methods of mea sur ing milk composition and yield in small ... · study design and objectives...

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SM A LL SPECI E S A R E ACU T ELY U N DER R EPR E SEN T ED in studies of milk composition and yield (Oftedal, 1984a; Oftedal and Iverson, 1995). This neglect is in part associated with the diffi culties of collecting and

analyzing milk samples of small volume. Standard methods of milk analysis typi-cally require greater volumes than can be collected from an individual of small body size at a single milking; therefore, most published data on milk composition in bats represent pooled samples from several individuals (Kunz et al., 1995; Stern et al., 1997; Korine and Arad, 1999), and often these have been pooled without re-spect to lactation stage (Jenness, 1974; Jenness and Studier, 1976; Kunz et al., 1983; Quicke et al., 1984). Of 20 bat species for which milk composition has been re-ported, stage of lactation is presented for 13 species, but in only 8 of these are data on unpooled samples given (some species are represented by multiple studies; Hood et al., 2001; Hood, 2001; Kwiecinski, unpublished data; Kunz and Hood, 2000, and references therein). Milk yield is even more poorly known: fi ve studies of fi ve bat species, with changes in milk production across lactation reported for only three (Hood 2001; Kunz and Hood, 2000, and references therein). However, we have modifi ed existing methods and developed new methods of analysis that can cope with small milk volumes and thus make it possible to analyze individual milk samples from bats and other small mammals, making it possible for future studies to answer more detailed questions about patterns of lactation in bats.

In this chapter, we describe factors infl uencing milk composition that should be considered when designing a study, methodologies for milk collection and analy-sis, and methods for mea sur ing milk yield. In keeping with the theme of this book, the primary focus is to make recommendations for investigators interested in bat lactation, yet these recommendations also are applicable to other small mammals, including rodents, insectivores, small primates, and small marsupials. We provide

Methods of Mea sur ing Milk Composition and Yield in Small Mammalswendy r. hoodmary beth volturaolav t. oftedal

550-38447_ch04_1P.indd 529550-38447_ch04_1P.indd 529 1/29/09 12:29:11 AM1/29/09 12:29:11 AM

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530 W. R . Hood , M. B . Voltura, and O. T . Oftedal

detailed descriptions of analytical methods that we cur-rently use for determining the proximate composition of small volume milk samples and for mea sur ing milk yield. Less detailed descriptions are given for other methods that have been commonly used. Although this chapter does not address mammary secretion or many details of milk biochemistry, this knowledge can be critical to under-standing and interpreting observed patterns of milk com-position and production. Readers are referred to standard texts on lactation and mammary function, including Jen-ness, 1974; Davies et al., 1983; Larson, 1985; Jensen, 1995; Akers, 2002; and review articles in the Journal of Mammary Gland Biology and Neoplasia.

STUDY DESIGN AND OBJECTIVES

Considerable thought should be given to study design in examining milk composition and yield in bats or any other mammal. Haphazard collection of samples and in-adequate methods of analysis can lead to erroneous re-sults and conclusions. As we discuss below, several intrin-sic and extrinsic variables may contribute to diff erences in the composition of milk collected. Knowledge of these factors and careful consideration of volume limitations of chemical analysis will allow the investigator to minimize unintended eff ects and limitations and thus improve the statistical power to detect signifi cant changes in variables of interest.

The volume and composition of milk that mothers produce varies across lactation in all species that have been examined (Oftedal, 1984a). These changes in nutrient transfer are presumably correlated with the shifting de-mands of growing off spring. The direction and extent of change diff ers among mammals, although some common trends are apparent. The volume of milk produced typi-cally increases to a peak and then declines, at least in ani-mals that are adequately nourished. In mid- to late- lactation, the total dry matter, energy, fat, and protein contents of milk usually rise, whereas the carbohydrate content typi-cally decreases (Oftedal, 1984a). In those bats species that have been examined to date, which include pteropodids, vespertilionids, a phyllostomid, and a molossid (Kunz et al., 1995; Messer and Parry- Jones, 1997; Stern et al., 1997; Korine and Arad, 1999; Hood et al., 2001; Hood, 2001), no changes in the concentration of carbohydrate or protein have been noted. Changes in fat, dry matter, and energy were more variable. Increases in these constituents ap-pear to be common to microbats, and in some cases the change is substantial (Kunz et al., 1995; Hood, 2001). For example, the average dry matter, energy, and fat contents of pooled milk samples from the Brazilian free- tailed bat (Tadarida brasiliensis) increase from parturition to wean-ing by an average of 1.3-, 1.3-, and 1.5- fold, respectively (Kunz et al., 1995; Fig. 25.1). Even greater changes in dry matter, energy, and fat were found in a comparison of the

composition of individually analyzed samples in the big brown bat, Eptesicus fuscus (2.0- fold increase in dry mat-ter, 2.8- fold increase in energy, and a 4.7- fold increase in fat; Fig. 25.2). In the pteropodid megabats, concentration of fat appears to increase during lactation in Pteropus hy-pomelanus, dry matter and energy increase in Pteropus pumi-lus, and all three components appear to increase in Rouset-tus aeygptiaicus (Korine and Arad, 1999; Hood et al., 2001), but no changes in fat, dry matter, or energy were found for Pteropus rodricensus or Pteropus vampyrus. Few studies have analyzed milk samples collected from bats in de pen-dently. When data from in de pen dent samples are evalu-ated relative to day of lactation, considerable variation is apparent (Fig. 25.2; Stern et al., 1997; Hood et al., 2001; Hood 2001, suggesting that individual patterns of nutrient provisioning may diff er considerably from regressions based on several individuals.

Milk composition and yield may vary with individual, litter size, parity, maternal body condition, plane of nutri-tion, water intake, weather conditions, and glandular fi ll. Substantial individual variation in milk composition has been described in two rodents, the rock cavy (Kerodon rup-estris) and the common spiny mouse (Acomys cahirinus), with the fat content of milk varying threefold among Ker-odon mothers and the protein content varying twofold among Acomys mothers (Derrickson et al., 1996). House mouse mothers (Mus musculus) suckling 10 or 18 pups pro-duced over 50% more milk than those suckling four pups, and multiparous mothers with one prior litter produced up to 34% more milk than primiparous mothers (Knight et al., 1986). In the common marmoset (Callithrix jacchus), mothers of high body mass produced milk that was higher in fat than that of low- mass mothers, and mothers suck-ling singletons produced higher- fat milk than mothers with twins (Tardiff et al., 2001). In agriculturally impor-tant species, many studies have shown that maternal con-dition can infl uence milk yield and/or milk composition. For example, mares (Equus caballus) with high body fat tend to produce milk that is lower in protein but higher in fat (Doreau et al., 1992). In Sarda goats (Capra hircus), milk yield is positively correlated with maternal body condi-tion (Vacca et al., 2004). Yet in Norway rats (Rattus norvegi-cus), initial maternal body condition had no eff ect on milk composition or yield for animals fed ad libitum while lac-tating. Milk yield (as estimated from pup growth) was re-duced, but milk composition was unaff ected, when lactat-ing rats were fed 50% less food (Rasmussen and Warman, 1983; Warman and Rasmussen, 1983).

The eff ect of low nutrient intakes on milk production is more pronounced when the diet is not nutritionally bal-anced. In albino rats (strain not given), mothers consum-ing a diet defi cient in protein (7% protein) during either gestation or lactation, or during both periods, produced milk similar in composition to the milk of rats fed a nutri-tionally complete diet (18% protein), but milk output (as


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Mea sur ing Milk Composition and Yield 531

assessed by milk expression) was severally reduced. Re-duced milk output was correlated with low rates of post-natal growth and high pup mortality (Venkatachalam and Ramanathan, 1964). Water restriction in house mice, spin-ifex hopping mice (Notomys alexis), fawn hopping mice (Notomys cervinus), and plains rats (Pseudomys australis) has been reported to increase the dry matter and fat content of milk while percent protein or carbohydrate remained unchanged (Baverstock et al., 1976).

Although it is generally impossible to thoroughly char-acterize nutrient intake of free- ranging animals as can be done for captive, laboratory, and domestic species, normal fl uctuations in environmental conditions may infl uence foraging success and thus aff ect the composition or vol-

ume of milk produced. Postnatal growth rates of insec-tivorous bat pups are often reduced during prolonged bouts of rain or unseasonably cool temperatures (Hol-royd, 1993; Hoying and Kunz, 1998; Reynolds, 1999). It is likely that these inclement conditions may prevent bats from foraging and promote torpor. Milk protein synthe-sis, and likely milk output, may be reduced as a conse-quence of torpor (Wilde et al., 1999).

Diurnal variation in milk composition and yield has also been reported in some species. For example, Saxema et al. (1997) compared milk composition and yield in cattle in the morning versus the eve ning. Cattle produced 21% more milk in the morning, but eve ning milk had 8% more fat and 2% more protein. This variation is likely correlated

Figure 25.1 Patterns of milk composition during lactation in Tadarida brasiliensis, including change in (A) energy content, (B) dry matter ( ) and fat ( ) and (C) protein ( ) and carbohydrate ( ) concentration. Note: samples were pooled by week of lactation. There was a signifi cant increase in energy, dry mass, and fat but not protein and carbohydrates. Redrawn from Kunz et al., 1995.

Figure 25.2 Patterns of milk composition during lactation in Eptesicus fuscus, including change in (A) energy content, (B) dry matter ( ) and fat ( ), and (C) protein ( ), and carbohydrate ( ) concentration. Note: all samples were analyzed individually. There was a signifi cant increase in energy, dry mass, and fat but not protein and carbohy-drates. Adapted from Hood 2001).


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paper does not describe methods of analysis, nor is stage or day of lactation given. Many of the results described by Ben Shaul are very diff erent from data presented in later studies (Ben Shaul, 1962; Knight et al., 1986; Oftedal and Jenness, 1988; Oftedal et al., 1993).

Most investigators who examine milk composition and yield in free- ranging animals are interested in ecological or evolutionary questions associated with patterns of pa-rental care and investment. Thus, a primary objective may be to characterize the “typical” pattern of nutrient trans-fer from mother to off spring. It is important to minimize the disturbance of normal nursing behavior during collec-tion procedures. For example, it may be advisable to collect milk samples from diff erent animals than those for which milk yield is being mea sured, since restraint and/or chem-ical immobilization could impact subsequent nursing.

Ideally, milk composition and yield should be described at multiple points across the lactation period, with day of lactation serving as a covariate. Unfortunately, births are rarely observed in wild populations of bats. Investigators often use growth equations to estimate the age of off -spring (Kunz and Anthony, 1982; Anthony, 1988; Kunz and Robson, 1995; Stern and Kunz, 1995; Hoying and Kunz, 1998; Hood et al., 2002 Kunz, Adams, and Hood, this vol-ume). Although the use of age predictive equations may be unavoidable, the accuracy of equations relating milk yield and composition to age will be undermined if pup growth rates vary substantially between individuals. An alternative that does not rely on uniformity of growth is to present data in relation to the mass of the pup or simply defi ne broad stages of lactation that acknowledge uncer-tainties in age estimation.

