USING EXOSOMES FOR RESOLVING MARE ENDOMETRITIS
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FIGURE 1 The diagrammatic scheme of the treatment protocol using exosomes.
from the uterine body using a set of 60 cm alligator forceps. The goal of biopsy was to determine the mare ' s initial state, and the results indicated that all endometritis mares had degenerative chronic endometritis compared to normal healthy mares ' uterine biopsies (class IIB according to the categorisation of endometrial score proposed by Kenny and Doig ( 1986 ) as Moderate endome- tritis OR Moderate endometriosis OR Moderate lymphatic lacu- nae OR Bareness >2 years OR A combination of two categories from IIa findings).
Ultrasound and uterine blood supply assessment
All alterations in uterine vascular perfusion were assessed via a Doppler ExaGo device (made in France) with a 5– 7.5 MHz linear probe. The uterus was examined with a slow scan of the entire uterus by B- mode and then colour mode was activated to cap- ture a 15– 30 s video for later recording (Abdelnaby & Abo El- Maaty, 2021 ). The evaluation protocol includes the presence of pixels of the uterine body (using Image J program) represented by the blue and red colours of the colour Doppler, and the vas- cular distribution of the uterus, including all layers (Abdelnaby, El- Maaty, & El- Badry, 2021 ). Uterine arteries vascular perfusion was determined via b- mode and spectral mode in all females be- fore treatment (day −1 during oestrous), during treatment (day 0 = 1st ovulation, and day 0 = 2nd ovulation), and after treatment (day 0 = 3rd ovulation) as shown in Figure 1 . Doppler parameters were automatically calculated as follows: both Doppler important indices in the form of pulsatility and resistive (PI and RI), Doppler velocity readings (centimetres per second), such as uterine peak velocity (PV) of contraction and uterine end velocity (EV; centi- metre per second) of relaxation. In addition, uterine blood flow rate was measured (R/bpm). The device settings are Doppler filter
(200 Hz), PRF (3500 KHz), angle (40°) and the maximum speed (35 cm/s) using two- colour maps (red + blue, Figure 2 ).
Exosomes preparation
Aspiration of bone marrow from the iliac crest was performed in healthy horses to isolate CD70 and CD105 positive mesenchymal stem cells (MSCs), which were cultivated at 37°C in 5% CO2 environment. After 3rd passage, MSCs were cultured in Dulbecco ' s modified Eagle medium (DMEM) deprived of fetal bovine serum and supplemented with 0.5% of bovine serum albumin (Sigma). Then, ultracentrifugation of the media was performed to collect the exosomes. Exosome con- centration was determined by Bradford assay and 400 μg/mL of ex- osomes was added to the sterilised carboxymethylcellulose (CMC) at a concentration of 22 mg/mL. Each 1 mL of the gel contained 200 μg ex- osomes (Bahr et al., 2021 ). CMC gel- loaded MSCs- derived exosomes were preserved by freeze- drying (lyophilisation) method, which is ap- propriate to preserve thermo- liable materials as exosomes, as lyoph- ilised exosomes can be easily kept in a constantly storable state and can be simply reconstituted by adding water (Bahr et al., 2020 ). The treatment protocol proposed to treat this case was an intra-
uterine administration of lyophilised exosomes solution diluted with 0.9% for intrauterine injection and then with 50 mL NaCl 0.9% (Bahr et al., 2020 ). The exosome management protocol was developed as shown in Figure 1 . The protocol started on the day of ovulation (day 0) on mares treated with MVs and was detected transrectally by Doppler ultrasonography and clinical examination. The first treat- ment was intrauterine administration on day 0 (first ovulation) by exosomes diluted with 50 mL normal saline (NaCl 0.9%) as intra- uterine infusion only. The second dose was given as 50 mL on day 0 (second ovulation), 21 days later. The mares were then ultrasonically monitored every 48 h to detect the first ovulation after treatment
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