Citation: Christodoulou, C.; Mavrommatis, A.; Loukovitis, D.; Symeon, G.; Dotas, V.; Kotsampasi, B.; Tsiplakou, E. Inclusion of Camelina sativa Seeds in Ewes’ Diet Modifies Rumen Microbiota. Animals 2023, 13, 377. https://doi.org/10.3390/ ani13030377 Academic Editor: Adriana Bonanno Received: 14 November 2022 Revised: 30 December 2022 Accepted: 17 January 2023 Published: 22 January 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). animals Article Inclusion of Camelina sativa Seeds in Ewes’ Diet Modifies Rumen Microbiota Christos Christodoulou 1 , Alexandros Mavrommatis 1 , Dimitris Loukovitis 2 , George Symeon 3 , Vassilios Dotas 4 , Basiliki Kotsampasi 3 and Eleni Tsiplakou 1, * 1 Laboratory of Nutritional Physiology and Feeding, Department of Animal Science, School of Animal Biosciences, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece 2 Department of Animal Production, Fisheries and Aquaculture, School of Agricultural Sciences, University of Patras, 30200 Messolonghi, Greece 3 Research Institute of Animal Science, ELGO ‘DIMITRA’, Paralimni Giannitsa, 58100 Pella, Greece 4 Department of Animal Production, Faculty of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece * Correspondence: eltsiplakou@aua.gr Simple Summary: Modern livestock research has focused on the evaluation of feeding strategies that led to modify the rumen microbiome to achieve optimum productivity without compromising ruminants’ physiology and health. For this reason, the supplementation of unconventional feedstuffs is extensively studied. In our study, we investigated the effect of Camelina sativa seeds supplementa- tion on ewe’s rumen microbiota. Our results suggested that supplementing Camelina sativa seeds, especially in the highest studied level (160 g·kg −1 of concentrate), resulted in significant alterations in the relative abundance of the rumen microorganisms, with those reported in methanogens being considered the most promising. Abstract: Supplementing ruminant diets with unconventional feedstuffs (Camelina sativa seeds; CS) rich in bioactive molecules such as polyunsaturated fatty acids, may prove a potential eco-efficient strategy to manipulate rumen microbiome towards efficiency. Forty-eight ewes were divided into four homogenous groups (n = 12) according to their fat-corrected milk yield (6%), body weight, and age, and were fed individually with concentrate, alfalfa hay, and wheat straw. The concentrate of the control group (CON) had no CS inclusion, whereas the treated groups were supplemented with CS at 60 (CS6), 110 (CS11), and 160 (CS16) g·kg −1 of concentrate, respectively. Rumen digesta was collected using an esophageal tube and then liquid and solid particles were separated using cheesecloth layers. An initial bacteriome screening using next-generation sequencing of 16S was followed by specific microbes targeting with a RT-qPCR platform, which unveiled the basic changes of the rumen microbiota under CS supplementation levels. The relative abundances of Archaea and methanogens were significantly reduced in the solid particles of CS11 and CS16. Furthermore, the relative abundance of Protozoa was significantly increased in both rumen fluid and solid particles of the CS6, whereas that of Fungi was significantly reduced in the rumen particle of the CS16. In rumen fluid, the relative abundance of Fibrobacter succinogens and Ruminobacter amylophilus were significantly increased in the CS6 and CS11, respectively. In the solid particles of the CS11, the relative abundance of Ruminococcus flavefaciens was significantly reduced, whereas those of Butyrivibrio proteoclasticus and Ruminobacter amylophilus were significantly increased. Additionally, the relative abundance of Selenomonas ruminantium was significantly increased in both CS11 and CS16. Consequently, the highest CS content in the concentrate reduced the relative abundance of methanogens without inducing radical changes in rumen microorganisms that could impair ruminal fermentation and ewes’ performance. Keywords: ewes; oilseeds; rumen; microorganisms; methane Animals 2023, 13, 377. https://doi.org/10.3390/ani13030377 https://www.mdpi.com/journal/animals