Phosphatidylcholine and cholesterol-loaded-cyclodextrins can replace egg yolk in the cryopreservation of equine spermatozoa

Abstract

In the horse breeding industry, cryopreservation of spermatozoa has become an integral aspect of modern-day protocols. To avoid sperm damage during the freezing process, freezing media containing egg yolk are used. Recently, urgency to find an alternative to egg yolk has been increased due to the prevalence of Avian Influenza Virus (AIV). In the UK, the World Organization for Animal Health reports that between July and December 2022, an AIV outbreak led to the death of 4.62 million birds (56.73 million globally), with substantial consequences for agriculture, environment and egg availability (WAHIS. 2023; https://wahis.woah.org/#/dashboard/qd-dashboard: Accessed: 12 January 2023). Recent research has also called into question the biosafety of egg yolk, with RNA from highly pathogenic and low pathogenic AIV found on and in eggs (Stephens.et.al. Avian Diseases. 2020; 64:143-148). The cryoprotectant potential of phosphatidylcholine, the main phospholipid in egg yolk, has been investigated in diluents used for cryopreservation of human spermatozoa with some success (Sicchieri.et.al. Translational Andrology and Urology. 2021; 10:397-407). The chemically defined origin and increased biosafety make phosphatidylcholine a desirable alternative to egg yolk for use in cryopreservation. Ejaculates from four horses (n=4) were frozen using four different freezing extenders: a reference medium containing egg yolk and 3% dimethylformamide (control) and media without egg yolk supplemented with 1.5mg/ml2-hydroxypropyl-β-cyclodextrin cholesterol complex, 3% dimethylformamide and either 2% (P1), 4% (P2) or 6% phosphatidylcholine (P3). Samples were diluted and centrifuged on a density gradient medium (1000xg’ for 20 minutes). The cell band was aspirated and resuspended (200 × 10⁶ cells/ml) with allotted treatment extender. Samples were packed into 0.25ml straws, cooled to 5˚C for 25 minutes, then frozen using a programmable freezing unit (IceCube14s, Minitube, Germany). Temperature of the straws was reduced from 4 ˚C to -143˚C in 9 minutes, after which they were plunged directly into liquid nitrogen (-196˚C). Viability was assessed using NucleoCounter® NC-100™. Total motility analyses were performed with computer assisted semen analysis (Hamilton Thorne, USA). No difference in post-thaw viability was found between any of the media tested (P>0.05). No difference in total motility was found between the control (36.75±1.93σM), P1 (29.25±3.82σM) or P2 media (19.5±6.35σM) (P>0.05). However, post-thaw total motility of sperm cryopreserved in P3 media (16±6.01σM) was lower than the control (P<0.05). Media supplemented with 2-4% phosphatidylcholine and 1.5mg/ml cholesterol was effective in limiting loss of total motility and membrane integrity of equine spermatozoa, establishing the need for further breeding trials into the use of phospholipids for egg yolk lipid replacement to improve biosafety. The authors wish to recognize the support of Tullis Matson and Stallion AI Services Ltd in this study.