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A technique for quantitating suckling behavior in brooder reared kittens
Physiology &Behavior,Vol. 27, pp. 953-956. PergamonPress and BrainResearch Publ., 1981. Printedin the U.S.A.
A Technique for Quantitating Suckling Behavior in Brooder Reared Kittens ' PATRICIA VETULA GALLO
Division of Human Nutrition, University of British Columbia, Vancouver, B.C., Canada V6T IW5 AND KIRVIN KNOX
Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06268 R e c e i v e d 25 M a r c h 1981 GALLO, P. V. AND K. KNOX. A techniquefor quantitating suckling behavior in brooder reared kittens. PHYSIOL. BEHAV. 27(5) 953-956, 1981.--A brooder chamber and pressure-transducer system used in polygraphic recording of suckling behavior in newborn kittens is described. Also detailed, is the formulation of a semi-purified diet to be used with the feeding system. This apparatus can be employed to monitor the duration, frequency and intensity of individual rooting and sucking bouts as well as overall patterns of rooting and suckling behavior. When utilized in conjunction with olfactory (or tactile) cues, it can also be beneficial in investigations of early learning in kittens. In addition, use of the semi-purified diet in combination with the feeding apparatus allows for accurate manipulation of dietary intake with minimal handling and disturbance of the young animal. Thus this feeding system could be especially useful in expanding research on behavioral and physiological consequences of neonatal nutritional or toxicological insult to the newborn kitten. Artificial feeding
Kitten
Suckling
Behavior
S U C K L I N G is a behavior fundamental to the survival of all mammals. Although the controls of suckling behavior have been well documented in the neonatal rat pup [1], little is known regarding the ontogeny of suckling in other species. Kovach and Kling [5] first presented data on the maturational factors associated with the onset of suckling behavior in brooder fed kittens. However, the design of their brooder may have hindered the newborn kittens in locating the nipple and attaching [8]. Utilizing a brooder designed to approximate closely the natural suckling environment of neonatal kittens, Rosenblatt [8] examined the role of tactile and olfactory stimuli in the development of suckling behavior. Stimulated by his findings, we became interested in expanding his approach in order to quantify specific components of kitten suckling behavior and to investigate the usefulness of our apparatus for testing learning in newborn kittens. In addition, we were interested in observing the extent to which the development of suckling behavior and early learning in kittens could be altered by neonatal protein restriction. METHOD AND RESULTS
Apparatus The brooder chamber was a modification of that described by Rosenblatt [8]. It consisted of 37 × 37 × 25 cm clear
Liquid diet
Plexiglas box with a 36× 18 cm flat surface (the brooder) placed against the back wall at a 45° angle to the floor. The front surface of the brooder was covered with artificial fur material which was tacked in place from behind. The brooder was secured by screws through the side walls of the chamber and was in direct contact with the floor. Two circular openings 9 cm in diameter, were cut on the horizontal midline and equidistant from the verticle midline of the brooder for insertion of 2 nipple-flange assembly units (Fig. 1). Each nipple-flange assembly unit was a 9×6 cm Plexiglas water-filled cylinder with a solid Plexiglas bottom. A 9 cm × 2 mm circle of rubber was cemented to the top end of the cylinder to form the flange. Each nipple-flange unit was inserted into the brooder and secured by a U-shaped bracket on the back of the brooder. Nipples were handmade by dipping fire sealed microhematocrit tubes for 1 sec into room temperature 20% calcium chloride (in absolute ethanol), waving in the air for 5 sec, holding in liquid latex (Naugatuck Chemicals Co., Div. of Uniroyal Chemical Inc., Naugatuck, CT) for 20 sec, redipping (1 sec) in the calcium chloride solution and air drying over night. Rubber nipples were removed from the hematocrit tubes by immersing in soapy water and twisting gently. The nipples were then rinsed in water and cured in a boiling water bath for 20 min (Robert Wall, personal com-
~This work was supported by a grant from the University of Connecticut Research Foundation.
C o p y r i g h t © 1981 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/81/110953-04502.00/0
954
G A L L ( ) AND KNOX
| ! --
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..J
k
~
CrossSectionof NippbeflangeUni~
-I - i ~ f • i
1 L
J
°
-
!
I
Trans~Sight BrooderChamber
/
Sucking Ttansd~m-y
/
~
J
.