For comparative purposes, lactation has often been di-vided into early, mid, and late stages: early lactation in-cludes colostrum and the transitional period prior to peak yield, mid lactation includes a period during and immedi-ately after peak production when milk composition is rel-atively stable, and late lactation encompasses the terminal period of declining production (and often changing com-position) as the young are weaned to solid foods (Oftedal, 1984a). Still, comparisons among species are complicated because the divisions between early, mid, and late lacta-tion are subjective and not easily distinguished in many species. Perhaps even more critical, species may diff er greatly in relative stage of development at birth and at weaning. Thus, “early lactation” may be prolonged and characterized by great changes in milk composition in species with altricial young, such as bears and marsupials, whereas “late lactation” may be virtually non ex is tent in species such as phocid seals and many mysticete whales that wean their young abruptly when they have had little or no chance to consume solids (Oftedal and Iverson, 1995; Oftedal, 1997). Given that most bats suckle their young to approximately 70% of adult mass, whereas many other mammals wean at 40% (Barclay, 1994), late lactation in

with the pattern of milk removal by suckling off spring. Typically, more milk is expressed when the young have not suckled for a substantial period of time, as a reduction in suckling frequency is associated with increasing degree of glandular fi ll (Daly et al., 1993). Moreover, in many large mammals, the initial milk expressed may be substan-tially lower in fat than the last milk obtained (Daly et al., 1993). This is accentuated if the mammary gland is full (Oftedal, 1984a). For example, milk collected from the full gland of human females prior to suckling had 75% less fat than milk collected after the gland had been evacuated by a suckling child (Daly et al., 1993). Thus, if off spring suckle periodically throughout the day and little during the night, milk samples expressed in the morning are likely to be greater in volume but lower in fat than milk expressed in the eve ning, which will have a greater percentage of hindmilk and thus a greater fat content. Hood et al. (2001) found no diurnal eff ect on the composition of Pteropus spp. milk collected from captive animals between 0900 and 1930 h, during which time pups likely suckle at frequent and regular intervals. in Rouettus eqyptiacus, however, Ko-rine and Arad (1999) found that the dry matter content of milk at peak lactation was 26% higher in late afternoon than in early morning, suggestive of higher fat concentra-tion in the hindmilk, assuming variation in dry matter is associated with change in fat and that glandular fi ll is greatest as females return from predawn foraging bouts. Nonetheless, the relationship between milk composition and glandular fi ll has not been as explicitly tested in small species as it has in larger mammals. Small alveolar vol-ume in bats and mice (Francis et al., 1994; Evarts et al., 2004) will correlate with a high luminal epithelial surface area relative to the volume of milk produced. Thus, fat may not separate to the same degree as observed in hu-mans. The relationship between fat content of the milk samples and glandular fi ll warrants further investigation.

Investigators should beware of drawing conclusions that are not supported by mammary function or complete data. For example, several studies have suggested that in-terspecifi c diff erences in milk composition among bats are consistent with the nutrient composition of the diet (Kunz and Stern, 1995; Korine and Arad, 1999; Kunz and Hood, 2000). Although there may be a correlation between the proximate composition of the milk produced and diet consumed by those bats studied to date, this correlation is commonly misinterpreted. The proximate components of milk are synthesized under endogenous control within the epithelial cells lining the mammary gland (Akers 2002). Although these cells need the subunits necessary to synthesize fat, protein, and carbohydrates, dietary fats, proteins, and carbohydrates are not transferred directly into milk. Investigators should also be wary of making comparisons to studies with incomplete data. For exam-ple, investigators commonly compare their results to the nursing categories described by Ben Shaul, 1962, yet this



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bats may not be comparable to late lactation in other taxa. Oftedal, 1984a, and Oftedal and Iverson, 1995, limited in-terspecifi c comparisons to mid lactation, i.e., the period of maximal lactational per for mance with reference to milk volume and milk energy (Oftedal, 1984a).

Evaluation of interspecifi c variation in milk composi-tion at mid lactation can be extremely valuable for qualita-tively comparing many species when insuffi cient data is available for a statistical analysis. As far as possible, stud-ies should identify this period of peak lactation to facili-tate comparisons with other studies. Yet, a mid lactation dataset will likely include milk consumed by young at dif-ferent stages of development and will not directly refl ect cumulative maternal eff ort, which may span a few days or a few years. Alternative comparisons should be con-sidered. For example, investigators may fi nd it more ap-propriate to compare milk composition between species with respect to the relative mass of the off spring (i.e. off -spring mass/average non- reproductive mass of adults), rate of change in milk composition between select points during lactation (i.e. (the fat content of milk at mid- lactation—the fat content of milk at birth)/number of day between birth and mid- lactation), or mass- specifi c en-ergy and nutrients invested in off spring for the duration of lactation.

MILK COLLECTION

The goal in milk collection must be to obtain samples that closely approximate the milk actually ingested by suckling young (i.e., samples that are not contaminated, that contain a representative fat content, etc.), and that are collected and stored in a manner that preserves the integ-rity of the sample until it is analyzed. In bats and other small mammals, the small amounts of milk obtained pres-ent special diffi culties that need to be addressed in the collection methods. Inattention to detail can result in con-taminated or dehydrated samples that are very diffi cult to homogenize for analysis.

Avoidance of ContaminationWhen collecting milk samples from small species, in-

vestigators should take care to avoid contamination from contact of milk with skin or fur. For example, most bats are highly gregarious, so the skin and fur surrounding the nipple of females can easily come in contact with urine and feces from contact with other individuals, the sub-strate animals are roosting on, and/or the cage or bag in which animals are held prior to milking. Based on the typical composition of milk and feces of the big brown bat (Hood, 2001), we estimate that one milligram of feces in-advertently collected with a milk sample of 500 mg (0.5 mL) would have only a minor eff ect on the composition of the milk, causing the concentrations of nitrogen, sodium, phosphorus, and calcium to increase by 2.0%, 0.4%, 0.8%

and 0.8%, respectively. However, if 1 mg of feces contami-nated a 50 mg (0.05 mL) sample of milk, the error attribut-able to the contaminant would be substantial, with the nitrogen content increasing by 17%, sodium by 3.8%, and phosphorus and calcium by 7.4 percent.

A number of mea sures can be taken to reduce contami-nation. Milk often wicks into the surrounding hair during collection. This can be minimized by trimming the fur around the nipple. We recommend cleaning both the nip-ple and the surrounding skin and hair with isopropyl alco-hol or a similar solvent that will evaporate quickly so that residual solvent does not contaminate the sample. A labo-ratory tissue such as Kimwipe can be used to dry the area before milking. Latex gloves worn by the individual col-lecting the sample will prevent transfer of oils or other matter from the hands to the sample, but leather gloves may be needed for restraining nonsedated bats or other animals that may infl ict a bite. There is also a risk of uri-nation or defecation by the animal during restraint, and placement of a dampened ball of cotton over the anal- genital area will reduce the risk of this material contami-nating the milk sample. Although it is possible that suck-ling young may occasionally consume contaminants with the milk, this would likely apply only to contaminants on the nipple itself, not contaminants on fur. If investigators are specifi cally interested in exogenous contaminants that young might consume with milk, material rinsed from the nipples should be retained and analyzed separately.

In general, investigator success in expressing milk di-rectly into collection tubes improves with practice, so opportunities for contamination become reduced. It is good practice to record whether collected milk has contacted hair or the surrounding skin so that analytic data can sub-sequently be assessed for the likelihood of contamination.

Gland EvacuationPractices associated with milk collection should aim to

obtain samples that represent milk typically consumed by suckling young. As discussed previously, because milk fat content may increase during expression, milk collected from an engorged gland, with a greater percentage of fore-milk, is likely to be lower in fat, whereas milk obtained shortly after suckling may be composed of high- fat resid-ual milk. Thus, milk samples collected without respect to suckling schedules and degree of glandular fi ll may be highly variable in fat content. Although the extent of glan-dular fi ll and thus degree of emptying was not mea sured directly, Hood et al. (2001) found that when smaller vol-umes were expressed, milk fat content was greater. It is possible that smaller volumes were obtained from moth-ers whose pups had recently suckled.

In small mammals with a branching ductal system and minimal development of collection sinuses or cisterns (Evarts et al., 2004), it may be diffi cult to empty any mam-mary gland to the same extent that the young achieve.


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Incomplete milking is likely to produce milk with reduced fat content, which could produce a systematic bias. Thus, it is important to strive for the most complete gland evac-uation possible. In small mammals with large litters and numerous nipples, there is often a trade- off between gland emptying and the numbers of glands milked. It may be tempting to seek to obtain a modest amount of milk from many glands, since this usually yields more milk overall than attempts to completely evacuate one gland. Although this has not been directly evaluated, it is likely that such a procedure would result in milk of lower fat content, which may not be representative of what suckling young ingest (Daly et al., 1993; see description of glandular fi ll, above).

Taking the normal suckling patterns of the animal into consideration, milk should be collected at a time when glandular fi ll is typical of that which accumulates during normal intersuckling intervals. For example, bats often leave dependent young in the roost while they forage and thus milk accumulates during the foraging period. Moth-ers trapped as they return from foraging bouts may carry greater volumes of milk than mothers captured during roosting, when the young may be suckling frequently. Since pups generally suckle immediately after the mother returns from foraging (Kunz and Hood, 2000), samples at this time represent what pups were prepared to consume. This milk may also be more representative, since it will not contain the high levels of residual milk found in glands that have recently been suckled (Daly et al., 1993).

Excessively prolonged separation of mother and young may lead to excessive intramammary pressure, accumula-tion of inhibitory compounds, and loss of integrity of the mammary epithelium, leading to changes in milk compo-sition and potential disruption of subsequent mammary function (Oftedal, 1984a). In a captive setting, mothers and young must usually be separated prior to milking to allow milk to accumulate, especially when the timing of the last suckling bout cannot be determined. A useful rule is to keep the duration of this separation to that of a nor-mal intersuckling interval. If this interval is not known, 2– 3 hours is probably safe in most cases. The length of time mothers and young are separated should be consis-tent between samples. Without separation, the amount of milk that can be expressed will be reduced, and this milk may be atypically high in fat due to the preponderance of residual milk (Daly et al., 1993).

Eff orts should be made to evacuate glands as completely as possible to avoid overrepre sen ta tion of the foremilk. Unfortunately, this can be diffi cult to do with an animal that is struggling and/or undergoing high levels of stress, since stress can inhibit milk letdown. To maximize vol-ume of the milk sample, it may be necessary to administer both sedatives and oxytocin to lactating females just prior to collection. Although some bats can be milked by man-ual restraint, many small mammals (e.g., large megachi-

ropterans, rodents) can be very diffi cult to restrain manu-ally, and sedation is necessary to collect a usable sample. Proper delivery of anesthetics (particularly inhalants) can be diffi cult with small mammals (see Barnard, this vol-ume), especially under fi eld conditions in which appropri-ate equipment for control of gas delivery is not available. We have used both inhalants such as isofl urane in liquid and gaseous forms and a variety of injectable sedatives and immobilizing drugs, but make no drug recommenda-tions here. Researchers should consult the literature, dis-cuss issues of sedation with colleagues studying similar taxa, and consult veterinarians with experience in lab ro-dent and/or exotic animal medicine.

Oxytocin is naturally released into maternal circulation in response to a sucking stimulus and induces contraction of myoepithelial cells and small ducts in the mammary gland, resulting in “milk letdown” (Goodman, 1996). Milk letdown involves the redistribution of milk from the al-veoli and small ducts to larger ducts and collection si-nuses, where it is more easily removed by suckling young (or an investigator). Thus, milk letdown is essential for mammary evacuation. In some domestic or semi- domestic animals, such as camels, milk letdown can be achieved by allowing the young to suckle on one side while the milker manually collects milk on the other. This is generally not feasible in most small mammals; hence, milk letdown has to be induced by injecting the animals with exogenous oxytocin prior to milking. While a small amount of milk can sometimes be obtained without oxytocin, the admin-istration of oxytocin usually results in collection of sub-stantially greater amounts of milk, particularly after a long period of maternal separation from her young. In Pteropus vampyrus, up to 4 mL of milk could be collected from ani-mals that were anesthetized with isofl urane gas and given a 0.4 IU/g injection of oxytocin (Hood et al., 2001). When oxytocin was not used, the average volume collected dropped to 0.5 mL (W. R. Hood, personal observation). We recommend use of exogenous oxytocin whenever possible.

