Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

摘要

牛奶是新生儿的主要营养来源。对牛奶成分的分析可能有助于深入了解影响后代健康的母体因素。该方案描述了一种从哺乳大鼠收集牛奶样品的手动方法，然后可用于进一步的下游分析。

摘要

牛奶作为新生哺乳动物的唯一营养来源，为后代的生长发育提供必要的营养和能量。它还含有大量对新生儿发育有很大影响的生物活性化合物。牛奶成分的分析将有助于阐明将母体新陈代谢和健康与后代生长发育联系起来的关键因素。实验室大鼠是母体研究的一种流行的模式生物，鼠奶可用于检查各种母体生理、营养和药物干预对乳汁成分的影响，这可能会影响后代的健康。这里描述了一种手动从哺乳大鼠收集牛奶的简单方法，该方法可以由单个研究人员进行，不需要专门的真空或抽吸设备，并为后续的下游分析提供足够的牛奶。还提出了一种通过测量牛奶样品中奶油的百分比来估计牛奶脂肪含量的方法，称为奶油比容。这些方法最终可用于增加对母婴健康的了解，并阐明与后代正常生长发育有关的母体因素。

引言

Milk is the sole source of nutrition for newborn mammals, providing energy and nutrients for infant growth and development^1,2. While milk mainly consists of cells, lipids, and protein¹, it also contains a plethora of bioactive compounds that modulate early life development of offspring including enzymes, carbohydrates, hormones, antibodies, growth factors, cytokines, exosomes, microvesicles, and small RNAs such as microRNA^1,2. The fundamental role of maternal milk in the establishment of offspring immune and intestinal health³, coupled with evidence that breastfed infants are less susceptible to disease², highlights the importance of identifying the milk constituents associated with disease processes in early life and the molecular mechanisms involved in their actions. The developing rat is a popular model for investigating the effect of various nutritional, physiological, and chemical interventions on early-life development⁴. The analysis of rat milk may therefore provide novel insight into maternal and offspring health.

Current scientific advances now provide increasing opportunities for in-depth investigations of the effects of specific milk constituents on health and disease. For example, sequencing of milk bacterial profiles has elucidated their role in early intestinal colonization of the infant gut⁵, mass spectrometry analysis of milk oligosaccharides have provided insight into the alteration of milk oligosaccharide profiles via maternal diet⁶, and deep sequencing of microRNA secreted in the fat globules of breast milk highlights possible roles in gene transcription, metabolism, and immune function⁷.

Rat models represent one of the most popular model organisms used in maternal studies^8,9. One advantage is their short gestation and lactation periods, lasting only approximately 21 days each; therefore the total time from the start of pregnancy to lactation represents a short period of time in which valuable data can be generated. The larger size of rats compared to mice, in the context of milk collection, may provide a significant advantage with respect to volume of milk and ease of milk collection; milk production in the mouse, for example, seems to be dependent on total body weight with heavier mice producing more milk¹⁰.

Here, a general description for the manual collection of milk from lactating rats is provided. This protocol requires minimal equipment, is non-invasive, inexpensive, and can be used to collect adequate volumes of milk for further downstream analyses. In brief, the dam is anesthetized with isoflurane, milk letdown is stimulated by oxytocin, and milk is collected into capillary tubes via manual expression of the milk. Finally, as two major components of milk are fat and proteins, a brief description of estimating milk fat content using creamatocrit measurements¹¹ and quantification of total protein concentration using a standard protein assay is presented.

方案

This protocol was approved by the University of Calgary Animal Care Committee and conformed to the Guide for the Care and Use of Laboratory Animals.

1. Separate Dam from Offspring

1. Separate the dam from her offspring for a minimum of 5 min prior to milking¹².
   NOTE: The dam can be milked up to 5-6 hr after separation^1,6,13, however periods of separation longer than 4 hr may alter milk composition¹⁴. While separation time does not appear to impact milk collection volume¹², it is advised that a consistent separation time be maintained throughout the study. Milk composition may change throughout lactation¹⁵, therefore attempts should be made to keep the day of milk collection consistent. Maximum milk production is suggested to occur on lactation day 14¹².
2. Using a warming chamber, ensure the pups are able to maintain proper body temperature without the presence of their mother for the duration of the milking procedure.
   NOTE: In the study presented below, milking was performed at weaning, when the dams were approximately 22 weeks old and the offspring 21 days old, therefore no warming chamber was used.

2. Set-up and Preparation

1. Collect all materials required for the milking procedure.
   NOTE: All materials can be found in the materials and equipment table.
2. Place a heating pad on the bench where milking will take place and cover the pad with an absorbent bench under-pad.
3. Set up the anesthetic system. Ensure that the system has sufficient oxygen and isoflurane prior to beginning. Attach the anesthetic mask that will be used for initial anesthesia induction to the machine. Place the mask that will be used for anesthesia maintenance nearby if different than the initial anesthetic mask.
4. Attach a 25 G needle to a 1 ml syringe for oxytocin injection using aseptic technique.
5. Turn on the heating pad so that maternal body temperature is maintained during the milking procedure. NOTE: Monitor the temperature of the heating pad to ensure the pad does not become too hot and cause burns. Alternatively, use a heat source, such as a heated surgical table, that can be set to a specific temperature. 

