This work was supported by the Nature Science Foundation grant of Hubei Province, China [Grant No. 2018CFB761].

1. Husbandry
<ol>
	<li>Use 12 week old Kunming mice. 
	NOTE: We used mice (n = 40, 20 males, 37&#8211;46 g, mean 42.38 &#177; 2.39 g) from the Experimental Animals Center of&#160;Tongji&#160;Medical&#160;College.</li>
	<li>House the mice under standard conditions with free access to food and drinking water. Keep two mice in a 530 cm2 cage with wood shaving bedding.</li>
	<li>Maintain a room temperature between 21&#8211;23 &#176;C.</li>
	<li>Feed mice with a normal salt diet (0.3% NaCl) throughout the study.</li>
</ol>
2. Blood sample collection
<ol>
	<li>Prepare the following equipment: vacuum tube (1 mL), butterfly needle, eyeglass magnifier, and plastic restraining holder. Place them on a sterile surface (Figure 1).</li>
	<li>Place a mouse in a plastic restraining holder and wash its tail with warm water (20&#8211;30 &#176;C). Wipe the tail with 70% ethanol-saturated cotton balls to expand the vein. Select the right or left tail vein for blood sampling. Grasp the lower portion of the tail gently and keep the tail straight during blood sample collection.</li>
	<li>Collect the blood. If comparing methods, collect blood in two groups: the &#8220;experimental&#8221; group using the procedure we have developed, presented below, and &#8220;conventional&#8221; group using a conventional method, also presented below.
	<ol>
		<li>Experimental collection:
		<ol>
			<li>Wear an eyeglass magnifier to improve the viewing for puncture of the tail vein. Insert the 22 G butterfly needle tip into one of the lateral tail veins at a position approximately half the distance distally from the tip of the tail at an angel approximately 10&#176;, moving towards the base of the tail for multiple samples.</li>
			<li>Insert the rubber end of the butterfly needle into the vacuum blood collection tube to collect blood (Figure 2). 
			NOTE: If blood stops flowing out during blood collection, the needle angle should be quickly adjusted. To avoid blood coagulation in the needle, another puncture position should be selected if blood stops flowing out after 15 s.</li>
		</ol>
		</li>
		<li>Conventional method:
		<ol>
			<li>Insert the needle connected to a syringe into one of the lateral veins approximately one-third of the distance distally from the tip of the tail.</li>
			<li>When blood appears in the hub, pull back the plunger slowly to collect blood (Figure 3)7. 
			NOTE: To further elucidate the effects of varying experience with blood collection, a beginner and expert were chosen to collect blood samples using experimental and conventional methods simultaneously.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>After blood collection, remove the needle and press the puncture point to stop bleeding. Then, release the mouse from the plastic restraining holder and return the mouse to its cage. 
	NOTE: It has been reported that up to 10% of total blood volume can be safely removed from a healthy animal at 2 week intervals8, so about 175 &#181;L of blood was collected each time in accordance with ethical principles.</li>
	<li>Use tubes with EDTA to collect plasma and use tubes without anticoagulants to collect serum. Gently invert the tube several times and put them on ice vertically.</li>
	<li>Centrifuge the blood sample collection tubes in a refrigerated centrifuge at 1,000 x g for 10 min to separate plasma and serum. 
	NOTE: Successful blood collection is defined as obtaining a volume of 175 &#181;L each time. No more than three punctures should be attempted, and a failed blood collection is defined as a total blood volume of less than 175 &#181;L after the third puncture. The sampling duration is defined as the time from the tail vein puncture to removal of the needle after blood collection.</li>
	<li>Collect blood twice at intervals of 2 weeks8.</li>
</ol>
3. Statistical analysis
<ol>
	<li>Use commercially available statistical software for analysis. Present data as mean value &#177; standard deviation, using p &#60; 0.05 as the cutoff for statistical significance.</li>
</ol>

