We would like to thank Chunshen Shen, Hiroko Akutsu, Fumiyo Sugiki, Yuuna Hashimoto, Hina Naritomi, Yuuki Sakamoto,&#160;and Mikiko Horigome for their support in establishing this protocol and in the day-to-day care of marmosets; Takayuki Mineshige for comments on the manuscript; Yukiko Abe and the members of Aiba lab for sharing zygote collection techniques; CIEA for sharing the information on marmosets housing and experiments that they have cultivated over 40 years. This research was supported by AMED, JST, and KAKENHI under the grant Nos. JP19gm6310010, JP20gm6310010, JP21gm6310010, and JP22gm6310010 (AMED), JPMJPR228B (JST), 20H05764, 20H03177, and 22K18356 (KAKENHI).

All methods involving marmosets utilized high ethical and welfare standards and were approved by the Institutional Animal Care and Use Committee at the National Center for Child Health and Development. Animals used here were single-housed or paired-housed (one female and one male) with 12 h of light per day.
1. Blood sampling&#160;of the femoral vein
<ol>
	<li>Prepare a 1 mL syringe (the short type is easy to use) with a 25 G needle attached blade-side up. To avoid blood clogging, heparinize the syringe by drawing 200 &#181;L of undiluted heparin sodium solution into the syringe. Coat the inside of the syringe evenly by pulling it up and down several times; then, expel the heparin solution from the syringe. 
	NOTE: Since changing to a new syringe is often necessary, prepare a few additional heparinized syringes.</li>
	<li>Prepare absorbent cotton and alcohol swabs. Switch on an adjustable lamp to illuminate the area where the marmoset will be placed for blood sampling.</li>
	<li>Prepare the restraint device (420 x 85 x 85 mm, Figure 1A), which is commercially available (see Table of Materials for details). To place a marmoset into the device, open the retention part with the sponge belt, which secures marmosets. Insert the marmoset into the restraint device facing up. 
	NOTE: Marmosets are usually calm in restrain devices which may negate the need for restraint acclimation or training.</li>
	<li>Capture the marmoset; insert the marmoset into the cylindrical part; and secure it by pressing down the sponge belt. Place the leg from which blood is to be collected on top of the other (Figure 1A). Hold the legs using the non-dominant hand; place the middle and ring fingers inside each leg to fasten them and the other fingers outside each leg to fix them. 
	NOTE: Bite gloves are recommended when capturing marmosets. If a restraining device is not present, perform blood collection under anesthesia or with another person restraining the marmoset.</li>
	<li>Check if the femoral vein is visible near the base of the thigh. If not, palpate to find the pulsating artery and use it as a landmark to check that the vein runs inside it (Figure 1B-D). 
	NOTE: Desk illumination and hair shaving are recommended to improve visibility. Visibility can also be improved by rubbing with alcohol swabs. Lymph nodes in the triangle are often located close to the vein and show a dark blue color as a vein. How to distinguish them is described in the Representative Results section.</li>
	<li>Disinfect the puncture site using an alcohol swab. Insert the needle blade-side up at an angle of 15&#176;-20&#176;. To prevent the needle from slipping out of the blood vessel during blood collection, stabilize the hand holding the syringe, for example, by resting it on the other hand.</li>
	<li>Gently pull back the plunger to apply negative pressure (Figure 1E). Push the needle tip forward. Once blood enters the syringe, maintain the position of the needle tip until the required volume (500-700 &#181;L) has been collected.
	<ol>
		<li>When blood does not enter the syringe, change the site of puncture. If the blood comes out when pulling out the needle, stop bleeding by applying pressure to the puncture site for 3 min. After stopping the bleeding, restart the venipuncture.</li>
		<li>If the blood being drawn into the syringe stops flowing during the drawing process, slowly push the needle tip forward and then pull it back to find the blood vessel. This may restore the flow of blood into the syringe. If not, pull out the needle and perform venipuncture using a new syringe. 
		NOTE: Use the thigh on the other leg when it is difficult to collect blood from the same side.</li>
	</ol>
	</li>
	<li>Carefully pull out the needle while pressing lightly on the puncture site using the little finger. Then, using an absorbent cotton swab, immediately apply pressure to the puncture site for 3 min to stop the bleeding. Invert the syringe to mix blood and heparin. 
	NOTE: Apply pressure for a longer time (5 min) while cooling when arterial blood is drawn, and carefully confirm the ceasure of bleeding to prevent the formation of hematoma, which can sometimes result in a fatal outcome.</li>
	<li>After confirming that bleeding has ceased, remove the sponge belt, hold the animal&#39;s waist using one hand, and rotate the animal so that one can hold the animal&#39;s underarm using another hand from the backside.</li>
	<li>Return the marmoset to its cage. To reduce stress and facilitate repeated blood sampling, provide the marmoset with its favorite food (e.g., biscuits, marshmallows, or sponge cake). Check the occurrence of hematoma occasionally. 
	NOTE: When hematoma is found at the early stage, apply a pressure bandage to prevent the progression of hematoma. When it is found after a long time, surgical removal of hematoma with ligation of the femoral artery and blood transfusion may be needed9,13.</li>
	<li>Detach the needle from the syringe to prevent hemolysis. Then, slowly expel the collected blood along the inside wall of a 1.5 mL microtube. 
	NOTE: The collected blood can be stored at 4 oC for up to 24 h before plasma separation for measuring P4/E2 levels.</li>
</ol>
2. Separation of plasma and determination of hormonal levels
<ol>
	<li>Centrifuge the blood in a 1.5 mL tube at 1,100 &#215; g for 5 min at 4 oC.</li>
	<li>Transfer the separated plasma (supernatant) from the 1.5 mL tube to a new tube/cup, carefully avoiding the inclusion of blood cells (sediment).</li>
	<li>Measure the P4 and E2 levels using an ELISA kit or an automatic analyzer. If the amount of plasma is not enough for the automatic analyzer, use a sample dilution solution. 
	NOTE: For the measurement using an automatic analyzer, &#62;175 &#181;L of plasma is required for determining only a P4 level, and &#62;250 &#181;L of plasma is required for determining both P4 and E2.</li>
</ol>
3. Urine CG measurement to detect ovulation and pregnancy
NOTE: CG levels in marmosets can be measured using an immunochromatographic kit test for both ovulation and pregnancy. In the case of ovulation, a positive result can be obtained 0-2 days before ovulation. In the case of pregnancy, a positive result is detected from days 15-20 to approximately day 100 of pregnancy. The test requires a small amount (90 &#181;L) of urine (one drop of urine is ~30 &#181;L).
<ol>
	<li>Tray method: If more than two animals are in the same cage, move the target marmoset (or the other animals) to another cage the day before. Place a clean tray at the bottom of the cage the night before or before lighting. Urine is not drained from the bladder during the night. Therefore, urine is usually released from the bladder shortly after lighting. 
	NOTE: Entering the room in the morning before lighting may disrupt the cohort&#39;s sleep cycle. Urine can usually be collected within approximately 30 min after lighting.</li>
	<li>Squeezing method: Prepare the washed trays for urine collection. After locating the marmoset&#39;s bladder, carefully squeeze it from the front and both sides using the entire length of the fingers (Figure 1F). Collect urine just before lighting up the room in the morning. 
	NOTE: If urine collection is unsuccessful due to the absence of urine in the bladder, wait a while and try again. Be careful not to use excessive force because this will injure the animal.</li>
	<li>Immediately after collection, place the urine sample in the well of the immunochromatographic test kit. Read the result after 10 min according to the manufacturer&#39;s instructions.</li>
</ol>
4. Control and determination of the ovarian cycle stage for the collection of oocytes, zygotes, and embryos
<ol>
	<li>Germinal vesicle (GV) oocyte collection from the ovaries
	<ol>
		<li>Administer an intramuscular injection of 3 &#181;L of 0.263 mg/mL cloprostenol (a synthetic PGF2&#945; analog) diluted in 150 &#181;L of saline14 (Figure 2) to reset the ovarian cycle at the end of the luteal phase (i.e., &#8805;10 days after the initiation of the luteal phase). 
		NOTE: Making the diluted solution in a larger volume may be convenient as the diluted cloprostenol stays stable for, at least, several weeks at +4 &#176;C.</li>
		<li>On the next day (day 1), confirm the initiation of the follicular phase by checking that the P4 level has dropped. 
		NOTE: Cloprostenol injection has been reported to dramatically decrease P4 levels (usually &#60;10 ng/mL) within 24 h3.</li>
		<li>From day 1, inject FSH (25 IU, intramuscular) once every 2 days for a total of 5x (days 1, 3, 5, 7, and 9). On day 10, inject hCG (75 IU, intramuscular) in the afternoon.</li>
		<li>Collect GVs from the ovaries on day 11 by follicular aspiration under anesthesia according to the literature15,16. 
		NOTE: Sometimes, ovulation occurs earlier than expected. Thus, it is recommended to check CG levels from day 8. If the CG test is positive, perform GV collection on this day.</li>
	</ol>
	</li>
	<li>Collection of metaphase II (MII) oocytes, zygotes, and early embryos
	<ol>
		<li>Reset the ovarian cycle using cloprostenol as described in step 4.1.1.</li>
		<li>On the next day (day 1), confirm the initiation of the follicular phase by checking that the P4 level has dropped.</li>
		<li>For the collection of zygotes and embryos, house female marmosets together with male marmosets for mating from day 6.</li>
		<li>From day 7, check the blood P4/E2 levels and urine CG levels of the females. Detection of CG is an indication of ovulation within a few days (usually the next day). On the day that P4 levels increase and E2 levels decrease compared with the previous day, collect MII oocytes or embryonic day 0 (E0) zygotes from the oviducts17,18.</li>
		<li>For embryo collection, as described in the literature, perform flushing either from the oviducts (E1-E3, 1-8 cells)17,18 or the uterus (E5-E10, 8 cells-blastocyst)19,20,21,22 at the appropriate time point, depending on the targeted stage.</li>
	</ol>
	</li>
</ol>

Assessing Ovarian Cycle in Marmosets by Blood Collection and Hormone Analysis

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+follicle-stimulating+hormone+and+follicle+populations+during+the+ovarian+cycle+of+the+common+marmoset.">Changes in follicle-stimulating hormone and follicle populations during the ovarian cycle of the common marmoset.</a> Biol Reprod. 64 (1), 127-135 (2001).</li><li>Gromoll, J., 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+new+subclass+of+the+luteinizing+hormone/chorionic+gonadotropin+receptor+lacking+exon+10+messenger+RNA+in+the+New+World+monkey+(Platyrrhini)+lineage.">A new subclass of the luteinizing hormone/chorionic gonadotropin receptor lacking exon 10 messenger RNA in the New World monkey (Platyrrhini) lineage.</a> Biol Reprod. 69 (1), 75-80 (2003).</li><li>M&#252;ller, T., 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=Chorionic+gonadotrophin+beta+subunit+mRNA+but+not+luteinising+hormone+beta+subunit+mRNA+is+expressed+in+the+pituitary+of+the+common+marmoset+(Callithrix+jacchus).">Chorionic gonadotrophin beta subunit mRNA but not luteinising hormone beta subunit mRNA is expressed in the pituitary of the common marmoset (Callithrix jacchus).</a> J Mol Endocrinol. 32 (1), 115-128 (2004).</li><li>Pacchiarotti, A., 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=Ovarian+stimulation+protocol+in+IVF:+an+up-to-date+review+of+the+literature.">Ovarian stimulation protocol in IVF: an up-to-date review of the literature.</a> Curr Pharm Biotechnol. 17 (4), 303-315 (2016).</li><li>Ezcurra, D., Humaidan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+luteinising+hormone+and+human+chorionic+gonadotropin+when+used+in+assisted+reproductive+technology.">A review of luteinising hormone and human chorionic gonadotropin when used in assisted reproductive technology.</a> Reprod Biol Endocrinol. 12, 95 (2014).</li><li>Lopez-Gatius, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+response+to+prostaglandin+F(2alpha)+in+lactating+dairy+cows:+A+clinical+update.">Ovarian response to prostaglandin F(2alpha) in lactating dairy cows: A clinical update.</a> J Reprod Dev. 68 (2), 104-109 (2022).</li><li>Marini, R. 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M., Shephard, A. M., Taylor, C. T., Hearn, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+cryopreservation+and+transfer+on+embryonic+development+in+the+common+marmoset+monkey,+Callithrix+jacchus.">The effects of cryopreservation and transfer on embryonic development in the common marmoset monkey, Callithrix jacchus.</a> J Reprod Fertil. 79 (1), 241-250 (1987).</li><li>Thomson, J. A., Kalishman, J., Hearn, J. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+ovulation+in+the+marmoset+monkey+(Callithrix+jacchus)+with+human+chorionic+gonadotropin+following+prostaglandin-induced+luteal+regression.">Controlled ovulation in the marmoset monkey (Callithrix jacchus) with human chorionic gonadotropin following prostaglandin-induced luteal regression.</a> Fertil Steril. 48 (2), 299-305 (1987).</li><li>Barrett, J., Abbott, D. H., George, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extension+of+reproductive+suppression+by+pheromonal+cues+in+subordinate+female+marmoset+monkeys,+Callithrix+jacchus.">Extension of reproductive suppression by pheromonal cues in subordinate female marmoset monkeys, Callithrix jacchus.</a> J Reprod Fertil. 90 (2), 411-418 (1990).</li><li>Barrett, J., Abbott, D. 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The authors have no conflicts of interest to declare.

Locating the vein is the most critical step in blood collection. Based on anatomical observations, this protocol introduces the proximal area in the femoral triangle as an easy site for blood collection in marmosets. Using this area, blood sampling from a large vein can be easily performed. However, even using this protocol, injury to an artery sometimes occurs. When injuring an artery, complete stopping of bleeding by applying pressure for &#62;5 min is suggested to prevent hematoma. In addition, while applying pressure...

The common marmoset (Callithrix jacchus) is a small New World monkey with many characteristics similar to those of humans, and the duration of its ovarian cycle is 26-30 days1,2. Studies on the early development and the generation of genetically modified marmosets require the harvesting of oocytes and zygotes at specific stages in the ovarian cycle. Thus, accurate determination of the stage is crucial and can be estimated by measuring the blood levels of the hormones progesterone (P4) and estradiol (E2)2,3. These hormones promote endometrial growth, which is necessary for implantation. P4 is produced from the corpus luteum, which forms in ovaries immediately following ovulation. E2 is secreted by the ovarian follicles in response to follicle-stimulating hormone (FSH) from the hypothalamus-pituitary complex in the brain. E2 levels increase as the follicle matures, peaking before ovulation3. High E2 levels cause the pulsed release of luteinizing hormone (LH) via the hypothalamus-pituitary complex in humans; this LH surge induces ovulation. However, in marmosets, the LH gene underwent degeneration during evolution, and ovulation is instead induced by the release of chorionic gonadotropin (CG), which has a similar structure to LH&#39;s, from the pituitary gland4,5.
The ovarian cycle can be controlled by hormone injections. FSH injections, in humans, act on ovarian FSH receptors and are used to promote estrogen synthesis and follicle growth6. The injection of human CG (hCG) as a substitute for LH at the end of the follicular phase is used to stimulate ovulation in humans7. CG injections are also used to treat human infertility because CG stimulates the corpus luteum in early pregnancy, resulting in increased P4 production. Prostaglandin F2&#945; (PGF2&#945;) injections reset the ovarian cycle8. In domestic cattle, PGF2&#945; injection is used to shorten the luteal phase and synchronize the estrus cycle for reproductive management.
Although marmosets and humans have similar biological mechanisms, making them ideal model animals, there is a lack of literature describing basic methods for many often-used techniques. Blood sampling is one of the most often used techniques9,10,11,12. However, beginners sometimes have trouble finding the vein. Hence, this study conducted anatomical analyses of the femoral vein region. Based on anatomical observations, this protocol introduces the proximal region of the femoral triangle as an easy site for venipuncture.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AIA-360</td>
			<td>Tosoh Corporation</td>
			<td>0019945</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>AIA-PACK DILUENT CONCENTRATE</td>
			<td>Tosoh Corporation</td>
			<td>0020956</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>AIA-PACK SUBSTRATE SET II</td>
			<td>Tosoh Corporation</td>
			<td>0020968</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>AIA-PACK WASH CONCENTRATE</td>
			<td>Tosoh Corporation</td>
			<td>0020955</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>CMS-1</td>
			<td>CLEA Japan</td>
			<td></td>
			<td>Marmoset food</td>
		</tr>
		<tr>
			<td>Estrumate</td>
			<td>MSD Animal Health</td>
			<td></td>
			<td>PGF2alpha analog (cloprostenol)</td>
		</tr>
		<tr>
			<td>Gonal-f Subcutaneous Injection 150</td>
			<td>Merck Biopharma Co., Ltd.</td>
			<td></td>
			<td>FSH</td>
		</tr>
		<tr>
			<td>Gonatropin for intramuscular injection 1000</td>
			<td>ASKA Pharmaceutical Co., Ltd.</td>
			<td>872413</td>
			<td>hCG</td>
		</tr>
		<tr>
			<td>Heparin sodium injection solution 5,000 units/5 mL</td>
			<td>Mochida Pharmaceutical Co., Ltd.</td>
			<td>224122458</td>
			<td>Blood collection</td>
		</tr>
		<tr>
			<td>Immunochromatographic Test Kit for Detection of Common Marmoset Chorionic Gonadotropin (Dual Checker)</td>
			<td>CLEA Japan, Inc.</td>
			<td></td>
			<td>Determining CG level</td>
		</tr>
		<tr>
			<td>Low-profile double-arm microscope illumination LPF-SD</td>
			<td>SHIOKAZE GIKEN</td>
			<td></td>
			<td>Desk lamp for blood collection</td>
		</tr>
		<tr>
			<td>Marmoset blood collection restraint device</td>
			<td>JIC Japan</td>
			<td>JM-1006</td>
			<td>Blood collection 
			http://www.jic-japan.jp/prd/marmoset/prd016.html 
			email: vi@jic-japan.jp</td>
		</tr>
		<tr>
			<td>Metacam 0.05%</td>
			<td>Boehringer Ingelheim Animal Health Japan Co., Ltd.</td>
			<td></td>
			<td>Hematoma treatment</td>
		</tr>
		<tr>
			<td>Sample Cup, 3 mL, PS, for Tosoh 360 and AIA-600 II, 1000/Bag</td>
			<td>Globe Scientific</td>
			<td>110913</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>ST AIA-PACK iE2</td>
			<td>Tosoh Corporation</td>
			<td>0025224</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>ST AIA-PACK iE2 CALIBRATOR SET</td>
			<td>Tosoh Corporation</td>
			<td>0025324</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>ST AIA-PACK iE2 SAMPLE DILUTING SOLUTION</td>
			<td>Tosoh Corporation</td>
			<td>0025524</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>ST AIA-PACK PROGIII</td>
			<td>Tosoh Corporation</td>
			<td>0025240</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>ST AIA-PACK PROGIII CALIBRATOR SET</td>
			<td>Tosoh Corporation</td>
			<td>0025340</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>ST AIA-PACK PROGIII SAMPLE DILUTING SOLUTION</td>
			<td>Tosoh Corporation</td>
			<td>&#160;0025540</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
		<tr>
			<td>Syringe with 25 G (0.50 x 25 mm) needle</td>
			<td>TERUMO</td>
			<td>SS-01T2525</td>
			<td>Blood collection</td>
		</tr>
		<tr>
			<td>Tensolvet 5.000 I.E. gel.</td>
			<td>Dechra Pharmaceuticals</td>
			<td>14033492</td>
			<td>Hematoma treatment</td>
		</tr>
		<tr>
			<td>TOSOH MULTI-CONTROL SET</td>
			<td>Tosoh Corporation</td>
			<td>0015965</td>
			<td>Hormone measurement (P4/E2)</td>
		</tr>
	</tbody>
</table>

blood sampling and hormone measurement for determining the stage in the ovarian cycle in marmosets

Common marmosets are small New World monkeys. Since many of their biological mechanisms are similar to those of humans, marmosets are potentially useful for medical and human biology research across a range of fields, such as neuroscience, regenerative medicine, and development. However, there is a lack of literature describing methods for many basic experiments and procedures. Here, detailed methods for determining the levels of sex hormones (progesterone, estradiol, and chorionic gonadotropin) in marmosets are described. The measurement of these hormones enables the prediction of the stage in the ovarian cycle, which is typically 26-30 days in marmosets; accurate determination is essential for the harvesting of oocytes/zygotes at the correct time point and for the preparation of host females for the generation of genetically modified marmosets. 
Additionally, the measurement of sex hormone levels is useful for endocrinology, ethology, early development, and reproductive biology studies. This protocol provides a detailed description of the methods for blood sampling from the femoral vein, separation of plasma for hormone measurement, measuring chorionic gonadotropin levels using urine and plasma, resetting the ovarian cycle using injections of a prostaglandin F2&#945; analog to shorten and synchronize the cycle, and promoting follicular growth and ovulation by injecting follicle-stimulating hormone and chorionic gonadotropin. Using these protocols, the stages in the ovarian cycle can be determined for the timely collection of oocytes/zygotes.

Author Spotlight: Advancing Primatology Through Germ Cell Research and Genetic Modification Techniques

Blood Sampling and Hormone Measurement for Determining the Stage in the Ovarian Cycle in Marmosets

We are working on two main aspects. First, we aim to understand male germ cells in primates, including humans. We are focusing on spermatogonial stem cells and the establishment of DNA methylation.Second, we aim to develop new techniques for making genetically modified marmosets. The generation of genetically modified marmosets requires a lot of effort. One of the challenges is developing more efficient method for making genetically modified marmosets.Our blood sampling protocol makes it easier to find veins and reduce the risk of drawing blood from arteries. Moreover, our protocol also measures three types of hormones and bring more accurate prediction and determination of ovulation date. We are now developing a technique for making sperm from iPS cells in marmosets.This technique will contribute to the study of germ cells and the generation of genetically modified marmosets.

Details related to the animals used in this study are listed in Table 1.
Anatomical analyses of the femoral vein 
Anatomical analyses of the femoral vein were performed using a 2-year-old male common marmoset (I 7713M) undergoing euthanasia. The femoral veins and arteries are located in the femoral triangle. The femoral triangle is formed at the boundaries between the abdominal wall and thigh muscles (Figure 1B-</stro...

This protocol describes blood and urine sampling for measuring progesterone/estradiol and chorionic gonadotropin levels to determine the ovarian cycle stage. The hormone levels are used to predict and determine the timing of ovulation and hormones are injected to regulate the ovarian cycle and oocyte growth.

Common marmosets are small New World monkeys. Since many of their biological mechanisms are similar to those of humans, marmosets are potentially useful ...

blood-sampling-hormone-measurement-for-determining-stage-ovarian

Research

JoVE Journal

Biology

542 Views.  National Center for Child Health and Development. We are working on two main aspects. First, we aim to understand male germ cells in primates, including humans. We are focusing on spermatogonial stem cells and the establishment of DNA methylation.Second, we aim to develop new techniques for making genetically modified marmosets. The generation of genetically modified marmosets requires a lot of effort. One of the challenges is developing more efficient method for making genetically modified marmosets.Our blood sampling protocol...

Video: Author Spotlight: Advancing Primatology Through Germ Cell Research and Genetic Modification Techniques

Collection of Blood from Marmosets for Hormone Analyses

To begin, prepare a one-milliliter syringe attached to a 25-gauge needle blade side up. Heparinize the syringe by drawing 200 microliters of undiluted heparin sodium solution and pulling it up and down several times. Then, expel the heparin solution from the syringe.Prepare absorbent cotton and alcohol swabs. Also, turn on an adjustable lamp to illuminate the area where the marmoset will be placed for blood sampling. Prepare the restraint device.Then, open the retention part with the sponge belt. After inserting the marmoset into the restraint device, hold and advance it into the cylindrical part. To secure it, press down the sponge belt.Place the leg from which blood is to be collected on top of the other. Hold the legs using the non-dominant hand, placing the middle and ring fingers inside each leg to fasten them and the other fingers outside each leg to fix them. Check if the femoral vein is visible near the base of the thigh.If not, palpate to find the pulsating artery and use it as a landmark to check if the vein runs inside it. Disinfect the puncture site using an alcohol swab. Insert the needle blade side up at an angle of 15 to 20 degrees.Stabilize the hand by resting it on the other hand to prevent the needle from slipping out of the blood vessel during collection. Gently, pull back the plunger to apply negative pressure. Push the needle tip forward.Once blood enters the syringe, maintain the position of the needle tip until the required volume has been collected. Carefully pull out the needle while pressing lightly on the puncture site using the little finger. Then, using an absorbent cotton swab, immediately apply pressure to the puncture site for three minutes to stop the bleeding.Invert the syringe to mix blood and heparin. After the bleeding has stopped, remove the sponge belt. Hold the animal's waist using one hand and rotate the animal to hold its underarm from the backside using another hand.Return the marmoset to its cage and provide the marmoset with its favorite food. Occasionally, check the occurrence of hematoma. Next, detach the needle from the syringe to prevent hemolysis.Then, slowly expel the collected blood along the inside of the wall of a 1.5-milliliter microtube. Centrifuge the tube at 1, 100g for five minutes at four degrees Celsius. Transfer the supernatant containing the separated plasma to a new tube, carefully avoiding the blood cells.Measure the progesterone and estradiol levels using an ELISA Kit or an automatic analyzer. Using this protocol, progesterone and estradiol levels were monitored in a three-year-old female marmoset for over 38 days. The initial luteal phase showed a high and stable P4 level, sharply decreasing on day 12, signaling the start of the follicular phase.This transition was also marked by a decline in E2 levels. During the follicular phase, P4 levels were consistently low with slight increases towards end, while E2 levels peaked sharply and then declined, marking possible ovulation. The luteal phase showed high levels of P4 until a sharp decline marked the transition from luteal to the follicular phase, paralleled by a reduction in E2 levels.Additional studies on marmosets highlighted the average durations of the follicular and luteal phases, helping to frame the typical hormonal cycle duration. The hormonal changes around ovulation include a rise in CG levels, a drop in E2 levels, and an increase in P4 levels. The increase in CG levels occurs prior to ovulation, suggesting that CG can serve as a predictor of impending ovulation.In contrast, the drop in E2 levels happens post-ovulation, making it the most promising marker for confirming that ovulation has occurred.