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.

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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.

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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 with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

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Improved Blood Sampling Technique to Assess Gut Microbial Metabolites in Mice

Sequential Blood Collection from Inferior Vena Cava Followed by Portal Vein to Evaluate Gut Microbial Metabolites in Mice

Gut microbial products are known to act both locally within the intestine and get absorbed into circulation, where their effects can extend to numerous distant organ systems. Short-chain fatty acids (SCFA) are one class of metabolites produced by gut microbes during the fermentation of indigestible dietary fiber. They are now recognized&#160;as important contributors to how the gut microbiome influences extra-intestinal organ systems via the gut-lung, gut-brain, and other gut-organ axes throughout the host. SCFAs are absorbed from the colon, through intestinal tissue,&#160;into the portal vein (PV). They then pass through the liver, and are consumed in various organs such as the brain, muscle, adipose tissue, and lungs. SCFAs are most easily measured in the expelled fecal material however, more accurate measurements have been obtained from intra-colonic fecal contents. Here we propose that sampling PV and systemic circulating plasma of a single subject may be preferable for studying the&#160;absorption, transport, and systemic levels of SCFAs in mice. We present a new technique for efficient blood sampling from the PV and inferior vena cava (IVC) that allows for the collection of relatively large volumes of blood from the portal and systemic circulations. This is accomplished by ligating the PV, thereby allowing&#160;for the dilation or enlargement of the PV as it backfills from the mesenteric veins that drain into it. Using this method, we were able to improve the rate of successful collection as well as the total amount of blood collected (up to 0.3 mL from IVC and 0.5 mL from PV).

Author Spotlight: Gut Microbiome-Lung Tissue Communication Through Short-Chain Fatty Acid Analysis

The protocol demonstrates a method to collect blood from portal veins and inferior vena cava from mice sequentially to evaluate the production and absorption of gut microbial metabolites.

Gut microbial products are known to act both locally within the intestine and get absorbed into circulation, where their effects can extend to numerous ...

Immunology and Infection

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 ...

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 ...

Education

JoVE Science Education

Basic Biology

Lab Animal Research

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 ...