<ol>
	<li>Grouzmann, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sampling+methodology+is+crucial+for+precise+measurement+of+plasma+catecholamines+concentrations+in+mice.">Blood sampling methodology is crucial for precise measurement of plasma catecholamines concentrations in mice.</a> European Journal of Physiology. 447 (2), 254-258 (2003).</li><li>Heimann, M., Roth, D. R., Ledieu, D., Pfister, R., Classen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sublingual+and+submandibular+blood+collection+in+mice:+a+comparison+of+effects+on+body+weight,+food+consumption+and+tissue+damage.">Sublingual and submandibular blood collection in mice: a comparison of effects on body weight, food consumption and tissue damage.</a> Lab Animal. 44 (4), 352-358 (2010).</li><li>Heimann, M., Kasermann, H. P., Pfister, R., Roth, D. R., Burki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+collection+from+the+sublingual+vein+in+mice+and+hamsters:+a+suitable+alternative+to+retrobulbar+technique+that+provides+large+volumes+and+minimizes+tissue+damage.">Blood collection from the sublingual vein in mice and hamsters: a suitable alternative to retrobulbar technique that provides large volumes and minimizes tissue damage.</a> Lab Animal. 43 (3), 255-260 (2009).</li><li>Hoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+blood+collection+in+the+mouse.">Methods of blood collection in the mouse.</a> Lab Animal. 29 (10), 47-53 (2000).</li><li>Zou, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+Blood+Collection+from+Tail+Vein+of+Non-Anesthetized+Rats+with+a+Vacuum+Blood+Collection+System.">Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System.</a> Journal of Visualized Experiments. (130), (2017).</li><li>Kilkenny, C., Altman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+bioscience+research+reporting:+ARRIVE-ing+at+a+solution.">Improving bioscience research reporting: ARRIVE-ing at a solution.</a> Lab Animal. 44 (4), 377-378 (2010).</li><li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sample+collection+in+small+laboratory+animals.">Blood sample collection in small laboratory animals.</a> Journal of Pharmacology and Pharmacotherapeutics. 1 (2), 87-93 (2010).</li><li>Diehl, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+good+practice+guide+to+the+administration+of+substances+and+removal+of+blood,+including+routes+and+volumes.">A good practice guide to the administration of substances and removal of blood, including routes and volumes.</a> Journal of Applied Toxicology. 21 (1), 15-23 (2001).</li><li>Wollowitz, A., Bijur, P. E., Esses, D., John Gallagher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+butterfly+needles+to+draw+blood+is+independently+associated+with+marked+reduction+in+hemolysis+compared+to+intravenous+catheter.">Use of butterfly needles to draw blood is independently associated with marked reduction in hemolysis compared to intravenous catheter.</a> Academic Emergency Medicine. 20 (11), 1151-1155 (2013).</li><li>Eder, J. M., Cutter, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+device+for+collecting+cord+blood.">A new device for collecting cord blood.</a> Obstetrics and Gynecology. 86 (5), 850-852 (1995).</li></ol>

The authors have nothing to disclose.

The present study describes an easy-to-learn blood collection method in mice that is superior to the conventional techniques. First, the method can be easily mastered with a high success rate. Second, it is based on the vacuum negative pressure principle and allows for continuous drawing of blood with a reduced risk of direct blood exposure, which also reduces the chance of contamination and hemolysis9. Third, this method is feasible for frequent sampling of blood with adequate volumes from mice over a short period of time for various research purposes. Moreover, the procedure inflicts only minimal injury upon mice, and blood collection can be performed without the use of anesthetics; thus, the influence of the stress response and anesthetics on blood samples can be avoided.
The tail vein is a superior location for blood sampling according to the approved protocol7. However, it is not always easy to obtain sufficient blood volume from thin tail veins with low blood flows. In this case, the skin is usually cut open and vein is penetrated by a lancet, or the end of the tail is removed quickly by a razor.
This protocol aims to improve the methodology of blood collection from mice using the vacuum blood collection system, which requires a vacuum blood sample collection tube, butterfly needle, and eyeglass magnifier. This vacuum blood sampling system is usually used for collecting blood samples from patients in daily clinical practice10. With the help of an eyeglass magnifier, the perfect puncture point of a tail vein is easier to locate. When the tip of a needle is inserted into the tail vein, blood will automatically flow into the vacuum tube due to negative pressure. After withdrawing the needle from the tail vein, the blood that is blocked in the catheter will flow into the collecting vacuum tube due to the vacuum.
The following tips are important for successful application of the method. First, the body weight of each mouse should be 40 g or higher to decrease difficulty in puncturing and obtaining enough blood. Second, in the case of failed blood collection, experimenters should try to withdraw the needle slowly until blood continues to flow out. Third, it is vital to extend the tail to avoid any movement during blood sampling. Holding the needle gently can help to keep the needle tip in the vein as the mouse tail moves. Fourth, if blood stops flowing out during blood collection, the needle angle should be adjusted in a timely manner. In order to avoid blood coagulation in the needle, another puncture position should be selected if blood stops flowing out after 15 s. Finally, the cooperation of two operators is recommended while using this technique to collect blood from mice.&#160;
In short, the adopted vacuum blood collection method for use in mice is safe, feasible, and easy to practice. This method enables frequent sampling of blood with adequate blood volumes from non-anesthetized mice.