NuzzlingTransducer
FIG. 1. Diagram of main features of the brooder, milk delivery and pressure monitoring systems. munication). Defective nipples (i.e. air bubbles, etc.) were discarded. The cured nipple was cemented to polyethylene tubing which passed through the Plexiglas cylinder and out through the center of the rubber flange. To avoid water leaks, the nipple was secured by cement on the inside of the flange. The polyethylene tubing communicated with a pressure transducer (Statham, P23A Series, Statham Labs Inc,, Div. of Statham Instrument Co., P.O. Box 1178, Hato Rey, PR) and from there to a modified 50 ml buret which held the milk in a manner described by Kron et al. [6]. This transducer (sucking transducer) monitored pressure changes in the milk system and thereby served to indicate sucking. Another section of polyethylene tubing was inserted into a plastic water inlet valve mounted on the back of each nipple-flange assembly unit and communicated with a second pressure transducer and from there to a 500 ml roundbottom glass flask (water reservoir). The bottom of the flask was modified to accept a glass tip which attached to the polyethylene tubing from the second pressure transducer. This second transducer (rooting transducer) was used to monitor changes in the water system when pressure was placed on the rubber flange by the kitten during rooting bouts as it attempted to locate the nipple. Associated with the water reservoir and the transducers connected to each nipple-flange unit, was a meter stick used for measuring adjustments made in the height of the water reservoir and transducers during calibration of the apparatus. Since we were interested in investigating the usefulness of our apparatus for testing learning in newborn kittens, two nipple-flange assembly units were inserted into the brooder; one with an open nipple communicating with the milk buret and a second with a blind nipple communicating with a water
filled buret. In addition, each nipple-flange unit was attached to a water reservoir. Thus, the 4 corresponding pressure transducers (1 sucking and 1 rooting for each of 2 nipple flange units) were attached to a Grass Model 7 polygraph, volts 15, frequency-60 (Grass Instrument Co., Quincy, MA). All tubing used in the nipple-flange assembly units and connecting with milk or water reservoirs was 1.57x2.08 mm intramedic polyethylene tubing. Tubing was connected to the pressure transducers via 16 gauge Luer-Stub adapters and one-way Luer-Lok stopcocks (Clay Adams, Div. of Becton, Dickinson and Co., Parsippany, NJ). The floor of the brooder chamber was warmed by a standard electric heating pad operated at lowest setting and covered with disposable soft paper pads. Continual operation of the heating pad in combination with a 60 W incandescent light suspended 10 cm above the brooder side of the chamber maintained the chamber air temperature at 22°C.
Behavioral Measures The parameters of suckling behavior measured on each nipple-flange unit were divided into the categories of rooting and sucking. Rooting is a behavior exhibited by kittens actively searching for a nipple prior to attachment. We believed that quantifying rooting behavior could serve as an index of a kitten's motivation to locate a nipple and attach. Rooting. (1) Number of bouts; (2) Duration of each bout; (3) Amplitude of each bout i.e. peak height could be used as a quantitative index of intensity of motivation to locate the nipple and attach; (4) Total time rooting. Sucking. (1) Total time sucking; (2) Sucking frequency; (3) Total diet consumed per feeding (direct reading from
SUCKLING BEHAVIOR OF KITTENS
955
TABLE 1 COMPOSITION OF THE DIETS Proportion in Diet (g/100 g) Component Enfamil, undiluted~; Water Casein, vitaminfree micropulverized¢ Cerelose§ Mineral Mixture¶# Vitamin Mixture** Choline Chloride# Inositol#
36% Protein Diet*
18% Protein Diet*
45.500 45.500 6.000
45.500 45.500 2.300
2.000 0.510 0.225 0.250 0.015
5.700 0.510 0.225 0.250 0.015
*Dry matter basis. The dry matter content of these diets was 18.9%. ?Mead Johnson and Company, Evansville, IN. *ICN Nutritional Biochemicals, Cleveland, OH. §Corn Products International, Englewood Cliffs, NJ. ¶Supplied the following per 100 g diet: 313.4 mg K2HPO4, 131.7 mg NaC1, 39.1 mg MgSO4, 21.6 mg ferric citrate, 3.0 mg MnSO4'H20, 0.6 mg KI, 0.4 mg ZnSO4.7H20, 0.2 mg CuSO4-5H20. #J. T. Baker Co., Phillipsburg, NJ. **Vitamin Mix Teklad, Teklad Test Diets, Madison, WI.
buret); (4) Pressure of each suck could also be monitored with appropriate calibration of the milk system transducer.
