Simple and noninvasive method for assessment of digestive efficiency: Validation of fecal steatocrit in greenfinch coccidiosis model

Abstract Animals’ capability to absorb energy and nutrients from food poses a major internal constraint that affects the amount of resources available for allocation to maintenance, growth, signaling, and reproduction. Intestinal surface is the largest area of contact between immune system and microbial antigens; gut thus appears the main arena where trade‐offs between immune function and other components of fitness arise. Assessment of the integrity of digestive machinery should therefore be of high priority in ecophysiological research. Traditional methods of digestive physiology, however, appear unsuitable for most ecological applications due to lethality or complexity of the procedure. Here, we test the reliability of a simple, cheap, and noninvasive procedure, an acid steatocrit that assesses fat content in feces. It is based on centrifugation of a fecal sample, diluted in acid medium, in hematocrit capillary tube and quantifying the percentage of fat in fecal matter. The method has been previously validated in humans and mice; here, we apply it for the first time in birds. When applied to captive wild‐caught greenfinches, the method showed reasonable internal consistency (r s = 0.71 for steatocrit values, sampled from the same fecal aliquot in duplicate but processed separately). Individual steatocrit values were significantly repeatable in time in different intervals from eight to at least 20 days (r s = 0.32–0.49). The relationship between intestinal health and steatocrit values was tested by experimental manipulations. Medication against coccidiosis (a naturally pervasive intestinal infection) reduced, and experimental infection with heterologous coccidian strains increased steatocrit. Individual changes in steatocrit correlated negatively with changes of two markers of nutritional state—plasma triglyceride levels and body mass. Findings of this study suggest that steatocrit has a wide application potential as a marker of intestinal health in ecophysiological research. In particular, we see the perspective of this method for increasingly popular immunoecological research, conservation medicine, and studies of animal coloration.

