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The present study aimed to investigate the effect of storage duration of frozen inoculum on fermentation parameters<br />obtained with the in vitro gas production technique. Two non-predigested diets differing in chemical composition and<br />especially crude fibre content (low fibre diet: 13.8%; high-fibre diet: 22.6%) were ground to pass a 1 mm screen and<br />subjected to fermentation with the same inoculum frozen for different periods: after 1 month (inoculum 1), after 2<br />months (inoculum 2) and after 3 months (inoculum 3). The inoculum used was obtained from the caecal content of 75-<br />day-old NZW rabbits. After defrosting, the caecal content was diluted with the medium 1:1 (V/V) and squeezed through<br />six layers of gauze to obtain the inoculum. The substrate affected several fermentation parameters. In particular, the<br />high-fibre diet had lower potential and cumulative gas production (A = ml/g 220 vs 256; P < 0.01 and OMCV ml/g 185<br />vs 221; P < 0.01), lower organic matter degradability (OMl 67.2% vs 58.0%; P < 0.01) and production of volatile fatty<br />acids (mmol/g 56.2 vs 49.8; P < 0.01), and took more time to obtain gas production equal to A/2 (B = h 11.8 vs 10.1;<br />P < 0.01) compared with the low-fibre diet. However, the three inocula had very similar gas production kinetics, overlapping<br />values of degraded organic matter (62.4%, 62.7% and 62.7% respectively for inocula 1, 2 and 3) and similar<br />production of VFA (54.0, 52.2 and 52.8 mmol/g, respectively for inocula 1, 2 and 3). This research showed it is possible<br />to use frozen inoculum for at least 3 months and in this time interval obtain the same parameters of in vitro fermentation.