The Diet Because we were interested in manipulating the protein intake of our kittens, a purified liquid control diet (Table 1) which met the nutritional requirements of the growing kitten[7] was formulated in our laboratory. To meet the high nitrogen requirements o f the growing kitten, a minimum of 30% of the diet must be protein. Although a variety of protein sources (dry milk powder, lactalbumin, vitamin-free casein) were investigated, problems with clogged feeder tubing, kitten diarrhea and inadequate weight gain were encountered. However, when Enfamil (Mead Johnson and Company, Evansville, IN ) was used as the primary protein source and supplemented with micropulverized casein and a vitamin and mineral mix, the nutritional requirements of the kitten were met. Kittens fed the 36% protein diet were found to attach and suck readily, urinate and defecate regularly and grow at at rate (9.5--_ 1.6 g/day) comparable to nursing kittens [4]. A low protein diet which provided 1/2 the amount of protein, but was isocaloric to the control diet by the addition of Cerelose, was also formulated (Table 1). The nitrogen content of our diets was routinely confirmed by Kjeldahl analyses [9]. To prepare the liquid diets, heat was applied to an aqueous solution of the minerals. This mixture was stirred until the salts were solubilized. The solution was then cooled in running water and Enfamil and the other nutrients were added, stirring after each addition. The liquid diet was made daily and refrigerated between feedings. Before each feeding, the diet was warmed to 22°C.
Operation Summary Prior to each behavioral observation, the pressure trans-
ducers were precisely positioned and calibrated. This procedure was necessary in order to quantitate the amplitude of the readout from the rooting transducers. Using a transit sight, the upper surface of each rooting transducer was aligned with the nipple in its respective nipple-flange unit and 0 mm on the meter stick. Then the corresponding water reservoir was lowered until its meniscus was aiso at the level of the transducer and 0 mm on the same meter stick. With this alignment, a baseline or 0 mm water pressure reading was obtained. Subsequent readings were made with the water reservoir raised 136 mm and 272 mm above the baseline. This calibration procedure was followed for each of the 2 rooting transducers before testing each animal. Thus the amplitude of the peaks on the readout from the transducers could be quantified in terms of mm of water pressure. Thereafter the 2 transducers communicating with the burets and monitoring sucking were similarly aligned with the nipple opening in their respective nipple-flange units. The height of the milk-filled buret was also adjusted so that the side arm was at the level of the nipple opening in its unit, thereby maintaining the delivery pressure at the nipple at 1 atmosphere. By positioning the buret at this level, the milk was just kept from flowing out of the nipple; a lower milk reservoir would allow the milk to run freely and a higher reservoir would require that the kitten suck harder to obtain milk. To assure equal pressure within the brooder nipples, the side arm of the water filled buret was similarly aligned with the blind nipple in its unit. Because olfactory stimuli are known to be used by the kitten in learning to locate the nipple and attach [8], ethyl benzoate was applied to the flange of the milk assembly unit and acetophenome applied to the flange of the blind assembly unit. Through use of the olfactory-cues in combination with reversal of the positions of the 2 nipple-flange assembly units in the brooder, we could monitor olfactory discrimination and learning in our brooder-fed kittens. To facilitate the adoption of brooder feeding, immediately after birth, all kittens were removed from their mothers and placed in a holding box lined with an electric heating pad which maintained the air temperature at 22°C. After an initial 6 hr waiting period, each kitten was individually removed from the holding box and held by the scruff of the neck over the milk nipple-flange assembly unit and allowed to root on the flange and attempt to locate the nipple. Once nipple attachment occurred, the kitten was gently lowered to rest on the brooder surface and allowed to suck ab lib. After feeding, kittens were stimulated to urninate and defecate, weighed and returned to the holding box. This procedure was followed every 4 hrs. By three days of age, the majority of kittens, when placed in the center of the brooder, could locate the milk flange assembly unit and, after rooting, attach to the nipple and self-feed. Polygraph recordings were made twice daily for each kitten at the 0900 and 2100 feedings. The components of rooting behavior recorded included: number of bouts, duration of each bout, bout amplitude at specified intervals and total time spent rooting prior to each nipple attachment. The components of sucking behavior monitored included: total time spent sucking, total number of sucks, milk consumed in a feeding (ml)/total sucking time per feeding and average volume of milk per suck, i.e. total milk in a feeding/total sucks per feeding. Kittens fed every 4 hrs using our feeding procedure and control diet consumed 4.8±0.2 ml (mean-+SEM) per feeding and gained 9.5+ 1.6 g/day. These values compared well with those previously reported for
