Extended Data Fig. 1: Paternal microbiome and physiological responses to nABX-induced dysbiosis.
From: Paternal microbiome perturbations impact offspring fitness
(a) Principal component analysis (PCA) showing gut microbiota composition (Bray–Curtis) in males after 6 weeks of nABX or in controls. Additional panels show gut microbiota composition after a 4- or 8- weeks recovery period following nABX withdrawal, demonstrating rescue of gut microbiota after 8 weeks nABX withdrawal. (b) qPCR quantifying the total abundance of microbes after 6wk nABX and following 4- and 8- weeks recovery (+rec), determined by 16S rRNA. (CON t0 = 8, 6wk = 8, 6wk + 4rec = 8, 6wk + 8rec = 7; nABX t0 = 7, 6wk = 8, 6wk + 4rec = 8, 6wk + 8rec = 7 individuals per timepoint). Error bars indicate S.D. (c) Boxplot showings body weights of males after 6wk nABX treatment. Bar represents median and whiskers indicate 5-95th percentile, p-value by unpaired two-tailed t-test. (d) Survival curve of males treated with nABX showing no subsequent effect on mortality. p-value by Mantel–Cox (log-rank). (e) Ratio of ceca to body weight in males treated with 6wk nABX. An enlarged ceca is symptomatic of major dysbiosis and/or reduced microbial abundance, further demonstrating changes in gut communities in nABX males. Bar represents median and whiskers indicate 5-95th percentile, p-value by unpaired two-tailed t-test (CON n = 25 males; nABX n = 26 males) (f) Number of pups per litter derived from nABX-treated male sires. Bar represents median and whiskers indicate 5-95th percentile. p-value by unpaired two-tailed t-test (CON n = 31 litters; nABX n = 36 litters). (g) Likelihood of males siring offspring following 6wk nABX, illustrating no difference in fertility relative to control (CON = 41 conceived from 51 plugs positive; nABX = 43 conceived from 55 plugs positive). (h) Sperm viability as judged by scoring the number of motile sperm per million after 6wk nABX exposure (CON n = 6 males; nABX n = 6 males, harvested sperm samples). (i–k) Mass spectrometry analysis of each component of the nABX cocktail in tissues of treated male mice to confirm they are non-absorbable and do not reach distal tissues. Shown are standard curves demonstrating the high sensitivity and limit of detection (LoD). We were unable to detect (i) Bacitracin (j) Neomycin and (k) Pimaricin in either testis or blood (serum) of nABX-treated mice (l) Functional assay for the detection of antibiotic residues in testis of nABX-treated males indicates no bioavailable nABX can be detected (LoD detection threshold is below maximum residual level (MRL)). All assays indicate systemic and testicular responses to nABX are not due to direct chemical interactions with drugs, because nABX remain in the gastrointestinal tract and do not reach the systemic level or the testes.
