Use of hypometabolic TRIS extenders and high cooling rate refrigeration for cryopreservation of stallion sperm: presence and sensitivity of 5′ AMP-activated protein kinase (AMPK).

The research article examines how using hypometabolic TRIS extenders with or without AMPK activators, in combination with high cooling rates, can impact the freeze resistance (freezability) of stallion sperm. The findings suggest utilizing a hypometabolic TRIS extender, with or without AMP or metformin, substantially improves stallion sperm’s ability to be frozen, when compared to a commercial extender.

Key Concepts

• Hypometabolic TRIS extenders are a type of diluent or additive used in sperm preservation to decrease the metabolic rate of sperm cells, therefore enabling better survival during freezing and thawing processes.
• AMPK activators (like metformin, AICAR, and AMP) are substances that stimulate the action of AMP-activated protein kinase, a crucial energy regulator in cells.
• A high cooling rate in the context of cryopreservation refers to rapidly cooling the samples to very low temperatures, which can help minimize ice crystal formation and potential damage to the cells.

Methodology

• The experiment involved cryopreserving twelve ejaculates using a commercial extender (Botucrio®) as a control group, and a basic TRIS extender (termed HM-0) with various supplements including metformin, AICAR, AMP, compound C AMPK inhibitor, or a combination of AMP and compound C.
• Metformin, AICAR, and AMP are recognized as activators of the AMPK, while compound C is used to inhibit the AMPK reaction.

Findings

• The study found that employing a hypometabolic TRIS extender, supplemented or not with AMP or metformin, notably enhances stallion sperm freezability when compared to a commercial extender.
• Fast cooling rates surprisingly don’t influence the quality of stallion sperm after cooling and thawing, implying that this factor doesn’t harm the sperm or reduce its freezability.
• Multiple putative AMPK sperm isoforms were identified in the stallion spermatozoa. Interestingly, these isoforms don’t seem to respond to the traditional AMPK activators, but do show reactions to the Compound C inhibitor. This discovery may hint at unique biological processes in sperm cells concerning energy regulation.