Blood sampling from animals involved in experiments is a basic research technique. There are some available techniques for blood collection from mice, including tail snips and puncturing of the heart, retro-orbital plexus, jugular vein, caudal vein, and vena cava. Ideally, blood should be collected in a minimally invasive manner, with minimal impact on the animal&#8217;s health. However, the most commonly used techniques often inflict stress upon animals and can impact research outcomes1. Blood collection from the retro-orbital plexus can be used to obtain enough blood volume from mice2. However, it can result in severe tissue damage and does not allow for obtaining blood repeatedly in short time intervals3.
The caudal vein is a superior location for blood collection, which inflicts minimal injury upon mice. However, the tail veins of mice are thin, and it is sometimes difficult to obtain enough blood through the conventional puncture technique. In some cases, repeated punctures are required to obtain the desired blood volume. Anesthesia is also commonly recommended to facilitate blood sampling from the tail veins of mice.&#160; Moreover, a scalpel, straight edge razor, or sharp scissors may be needed to remove the ends of the tails to obtain the required blood samples4. We have previously reported successful blood collection from the tail veins of non-anaesthetized rats by the vacuum blood sample collection system, which reduced the risk of blood contamination and avoided the need for repeated punctures5. This study reports a similar blood collection method in non-anesthetized mice.

<table><tbody><tr><td>Double-pointed needle</td><td>Shanghai Kang Nong medical instrument co., LTD, China</td><td>20163151718</td><td>22G (0.7 mm x 25 mm)</td></tr><tr><td>Eyeglass magnifier</td><td>Vergroberung</td><td></td><td>1.5x</td></tr><tr><td>Normal salt diet for mice</td><td></td><td></td><td>Mice received a normal salt diet (0.3% NaCl) throughout the study.</td></tr><tr><td>Plastic holder</td><td>Shanghai Kang Nong medical instrument co., LTD, China</td><td></td><td>35-45 g rat hoder</td></tr><tr><td>SPSS software for statistical analysis</td><td>SPSS Inc, Chicago; USA</td><td>Version 17.0.</td><td></td></tr><tr><td>Syringe</td><td>Shandong wego Medical polymer products co. LTD., China</td><td>20160911A</td><td>1 mL (Matching needle size: 0.45&amp;times;16RW LB)</td></tr><tr><td>Vacuum blood collection system</td><td>Wuhan Zhi Yuan, medical science and technology co., LTD, China</td><td>20171222</td><td>1 mL (&amp;Phi;12.4&amp;times;L75)</td></tr></tbody></table>

modified blood collection from tail veins of non-anesthetized mice with a vacuum blood collection system and eyeglass magnifier

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. The tail veins of mice are small, and it is sometimes difficult to obtain the required blood volume by conventional puncture methods. This study investigates the superiority of repeated blood sample collection from tail veins of mice through use of a vacuum blood collection system and eyeglass magnifier (experimental group) compared to conventional blood sampling methods (conventional group), performed by beginners and experts, respectively. With the help of an eyeglass magnifier, a butterfly needle tip is inserted into the tail vein of each mouse in the experimental group. When the vein is penetrated successfully, a blood sample is collected in the vacuum collection tube by insertion of the rubber end of a butterfly needle into the vacuum blood collection tube. The plunger is then used to collect blood without the help of the eyeglass magnifier in the conventional group. Success rates of blood sample collection by the beginners and experts were shown to be 70% vs. 100% (p &#60; 0.01) in the experimental group and 35% vs. 85% (p &#60; 0.01) in the conventional group. For both beginners and experts, puncture times required for obtaining required blood sample were significantly lower in the experimental group compared to the conventional group (2.40 &#177; 0.75 vs. 2.90 &#177; 0.31, p &#60; 0.05; 1.15 &#177; 0.37 vs. 1.55 &#177; 0.76, p &#60; 0.05). In conclusion, the presented blood sampling technique is feasible and easy to practice and enables frequent sampling of adequate blood volumes from non-anesthetized mice.&#160;

