Sweating in the guanaco (Lama guanicoe)

Journal of Thermal Biology 26 (2001) 77–83

Sweating in the guanaco (Lama guanicoe) Daniel A. de Lamoa,*, Daniel Lacollab, James E. Heathc a

Universidad Nacional de la Patagonia San Juan Bosco, Sede Puerto Madryn and Centro Nacional Patagonico (CENPAT-CONICET), Almirante Brown 3700, (9120) Puerto Madryn, Chubut, Argentina b Facultad de Ciencias Veterinarias, Universidad Nacional de la Pampa, (6360) General Pico, La Pampa, Argentina c Route 1, Box 217, Buchanan Dam, TX 78609, USA Received 4 February 2000; accepted 29 April 2000

Abstract Sweat glands are present all over the skin, where sweat production varies from 4.98 to 73.36 g m ÿ 2 h ÿ 1 of skin. Ambient temperatures between 20 and 338C are the main stimuli for activation of sweat glands, generating a heat loss ranging from 11.9 to 37% of standing basal metabolic rate. Respiratory water loss is not an important mechanism for heat dissipation. Water loss is controlled by postural changes in the guanaco. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Guanaco; Sweat glands; Thermal stress; Thermoregulation; Patagonia; Argentina

1. Introduction The guanaco (Lama guanicoe) is the widest ranging wild ungulate from South America. Its habitats are diverse, ranging from arid to semi-arid and from sea level to altitudes of about 4500 m. At present, most of the wild populations are in Argentina and the animals are particularly abundant in fragile ecosystems of low productivity. It is well adapted to dry environments such as those found in the Argentina Patagonia. Thus, it must control its water economy closely. Animals use several means of evaporation for cooling: behavior such as wallowing; autonomic and behavioral mechanisms such as salivating and saliva spreading; and the autonomic process of panting or sweating (Bligh, 1972). Wallowing is a primitive evaporative process since it does not involve the evolution of specialized evaporative organs. The same is true for saliva spreading which is used by some marsupials and bats (Robinson and Morrison, 1957). Panting, which also occurs in *Corresponding author. Fax: +54-2965-451543. E-mail addresses: [email protected] (D.A. de Lamo), [email protected] (D. Lacolla), [email protected] (J.E. Heath).

reptiles (Heath, 1965) and birds (Bartholomew et al., 1968), seems to be the most common means of evaporative heat loss in mammals. According to Hammel (1968), panting preceded the evolution of sweating as an evaporative mechanism. Panting has the advantage over sweating in that it is not influenced by the presence of pelage that may insulate the animal against heat transfer and evaporative heat loss. Sweating also generates the loss of some electrolytes which are secreted in the sweat fluids. On the other hand, panting may conflict in the use of the respiratory system for maximum gaseous exchange, thus restricting evaporative heat loss during sustained exercise in both predator and prey. The presence of sweat glands suggests a thermoregulatory function. The histological structure and spatial distribution in the skin underlie the pattern of sweat production. However, There is no evidence of thermoregulatory function of the sweat glands of the pig (Sus scrofa) (Ingram, 1967), sheep (Ovis aries) and goat (Capra hircus) (Allen and Bligh, 1969). The horse (Equus caballus), donkey (Equus asinus), ox (Bos taurus), camel (Camelus dromedarius) and llama (Lama glama), respond to heat stress and infused adrenaline with a continuous output of sweat. In the alpaca (Lama pacos),

0306-4565/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 6 - 4 5 6 5 ( 0 0 ) 0 0 0 1 4 - 0

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the function of epitrichial sweat glands showed no evidence of apocrine secretion (Montalvo and Cevallos, 1973). Guanacos possess two regions, axillary and inguinal, of very short, sparse pelage (Morrison, 1966) and under heat stress they produce copious fluid in these regions. The guanaco is a close relative or ancestor of the domestic llama (Wheeler, 1995). Do the axillar and inguinal bare patches in the guanaco behave as those in llamas and other camels producing sweat in response to thermal stress? Guanacos are not known to pant except during severe dehydration (Rosenmann and Morrison, 1963). We have never seen guanacos panting in nature even at ambient temperatures as high as 408C. The objectives of this paper are to give a general description of the structure of sweat glands in guanacos and to relate sweat gland activity to thermal stress.

