Chapter 15
Cryopreservation of Semen from Domestic Livestock:
Bovine, Equine, and Porcine Sperm
Harrie ¨ tte Oldenhof, Willem F. Wolkers, and Harald Sieme
Abstract
In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm
cryopreservation allows for long-term storage of insemination doses and secures reproduction at a desired
time point. In order to cryopreserve semen, it needs to be carefully processed to preserve its vital functions
after thawing. In this chapter, we describe the processes involved in cryopreservation of bull, stallion, and
boar sperm. These include preparation of diluents, dilution of sperm in primary and freezing extender, slow
cooling from room temperature to 5
C, packaging of insemination doses in straws, freezing at a defined
cooling rate in liquid nitrogen vapor, cryogenic storage, and thawing. Two-step dilution approaches, with
commonly used diluents, are presented, namely, TRIS-egg yolk (TEY) extender for bull sperm, skim milk
(INRA-82) extender for stallion sperm, and lactose-egg yolk (LEY) extender for boar sperm. Furthermore,
simple methods are presented for cooling and freezing of sperm at defined cooling rates.
Key words Bull, Cryopreservation, Lactose-egg yolk extender, Skim milk extender, Pig, Semen,
Spermatozoa, Stallion, TRIS-egg yolk extender
1 Introduction
Cryopreserved sperm can be used irrespective of locations of the
donor and recipient, stored for long durations, and serve as genetic
reserve even after sires have deceased. In modern livestock breeding, the use of cryopreserved semen for artificial insemination is
favored above use of cryopreserved embryos and oocytes. This is
related to the relatively low costs and minimal efforts needed for
cryopreservation of semen. In the cattle breeding industry, insemination almost entirely relies on using cryopreserved semen. In
equine breeding, cold-stored (5
C) diluted semen is predominantly used; however, there is growing interest to use cryopreserved semen. For porcine species, insemination is done with
diluted semen, which is stored and shipped at 16–20
C. In all
cases, sperm cryopreservation is used to secure valuable genetic
resources [1–3].
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021
365
Cryopreservation of Semen from Domestic Livestock:
Bovine, Equine, and Porcine Sperm
Harrie ¨ tte Oldenhof, Willem F. Wolkers, and Harald Sieme
Abstract
In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm
cryopreservation allows for long-term storage of insemination doses and secures reproduction at a desired
time point. In order to cryopreserve semen, it needs to be carefully processed to preserve its vital functions
after thawing. In this chapter, we describe the processes involved in cryopreservation of bull, stallion, and
boar sperm. These include preparation of diluents, dilution of sperm in primary and freezing extender, slow
cooling from room temperature to 5
C, packaging of insemination doses in straws, freezing at a defined
cooling rate in liquid nitrogen vapor, cryogenic storage, and thawing. Two-step dilution approaches, with
commonly used diluents, are presented, namely, TRIS-egg yolk (TEY) extender for bull sperm, skim milk
(INRA-82) extender for stallion sperm, and lactose-egg yolk (LEY) extender for boar sperm. Furthermore,
simple methods are presented for cooling and freezing of sperm at defined cooling rates.
Key words Bull, Cryopreservation, Lactose-egg yolk extender, Skim milk extender, Pig, Semen,
Spermatozoa, Stallion, TRIS-egg yolk extender
1 Introduction
Cryopreserved sperm can be used irrespective of locations of the
donor and recipient, stored for long durations, and serve as genetic
reserve even after sires have deceased. In modern livestock breeding, the use of cryopreserved semen for artificial insemination is
favored above use of cryopreserved embryos and oocytes. This is
related to the relatively low costs and minimal efforts needed for
cryopreservation of semen. In the cattle breeding industry, insemination almost entirely relies on using cryopreserved semen. In
equine breeding, cold-stored (5
C) diluted semen is predominantly used; however, there is growing interest to use cryopreserved semen. For porcine species, insemination is done with
diluted semen, which is stored and shipped at 16–20
C. In all
cases, sperm cryopreservation is used to secure valuable genetic
resources [1–3].
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021
365