Oxytocin has a very short half- life (<2– 3 min), and plasma levels vary widely with species but are typically low (20– 300 pmol) after sucking stimulus in rats and rab-bits (Wakerly et al., 1994). Thus, high circulating oxytocin concentrations are not required for milk letdown. How-ever, in bats and small non- laboratory mammals, it is usu-ally not feasible to administer oxytocin by an intravenous route, and much larger (pharmacologic) doses may be needed to achieve adequate circulating levels when oxyto-cin is administered via an intramuscular or intraperito-neal route. In the United States, oxytocin is typically avail-able at concentrations of 10 or 20 IU/mL for veterinary purposes. Dosages previously used in bats and rodents (about 0.1– 0.4 IU/g; Kunz et al., 1995; O.T. Oftedal, unpub-lished) may be in excess of the minimal eff ective dose but have been used without complication. Animals that have not been sedated may also require higher doses due to the



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eff ects of stress on the milk letdown response (Denamur, 1965). If oxytocin is administered by intramuscular (IM) injection, the best location in bats is probably the upper pectoralis, the largest muscle in the body (M. Mendonca, pers. comm.); for other small mammals hindlimb muscles are likely more suitable.

Milk Expression and Sample StorageMilk samples should be expressed from animals by man-

ual palpation, using fi ngertips to lightly press the glands from the edges toward the nipple (see Fig. 25.3). In small mammals, attempts to squeeze the nipple per se are usually unsuccessful, and investigators may need to practice milk-ing using diff erent positions and movements before they become adept. Vacuum- assisted collection methods have been attempted in small mammals (e.g., Baverstock et al., 1976), but there is risk of substantial moisture loss in such systems, leading to analytic error (Oftedal, 1984a).

When only small volumes can be expressed, it is sim-plest to collect milk directly into a glass capillary tube as it wells up on the surface of the nipple. The capillary tubes should be suitably clean (e.g., acid- washed if milk is to be assayed for trace elements) and should not contain any additives. For example, heparin is a polysaccharide that could interfere with sugar analyses in some methods of analysis. The sample should be immediately transferred to an airtight tube or vial to prevent water loss. We use a capillary pipette bulb or fi ltered mouth pipette to care-fully blow the sample from the capillary tube to the vial for storage.

When dealing with small samples of milk it is critical that these are placed in tubes or vials that will be mostly fi lled by the milk sample. Many investigators have had the

shock of fi nding a small, mostly dry disk of milk solids on the bottom of a vial after frozen storage; the water in the milk has sublimated and recondensed as ice crystals on the sides of the tube. Unfortunately, such samples are very diffi cult to homogenize, because the proteins in the dried samples have been denatured by the salts in the milk. This problem can be minimized by (1) using small tubes with-out much airspace, (2) storing of samples in a non- frost- free freezer at less than −20°C, (3) avoiding exposure to temperature gradients (e.g., during shipment or from be-ing moved about in the freezer), (4) minimizing the time period between collection and analysis, and (5) quantita-tive dilution of the samples with water (1:1 or 2:1) prior to freezing (see below).

Tubes for sample storage should have a narrow mouth and total volume similar to that of the sample. Narrow, 0.5- mL eppendorf tubes may work well for samples col-lected from mammals < 50 g. Tubes should be acid washed prior to collection if the mineral content of milk is to be mea sured. Even with the proper tube, milk samples that are naturally high in fat, dry matter, or protein may be diffi cult to resuspend and subsample after thawing. Such samples may be diluted with a known and precisely mea-sured (by mass) volume of distilled deionized water prior to freezing (accurate mea sure ment of mass of both milk and added water is critical for fi nal calculations). Dilution can be used both as a way to avoid the air space problem and may also facilitate subsequent removal of thick or high- fat samples from the tube for analysis. For example, milk from white- footed mice (Peromyscus leucopus) thick-ens considerably after freezing; diluting the milk with two parts water and vortexing prior to freezing resulted in much improved subsampling after thawing (M. B. Vol-tura, pers. obs.).

Samples should be stored at less than −20°C immedi-ately after collection. In the fi eld, samples should be fro-zen as soon as possible and transported on dry ice or liq-uid nitrogen. When using liquid nitrogen, be sure vials can withstand rapid cooling.

Collection FrequencyTo examine changes in milk composition through lacta-

tion, it is necessary to collect serial samples. For mammals with short lactation periods, this can mean administering exogenous oxytocin and anesthetizing females every few days. Although necessary for suffi cient data collection, this pro cess can potentially infl uence milk composition and the health of the mother. Serial milking with exoge-nous oxytocin can infl uence milk composition in rabbits, ruminants, and other taxa (Allen, 1990; Oftedal, 1984a; but see Ballou et al., 1993). Potential changes in milk com-position, however, may also be related to diff erences in gland evacuation after oxytocin administration, and thus can be diffi cult to interpret. Keen et al. (1980) found that the zinc content of milk and the zinc stored in the liver of

Figure 25.3. Milk expression from anesthetized Pteropus hypomelanus. Milk is expressed by carefully pressing milk from the edges of the gland toward the nipple. Milk is drawn into a nonheparinized capillary tube as it is exuded. The sample is then transferred to a 0.5- mL tube for storage.


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suckling rat pups was higher for females milked weekly (3 times total) versus mothers only milked once. The mechanism is unknown but could be associated with dis-ruption of the secretory epithelium or stress associated with more frequent mother- pup separation.

Repeated milking events spaced closely together may be diffi cult for some small animals (due to mechanical in-sult associated with milk expression) and high- strung wild animals that react poorly to manual or chemical immobi-lization. In white- footed mice (Peromyscus leucopus), one of us (MBV) observed an apparent increase in the incidence of capillary damage and mastitis (or perhaps clogged ducts) when mothers were milked repeatedly; apparent damage was observed when animals were milked every 5 days but rarely occurred when females were milked only once during the 20- day lactation period.

These observations indicate that special attention should be given to animals that are milked serially. Because no other investigators have reported mammary damage in small species, we assume this occurrence is relatively rare. Investigators should be gentle while expressing samples and watch for damage or unusual swelling of the nipple and surrounding tissue. If any blood is expressed with the milk, milking should be ceased immediately, the sample discarded if the blood will contaminate the compounds to be analyzed, and the mammary condition should be care-fully examined before additional samples are taken at fu-ture milking events. In most cases, if researchers design their experiments with milking intervals that minimize impact of milking on dependent young (ASM, 1998), the eff ect of serial milking on the mother will likely be mini-mal. Researchers should take into consideration lactation duration, suckling pattern, and suckling frequency when deciding on an appropriate milking interval. Although the eff ects of milking on pup growth has not been exam-ined in bats, we would recommend milking no more than once per week in free- ranging bats and carefully monitor-ing gland condition before considering an increase in milking frequency.

CHEMICAL ANALYSIS OF MILK CONSTITUENTS

Numerous methods have been employed for charac-terizing the nutritional content of milk, especially of dairy species; however, most of these require one or more mil-liliters of milk, or they may be valid only if calibrated for the par tic u lar species being studied (e.g., dye- binding methods). For this reason, only a limited number of meth-ods are suitable for small mammals such as insectivorous bats or rodents. Precaution and planning are critical when analyzing small volumes of milk since little, if any, resid-ual milk will be available to repeat analyses if an error is made or if high variance indicates a need to analyze addi-tional replicates.

When designing a study and selecting analytic meth-ods, it is important to consider both the volume and com-position of milk to be collected, either from preliminary data or from previous studies on the same or closely related species. For most analyses, there is a minimum nutrient amount that can be mea sured with accuracy. It may be pos-sible to assay smaller quantities of milk if the concentration is high. Analysis of reference materials, such whole and nonfat milk powder available from the National Institute of Standards (reference numbers 8435 and 1549, respec-tively), or even fresh cow’s milk purchased locally, are valu-able for testing methodologies, assessing the variance be-tween replicates, and determining the minimum amounts of milk that can be assayed. However, cow’s milk and the powder derived from it may not be appropriate standard materials for all types of assays due to diff erences in milk constituents among species; for example, reducing sugar methods work well with cow’s milk but may give biased results for milks containing sugars other than lactose (re-ducing sugar methods rely on the oxidation of the ketone or aldehyde groups present in many sugars and an indica-tor that is the recipient of electrons in a redox reaction; not all sugars have these reducing groups; Oftedal and Iverson, 1995). Thawed samples of small volume may also have much higher analytic variance than fresh cow’s milk because of problems of homogenization.

All assays should be run in replicate so that within- sample analytic variation can be assessed. Two replicates are normally suffi cient if the within- sample coeffi cient of variation (CV) is less than 5%, but additional replicates are appropriate if the CV is higher. Unfortunately, although low CVs may be relatively easy to obtain with fresh milk and macromethods that use large volumes, this is not al-ways true for thawed samples assayed by micromethods. Adequate storage (see above) and rapid thawing mini-mize, but do not eliminate, sample heterogeneity that makes representative sampling diffi cult. For frozen cow’s milk, the Association of Offi cial Analytical Chemists (AOAC) recommends that milk samples be thawed to room temperature (20°C) for subsampling, but that the temper-ature be increased to 38°C if lumps do not disperse (AOAC, 1984). Our experience is that rapid thawing in a warm water bath, followed by immediate vortexing and prompt subsampling, reduces subsampling problems. If possible, all subsampling should be done shortly after thawing; samples that are allowed to thaw slowly or to sit after thaw-ing may begin to separate into discrete layers that resist remixing. Samples should be gently vortexed between subsamples, but extreme shaking or mixing may destabi-lize the cream fraction, causing “butter” to adhere to the sample vial. Finally, milk samples should not be thawed and refrozen repeatedly, as this may disrupt the milk fat globules so that samples end up with beads of oil fl oating on top. This fl oating oil is virtually impossible to reho-mogenize into milk without using added emulsifi ers.



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The amount of subsample used for each method should be determined by mass; volume mea sure ments are not only less precise, but will also be infl uenced by the specifi c gravity of milk. The selected analytical balance should permit mea sure ment of four signifi cant digits, as the fi nal digit is often approximate because of mass change due to evaporation. Thus, when mea sur ing mg quantities of milk, it is critical to use a microbalance that reads to the nearest 0.01 mg. At these low masses, temperature can af-fect the tare weight of the empty weighing pan, causing errors when milk mass is estimated by diff erence (pan with milk minus empty pan); therefore, both the milk and the weighing pan should be at room temperature during weighing. Similarly, samples heated to determine dry matter content should be cooled in a desiccator before weighing. Evaporative water loss before and during weigh-ing can be minimized by keeping samples covered, weigh-ing samples quickly, and using the balance in an area that is neither drafty nor hot.

For all methods discussed below, researchers are ad-vised to consult the original papers describing these meth-ods, to be familiar with hazards described in material safety datasheets (MSDS) for all chemicals used, and to learn instrument operation from a trained technician be-fore attempting these analyses.

Proximate CompositionAnalytical methods that have been used to mea sure

the proximate, or macronutrient, components of milk have been reviewed by Oftedal, 1984a, and Oftedal and Iver-son, 1995. In this chapter, we specifi cally discuss methods that can be applied to small milk volumes and will present our preferred methods of analysis based on accuracy, pre-cision, and volume required.