Digestion is important to ecologists also for other reasons.
Digestive organs, given their high metabolic activity and cell turnover rates, contribute importantly to individual variation in metabolic rates (Dibner & Richards, 2005;Killpack & Karasov, 2012) which eventually determine organisms' maximum aerobic capacity. Precursors of pigments used for signaling and crypsis are absorbed from food, so that expression of animal coloration can provide information on the integrity of digestive machinery (Hill, 2006;Roulin, 2009). Finally, intestinal surface is the major contact zone between immune system and food-borne and microbial antigens as the vertebrate gut is home to one of the most densely populated microbial populations on earth (Ley, Peterson, & Gordon, 2006). Functioning of the digestive system therefore provides information about the animals' capability to resist infections. To study all these topics, ecologists need feasible proxies for assessment of digestive efficiency in wild animals.
One of such simple, cheap, and noninvasive techniques comprises gravimetric assessment of fat content in feces. The method was initially developed as a quick technique for diagnosing fat malabsorption in 5-day-old infants in India (Phuapradit, Narang, Mendonca, Harris, & Baum, 1981). After implementation by Tran et al. (1994), the method became known as an acid steatocrit. It is based on centrifugation of a fecal sample, diluted in acid medium, in hematocrit capillary tube and quantifying the percentage of fat in fecal matter. Both fresh and frozen samples can be used.
The purpose of this study was to examine the reliability of the steatocrit measure and to apply it for the first time in birds, favorite study objects of immunoecological research (e.g., Hasselquist, 2007). First, we aimed to test whether this is a reliable technique as to enable to detect between-individual differences in fat absorption capacity that are persistent (repeatable) in time. Second, does this technique adequately reflect experimental manipulations expected to change fat absorption? The rationale of our approach is based on an assumption that individuals differ in their ability to digest fat and that this ability is related to various microbial infections that can be manipulated by experimental infection and antimicrobial treatments. This assumption is largely based on previous work on wild-caught captive greenfinches (Carduelis chloris), our model system.
In order to manipulate diverse components of intestinal microbiota, we treated greenfinches by administration of three different antimicrobials and also performed experimental coccidian infection. One of the antimicrobials-toltrazuril-is designed specifically for treatment of coccidiosis and lacks known effects on microbes other than apicomplexans (see Sepp et al., 2012). Metronidazole has wider spectrum of activity against anaerobic bacteria and protozoans and is widely used for medication of trichomonosis (Samuelson, 1999). Sulfadimethoxine is a broad-spectrum sulfonamide drug of antiprotozoal (including anticoccidial) and antibacterial efficacy (Mitrovic & Bauernfeind, 1971).
All the greenfinches in our study population have been naturally infected by isosporan coccidians (Sepp et al., 2012), and occasionally, some birds in the aviary have died with symptoms characteristic to trichomonosis (Männiste & Hõrak, 2014). Both toltrazuril (Sepp et al., 2012) and sulfonamide drug Vetacox (Hõrak et al., 2004) reduce the intensity of coccidian infection; birds treated with sulfonamide have higher plasma triglyceride levels (a marker of nutritional state) and body mass than birds experimentally infected with coccidians (Hõrak et al., 2004). We thus predicted that (i) treatments with toltrazuril and sulfadimethoxine result in decrease in steatocrit values as compared to untreated control birds. We have also shown that infecting greenfinches with novel coccidian strains originating from multiple hosts cause decline in body mass and plasma triglyceride levels (Hõrak, Saks, Karu, & Ots, 2006). Hence, we predicted that (ii) such experimental infection will result in increase in steatocrit as compared to untreated control birds. We also predicted that (iii) if any of the microbes that are sensitive to metronidazole cause fat malabsorption, we will see decline in steatocrit among metronidazole-treated birds. Additionally, we asked whether coccidian infection intensities correlate with steatocrit at individual level and whether individual steatocrit values correlate with body mass and plasma triglycerides. Detection of strong correlations between steatocrit vs intensity of coccidiosis, plasma triglyceride content and body mass would confirm that inhibition of fat absorption by intestinal parasites appears a major pathway affecting nutritional state under current experimental conditions. with their neighbors. The average temperature in the aviary during the experiment was 15.5 ± 1.0° (SD) °C, and average humidity was 57 ± 7 (SD) %. The birds were supplied ad libitum with sunflower seeds and tap water and were exposed to a natural day-length cycle using artificial lighting by luminophore tubes. They were released back to their natural habitat on 3rd (males) and 23rd (females) March 2015. Experimental treatment of females involved medication with Sulfadimethoxine (14 birds) and infection with unfamiliar coccidian strains (14 birds); 13 females served as controls. On the 54th day of the study (9 March), fecal samples were collected for determination of pretreatment steatocrits and on day 56, for determination of coccidian infection intensity. All the birds were blood sampled and weighed in the morning of the day 58. Thereafter, they were divided into three groups on the basis of similar age composition, body mass, and coccidian infection intensity. In the evening of day 59, a dose of 2000 sporulated oocytes, diluted in 1 ml tap water, was administered F I G U R E 1 Male greenfinch at winter-feeding site where intestinal infections likely spread due to contamination of food with feces and saliva. Photograph by Arne Ader, http://www.loodusemees.ee/en/ picture-library F I G U R E 2 Timeline of the experiment with male greenfinches. Day 1 = 15th January 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

| Acid steatocrit
Fecal fat content was estimated on the basis of the acid steatocrit method according to Tran et al. (1994). Bird droppings were diluted    F I G U R E 5 Effects of medication on intensity of coccidiosis (a) and steatocrits in male greenfinches. Means ± SE in (a), medians ± interquartile ranges in (b). Twenty-two birds in control group and 23 birds in both medicated groups in the analysis of infection intensity. Twenty-one birds in control and metronidazole group, 23 birds in toltrazuril group in the analysis of steatocrit. See Table 1 for p-values for coccidiosis and text for steatocrit We tested the hypothesis that causes of individual fat absorption capacity were reliably captured by the steatocrit measure. Our experimental manipulations provided some insight about the causes of individual variation in fat absorption capacity. Male greenfinches that were administered toltrazuril against coccidiosis showed reduced values of steatocrit 7 days after the end of medication. Interestingly, there was no decline in steatocrit immediately after the end of medication period despite the significant reduction of oocyst shedding on day 15 ( Figure 5). This suggests that repair of damage to intestinal surface, exerted by coccidians, takes at least a week in greenfinches.

