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Procedures for the collection of milk from the rat dam
Physiology & Behavior, Vol. 22, pp. 795-797. Pergamon Press and Brain Research Publ., 1979. Printed in the U.S.A.
Procedures for the Collection of Milk from the Rat Dam S T E P H E N C. B R A K E
Department o f Psychiatry, Albert Einstein College o f Medicine, Montefiore Hospital and Medical Center 111 East 210th Street, Bronx, N Y 10467 ( R e c e i v e d 26 O c t o b e r 1978) BRAKE, S. C. Procedures for the collection o f milk from the rat dam. PHYSIOL. BEHAV. 22(4) 795-797, 1979.--Three procedures for the collection of milk from the rat dam were investigated. Before each procedure, dams were separated from
their litters for either 4-6 or 22-24 hr and half received oxytocin injections (IP) prior to milking. The most productive and efficient milking procedure involved cannulation of the mammary tissue following 22-24 hr separations and oxytocin injections. Milk collection
Lactation
Oxytocin
Rat's milk
I N V E S T I G A T I O N S of suckling and ingestion of infant rats have increased substantially in recent years. It is thus somewhat surprising that experimenters interested in administering nutrient to infant rat pups usually employ heavy cream, bovine milk, or various milk formulas rather than rat milk itself [5, 7, 8, 9]. The composition and viscosity of the most widely used formulas [4,12] are similar to that of rat milk [2, 4, 11], but even they result in less than normal weight gains or altered body-fat composition in pups reared on them [4, 13, 15]. It may also be desirable to avoid the use of milk substitutes in the study of preferential consummatory behavior, as it has been shown that rat pups are capable of discriminating different milk tastes [6]. The relative inefficiency of most milk collection techniques has made the use of rat milk prohibitively troublesome. One of the earliest procedures involved the use of rather elaborate milking machines, originally designed for use with other species [2, 13, 14]. These aspiration devices were usually employed in conjunction with IP oxytocin injections given 5-10 rain prior to milking. Oxytocin, in physiological doses, initiates natural milk-ejection [3,15] and the larger IP doses (0.2-0.4 ml) had the effect of "flooding" the ducts of the mammary glands with far more milk than is usually available to suckling pups [10]. Further, overnight separation of the lactating dam and pups results in engorgement of mammary tissue [1,5], and so separations were often employed as well. Apparently, 3-7 ml could be collected by using the machines in this manner, but it required considerable time and effort [13]. Further, Luckey et al. [11] reported that the machines were ineffective with difficult milkers. Another common procedure known as stripping consists of repetitive manual kneading of the teat [6,11 ]. This protocol usually involves overnight separations as well, but, in this case, prior oxytocin administration apparently does not result in increased milk yields [6]. Stripping also involves considerable time and effort. In a relatively productive effort, Galef and Sherry [6] obtained up to 2 mi of milk from previously
separated dams, but every nipple was milked and the procedure took 40 min. In this report I describe an efficient technique for the collection of rat milk which involves simple cannulation of mammary tissue. This technique is compared to stripping and simple aspiration. Each of the three procedures are evaluated with 'and without prior lengthy separation of the dam from its litter, and with and without prior oxytocin injections. METHOD
Animals and Housing Thirty-six adult Wistar dams, 10-13 days post-partum, served in the experiment. Each was nursing a litter of eight-nine pups at the time of testing. Each dam and litter was housed in a 40 x 20 x 24 cm Plexiglas terrarium with food and water continuously available. A 12/12 light-dark regimen was in effect, with light onset at 7 a.m.
Design and Procedure Six animals were assigned to each of six groups. Animals in three groups were separated from their litters 4--6 hr prior to milking (Short Separation) and animals of the remaining three groups were separated from their litters 22-24 hr prior to milking (Long Separation). One Short Separation and one Long Separation group were then assigned to each of three different milking conditions, Stripping, Aspiration and Cannulation. All animals were anesthetized (Urethane, 2 g/10 mi, 1 cc/100 g body weight) immediately prior to milking. Stripping involved manual kneading of the teat with repetitive bottom-to-top stroking motions. Expressed milk was collected in graduated cylinders. Aspiration consisted simply of applying suction to the teat. One end of a hollow glass tube was placed over the teat and the other run into a stoppered graduated cylinder, itself attached to an aspirator
C o p y r i g h t © 1979 Brain R e s e a r c h Publications Inc.--0031-9384/79/040795-03502.00/0
796
BRAKE
FIG. 1. An anesthetized dam with mammary cannula. The tube is filled with milk and milk has dripped onto the table beneath the end of the tube.
