Semen Storage and Dilution

Semen Dilution and Storage Dr. Salah M. Salih  Semen processing (Semen Handling) Including: 1. Dilution (Extension). 2. Package of semen. 3. Preservation (Storage). 4. Insemination (Usage). Dilution or Extension: It is addition of suitable media to the semen for certain reasons: a. Incr...

Semen Dilution and Storage Dr. Salah M. Salih  Semen processing (Semen Handling) Including: 1. Dilution (Extension). 2. Package of semen. 3. Preservation (Storage). 4. Insemination (Usage). Dilution or Extension: It is addition of suitable media to the semen for certain reasons: a. Increases the semen volume leading to inseminating larger number of females. b. Supply proper substances to protect spermatozoa from sudden variations in temperature, also nutrients supplements. c. Improving spermatozoa fertilization ability especially by decreasing capacitating time.  Factors affecting dilution rate calculation 1- Species of animal; diluting rate of bull semen ranged from 1:15 to 1:50 while for ram and buck semen ranged from 1:3 to 1:5. 2- Semen properties; progressive motility percentage and sperm concentration. 3- Inseminating dose. 4- Temperature of semen preservation. In deep- freezing preservation with optimum conditions approximately 20 – 30% of sperms die.  TYPES OF DILUTERS Many different types of extenders have been used in different species. The choice of diluter depends upon the species and the way semen would be stored (Liquid/Frozen).  Procedure of semen dilution Semen is kept at 37 C in a water bath The diluter is also kept at same temperature Check the diluter for pH etc. Add the semen to diluter in small parts and check the motility after each  SEMEN DILUTION RATES The dilution rates for semen would depend upon the sperm concentration and the use(Liquid/Frozen) For Frozen semen Dilution is done in such a way that the final volume of semen straw will have 20-30 million sperms (bull semen) as the final minimum number of sperms that are required for insemination is 10 million  Package of semen or semen canning The common forms of semen packaging are; Straw, Ampoule, Tube and Pellet. Each package should be clean, dried and sterilized before using and label with some data such as, name of producer center, country of origin, name of bull, date of manufacturing, breed of bull……etc  SEMEN CRYOPRESERVATION Cryopreservation is the preservation of sperm cells at ultra low temperatures. At ultra low temperatures, (-196 C) any enzymatic or chemical activity and metabolic activities of cells are stopped. Cryopreserved semen can be ideally kept for indefinite periods without loss of fertility All the metabolic activities of cells stored at ultra low temperatures are stopped  Causes of sperm damage during cryopreservation 1-Solution effects Solution effects caused by concentration of solutes in non-frozen solution during freezing as solutes are excluded from the crystal structure of the ice. High concentrations can be very damaging. 2-Extracellular ice formation When tissues are cooled slowly, water migrates out of cells and ice forms in the extracellular space. Too much extracellular ice can cause mechanical damage due to crushing 3-Dehydration The migration of water causing extracellular ice formation can also cause cellular dehydration. The associated stresses on the cell can cause damage directly. 4-Intracellular ice formation When cryopreservation is done rapidly intracellular ice forms in the cells  CRYOPROTECTANTS Cryoprotective agents can be divided into permeating and non-permeating agents. 1. Permeating agents (penetrate the plasma membrane) such as glycerol, dimethyl acetaldehyde; dimethyl sulfoxide, ethylene glycol. They stabilize cell plasma membrane proteins and reduce concentrations of electrolytes. 2. Non permeating agents (unable to penetrate the plasma membrane) such as albumins, dextrans, egg yolk citrate, hydroxyethyl, polyethylene glycols, polyvinyl pyrollidone, fructose and sucrose. They minimize intracellular crystallization by increasing viscosity of the sample.  Packaging of semen: There are many types of packaging semen which include: 1- Ampoule 2- Pellets 3- straw  Ampoule Glass ampules with 0.5-ml, 0.8-ml, and 1.0-ml capacities were used almost exclusively for packaging from the onset of frozen semen until about 1970. The ampules were filled through a small opening and heat was used to melt the glass to form a seal  Pellets Semen containing about 10 times the usual number of motile sperm is placed in the depression to freeze. The usual volume is about 0. I ml. For insemination, the pellets are thawed in enough warm diluter to provide adequate volume for insemination.  Disadvantages of the pellet Two major disadvantages of the pellet are: 1- Difficult identification of the individual pellet and microbial contamination incurred during handling. 2- There is also some possibility of mixing sperm from different bulls from forceps used to handle the pellets. Difficult to handle and transport, dry ice must be replenished frequently.  straw The 0.5-ml plastic straw has been the package of preference since about 1970. Plastic straws with 0.25- ml and 0.5 ml capacities are used One end of the straw contains a three-part plug. A small amount of polyvinyl alcohol powder is placed between two small cotton plugs. The straws are filled by applying a vacuum to the end with the plugs. The powder allows air to pass through as long as it remains dry, but when aqueous material (the semen) comes in contact with the powder it forms a seal which will not allow liquid or air to pass through. The opposite end of the straw is sealed ultrasonically after filling. The standard procedure for freezing is to place a single layer of straws on a tray. The tray is placed about 5.5 cm above the liquid nitrogen level of a large storage unit. The cold nitrogen vapors in this area will freeze the semen at about the desired rate. Straws will reach the vapor temperature in about 2 minutes.  major advantages of the straw over the ampule is the conservation of storage space. Up to three times as many straws can be stored in a field or storage unit as ampules. Most studies indicate that the straw offers the added advantage of increased sperm survival and a slight increase in conception rate over the ampule Packaging of semen