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INTRODUCTION Although a healthy uterine environment
aids proper embryo
growth and development, clinical or subclinical abnormalities may contribute to pregnancy failure (LeBlanc & Causey, 2009 ; Sikora et al., 2015 ). Since the pre- implantation phase is so important in mares (Koot et al., 2011 ; LeBlanc, 2010 ), endometritis is one of the most common causes of decreased mare fertility (Canisso et al., 2020 ; Hurtgen, 2006 ; Troedsson & Woodward, 2016 ). It is a chronic inflammatory change of the endometrium that decreases the pregnancy rate (Hanlon et al., 2012 ; Traub- Dargatz et al., 1991 ) resulting in conception failure. However, it is a natural inflammatory response that incorporates numerous mechanical channels and im- munological reactions to eliminate semen and microorganisms from the uterus within 48 h of breeding (Woodward & Troedsson, 2015 ). Chronic endometritis is considered an inflammatory disease of the uterine mucosa (inner layer) that impairs the animal ' s fertility. Chronic endometritis can occur because of unresolved post- mating endometritis or weakened intrinsic uterine defences,
allowing
local microorganisms to proliferate uncontrollably, resulting in chronic inflammation (Morris et al., 2020 ). Therefore, chronic en- dometritis is one of the most common causes of infertility, early embryo mortality and embryo death in mares (Canisso et al., 2016 ; Varadin, 1975 ). Despite the large range of therapeutics available, their usefulness has been questioned due to the variability of re- sponses (Scoggin, 2016 ), and no effective treatments have been able to renew or halt the harmed tissue. However, regenerative medicine therapy might enhance the embryo- maternal relationship. Stem cell therapy has recently been offered as an alternate treat- ment for uterine illness (Mambelli et al., 2013 ; Rink et al., 2018 ). Although transplantation of stem cells (SCs) has been reported (Kotton et al., 2001 ), only a small portion of the injected stem cells is successfully transplanted into various disease models (Chimenti et al., 2010 ). In an irradiated mouse model, SCs derived from bone marrow were examined for endometrial regeneration and showed a small number of cells transplanted into the regenerating endome- trium (Gil- Sanchis et al., 2015 ). Blood flow also plays an important role in inflammation via
vasodilation action (Debertolis et al., 2016 ; Foster, 2007 ). Colour Doppler ultrasonography has been used to noninvasively inves- tigate the blood flow to the reproductive tracts of healthy and diseased mares (Abdelnaby, Emam, Salem, et al., 2020 ), in addi- tion to its application in buffalos (El- Sherbiny et al., 2022 ), and she donkeys (Abdelnaby et al., 2022 ). The most common approach to this technique involves examining blood flow in the corpus luteum or follicle in colour mode (Miyamoto et al., 2005 ). The spectral mode, on the contrary, is shown to examine the uterine artery (Abdelnaby, 2020a ). In colour Doppler examinations, angiogenesis can be determined by quantifying the number and size of dots or coloured areas (objective assessment) and assessing the dots (such as subjective assessment using the magnetic lasso tool in Adobe Photoshop program). The main advantage of this type of evalua- tion is the direct evaluation of the characteristics of blood vessels
ABDELNABY ET AL.
within the structure (Sá et al., 2020 ). Because uterine glandular se- cretion is thought to be critical for embryo implantation, this study proposes a novel therapeutic approach based on the endometrium regeneration and restoration using exosomes that were derived from bone marrow SCs through a paracrine communication mech- anism. However, a scarce number of papers are available regarding the therapeutic application of exosomes in Arabian mare endo- metritis linked with the Doppler investigation. This novel study used exosomes to treat chronic endometritis as an intrauterine infusion to determine the alterations in uterine haemodynamics, hormonal levels and histopathological evaluations before and after treatment.
MATERIALS AND METHODS Study population, plan and management
Fourteen Arabian mares aged 13– 15 years were divided into two groups: control group ( n = 7) with neutrophils to epithelial cells ratio of ≤2% and an endometritis group (chronic type; n = 7). No previous gynaecological problems were reported in all mares. For each mare, multiple breedings via a combination of either
live cover and artificial insemination had been performed without success and with no attempts performed within the last 3 years prior to enrolment in this study. All mares are pluriparous that previously mated physiologically by
a mature adult stallion. All females weighed between 300 and 500 kg (average, 410 ± 20 kg) with a body condition score of 4 (Garlinghouse & Burrill, 1999 ). Samples were collected days before treatment (day −1, during oestrus), during treatment (day 0 of the 1st cycle and day 0 of the 2nd cycle) and after treatment (day 0 of the 3rd cycle) as shown in Figure 1 , and samples were examined from all animals during oestrus phase. Those days were standardised in all mares.
Clinical examination
An examination was done, and then history and clinical signs were determined for each Arabian mare. Palpation of the reproductive tract was done rectally. There was no relevant history, and the gynaecological examinations were normal. Visual examination of external genitalia determines the amount of separation be- tween the anus and vulva, with past lacerations or Caslick opera- tions considered. A physical assessment of the vestibular- vagina sphincter ' s integrity was also done to rule out subsequent infec- tions caused by pneumovagina or faecal contamination. A sterile tubular speculum was used to examine the vagina and cervix for changes that might suggest endometritis, such as purulent leak- age, cervical
lesions or urine stagnation. The vestibule- vaginal
characteristics were also examined to determine the oestrus cycle stage. The uterus, particularly the cervix, showed a small increase in tone and consistency. Finally, endometrial samples were taken
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