via a rubber tube. During aspiration the teat was sucked into the tube and milk was collected in the graduated cylinder, Cannulation consisted of collecting milk from a wound in the animal's flank following cannulation of the mammary tissue. A 4-5 cm band was shaved along both flanks, lateral to each row of nipples, from forelimbs to hindlimbs. A 17 ga needle was then inserted into the flank within the shaved band, 1.5-2.0 cm from the nipple, and run through the mammary tissue to create a channel. The needle was then removed. A length of PE 160 tubing with one flanged end was inserted beneath the skin (through an incision) on the side of the teat opposite the puncture wound and drawn under the skin and out the puncture wound so that the flanged end caught against the inner wall of the skin and made a seal. Milk flowed down the channel and out the tube and was collected in graduated cylinders. An animal with one Cannulation tube in place is depicted in Fig. I. Two milk collections were taken from each of the animals. Milk was collected for 15 min both prior and subsequent to an IP injection of 0.4 ml oxytocin (Pitocin). The second collection began 18 min after the first and 3 min following oxytocin administration. Milk was collected from the middle four nipples of each row. For half the animals of each group the first collection was made from the left row and for half the first collection was made from the right row.
RESULTS AND DISCUSSION The mean amount of milk collected from the four teats in each condition is shown in Fig. 2. As is immediately obvious, the Cannulation procedure, in conjunction with oxytocin injections, was the most efficient milk collection procedure.
c3
3.0--
tu i.-ulid 2.5 -~ _..i O 2.0 u ~, 1.5
[ ] Oxytocin [ ] No Oxytocin
_~ 1.0 IX
0.5 0
LONG
SHORT
GANNULATION
LONG
SHORT
STRIPPING
LONG
SHORT
ASPIRATION
FIG. 2. Mean amount of milk collected with three milking procedures following Long (22-24 hr) or Short (4--6 hr) Separations from pups and with or without prior oxytocin administration.
The 2.7 ml average is somewhat less than the 3-7 ml collected by Miller and Dymsza with the use of a milking maching [ 13], but it should be remembered that they milked every teat over a 40-min period while we milked only four over a 15-min period. It was quite typical, particularly in the Long Separation condition, for milk to flow profusely from the tubes until each gland had been drained, usually within 10 min. A two-tailed independent t-test revealed that the amount of milk obtained with Cannulation + oxytocin in the Long Separation animals was greater than the amount obtained in all other Long Separation conditions, t(34)=7.61, p<0.001. This same comparison, Cannulation + oxytocin
MILK C O L L E C T I O N IN RATS
797
vs. all other conditions, was significant for the Short Separation groups as well, t(34)=4.38, p<0.001. Still, engorgement of the mammary glands does increase the yield of milkings as the Cannulation + oxytocin condition resulted in more milk from the Long Separation animals than from the Short Separation animals, t(10)= 1.82, p<0.05. The amount of milk obtained via Cannulation without the use of oxytocin administration was negligible. Apparently, the milk-releasing function of oxytoein [13] causes considerably more milk to be dumped into the wound created by cannulation than would accumulate there otherwise. The effects of engorgement resulting from differential separation is less pronounced in these groups. It should be mentioned that traces of blood appeared in the milk collected in the Cannulation procedures, but only in about 15% of all cannulations. The Stripping procedure resulted in considerably less milk than did the Cannulation procedure. In addition, the oxytocin manipulation proved ineffective in the Long Separation animals, as previously reported by Galef and Sherry [6]. In the Short Separation animals, however, oxytocin was apparently necessary for the expression of milk; Short Separation animals which did not receive oxytocin yielded less milk than did the other Stripping animals, t(22)=6.52, p<0.001. Thus, it makes little difference whether oxytocin injection or lengthy prior separation is employed in conjunction with the Stripping procedure, as long as one of them is. Even so, the yields are meager when compared to those of the Cannulation + oxytocin groups. It should be mentioned here that oxytocin injections are effective whether they precede or follow milking without oxytocin. For both the Cannulation and Stripping groups we departed from the normal procedure on occasion and con-
