Two manual non-commercial preparation methods for platelet-rich plasma in donkeys (Equus asinus)

Abstract

Platelet-rich plasma (PRP) is a biologic, whole blood (WB) derived, immunomodulatory therapeutic that has gained popularity in brood mares for its potential to mitigate post-breeding uterine inflammation/infection and enhance fertility rates. In jennies, increased susceptibility to post-breeding endometritis and poor fertility rates have been associated with insemination using frozen-thawed jack semen. Because many donkey breeds are threatened with extinction, frozen semen technology offers a viable chance at recovery. Thus, PRP has potential in similarly benefitting donkey breeding programs. However, the majority of PRP protocols previously described in donkeys have not undergone critical assessment. Commercial and noncommercial PRP production methods exist, but the latter confers advantages (e.g., field applicability and decreased cost) for a species more likely to receive veterinary care in resource-limited settings. The objective of this study was to evaluate two manual noncommercial methods of producing PRP (double centrifugation [DC] and single centrifugation [SC]) in six healthy adult donkeys (n=3 jennies; n=3 johns; ages 4-8 years old) undergoing three blood collections two weeks apart. For DC-PRP, WB collected into a 150 mL blood transfusion bag containing citrate-phosphate-dextrose solution with adenine was split into 50 mL tubes, centrifuged at 400 × g/15 min, then plasma transferred into 15 mL conical tubes and centrifuged at 1000 × g/10 min. Afterwards, the bottom 2.5 mL of plasma per tube waspreserved as DC-PRP with the above supernatant as DC platelet-poor plasma (PPP). For SC-PRP, WB was collected into 2.7 mL sodium citrate vacutainer tubes, then centrifuged at 120 × g/10 min. Post-centrifugation, the top third layer of plasma was deemed SC-PPP with the remaining portion as SC-PRP. Concentrations of platelets, leukocytes (WBC), and erythrocytes (RBC), and platelet-derived growth factor-BB (PDGF-BB) were assessed in the WB, and extracted PRP and PPP. Data was analyzed by Friedman and Dunn's as a post hoc test with significance as P<0.05. Both protocols concentrated platelets 1.7–5.2-fold, reduced WBC 1.1–50.4-fold, and decreased RBC at least 829-fold compared to WB (P<0.0001). Activity of PDGF-BB in DC-PRP and SC-PRP increased by 1.2–7.0-fold in comparison to WB (P=0.0003). Platelet concentration and PDGF-BB activity rose 3-fold (P<0.0001) and 5.8-fold (P=0.0007), respectively, in DC-PRP than SC-PRP. Leukocyte concentration was wider in DC-PRP than SC-PRP (P<0.0001), but did not differ between WB and DC-PRP (P=0.61). Reduction in WBC and RBC was 42-fold and 22-fold greater, respectively, in SC-PRP than DC-PRP (P<0.0001). These results indicate that noncommercial methods of PRP preparation described in horses are applicable in donkeys. Double centrifugation yielded higher platelet concentration and PDGF-BB activity despite greater WBC and RBC contamination than PRP processing via SC. Future studies are needed to determine in vivo efficacy of PRP in donkeys.