生化过程有抑制作用, UV-B辐射既可以诱导虾青素的合成又会消耗一部分虾青素, 对虾青素含量的影响与其强度有关, 而利用虾青素来清除细胞内的ROS可能是雨生红球藻抵御这种不利环境条件的最重要的途径。       

The Spectral Sensitivity of Crayfish and Lobster Vision

(1) The spectral sensitivity function for the compound eye of the crayfish has been determined by recording the retinal action potentials elicited by monochromatic stimuli. Its peak lies at approximately 570 mµ. (2) Similar measurements made on lobster eyes yield functions with maxima in the region of 520 to 525 mµ, which agree well with the absorption spectrum of lobster rhodopsin if minor allowances are made for distortion by known screening pigments. (3) The crayfish sensitivity function, since it is unaffected by selective monochromatic light adaptation, must be determined by a single photosensitive pigment. The absorption maximum of this pigment may be inferred with reasonable accuracy from the sensitivity data. (4) The visual pigment of the crayfish thus has its maximum absorption displaced by 50 to 60 mµ towards the red end of the spectrum from that of the lobster and other marine crustacea. This shift parallels that found in both rod and cone pigments between fresh water and marine vertebrates. In the crayfish, however, an altered protein is responsible for the shift and not a new carotenoid chromophore as in the vertebrates. (5) The existence of this situation in a new group of animals (with photoreceptors which have been evolved independently from those of vertebrates) strengthens the view that there may be strong selection for long wavelength visual sensitivity in fresh water.