Comparación de dos métodos de extracción de líquido ruminal (fístula vs sonda) a partir de la población bacteriana obtenida en vacas Primiparas durante el periodo de transición

The quest to improve the efficiency and sustainability of dairy production has led to research into the complex ruminal microbiome system, and in this context, the extraction of ruminal fluid samples is a critical step. The standard method for extraction is through the ruminal fistula, which offers accurate sampling but raises methodological questions regarding animal welfare and makes it more difficult to perform mass sampling, as it involves surgery in the flank area. As an alternative, the oro-ruminal probe is a less invasive and more practical method for obtaining multiple samples. The objective of this study was to compare the use of the oro-ruminal probe versus the ruminal fistula as a sampling methodology to characterize the prokaryotic microbial community of the rumen in primiparous Holstein cows during the transition period, evaluating similarities and differences in microbial composition and structure at three time points around calving (-30, +35, and +65 days). Nine Holstein-American heifers cannulated at the rumen level underwent ruminal fluid extraction using both methods at the three time points around calving. Samples were taken in the morning when the animals were fasting. Each sample was placed in a sterile, labeled 50 ml tube. These samples were centrifuged at 10,000 rpm, 4°C, for 10 minutes, and the sedimented material was extracted using sterile pipettes, transferring it to two sterile, properly labeled 2 ml bottles. These small vials are the sample from which the microbial population was evaluated and were stored at -80°C. The microbial population of each sample was detected by amplifying the variable regions V3-V4 of the 16S gene and paired-end sequencing with the Illumina MiSeq platform. The objective was to group the sequences at the ASV (Amplicon Sequence Variants) level to characterize the taxonomic groups at the microbial genus level using the public Silva database (version 138.2). The DADA2 package of the R statistical software (version 4.3.1) was used for statistical analysis. Rarefaction curves showed that the sequencing depth was adequate. The richness and diversity of ASVs and microbial genera did not show significant differences between sampling methods (p >0.05). The mean Pearson and Spearman correlations according to sampling time for the relative abundances of microbial genera using fistula and probe were high (r >0.7) but decreased from prepartum to postpartum. Differential expression analysis showed that none of the evaluated genera presented differences between methods (padj >0.05). The %CV of relative abundances of genera between sampling methods increased according to sampling time, from 8.7% at time -30 to 15.4% at time +35, and 26.6% at time +65. On average, the probe shared 85.8% of the central genera detected by the fistula extraction method. Therefore, the use of the oro-ruminal probe was able to characterize and provide a good biological signal about the microorganisms in the ruminal fluid, which suggests that it is a reliable alternative for evaluating the ruminal environment.