956
GALL() AND KNOX
hand-reared and nursing kittens [4] and those of nursing kittens in earlier studies in our laboratory 131. DISCUSSION The above method of artificial rearing allows for quantitative assessment of several components of suckling behavior in the newborn kitten. Through modification of the nipple-flange assembly units, this technique can also be used to study the role of olfactory, tactile or temperature cues in controlling the ontogeny of suckling behavior and in studies of early learning in kittens. In addition, use of a semi-purified diet in combination with our method of feeding could expand research on the behavioral consequences of neonatal nutritional insults. In the past, research on the effects of specific
dietary deficiencies on the nursing animal has been hindered because both quantitative and qualitative 12} changes have been observed in the milk of nutrient deprived females. Thus direct manipulation of the neonate's dietary intake was possible only by intubation--a tedious and obtrusive tech+ nique, especially when the animal's behavior is under study. However, our method of feeding allows for accurate manipulation of dietary intake with minimal handling and disturbance to the suckling animal. Furthermore, since kittens fed by our procedure gain weight at a rate comparable to that of nursing kittens, the feeding system alone could be used in toxicological and nutritional paradigms as a more "'natural" method of maintaining newborn kittens on a diet of specified composition while monitoring physiological and biochemical changes during early postnatal life.
REFERENCES
1. Blass, E. M. and M. H. Teicher. Suckling. Science 210: 15-22, 1980. 2. Crnic, L. S. and P. Chase. Models of infantile undernutrition in rats: effects on milk. J. Nutr. 108: 1755-1760, 1978. 3. Gallo, P. V., J. Werboff and K. Knox. Protein restriction during gestation and lactation: development of attachment behavior in cats. Behav. Neural Biol. 29: 216-223, 1980. 4. Hafez, E. S. E. Reproduction and Breeding Techniques fi~r Laboratory Animals. Philadelphia: Lea and Febiger, 1970, pp. 192208. 5. Kovach, J. K. and A. Kling. Mechanisms of neonate sucking behavior in the kitten. Anim. Behav. 15: 91-101, 1967.
6. Kron, R. E., M. Stein and K. E. Goddard. A method of measuring sucking behavior of newborn infants. Psychosom. Med. 25: 181-191, 1963. 7. National Research Council. Nutrient requirements of the cat. In: Nutrient Requirements of Laboratory Animals, 2nd revised edition. Washington, DC: National Academy of Science, 1972, pp. 1-8. 8. Rosenblatt, J. Suckling and home orientation in the kitten: a comparative developmental study. In: The Psychobiology of Development, edited by E. Tobach, L. R. Aronson and E. Shaw. New York: Academic Press, 1971, pp. 345-410. 9. Triebold, H. O. Quantitatiw, Analysis with Applications to Agricultural and Food Products. New York: D. Van Nostrand Co.. 1946, pp. 151-158.
A Technique for Quantitating Suckling Behavior in Brooder Reared Kittens ' PATRICIA VETULA GALLO
Division of Human Nutrition, University of British Columbia, Vancouver, B.C., Canada V6T IW5 AND KIRVIN KNOX
Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06268 R e c e i v e d 25 M a r c h 1981 GALLO, P. V. AND K. KNOX. A techniquefor quantitating suckling behavior in brooder reared kittens. PHYSIOL. BEHAV. 27(5) 953-956, 1981.--A brooder chamber and pressure-transducer system used in polygraphic recording of suckling behavior in newborn kittens is described. Also detailed, is the formulation of a semi-purified diet to be used with the feeding system. This apparatus can be employed to monitor the duration, frequency and intensity of individual rooting and sucking bouts as well as overall patterns of rooting and suckling behavior. When utilized in conjunction with olfactory (or tactile) cues, it can also be beneficial in investigations of early learning in kittens. In addition, use of the semi-purified diet in combination with the feeding apparatus allows for accurate manipulation of dietary intake with minimal handling and disturbance of the young animal. Thus this feeding system could be especially useful in expanding research on behavioral and physiological consequences of neonatal nutritional or toxicological insult to the newborn kitten. Artificial feeding
Kitten
Suckling
Behavior
S U C K L I N G is a behavior fundamental to the survival of all mammals. Although the controls of suckling behavior have been well documented in the neonatal rat pup [1], little is known regarding the ontogeny of suckling in other species. Kovach and Kling [5] first presented data on the maturational factors associated with the onset of suckling behavior in brooder fed kittens. However, the design of their brooder may have hindered the newborn kittens in locating the nipple and attaching [8]. Utilizing a brooder designed to approximate closely the natural suckling environment of neonatal kittens, Rosenblatt [8] examined the role of tactile and olfactory stimuli in the development of suckling behavior. Stimulated by his findings, we became interested in expanding his approach in order to quantify specific components of kitten suckling behavior and to investigate the usefulness of our apparatus for testing learning in newborn kittens. In addition, we were interested in observing the extent to which the development of suckling behavior and early learning in kittens could be altered by neonatal protein restriction. METHOD AND RESULTS