Body mass, blood collection volumes, and sampling durations of the two groups&#160; 
Blood samples were collected from 20 mice (10 males) twice at 2 week intervals in each group. The mean body mass of mice was similar between the experimental and conventional groups for beginners and experts, respectively (42.40 g &#177; 1.42 g vs. 42.65 g &#177; 1.14 g, p &#62; 0.05; 42.55 g &#177; 2.91 g vs. 43.20 g &#177; 2.69 g, p &#62; 0.05). Collected blood volumes and sampling durations were similar between the two groups in experts (184.25 &#181;L &#177; 11.95 &#181;L vs. 171.75 &#181;L &#177; 25.61 &#181;L, p &#62; 0.05; 1.85 min &#177; 0.68 min vs. 2.17 min &#177; 0.80 min, p &#62; 0.05). However, higher collected blood volumes and shorter sampling durations were seen in the experimental group compared to the conventional group in beginners (172.00 &#181;L &#177; 15.17 &#181;L vs. 148.50 &#181;L &#177; 30.22 &#181;L, p &#60; 0.01; 3.11 min &#177; 0.44 min vs. 4.08 min &#177; 0.61 min, p &#60; 0.01). Compared to beginners, experts collected higher blood volumes and showed lower sampling times using experimental and conventional methods (184.25 &#181;L &#177; 11.95 &#181;L vs. 172.00 &#181;L &#177; 15.17 &#181;L, p &#60; 0.01; 171.75 &#181;L &#177; 25.61 &#181;L vs. 148.50 &#181;L &#177; 30.22 &#181;L, p &#60; 0.05; 1.85 min &#177; 0.68 min vs. 3.11 min &#177; 0.44 min, p &#60; 0.01; 2.17 min &#177; 0.80 min vs. 4.08 min &#177; 0.61 min, p &#60; 0.05) (Table 1).&#160;
Success rates and puncture times of the two groups&#160; 
The comparison of success rates between beginners and experts was 70% (14/20) vs. 100% (20/20) (p &#60; 0.01) in the experimental group and 35% (7/20) vs. 85% (17/20) (p &#60; 0.01) in the conventional group. Higher success rates were also seen in the experimental group compared to the conventional group in beginners [70% (14/20) vs. 35% (7/20), p &#60; 0.05]. In both beginners and experts, the number of punctures was significantly lower in the experimental group compared to the conventional group (2.40 &#177; 0.75 vs. 2.90 &#177; 0.31, p &#60; 0.05; 1.15 &#177; 0.37 vs. 1.55 &#177; 0.76, p &#60; 0.05). Compared to beginners, lower puncture times for experts were observed using experimental and conventional methods. (1.15 &#177; 0.37 vs. 2.40 &#177; 0.75, p &#60; 0.01; 1.55 &#177; 0.76 vs. 2.90 &#177; 0.31, p &#60; 0.01) (Table 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59136/59136fig1.jpg" /> 
Figure 1: Equipment. Shown are 1 mL vacuum blood collection tubes and a 22 G butterfly needle (left), an eyeglass magnifier (middle), and a plastic restraining holder (right). <a href="https://www.jove.com/files/ftp_upload/59136/59136fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59136/59136fig2.jpg" /> 
Figure 2: Successful blood collection in the experimental group. <a href="https://www.jove.com/files/ftp_upload/59136/59136fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59136/59136fig3.jpg" /> 
Figure 3: Successful blood collection in the conventional group. <a href="https://www.jove.com/files/ftp_upload/59136/59136fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">Experimental group</td>
			<td colspan="2">Conventional group</td>
		</tr>
		<tr>
			<td></td>
			<td>beginner</td>
			<td>expert</td>
			<td>beginner</td>
			<td>expert</td>
		</tr>
		<tr>
			<td>Body mass (g)</td>
			<td>42.4&#177;1.42</td>
			<td>42.55&#177;2.91</td>
			<td>42.65&#177;1.14</td>
			<td>43.20&#177;2.69</td>
		</tr>
		<tr>
			<td>Collected blood volume (&#181;L)</td>
			<td>172.00&#177;15.17</td>
			<td>184.25&#177;11.95##</td>
			<td>148.50&#177;30.22**</td>
			<td>171.75&#177;25.61#</td>
		</tr>
		<tr>
			<td>Sampling duration (min)</td>
			<td>3.11&#177;0.44</td>
			<td>1.85&#177;0.68##</td>
			<td>4.08&#177;0.61**</td>
			<td>2.17&#177;0.80##</td>
		</tr>
		<tr>
			<td>Blood collection times</td>
			<td>20</td>
			<td>20</td>
			<td>20</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Average number of punctures&#160;</td>
			<td>2.40&#177;0.75</td>
			<td>1.15&#177;0.37##</td>
			<td>2.90&#177;0.31*</td>
			<td>1.55&#177;0.76##*</td>
		</tr>
		<tr>
			<td>One time puncture</td>
			<td>3</td>
			<td>17</td>
			<td>0</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Two times puncture&#160;</td>
			<td>6</td>
			<td>3</td>
			<td>2</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Three times puncture&#160;</td>
			<td>5</td>
			<td>0</td>
			<td>5</td>
			<td>0</td>
		</tr>
		<tr>
			<td>Failed</td>
			<td>6</td>
			<td>0</td>
			<td>13</td>
			<td>3</td>
		</tr>
		<tr>
			<td>Success rate</td>
			<td>70%</td>
			<td>100%##</td>
			<td>35%*</td>
			<td>85%##</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of results between the experimental and conventional groups. *p &#60; 0.05, **p &#60; 0.01, experimental method vs. conventional method. #p &#60; 0.05, ##p &#60; 0.01, beginner vs. expert.