Dry MatterThe dry matter content of samples is determined by

comparing the wet to dry mass of milk aliquots that have been dried to constant mass (see Box 25.1). Oven drying (forced air or vacuum oven) is recommended over freeze- drying, as freeze- dried samples typically contain small amounts of residual water, and thus will overestimate dry matter content (Sherbon et al., 1978; Oftedal and Iverson, 1995). However, even oven- dried samples contain some

water that becomes chemically bound during drying; for example, under typical oven- drying conditions lactose comes out of solution as lactose monohydrate. In addition, lactose may react with certain amino acids to form Mail-lard products (non- enzymatic browning). Thus, it is im-portant that drying methods be calibrated against a stan-dard procedure. The AOAC (1990) recommends drying 1 g of cow’s milk for 4 hours at 100°C; however, with very small samples the more rapid heating that occurs will cause heat damage and potential volatilization of compounds other than water. For small samples (e.g., 20– 100 μL), dry-ing for 2.5 hours at 100°C in a forced convection oven yields comparable values to the AOAC method in the Na-tional Zoological Park (NZP) Nutrition Laboratory (Hood, unpublished data). Investigators are encouraged to calibrate drying time for small samples to the AOAC method under their own conditions, as heating and air circulation may vary among ovens.

Avoidance of excessive heat damage is also important if dried subsamples will be used for other analyses, such as energy determination by microbomb calorimetry, nitro-gen determination by CHN elemental gas analysis, or mineral analysis by atomic absorption spectrometry. Such “piggybacking” of other assays onto dry matter analysis reduces the total amount of sample material required. However, oven- dried samples are not suitable for com-pounds that may be damaged or altered by heat, including amino acids, fatty acids, and most vitamins.

The amount of subsample required to mea sure dry matter content is limited by the accuracy with which the dry mass of the subsample can be mea sured and the ho-mogeneity of the subsample. The CV can be expected to be higher for samples that are not well homogenized, which may necessitate use of larger subsample volumes or more replicates.

CarbohydratesThe carbohydrate constituents of milk may include a

variety of diff erent types of sugars, including lactose, monosaccharides, oligosaccharides, and protein- bound and lipid- bound carbohydrates (Jenness, 1974; Messer and Urashima, 2002; Newburg, 1996). Although lactose is often assumed to be the most abundant sugar in milk, the con-tribution of other sugars to the total carbohydrate content

box 25.1 . Dry matter content of milk

1. All mea sure ments of mass should be made in the same units (i.e., grams).2. Weigh aluminum pan or other object on which sample will be dried (P).3. Deliver milk to pan and immediately weigh the pan with milk (PM).4. Dry to reference mass at 100°C (e.g., 2.5 hours for 20– 100 μL).5. Weigh the pan with dried sample (PDM).6. Percent dry matter (%DM) = [(PDM − P)/(PM − P)] × 100.


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of milk may be substantial (Messer and Urashima, 2002). For example, tri- and oligosaccharides are well character-ized and prominent in milks of at least some species within the orders Monotremata, Marsupialia, Primates, and Car-nivora. The relative abundance of sugars other than lac-tose in the milks of bats and most small mammals is unknown.

A variety of methods have been employed to deter-mine sugar content of milk samples, but most have short-falls that can lead to underestimation of total sugar con-tent. These methods include enzymatic assay of lactose and high- performance liquid chromatography using a lac-tose specifi c column, both of which have reduced detec-tion of non- lactose sugars; reducing sugar methods that underestimate sugars, such as sucrose, that do not have prominent ketone or aldehyde groups; and the anthrone procedure, which severely underestimates galactose (Oft-edal and Iverson, 1995; Korine and Arad, 1999).

The phenol- sulfuric acid method (Dubois et al., 1956; Marier and Boulet, 1959) is based on the hydrolysis of di- and oligosaccharides with sulfuric acid; the hydrolyzed sugars react with phenol to form a relatively stable yellow- orange chromogen that can be quantifi ed with an ultravi-olet spectrophotometer using a lactose monohydrate standard. Thus, this yields the monosaccharide equiva-lent of the total sugar content of the sample, a value can be corrected for the hydration of lactose monohydrate (by dividing by 0.95), thereby providing the disaccharide equivalent.

The phenol- sulfuric acid method (Dubois et al., 1956; Marier and Boulet, 1959) is generally preferred for mea-sur ing the total concentration of carbohydrates in milk because the assay is relatively insensitive to the type of sugar mea sured. It encompasses mono-, di-, and oligosac-charides, whether free or bound, and whether reducing or nonreducing, This method may fail to detect amino sug-ars; however, amino sugars are typically of very low con-centration in milk and should not lead to signifi cant error (Oftedal and Iverson, 1995). A minor modifi cation to this method has been recommended for milks with oligosac-charides composed predominantly of galactose (such as most marsupial milks; Messer and Green, 1979).

The phenol- sulfuric acid method is sensitive to μg quantities of sugars and thus requires extensive dilution of milk to ensure that assayed amounts fall within the range of the standard curve. The range of concentrations used to generate the standard curve, 10– 50 μg lactose per gram of solution, provide a linear response in UV absor-bance to color development, which, in turn, is propor-tional to sugar concentration. This method can be limited by the ability to weigh small samples, but if mea sure-ments are suffi ciently precise, the procedure can be used with very small amounts of milk. Bat milk samples as small as 5 mg have been analyzed (Hood et al., in prep.a);

however, precision was not ideal for all samples. We rec-ommend analyzing a small subset of samples and calculat-ing the coeffi cient of variation between replicates before fi nalizing replicate mass for critical samples. The phenol- sulfuric acid method is summarized in Box 25.2.

To obtain optimal color development, phenol and sul-furic acid must be added to the diluted sample in volumes that will provide a fi nal solution containing 1.1% phenol and 74% sulfuric acid. The volumes given in Box 25.2 are recommended, but they can be modifi ed as long as appro-priate reagent concentrations are maintained. For consis-tency and to save time when pro cessing large sets of sam-ples, standards can be prepared in a large volume and then frozen for use with all samples for a given study. We nor-mally prepare a stock solution of 1:1000 (w/w) lactose to distilled water. Aliquots of 1– 5 g of the stock are diluted to 100 g to produce standard solutions of 10– 50 μg/g. Samples should be diluted to a target dilution of approximately 25 μg/g so that they fall near the middle of the standard curve. Milk samples of related species can be used to pre-dict the expected sugar concentration of the milk. The color is stable, provided tubes are read in the UV spectro-photometer within two hours of the reaction with phenol and sulfuric acid. If layering develops within sample tubes it may be necessary to vortex them gently prior to reading in the UV spectrophotometer, but this can also introduce minute air bubbles that interfere with UV mea sure ments and should be undertaken with caution. Because of the extensive dilution and high sensitivity of the reaction, it is common for within- sample CVs to be high. Thus, we rec-ommend assay of at least three replicates per milk sample, and mea sur ing the absorbance of each replicate three times.

ProteinThe standard method for mea sur ing the crude protein

content of milk involves an assay of nitrogen content, which is multiplied by a standard conversion factor of 6.38, based on the assumption that milk proteins contain on average 15.67% nitrogen (Jones, 1931). It is not known if this factor is appropriate for the wide variety of mam-malian species, but its use is a widely accepted convention (Oftedal and Iverson, 1995). Crude protein can be cor-rected to true protein by subtracting non- protein nitrogen (NPN), where this is assayed followed by precipitation of the proteins in milk. In most mammals NPN is in the range of 0.03– 0.08% and accounts for only 3.4– 7.8% of the total milk nitrogen, but it may be higher in species with very low protein milks (such as perissodactyls and some primates) and in Carnivora (Oftedal, 1984a). NPN ac-counts for about 6% of milk nitrogen in lab rats (Rattus norvegicus, Luckey et al., 1955). Although NPN has not been mea sured in bat milk, the error in ignoring it is prob-ably small.



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An alternative approach is to mea sure milk with the Lowry and dye- binding methods (see Oftedal and Iverson, 1995 for a review). While these methods are often rapid and require little milk, they are plagued by unequal sensi-tivity to diff erent proteins and may produce errors of 20% or more. These methods are therefore not recommended unless they are calibrated by more reliable methods for the species to be studied (Oftedal, 1984a).

We have been particularly interested in the relative suit-ability of three methods for mea sur ing the nitrogen con-tent of small milk samples: the micro- Kjeldahl method, the Nesslerization procedure, and elemental gas analysis fol-lowing combustion. Theoretical descriptions will be given for each methods, but a detailed description will only be provided for our preferred method, elemental analysis.

The Kjeldahl method has been a standard approach for estimating protein from the nitrogen content of biological samples for more than 100 years (Bradstreet, 1965). Sam-ples are digested in strong acid with salts and catalysts to

produce ammonia, which is collected by steam distilla-tion. With proper calibration to give 98– 100% N recovery, this method can be both accurate and precise, with low within- sample CVs.

The microadaptation of the Kjeldahl method (i.e., micro- Kjeldahl) is scaled down to smaller volumes of sam-ple and reagents, and the fi nal titration step may be re-placed by colorimetric determination of nitrogen; due to sampling and analytic error, the within- sample CV is usu-ally higher than the conventional Kjeldahl method. The required volume of milk for the micro- Kjeldahl technique is limited by the ability to consistently collect and assay ammonia. Researchers in the NZP nutrition lab have analyzed 75 mg milk per replicate for small primate milk samples containing approximately 3% protein, but lower assay volumes are possible with higher concentrations of protein (M. Power, pers. comm.).

In the micro- Kjeldahl method, samples are heated on a micro digestion unit (Micro Digestor, Labconco, Kansas

box 25.2 The phenol- sulfuric method for the carbohydrate content of milk

1. All mea sure ments of mass should be made in grams, or adjust calculations as appropriate.

2. Prepare samples by diluting milk with distilled water to achieve a sugar concentration of approximately 25 μg/g (e.g., if the sample is predicted to have 5% sugar, dilute approxi-mately 25 mg of milk in 50 g of water). Record mass of milk (M) in μg and the mass of the fi nal solution (F1) in grams to determine diluted milk concentration (C, μg/g):

C = (M/F1).

3. Prepare fi ve standard solutions ranging in concentration from 10 to 50 μg/g using dried α- lactose monohydrate powder and distilled water. Determine the mass of the standard (S) in μg and the mass of fi nal solution (F2) in grams. Calculate each standard concentra-tion (SC, μg/g):

SC = (S/F2).

These concentrations will be used to generate a standard curve. 4. Prepare 11% phenol solution in an Erlenmeyer fl ask (enough to add 1 mL to each test

tube, plus extra). The phenol solution should be left on a 50°C hot plate with stir bar to ensure that it is remains completely dissolved.

5. Prepare standards in duplicate, pipetting 1.6 mL of each of the fi ve standard solutions into two 10- mL test tubes. Prepare three “blank” tubes that contain only 1.6 mL distilled water each.

6. For milk samples, pipette 1.6 mL of diluted sample into each of three 10 mL test tubes. 7. For each tube, add 1 mL of phenol solution and vortex for 10 seconds. Add 7.4 mL of

concentrated sulfuric acid and vortex again. After the addition of acid, the tubes will be very hot and must be handled with care. Record the time of the acid addition.

8. Exactly 10 minutes after addition of sulfuric acid, stop the reaction by placing tube in a water bath at room temperature.

9. The three blanks and fi ve standards are used to establish the linear regression curve for absorbance at 490 nm versus known sugar concentration (SC).

10. Each sample tube is read by the UV spectrophotometer three times, and the average (A) of those readings is used to determine sugar concentration of the tube:

% Sugar = (A/C) × 100.