Individual
In 2-week-old chickens, the turnover time of epithelial cells is approximately 5 days (Fernando & Mccraw, 1973). It may thus appear that epithelial cell regeneration in adult passerines takes longer than in growing chickens. Such explanation would also clarify why we did not detect a decrease of steatocrit in sulfadimethoxine-treated females that were sampled for steatocrit after 6 days of medication. It should be noted, however, that direct inhibition of fat absorption due to rupture of epithelial cells is just one possible pathway how coccidians can cause steatorrhea. Other possible mechanisms include impairment of intraluminal hydrolysis or solubilization of fat, decreased uptake of the products of fat hydrolysis by the epithelial cells, or abnormalities of mucosal cell transport (Sharma & Fernando, 1975). For instance, in turkeys coccidian infections that produce several weight depression and mortality can occur without much damage to intestinal mucosa (Madden & Ruff, 1979;Ruff, Augustine, & Madden, 1981).
Perhaps the most interesting finding of this study was detection of increase in steatocrit among the females infected with heterologous parasite strains. This treatment did not affect infection intensity, body mass, or circulating triglycerides, yet the effect on fat absorption was detected. This finding implies that steatocrit enables to identify much more subtle effects on digestion than measurement of oocyst shedding or circulating fats. It also confirms the previous finding in the same model system that new, heterologous parasite strains appear more virulent than familiar strains (Hõrak et al., 2006).
The finding that higher virulence (assessed on the basis of increased steatocrits) was obtained without eventual increase in infection intensities suggests that some parasite-induced host response rather than the direct damage by parasites per se was responsible for the impaired fat absorption. Similarly, it has been observed in turkeys that decrease in absorption was not always related to the number of parasites in the cells or the extent of damage to mucosa (Ruff et al., 1981 Infected Sulfadimethoxine normal differentiation into absorptive cells (Fernando & Mccraw, 1973). Experimental coccidian infection in chickens can also reduce the intestinal acidity and pancreatic production of digestive enzymes (Major & Ruff, 1978).
Differently from previous studies (Hõrak et al., 2004(Hõrak et al., , 2006 Although the utility of acid steatocrit has been successfully demonstrated in at least two species of mammals (see Introduction), we suggest that this method has particularly broad applicability in avian studies. For instance, unlike most of mammal species, birds possess tetrachromatic vision which enables extensive use of vivid integument coloration for signaling purposes (Hill & Mcgraw, 2006). Functions and mechanisms of such signals may thus largely depend on integrity of digestive machinery, which has so far been assessed only on the basis of distant endpoints such as circulating or deposited pigment levels.
Similarly, it would be useful to test whether fecal fat content in freeranging birds, [in parallel with measurement of fecal parasite load and hormone metabolites (see e.g., Martínez-Padilla, Redpath, Zeineddine, & Mougeot, 2013)] could be used for assessment of population health.
Using steatocrit for quick diagnosis of fat malabsorption could be also of practical use for practitioners and veterinarians, including those working with endangered species in captivity.
To summarize, acid steatocrit is a simple, cheap, and noninvasive method for measuring of fecal fat content that can be used as a proxy for digestive efficiency in birds. In greenfinch coccidiosis, steatocrit appeared more sensitive to experimental manipulation of infection than plasma triglyceride levels, body mass, and in some occasions, infection intensity. This suggests that the method has wide applicability in avian ecophysiological research and calls for further testing of its use in different manipulations of intestinal health.

ACKNOWLEDGMENTS
We thank Elin Sild, Tuul Sepp, Janek Urvik, Helena Pahtma, Liina Ots, and Kristiina Stseglova for the help with bird catching and maintenance and an anonymous reviewer for the constructive comments. The study was financed by the Estonian Science Agency (Grants IUT34-8 and PUT653).