Arresting calcium-regulated sperm metabolic dynamics enables prolonged fertility in poultry liquid semen storage

Abstract

The preservation of liquid semen is pivotal for both industrial livestock production and genetic management/conservation of species with sperm that are not highly cryo-tolerant. Nevertheless, with regard to poultry semen, even brief in vitro storage periods can lead to a notable decline in fertility, despite the in vivo capacity to maintain fertility for several weeks when within the hen's sperm storage tubules. For fertility in sperm, intracellular calcium ions ([Ca²⁺]i) play a key role in signaling towards modifying energy metabolism. While reducing [Ca²⁺]i has been found to enhance the preservation of sperm fertility in some mammals, the connection between semen fertility and calcium availability in avian sperm has received limited attention. In this study, we demonstrate that the use of extracellular and intracellular calcium chelators in liquid semen extenders, specifically EGTA and EGTA-AM, has distinct effects on prolonging the fertility of chicken sperm. These results were validated through in vivo fertility tests. Mechanistically, the effects observed were linked to coordination of mitochondrial metabolism and ATP catabolism. Despite both calcium chelators inducing hypoxia, they differentially regulated mitochondrial respiration and ATP accumulation. This regulation was closely linked to a bimodal control of dynein ATPase activity; a direct initial activation with reduction in [Ca²⁺]i, and subsequent suppression by cytoplasmic acidification caused by lactic acid. These findings not only contribute to advancing poultry liquid semen preservation techniques, but also elucidates biologically relevant mechanisms that may underlie storage within the female reproductive tract in birds.

Figure 1

Chicken sperm viability and motility during liquid storage. Sperm were incubated at 4 °C for 0–96 h in BPSE supplemented with 2 mM EDTA, 2 mM EGTA, 10 µM EGTA-AM, or 10 µM TPEN, and subjected to viability assay (a). Viability decreased in a storage period–dependent manner, with a notable reduction under BPSE and TPEN conditions (^a–cP < 0.05 in storage periods). Poststorage sperm of 72 h were analyzed using SMAS after activation with 5 mM Ca²⁺ addition (b). Multiple motility parameters in the EDTA, EGTA, and EGTA-AM groups were enhanced upon Ca²⁺ addition, showing significant differences compared with the control (BPSE) (^*P < 0.05 in treatments; ^‡P < 0.05 vs BPSE alone). Data are expressed as means ± SEM (n = 5, respectively).

Figure 2

Changes in [Ca²⁺]i, IPVL penetrability, and spontaneous AR in chicken sperm during liquid storage. Sperm were incubated at 4 °C in BPSE for 0, 24, 72, or 96 h, followed by [Ca²⁺]i quantification and an IPVL penetration assay. [Ca²⁺]i levels increased and IPVL penetrability decreased in a storage period–dependent manner (a and b). At 72 h poststorage, IPVL penetrability and spontaneous AR were examined in groups treated with 2 mM EDTA, 2 mM EGTA, 10 µM EGTA-AM, or 10 µM TPEN. EGTA and EGTA-AM prolonged IPVL penetrability (c) while inhibiting spontaneous AR (d). Data are expressed as means ± SEM (n = 5, respectively). ^a–dP < 0.05.

Figure 3

Mitochondrial redox and OxPhos of chicken sperm during liquid storage. Sperm were incubated at 4 °C for 72 h with 2 mM EDTA, 2 mM EGTA, or 10 µM EGTA-AM and subjected to quantification of ROS, ΔΨ_M, oxygen consumption and ATP levels. ROS content increased during storage but was reduced in the presence of EGTA-AM (a). Both EDTA and EGTA treatments decreased ΔΨ_M (b) and increased ATP content (d), whereas EGTA-AM had no effect on ATP. Oxygen consumption increased by EGTA and EGTA-AM treatments (c). Data are expressed as means ± SEM (n = 5, respectively). ^a–dP < 0.05.

Figure 4

Changes in energy metabolism in chicken sperm during liquid storage. Sperm were incubated at 4 °C for 72 h with 2 mM EDTA, 2 mM EGTA, 10 µM EGTA-AM or 10 µM TPEN and then [pH]i, lactic acid content, dynein ATPase, ADP/ATP ratio were measured. Liquid preservation increased [pH]i, but this was cancelled by EGTA and EGTA-AM (a), concomitantly with increase in lactic acid content (b). ATPase activity increased upon EDTA and EGTA-AM treatments but was reduced by EGTA (c). EGTA-AM treatment increased ADP/ATP ratio (d). Data are expressed as means ± SEM (n = 5, respectively). ^a–dP < 0.05.