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City, MO) with sulfuric acid and an appropriate catalyst. Several salt- catalyst combinations have been described (Bradstreet, 1965) and many can be purchased premixed. A 33:1 mixture of potassium sulfate and copper sulfate (e.g., FisherTab CT- 50 Kjeldahl Tablets, Fisher Scientifi c, Fair Lawn, NJ) works well for milk samples. During di-gestion, proteins and other sources of organic nitrogen are converted to carbon dioxide, water, and ammonium hydrogen sulfate (NH4SO4). The digest and distilled water are then boiled in the presence of a strong base such as sodium hydroxide (RapidStill I, Labconco, Kansas City, MO). Ammonium gas liberated from the NH4SO4 is cap-tured by steam distillation into an indicator solution of boric acid (e.g., Kjel- sorb solution, containing 4% boric acid, bromocresol green, and methyl red indicator dyes; Fisher Scientifi c, Fair Lawn, NJ). The steam distillation system must be a closed system to prevent ammonia loss. The distillate is typically titrated with a known hydro-chloric acid standard solution to determine the amount of ammonia captured, as determined by appearance of a pink color due to pH change. Errors can occur due to incom-plete digestion (e.g., inadequate acid, temperature, or time for digestion), ammonia losses prior to or during distilla-tion (e.g., excessive heating, gas escape), and diffi culties in determining the endpoint during titration (especially if ammonia amounts are small). Thus, determining recov-ery with test compounds and testing standard materials of known protein content, such as NIST (National Institute of Standards and Technology) milk powders, are critical to ensuring that results are accurate.

The Nesslerization procedure may seem an attractive alternative to the micro- Kjeldahl method because much smaller volumes of milk are required (Folin and Denis, 1916; Koch and McMeekin, 1924). For many years, it was the method of choice for small milk samples in the NZP Nutrition Laboratory. Nesslerization is initially similar to micro- Kjeldahl, in that milk samples are digested in sulfu-ric acid to form ammonium hydrogen sulfate. The digest is heated for 90 minutes in a heating block and then quickly fl amed over a Bunsen burner until sulfuric acid fumes are observed. Hydrogen peroxide (H2O2) is added to the sample to oxidize the ammonium hydrogen sulfate to ammonium sulfi de. The H2O2 addition and fl aming are repeated twice at 135- second intervals. Finally, the sam-ples are diluted and Nessler’s reagent is added to produce a yellow- orange solution that varies in intensity in relation to the concentration of nitrogen. The concentration of ni-trogen (N) is determined by spectrometry, comparing the absorbance of samples at 500 nm to a standard curve ob-tained for known concentrations of N in ammonium sul-fate solutions. The standard curve is linear at low N con-centrations (e.g., 0.04 −0.2 mg N per g of Nessler’s solution), so a suitable milk sample quantity is one that falls in the middle of this range, such as 0.1 mg/gram. Thus, this

method requires as little as 8 mg of milk for a sample with 8% protein, as observed in many insectivorous bats (Kunz and Hood, 2000).

One drawback of Nessler’s method is that it requires uniform timing for the heating and cooling steps, with all standards and samples treated identically. This can be diffi cult to achieve and may result in operator bias. It is necessary to demonstrate that a given operator can achieve results with Nessler’s that are equivalent to other methods (e.g., micro- Kjeldahl) and have acceptable CVs. Additionally, Nessler’s reagent is toxic, containing 50 g mercuric iodide per 1000 mL of Nessler’s solution, thus creating potential hazard and the necessity of appropriate hazardous waste disposal. The color of the solution be-gins to fade approximately one hour after the Nessler’s reagent is added, therefore, careful attention to timing is critical.

Dissatisfaction with the potential operator eff ect in Nessler’s method, despite attempts to standardize heating and cooling protocols, led the NZP Nutrition Laboratory to switch to elemental analysis, a combustion method in which organic nitrogen is converted to gaseous form and mea sured in a Carbon- Hydrogen- Nitrogen (CHN) elemen-tal gas analyzer (Perkin Elmer, 1996). In this method, small milk samples are dried in tin vials, which are rolled into balls and placed in an autosampler tray. The autosampler system sequentially delivers the samples, interspersed with standard material (acetoanilide 10.36% N) and blanks, into a high- temperature (1800°C) combustion chamber fi lled with silver tungstate on magnesium oxide, silver vanadate, and other catalytic material. Boosts of pure oxy-gen are provided to ensure complete fl ash combustion. The initial combustion products are scrubbed through a reduction chamber, and the gases produced are segre-gated into sequential wave fronts that are mea sured by a thermal conductivity detector. Initial validation with a variety of biologic materials required adjustment of com-bustion temperatures and supplemental oxygen to pro-duce values equivalent to Kjeldahl procedures. When ap-plied to a variety of milk samples and NIST standard materials, this CHN elemental analysis has proven to be accurate, precise, labor- effi cient and suitable for samples with dry masses of just 1– 3 mg (W.R. Hood, M.B. Voltura, O.T. Oftedal, unpublished. data). The primary constraint is the necessity of homogeneity in the sample material be-ing tested; with homogenous milk samples, the within- sample CVs may be even lower than for micro- Kjeldahl or the Nessler’s method (W.R. Hood, M.B. Voltura, O.T. Oft-edal, unpublished data). Milk that is high in fat or improp-erly stored may lack adequate homogeneity; the resulting high within- sample CV may require an increase in the number of replicates per sample or the switch to a method that uses larger subsamples, such as micro- Kjeldahl. Due to the limitations of the CHN combustion system, it is not



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possible to increase the amount of milk assayed per repli-cate because incomplete combustion and erroneous data may result.

All of the above methods are used to assay total nitro-gen. Crude protein is calculated by a standard conversion factor (6.38), assuming that non- protein nitrogen is insig-nifi cant (see Box 25.3). Until actual NPN levels in the milks of bats and other small mammals have been mea sured, and the amino acid concentrations in milk proteins for these taxa have been examined, we cannot assess the er-rors involved in these assumptions. This would be a fruit-ful fi eld for further investigation.

LipidsA variety of methods have been used to determine the

fat content of milk, including the Roese- Gottlieb and Folch extraction methods, a semiquantitative “creamat-ocrit” approach, quantitative gas chromatography, and various colorimetric methods that mea sure glycerol or fatty acids (Oftedal and Iverson, 1995). The “creamatocrit” method involves drawing milk into hematocrit tubes sep-arating the lipid- containing cream layer by centrifuga-tion and mea sur ing the relative length of the cream layer. Although this can be completed with microliter volumes of milk, the packing characteristics of lipid droplets are species- specifi c, requiring development of individual regression lines between percent cream and percent fat for each species (Linzell and Fleet, 1969; Oft-edal and Iverson, 1995). Colorimetric methods, includ-ing sulfuric acid- phosphoric acid- vanillin reaction (Zöll-ner and Kirsch, 1962), must also be calibrated for the species under investigation because assay results depend on the fatty acid composition of the milk (Oftedal and

Iverson, 1995). Even within a species, the fatty acid com-position of milk may change across the lactation period (Iverson and Oftedal, 1995), confounding eff orts at accu-rate calibration. Finally, it is possible to quantify milk fat from fatty acid composition, so long as (1) fatty acid stan-dards of known amount are used, (2) the proportion of lipids as triacylglycerols is known, and (3) the contribution of glycerol to lipid mass can be calculated (Glass et al., 1968; Oftedal and Iverson, 1995). Fatty acid analysis by gas- liquid chromatography (GLC) requires only a few mi-croliters of milk, but the assays themselves require careful attention to sample handling, column selection, and assay conditions (Iverson and Oftedal, 1995), and any unknown or misidentifi ed peaks may produce error in the fi nal result.

The most direct method of determining lipid content in milk samples is solvent extraction. The par tic u lar lipid classes extracted depend on the solvents used; although in some applications only neutral lipids are of interest, we recommend use of multiple- solvent systems that extract both neutral and polar lipids, preferably avoiding the ex-traction of sugars or other constituents. Although the Folch method involving chloroform and methanol is suit-able for lipid class analysis, at NZP nutrition laboratory we use the standard Roese- Gottlieb procedure developed for milk, which relies on sequential extractions with diethyl and petroleum ethers (Horwitz, 1980; Box 25.4). For small samples, a microadaptation of the Roese- Gottlieb assay is used, as developed by R. Jenness at the University of Min-nesota. It does not require specialized equipment other than a small centrifuge and accurate microbalance (to nearest 0.01 mg) and will accurately recover as little as 3 mg of fat. Milk subsamples as small as 50 mg from big

box 25.3 Elemental analysis for the protein content of milk

1. All mea sure ments of mass should be made in milligrams, or calculations adjusted as appropriate. Before running irreplaceable samples, validate combustion temperature and oxygen boost with milk standards or samples analyzed using the micro- Kjeldahl method.

2. Weigh sample tin (T).3. Deliver milk to tin and weigh immediately (TM). Calculate wet mass of sample (M):

M = (TM − T).

4. Dry to constant mass at 100°C.5. Weigh tin with dry milk (TDM) for input into the instrument and calculate dry matter

concentration (percent dry matter in milk):

%DM = [(TDM − T)/M] × 100.

6. Load samples and then program and run the elemental analyzer as described by the manufacturer, adjusting the appropriate combustion settings for the milk.

7. Retrieve %N (dry matter basis) from analyzer.8. Percent protein = (%DM/100) × %N × 6.38.


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brown bats (Hood 2001) and 70 mg for small mice have been assayed (Peromyscus spp.; M.B. Voltura, unpubl. data). Since the solvents may extract lipids in plastics, it is im-portant to use glass centrifuge tubes and aluminum pans to prevent contamination.

The Roese- Gottlieb procedure begins with sequential addition of ammonium hydroxide and ethyl alcohol to the milk sample to disrupt the polar membrane that sur-rounds lipid droplets in milk and to facilitate clear separa-tion of layers during subsequent ether extractions. Ethyl ether and petroleum ether are then added to the sample to extract the lipids. The tubes are briefl y centrifuged to fa-cilitate separation of the liquid phases. A line of demarca-tion between the two layers should be visible, and the top layer containing the extracted lipids and associated sol-vents can be decanted using a positive displacement pi-pette (these pipettes have a piston within the tip that cre-ates negative pressure allowing volatile solvents to be aspirated and accurately delivered). The lipid- containing layer is carefully pipetted into a labeled and preweighed aluminum weighing container, and the solvents allowed to evaporate under an explosion- proof fume hood. The pro cess is repeated with the alcohol and both ethers, and a third and fi nal extraction is performed with the ethyl and petroleum ethers alone. After each centrifugation, the top layer is transferred into the aluminum weighing con-tainer. After all the solvent has evaporated, the pans are

dried at 100°C for 20 minutes, cooled in a desiccator, and weighed.

At this point, the pans typically contain lipids from the milk and a small amount of particulate precipitate, which may be visible upon inspection. Inclusion of this particu-late matter would result in overestimation of the fat; hence, the fat is fi rst solubilized by rinsing the pans carefully sev-eral times with warm petroleum ether. Great care must be taken not rinse the particulate matter out of the pan. The ether can be discarded or, if desired, collected into a clean container (e.g., a test tube) in order to capture the extracted fat for other analyses. Once the pans have been thoroughly rinsed and any residual petroleum ether is al-lowed to evaporate under the hood, the pans are dried again for 20 min in a 100°C oven and reweighed after cooling in a desiccator. The diff erence between the fi rst mass (pan plus fat and particulate) and the second mass (pan plus particulate) represents the amount of fat in the sample.