3. Anesthetize the Dam Using Isoflurane

1. Open the oxygen tank and turn the flow to 1 L (1,000 cc) per min. Turn on the flow of isoflurane and set to 5%. CAUTION: Avoid direct inhalation of anesthetic and prevent accumulation of anesthetic vapors.
2. Anesthetize the dam.
3. Switch over to the maintenance mask if required, placing the dam supine on the absorbent bench pad. Confirm anesthetization by lack of pedal reflex.
4. Reduce the flow of isoflurane to 2-3% for maintenance of anesthesia. Continually monitor dam throughout the procedure to ensure depression of respiration does not occur. NOTE: Once under anesthesia, the dam’s eyes should be protected using a sterile eye lubricant to prevent the eyes from drying out or becoming scratched. 

4. Oxytocin Injection

1. Ensure the oxytocin (20 USP Units/ml) has not passed its expiry date. Disinfect the vial of oxytocin with a sterile alcohol wipe/alcohol cleansing pad.
2. Using aseptic technique, draw up 2 IU (0.1 ml) of oxytocin into the syringe. Use a new needle and syringe for each dam that will be milked.
   NOTE: Oxytocin doses generally range from single injections of 1 to 5 IU^1,6,12,16. Alternatively, a dose of 4 IU/kg body weight can be used¹². A single dose of 2 IU can be repeated once if difficulty milking is encountered.
3. Inject the oxytocin intraperitoneally. Insert the needle into the lower right quadrant of the abdomen with the needle pointing towards the head, at an angle of 15-30°, about 0.5 cm deep.
4. Pull back on the plunger to ensure negative pressure prior to injection. If any fluid (blood, urine, intestinal contents, etc.) is aspirated into the syringe, remove the needle and attempt the injection with a new needle and syringe. If no fluid is aspirated inject the oxytocin and discard the needle and syringe immediately into a biohazard container.
5. Wait approximately 5-15 min for the oxytocin to stimulate milk letdown.

5. Preparation of Milking Sites

1. Choose the sites/teats from which milk will be collected. Milk can be collected from any teat¹².
2. Gently remove the fur around the teats to be milked with the trimmers, as fur may cause difficulty in sample collection due to wicking of the milk. Be gentle - the skin around the teats is extremely sensitive and may be dry and as such is susceptible to scratches and tears.
3. Sterilization of the teat is not necessary, but optionally, clean the teat with lukewarm water after the fur is removed. Prepare at least two sites as more than one site may be required for milk collection.
   NOTE: If the milk analysis includes microbial profiling, the teat area may require sterilization with iodine⁵.

6. Milk Collection

1. Gently squeeze the base of the teat, manually expelling the milk for collection.
   NOTE: If the milk analysis includes microbial profiling, the first few drops of milk should be discarded.
2. Collect the milk droplets into a capillary tube, filling the capillary tube. Capillary tubes that accommodate larger volumes (e.g., 50 µl) ease the process.
   NOTE: If difficulty in collecting milk is encountered, a second dose of oxytocin can be administered. It is recommended the dose not exceed 4 IU total.
3. Dispense the milk from the capillary tube into a sterile microcentrifuge tube by touching the end of the tube that was used to draw milk from the teat to the side of the microcentrifuge tube - observe the milk being drawn out of the capillary tube via capillary action.
   NOTE: 'Blow out' the milk that is not drawn into the tube using an 18 G needle attached to a 1 ml syringe.
4. Continually monitor the dam for signs of pain or respiratory depression and adjust the flow of isoflurane accordingly.
5. Continue to collect milk as described in this section until sufficient milk has been collected for the chosen milk analysis. For the creamatocrit and protein concentration determination described below, collect 0.25 ml of milk.
   NOTE: Use a different milking site if milk letdown slows or the chosen site does not expel milk sufficiently. A maximum of approximately 2.5 ml of milk per animal can be collected¹⁷, or up to 0.5 ml per teat. The authors recommend that total time under anesthetic be limited to approximately 45-60 min, or 45 min of milking.
6. Collect milk into a microhematocrit tube for creamatocrit measurement from fresh milk samples, and seal the end of the tube with clay sealant. Label the microhematocrit tube with the dam ID and store the milk sample upright.
7. When milking is complete, turn off the flow of isoflurane and oxygen. Remove the mask from the dam and continue to monitor dam until awake. It is recommended that if not fully conscious, the dam should be placed on an absorbent bench pad during the recovery period, rather than directly on the cage bedding, to prevent the bedding from being aspirated or scratching the dam’s eyes during recovery. 
   NOTE: Do not leave the dam unattended until it has regained sufficient consciousness to maintain sternal recumbancy.
8. If no further analyses are required, freeze milk at -80 °C. Others have suggested that milk can be stored for up to 3 hr at 4 °C or 5 months at -20 °C¹⁸.