Watch this Scientific Journal Video about Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier at JoVE.com

Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier

This study reports blood sampling from tail vein in mice using a vacuum extraction tube system with eyeglass magnifier. Our method is easy to practice and could be used for repeat blood sampling in mice.

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. ...

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17.8K Views.  Huazhong University of Science and Technology. This study reports blood sampling from tail vein in mice using a vacuum extraction tube system with eyeglass magnifier. Our method is easy to practice and could be used for repeat blood sampling in mice.

Video: Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier

Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System

Blood can be collected from rats in a number of sampling locations. For instance, the tail vein is a superior location for blood sampling. However, the tail vein is thin so that it is sometimes hard to puncture. In addition, the tail vein has low blood flow and requires a long sampling time to get sufficient blood. The present report describes a simple blood sampling method, the vacuum blood collection method, which is usually used to obtain blood samples from patients, here used for non-anesthetized rats. The 22 G butterfly needle tip was inserted into one of the lateral tail veins approximately 2-3 cm from the tip of the tail at an angle of approximately 20&#176;, and blood was collected in the vacuum collection tube by inserting the rubber end of the butterfly needle into the vacuum blood collection tube. The present experimental results show that the success rate was 95% in the experimental group and 90% in the beginner group. The success rate and puncture times were similar between two groups. The sampling duration was significantly shorter in the experimental group compared to beginner group. In conclusion, this vacuum blood collection method for sequential blood sampling from the tail vein of non- anesthetized rats is feasible and easy-to-learn, which might serve as a reliable alternative to other conventionally used blood sampling techniques for rats.

Here, we describe a simple tail vein blood sampling method in non-anesthetized rats using a vacuum extraction tube system. This method reduces the risk of direct exposure to blood and simplifies taking multiple samples from a single venipuncture.