EnergyThe gross energy content of milk may be mea sured

directly using bomb calorimetry or may be calculated from the estimated energy content of the milk’s proxi-mate components (Oftedal, 1984a). Most published studies mea sur ing the energy content of small milk samples have utilized a Phillipson, Gentry Instruments, or Parr mi-

box 25.4 The modifi ed Roese- Gottlieb method for determining fat content

1. All mea sure ments of mass should be made in grams, or adjust calculations as appropri-ate. Ether is highly fl ammable; therefore, the evaporation of all ether samples should take place in an explosion- proof fume hood.

2. First, weigh a 2- ml centrifuge tube (CT). Then pipette a known mass of milk (0.100 g) into the bottom of the tube and weigh the tube after addition (CFM) to determine the exact sample mass (milk mass, M):

M = CFM − CT.

3. Add (in order) 20 μL NH4OH, 100 μL ethyl alcohol, 250 μL ethyl ether and 250 μL petroleum ether. Vortex carefully for 10 seconds after each addition.

4. Cover each tube securely with foil and then parafi lm. Centrifuge for 5 min at 1,000 G. 5. Carefully transfer top layer, which contains the extracted fat, into aluminum collection

pans and allow solvents to evaporate under hood. 6. Repeat steps 2– 4, but use 50 μL ethyl alcohol, 150 μL ethyl ether, and 150 μL petroleum

ether, and decant into same collection pans. 7. Repeat steps 2– 4, but use 150 μL ethyl ether and 150 μL petroleum ether, and decant into

same collection pans. 8. Once no ether smell is detectable from pans, dry them for 20 min at 100°C in an oven,

cool in desiccator, and weigh (P1). 9. Using a disposable pipette, rinse each pan three times with warm petroleum ether, being

careful not to disturb any particulate matter. These rinses can be discarded.10. Evaporate residual ether in the rinsed pan under the fume hood and dry pans for 20 min at

100°C. Cool in desiccator and weigh again (P2).11. Percent fat = [(P1– P2)/M] × 100.



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crobomb calorimeter. This instrument mea sures the rise in the temperature of the chamber following ignition of a 1– 20 mg sample in the presence of pure oxygen; the out-put is recorded by a potentiometer (Phillipson, 1964; Gen-try Instruments, Inc). Sample energy content is calculated from a benzoic acid standard curve relating signal re-sponse to energy content for standards (at least 10) be-tween 1 and 20 mg. The signal response for both sample and standard must be corrected for heat fl ux to the envi-ronment before, during, and after combustion. Energy estimates are most accurate if they are also corrected for the nitric acid that is produced during the combustion as well as the heat input from the fuse wire. Correction for sulfuric acid production during combustion is usually not required since the sulfur content of milk is typically low.

Unfortunately, microbomb calorimeters are no longer manufactured. Parr Instruments makes a semi- microbomb calorimeter that will mea sure the energy contents of sam-ples as small as 25 mg dry mass. Since this instrument will require at least 100 mg of milk on a wet mass basis, inves-tigators may prefer to estimate energy based on the en-ergy equivalents of the carbohydrate (3.95 kcal or 16.5 kJ), fat (9.11 kcal, 38.1 kJ), and protein fractions (5.89 kcal, 24.5 kJ) of the milk (Oftedal, 1984a). However, it is impor-tant to realize that there will be some error associated with these calculations because the estimated energy con-tents of milk carbohydrate, fat, and protein are generated from the milks of domestic animals. This error is not likely to be substantial (Perrin, 1958).

Minerals and Other ConstituentsThe minimal volume of milk required to complete min-

eral analyses is limited by the detection limits of the assay, the mineral content of the digested sample, and the num-ber of elements to be analyzed. We have run fi ve macrom-inerals on samples as small as 10 mg wet mass. For most elemental mineral assays, samples are fi rst acid- digested in strong acid, such as sulfuric acid combined with per-chloric acid (Studier et al., 1995) or nitric plus perchloric acid. Perchloric acid has the disadvantage of being explo-sive in its crystal form, and hence requires the use of spe-cial perchloric acid hoods with built- in washdown fea-tures. Perchloric acid digestion of high- fat milks is also diffi cult, as the lipid material is combustible. An alterna-tive approach is to digest samples with nitric acid in a high- pressure microwave- accelerated digestion system. Micro-wave energy is provided to digestion vessels to maintain a preprogrammed temperature or pressure profi le, as as-sessed via sensors in a vessel. The elevated pressure al-lows an increase in the boiling temperature of the acid, increasing reaction rates. In the NZP Nutrition Labora-tory, milks from bats and other mammals are thoroughly digested in 10 mL nitric acid in a micro wave system with the temperature ramped to 220°C over 15 minutes and held at 220°C for an additional 15 minutes. The nitric acid

digest (milk plus acid) is then diluted with deionized dis-tilled water to achieve the desired volume and concentra-tion necessary to quantify concentration.

Mineral concentrations of solutions are often deter-mined by atomic absorption spectrophotometry (AAS; Haswell, 1991). Flame AAS detects the concentration of a single mineral per run by introducing sample digests into a nitrous oxide- acetylene or air- acetylene fl ame. A beam of light (including spectral lines that are absorbed by the element of interest) is passed through the fl ame, and the absorbance at a specifi ed wavelength is mea sured and compared to that of standard solutions. Interferences by elements other than the target can usually be minimized by the use of modifi ers added to the sample and standard solutions. Nonetheless, it is important to monitor instru-ment per for mance using quality control solutions of known concentration, and to assay reference materials (such as NIST milk powders). Most fl ame AASs mea sure elements in parts- per- million concentrations (μg of mineral per g of sample) with 1– 3% accuracy (McGuire, 2004), which should be suffi cient for most macrominerals in milk (e.g., calcium, potassium, and magnesium, although sodium may need to be assayed by atomic emission and phospho-rus by chemical methods, see below). Trace elements (e.g., iron, zinc, manganese, and copper) may require a more sensitive assay such as graphite furnace AAS, that can de-tect parts- per- billion concentrations (0.001 μg/g) of min-eral in the sample. The graphite furnace method diff ers from fl ame AAS in that the digested sample is volatilized by shock heating to a very high temperature (as high as 2700°C) in a special graphite tube rather than being intro-duced into a fl ame.

To avoid contamination, all tubes or glassware that comes in contact with samples must be acid- washed. Many common fi eld and laboratory materials (including rubber, many types of glass, and some plastics) will bleed contami-nants into samples. For example, borosilicate glass test tubes that have been repeatedly acid washed will continue to leach sodium resulting in substantial infl ation of the sodium concentration of the sample (W. R. Hood, pers. obs.). Fastidious eff orts are required to avoid contamina-tion when working with any trace element. In fl ame and graphite furnace AAS, each element is assayed separately using the same acid digests. An alternative instrumental approach, inductively coupled plasma (ICP) spectropho-tometry, allows investigators to mea sure multiple ele-ments simultaneously, which saves both sample and time. However, the cost of an ICP and its maintenance can be prohibitive and thus ICP mea sure ments of milk mineral concentrations are generally conducted by commercial laboratories. As with AAS, validation of results with refer-ence materials is critical. More detail on these methods can be found in Hill (2000).

Phosphorus concentration is mea sured using a colori-metric procedure rather than AAS. A reagent is prepared


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from ammonium metavanadate, ammonium molybdate, and perchloric acid. This reagent is added to sample di-gests and the resulting solution varies in color according to the phosphorus concentration, in a very similar man-ner to the colorimetric reactions of the Nessler’s and phenol- sulfuric methods. The AOAC- modifi ed Gomorri method is described by the AOAC (Horwitz, 1980).

In earlier publications, ash content was often used as an index of total mineral content, but these values are dif-fi cult to interpret. Most minerals in the ash are in the form of oxides rather than pure elemental forms. Thus, the mass of ash exceeds the total mineral mass.

Other nutrients that may be of interest include protein- bound and free amino acids, lipid classes, fatty acids, and water- and fat- soluble vitamins. A variety of methods are available for these constituents but are beyond the scope of this chapter. Investigators are referred to standard ref-erence works, including the Handbook on Milk Composition ( Jensen, 1995), the Handbook of Vitamins (Machlin, 1991) and books on food analysis (e.g., AOAC publications). In-vestigations should also be aware that short and long- term changes in diet may infl uence the concentrations of both fatty acids and vitamins in milk, complicating interpreta-tion of results.

Units for Pre sen ta tionIn most cases, milk constituents are presented on a

fresh (also termed wet mass) basis, as this is how milk is secreted (Jenness, 1974; Oftedal, 1984a; Jensen, 1995). We discourage presenting results per unit volume, as the spe-cifi c gravity of milk varies with composition, both within and between species. However, it may be instructive, in comparing among species or stages of lactation, to exam-ine soluble constituents (e.g., lactose, electrolytes) in rela-tion to water so as to understand the physiology of milk secretion or to compare major constituents (e.g., fat, pro-tein, carbohydrate) or major minerals in relation to milk dry mass to remove the diluting eff ect of water. It can also be instructive to examine the estimated energy in protein or other constituents in relation to the total gross energy content of milk, as this partially corrects for diff erences in intake among suckling young associated with the energy densities of milk (Oftedal, 1984a). However, in all cases it is essential to provide enough information to allow read-ers to convert units from fresh to dry mass bases.

MILK YIELD

Methods for mea sur ing the volume of milk produced include weight- diff erential, timed milking, isotope dilu-tion, and isotope transfer (Oftedal, 1984a). Weight diff er-ential methods determine milk yield as the change in off -spring mass during a suckling bout, but it may be inaccurate if either suckling behavior or the intersuckling interval is disturbed, or if the young produce excreta be-

tween weighings. The timed milking method is based on an estimate of volume of milk produced per unit time. To complete this mea sure ment, the mother is separated from her off spring, the mammary gland is manually emptied, and then milk is allowed to accumulate within the gland. After an appropriate interval, the gland is emptied again. The volume of milk collected at the second milking is as-sumed to be equivalent to the volume of milk that would be produced for the off spring during that time interval. Daily milk yield is determined by multiplying the volume collected by 24 hours and dividing by the time between milking events (in hours). The interval between milkings and calculation of daily yield should be modifi ed based on knowledge of the animals suckling pattern. For example, if it is known that mothers suckle their young every 2 hours, then 2 hours would be an appropriate interval between milkings. If the investigator knew that mothers never nurse their young between 2100 and 0600 h, 9 hours could be subtracted from 24 in the yield estimate. However, this method requires that the mammary glands are emptied to a substantial and equivalent degree at each milking. More importantly, this method assumes that the investi-gator expresses a volume of milk that is equivalent to that which would be consumed by the off spring. The stress of frequent capture and handling for milking or weighing is likely to dramatically alter suckling behavior, milk trans-fer to the young, and milk expression during milk collec-tion (Oftedal, 1984a). Thus, although weight diff erential and timed milking methods can yield comparable results to isotope methods in large, trained domestic animals, this is highly unlikely in bats or other small mammals. One or more isotope methods appear to be the only suitable meth-ods for mea sur ing milk yield in small mammals.

Overview of Isotope DilutionIsotopic methods are particularly well suited to fi eld

studies of animals exhibiting strong site fi delity, such as many bats (Oftedal, 1984a; Oftedal and Iverson, 1987). The most common approach is to use the stable or radioactive isotopes of hydrogen (deuterium and tritium) as markers of body water kinetics; other isotopes, such as those of so-dium, potassium, cesium and iodine, have been used oc-casionally but will not be discussed herein (see Oftedal, 1984a). With the hydrogen isotope dilution method, a pup’s body water is labeled with a known amount of isotope and then an initial sample of body water is taken after a period of equilibration. These samples allow the total vol-ume of body water to be mea sured. The pups are then re-turned to their mothers. Subsequently any water consumed by the pup will act to dilute the isotopic label. One or more subsequent blood samples are collected from isotope- labeled pups after one or more days and concentrations of isotopes in these samples are compared to the equilibra-tion sample to determine isotope turnover, and hence wa-ter fl ux. If milk is the only source of water consumed and



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the free and metabolic water content of the milk is known, total milk consumption can be calculated based on the extent the isotope is diluted. Finally, milk intake of the pup is determined by dividing the total water consumed by the total water content of the milk at the stage of lacta-tion at which milk intake was mea sured. Milk intake is converted to estimated milk output by the mother by multiplying pup intake by the total litter size. As most bats produce a single young per reproductive event (Kunz and Hood, 2000), volume of milk intake by pups and milk output by mothers are generally equivalent.

