44


| IMAGING AND DIAGNOSIS


Samples from a total of 48 jenny cases collected between 1 January 2019 and 31 December 2021 were used and analysed. All of the jennies were Yangyuan donkeys from Hebei province, China. Their ages ranged from 4 to 8 years (average 4.6 years old, SD 1.06). Examinations including reproductive history, reproductive tract ex- amination, low volume lavage (LVL) of the uterus, endometrial cytol- ogy, bacterial culture of endometrium, and endometrial biopsy were performed in all cases. Jennies with two or more of the following five criteria present were classified positive for endometritis: (1) abnormal breeding history and abnormal findings of reproductive examination, (2) abnormal appearance of LVL fluid, (3) positive endo- metrial cytology (the proportion of PMN >5%), (4) positive bacterial culture results of endometrial samples, (5) inflammation detected in the endometrial biopsy (Card, 2005 ; de Amorim et al., 2016 ). Jennies involved in this study were diagnosed with endometritis according to the checklist. Oestrus stage upon diagnosis was confirmed via transrectal ultrasound and listed in Table S1 . In all cases, uterine LVL was performed as described previously


for mares (Card, 2005 ). Basically, the jenny was restrained with the tail wrapped and tied to the body to fully expose the vulva. The vulva and perineal region were scrubbed with soap at least 3 times until the gauzes looked clean. A sterile obstetrical sleeve and sterile lube were applied for sampling. With the end covered by the sleeved hand, a uterine catheter (Animal Reproduction Systems) was carried to the external os of the cervix, then passed transcervically into the caudal uterine body and the balloon of catheter was then inflated. Approximately 150 mL sterile saline was infused into the uterus, and after a 30s transrectal massage to disperse the lavage fluid, it was recovered by gravity flow into a sterile 50 mL conical tube(s). The lavage sample was centrifuged at 400 × g for 10 min. The pellet was recovered with 3 mL supernatant retained and then resuspended via


TABLE 1 Endometrial biopsy results of 48 jennies.


Category of endometrium


I IIA


Numbers of animals


2 24 Remark


No abnormal findings in their luminal epithelium, endometrial glands, lymphatics, and vessels; inflammatory cells were sparsely scattered in both stratum compactum and stratum spongiosum; 1 of 2 jennies had mild stromal oedema


18 of 24 jennies had mild cellular infiltration of stratum compactum; 6 of 24 jennies had multifocal mild fibrotic changes (fibrosis of individual gland was 1– 3 layers or the average of fibrotic nests <2); 7 of 24 jennies had mild stromal oedema; no abnormal findings in all jennies ' luminal epithelium and vessels; no jennies had lymphatic lacunae or endometrial atrophy


IIB 15


6 of 15 jennies had diffuse and moderately severe foci of inflammation; 3 of 15 jennies had mild cellular infiltration of stratum compactum coexisting with moderate fibrosis (fibrosis of individual gland was >4 layers); 6 of 15 jennies had widespread fibrosis of individual gland branches or an average of 2– 4 fibrotic nests per high- power field (hpf); 7 of 15 jennies had stromal oedema; no abnormal findings in all jennies ' luminal epithelium and vessels; no jennies had lymphatic lacunae or endometrial atrophy


III 7


5 of 7 jennies had widespread and moderately severe foci of inflammation coexisting with widespread fibrosis; 2 of 7 jennies had widespread and uniformly distributed fibrosis of individual gland branches and an average of 2– 4 fibrotic nests per hpf, coexisting with lymphatic lacunae; no abnormal findings in their luminal epithelium and vessels except 1 jenny ' s endometrial luminal epithelium was lost due to artefact; no jennies had endometrial atrophy


WU ET AL.


vortexing. Smears of the sediment were made on at least 2 slides, air- dried and stained with Diff- Quick stain (Regal). After uterine lavage, endometrial biopsy samples were collected


using a uterine biopsy punch (CMF Medicon Surgical Inc.), which was performed as described previously for the mare (Snider et al., 2011 ). With transrectal guidance, the biopsy punch was passed through the cervix into the uterine body. The specimen was taken from the cranial aspect of the uterine body (Vilés et al., 2013 ). Tissues were carefully recovered from the punch and immediately stored in a 10% formalin solution until further processing for histopathology. The endometrial histology sections were stained with haematoxylin and eosin (H&E). Diagnostic samples were submitted to the pathologist at the


Equine Clinical Diagnostic Centre along with the jennies ' signalment, breeding history, and relevant clinical findings. The cytology slides were evaluated and graded for severity of inflammation based on the proportion of PMN as previously described: <5% PMN, non- inflammation; 5– 15% PMN, mild inflammation; 15– 30% PMN, mod- erate inflammation; >30% PMN, severe inflammation (Card, 2005 ). A standardised rubric for the histopathology interpretation was based on the grading scheme of Kenney and Doig (Canisso et al., 2019 ; Kenney & Doig, 1986 ; Snider et al., 2011 ). The rubric included the luminal epithelium, the endometrial glands arranged in stratum com- pactum and the stratum spongiosum, stromal tissue, lymphatics as well as blood vessels. Case information of all 48 jennies is summarised in Table S1 .


Two out of 48 jennies did not have the evidence indicative of endo- metritis in the biopsy samples and were classified as grade I, while 24/48 (54.16%), 15/48 (31.25%) and 7/48 (14.58%) jennies were cat- egorised as grade IIA, IIB, and III respectively (Table 1 ). In order to present the histopathology results more effectively and concisely, one representative endometrial biopsy sample of each category was selected for a detailed description. The biopsy with the most


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