Practical aspects of milk collection in the rat

Practical aspects of milk collection in the rat C. T. Rodgers Safety of Medicines, ZENECA Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire SK 4TG, UK Summary It is a requirement as part of the safety evaluation of novel compounds, which are intended to be administered to pregnant women or women with young children, that the potential for the compound or its metabolites to be transferred from a dam to its litter during lactation is measured. The data obtained from the rat milking studies are also used to support the rat peri- and post-natal studies. Therefore it is necessary to collect multiple milk samples from lactating rats. In this paper it was decided to concentrate on three areas: 1. The factors which had to be considered during the development of the rat milking technique; 2. The outline of the basic study design for a rat milking study; 3. The technique for milking rats. Keywords Rats; milking; oxytocin; parturition Direct and indirect methods of milk collection were considered, e.g. milking by hand into the collecting tube or euthanasia of pups and taking milk from their stomachs. Previously published methods were also considered (see references). None of the methods considered were deemed to be entirely suitable, due to the generally complicated nature of the equipment and procedures involved or because most methods involved continuous restraint or anaesthesia. The majority of the previously published methods also concentrated on obtaining one large milk sample per day; for the purpose of metabolism studies we required multiple milk samples from the same animals over a 4-day period. Therefore it was necessary to establish an improved method of collecting multiple milk samples from lactating rats, using a quick and simple technique. The method was developed and validated using a Zeneca test compound. All procedures were conducted in compliance with the Animals (Scientific Procedures) Act 1986. Accepted 6 December 1994 Materials The development technique and methods of the rat milking Several points were considered when developing the technique for milking rats. The main areas under debate were: fi) The litter size, i.e. should the size of the litter be reduced to improve the milk yield? (ii) The optimum time for milking after parturition? (iii) The length of separation of the dam from the litter prior to milking? fiv) How to stimulate 'let down' of the dam's milk? (v) How to collect the milk, and whether an anaesthetic should be used? (vi) The time to return the dam to its litter after milking? Afterresearchand initial sightingwork it was decided to proceed on the following basis: (i) In an effort to increase the milk yield the litter size was reduced to 6 pups per litter within 48 h of parturition. Laboratory Animals (1995) 29. 450-455

Milk collection in the rat 451 lii) The optimum time for milk collection was considered to be 14 days after parturition due to the amount of milk being produced. (iii) The dam was removed from the litter for approximately 5 min prior to milking. Longer periods of removal did not increase the milk yield. (iv) To stimulate 'let down' each dam was administered a single intraperitoneal injection of oxytocin. The optimum dose was considered to be 4 IU/kg body weight administered approximately 5 min prior to milking. Raising the dose of oxytocin did not improve the milk yield. (vj The milk was collected using a vacuum system with no anaesthetic beingrequired. [vi) The dam was returned to its litter immediately after milking. Study design Ten Alderley Park, Alpk:APfSDoutbred, Wistar derived rats, body weight 280 to 380 g, were supplied by the Zeneca Barriered Animal Breeding Unit on day 1 of pregnancy. The rats were housed in a conventional animal facility in individual clear polycarbonate solid bottom cages (44x 28x 20cmJ. Sawdust [5 mesh) supplied by Wood Treatment Ltd (Bosley, Cheshire) was provided for bedding. Two days prior to littering nesting material in the form of strips of soft tissue paper was provided for each dam. The animals were fed ad libitum with modified R &. M Number 1 irradiated pelleted diet, supplied by Special Diets Services Ltd (Witham, Essex) to Zeneca specifications. Water was available ad libitum via gravity-fed water bottles. The animal room temperature and relative humidity were maintained at 19-23 C and 45-70%, and the lighting was on a l2h dark, 12 h artificial light cycle. The animals were observed at least twice daily from arrival until the end of the study and any abnormalities recorded. Within 48 h of parturition each litter was reduced to 6 pups. All extra pups were killed by inhalation of Halothane (Fluothane Zeneca Ltd). The optimum time for dosing and milking was considered to be day 14 after parturition, due to the amount of milk being produced. The day of parturition was classed as day O. Nine of the dams delivered were allocated to the study which consisted of 3 groups of 3 dams. One control group (Group IJ and 2 test groups (Groups II and III). Milk samples were obtained from all the dams, in all 3 groups, on the day prior to dosing to provide control samples. The control Group I animals remained undosed to provide additional control milk samples. All animals in Groups II and III, received a single oral dose of the test compound, at identical dose levels and dose volumes, on day one of the study [14 days after parturition). Milk samples were collected twice on each of days 1 and 2, from all Group I animals, to provide extra control samples. Milk samples were collected from Group II animals at 30 min, 3, 9, 24 and 48 h after dosing and from Group III animals at I, 6, 12, 24 and 48 h after dosing. The sample time points on day one were split; 3 time points for Group II animals and 3 time"points for Group III animals; this was to ensure a wide spread of time points. In addition it was deemed impractical to collect more than 3 milk samples from anyone dam, within a 12-h period. Milk samples (0.75ml) were taken from each dam at each appropriate time point and diluted 50 : 50 with sterile physiological saline, prior to being stored at - 20 C. A small blood sample [approximately 0.3 ml) was also taken at each time point, immediately after milking, from all Group II and III animals. These blood samples were taken via a lateral tail vein, using a 21G butterfly needle, Micro-Flow, LDKS oversan, (International Market Supplies, CheshireJ and stored, divided between 3 combustacones per sample, at + 4 C. To enable the analysts to identify the quantity of compound, or its metabolites present in the milk and blood samples, the test compound administered was radiolabelled with Carbon 14. All samples were submitted to the Zeneca Drug Kinetics Group for analysis.