Isotope Dilution MethodsMuch of the methodology used to mea sure milk yield

by isotope dilution parallels methods to mea sure energy expenditure with doubly labeled water (DLW); in fact, milk yield and pup energy expenditure can be mea sured simultaneously with a single injection of DLW solution. For discussion of the DLW method, see Voigt and Cruz- Neto, this volume.

At present, most of the isotopes available to mea sure milk intake, except deuterium, are radioactive. Due to the problems of environmental contamination and restric-tions by state and/or federal regulations in many coun-tries, radioisotopes are rarely used in fi eld studies. This is particularly an issue for radioisotopes that emit high- energy gamma radiation, such as sodium and potassium radioisotopes (Chapman, 1981). However, tritium is a weak beta emitter and thus can be used safely with little extra precaution other than wearing latex gloves (Chapman, 1981). There is an economic benefi t of using tritiated wa-ter for mea sure ments of milk yield: tritium is inexpensive to buy and to assay if one has access to a liquid scintilla-tion counter. Deuterated water (“heavy water”, D2O) is also relatively inexpensive to purchase, but small volume samples, as is typically collected from small animals, must be assayed by isotope ratio mass spectrometry. The initial cost and maintenance necessary to run an isotope ratio mass spectrometer is prohibitive for most laboratories, and thus most investigators must send samples to a com-mercial or university- based stable isotope laboratory for analysis. Analyses by external laboratories are generally expensive and the researcher(s) may have to wait several months or longer for the analyses to be completed. Where sample volume is not limiting, deuterium can be assayed by infrared spectrophotometry, which is inexpensive and rapid (Oftedal and Iverson, 1987). In bats, tritium has been used to mea sure milk yield in Eptesicus fuscus, Myotis lu-cifugus, Nycticeius humeralis, and deuterium in Phyllostomus hastatus and a separate study on Eptesicus fuscus (Kunz, 1987; Steele, 1991; Stern et al., 1997 ; Hood, 2001); McLean and Speakman (2000) used both tritium and deuterium to mea sure isotope dilution and isotope transfer in Plecotus auritus as described below under Error Associated with Isotope Dilution.

Isotope dosage should be adjusted for body mass of the animal and the sensitivity of the assay to be used. Dosage rates for milk intake mea sure ments may follow those given for DLW, as described in Voigt and Cruz- Neto, this volume, and Speakman, 1997. In most studies on small mammals, isotope is introduced into the body by intrap-eritoneal (IP) injection, since other routes of administra-tion (such as intravenous or intragastric injection) are dif-fi cult. With IP injection, care should be taken to prevent leakage from the injection site (Voigt and Cruz- Neto, this volume). Loss of isotope during administration results in an overestimation of total body water and hence an over-estimation of milk production.

Before the isotope is introduced, an initial body water sample should be taken to determine preinjection or base-line isotope levels. To avoid excessive disruption of study animals, baseline samples can also be collected from other animals in the colony, so long as they have not been ex-posed to sources of isotope enrichment (such as by prox-imity to isotope- labeled animals). After injection, the iso-topic label must be allowed to come to equilibrium within the body before sampling (Voigt and Cruz- Neto, this vol-ume, describes the method for determining time to reach equilibrium). It is generally reasonable to utilize equili-bration times determined for closely related species of comparable body size. Tritium and deuterium reach equi-librium after one hour in microbats that have been stud-ied (Kunz and Nagy, 1988; Voigt and Cruz- Neto, this vol-ume) and after 1.5 hours for larger megabats (Korine et al., 2004). Body water samples may be collected from blood, saliva, or urine. However, saliva samples are unlikely to provide suffi cient sample for analysis in small mammals.

Urine collection is simple in captive studies in which it can be collected immediately after urination, so long as there is no contamination from the surface on which it lands. This reduces the stress of handling, but animals may not cooperate in producing urine when it is needed. There is also concern that urine may not be completely equilibrated with other body fl uids, primarily because urine is not excreted immediately upon production, but rather is stored in the bladder. If, for example, the animal’s bladder is full of nonlabeled urine at the time of isotope administration, the fi rst sample of urine collected may re-fl ect additional dilution by this fl uid over and above that due to other body fl uids, leading to overestimation of body water pool. If urine is to be collected, we recom-mend initial validation against blood samples to demon-strate that urine provides equivalent results. In general, we recommend sampling blood rather than other fl uids. Methods of blood collection and sample storage are de-scribed in Voigt and Cruz- Neto, this volume, and Reeder and Widmaier, this volume.

If possible, isotope injection, equilibration, and initial blood sampling should take place at a time when the pup is unlikely to be suckling, so as to minimize interruption of


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the normal suckling pattern of the animal. In free- ranging bats, this is possible when mothers leave their pups at the roost while foraging. When working with free- ranging animals isotopically labeled bats can be marked with a temporary plastic band covered with refl ective tape that can easily be seen in the beam of a fl ashlight, simplifying subsequent recapture. After the post- equilibration sam-ples are taken, the pups are returned to their cage or roost so they can freely suckle and interact with their mother.

Milk yield calculations assume that isotope concentra-tions decline in a log- linear fashion, and this can be dem-onstrated if sequential blood samples are obtained, such as repeated sampling at 24- h intervals (Oftedal and Iver-son, 1987; Scantlebury et al., 2000). However, repeated captures may be stressful, especially to free- ranging ani-mals, and this in itself could reduce milk yield. Thus, most studies in bats have used a 2- point model, in which an ini-tial equilibrated sample is compared to a single subse-quent sample, making the assumption that isotopic de-cline has followed expected kinetics. The degree of error that may be introduced by this assumption warrants fur-ther investigation. In the 2- point method, the second bleed-ing can be timed such that the mea sure ment encompasses water turnover during a full 24- h day of suckling or mul-tiples thereof (mea sure ments are typically expressed in mL/day). Since pups will not normally suckle after moth-ers leave the roost, labeled pups can be recaptured after the mothers depart.

Water intake calculations are based on the washout rate of isotope due to water fl ux, but in growing young, an in-crease in pool size will also contribute to the change in isotope concentration between sequential blood samples. This is quite diff erent from DLW studies in adults, where constant pool size is usually assumed (Speakman, 1997; Voigt and Cruz- Neto, this volume). One approach to ad-justing for this change is to reinject the pup at the second capture, so that a second pool size estimate can be ob-tained. However, this requires holding the pup for a sec-ond equilibration period. So long as the interval between fi rst and second bleeds is short, it is reasonable to assume that the ratio of water pool:body mass will be relatively constant, in which case the pool size at the second capture can be estimated from body mass (Box 25.5). It may also be possible to assess the relationship between pool size and body mass by regression of pool size on body mass for several individuals, and to use this regression to predict pool size at the second capture (e.g., Oftedal et al., 1996). Other methods of calculating the eff ect of pool size have been described by Nagy and Costa, 1980, and Speakman, 1997. Consult Nagy, 1983, and Kunz and Nagy, 1988, for methods of tritium analysis, and Voigt and Cruz- Neto, this volume, for recommendations on blood collection and on preparing deuterium samples for external analysis.

Milk intake by pups is equivalent to water intake/wa-ter content of milk, where the water yield is the amount of

free plus metabolic water derived from each gram of milk (Box 25.5). The amount of metabolic water produced is infl uenced by the composition of the milk, since diff erent constituents yield diff erent amounts of water when me-tabolized. However, some fraction of the milk protein and fat are not metabolized, but rather are deposited in new tissue during growth. Thus, metabolic water cannot sim-ply be calculated from milk composition when mass is gained.

One approach is to account for the amounts of protein and fat deposited during growth, and then to calculate by diff erence the proportion of milk protein and fat that has been metabolized. An equation to this eff ect is provided in Box 25.5, based on a derivation of equations published by Oftedal et al. (1987); this requires estimates or assump-tions about the composition of body mass gain in pups. Alternatively, metabolic water can be calculated from doubly labeled water (DLW) estimates of carbon dioxide production (see Voigt and Cruz- Neto, this volume). In Ple-cotus auritus metabolic water production increased water fl ux by 8.2% (McLean and Speakman, 2000). Additional metabolic water will be produced if pups are forced to catabolize body fat due to inadequate energy intake. Since cold temperatures or rain could reduce the total volume of milk that a mother provides to the pup, forcing the pup to utilize stored fat, data obtained during inclement weather should be considered suspect.

Finally, milk yield can be estimated from litter size if one assumes that a similar amount of milk is consumed by each pup (Box 25.5). This assumption may introduce error if littermates diff er greatly in size and growth or in situations where males consume more milk than females (as mea sured in California sea lions by Oftedal et al., 1987). A comparison of variation in growth rates between male and female pups is a good check on this assumption; if one gender grows at a rate greater than the other, intake of male and female pups should be mea sured in de pen dently.

Error Associated with Isotope DilutionAs with all isotopic tracer studies, there is concern that

the label may disperse to other compounds than the one to be traced, and that the isotopically labeled compound may behave diff erently than one that has not been labeled. A known problem of hydrogen isotope studies is that the deuterium or tritium label may exchange with hydrogen in organic molecules, particularly hydrogen, which is weakly bound to electrophilic elements such as oxygen and nitrogen. This may be particularly common during growth when isotopes can be incorporated into nonex-changeable sites in new tissues and thus disappear from body fl uids (although still in the animal; Ussing, 1938). This is a primary cause for the typical 2– 5% overestima-tion of the body water pool by hydrogen isotope dilution (Oftedal and Iverson, 1987; Speakman et al., 2001). This source of error has not been adequately evaluated for small



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box 25.5 Milk yield calculation based on a two- point method

1. Calculate initial water pool size (P1, in grams) at the initial dosing as:

P1 = [(Id − Ib) × Q1/(I1 − Ib)]

whereId is the isotopic enrichment of the dose solution (ppm),Ib is the background level of isotope enrichment (ppm) determined from a predose bleed,I1 is isotope enrichment after equilibration (ppm),andQ1 is the mass of initial dose solution (g).

2. Calculate fi nal water pool size (P2, in grams) at the fi nal dosing as:

P2 = [(Id − Ib) × Q2/(I3 − I2)]

whereI2 is the isotope enrichment (ppm) at the second bleed, before dosing,I3 is isotope enrichment (ppm) at the third bleed, after second equilibration, andQ2 is the mass of second dose solution (g).

Note: If isotope injection is only done at the beginning of the study, fi nal water pool size (P2) must be calculated from fi nal pup mass on the assumption that the ratio of pool size to body mass is constant, P2 = (P1/BM1) × BM2, where BM1 and BM2 are the initial and fi nal body mass of the pup (g), respectively.

