Breed Diagnostics

The SpermSafe™ Story

The Problem

The traditional sperm storage methods of chilling and cryopreservation can inflict severe damage to sperm cells, so much so, that cells have a reduced lifespan once warmed up, and fertility is markedly reduced. Now, researchers from the University of Newcastle have developed a solution to this problem; a 17 °C sperm storage medium, SpermSafe™,that maintains sperm viability and fertility for at least 7 (and up to 14) days, without inflicting cold shock.

The Research

Dr Zamira Gibb and Dr Aleona Swegen carried out extensive research to firstly understand how stallion sperm cells metabolise energy [1], and then to identify the nutrients, antioxidants, and storage conditions required for optimal longevity [2-5]. During storage, sperm cells are actively metabolising, and in doing so, produce a great deal of harmful reactive oxygen species and waste products [1, 2]. Following repeated experimentation, these researchers devised the optimal formulation that works to neutralise these toxic by-products, thereby supporting sperm longevity. The result has been the development of a synthetic sperm medium that maintains cell motility, viability, and fertilising ability of cells up to 14 days [6]. The first foal bred using the sperm extender was born on Christmas Day 2018, with many more on the ground to date.

The Benefits

As an alternative to traditional semen storage methods, SpermSafe™ is more cost-effective, less labour-intensive, and less invasive for mares―offering greater flexibility in insemination regimens. SpermSafe™ is free of any biological materials, such as egg or milk, thereby eliminating the biosecurity threat that accompanies international importation, and reducing the risk of processing delays at international borders. Furthermore, this protein-free formulation produces significantly less breeding-induced endometritis compared to AI with frozen semen.

The Usage

Following collection, semen should be diluted (1:1) in any commercial milk-based extender.

For 7- to 14-day storage; extended ejaculates should be processed through a single layer colloidal gradient (such as EquiPure) before diluting the sperm pellet in SpermSafe™ at a final concentration of 50 million sperm/mL, for storage at 17 ⁰C.
For 3-day storage; extended ejaculates should be centrifuged, with the sperm pellet extended in SpermSafe™ at a final concentration of 50 million sperm/mL, for storage at 17 ⁰C.

References

1. Gibb, Z., Lambourne, S.R., and Aitken, R.J. (2014) The Paradoxical Relationship between Stallion Fertility and Oxidative Stress. Biology of Reproduction. 91 (3): p. 77.

2. Gibb, Z., Lambourne, S.R., Curry, B.J., Hall, S.E., and Aitken, R.J. (2016) Aldehyde Dehydrogenase Plays a Pivotal Role in the Maintenance of Stallion Sperm Motility. Biology of Reproduction. 94 (6): p. 133.

3. Swegen, A., Lambourne, S.R., Aitken, R.J., and Gibb, Z. (2016) Rosiglitazone Improves Stallion Sperm Motility, ATP Content, and Mitochondrial Function. Biology of Reproduction. 95(5): p. 107.

4. Gibb, Z., Lambourne, S.R., Quadrelli, J., Smith, N.D., and Aitken, R.J. (2015) L-Carnitine and Pyruvate are Prosurvival factors During the Storage of Stallion Spermatozoa at Room Temperature. Biology of Reproduction. 93 (4): p. 104, 1-9-104, 1-9.

5. Gibb, Z., Holt, B., Swegen, A., Lambourne, S.R., and Aitken, R.J. (2017) Mitochondrial permeability transition pore formation during chilling and cryopreservation of stallion spermatozoa, in 48th Annual Scientific Meeting of the Endocrine Society of Australia and the Society for Reproductive Biology. Perth, WA, Australia. p. 90.

6. Gibb, Z., Clulow, J.R., Aitken, R.J., and Swegen, A. (2018) First Publication to Describe a Protocol for the Liquid Storage of Stallion Spermatozoa for 7 Days. Journal of Equine Veterinary Science. 66: p. 37-40.

Protocol For Use

Prior to Use:

1. Upon arrival; store in -20 ⁰C freezer

2. Prior to use; reconstitute product by filling to 50 mL mark with the supplied sterile injection water or MilliQ water

Invert the bottle until all contents are dissolved – do not shake
Use at room temperature (RT) and keep out of direct sunlight

Following Semen Collection:

1. Dilute semen 2:1 with warm milk-based extender (such as EquiPlus, INRA96, or Kenneys), and allow to cool to room temperature.

Do not dilute raw semen with SpermSafe™ medium. SpermSafe™ is protein-free and cannot scavenge toxic seminal plasma proteins – milk components are required for this

2. Centrifuge extended semen as per usual protocol‍

‍For long-term storage, spermatozoa should be isolated using an EquiPure gradient to exclude dead and dying cells, seminal plasma, and microbial contaminants
For short-term storage (without EquiPure isolation), we recommend the use of a cushion during centrifugation to tightly pellet cells and allow the removal of all the seminal plasma
Remove all of the supernatant, down to the sperm pellet (for optimal storage it is vital that all seminal plasma is removed)

3. Resuspend sperm pellet in reconstituted RT SpermSafe™ medium to a final sperm concentration between 20 and 50 million sperm/mL

4. Store and ship in flat-bottom specimen pots at 17 ⁰C ‍(Note: Do not use semen syringes for storage)

5. Load into semen syringe immediately before insemination, ensuring semen is not left in the syringe for more than a few minutes

N.B. Use reconstituted product within 2 days and store at 4 ⁰C between use.

DO NOT REFREEZE!