Cryopreservation of reproductive and germ cells of animals and humans

Cryopreservation is a process where cells, whole tissues, or any other substances susceptible to damage caused by chemical reactivity or time are preserved by cooling to sub-zero temperatures. At low enough temperatures, any enzymatic or chemical activity which might cause damage to the material in question is effectively stopped. Cryopreservation methods seek to reach low temperatures without causing additional damage caused by the formation of ice during freezing. Traditional cryopreservation has relied on coating the material to be frozen a class of molecules termed cryoprotectants. New methods are constantly being investigated due to the inherent toxicity of many cryoprotectants.

Human oocyte cryopreservation (egg freezing) is a novel technology in which a woman’s eggs (oocytes) are extracted, frozen and stored. Later, when she is ready to become pregnant, the eggs can be thawed, fertilized, and transferred to the uterus as embryos.

Method

The egg retrieval process for oocyte cryopreservation is the same as that for in vitro fertilization. This includes one to several weeks of hormone injections that stimulate ovaries to ripen multiple eggs. When the eggs are mature, a medication to trigger ovulation is given and the eggs are removed from the body using an ultrasound-guided needle through the vagina. The procedure is usually conducted under sedation. The eggs are immediately frozen.

The egg is the largest cell in the human body and contains a great amount of water. When the egg is frozen, the ice crystals that form can destroy the integrity of the cell. To prevent this, the egg must be dehydrated prior to freezing. This is done using cryoprotectants which replace the water within the cell and inhibit the formation of ice crystals.

Eggs (oocytes) are frozen using either a controlled-rate, slow-cooling method or a newer flash-freezing process known as vitrification. Vitrification is much faster but requires higher concentrations of cryoprotectants to be added. The result of vitrification is a solid glass-like cell, free of ice crystals. Vitrification is associated with higher survival rates and better development compared to slow-cooling when applied to oocytes in metaphase II (MII).[2] Vitrification has also become the method of choice for pronuclear oocytes, although prospective randomized controlled trials are still lacking.[2]

Once frozen, the zona pellucida, or shell of the egg hardens. Thus, currently, when eggs are thawed, a special fertilization procedure is performed by an embryologist whereby sperm is injected directly into the egg with a needle rather than allowing sperm to penetrate naturally by placing it around the egg in a dish. This injection technique is called ICSI (Intracytoplasmic Sperm Injection) and is also used in IVF.

Semen cryopreservation is a procedure to preserve sperm cells. Semen can be used successfully indefinitely after cryopreservation. For human sperm, the longest reported successful storage is 21 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.

Freezing

The most common cryoprotectant used for semen is glycerol (10% in culture medium). Often sucrose or other di-, trisaccharides are added to glycerol solution. Cryoprotectant media may be supplemented with either egg yolk or soy lecithin, with the two having no statistically significant differences compared to each other regarding motility, morphology, ability to bind to hyaluronate in vitro, or DNA integrity after thawing. [1]

Semen is frozen using either a controlled-rate, slow-cooling method (slow programmable freezing or SPF) or a newer flash-freezing process known as vitrification. Vitrification gives superior post-thaw motility and cryosurvival than slow programmable freezing.[2]

Thawing

Thawing at 40°C seems to result in optimal sperm motility. On the other hand, the exact thawing temperature seems to have only minor effect on sperm viability, acrosomal status, ATP content, and DNA.

Refreezing

In terms of the level of sperm DNA fragmentation, up to three cycles of freezing and thawing can be performed without causing a level of risk significantly higher than following a single cycle of freezing and thawing. This is provided that samples are refrozen in their original cryoprotectant and are not going through sperm washing or other alteration in between, and provided that they are separated by density gradient centrifugation or swim-up before use in assisted reproduction technology.

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10) Vector systems used in animal biotechnology
In molecular cloning, a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.
The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector which transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell. Vectors called expression vectors (expression constructs) specifically are for the expression of the transgene in the target cell, and generally have a promoter sequence that drives expression of the transgene. Simpler vectors called transcription vectors are only capable of being transcribed but not translated: they can be replicated in a target cell but not expressed, unlike expression vectors.
Plasmids are double-stranded generally circular DNA sequences that are capable of automatically replicating in a host cell. Plasmid vectors minimalistically consist of an origin of replication that allows for semi-independent replication of the plasmid in the host and also the transgene insert. Modern plasmids generally have many more features, notably including a "multiple cloning site" which includes nucleotide overhangs for insertion of an insert, and multiple restriction enzyme consensus sites to either side of the insert. In the case of plasmids utilized as transcription vectors, incubating bacteria with plasmids generates hundreds or thousands of copies of the vector within the bacteria in hours, and the vectors can be extracted from the bacteria, and the multiple cloning site can be cut by restriction enzymes to excise the hundredfold or thousandfold amplified insert. These plasmid transcription vectors characteristically lack crucial sequences that code forpolyadenylation sequences and translation termination sequences in translated mRNAs, making protein expression from transcription vectors impossible. Plasmids may be conjugative/transmissible and non-conjugative:
-conjugative: mediate DNA transfer through conjugation and therefore spread rapidly among the bacterial cells of a population; e.g., F plasmid, many R and some col plasmids.
-nonconjugative- do not mediate DNA through conjugation, e.g., many R and col plasmids.
Viral vectors
Viral vectors are generally genetically-engineered viruses carrying modified viral DNA or RNA that has been rendered noninfectious, but still contain viral promoters and also the transgene, thus allowing for translation of the transgene through a viral promoter. However, because viral vectors frequently are lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection. Viral vectors are often designed for permanent incorporation of the insert into the host genome, and thus leave distinct genetic markers in the host genome after incorporating the transgene. For example, retroviruses leave a characteristic retroviral integration pattern after insertion that is detectable and indicates that the viral vector has incorporated into the host genome.