7. Creamatocrit Measurement

1. Estimate the fat content in the milk by calculating the milk creamatocrit (the percentage of cream in the milk sample)¹⁹.
   NOTE: Measurements with human milk have demonstrated that either fresh or frozen milk can be used for a creamatocrit measurement, however fresh milk is more highly correlated (r = 0.92 versus r = 0.90) with lipid concentration¹¹. The use of fresh or frozen milk for creamatocrit measurements should be kept consistent across the study, as thawed milk is associated with a small decrease in creamatocrit values¹¹.
2. For fresh milk, collect a sample of milk from the teat into a microhematocrit tube; fill at least ¾ full (approximately 15-20 µl). Alternatively, draw fresh milk from the collected sample into the capillary tube after mixing well. Seal the end with clay sealant.
3. Place the capillary tube into the hematocrit spinner, with the sealed end pointing towards the outside, ensuring the centrifuge is balanced.
4. Begin the hematocrit spin (120 sec at 13,700 x g).
   NOTE: Spin time or speed may change depending on the model of centrifuge used.
5. Remove the tube from the centrifuge after the spin is complete and perform the measurements for calculating the creamatocrit. Observe the sample's separation into a cream layer and a clear layer.
6. Measure and record the total length of fluid in the tube and the length of the fat (cream) layer using calipers or a ruler.
   NOTE: The creamatocrit is expressed as the percentage of the cream layer within the the milk sample¹⁹, calculated as (length of cream layer/total length of milk column) x 100 (Figure 1A). Calculate fat concentration and energy values from the creamatocrit measurement as follows: Fat concentration (g/L) = (creamatocrit (%)-0.59)/0.146 (Figure 1B)¹⁹; Energy value (kcal/L) = 290 + (66.8*creamatocrit (%) (Figure 1C)¹⁹.

8. Protein Concentration Determination

1. Using Bovine Serum Albumin as a protein standard, determine total milk protein concentration using a standard protein assay, such as a Lowry protein assay⁶.
   NOTE: Dilution of the milk may be necessary for the milk protein measurements to fall within the standard curve of the assay.

结果

Milk was collected as described at weaning from Wistar dams (approximately 22 weeks old, weighing 350 to 400 g) that consumed a control (AIN-93G, n = 5), high protein (40% casein wt/wt, n = 5), or high prebiotic fibre (21.6% wt/wt, 1:1 ratio of oligofructose and inulin, n = 4) diet throughout pregnancy and lactation. The oxytocin dose was 2 IU. Milk was collected using capillary tubes, and one tube was spun using a hematocrit spinner to determine creamatocrit (Figure 1A), which was then used to estimate ...

讨论

随着人们对生命早期发展研究的兴趣增加，对母乳成分的研究也有所增加。作为新生儿期唯一的营养来源，牛奶中的生物活性化合物对于理想的生长发育至关重要，尤其是在肠道和免疫健康方面³.这里介绍的是一种简单的非侵入性方法，从哺乳期大鼠身上收集足以进行下游分析的乳汁，例如低聚糖分析⁶。该方法不需要专门的真空设备，可以由一个人执行。

虽然该协议旨在在哺乳期间的单个时间点使用，但其他人在整个研究过程中进行了连续母乳收集^12,15。然而，由于连续挤奶可能会影响牛奶成分¹⁷，因此建议研究人员根据感兴趣的结果确定最适合其研究的牛奶收集频率和数量。此外，虽然其他人在大鼠^14,17的挤奶过程中使用了注射麻醉剂，但该协议涉及使用异氟醚对大鼠进行麻醉。异氟醚是一种可吸入麻醉剂，其优点包括快速诱导和恢复率²⁰ 以及能够轻松调整动物麻醉时间。然而，在小鼠中，与注射麻醉剂¹⁰相比，异氟醚可能会导致产奶量降低，尽管这似乎尚未在大鼠身上进行测试。此外，涉及真空抽吸的方案...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
设备 - 挤奶
1 ml 注射器	BD-Canada	309602
25 G 针头	BD-Canada	305122
18 G 针头	BD-Canada	305196
50 μl 微量分配器 毛细	Fisher Scientific	21-169D
催产素（20 USP 单位/ml）	Bimeda-MTC	1OXY015
PPC 兽医异氟醚吸入麻醉剂，250 ml	费森尤斯 Kabi	M60302	根据兽医的订单使用
酒精制备垫	Dukal	853
吸收剂工作台垫	VWR	82020-845
Maxi-Therm Hyper/Hypothermia毯	辛那提Sub-Zero	274
啮齿动物麻醉机配有蒸发器	Benson Medical Industries Inc.		根据个人实验室需求
，动物面具	Benson Medical Industries， Inc.	50100/50102
离心管	Axygen	MCT-060-C
ChroMini专业修剪器	Wahl
设备 - Creamatocrit
StatSpin SafeCrit塑料微血细胞比容管（未处理）	Fisher Scientific	22-274-914
Critoseal毛细管密封剂托盘	VWR	470161-478
StatSpin CritSpin 微红细胞比容离心机	Beckman Coulter， Inc	X00-004999-001