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.
