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Uploaded byAbhishek Singh
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Cryobanking of germplasm, sperm

This document provides information on cryopreservation of germplasm, sperm, and oocytes. It defines cryopreservation as the preservation of biological tissues at sub-zero temperatures, typically -196°C, to effectively stop all biological activities. Cryopreservation benefits include conserving germplasm of endangered species, fertility preservation, and creating stocks that remain viable indefinitely. The process involves using liquid nitrogen, cryoprotectants, and cryofreezers. It then describes the specific processes for cryopreserving sperm and oocytes, including the steps of collection, analysis, freezing, thawing, and potential risks and results.

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CRYOPRESERVATION OF GERMPLASM, SPERM AND OOCYTES SUBMITTED BY ABHISHEK ROLL NO. 47 M.SC FINAL SUBMITTED TO; DR. ANITA YADAV Department of biotechnology, Kuk
CRYOPRESERVATION Cryopreservation Drives From The Greek Word cryo meaning ‘cold’. Thus it refers to the preservation of biological tissue in sub zero temperature, typically -196 C At these temperatures, all biological activities of cells and tissues is effectively stopped or ceased. Cryogenic storage at very low temperature is presumed to provide an indefinite, if not near infinite, longevity to cells. Pre –implantation embryo, oocytes, spermatozoa, ovarian tissue can be cryopreserved.
BENEFITS OF CRYOPRESERVATION Effective mean to conserve the germ plasms of endangered species. Fertility preservation. Methods to reduce multiple pregnancies. Large range of stocks available Easy disease- free exchange of stocks, nationally and internationally Stocks remain viable indefinitely. Safety from disease, genetic contamination and breeding failure
WHAT YOU NEED FOR CRYOPRESERVATION Liquid nitrogen (liquid phase of vapor phase) • Characteristics of liquid nitrogen: - Chemically inert, Relatively low cost -Non-Toxic, Non-flammable, Readily available Cryoprotectant: organic or inorganic additive which will protect the cell from freezing injuries during cryopreservation. •Characteristicss of cryoprotectants: -should easily penetrate into cell -Non- electrolyte, Easy misible with water -E.g: Glycerol, DMSO, PVP, PEG etc. Cryofreezer
CRYOPRESERVATION OF SPERM •It is commonly known as sperm banking. •Sperm banking is a procedure to preserve sperm cells for future use. •The first successful cryopreservation of spermatozoa was initiated over 50 year ago. •For human sperm, the longest reported successful storage is 22 years. •It can be used for sperm donation where the recipient wants the treatment in a different time or place or for men undergoing a vasectomy to still have the option to have children. •It is also useful for men suffering from azoosprmia (Lack of motile sperm) and Gonadial cancer. •These frozen sperm can be used in association with one of the Assisted Reproductive Techniques(ART) to induce pregnancy.
PROCESS OF SPERM CRYOPRESERVATION Obtaining semen sample: - Semen sample are collected in sterile container. -The semen so collected is suspended in 10-20% glycerol in egg yolk buffer. Semen analysis: -Semen samples are analyzed for volume, viscosity and PH level, and microscopically evaluated to determine motility, sperm count and morphology. Freezing: -Freezing of semen is done either in straw or ampoules. -Freezing can be achieved in three ways: 1. Slow freezing 2. Rapid freezing
1.Slow freezing: liquid nitrogen is poured into the tank and cooling rate is obtained from 20- 80 degree C at rate of 1.5 degree C/min and then at 6 degree C/min at completion of the freezing the straws are removed and stored into nitrogen at -196 degree C. Thus take about 40 min. 2.Rapid freezing: Required direct contact between straws and the nitrogen vapors for 8-10 min. and immersion in liquid nitrogen at -196 degree C. . The sample is initially mixed drop wise manner with equal volume cold cry protectant the mixture is loaded into the straws and left to incubate at 4 degree C for 10 min. .The straws are then placed at distance of 15-20 cm above the level of liquid nitrogen (-80 degree C )for 15 min. after the stage, the straws are immersed in liquid nitrogen. Thawing: . It is done by putting ampoule containing the sample in a warm water bath (35 to 40 degree C) . Frozen tips of the sample in tubes or ampoules are plunged into the warm water with a vigorous swirling action just to the point of ice disappearance. . Just a point of thawing quickly transfer the tubes to a water bath maintained at room temperature and contained the swirling action for 15 sec to cool the warm walls of the tube.
CRYOPRESERVATION OF OOCYTES • Cryopreservation of oocytes is also known as Egg freezing is a method used to save women’s ability to get pregnant in the future. • Eggs harvested from your ovaries are frozen unfertilized and stored for later use. • A frozen eggs can be thawed, combined with sperm in a lab and implanted in your uterus (in vitro fertilization)
WHY IT’S DONE • Egg freezing might be an option if you’re not ready to become pregnant now but want to try to make sure you can get pregnant later. • unlike with fertilized egg freezing (embryo cryopreservation), eggs freezing doesn’t require sperm because the eggs aren’t fertilized before they are frozen.
YOU MIGHT CONSIDER EGG FREEZING IF; • you have a condition or circumstance that can affect your fertility. like sickle cell anemia, autoimmune disease • you are undergoing in vitro fertilization. when undergoing in vitro fertilization, some people prefer egg freezing to embryo freezing. • you need treatment for cancer or another illness that can affect your ability to get pregnant. such as radiation or chemotherapy – can harm your fertility • you wish to preserve younger eggs now for future use. freezing eggs at younger age might help you get pregnant when you’re ready.
RISKS • Condition related to the use of fertility drugs: Rarely use of injectable fertility drugs can cause your ovaries to become swollen and painful soon after ovulation or egg retrieval. • Egg retrieval procedure complication: Rarely, use of an aspirating needle to retrieval eggs causes bleeding, infection or damage to the bowel bladder or a blood vessel • Emotional risks: Egg freezing can provide hope for a future pregnancy, but there’s no guarantee of success.
PROCEDURE OVARIAN STIMULATION Synthetic hormones to stimulate ovaries to produce multiple eggs. EGG RETRIEVAL Egg retrieval is done under sedation. A common approach is transvaginal ultrasound aspiration. FREEZING Shortly after your unfertilized eg are harvested, they’re cooled to sub zero temp. to preserve them for future use.
RESULTS • Use frozen eggs, they’ll be thawed, fertilized with sperm in lab and implanted in your or a gestational carrier’s uterus. • Using a fertilization technique called intracytoplasmic sperm injection (ICSI)
REFERENCE • Molecular biotechnology; principle and application of recombinant DNA, Bernard R.Glick, jack j. Pasternak, and Chery L. Patten • Pegg DE (January 1, 2007). "Principles of cryopreservation". Methods in Molecular Biology. • Mazur P (May 1970). "Cryobiology: the freezing of biological systems". Science. • Deller RC, Vatish M, Mitchell DA, Gibson MI (February 3, 2014). "Synthetic polymers enable non-vitreous cellular cryopreservation by reducing ice crystal growth during thawing". Nature Communication
Cryobanking of germplasm, sperm