2. Materials and methods 2.1. Sweat glands To determine the presence, distribution and density of sweat glands, skin samples were taken from newly dead animals in the field. The animals and areas of sampling are presented in Fig. 1 and Table 1. The skin samples (100 mm2) were fixed in buffered formaldehyde and refixed 24 h later. The samples were

processed by the paraffin inclusion technique and stained with hematoxilin-eosine, Per-Iodic Acid Schiff (P.A.S.) (Martoja and Martoja-Pierson, 1970), orcein and trichromic of Mallory (Lillye, 1954). The density of sweat glands was determined by counting stained glands in a low-power microscope (10X) with a reticulate ocular of 0.48 mm (Leitz Wetzlar). The reconstruction of 3-D images was performed with an ABBE chamber (Luna, 1975). Black and white pictures of selected areas were taken with a Zeiss photo-microscope using an 100 ASA panchromatic film. 2.2. Sweat production The guanacos used for experimentation were kept in individual stalls before and during measurements. Details of the animals are given in Table 2. The apparatus used to measure sweat production consisted of a Lucite cylinder, 5.4 cm in diameter and 4.0 cm in height. The open mouth of the capsule was in contact with the skin and the other end was sealed with Lucite and connected to the collection chamber through a small acrylic tubing (5 cm long). Four holes were drilled in the cylinder (0.5 cm), to allow ventilation of the capsule. The collection chamber used was a cylinder (12 cm long; 2.87 cm in diameter) filled with ‘‘Drierite’’. The desiccant substance was changed before each experiment. One side of the chamber was connected to the sweat capsule and the other to a pump that provided a steady flow of 3.2 l/min at a pressure of 3.5 cm water below atmospheric. The control capsule was similar in dimension to the sweat capsule, but the open end was sealed with Lucite. Passive transfer of water vapor across the skin is negligible when the skin lies beneath a heavy pelage. Thus, the humidity of the air immediately above the skin is high enough to preclude significant water vapor transfer unless the fur is strongly ruffled. In the axillary and inguinal regions the hair is short enough that both sensible and insensible water loss is possible to measure.

Table 1 Characteristics of guanacos and season from which pelt samples were taken to determine the presence of sweat glands

Fig. 1. Scheme of a guanaco and the topographic areas where skin samples were taken to determine the presence and distribution of sweat glands. 1: inner surface of the upper limb (axillar); 2: lower flank (flank); 3: shoulder; 4: head (forehead).

Sample

Sex

Age (years)a

Season

Anatomical regionb

11 12 14 24 26

Male Female Female Male Female

4 5 3 6 2

Summer Winter Winter Winter Summer

1–2–3–4 1–2–4 1–2–3 1–2 1–2–3–4

a b

Determined by tooth wear. From Fig. 1.

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D.A. de Lamo et al. / Journal of Thermal Biology 26 (2001) 77–83 Table 2 Experimental animals used to determine sweat productiona ID

Sex

Age (years)

Weight (kg)

Ta range (8C)

RH range (%)

Tb range (8C)

M1 M3 H1 H3

Male Male Female Female

2 2 5 2

85 77 110 65

15.8–29.8 16–33 15.2–28.0 7.0–32.0

32–70 37–70 40–68 38–60

37.7–38.8 37.7–38.8 37.6–38.1 37.9–38.4

a

Ta ¼ambient temperature; RH=relative humidity; Tb ¼body temperature.

To secure the capsule, the sparse fur was clipped and the skin surface dried with facial tissue. The sweat capsule was applied to the skin for a period of 10 min, while the pump pulled air across the skin. The guanacos were tranquilized with xylazine 20% (0.5 ml I M) or Promazine (10 mg/ml) (de Lamo and Defosse´, 1984), to facilitate holding the capsule in place for 10 min. During the experiments the rectal temperatures were recorded continually. Only the data collected when the rectal temperature was between 37.5 and 38.58C were analyzed because that is the normal average core temperature in the morning when the samples were taken (de Lamo, 1990). The experiments were performed at the same time of the day with the four animals. Each animal was studied not more than twice a week. The samples were taken with the sweat capsule applied to the skin of the areas where the sparse fur is about 0.5 cm in length and very little longer than a droplet of sweat. When possible, a sample was taken from each side of the animal. After the 10 min procedure, the control capsule was attached to the pump the same period of time near the animal in the stall. Both the collecting cylinders were weighed and the sweat production determined by weight differences using the following equation: SP ¼ ðWsa ÿ Wsb Þÿ ðWca ÿ Wcb Þ, where SP is the sweat production in grams; Wsb is the weight of the collecting sample before the experiment; Wsa is the weight of the sample after the experiment; Wcb is the weight of the control cylinder before the experiment and Wca is the weight of the control cylinder after the measurement. During the procedure, ambient temperature (Ta ), rectal temperature (Tb ) and surface temperature of the axillar region (Ts ) were recorded with thermocouples connected to a BAT-12 digital thermometer (Bailey). Relative humidity in the stall was recorded on a Vaisala hygrothermograph. The 10 min sweat production from the sample area was transformed to grams of sweat per square meter per hour (g m ÿ 2 h ÿ 1). The expected or theoretical evaporative water loss (Qe ) was calculated by Qe ¼ LE; where L is the latent heat of vaporization, 2.45  106j kg ÿ 1 at 20–308C of Ta (Gates, 1980). E is the amount of sweat produced in g m ÿ 2 h ÿ 1. Qe is expressed in J m ÿ 2 s ÿ 1.

