Stable isotope analyses of animal tissues provides information about an animal’s life during tissue growth. Some tissues, such as teeth, hair/fur and nails are
metabolically inactive; that is, once the tissue is formed, its composition is fixed and stable isotope analysis will provide information about the period of time when it was
grown (Voigt et al. 2003). Other tissues, such as blood, muscle, organs, etc., are
metabolically active and are constantly being replaced. The time period represented by stable isotope analysis of metabolically active tissues varies depending on the rate of tissue replacement (turnover) and the species being studied. Among birds, stable carbon isotope turnover rates in various tissues range from several days for blood plasma (Hobson and Clark 1993) to several months for bone collagen (Hobson and Clark 1992).
The timing of tissue growth combines with a host of other important
considerations, such as the logistics of tissue collection, storage and analysis, to make the choice of tissue an important decision. Other researchers have conducted stable isotope analyses on a wide variety of tissues, including whole blood (Mirón et al. 2006), blood plasma (Jenkins et al. 2001), the cellular fraction of blood (Mazerolle and Hobson 2005), muscle (Lohuis et al. 2007), liver (Wolf and Martinez del Rio 2000), wing
membrane (Voigt and Kelm 2006), breath (Voigt et al. 2008), milk (Miller et al. 2011), bone collagen (Cormie et al. 1994), teeth (Metcalfe et al. 2010), feathers (Langin et al. 2007), hair/fur (Cryan et al. 2004), nails (Fraser et al. 2010), stomach contents (Hwang et al. 2007) and fecal matter (Des Marais et al. 1980). Migration studies using stable
isotope techniques are frequently conducted using keratinous tissues, such as feathers, fur or claws (e.g. Clark et al. 2006), although some researchers have used metabolically active tissues (e.g. Marra et al. 1998).
All of the analyses reported in this dissertation were conducted on bat fur samples. I selected fur as the tissue for analysis for four reasons. First, it is consistent
with the work that has already been done in this field. The majority of stable hydrogen isotope studies investigating bird migration analyze feathers, a keratinous tissue to which fur is the best mammalian analogue. All of the current published literature using stable hydrogen isotope analysis to investigate bat migration has used fur (Cryan et al. 2004; Britzke et al. 2009; Fraser et al. 2010), and several other bat researchers are currently using fur for this purpose. A key contribution of my dissertation is to describe natural variation in wild populations of resident bats to provide baseline data for future research, so it is logical to use the most frequently analyzed tissue type. Previous research indicates that temperate bats moult once annually during the summer (Cryan et al. 2004; Quay 1970; Tiunov and Makarikova 2007), and analyses of bat fur should provide information about the bat’s life during the previous summer.
A second reason to use fur for stable isotope analyses is that turnover rates in metabolically active bat tissues are not well understood. Currently, no studies exist examining stable hydrogen isotope turnover in bat tissues. However, several lab studies investigated blood carbon turnover rates in nectarivorous bats and found widely varying results. Mirón et al. (2006) estimated a carbon half life in Glossophaga soricina whole blood of approximately 19 to 44 days and a nitrogen half life of approximately 20 to 34 days; Voigt et al. (2003) reported blood carbon turnover values of 120 to 126 days in both G. soricina and Leptonycteris curasoe; and Voigt and Matt (2004) estimated blood nitrogen turnover values of 274 days in G. soricina and 514 days in L. curasoe. Mirón et al. (2006) cited differences in diet as the main causal factor in these widely varying
estimates. They showed that bats fed a diet with higher nitrogen content had shorter blood carbon turnover times than those fed a diet with lower nitrogen content. Voigt et al. (2003)fed their bats a diet containing low amounts of nitrogen and protein and reported much longer carbon turnover times. A further conclusion by Mirón et al. (2006) was that turnover rates of individual elements within tissues (in this case, carbon and nitrogen) are not necessarily synchronous. Voigt et al. (2003) also suggested torpor use and nutritional stress as potential factors in determining tissue half life.
Although turnover rate estimates for bat tissue hydrogen do not exist, the aforementioned results for carbon and nitrogen suggest that turnover rates in metabolically active bat tissues are plastic and highly related to individual-specific variation in life history. Uncertainty about the timing of tissue turnover poses difficulties to research projects aiming to use stable isotope analysis to learn about migratory origin within a specific timeframe and is an argument for selecting non-metabolically active tissues for analysis. Inert, non-lethal tissues that can be sampled from bats are limited to claws and fur. Bats use their hind claws during roosting and sampling enough of this tissue to conduct stable isotope analyses could have a negative effect on the bat’s roosting abilities. Further, recent evidence suggests that claw keratin is not formed in a linear fashion from the base of the claw to the tip in some mammal species, but rather that the claw tip has both old and new tissue, while the lateral walls may present linear time-series data (Ethier et al. 2010). These results indicate that sampling from the claw tip may in fact present an integrated sample of the bat’s life over an extended period of
time. In contrast, enough fur for several isotope analyses can easily be sampled from a live bat with little potential for adverse effects on the bat’s health.
The third and fourth reasons for selecting fur as the tissue to analyze are logistical, both from the perspectives of sample collection and analysis. In terms of sample acquisition, fur is much easier to collect, store and ship than metabolically active tissues, which must be frozen for storage and dried prior to analysis. Many migration studies employing stable hydrogen isotope analysis take place across a large geographic scale, requiring cooperation and sample collection from a large and varied team of researchers (e.g. Britzke et al. 2009; Cryan et al. 2004). It is sensible to concentrate efforts on a tissue that can be easily and non-lethally sampled, and that can be stored at ambient temperature.
In terms of laboratory analysis, tissue choice for stable hydrogen isotope analysis is further complicated by analytical considerations. In order to account for the presence of exchangeable hydrogen in complex organic tissues (for more detail, see appendix A), it is necessary to use tissue-specific standards to calibrate raw sample results to an internationally accepted standard value (Vienna Standard Mean Ocean Water, VSMOW) (Wassenaar and Hobson 2003). Tissue standards are not widely available and must be experimentally developed on a case by case basis in individual laboratories. The experimental procedure for developing these standards requires some assumptions about the chemical and physical properties of the samples and these assumptions are better understood for some materials (such as keratin) than others (Schimmelmann
1991; Schimmelmann et al. 1999). The use of non-tissue specific standards increases the potential for error in determining final stable hydrogen isotope results (Chesson et al. 2009).