Apparatus The brooder chamber was a modification of that described by Rosenblatt [8]. It consisted of 37 × 37 × 25 cm clear
Liquid diet
Plexiglas box with a 36× 18 cm flat surface (the brooder) placed against the back wall at a 45° angle to the floor. The front surface of the brooder was covered with artificial fur material which was tacked in place from behind. The brooder was secured by screws through the side walls of the chamber and was in direct contact with the floor. Two circular openings 9 cm in diameter, were cut on the horizontal midline and equidistant from the verticle midline of the brooder for insertion of 2 nipple-flange assembly units (Fig. 1). Each nipple-flange assembly unit was a 9×6 cm Plexiglas water-filled cylinder with a solid Plexiglas bottom. A 9 cm × 2 mm circle of rubber was cemented to the top end of the cylinder to form the flange. Each nipple-flange unit was inserted into the brooder and secured by a U-shaped bracket on the back of the brooder. Nipples were handmade by dipping fire sealed microhematocrit tubes for 1 sec into room temperature 20% calcium chloride (in absolute ethanol), waving in the air for 5 sec, holding in liquid latex (Naugatuck Chemicals Co., Div. of Uniroyal Chemical Inc., Naugatuck, CT) for 20 sec, redipping (1 sec) in the calcium chloride solution and air drying over night. Rubber nipples were removed from the hematocrit tubes by immersing in soapy water and twisting gently. The nipples were then rinsed in water and cured in a boiling water bath for 20 min (Robert Wall, personal com-
~This work was supported by a grant from the University of Connecticut Research Foundation.
C o p y r i g h t © 1981 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/81/110953-04502.00/0
954
G A L L ( ) AND KNOX
| ! --
~ .--1
..J
k
~
CrossSectionof NippbeflangeUni~
-I - i ~ f • i
1 L
J
°
-
!
I
Trans~Sight BrooderChamber
/
Sucking Ttansd~m-y
/
~
J
.
NuzzlingTransducer
FIG. 1. Diagram of main features of the brooder, milk delivery and pressure monitoring systems. munication). Defective nipples (i.e. air bubbles, etc.) were discarded. The cured nipple was cemented to polyethylene tubing which passed through the Plexiglas cylinder and out through the center of the rubber flange. To avoid water leaks, the nipple was secured by cement on the inside of the flange. The polyethylene tubing communicated with a pressure transducer (Statham, P23A Series, Statham Labs Inc,, Div. of Statham Instrument Co., P.O. Box 1178, Hato Rey, PR) and from there to a modified 50 ml buret which held the milk in a manner described by Kron et al. [6]. This transducer (sucking transducer) monitored pressure changes in the milk system and thereby served to indicate sucking. Another section of polyethylene tubing was inserted into a plastic water inlet valve mounted on the back of each nipple-flange assembly unit and communicated with a second pressure transducer and from there to a 500 ml roundbottom glass flask (water reservoir). The bottom of the flask was modified to accept a glass tip which attached to the polyethylene tubing from the second pressure transducer. This second transducer (rooting transducer) was used to monitor changes in the water system when pressure was placed on the rubber flange by the kitten during rooting bouts as it attempted to locate the nipple. Associated with the water reservoir and the transducers connected to each nipple-flange unit, was a meter stick used for measuring adjustments made in the height of the water reservoir and transducers during calibration of the apparatus. Since we were interested in investigating the usefulness of our apparatus for testing learning in newborn kittens, two nipple-flange assembly units were inserted into the brooder; one with an open nipple communicating with the milk buret and a second with a blind nipple communicating with a water
filled buret. In addition, each nipple-flange unit was attached to a water reservoir. Thus, the 4 corresponding pressure transducers (1 sucking and 1 rooting for each of 2 nipple flange units) were attached to a Grass Model 7 polygraph, volts 15, frequency-60 (Grass Instrument Co., Quincy, MA). All tubing used in the nipple-flange assembly units and connecting with milk or water reservoirs was 1.57x2.08 mm intramedic polyethylene tubing. Tubing was connected to the pressure transducers via 16 gauge Luer-Stub adapters and one-way Luer-Lok stopcocks (Clay Adams, Div. of Becton, Dickinson and Co., Parsippany, NJ). The floor of the brooder chamber was warmed by a standard electric heating pad operated at lowest setting and covered with disposable soft paper pads. Continual operation of the heating pad in combination with a 60 W incandescent light suspended 10 cm above the brooder side of the chamber maintained the chamber air temperature at 22°C.