2.3. Respiratory water loss Two male guanacos were used to determine respiratory heat exchange. The animals were restrained but not anesthetized. Exhaled air (Te ), Ta and Tb were measured with copper–constantan thermocouples and digital thermometer (Cole-Parmer 8500-40) sensitive to 0.18C. Relative humidity was measured with hygrothermograph (Vaisala HMT 13). Exhaled air was measured with a thermocouple inside a piece of plastic tube placed in one nostril. During the experiments, the mouth of the animals was kept closed, to make them breathe through the nose. Water loss was calculated as the difference between water content in lung air (saturated with water vapor) at that Tb and the water content of exhaled air at the temperature it was measured within the nostrils (Langman et al., 1979). Curves for single and multiple regression were fitted using the least-squares method. Significant differences were determined by analysis of variance (Sokal and Rohlf, 1986).

3. Results and discussion Sweat glands in all the areas are present sampled (Table 3). Results presented here are from axillar and flank regions because these areas are covered with short hair (Morrison, 1966) and are potentially more effective in evaporative cooling. The glands are simple and glomerular. The secretory portion is extremely curled in areas where hair is short and the epithelium is of the simple cubic type (Fig. 2). The secretion is apocrine and the high P.A.S. (+) reaction indicates the high glycogen content. The secretory ducts are straight; the lumen is small and characterized by a cubic simple epithelium. The mouth of the ducts surface close to the hair follicle. In areas where the hair follicles are grouped, only one sweat gland per group is present. In other areas where hair follicles are sparser, sweat glands are associated to the big hair follicles where a single tube may be folded into a ball-like shape (Fig. 2). The average count was 925 and 750 glands/cm2 in the axillar and flank regions, respectively. In both areas the

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Table 3 Presence and number of hair follicles and sweat glands in guanacos determined by histological analysis Anatomical regiona

Hair follicles (  SD)/cm2

Sweat glands(  SD)/cm2

Samples (N)

Axillar (1) Flank (2) Shoulder (3) Face (4)

2875 (26) 2675 (32) 11175 (98) 7300 (54)

925 (48) 750 (69) 1260 (140) 603 (23)

20 16 17 10

a

From Fig. 1.

Fig. 2. (a) Microphotograph (100X) of a skin sample from the axillar region. Arrows mark the sweat glands surrounding a hair follicle (f) or unevenly distributed in a deeper plane (arrow, center of the picture); (b) Details of (a) (250X), showing a transversal cut of an extremely curled adenomere of one sweat gland (arrow). The darker spots are hair follicles surrounded by sweat glands and the big one to the right (F) is the bulb of a hair follicle.

glands are epitrichial, with their secretory portion deep in the dermis and beneath the hair follicles. The adenomers are extremely curled and surrounded by myoepithelial tissue. No difference in distribution or number of sweat glands was found (p50:05) in samples taken from males and females in different times of the year (summer–winter) (Table 3). 3.1. Sweat production Sensible and insensible water loss ranged from 4.98 to 73.36 g m ÿ 2 h ÿ 1 under the conditions studied. Individual perspiration values showed non-significant differences (p50:001) at the body temperatures analyzed. In order to analyze dependence of water loss, Tb ’s were grouped in 0.58C intervals. A normal distribution (skewness ÿ 1.5) was obtained, showing that sweat formation is not dependent on Tb ’s between 37.5 and 38.58C in guanacos. We infer that only a few glands are active in the first case (4.98 g m ÿ 2 h ÿ 1) and most of the glands of the area were active in the second case (73.36 g m ÿ 2 h ÿ 1). Bligh (1967) suggested that all epitrichial sweat glands might have slow secretion and periodic emptying by

