The Kacang goat is one of Indonesia’s germ-plasm, where its body shape is small and may not be profitable economically compared to foreign goats, but it has several advantages, including being easy to adapt because it is native to Indonesia and maintenance costs are not high. The breakthrough technology to produce goat embryos quickly can be done in vitro using the In vitro fertilization (IVF) method, either conventionally by adding mature eggs with spermatozoa outside the body or using the intra-cytoplasmic sperm injection (ICSI) method. The main obstacles to goat embryo production through conventional IVF are low oocyte quality and limited oocyte sources for in vitro goat embryo production.
Oocytes, as a source of female gamete cells, can be stored before maturation as an oocyte bank. The oocyte bank is a breakthrough for providing gamete cells for in vitro fertilization. Storage of oocytes for a long time for oocyte bank purposes can be done by cryopreservation. The best oocyte cryopreservation method is the vitrification method. The vitrification method is often used because there is no crystal ice formation, and the processing time is faster than the slow freezing method.
During the vitrification process, cryoprotectants are needed to protect oocytes both extracellularly and intracellularly from drastic temperature changes. This temperature change will cause an increase in Reactive Oxygen Species (ROS). Likewise, when warming up, a heat shock will occur. The heat shock occurs when there is a change in temperature from cold to warm. Increased ROS during thawing will cause damage to the mitochondrial function and changes in Ca2+ ion influx.
Changes in temperature due to vitrification and during thawing will highly affect the intensity of Ca2+ ions. The role of Ca2+ ions in oocytes is very important and is related to the oocyte maturation process. In the maturation process, there will be maturation of the nucleus and cytoplasm. In the nucleus maturation process, there will be an increase in Ca2+ influx into the oocyte nucleus, so protein regulators will run for the nuclear maturation process to occur. Calcium (Ca2+) is necessary for the fertilization process and embryo development.
The purpose of this study was to measure the expression of Ca2+ and the intensity of Ca2+ in cumulus-oocyte complex. The immunocytochemistry (ICC) method indicates an increase in Ca2+ expression. The extracellular Ca2+ expression can only be observed using the ICC method, while the intracellular Ca2+ intensity requires the use of the Confocal Laser Scanning Microscope (CLSM) method. The combination of the immunocytochemistry method and the (CLSM) can accurately measure the content and position of Ca2+ in oocytes due to the freezing process using the vitrification method.
The study was divided into two groups, the control group (C) of fresh COCs, and the treatment group (T) of COCs after vitrification. After vitrification for 24 hours, then thawing, the expression of Ca2+ was examined using the Immunocytochemistry (ICC) method and the intensity of calcium (Ca2+) with a Confocal Laser Scanning Microscope (CLSM). The research data obtained were analyzed statistically by T-Test. The results showed that the expression of Ca2+ in the control group (12.00±0.00) was different from the treatment group (0.35±0.79). The intensity of Ca2+ in the control group (1059.43±489.59) was different from the treatment group (568.21±84.31). The conclusion of this study is that cryopreservation affects calcium in COCs; there were differences in the expression and the intensity of Ca2+ between fresh COCs and COCs after vitrification. Ca2+ intensity of COCs after vitrification was concentrated in the nucleus, while in fresh COCs, it was concentrated in the cytoplasm.
Author: Widjiati
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