452 Rodgers Individual body weights and animal observations were recorded daily for all the control and test dams throughout the study period. There was no effect on body weight and there were no atypical observations recorded throughout the study period. At the end of the study all dams and litters were killed by inhalation of halothane. All carcasses were discarded without further examination. The sample time points, route of administration and the dose level of the test compound administered may be varied on individual studies, depending on the specific action of the compound. Milk collection To stimulate the 'let down' of milk, each dam was removed from its litter approximately 5 min prior to milking and administered a single intraperitoneal dose of oxytocin. The oxytocin was prepared as a 4IU/ml solution and administered at a dose level of 4IU/Kg body weight, at a standard dose volume of 1 ml/kg body weight. Care was taken to avoid damaging any mammary tissue when dosing lactating dams by the intraperitoneal route. Milk samples were collected using a vacuum milking system, the equipment is presented ISlUCONe!" SUC'l1ON cul I TO RAT8 NIPI'IL TUse HOLDeR. GI.AISllJAA. I COl.L!CTION TUSe. TO VACUUM UNe. Fig 1 Diagrammatic representation of rat milking equipment in Fig. 1. The equipment consisted of a soft moulded silicone suction cup llength 17mm,ID 16mm tapering to ID Imm, wall 1.5 mm) made by Zeneca engineers, connected to a 500 mm length of polythene tubing (ID 1.14mm, OD 1.57mm). One end of the polythene tubing was inserted 7 mm into the silicone suction cup and secured with a small amount of super glue (Loctite UK). The other end of the polythene tubing was passed through a size 49 rubber bung. This was achieved using a 2.mm gauge trocar and cannula to ensure an airtight seal was maintained between the tubing and the rubber bung. The rubber bung, with a 50 mm length of tubing extending from its base, was placed firmly in the top of a glass vacuum flask. The glass vacuum flask (height 145 mm, neck ID 52.mm, base OD 60 mm), was made by Zeneca glass blowers, with two side arms (ID 8 mm) and a glass tube holder (height 0mm, ill mm) moulded to the flask. Only the tip of the tubing was in the collection tube (1.5 ml Eppendorf plain blood tube), this was to ensure a constant flow of milk could be maintained, even when the collection tube was almost full. Two technicians were required to carry out the milking procedure. One technician was responsible for restraining the dam and the second technician was responsible for milking the dam. The fur surrounding each nipple was moistened with lukewarm water to allow a good seal with the silicone suction cup. A small amount of milk was then expressed from an upper thoracic nipple, by gently squeezing the nipple between the thumb and forefinger (See Fig. 2). The silicone suction cup was placed over the nipple and the nipple 'milked' by very gentle thumb and forefinger pressure applied on and off the silicone suction cup. The technician restraining the dam had to ensure that the area of skin around the nipple being milked was drawn quite tight; this was so that the technician milking the dam could maintain a good seal between the skin and the silicone suction cup.

Milk collection in the rat 453 Fig 2 Expression of milk immediately prior to milk collection Fig 3 Milking apparatus in use The milk was drawn through the plastic tubing and into the sample collection tube. This was achieved by the vacuum created in the glass flask. The vacuum was created by using a vacuum line. A flow meter was placed between the vacuum line and the vacuum flask to enable an increase or reduction of the vacuum. The vacuum was controlled at 200 m bar. A pulsation of vacuum was created by placing a finger on the control hole and repeated slight movements of the finger off the control hole would allow small amounts of air into the flask, therefore increasing and decreasing the vacuum. Excessive vacuum was avoided as this would have resulted in the nipple becoming attached to the inside of the silicone suction cup and cessation of the milk flow. Milk collection is shown in Fig. 3. Between each sample the silicone suction cup and plastic tubing were flushed through with warm water to prevent any cross contamination of samples. The dam was returned to its litter immediately after sampling. Results It was found that milk could be collected from all nipples. When commencing milking we started with the 2 upper thoracic nipples, as the skin could be drawn quite tight around these nipples to achieve a good seal with the silicone suction cup. After the 2 upper thoracic nipples had been milked, the lower thoracic, upper abdominal and lower abdominal nipples were milked. It was harder.to achieve an air tight seal between the skin and the silicone suction cup on the lower abdominal nipples owing to the amount of loose skin in this region; this increased difficulty in obtaining samples from these nipples. However, when proficient at the technique, it was possible to obtain milk samples from the abdominal and thoracic nipples with a similar degree of success. The skill in the rat milking procedure was to ensure a constant flow of milk, by pulsing the vacuum and using a gentle 'milking' technique. It was not possible to wear gloves during milking procedure as the vacuum tended to suck the end of the glove to the control hole or the silicone suction cup.

