Semen freezing: a brief history

In the 17th century an Italian priest named Lazzaro Spallanzani first documented the effect of cold on sperm, noting that sperm cooled by snow became motionless, but if reheated, motion would be restored. The next major breakthrough following Spallanzani’s observations happened in 1949 when A.S. Parkes and two British scientists discovered that glycerol could protect sperm from damage during freezing.

The use of glycerol as a cryoprotectant and the development of artificial insemination in dairy cattle during the 1950’s pushed for the long term storage of bull semen to be developed on a commercial scale. In 1953 the first successful human pregnancy was produced using frozen sperm. 

In the 1960’s the use of liquid nitrogen for long term storage of bull semen was adopted. Liquid nitrogen allowed for the widespread use of cryopreserved semen and also made it more reliable across the industry. In the 70s, the sperm bank industry grew across the United States and protocols for freezing semen across different species were developed. Today, cryopreserved semen is used across the world in the livestock industry as well as in human medicine. 

Step 1: Semen Collection

Sterling Sire Services always tries to collect males by jumping them and using an artificial vagina(AV). Those rams who do not jump using an AV will be collected using an electro-ejaculator, with consent from the owner. Use of an AV has been shown to improve ejaculate concentration and uniformity while also minimizing the risk of contamination. 

Step 2: Pre-freeze processing 

Following collection, the sample is immediately taken into the lab for processing. Processing the sample starts with a concentration reading using a photometer and the initial dilution with Sterling’s part A extender. Once extended, an initial motility and morphology check is done utilizing a phase contrast microscope. 

If the ejaculate passes the pre-freeze motility and morphology check, the extended sample begins the chilling phase.   

Step 3: Chilled Processing

Slowly chilling the sample utilizing a cold room minimizes the amount of damage done to cells from temperature change. After being chilled and allowed to equilibrate, the sample is extended with Sterling’s part B extender. 

Following another equilibration period after the sample is extended with part B, the semen is loaded into 1/4cc straws and sealed using ultrasonic sealing technology.  Straws are printed each freeze day with the name of the ram, his unique cane code, and the date. The straws are then loaded onto racks and ready to be frozen.

Step 4: Semen Freezing and Post Thaw evaluation 

Loaded racks are then lowered into a vat and held above the surface of liquid nitrogen. After freezing in nitrogen vapors, the straws are plunged into nitrogen as the final step in the freeze process. 

Following freezing, a straw from each sample frozen on that day will be subjected to a post thaw evaluation.  The following standards must be met to pass the post thaw evaluation: 

• >30 million live cells per straw
• >50% motility 
• <15% abnormalities

Straws that pass the post thaw evaluation will be caned and ready for use.