3. Water loss of the pup (L, g/d) due to water fl ux is:

L = | k × (P1 + P2)/2 |

where the fractional turnover rate of total isotope burden(k, fraction/d) is calculated as

k = {ln [ P2 × (I2 − Ib)] − ln [P1 × (I1 − Ib)]}/t

and t is the elapsed time between initial and second bleeds.Note: If isotope recycling is confi rmed by mea sure ment of enriched isotope concentra-

tions in a littermate, I2 can be corrected for recycling by subtraction of the isotope concentration (I2L) mea sured in the littermate at the same time (see text). The equation would then be: k = {ln [P2 × (I2 − I2L)] − ln [P1 × (I1– Ib)]}/t

4. Water gain of the pup (G, g/d) due to growth is

G = P2 − P1

5. Total water intake (TWI, g/d) of the pup is:

TWI = L + G

TWI includes not only water from milk but also water from metabolism of milk constitu-ents and water obtained from non- milk sources (if any).

6. Milk intake (MI, g/d) can be calculated from TWI if metabolic water is accounted for and there is no other source of water intake. There are two alternative approaches to estimating metabolic water production.

A. If metabolic water production (MeWP, g/d) is estimated from a concurrent doubly labeled water study (see Voigt and Cruz- Neto, this volume), this can be subtracted from TWI and then milk intake can be calculated as:

MI = 100 × (TWI − MeWP)/%Wm

where %Wm is the percentage of water in milk.

B. An alternative approach (see Oftedal et al., 1987 for derivation) accounts for metabolic water from the catabolism of milk nutrients, based on mea sured or assumed rates of fat deposition (FD) and protein deposition (PD):

MI = 100 × (TWI + 1.07 × FD + 0.42 × PD)/(%WM + 1.07 × %FM + 0.42 × %PM + 0.58 × %SM)

(continued)


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where FD = [(BM2- BM1)/100] × %fat in gain and PD = [(BM2- BM1)/100] × %protein in gain (thus, deposition is zero when there is no change in mass or negative if mass is lost) and %FM, %PM, and %SM refer to the percentages of fat, protein and sugar in milk, respectively. If changes in body composition are not known, they can be estimated from pup growth rate (BM2 − BM1)/t) and assumed proportions of fat and protein in gain. For example, in Eptesicus fuscus, mass gain of suckling pups contained 17% protein and 3% fat (Hood et al., in prep- c). Application of these values to growth in other bat species will not produce much error in milk intake estimates since in most mammals FD and PD are small compared to TWI.

7. Milk yield (MY, g/d) of the mother is equal to milk intake of the pup if she has only one pup. For mothers with litters,

MY = MI × LS

where LS is number of young in the litter.

Calculations adapted from Oftedal, 1984b,and Oftedal and Iverson, 1987. For additional comments, seeOftedal et al., 1987, 1993, 1996.

rapidly growing young, but likely remains small when the study period is relatively brief (one or a few days).

Another problem is potential fractionation of isotopes during evaporative water loss, since the greater mass of deuterium and tritium than hydrogen requires a bit more energy for evaporation, and thus label is lost somewhat less rapidly than unlabeled water (Fjeld et al., 1988). This will cause underestimation of water fl ux. It is common to correct for suspected fractionation in DLW calculations (Speakman, 1997; Voigt and Cruz- Neto, this volume) be-cause fractionation aff ects isotopes of hydrogen and oxy-gen diff erently, and because the diff erence between the fl uxes of these two isotopes is used to estimate CO2 pro-duction and energy expenditure in the DLW procedure. However, evaporative water loss accounts for less of total water fl ux in rapidly growing suckling young than in adults, and hence the bias produced by fractionation is re-duced (Oftedal and Iverson, 1987). It appears that the vari-ous biases associated with isotope behavior largely cancel out in milk intake studies, based on the few direct valida-tions that have been done (Oftedal, 1984a; Oftedal and Iver-son, 1987; Arnould, et al., 1996). While these do not nor-mally introduce large error into milk intake mea sure ments, investigators should be aware that minor errors can arise.

Consideration should also be given to error associated with the two- point model. In the event that there has be an insuffi cient decrease in the isotope concentration (as when the interval between mea sure ments is short) or if the fi nal isotope concentration is too close to background (if the interval between mea sure ments is too long), sub-stantial error in mea sure ment of water turnover can oc-cur (Arnould et al., 1996; Oftedal et al., 1987; Gales, 1989). Small mammals are expected to consume larger volumes of milk relative to their size than large mammals, and spe-

cies ingesting dilute milks will have greater water turn-over; thus, the rate of decline in isotope concentration, and the optimal period between blood collections, will vary among species. If no adequate model species is avail-able (e.g., a related species of similar size, milk composi-tion, and pup growth rate), it may be necessary to validate the rate of isotope decline in a pi lot study before deter-mining study design. It would be reasonable to target the second bleed at a time when more than 40% but less than 90% of the isotope has been lost. Furthermore, a short in-terval between isotope mea sure ments (i.e., 24 h) may un-derestimate milk intake in the event that stress associated with handling reduces milk intake. This can be evident by mass loss in growing pups during the mea sure ment inter-val (Hood et al., in prep.a). Thus, increasing the duration between blood samples will allow the fi rst day to be aver-aged with days without disturbance, thus decreasing the error associated with handling.

There are two additional sources of error that can arise specifi cally when studying rapidly growing, suckling young. These are (1) intake of water from sources other than milk and (2) recycling of isotopes from young to mother to young via maternal ingestion of pup urine or feces (Oftedal, 1984a; Oftedal and Iverson, 1987; Kunz and Nagy, 1988; Scantlebury et al., 2000).

Intake of water from sources other than milk can lead to major error in any milk intake study employing isotope dilution. Most mammals, including bats, experience a pe-riod of transition where suckling pups begin taking in solid food. Water associated with solid food will increase isotope dilution and thus erroneously infl ate mea sure-ments of milk intake. We typically abort mea sure ments of intake when it is apparent that pups are foraging. Food intake by pups can be monitored by observation of indi-


Box 25.5(continued)


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vidual pups in captivity or monitoring fl ight away from the roost as pups begin foraging. Monitoring fecal color-ation can also be a valuable check on intake of food other than milk. The feces produced by suckling pups are dis-tinct. In insectivorous bats, the feces of pups feeding ex-clusively on milk are green and relatively transparent. As pups begin to feed, insect remains will be evident in the feces. A modifi cation of the isotope dilution method has been used to account for non- milk water intake. Usually this involves mea sure ment of water fl ux in the pups, as described above, and the use of a second isotope to mea-sure water transferred from mother to pup via the milk (although Oftedal (1981) described a method of using a single isotope to mea sure both pro cesses in species with litters, such as guinea pigs). The double- isotope approach was used by McLean and Speakman (2000) in Plecotus auri-tus. The water intake in the pup using one isotope (A) is compared to the acquisition of a second isotope (B) via the milk, associated with the maternal label. If A is greater than B by an amount more than can be accounted for by metabolic water production in the pup, the additional wa-ter must come from sources other than milk.

Isotope recycling may be a substantial source of error in milk yield mea sure ments for many mammals in which mothers routinely ingest urine and feces of suckling young, since these excreta contain isotope if the young have been injected with it. If a mother ingests isotope from a labeled pup, the isotope becomes incorporated into maternal body water and thus may be returned to the pup via milk. This scenario leads to an underestimation of milk intake (estimated at 14% in bear cubs; Oftedal et al., 1993). Addi-tionally, water from urine might be absorbed through the thin skin of siblings, especially before young become furred. In puppies, Scantlebury et al. (2000) found that most recycling appeared to be due to young- mother- young vs. young- young transfer, but this has not been mea sured in bats. Recycling can be documented by the appearance of isotope in the blood of the mother and/or a litter mate, since these animals should not have enriched isotope lev-els in the absence of recycling. In litter- bearing mammals, it is possible to correct for isotope recycling by monitoring isotope accumulation in an unlabeled pup, assuming the unlabeled pup consumes an equivalent volume of milk and thus receives the same amount of recycled isotope (Of-tedal, 1984b; Box 25.5). Thus, in litter- bearing mammals, blood samples should be collected from unlabeled litter-mates at the same time as blood taken from labeled pups. Unfortunately, this is not possible in bats that rear single young, and the best that can be done is to obtain blood samples from the mother to determine if isotope recycling is potentially biasing milk intake estimates.

Units for Pre sen ta tionData on milk yield in bats is generally presented as vol-

ume or amount transferred per day, and these may be

converted to energy fl uxes if yield is multiplied by the en-ergy content (kJ) of the milk (Kunz and Hood, 2000). As described for milk composition, day of lactation is fre-quently estimated from pup growth curves, which may introduce potential error. The validity of age estimates should be considered when data are presented; it may be more appropriate to consider age classes or to present data relative to pup mass dividing when age estimates are likely to have a high degree of error. Eff ort should be made to determine milk yield at peak lactation to facilitate com-parison to other studies.

NUTRIENT TRANSFER

Mea sures of total nutrient transfer during lactation are critical to an understanding of the unique pattern of lacta-tion in bats, in which females give birth to one or a few large young that are weaned at a relative body size nearly twice that of other mammals. Yet, only two studies in bats and few studies in other small mammals have simultane-ously mea sured milk composition and milk yield (Stern et al., 1997; Hood 2001). Over 20 years ago, Oftedal (1984a) developed an equation that has been used to predict milk energy output relative to litter mass in mammals produc-ing litters. The smallest animal used in developing this equation was the approximately 500- g rat. With so few studies mea sur ing milk composition and yield simultane-ously, it is diffi cult to assess the suitability of this equation for bats and other small mammals. In fact, the estimated energy output for all fi ve bat species for which milk yield has been mea sured suggests that bats produce more milk than would be expected relative to their body mass and litter size (Kunz and Hood, 2000).

When both milk composition and milk yield are mea-sured, total energy transferred per day, or energy trans-ferred relative to pup mass, or stage of growth, should be presented. Eff ort should be made to mea sure energy transfer at peak lactation to facilitate comparisons to other species.

SUMMARY

It is our hope that the recommendations and method-ologies described in this chapter will encourage other in-vestigators to examine milk composition and production in bat and other small mammals. A brief summary of ma-jor considerations is provided below:

1. Limit the eff ects of those intrinsic and extrinsic variables that may impact milk composition and milk production but are not of interest in your study.

2. Use oxytocin to aid in complete evacuation of the mammary gland(s) during milking.

3. Small volumes of milk are especially prone to contamination and desiccation. Take special care to


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clean the gland before milk collection; be sure that samples are stored in vials that have a capacity closely matching the volume of sample collected; and store samples at a stable temperature (less than −20°C).

4. Plan nutrients to be assayed and methods of analysis before samples are collected. Take into consideration the volume of milk that will be collected and when-ever possible, plan to run more than one aliquot of each sample for each assay performed. Retain some residual sample that can be run in the event of error or equip-ment failure.

5. Regularly calibrate equipment and run standards every time samples are analyzed to confi rm accuracy.

6. Just as the collection and analysis of small volume samples required special care, similar precaution must be taken when mea sur ing milk production using isotopic techniques in small species. Take care to not disturb typical suckling patterns of young and avoid isotope leakage from site of injection.7. When mea sur ing milk yield, account for recycling

of isotopes associated with maternal ingestion of urine and feces whenever possible. Account for or avoid mea sure ments of milk yield when pups take in water from sources other than milk.

AC KNOW LEDG MENTS

We would like to thank all individuals that have contrib-uted to the testing, validation and improvement of meth-ods of milk and isotope analysis at the Nutrition Laboratory of the National Zoological Park, including M. Jakubasz, M. Power, M. Kretzmann, S. Iverson, R. Morgan, D. Hellinga, and J. Keene. We are especially indebted to the late dairy chemist R. Jenness of the University of Minnesota for pio-neering eff orts to develop assays suitable for small milk samples, for making unpublished descriptions of lab proce-dures freely available, and for permitting one of us (OTO) to investigate these methods in his laboratory.

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