ducted an oxytocin milking without first obtaining a nooxytocin milking. No difference was found in the amount of milk collected between the two orders. The Aspiration technique resulted in virtually no expressed milk in any condition. Indeed, only in the Long Separation + oxytocin condition was any measureable amount of milk collected. It should be pointed out that the nipples were not kneaded during this procedure and that aspiration provided the only stimulation of the teats. Apparently, aspiration is most effective in conjunction with stripping or more powerful milking machines [2, 13, 14]. Finally, we should mention that the Cannulation procedure is repeatable. Employing Long Separation and oxytocin injections in four dams, we were able to collect an average 1.7 ml from the same four nipples cannulated two days previously. Thus, by alternating nipple rows and allowing pups to remain with the dam for a day every other day, the dam might be milked over an extended period. In summary, Cannulation of mammary tissue following IP oxytocin administration is the most efficient and productive method for the collection of milk from the lactating rat. Lengthy prior separation of the dam from its litter further increases the volume of milk obtainable with this procedure. Stripping, in conjunction with either oxytocin injection or lengthy prior separation, results in approximately 31)% of the milk collected via Cannulation + oxytocin, while Aspiration (without kneading) yields no appreciable amount of milk. ANKNOWLEDGEMENTS The author wishes to thank Myron Hofer for a critical reading of the manuscript.
REFERENCES
1. Cowie, A. T. and J. S. Tindal. The Physiology o f Lactation. London: Arnold, 1971. 2. Cox, W. M. and A. J. Mueller. The composition of milk from stock rats and an apparatus for milking small laboratory animals. J. Nutr. 13: 24%261, 1937. 3. Drewett, R. F., C. Statham and J. B. Wakerley. A quantitative analysis of the feeding behavior of suckling rats. Anita. Behav. 22: 907-913, 1974. 4. Dymsza, H. A., D. M. Czajka and S. A. Miller. Influence of artificial diet on weight gain and body composition of the neonatal rat. J. Nutr. 84: 100, 1964. 5. Friedman, M. I. Some determinants of milk ingestion in suckling rats. J. comp. physiol. Psychol. 89: 636--647, 1975. 6. Galef, B. G. J. and D. F. Sherry. Mother's milk: A medium for transmission of cues reflecting the flavor of the mothers' diet. J. comp. physiol. Psychol. 83: 374-379, 1973. 7. Hall, W. G. and J. S. Rosenblatt. Suckling behavior and intake control in the developing rat pup. J. cornp, physiol. Psychol. 91: 1232-1247, 1977. 8. Houpt, K. A. and A. N. Epstein. Ontogeny of controls of food intake in the rat: GI fill and glucodeprivation. Am. J. Physiol. 225: 58-66, 1973.
9. Houpt, K. A. and T. R. Houpt. Effects of gastric loads and food deprivation on subsequent food intake in suckling rats. J. comp. physiol. Psychol. 88: 764-772, 1975. 10. Lincoln, D. W., A. Hill and J. B. Wakerley. The milk-ejection reflex of the rat: An intermittant function not abolished by surgical levels of anesthesia. J. Endocr. 57: 459-476, 1973. 11. Luckey, T. D., T. J. Mende and J. Pleasants. The physical and chemical characterization of rats' milk. J. Nutr. 54: 345-359, 1954. 12. Messer, M., E. B. Thoman, A. G. Terasa and P. R. Dallman. Artificial feeding of infant rats by gastric infusion. J. Nutr. 98: 404-410, 1969. 13. Miller, S. A. and H. A. Dymsza. Artificial feeding of neonatal rats. Science 141: 517, 1963. 14. Nelson, W. W., A. Kaye, M. Moore, H. Williams and B. L. Herrington. Milking techniques and the composition of guinea pig milk. J. Nutr. 44: 585-594, 1951. 15. Wakerley, J. B. and R. F. Drewett. Patterns of suckling in the infant rat during spontaneous milk-ejection. Physiol. Behav. 15: 277-281, 1975.
