Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction.
Mammalian spermatozoa utilize ATP as the energy source for key functions on the route to fertilization. ATP and its precursor nucleotides ADP and AMP are regularly investigated in sperm physiology studies, mostly by bioluminescence assays. Assay results vary widely, mainly due to different efficiencies in nucleotide extraction and prevention of their enzymatic degradation. Here, we describe a revised, validated protocol for efficient phosphatase inhibition and adenine nucleotide extraction resulting in consistently high ATP concentrations exceeding previously reported values for boar spermatozoa up to 20-fold. The revised assay is applicable for determining ATP concentrations and adenylate energy charge in extracts from fresh and frozen samples, thereby allowing simultaneous assessment of semen samples from long-term storage experiments. After validation, the assay was applied to liquid-preserved boar spermatozoa stored at 17°C and 5°C for 24 and 72 h. Cooling to 5°C, but not storage duration, reduced ATP concentration in spermatozoa (P<0.05), which was accompanied by the appearance of AMP and ADP in the preservation medium. ATP and energy charge were highly correlated to the proportion of membrane-intact spermatozoa, supporting the idea of nucleotides leaking through disrupted membranes in cold-shocked cells. The present assay allows highly standardized studies of energy metabolism in spermatozoa.
Unit for Reproductive Medicine of Clinics/Clinic for Pigs and Small Ruminants, University of Veterinary Medicine Hannover, Buenteweg 15, Hannover 30559, Germany.|Department of Animal Sciences, University of Göttingen, Albrecht-Thaer-Weg 3, Göttingen 37075, Germany.
Quynh Thu
QT
Nguyen
Unit for Reproductive Medicine of Clinics/Clinic for Pigs and Small Ruminants, University of Veterinary Medicine Hannover, Buenteweg 15, Hannover 30559, Germany.
Ulrike
U
Wallner
Clinic for Cattle, Endocrinology Laboratory, University of Veterinary Medicine Hannover, Hannover, Bischofsholer Damm 15, Hannover 30173, Germany.
Marion
M
Schmicke
Unit for Reproductive Medicine of Clinics/Clinic for Pigs and Small Ruminants, University of Veterinary Medicine Hannover, Buenteweg 15, Hannover 30559, Germany Dagmar.Waberski@tiho-hannover.de H.H.W.Henning@uu.nl.
Dagmar
D
Waberski
Unit for Reproductive Medicine of Clinics/Clinic for Pigs and Small Ruminants, University of Veterinary Medicine Hannover, Buenteweg 15, Hannover 30559, Germany Dagmar.Waberski@tiho-hannover.de H.H.W.Henning@uu.nl.|Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 112, Utrecht 3584 CM, The Netherlands.
Heiko
http://orcid.org/0000-0003-4064-7792
H
Henning
ATP; Energy charge; Spermatozoa