contraction of the myoepithelium in some species, and a uniform rate of secretion in response to an appropriate stimulus in other species. When sweat production is analyzed at different Ta ’s, there is an increased production with higher Ta (Fig. 3). The sweat production varies, but increases proportionately with Ta . The equation that best fits the data is y ¼1.119x1.086 (r ¼ 0:674; n ¼ 32). No significant sweat production (p50:001) was measured at Ta between 3 and 78C, where the lowest value of 4.98 g m ÿ 2 h ÿ 1 was obtained. Between 7 and 208C the production fluctuates between 4.98 and 39.3 g m ÿ 2 h ÿ 1; and between 20 and 338C the sweat production ranged from 23.58 to 73.36 g m ÿ 2 h ÿ 1. Allen and Bligh’s (1969) llama (Lama glama), did not show any water loss from the skin when exposed to a Ta of 20–258C. When Ta was raised up to 408C, the llama showed a gradual increase in sweat production until it peaked at 100–250 g m ÿ 2 h ÿ 1. From the graphical data in that paper, the maximal production is 83 g m ÿ 2 h ÿ 1 in the normal animal and 110 g m ÿ 2 h ÿ 1 with adrenaline injected. In contrast with its close relative, guanacos appear to sweat at lower Ta ’s, showing maximal production at Ta ’s

D.A. de Lamo et al. / Journal of Thermal Biology 26 (2001) 77–83

Fig. 3. Sweat production from the axillar region as a function of ambient temperature ðTa Þ measured between 3 and 368C. The line is the best fit regression for the measured data y ¼ 1:119x1:086 ðr ¼ 0:674; n ¼ 32Þ.

above 258C. In the experiments, the highest production never reached the 100 g m ÿ 2 h ÿ 1 detected in llamas, but at a Ta of 408C, the predicted water loss would be 62.24 (  3.1) g m ÿ 2 h ÿ 1 in guanacos. In one case an excited guanaco measured at Ta ¼ 78C, sweat production reached 6.28 g m ÿ 2 h ÿ 1, when Tb was near the upper accepted limit (38.58C). Perhaps this production is the result of the high level of circulating adrenaline, being the sweat production independent of ambient temperature. Breeders have selected llamas with a denser and thicker coverage than guanacos, so their higher production of sweat does not necessarily mean a more effective heat loss. Instead, llamas may have little evaporative cooling because there is a small moisture gradient across the pelage. Guanacos with shorter hair in the flank and axillar areas should be able to optimize the moisture gradient to cool more responsively the skin by sweating even under water restrictions and high ambient temperatures. The dromedary camel, a well-adapted desert dweller shows a high rate of sweat production when normally hydrated. Camels may evaporate more than 4% of body weight per day in the summer and around 1% of their body weight in the winter (Schmidt-Nielsen et al., 1957). In this species increased evaporative water loss increased as Ta rose as we found in the guanaco. Sweat production in guanacos shows no significant relation to relative humidity (RH) at the Ta ’s measured in our experiments (r ¼ 0:28; p > 0:10; n ¼ 17). To test the non-significant relationship, a model with several variables was developed to determine statistically the best correlation with any of the independent variables.