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Frequent Tail-tip Blood Sampling in Mice for the Assessment of Pulsatile Luteinizing Hormone Secretion

In many endocrine systems, circulating factors or hormones are not released continuously, but are secreted as a discrete pulse in response to a releasing factor. Single-point sampling measures are inadequate to fully understand the biological significance of the secretory pattern of pulsatile hormones either under normal physiologic conditions or during conditions of dysregulation. Luteinizing hormone (LH) is synthesized by the anterior pituitary gonadotrope&#160;cells and secreted in a pulsatile pattern which requires frequent collection of blood samples for pulse assessment. This has not been possible in mice until recently, due to the development of a high-sensitivity LH assay and advancement in a technique for frequent low-volume sample collection, initially described by Steyn and colleagues.1 Here we describe a protocol for the frequent peripheral blood sample collection from mice with sufficient handling acclimatization to detect pulsatile secretion of LH. The current protocol details an expanded acclimatization period that allows assessment of robust and continuous pulses of LH over multiple hours. In this protocol, the tip of the tail is clipped&#160;and blood is collected from the tail using a hand-held pipette. For assessment of pulsatile LH in gonadectomized mice, serial samples are collected every 5-6 min for 90-180 min. Importantly, the collection of blood and measurement of robust pulses of LH can be accomplished in awake, freely behaving mice, given adequate handling acclimatization and effort to minimize environmental stressors. Sufficient acclimatization can be achieved within 4-5 weeks prior to blood collection. This protocol highlights advances in the methodology to ensure collection of whole blood samples for assessment of pulsatile LH secretion patterns over multiple hours in the mouse, a powerful animal model for neuroendocrine research.

Here we present a tail-tip blood sampling protocol for frequent sample collection in unrestrained mice. This method is useful for assessing patterns of pulsatile luteinizing hormone secretion and could be adapted for analysis of other circulating factors.

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Blood Collection Through Subclavian Vein Puncture in Mice

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in research work. Tail blood collection is a popular method when a small amount of blood sample is needed. Orbital artery could be considered if a large amount of blood is needed but this blood collection method has ethical issues. Formerly, we demonstrated the feasibility and safety of blood sample collection through subclavian vein puncture in rats, and here we investigate whether this method could be used in mice. We report that this method is safe and practical for blood collection in mice. Blood collection through the subclavian vein puncture in mice can be a convenient method in daily research works.

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Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

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This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Biology

Cerebrospinal Fluid and Blood Withdrawal from Rats with Concurrent EEG Recording

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats During EEG Recordings

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many experimental contexts to measure molecules of interest, such as hormones, growth factors, proteins, or small non-coding RNAs. A specific example is the sampling of biological liquids in the research of biomarkers for epilepsy. In these studies, it is desirable to compare the levels of molecules in cerebrospinal fluid (CSF) and in plasma, by withdrawing CSF and plasma in parallel and considering the time distance of the sampling from and to seizures. The combined CSF and plasma sampling, coupled with video-EEG monitoring in epileptic animals, is a promising approach for the validation of putative diagnostic and prognostic biomarkers. Here, a procedure of combined CSF withdrawal from cisterna magna and blood sampling from the lateral tail vein in epileptic rats that are continuously video-EEG monitored is described. This procedure offers significant advantages over other commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, and reduced time of anesthesia. Additionally, it can be used to obtain CSF and plasma samples in both tethered and telemetry EEG recorded rats, and it may be used repeatedly across multiple days of experiment. By minimizing the stress due to sampling by shortening isoflurane anesthesia, measures are expected to reflect more accurately the true levels of investigated molecules in biofluids. Depending on the availability of an appropriate analytical assay, this technique may be used to measure the levels of multiple, different molecules while performing EEG recording at the same time.

Author Spotlight: Obtaining High-Quality CSF and Blood Samples for Epilepsy Biomarker Discovery 

The protocol shows repeated cerebrospinal fluid and blood collections from epileptic rats performed in parallel with continuous video-electroencephalogram (EEG) monitoring. These are instrumental for exploring possible links between changes in various body fluid molecules and seizure activity.

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many ...