Procedures for the Collection of Milk from the Rat Dam S T E P H E N C. B R A K E
Department o f Psychiatry, Albert Einstein College o f Medicine, Montefiore Hospital and Medical Center 111 East 210th Street, Bronx, N Y 10467 ( R e c e i v e d 26 O c t o b e r 1978) BRAKE, S. C. Procedures for the collection o f milk from the rat dam. PHYSIOL. BEHAV. 22(4) 795-797, 1979.--Three procedures for the collection of milk from the rat dam were investigated. Before each procedure, dams were separated from
their litters for either 4-6 or 22-24 hr and half received oxytocin injections (IP) prior to milking. The most productive and efficient milking procedure involved cannulation of the mammary tissue following 22-24 hr separations and oxytocin injections. Milk collection
Lactation
Oxytocin
Rat's milk
I N V E S T I G A T I O N S of suckling and ingestion of infant rats have increased substantially in recent years. It is thus somewhat surprising that experimenters interested in administering nutrient to infant rat pups usually employ heavy cream, bovine milk, or various milk formulas rather than rat milk itself [5, 7, 8, 9]. The composition and viscosity of the most widely used formulas [4,12] are similar to that of rat milk [2, 4, 11], but even they result in less than normal weight gains or altered body-fat composition in pups reared on them [4, 13, 15]. It may also be desirable to avoid the use of milk substitutes in the study of preferential consummatory behavior, as it has been shown that rat pups are capable of discriminating different milk tastes [6]. The relative inefficiency of most milk collection techniques has made the use of rat milk prohibitively troublesome. One of the earliest procedures involved the use of rather elaborate milking machines, originally designed for use with other species [2, 13, 14]. These aspiration devices were usually employed in conjunction with IP oxytocin injections given 5-10 rain prior to milking. Oxytocin, in physiological doses, initiates natural milk-ejection [3,15] and the larger IP doses (0.2-0.4 ml) had the effect of "flooding" the ducts of the mammary glands with far more milk than is usually available to suckling pups [10]. Further, overnight separation of the lactating dam and pups results in engorgement of mammary tissue [1,5], and so separations were often employed as well. Apparently, 3-7 ml could be collected by using the machines in this manner, but it required considerable time and effort [13]. Further, Luckey et al. [11] reported that the machines were ineffective with difficult milkers. Another common procedure known as stripping consists of repetitive manual kneading of the teat [6,11 ]. This protocol usually involves overnight separations as well, but, in this case, prior oxytocin administration apparently does not result in increased milk yields [6]. Stripping also involves considerable time and effort. In a relatively productive effort, Galef and Sherry [6] obtained up to 2 mi of milk from previously
separated dams, but every nipple was milked and the procedure took 40 min. In this report I describe an efficient technique for the collection of rat milk which involves simple cannulation of mammary tissue. This technique is compared to stripping and simple aspiration. Each of the three procedures are evaluated with 'and without prior lengthy separation of the dam from its litter, and with and without prior oxytocin injections. METHOD
Animals and Housing Thirty-six adult Wistar dams, 10-13 days post-partum, served in the experiment. Each was nursing a litter of eight-nine pups at the time of testing. Each dam and litter was housed in a 40 x 20 x 24 cm Plexiglas terrarium with food and water continuously available. A 12/12 light-dark regimen was in effect, with light onset at 7 a.m.
Design and Procedure Six animals were assigned to each of six groups. Animals in three groups were separated from their litters 4--6 hr prior to milking (Short Separation) and animals of the remaining three groups were separated from their litters 22-24 hr prior to milking (Long Separation). One Short Separation and one Long Separation group were then assigned to each of three different milking conditions, Stripping, Aspiration and Cannulation. All animals were anesthetized (Urethane, 2 g/10 mi, 1 cc/100 g body weight) immediately prior to milking. Stripping involved manual kneading of the teat with repetitive bottom-to-top stroking motions. Expressed milk was collected in graduated cylinders. Aspiration consisted simply of applying suction to the teat. One end of a hollow glass tube was placed over the teat and the other run into a stoppered graduated cylinder, itself attached to an aspirator
C o p y r i g h t © 1979 Brain R e s e a r c h Publications Inc.--0031-9384/79/040795-03502.00/0
796
BRAKE
FIG. 1. An anesthetized dam with mammary cannula. The tube is filled with milk and milk has dripped onto the table beneath the end of the tube.
