16S rRNA NGS sequencing of fecal bacterial microbiota in some obese and healthy horses of Ukrainian origin

Aim. The aim of this study is to characterize and possibly differentiate the lower gut (fecal) bacteriota of healthy and obese horses using Next Generation Sequencing (NGS) of the 16S rRNA gene. Methods. The study involved 7 horses (4 stallions and 3 mares) of different breeds, aged 8-17 years: horses 1-4 of Ukrainian Saddle breed (horse 1 sports horse stallion Rebus, 10 y.o., horse 2 stallion Santes, 15 y.o., horse 3 stallion Sens, 14 y.o., horse 4, mare Siren, 17 y.o.), horse 5 of Heavy Draft breed (stallion Tsyhan, 8 y.o.), and non-thoroughbred horses 6 and 7 (mare Snezhynka, 10 y.o., mare Rumba 12 y.o.) Horses 2, 4, 5 and 7 were obese and horses 1, 3 and 6 were healthy. All horses were kept at the equestrian centre of the State Biotechnological University the Ministry of Education and Science of Ukraine (Kharkiv, Ukraine). Total DNA from rectal fecal samples were extracted using the PureLink Microbiome DNA purification kit (Invitrogen, USA), according to the manufacturer's instructions. To prepare libraries of the 16s rRNA of the bacteriota, we used the 16S rRNA barcoding kit 1-24 (Oxford Nanopore, USA). To purify the libraries obtained, magnetic particles NucleoMag NGS Clean-up and Size Select (Macherey-Nagel, Germany were used according to the recommended protocol of the rapid sequencing amplicons – 16S barcoding (SQK-16S024). These conditions are based on standard protocols for 16S rRNA gene amplification, as described in Fujiyoshi et al (2020), and ensure robust amplification of bacterial DNA across a wide range of taxa. Results. Representatives of the bacterial phyla Actinomycetota (syn. Actinobacteriota), Fibrobacterota, Lentisphaerota, Spirochaetota (syn. Spirochaetes), Bacteroidota, Firmicutes (syn. Bacillota), Planctomycetota, Verrucomicrobiota (syn. Verrucomicrobia), Candidatus Melainabacteria, Kiritimatiellota and Proteobacteria (syn. Pseudomonadota) were detected. The dominating phylum was found to be Firmicutes, whose share was from 50 to 82 % of all the phyla detected. The number of Firmicutes, when compared to those of Bacteroidota varied considerably between healthy and obese horses. In the healthy horses 1,3 and 6 this was 2.5, 3.4 and 2.9 times higher for the Firmicutes and for the obese horses 2,4,5 and 7 it was 8.6, 8.2, 7.6 and 5.7 times higher. Increased numbers of Proteobacteria genera were observed in obese horses 2, 4, 5, and 7, ranging from 25 to 37 %, while in the healthy sport horses 1, 3 and 6 the level of Proteobacteria was between 1.07 and 3.43 %, which is typical for the microbiome of healthy animals. A low level of Actinomycetota (Actinobacteriota) was detected in the feces of the horses under study: 0.09 % in healthy sport horse 1, 0.09 % in healthy sport horse 3, and 0.15 % in healthy horse 6, respectively. In contrast, the level of this bacterial phylum varied in obese horses 2, 4, 5, and 7, ranging from 0.21 % to 0.48 %, respectively. It is important to note that the Actinomycetota phylum also includes the genus Bifidobacterium, which was not detected in any of the animals studied. Conclusions. For the first time in Ukraine, we sequenced the bacterial microbiota of the lower intestinal tract (fecal material) of seven horses of different ages, sexes, and breeds. In the feces of obese horses, there was a predominance of bacteria from the order Eubacteriales (phylum Firmicutes, class Clostridia), particularly from the families Oscillospiraceae and Lachnospiraceae, accompanied by a reduction in bacteria from the phylum Bacteroidota (FCB group clade) compared to healthy horses. These alterations may be related to fat accumulation in the animals, possibly due to increased energy synthesis from feed. Cluster analysis revealed a high degree of similarity in bacteriota composition among the samples. Further studies, including larger sample sizes and exploration of physiological characteristics, are needed to obtain more comprehensive information.