1. Nitrogen in Animals: Applications

2. Overview How are δ 15 N affected by the following processes: 1)Pregnancy 2)Nutritional Stress 3)Different diets & diet quality 4)Nursing 5)Fasting

3. Studies investigate : δ 15 N & δ 13 C values in body of pregnant women with and without nutritional stress (Fuller et al. 2004, 2005) Methods Examine δ 15 N & δ 13 C isotopes in the body from pre-conception to delivery by: 1)Conducting dietary surveys 2) Collect hair samples of mother post-delivery - Cut hair into 1-1.5 cm sections of hair to analyze isotope composition for each 4-6 week period Pregnancy and Nutritional Stress

4. Results – w/o nutritional stress Diet surveys: No real change in diet- some increased animal protein intake, others decreased, & others remained constant Hair analysis: 1) No difference in δ 13 C b/w conception and birth 2) Decrease in δ 15 N values in pregnant women during gestation (-0.3 to -1.1%) 3) Increase in maternal weight, drop in δ 15 N values

5. d13C d15N

7. Why? Well…not completely understood DECREASES IN δ 15 N due to maternal N conservation: Assimilation of greater percentage of dietary N with lower δ 15 N values results in a reduction of steady state diet to body trophic fractionation (0.5-1‰) Decreased urea synthesis and increased urea salvage in colon (peeing out heavy nitrogen) Mother decreases in N and fetus increases = fetus feeding off mom would result in an increase in trophic level, causing equilibrium b/w mom and offspring

8. Results – w/ nutritional stress Diet surveys: Reduced dietary intake and weight loss during episodes of morning sickness Hair analysis: 1) No uniform change in δ 13 C b/w conception & birth 2) Increase in δ 15 N values in pregnant women during episodes of nutritional stress / weight loss 3) Decrease in δ 15 N values as gestation progressed

9. Sick b/w 10-16 weeks Sick throughout 1 st trimester Negative nitrogen balance = increased fractionation

10. Reasoning for INCREASE in δ 15 N: As nutritional stress & weight loss continue, increase of muscle protein breakdown, lighter N is not replaced by dietary protein, & remaining tissues are enriched in heavier N (increased fractionation)

11. Diet quality 2 Studies: Oreochromis niloticus – “Nile tilapia” (Gaye-Siessegger 2004) Paradosa lugubris – “Wolf spider” (Oelbermann and Scheu 2001)

12. Nile tilapia Study investigates: Influence of different diets on δ 15 N & δ 13 C in fish bodies Methods 32 fish reared on same diet for 11 weeks to equilibrate on diet, then 7 were killed to estimate initial isotopic composition of body Provide fish with 3 different protein diets in controlled lab for 8 weeks Analyze all fish at end of experiment after 48 hour fast

13. Results No significant decrease in δ 15 N & δ 13 C, body mass, b/w averages of feeding groups Declining trophic shift for individuals in δ 15 N (6.5‰- 4‰) & δ 13 C (4‰-2.5‰) w/ increasing protein retention in individual fish Experiment demonstrates high influence of individual protein balance of δ15N & δ13C in animals – dietary protein was conserved differently in tissue of individual fish = less fraction.

14. Wolf spider Study investigates: Influence of diet quality on δ 15 N & δ 13 C in spiders on different quality diets Methods: Changes in δ 15 N & δ 13 C w/ time: - Adult females, hatchlings, spiderlings fed intermediate quality food for 3, 6, & 11 weeks Changes in δ 15 N & δ 13 C w/ trophic level and starvation: - Adult and juvenile spiders were fed single species diets (low, med, high quality), mixed (high & low), and starved

15. Results Changes in δ 15 N & δ 13 C w/ time: - Since 14N is preferentially excreted during metabolism, the hatchling N would be isotopically lighter. - As individuals become larger, more isotopically like food item, then become enriched over food item by ≈3‰

16. Changes in δ15N & δ13C w/ trophic level: 5.2‰ 2.5‰ 3.3‰ 2.2‰ Stepwise trophic level enrichment

17. V. High quality Spiders on high-quality diet were bigger and increased in size faster, & had higher δ15N & δ13C values High quality Intermediate qualityV. Low quality Low quality

18. Changes in δ 15 N & δ 13 C when starved: Starvation leads to enrichment of δ 15 N due to recycling body nitrogen (i.e. “eating yourself”)

19. Polar bears – Ursus maritimus Facts: Feed primarily on ringed seals (Phoca hispida) Peak feeding March-June -> when seal pups are weaned Adult males females w/ cubs, and subadults fast for 4 months (July-Nov.) while Hudson Bay freezes Pregnant females stay on land until spring and fast for 8+ months (gestation & 3 months lactation) Bears are not nutritionally stressed when they fast - they accumulate fat stores to sustain long fasts Cubs emerge from dens in spring, they are dependent on milk from mom Cubs stay with mom for 1-2 years

20. Study investigates (Polischuk et al. 2001) : 1)Whether cubs are at higher trophic level than mothers as a result of nursing 2) Impact of seasonal fasting on δ 15 N & δ 13 C values Methods: Examine δ 15 N & δ 13 C isotopes collected from plasma (≈ 4 days) and milk: 1)Females with cubs of the year (COY) 2)Females with yearlings 3) Females with 2 year old cubs

21. Cubs plasma enriched in δ 15 N and depleted in δ 13 C when compared to mom – reliance on mom’s milk (i.e. eating mom) δ15N δ13C

22. Increase in δ 15 N after birth during early lactation Female is 5 months into an 8 month fast and is catabolizing her own tissue for energy

23. - COY begin to feed on seals in spring and summer- by fall plasma was depleted in N, relative to mom - Summer and fall, no difference in mom and cub, indicating similar feeding habits or metabolism

24. Applications May lead to a method for investigating pregnancy and fertility patterns in populations of animals (past & present) Pregnancy, nutritional stress, diet quality findings need to be considered in diet studies (ca not always assume ≈3‰ fractionation factor for δ 15 N) Non-invasive monitoring of nitrogen balance in women during pregnancy – examine overall health Applied to medical studies of protein stress & N balance: anorexia, bulimia, exercise, disease, weight loss, & burns Do other tissues detect pregnancy, nutritional stress, changes in diet quality? More research to discover the biochemical reasons for δ 15 N increase and decrease in animals