INFLAMMATION, INFECTION, OR BOTH? ROOT CAUSES OF ENDOMETRITIS


are cytoplasmatic fragments of the megakaryocytes that carry numerous growth factors (e.g., vascular en- dothelial growth factor, transforming growth factor b), cytokines (e.g., TNF-a and CLX8), and antimicrobial peptides (e.g., platelet basic protein, RANTES, thymo- sin beta-4). These growth factors, cytokines, and anti- microbial peptides are released after platelet activation and act in an anti-inflammatory matter. Even though the mechanisms of PRP to mitigate inflammation are not yet well elucidated, the suppression of inflamma- torymarkers (e.g., COX-2,metalloproteinase-3, TNF-a, IL1b, IL6, CXCL8) has been described in several in vitro studies,16–18 as well as in mares treated with intrauterine infusion of PRP.9,13 In addition, PRP has demonstrated antimicrobial activity against microor- ganisms-causing endometritis19–21 and reduced post- breeding uterine infections in mares susceptible to endometritis.9 Many protocols can be used to produce PRP.22 Auto-


mated commercial and manual protocols have been described. These protocols differ in time to prepare, costs, practicality, volume of blood needed, and materi- als required for processing. Therefore, the protocol used will depend on the practitioner’s choice. In addition, variations of PRP quality can be observed between practitioners, methods, and animals. Although not all theprotocols availabletoproducePRP will beover- viewed in this study, the authors will comment on the most commonly used and proposed therapies in mares. Additional protocols can be reviewed in other manu- scripts.22,23 In addition, a commercially available ly- ophilized PRPa has been recently described for use in broodmare practice.24 Many clinicians have employed protocols using


vacutainer tubes or blood transfusion bags to produce PRP with good results for intrauterine infusion in mares. The vacutainer tubes protocol is themost com- monly used in the field. Using this protocol, blood is collected in 4.5mL vacutainer tubes containing 3.2% sodiumcitrate to produce PRP. Blood tubes are centri-


fuged at 120g for 10min. After centrifugation, the top third layer of the plasma is discarded, while the remaining plasma adjacent to the buffy coat is recov- ered as PRP (Fig. 1). The amount of blood collected to produce PRP by this method depends on the practi- tioner’s choice or the number of tubes placed in the centrifuge. To produce PRP using a blood transfusion bag, blood


is collected in a 450mL blood transfusion bag contain- ing 63mL of citrate-phosphate-dextrose solution with adenine as an anticoagulant (CPD-A). Four hundred milliliters of whole blood are split into eight 50-mL


tubes and centrifuged at 400g for 15min. After the first centrifugation, the plasma fraction is recovered and transferred into 15-mL conical tubes. This fraction


is submitted to centrifugation at 1000g for 10min. After the second centrifugation, 2.5mL of plasma at the bottom of each tube is preserved as PRP (Fig. 1). This method produces around 40 to 50mL of PRP for intrauterine infusion.


Practitioners have recently described another proto-


col that involves harvesting blood in a 60mL syringe prefilled with 7mL of CPD-A. The mixed-blood is transferred to a 50-mL falcon tube and centrifuged at


400g for 15min. After centrifugation, the whole plasma fraction (;30mL) is used for intrauterine infu- sion (Fig. 1). Many different protocols to obtain PRP have been used to treat mares with uterine problems. The number of treatments, the time that therapy was implemented, and the volume of PRP used for intrauterine infusion varies between studies.9,11–15,24 Independent of these differences, PRP therapy was able to reduce uterine inflammatory markers and improve the fertility rates ofmares in all reports.9,11–15,24 Results of PRP therapy in mares are highlighted in Table 1. In one of the authors’ studies,9 mares received four


uterine infusions of PRP (Fig. 2). In this study, the treatments were performed 48 and 24 h before and six and 24 h after breeding. The authors observed a sig- nificant benefit of those treatments in mitigating endometrial inflammation and uterine infections. However, implementing four treatments can be timing-consuming and expensive. Therefore, some practitioners have chosen only one or two treat- ments in a cycle to employ PRP therapy, as reported in other studies.11–15 In the author's experience, the best time to infuse PRP in mares to improve the uterine environment, mitigate post-breeding inflam- mation, and increase the chances of a mare becom- ing pregnant are 24 h before and 4-6 h after breeding. Practitioners can modify the protocol to best fit their practice.


3. Platelet Lysate


Platelet lysate (PL) is derived from freeze-thawing PRP. The PL contains all growth factors and cyto- kines released by the platelets after activation.25,26 Platelets are activated during cryopreservation, and the bioactive molecules released during this process are preserved in the PL.25,26 An advantage of PL com- pared to PRP is that this platelet-derived product can be frozen and stored long-term, making it available for immediate patient use. The processing of PL is simple. After PRP is obtained, it is diluted with platelet-poor plasma (PPP)


obtained during the processing protocol to bring a final volume of 10 to 20mL. Thereafter, the PRP alone


or the PRP+PPP should be frozen at 80°C. The freezing process will cause platelet disruption/activa- tion, and the PL containing platelet-released growth factors will be obtained.27 PL canbe frozenstored until needed. Before intrauterine infusion, PL must be at room temperature (20-25°C). Of interest, this therapy has been recently implemented to treat horses with osteoarthritis injuries.28,29 In a recent report, PL reduced PMN counts, intrauterine fluid accumulation, and edema score of mares susceptible to endometritis after breeding.30 Results of mares treatedwith PL are highlighted in Table 1.


AAEP PROCEEDINGS / Vol. 68 / 2022 47


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