Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood ...

University of Glasgow

Egg quality is a phenotype of, and can profoundly influence fitness in, both mother and offspring. However, the physiological mechanisms that underlie this maternal effect are poorly understood. Carotenoids are hypothesized to enhance antioxidant activity and immune function, and are responsible for the pigmentation of egg yolk. The proximate basis and consequences of this maternal investment, however, have not previously been studied in wild birds. In this supplemental feeding study of lesser black-backed gulls, Larus fuscus, carotenoid-fed females are shown to have increased integument pigmentation, higher plasma concentrations of carotenoids and antioxidant activity, and lower plasma concentrations of immunoglobulins (Igs) in comparison with controls. In turn, carotenoid-fed females produced eggs containing high carotenoid but low Ig concentrations (i.e. passive immunity), whereas control females produced eggs containing low carotenoid but high Ig concentrations. Within-clutch patterns of these resources varied over the laying sequence in a similar manner in both carotenoid-fed and control nests. Our results suggest that carotenoids could be one resource responsible for egg quality maternal effects in birds. We discuss the possible implications of carotenoid-mediated effects on phenotype for fitness in mothers and their offspring.

Carotenoids and egg quality in the lesser blackbacked gull Larus fuscus: a supplemental feeding study of maternal effects.

It has been proposed that the maternal androgens in avian egg yolk enhance offspring fitness by accelerating growth and improving competitive ability. Because egg quality is strongly influenced by maternal condition, we predicted that females in good condition would produce high-quality eggs with relatively high androgen content. We experimentally enhanced maternal condition by supplementary feeding lesser black-backed gulls (Larus fuscus) during egg formation and compared the concentrations of androstenedione (A4), 5alpha-dihydrotestosterone (DHT) and testosterone (T) in their eggs with those in eggs laid by control females. We also measured circulating levels of T in females immediately after laying. Egg androgens could affect offspring performance directly through chick development and/or indirectly through changes in the competitive ability of a chick relative to its siblings. To avoid confounding these two routes, and to separate effects operating through the egg itself with those operating through experimental changes in parental chick rearing capacity, we fostered eggs from both maternal treatment groups singly into the nests of unmanipulated parents. Contrary to expectation, mothers with experimentally enhanced body condition laid eggs with lower levels of androgens, while exhibiting higher circulating T concentrations post-laying. Despite these lower levels of egg androgen, offspring hatched from eggs laid by mothers in good condition did not show reduced growth or survival when reared in the absence of sibling competition. Our results demonstrate that yolk androgen concentrations vary with the body condition of the female at the time of egg formation and that females in good condition reduced the yolk androgen content of their eggs without altering offspring performance.

Maternal condition, yolk androgens and offspring performance: a supplemental feeding experiment in the lesser black-backed gull (Larus fuscus).

Maternal effects, in which differences in parental state cause differences in offspring fitness, are important in trade-offs influencing an individual's optimal reproductive strategy. In zebra finches (Taeniopygia guttata) we manipulated the nutritional state for four weeks before the start of breeding through protein supplementation. Zebra finches were kept on identical diets during the rest of the experiment. We then tested the effects of maternal state on offspring size, survival and fecundity. In order to separate the effects of maternal state occurring through egg production, incubation and chick-rearing, we used a cross-fostering experiment. We show that a protein-rich diet prior to laying improved maternal body weight prior to breeding compared with birds on a protein-poor diet. Poorer maternal state prior to breeding gave rise to offspring with lower fecundity than offspring from birds in a better nutritional state. Maternal state is thought to affect the conditions developing offspring experience through the bird's ability to produce and incubate eggs. Male and female embryos differed in their responses to conditions at different developmental stages. This shows that embryonic developmental conditions and sex differences in vulnerability to these conditions need to be incorporated into future models of selection, life-history evolution and sex-ratio theory.

Prenatal developmental conditions have long-term effects on offspring fecundity.

Several studies have demonstrated that poor early nutrition, followed by growth compensation, can have negative consequences later in life. However, it remains unclear whether this is attributable to the nutritional deficit itself or a cost of compensatory growth. This distinction is important to our understanding both of the proximate and ultimate factors that shape growth trajectories and of how best to manage growth in our own and other species following low birth weight. We reared sibling pairs of zebra finches on different quality nutrition for the first 20 d of life only and examined their learning performance in adulthood. Final body size was not affected. However, the speed of learning a simple task in adulthood, which involved associating a screen colour with the presence of a food reward, was negatively related to the amount of growth compensation that had occurred. Learning speed was not related to the early diet itself or the amount of early growth depression. These results show that the level of compensatory growth that occurs following a period of poor nutrition is associated with long-term negative consequences for cognitive function and suggest that a growth-performance trade-off may determine optimal growth trajectories.

Compensatory growth impairs adult cognitive performance.

Several techniques are available for quantifying the vertebrate immune response, information that is particularly useful for understanding the contribution of immunity to the evolution of life-history strategies. The most widely used is the phytohaemagglutinin (PHA) skin-swelling technique, which is usually regarded as an index of acquired immunity. However, our understanding of the effects of PHA in skin is poor, despite the fact that it has implications for what the test can tell us about immune activity. As we discuss here, a recent study by Martin and colleagues on the response to PHA at the cellular level in wild birds has highlighted the relative extent to which PHA-induced swelling, as most commonly applied, measures innate immunity versus acquired immunity.

The perils and prospects of using phytohaemagglutinin in evolutionary ecology.