via a rubber tube. During aspiration the teat was sucked into the tube and milk was collected in the graduated cylinder, Cannulation consisted of collecting milk from a wound in the animal's flank following cannulation of the mammary tissue. A 4-5 cm band was shaved along both flanks, lateral to each row of nipples, from forelimbs to hindlimbs. A 17 ga needle was then inserted into the flank within the shaved band, 1.5-2.0 cm from the nipple, and run through the mammary tissue to create a channel. The needle was then removed. A length of PE 160 tubing with one flanged end was inserted beneath the skin (through an incision) on the side of the teat opposite the puncture wound and drawn under the skin and out the puncture wound so that the flanged end caught against the inner wall of the skin and made a seal. Milk flowed down the channel and out the tube and was collected in graduated cylinders. An animal with one Cannulation tube in place is depicted in Fig. I. Two milk collections were taken from each of the animals. Milk was collected for 15 min both prior and subsequent to an IP injection of 0.4 ml oxytocin (Pitocin). The second collection began 18 min after the first and 3 min following oxytocin administration. Milk was collected from the middle four nipples of each row. For half the animals of each group the first collection was made from the left row and for half the first collection was made from the right row.
RESULTS AND DISCUSSION The mean amount of milk collected from the four teats in each condition is shown in Fig. 2. As is immediately obvious, the Cannulation procedure, in conjunction with oxytocin injections, was the most efficient milk collection procedure.
c3
3.0--
tu i.-ulid 2.5 -~ _..i O 2.0 u ~, 1.5
[ ] Oxytocin [ ] No Oxytocin
_~ 1.0 IX
0.5 0
LONG
SHORT
GANNULATION
LONG
SHORT
STRIPPING
LONG
SHORT
ASPIRATION
FIG. 2. Mean amount of milk collected with three milking procedures following Long (22-24 hr) or Short (4--6 hr) Separations from pups and with or without prior oxytocin administration.
The 2.7 ml average is somewhat less than the 3-7 ml collected by Miller and Dymsza with the use of a milking maching [ 13], but it should be remembered that they milked every teat over a 40-min period while we milked only four over a 15-min period. It was quite typical, particularly in the Long Separation condition, for milk to flow profusely from the tubes until each gland had been drained, usually within 10 min. A two-tailed independent t-test revealed that the amount of milk obtained with Cannulation + oxytocin in the Long Separation animals was greater than the amount obtained in all other Long Separation conditions, t(34)=7.61, p<0.001. This same comparison, Cannulation + oxytocin
MILK C O L L E C T I O N IN RATS
797
vs. all other conditions, was significant for the Short Separation groups as well, t(34)=4.38, p<0.001. Still, engorgement of the mammary glands does increase the yield of milkings as the Cannulation + oxytocin condition resulted in more milk from the Long Separation animals than from the Short Separation animals, t(10)= 1.82, p<0.05. The amount of milk obtained via Cannulation without the use of oxytocin administration was negligible. Apparently, the milk-releasing function of oxytoein [13] causes considerably more milk to be dumped into the wound created by cannulation than would accumulate there otherwise. The effects of engorgement resulting from differential separation is less pronounced in these groups. It should be mentioned that traces of blood appeared in the milk collected in the Cannulation procedures, but only in about 15% of all cannulations. The Stripping procedure resulted in considerably less milk than did the Cannulation procedure. In addition, the oxytocin manipulation proved ineffective in the Long Separation animals, as previously reported by Galef and Sherry [6]. In the Short Separation animals, however, oxytocin was apparently necessary for the expression of milk; Short Separation animals which did not receive oxytocin yielded less milk than did the other Stripping animals, t(22)=6.52, p<0.001. Thus, it makes little difference whether oxytocin injection or lengthy prior separation is employed in conjunction with the Stripping procedure, as long as one of them is. Even so, the yields are meager when compared to those of the Cannulation + oxytocin groups. It should be mentioned here that oxytocin injections are effective whether they precede or follow milking without oxytocin. For both the Cannulation and Stripping groups we departed from the normal procedure on occasion and con-