454 Rodgers Fig 4 Rat milking equipment and milk samples When conducted correctly it was possible to obtain approximately 0.5 ml of milk from one nipple. Therefore milk samples of 1 to 1.5 ml could be easily obtained from Concentration 3 0.3 (ug/ml). e- Gl Milk. Blood.."'" \, e + 'b",...... "'1;) ~ each dam at each time point (see Fig. 4), with no detrimental effect to either the dam or the litter. The success rate of milking was approximately 95% as occasionally samples could not be obtained. This was generally due to the pups suckling the dam immediately prior to sampling. Due to the procedures involved with the analysis of radio-labelled milk samples, and in an attempt to ensure that a sample could be obtained at each time point, 0.75 ml of milk diluted 50: 50 with sterile physiological saline, was deemed to be the appropriate sample volume to be taken at each time point on a study. Typical milk and blood profiles showing mean data for total radioactivity, generated from 3 animals (30min, I, 3, 6, 9 and 12h) and 6 animals (24 and 48 hj per time point are presented in Fig. 5. The whole procedure for milking and blood sampling one dam could be completed 5 2 0.1 Intravenous dose. ~""""'-'.~"""""'~""""'E) 0.5 o 5 15 20 25 30 35 40 45 50 Concentration 0.03 o 5 15 20 25 30 35 40 45 50 (ug/ml). Concentration (ug/ml). 3 5 \ \\ G. '0. 2 0.3 0.1 '.... ""E)._a_. Intravenous dose. -"'~-""~-''''''-{) _ 0.5 Oral dose. 0.03 o 5 15 20 25 30 35 40 45 50 0.2 o 5 15 20 25 30 35 40 45 50 Fig 5 Examplesof milk and blood profiles for total radioactivity, following single intravenous or oral doses of test compounds

Milk collection in the rat 455 within approximately 15min. Owing to the short amount of time the dam was removed from its litter, it was not deemed necessary to remove the pups from their cage. However, if the dam was required to be removed from its litter for longer than 15min, it was advisable to place the pups in a warming chamber set at approximately 37 C to ensure there was no reduction in their body temperature. 1 any other procedures were required to be carried out on the dam at the same time or during training, the procedure time could be extended without any detrimental effect to either the dam or the litter. Discussion This paper will be of benefit to Drug Kinetics scientists as the apparatus and procedure described allows serial collection of milk samples from lactating rat dams and a basic study design is also provided. This method of milk collection is an improvement on pre~iously published methods due to the simplicity of the equipment and the technique. This method also causes less stress to the animals due to the quick nature of the procedure and as samples can be obtained without the use of continuous restraint or anaesthesia. It was found that milk could be obtained from all the dam's nipples. However, the best results were achieved by taking a small amount of milk from each nipple at each time point to achieve the required sample volume, rather than a large sample from one or two nipples. Milking the two upper thoracic nipples produced the best results, as the skin could be drawn quite tight to attain a good seal with the silicone suction cup. The rat milking technique requires a great deal of patience and manual dexterity to achieve competencej however, with a very gentle touch and a good deal of patience any person could soon learn how to milk rats successfully. Acknowledgments The author gratefully acknowledges Dr A. Warrander, Mr M. Shim well and Mr L. Clarke, Zeneca Pharmaceuticals, for their considerable help during the initial setting up of the study design, analysing of samples and the development of the Rat Milking Technique. References Feller WF, Boretos J (1967) Semiautomatic apparatus for milking mice. [oumal of the National Cancer Institute 38/ 7-11 Fisher AN, Neale MG, Smith DA (1981) Methods for the estimation of the excretion of proxicromil and its metabolites in the milk of nursing rat mothers. Xenobiotica 11, 871-7 Goole JA, Taylor JC (19741 A simple method of milking small animals. [oumal of Physiology 242, 5-7 Hoffmann FA, Sawatzki G/ Schmitt H, Kubanek B (19821 A milker for mice. Laboratory Animal Science 32, 387-8 Kahler H (1942) Apparatus for milking mice. [oumal of the National Cancer Institute 2/ 457-8 Keen el, Lonnerdal B, Sloan MV, Hurley LS (19801 Effects of milking procedure on rat milk composition. Physiology evbehaviour 24/ 613-15 McBurney IT/ Meier H, Hoag WG 119641Device for milking mice. Journal of Laboratory Clinical Medicine 64, 485 Reddy RR, Danker (1965) Lactation studies. [ Dairy Science 48, 978-82 Waynforth HB, Flecknell PA (1992) Experimental and Surgical Technique in the Rat, 2nd Edn. London: Academic Press, pp 90-2