Comparative Study of Blood Collection Techniques in Rats

Modified Tail Vein and Penile Vein Puncture for Blood Sampling in the Rat Model

Blood samples are required in most experimental animal designs to assess various hematological parameters. This paper presents two procedures for blood collection in rats: the lateral tail vein puncture and the dorsal penile vein puncture, which offer significant advantages over other previously described techniques. This study shows that these two procedures allow for fast sampling (under 10 min) and yield sufficient blood volumes for most assays (202 &#956;L &#177; 67.7 &#956;L). The dorsal penile vein puncture must be done under anesthesia, whereas the lateral tail vein puncture can be done on a conscious, restrained animal.
Alternating these two techniques, therefore, enables blood draw in any situation. While it is always recommended for an operator to be assisted during a procedure to ensure animal welfare, these techniques require only a single operator, unlike most blood sampling methods that require two. Moreover, whereas these previously described methods (e.g., jugular stick, subclavian vein blood draw) require extensive prior training to avoid harm to or death of the animal, tail vein and dorsal penile vein puncture are rarely fatal. For all these reasons, and according to the context (e.g., for studies including male rats, during the perioperative or immediate postoperative period, for animals with thin tail veins), both techniques can be used alternately to enable repeated blood draws.

Author Spotlight: Context Dependent Blood Collection Methods in Rats 

Here, we present a protocol to offer rapid, easy, and reliable blood collection alternatives for the rat model. We describe three different blood sampling methods according to the context: tail vein puncture under anesthesia or on a conscious animal, and dorsal penile vein puncture under anesthesia.

Blood samples are required in most experimental animal designs to assess various hematological parameters. This paper presents two procedures for blood ...

Subclavian Vein Puncture Method for Repeated Blood Collection in Conscious Rats

Subclavian Vein Blood Sampling in Conscious Rats

There are several established methods for obtaining repeated blood samples from rats, with the most commonly employed methods being lateral tail vein sampling without anesthesia and jugular vein sampling with anesthesia. However, most of these methods require assistance and anesthetic equipment and sometimes pose difficulties in terms of blood collection or the poor quality of blood samples. In addition, these methods of blood collection consume significant time and human resources when repeated blood sampling is required for a large number of rats. This study presents a technique for repetitive blood sampling in non-anesthetized rats by a single proficient individual. Highly satisfactory blood samples can be obtained by puncturing the subclavian vein. The method demonstrated an impressive overall success rate of 95%, with a median time of merely 2 min from rat restraint to the completion of blood collection. Furthermore, performing consecutive blood collections within the designated range does not inflict any harm on the rats. This method is worth promoting for blood collection, especially in large-scale pharmacokinetic studies.

Author Spotlight: Developing an Efficient Blood Collection Method in Non-Anesthetized Rats 

Here we present a combination of effective rat restriction and subclavian vein puncture methods that enable rapid, safe, and repeated blood collection in rats without anesthesia.

There are several established methods for obtaining repeated blood samples from rats, with the most commonly employed methods being lateral tail vein ...

Blood Withdrawal I

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
Blood collection is a common requirement for research studies that involve mice and rats. The method of blood withdrawal in mice and rats is dependent upon the volume of blood needed, the frequency of the sampling, the health status of the animal to be bled, and the skill level of the technician.1 All methods discussed-retro-orbital sinus bleeds, initial tail snip bleeds, and intracardiac bleeds-require the use of a general anesthesia.

Retro Orbital, Tail and Intra-cardiac Blood Collection in Rodents

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
Blood collection is a common requirement for ...

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Blood Withdrawal II

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
The collection of blood from mice and rats for analysis can be done through a variety of methods. Each method of collection has variations in the type of restraint required, the invasiveness of the procedure, and the necessity of a general anesthetic.1Historically, the use of the retro-orbital sinus cavity has been used, but not without debate. The controversy related to the potential tissue damage,or even blindness,caused by retro-orbital bleeds has led to the development of facial and submandibular vein bleeding methods in mice.Blood collection from the saphenous vein in both mice and rats is another technique that has been developed. These procedures do not require anesthesia and therefore are suitable when the use of anesthetics may confound blood results or other data.

Collecting Blood from Facial, Submandibular, Saphenous &amp; Femoral Veins in Rodents

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
The collection of blood from mice and rats for ...

Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier

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Chapters in this video

Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System

Frequent Tail-tip Blood Sampling in Mice for the Assessment of Pulsatile Luteinizing Hormone Secretion

Blood Collection Through Subclavian Vein Puncture in Mice

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Sampling Blood from the Lateral Tail Vein of the Rat

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats During EEG Recordings

Modified Tail Vein and Penile Vein Puncture for Blood Sampling in the Rat Model

Subclavian Vein Blood Sampling in Conscious Rats

Blood Withdrawal I

Blood Withdrawal II