Fungal DNA in pooled equine uterine samples: an exploratory ITS2 amplicon sequencing pilot study

Fungal DNA in the equine uterus remains poorly characterized, particularly in mares without overt clinical uterine disease. This exploratory pooled pilot study used ITS2 amplicon sequencing to describe fungal DNA profiles in pooled uterine samples and to perform descriptive comparisons by subsequent pregnancy outcome and sampling method. Conventional microbiological culture findings were summarized as clinical context. samples were collected during naturally occurring estrus and, in mares that were subsequently mated, before the first mating of the studied estrous cycle. Low-volume uterine lavage (LVL) samples from 12 naturally mated Trakehner mares were pooled by outcome into a pregnant-LVL pool (1FR; n = 5) and a non-pregnant-LVL pool (2NFR; n = 7). The paired sampling-method comparison comprised a paired LVL pool (3LVL) and a paired SWAB pool (4SWAB), each constructed from samples collected from the same five mares. After bioinformatic processing, 82,862–130,870 reads and 27–111 final unique amplicon sequence variants were retained per pooled library. ITS2-target quantitative PCR detected 1,107–1,728 target copies/μL of pooled DNA extract. Neither the extraction blank nor the library-preparation blank crossed the qPCR detection threshold within 40 cycles; however, both yielded sequencing reads, and sequence-resolved control analysis could not exclude low-level laboratory or sequencing-related contamination. A targeted sensitivity analysis excluding the sequence feature shared by the library-preparation blank and two biological libraries did not materially alter the descriptive taxonomic composition, diversity, or ordination patterns. Ascomycota predominated across the pooled libraries (50.7–72.5%), followed by Basidiomycota (8.1–24.1%). The pregnant-LVL library had the lowest pool-level Shannon diversity value and occupied an ordination position that differed from the other libraries primarily along the first principal coordinate. The paired LVL and swab libraries also differed descriptively in taxonomic composition and ordination position. No fungal growth was reported in the available routine culture reports. These observations demonstrate the presence of diverse fungal DNA signals but do not establish viable uterine colonization, fungal infection, an association with subsequent pregnancy outcome, or equivalence between sampling methods. Confirmation requires sequencing of individual mares, comprehensive field and laboratory controls, standardized mycological culture, and integration with cytological, histopathological, and reproductive data.