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This model (SP ¼Ta þ Tb þ Ts þRH) contemplates all the variables measured; SP is the sweat production; Ta the ambient temperature; Tb the body temperature; Ts the skin temperature where the sample were taken and RH is the relative humidity, and was run by the general linear procedure (SAS, 1985) and showed a high R2 (0.8836). Only Ta showed a high significant correlation (p > F ¼ 0:001), the coefficients for the other variables were not significant at the 1% level. However, Ts showed significance at the 5% level. This relationship may be explained by the high dependence of Ts on ambient temperature (de Lamo, 1990). The water loss from the skin of guanacos seems to be independent of body temperature and of skin temperature, when animals are in the normal range of thermoregulation (37.5–38.58C). The temperature of the skin spot measured could be subject to a different temperature if sweat glands were present and active at the time and the temperature of that region of skin lowered by the effect of evaporation. Variations in relative humidity may affect the efficiency of evaporative cooling, but it does not seem to be the direct stimulus for sweat production in guanacos. A rise in ambient temperature raises the rate of secretion to a new steady rate. When Ta is above 208C the production increases, reaching its maximum at the highest Ta ’s obtained (368C). In ox and sheep, contraction of the myoepithelium increases secretion (Allen and Bligh, 1969). While in the horse, donkey and llama; the responses to heat do not seem to involve the contractile elements (Allen and Bligh, 1969). Myoepithelial secretion was not considered for guanacos in this study. The calculated evaporation rate for animals exposed to Ta ’s between 20 and 308C and 38–70 % RH was 30.79 (  3.33) J s ÿ 1 m ÿ 2. The flux density was 15.92 W/m2 at 208C and 49.52 W/m2 at 308C. Total heat loss by evaporation calculated at ambient temperatures of 20 and 308C represents 11.9 and 37%, respectively, of the standing metabolic heat production (de Lamo, 1990). The sweat production measured between 10 and 208C (Ta ) may be representative of cutaneous insensible water loss. When Ta ’s are above 208C the increased sweat production is an important factor on total heat loss. We obtained only three reliable measurements of respiratory water loss. In most cases, guanacos expired air through the mouth. The animals were frequently restless and excited during the experiments. Excitement may have caused changes in the blood flow to the nasal passages, masking a possible physiological mechanism of water recovery. That mechanism has been reported for small rodents (Jackson and Schmidt-Nielsen, 1964) and other ungulates (Langman et al., 1979). In the giraffe, an increase in respiratory rate with increased Ta led to an increase of respiratory water loss (Langman et al., 1979). In guanacos, the respiratory

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frequency did not change when animals were exposed to Ta ’s between 7.5 and 27.58C. It seems that the effectiveness of water recovery is dependent on the anatomical structure of the nasal region, which provides an area available for heat exchange. Guanacos show an identical nasal structure to those observed in the giraffe, waterbuck and other desert herbivores. In the three measurements obtained from guanacos, the temperature of the exhaled air was about 78C below Tb when ambient temperatures fluctuated between 11 and 208C. If the mechanism is as efficient as in the giraffe, the amount of water recovered would be about 50%. Losses from the respiratory tract will be 0.75 W/m2 at low Ta ’s (0–108C) and 1.35 W/m2 at Ta ’s above 108C. Guanacos do not gape at high ambient temperatures, and they increase breathing rate only in response to exercise (Rosenmann and Morrison, 1963). Since guanacos did not show changes in the respiratory rate, respiratory water loss is not an important mechanism for heat dissipation. As in other large ungulates the main channel for water loss is evaporation by sweating. The distribution of sweat glands in the guanaco showed no significant differences between areas, season or sex. However, the same number of active sweat glands in the areas covered with short hair may represent an important avenue of heat loss by evaporation if body temperature is stable. As was shown for radiative heat loss, postural changes by the guanaco modify the exposed surface area of the almost bare areas (de Lamo et al., 1998). This behavioral response may control not only heat and water loss by evaporation; depending on the exposed surface area, a standing guanaco should be able to optimize the moisture gradient to cool the skin more effectively by sweating. Under water restrictions and high ambient temperatures guanacos conserve water by reducing the effective sweating surface or by changing the avenue of heat loss to conduction or convection. This illustrates the complex interaction between temperature regulation and water conservation of animals as they cope with seasonal changes and daily fluctuations encountered in nature.

4. Summary We investigated the presence and characteristics of sweat gland and their activity in relation to thermal stress in the guanaco (Lama guanicoe). Sweat glands are present all over the skin, showing higher densities around the shoulder, lower part of the upper limb and lower flank. Sensible and insensible water loss varies from 4.98 and 73.36 g m ÿ 2 h ÿ 1 of skin. Ambient temperatures between 20 and 338C are the main stimuli for the activation of sweat glands, generating a heat loss

ranging from 11.9 to 37% of standing basal metabolic rate. Under these experimental conditions, respiratory water loss is not an important mechanism for heat dissipation in guanacos. As in other large ungulates the main channel for water loss is evaporation by sweating. The potential water loss may be regulated by postural changes as is with heat loss by conduction, convection or radiation in this species.

Acknowledgements Part of this research was derived from the doctoral dissertation of D.A. de L at the University of Illinois. Support for this research came from CONICET (Argentina) and the Department of Physiology and Biophysics at the University of Illinois at UrbanaChampaign. Mr. German Solari helped with animal care and Mr. Jorge Upton helped to control the guanacos during the experiments.

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