Many populations have recovered from severe bottlenecks either naturally or through intensive conservation management. In the past, however, few conservation programs have monitored the genetic health of recovering populations. We conducted a conservation genetic assessment of a small, reintroduced population of Mauritius Kestrel (Falco punctatus) to determine whether genetic deterioration has occurred since its reintroduction. We used pedigree analysis that partially accounted for individuals of unknown origin to document that (1) inbreeding occurred frequently (2.6% increase per generation; N(eI)= 18.9), (2) 25% of breeding pairs were composed of either closely or moderately related individuals, (3) genetic diversity has been lost from the population (1.6% loss per generation; N(eV)= 32.1) less rapidly than the corresponding increase in inbreeding, and (4) ignoring the contribution of unknown individuals to a pedigree will bias the metrics derived from that pedigree, ultimately obscuring the prevailing genetic dynamics. The rates of inbreeding and loss of genetic variation in the subpopulation of Mauritius Kestrel we examined were extreme and among the highest yet documented in a wild vertebrate population. Thus, genetic deterioration may affect this population's long-term viability. Remedial conservation strategies are needed to reduce the impact of inbreeding and loss of genetic variation in this species. We suggest that schemes to monitor genetic variation after reintroduction should be an integral component of endangered species recovery programs.

Inbreeding and loss of genetic variation in a reintroduced population of Mauritius Kestrel.

It has been proposed that female birds can influence the phenotype of their offspring by provisioning eggs with variable amounts of nutrients and maternal hormones. Egg quality is strongly influenced by maternal body reserves and the amount of food available at the time of egg formation. This study investigated the effects of maternal state and food availability on the capacity of female lesser black-backed gulls Larus fuscus to provision their eggs with macronutrients and steroid hormones. Maternal state was reduced by increasing egg-production effort, whereas extra food was provided to reverse this effect. Compared with eggs of first clutches, eggs of experimentally induced replacement clutches exhibited a lower yolk/albumen ratio and contained more yolk testosterone. During one of the three years in which the study was performed, replacement eggs also contained more 17β-estradiol. Food provisioning during the relaying interval did not affect changes in yolk/albumen ratio or steroid concentrations, but fed females produced bigger eggs in their replacement clutch. This study demonstrates significant within-female consistency in egg size, macronutrient content, and yolk steroid concentration, and it shows that these egg characteristics are influenced by maternal state, food availability, and the timing of breeding.

The effect of maternal state on the steroid and macronutrient content of lesser black-backed gull eggs.

In many incubating birds, heat transfer from parent to egg is facilitated by the brood patch, an area of ventral abdominal skin that becomes highly vascularised, swells and loses its down feathers around the time of laying. Only the female develops a brood patch in most passerine species, but males of some species can incubate and maintain the eggs at similar temperatures to females even without a brood patch. Here we used a novel application of infrared thermography to examine sex differences in parental care from a physiological perspective. Using incubating male and female zebra finches (Taeniopygia guttata), a species in which the male lacks a brood patch, we measured the surface temperature of the ventral plumage overlying the abdomen and a reference area that does not contact the eggs (thorax) twice per pair. In half of the pairs, clutch size was experimentally enlarged between the two sets of measurements to increase incubation demand. We found that the temperature differential between abdomen and thorax plumage was greater in females than in males, and that abdomen plumage was warmer after clutch enlargement than before in females but not in males. These findings are consistent with morphological sex differences in brood patch development and suggest that male and female zebra finches differ in the way they regulate abdomen versus general body surface temperature in response to variation in incubation demand.

Female but not male zebra finches adjust heat output in response to increased incubation demand.

Nests are structures built to support and protect eggs and/or offspring from predators, parasites, and adverse weather conditions. Nests are mainly constructed prior to egg laying, meaning that parent birds must make decisions about nest site choice and nest building behavior before the start of egg-laying. Parent birds should be selected to choose nest sites and to build optimally sized nests, yet our current understanding of clutch size-nest size relationships is limited to small-scale studies performed over short time periods. Here, we quantified the relationship between clutch size and nest size, using an exhaustive database of 116 slope estimates based on 17,472 nests of 21 species of hole and non-hole-nesting birds. There was a significant, positive relationship between clutch size and the base area of the nest box or the nest, and this relationship did not differ significantly between open nesting and hole-nesting species. The slope of the relationship showed significant intraspecific and interspecific heterogeneity among four species of secondary hole-nesting species, but also among all 116 slope estimates. The estimated relationship between clutch size and nest box base area in study sites with more than a single size of nest box was not significantly different from the relationship using studies with only a single size of nest box. The slope of the relationship between clutch size and nest base area in different species of birds was significantly negatively related to minimum base area, and less so to maximum base area in a given study. These findings are consistent with the hypothesis that bird species have a general reaction norm reflecting the relationship between nest size and clutch size. Further, they suggest that scientists may influence the clutch size decisions of hole-nesting birds through the provisioning of nest boxes of varying sizes.

Variation in clutch size in relation to nest size in birds.

Acute stress triggers peripheral vasoconstriction, causing a rapid, short-term drop in skin temperature in homeotherms. We tested, for the first time, whether this response has the potential to quantify stress, by exhibiting proportionality with stressor intensity. We used established behavioural and hormonal markers: activity level and corticosterone level, to validate a mild and more severe form of an acute restraint stressor in hens (Gallus gallus domesticus). We then used infrared thermography (IRT) to non-invasively collect continuous temperature measurements following exposure to these two intensities of acute handling stress. In the comb and wattle, two skin regions with a known thermoregulatory role, stressor intensity predicted the extent of initial skin cooling, and also the occurrence of a more delayed skin warming, providing two opportunities to quantify stress. With the present, cost-effective availability of IRT technology, this non-invasive and continuous method of stress assessment in unrestrained animals has the potential to become common practice in pure and applied research.

Ruedi Nager

Institute of Biodiversity,
Animal Health and Comparative Medicine,
College of Medical,
Veterinary & Life Sciences,
Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary & Life Sciences

University of Glasgow

Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds