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

<ol>
	<li>Kholkute, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+progesterone+levels+throughout+the+ovarian+cycle+of+the+common+marmoset+(Callithrix+jacchus).">Plasma progesterone levels throughout the ovarian cycle of the common marmoset (Callithrix jacchus).</a> Primates. 25 (1), 123-126 (1984).</li><li>Harding, R. D., Hulme, M. J., Lunn, S. F., Henderson, C., Aitken, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+progesterone+levels+throughout+the+ovarian+cycle+of+the+common+marmoset+(Callithrix+jacchus).">Plasma progesterone levels throughout the ovarian cycle of the common marmoset (Callithrix jacchus).</a> J Med Primatol. 11 (1), 43-51 (1982).</li><li>Gilchrist, R. B., Wicherek, M., Heistermann, M., Nayudu, P. L., Hodges, J. 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. P., Wachtman, L. M., Tardif, S. D., Mansfield, K., Fox, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Common Marmoset in Captivity and Biomedical Research. , (2018).</li><li>Schultz-Darken, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+collection+and+restraint+techniques+used+for+common+marmosets+(Callithrix+jacchus).">Sample collection and restraint techniques used for common marmosets (Callithrix jacchus).</a> Comp Med. 53 (4), 360-363 (2003).</li><li>Hopper, J., Kubik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+marmosets.">Common marmosets.</a> Handbook of Exotic Pet. , 27-42 (2020).</li><li>Harlow, C. R., Hearn, J. P., Hodges, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovulation+in+the+marmoset+monkey:+endocrinology,+prediction+and+detection.">Ovulation in the marmoset monkey: endocrinology, prediction and detection.</a> J Endocrinol. 103 (1), 17-24 (1984).</li><li>Ludlage, E., Mansfield, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+care+and+diseases+of+the+common+marmoset+(Callithrix+jacchus).">Clinical care and diseases of the common marmoset (Callithrix jacchus).</a> Comp Med. 53 (4), 369-382 (2003).</li><li>Summers, P. M., Wennink, C. J., Hodges, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloprostenol-induced+luteolysis+in+the+marmoset+monkey+(Callithrix+jacchus).">Cloprostenol-induced luteolysis in the marmoset monkey (Callithrix jacchus).</a> J Reprod Fertil. 73 (1), 133-138 (1985).</li><li>Takahashi, 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=Birth+of+healthy+offspring+following+ICSI+in+in+vitro-matured+common+marmoset+(Callithrix+jacchus)+oocytes.">Birth of healthy offspring following ICSI in in vitro-matured common marmoset (Callithrix jacchus) oocytes.</a> PLoS One. 9 (4), e95560 (2014).</li><li>Tomioka, I., Takahashi, T., Shimada, A., Yoshioka, K., Sasaki, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birth+of+common+marmoset+(Callithrix+jacchus)+offspring+derived+from+in+vitro-matured+oocytes+in+chemically+defined+medium.">Birth of common marmoset (Callithrix jacchus) offspring derived from in vitro-matured oocytes in chemically defined medium.</a> Theriogenology. 78 (7), 1487-1493 (2012).</li><li>Abe, Y., 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=Efficient+marmoset+genome+engineering+by+autologous+embryo+transfer+and+CRISPR/Cas9+technology.">Efficient marmoset genome engineering by autologous embryo transfer and CRISPR/Cas9 technology.</a> Sci Rep. 11 (1), 20234 (2021).</li><li>Summers, P. 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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonsurgical+uterine+stage+preimplantation+embryo+collection+from+the+common+marmoset.">Nonsurgical uterine stage preimplantation embryo collection from the common marmoset.</a> J Med Primatol. 23 (6), 333-336 (1994).</li><li>Hanazawa, K., 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=Minimally+invasive+transabdominal+collection+of+preimplantation+embryos+from+the+common+marmoset+monkey+(Callithrix+jacchus).">Minimally invasive transabdominal collection of preimplantation embryos from the common marmoset monkey (Callithrix jacchus).</a> Theriogenology. 78 (4), 811-816 (2012).</li><li>Ishibashi, 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=Efficient+embryo+transfer+in+the+common+marmoset+monkey+(Callithrix+jacchus)+with+a+reduced+transfer+volume:+a+non-surgical+approach+with+cryopreserved+late-stage+embryos.">Efficient embryo transfer in the common marmoset monkey (Callithrix jacchus) with a reduced transfer volume: a non-surgical approach with cryopreserved late-stage embryos.</a> Biol Reprod. 88 (5), 115 (2013).</li><li>Kishimoto, K., 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=Establishment+of+novel+common+marmoset+embryonic+stem+cell+lines+under+various+conditions.">Establishment of novel common marmoset embryonic stem cell lines under various conditions.</a> Stem Cell Res. 53, 102252 (2021).</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> J Appl Toxicol. 21 (1), 15-23 (2001).</li><li>Daskalaki, M., Drummer, C., Behr, R., Heistermann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+alfaxalone+for+short-term+anesthesia+can+confound+serum+progesterone+measurements+in+the+common+marmoset:+a+case+report.">The use of alfaxalone for short-term anesthesia can confound serum progesterone measurements in the common marmoset: a case report.</a> Primate Biol. 9 (2), 23-28 (2022).</li><li>Hodges, J. K., Cottingham, P. G., Summers, P. M., Liang, Y. 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. 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=Sensory+cues+and+the+suppression+of+reproduction+in+subordinate+female+marmoset+monkeys,+Callithrix+jacchus.">Sensory cues and the suppression of reproduction in subordinate female marmoset monkeys, Callithrix jacchus.</a> J Reprod Fertil. 97 (1), 301-310 (1993).</li></ol>

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><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&lt;br/&gt; http://www.jic-japan.jp/prd/marmoset/prd016.html&lt;br/&gt; 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>&amp;nbsp;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.

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...

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

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.

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 ...

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

Research

JoVE Journal

Biology

849 Views.  National Center for Child Health and Development. 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.

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

Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that neural mechanisms for certain behavioral tasks and response behaviors are different in females and males. Using females in research studies can represent a challenge because of the variation of gonadal hormones during their estrous cycle. This protocol describes well-established procedures that are useful in investigating the role of estrogen in fear extinction memory consolidation in female rats. Phase of the estrous cycle and exogenous estrogen administration prior to extinction training can influence extinction recall 24 hr later. The vaginal swabbing technique for estrous phase identification described here aids the examination and manipulation of naturally cycling gonadal hormones. The use of this basic rodent model may further delineate the mechanisms by which estrogen can modulate fear extinction memory in females.

Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration.

Extinction of conditioned fear has been extensively studied in male rodents. Recently, there have been an increasing number of studies indicating that ...

Behavior

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a lean PCOS-like mouse model that mimics women with PCOS is clinically meaningful. In this protocol, we describe such a model. By inserting a 4 mm length of DHT (dihydrotestosterone) crystal powder pellet (total length of pellet is 8 mm), and replacing it monthly, we are able to produce a PCOS-like mouse model with serum DHT levels 2 fold higher than mice not implanted with DHT (no-DHT). We observed reproductive and metabolic dysfunction without changing body weight and body composition. While exhibiting a high degree of infertility, a small subset of these PCOS-like female mice can get pregnant and their offspring show delayed puberty and increased testosterone as adults. This PCOS-like lean mouse model is a useful tool to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

We describe the development of a lean PCOS-like mouse model with&#160;dihydrotestosterone pellet to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a ...

Developmental Biology

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a promising future of using miRNAs in the diagnosis and care of lung diseases. Here, we define a protocol for miRNA profiling to measure the relative abundance of a group of miRNAs predicted to regulate inflammatory genes in the lung tissue from of an ozone-induced airway inflammation mouse model. Because it has been shown that circulating sex hormone levels can affect the regulation of lung innate immunity in females, the purpose of this method is to describe an inflammatory miRNA profiling protocol in female mice, taking into consideration the estrous cycle stage of each animal at the time of ozone exposure. We also address applicable bioinformatics approaches to miRNA discovery and target identification methods using limma, an R/Bioconductor software, and functional analysis software to understand the biological context and pathways associated with differential miRNA expression.

Here we describe a method to assess lung expression of miRNAs that are predicted to regulate inflammatory genes using mice exposed to ozone or filtered air at different stages of the estrous cycle.

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a ...

Immunology and Infection

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.

In many endocrine systems, circulating factors or hormones are not released continuously, but are secreted as a discrete pulse in response to a releasing ...

Neuroscience

Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-&beta;-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.

Here, we describe how to identify the stage of the murine reproductive (proestrus, estrus, metestrus, or diestrus) by simple, non-invasive collection and cytological assessment of vaginal smear samples. We further describe how vaginal cytology reflects circulating hormonal levels underlying transition through the murine reproductive cycle.

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the ...

DREADDs Technology and Automated Blood Sampling

Remote Neuronal Activation Coupled with Automated Blood Sampling to Induce and Measure Circulating Luteinizing Hormone in Mice

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of numerous inputs and neuronal populations in the modulation of LH release is still unknown. Measuring changes in LH levels in mice is often a challenge since they are easily disrupted by environmental stress. Current techniques to measure LH release and pulsatility require long-term training for mice to adapt to manipulation stress, certain restraint, the presence of the investigator, and working on individual animals, reducing its usefulness for many research questions.
This paper presents a technique to remotely activate specific neuronal populations using Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) technology coupled with automated sequential blood sampling in conscious, freely moving, and undisturbed mice. We first describe the stereotaxic surgery protocol to deliver adeno-associated virus (AAV) vectors expressing DREADDs to specific neuronal populations. Next, we describe the protocol for carotid artery and jugular vein cannulation and postsurgical connection to the CULEX automated blood sampling system. Finally, we describe the protocol for clozapine-N-oxide intravenous injection for remote neuronal activation and automated blood collection. This technique allows for programmed automated sampling every 5 min or longer for a given period, coupled with intravenous substance injection at a desired time point or duration. Overall, we found this technique to be a powerful approach for research on neuroendocrine control.

Author Spotlight: Automated Infusion and Blood Sampling for Precise Hormonal Analysis in Conscious Mice

Luteinizing hormone (LH) pulsatility is a hallmark of reproductive function. We describe a protocol for remote activation of specific neuronal populations linked to serial automated blood collection. This technique allows timed hormonal modulation, multiplexing, and minimizing manipulation effects on LH levels in conscious freely moving, and undisturbed animals.

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of ...

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive behaviors and the acquisition of gametes and embryos for physiological or molecular research. Amphibians contribute to one-quarter to one-third of vertebrate research, and of interest in this context is their contribution to the scientific community&#39;s knowledge of reproductive processes and embryological development. However, most of this knowledge is derived from a small number of species. In recent times, the decimation of amphibians across the globe has required increasing intervention by conservationists. The captive recovery and assurance colonies that continue to emerge in response to the extinction risk make existing research and clinical applications invaluable to the survival and reproduction of amphibians held under human care. The success of any captive population is founded on its health and reproduction and the ability to develop viable offspring that carry forward the most diverse genetic representation of their species. For researchers and veterinarians, the ability to monitor and control ovarian development and health is, therefore, imperative. The focus of this article is to highlight the different assisted reproductive techniques that can be used to monitor and, where appropriate or necessary, control ovarian function in amphibians. Ideally, any reproductive and health issues should be reduced through proper captive husbandry, but, as with any animal, issues of health and reproductive pathologies are inevitable. Non-invasive techniques include behavioral assessments, visual inspection and palpation and morphometric measurements for the calculation of body condition indices and ultrasound. Invasive techniques include hormonal injections, blood sampling, and surgery. Ovarian control can be exercised in a number of ways depending on the application required and species of interest.

The study of amphibian biology provides valuable information on the reproductive, physiological, embryological and developmental processes that drive organisms of many taxonomic groups. Here, we present a comprehensive guide on different methodologies that can be used to study ovarian control and monitoring in amphibians.

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive ...

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...

Rodent Estrous Cycle Monitoring Utilizing Vaginal Lavage: No Such Thing As a Normal Cycle

The current methodology establishes a reproducible, standardized, and cost-effective approach to monitoring the estrous cycle of female Sprague Dawley (SD) adolescent rats. This study demonstrates the complexity of hormonal cycles and the broad spectrum of understanding required to construct a reliable and valid monitoring technique. Through an in-depth examination of principal experimental design and procedural elements,&#160;this description of the cycle and its fundamental principles provides a framework for further understanding and deconstructs misconceptions for future replication.
Along with an outline of the sample collection process employing vaginal lavage, the procedure describes the mechanism of data categorization into the four-stage model of proestrus, estrus, metestrus, and diestrus. These stages are characterized by a new proposed approach, utilizing the 4 categorizing determinants of vaginal fluid condition, cell type(s) present, cell arrangement, and cell quantity at the time of collection. Variations of each stage, favorable and unfavorable samples, the distinction between cyclicity and acyclicity, and graphic depictions of the collected categorizing components are presented alongside effective interpretive and organizational practices of the data. Overall, these tools allow for the publication of quantifiable data ranges for the first time, leading to the standardization of categorization factors upon replication.

This study details the crucial factors to consider in experimental designs involving female rats. In a larger sense, these data serve to decrease stigma and assist in the development of more inclusive diagnostic and intervention tools.

The current methodology establishes a reproducible, standardized, and cost-effective approach to monitoring the estrous cycle of female Sprague Dawley ...

Enhancing Survival and Development of Marmosets Using a Hand-Rearing Approach

Hand-Rearing Method for Infant Marmosets

The common marmoset (Callithrix jacchus) is a small and highly social New World monkey with high reproduction rates, which has been proven to be a compelling non-human primate model for biomedical and neuroscience research. Some females give birth to triplets; however, the parents cannot raise all of them. To save these infants, we have developed a hand-rearing method for raising newborn marmosets. In this protocol, we describe the formula of the food, the time for feeding, the configuration of the temperature and humidity, as well as the adaptation of the hand-reared infants to the colony environment. This hand-rearing method significantly increases the survival rate of marmoset infants (without hand-rearing: 45%; with hand-rearing: 86%) and provides the opportunity to study the development of marmoset infants with similar genetic backgrounds raised in different postnatal environments. As the method is practical and easy to use, we anticipate that it could also be applied to other labs working with common marmosets.

Author Spotlight: Marmoset Research - Scope and Challenges 

Here, we describe a hand-rearing method for raising infant marmosets in an animal incubator. This method greatly increases the survival rate of marmoset infants, which provides the opportunity to study the development of marmoset infants with similar genetic backgrounds raised in different postnatal environments.