Behavioral Measures The parameters of suckling behavior measured on each nipple-flange unit were divided into the categories of rooting and sucking. Rooting is a behavior exhibited by kittens actively searching for a nipple prior to attachment. We believed that quantifying rooting behavior could serve as an index of a kitten's motivation to locate a nipple and attach. Rooting. (1) Number of bouts; (2) Duration of each bout; (3) Amplitude of each bout i.e. peak height could be used as a quantitative index of intensity of motivation to locate the nipple and attach; (4) Total time rooting. Sucking. (1) Total time sucking; (2) Sucking frequency; (3) Total diet consumed per feeding (direct reading from
SUCKLING BEHAVIOR OF KITTENS
955
TABLE 1 COMPOSITION OF THE DIETS Proportion in Diet (g/100 g) Component Enfamil, undiluted~; Water Casein, vitaminfree micropulverized¢ Cerelose§ Mineral Mixture¶# Vitamin Mixture** Choline Chloride# Inositol#
36% Protein Diet*
18% Protein Diet*
45.500 45.500 6.000
45.500 45.500 2.300
2.000 0.510 0.225 0.250 0.015
5.700 0.510 0.225 0.250 0.015
*Dry matter basis. The dry matter content of these diets was 18.9%. ?Mead Johnson and Company, Evansville, IN. *ICN Nutritional Biochemicals, Cleveland, OH. §Corn Products International, Englewood Cliffs, NJ. ¶Supplied the following per 100 g diet: 313.4 mg K2HPO4, 131.7 mg NaC1, 39.1 mg MgSO4, 21.6 mg ferric citrate, 3.0 mg MnSO4'H20, 0.6 mg KI, 0.4 mg ZnSO4.7H20, 0.2 mg CuSO4-5H20. #J. T. Baker Co., Phillipsburg, NJ. **Vitamin Mix Teklad, Teklad Test Diets, Madison, WI.
buret); (4) Pressure of each suck could also be monitored with appropriate calibration of the milk system transducer.
The Diet Because we were interested in manipulating the protein intake of our kittens, a purified liquid control diet (Table 1) which met the nutritional requirements of the growing kitten[7] was formulated in our laboratory. To meet the high nitrogen requirements o f the growing kitten, a minimum of 30% of the diet must be protein. Although a variety of protein sources (dry milk powder, lactalbumin, vitamin-free casein) were investigated, problems with clogged feeder tubing, kitten diarrhea and inadequate weight gain were encountered. However, when Enfamil (Mead Johnson and Company, Evansville, IN ) was used as the primary protein source and supplemented with micropulverized casein and a vitamin and mineral mix, the nutritional requirements of the kitten were met. Kittens fed the 36% protein diet were found to attach and suck readily, urinate and defecate regularly and grow at at rate (9.5--_ 1.6 g/day) comparable to nursing kittens [4]. A low protein diet which provided 1/2 the amount of protein, but was isocaloric to the control diet by the addition of Cerelose, was also formulated (Table 1). The nitrogen content of our diets was routinely confirmed by Kjeldahl analyses [9]. To prepare the liquid diets, heat was applied to an aqueous solution of the minerals. This mixture was stirred until the salts were solubilized. The solution was then cooled in running water and Enfamil and the other nutrients were added, stirring after each addition. The liquid diet was made daily and refrigerated between feedings. Before each feeding, the diet was warmed to 22°C.