ducted an oxytocin milking without first obtaining a nooxytocin milking. No difference was found in the amount of milk collected between the two orders. The Aspiration technique resulted in virtually no expressed milk in any condition. Indeed, only in the Long Separation + oxytocin condition was any measureable amount of milk collected. It should be pointed out that the nipples were not kneaded during this procedure and that aspiration provided the only stimulation of the teats. Apparently, aspiration is most effective in conjunction with stripping or more powerful milking machines [2, 13, 14]. Finally, we should mention that the Cannulation procedure is repeatable. Employing Long Separation and oxytocin injections in four dams, we were able to collect an average 1.7 ml from the same four nipples cannulated two days previously. Thus, by alternating nipple rows and allowing pups to remain with the dam for a day every other day, the dam might be milked over an extended period. In summary, Cannulation of mammary tissue following IP oxytocin administration is the most efficient and productive method for the collection of milk from the lactating rat. Lengthy prior separation of the dam from its litter further increases the volume of milk obtainable with this procedure. Stripping, in conjunction with either oxytocin injection or lengthy prior separation, results in approximately 31)% of the milk collected via Cannulation + oxytocin, while Aspiration (without kneading) yields no appreciable amount of milk. ANKNOWLEDGEMENTS The author wishes to thank Myron Hofer for a critical reading of the manuscript.
REFERENCES
1. Cowie, A. T. and J. S. Tindal. The Physiology o f Lactation. London: Arnold, 1971. 2. Cox, W. M. and A. J. Mueller. The composition of milk from stock rats and an apparatus for milking small laboratory animals. J. Nutr. 13: 24%261, 1937. 3. Drewett, R. F., C. Statham and J. B. Wakerley. A quantitative analysis of the feeding behavior of suckling rats. Anita. Behav. 22: 907-913, 1974. 4. Dymsza, H. A., D. M. Czajka and S. A. Miller. Influence of artificial diet on weight gain and body composition of the neonatal rat. J. Nutr. 84: 100, 1964. 5. Friedman, M. I. Some determinants of milk ingestion in suckling rats. J. comp. physiol. Psychol. 89: 636--647, 1975. 6. Galef, B. G. J. and D. F. Sherry. Mother's milk: A medium for transmission of cues reflecting the flavor of the mothers' diet. J. comp. physiol. Psychol. 83: 374-379, 1973. 7. Hall, W. G. and J. S. Rosenblatt. Suckling behavior and intake control in the developing rat pup. J. cornp, physiol. Psychol. 91: 1232-1247, 1977. 8. Houpt, K. A. and A. N. Epstein. Ontogeny of controls of food intake in the rat: GI fill and glucodeprivation. Am. J. Physiol. 225: 58-66, 1973.
9. Houpt, K. A. and T. R. Houpt. Effects of gastric loads and food deprivation on subsequent food intake in suckling rats. J. comp. physiol. Psychol. 88: 764-772, 1975. 10. Lincoln, D. W., A. Hill and J. B. Wakerley. The milk-ejection reflex of the rat: An intermittant function not abolished by surgical levels of anesthesia. J. Endocr. 57: 459-476, 1973. 11. Luckey, T. D., T. J. Mende and J. Pleasants. The physical and chemical characterization of rats' milk. J. Nutr. 54: 345-359, 1954. 12. Messer, M., E. B. Thoman, A. G. Terasa and P. R. Dallman. Artificial feeding of infant rats by gastric infusion. J. Nutr. 98: 404-410, 1969. 13. Miller, S. A. and H. A. Dymsza. Artificial feeding of neonatal rats. Science 141: 517, 1963. 14. Nelson, W. W., A. Kaye, M. Moore, H. Williams and B. L. Herrington. Milking techniques and the composition of guinea pig milk. J. Nutr. 44: 585-594, 1951. 15. Wakerley, J. B. and R. F. Drewett. Patterns of suckling in the infant rat during spontaneous milk-ejection. Physiol. Behav. 15: 277-281, 1975.