Operation Summary Prior to each behavioral observation, the pressure trans-
ducers were precisely positioned and calibrated. This procedure was necessary in order to quantitate the amplitude of the readout from the rooting transducers. Using a transit sight, the upper surface of each rooting transducer was aligned with the nipple in its respective nipple-flange unit and 0 mm on the meter stick. Then the corresponding water reservoir was lowered until its meniscus was aiso at the level of the transducer and 0 mm on the same meter stick. With this alignment, a baseline or 0 mm water pressure reading was obtained. Subsequent readings were made with the water reservoir raised 136 mm and 272 mm above the baseline. This calibration procedure was followed for each of the 2 rooting transducers before testing each animal. Thus the amplitude of the peaks on the readout from the transducers could be quantified in terms of mm of water pressure. Thereafter the 2 transducers communicating with the burets and monitoring sucking were similarly aligned with the nipple opening in their respective nipple-flange units. The height of the milk-filled buret was also adjusted so that the side arm was at the level of the nipple opening in its unit, thereby maintaining the delivery pressure at the nipple at 1 atmosphere. By positioning the buret at this level, the milk was just kept from flowing out of the nipple; a lower milk reservoir would allow the milk to run freely and a higher reservoir would require that the kitten suck harder to obtain milk. To assure equal pressure within the brooder nipples, the side arm of the water filled buret was similarly aligned with the blind nipple in its unit. Because olfactory stimuli are known to be used by the kitten in learning to locate the nipple and attach [8], ethyl benzoate was applied to the flange of the milk assembly unit and acetophenome applied to the flange of the blind assembly unit. Through use of the olfactory-cues in combination with reversal of the positions of the 2 nipple-flange assembly units in the brooder, we could monitor olfactory discrimination and learning in our brooder-fed kittens. To facilitate the adoption of brooder feeding, immediately after birth, all kittens were removed from their mothers and placed in a holding box lined with an electric heating pad which maintained the air temperature at 22°C. After an initial 6 hr waiting period, each kitten was individually removed from the holding box and held by the scruff of the neck over the milk nipple-flange assembly unit and allowed to root on the flange and attempt to locate the nipple. Once nipple attachment occurred, the kitten was gently lowered to rest on the brooder surface and allowed to suck ab lib. After feeding, kittens were stimulated to urninate and defecate, weighed and returned to the holding box. This procedure was followed every 4 hrs. By three days of age, the majority of kittens, when placed in the center of the brooder, could locate the milk flange assembly unit and, after rooting, attach to the nipple and self-feed. Polygraph recordings were made twice daily for each kitten at the 0900 and 2100 feedings. The components of rooting behavior recorded included: number of bouts, duration of each bout, bout amplitude at specified intervals and total time spent rooting prior to each nipple attachment. The components of sucking behavior monitored included: total time spent sucking, total number of sucks, milk consumed in a feeding (ml)/total sucking time per feeding and average volume of milk per suck, i.e. total milk in a feeding/total sucks per feeding. Kittens fed every 4 hrs using our feeding procedure and control diet consumed 4.8±0.2 ml (mean-+SEM) per feeding and gained 9.5+ 1.6 g/day. These values compared well with those previously reported for
956
GALL() AND KNOX
hand-reared and nursing kittens [4] and those of nursing kittens in earlier studies in our laboratory 131. DISCUSSION The above method of artificial rearing allows for quantitative assessment of several components of suckling behavior in the newborn kitten. Through modification of the nipple-flange assembly units, this technique can also be used to study the role of olfactory, tactile or temperature cues in controlling the ontogeny of suckling behavior and in studies of early learning in kittens. In addition, use of a semi-purified diet in combination with our method of feeding could expand research on the behavioral consequences of neonatal nutritional insults. In the past, research on the effects of specific
dietary deficiencies on the nursing animal has been hindered because both quantitative and qualitative 12} changes have been observed in the milk of nutrient deprived females. Thus direct manipulation of the neonate's dietary intake was possible only by intubation--a tedious and obtrusive tech+ nique, especially when the animal's behavior is under study. However, our method of feeding allows for accurate manipulation of dietary intake with minimal handling and disturbance to the suckling animal. Furthermore, since kittens fed by our procedure gain weight at a rate comparable to that of nursing kittens, the feeding system alone could be used in toxicological and nutritional paradigms as a more "'natural" method of maintaining newborn kittens on a diet of specified composition while monitoring physiological and biochemical changes during early postnatal life.
REFERENCES
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