A new sterilization and inoculation method in silage research K. Mogodiniyai Kasmaei*, V. Passoth†, R. Sp€ orndly* and P. Ud en* *Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, Uppsala, Sweden, †Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden Abstract The study aimed at evaluating an effective steriliza- tion–inoculation technique to facilitate silage research on the effect of forage microflora on fermentation variables. The sterilization effect of heating at 60°C for 3h + 103°C for 15 h was tested on samples of grass, grass–clover, white clover and maize, pre-dried at 60°C to a dry-matter (DM) content >900 g kg À1 . The ensilability of treated samples, reconstituted to original DM concentration (250–390 g kg À1 ), was assessed by inoculation with microfloras extracted from the origi- nal samples. Microfloral inoculants were obtained by a combination of centrifugation (15 500 g for 40 min) and filtration (0Á45 and 0Á22 lm pore sizes) of the supernatant. The sterilization treatment effectively sterilized the forage samples but decreased water solu- ble carbohydrates by 49% and N buffer solubility by 22% and increased the acid detergent insoluble N pro- portion of total N by 53% (P < 0Á05). The reconsti- tuted silages had 18% less lactic acid, 20% less ethanol and 37% less ammonia-N (P < 0Á05), but vol- atile fatty acids and 2,3-butanediol did not differ from the untreated silages (P > 0Á05). Counts of lactic acid bacteria, enterobacteria, clostridia, yeasts and moulds in the two silage treatments were also similar (P > 0Á05). It is concluded that, despite causing chemi- cal and physical alterations, the sterilization–inocula- tion technique evaluated could be a useful tool for future studies on the effects of microflora on ensiling results. Keywords: inoculation, microflora, silage, sterilization Introduction Fermentation quality of silage can affect intake, milk composition and milk flavour (Randby et al., 1999; Huhtanen et al., 2003, 2007). However, variation in fermentation results is often large and in many cases largely unexplained (Mogodiniyai Kasmaei et al., 2013). A further issue of concern is the variation often seen in aerobic stability of silages. Although micro- biological, biochemical and management factors may be known (Wilkinson and Davies, 2013) and inocu- lants (Driehuis et al., 2001; Kleinschmit and Kung, 2006) or chemical additives (Knicky and Sp€ orndly, 2011) are taken into account, the stability can still vary unexpectedly. There exists a definite problem in separating the effects of forage microflora and chemical composition on the ensiling results. An ability to sterilize forages and isolate field floras could make it possible to explain more of this variation. Such ability can also be of a great use in evaluation of silage additives. A few techniques have been tested to obtain sterile substrates in silage research, including gamma-irradia- tion (Heron et al., 1986), growing forages aseptically (Playne et al., 1967) or simulating chemical and physi- cal characteristics of forages using artificial substrates (Woolford and Wilkins, 1975). These techniques, however, have drawbacks as they are either expensive (gamma-irradiation), induce chemical alterations (aseptically grown forages) or have not been validated (artificial substrates). Heating (121°C for 20 min) of forages, previously freeze-dried, has recently been suggested as a promising and practical sterilization technique (Mogodiniyai Kasmaei et al., 2014). Samples were first dried to miti- gate the extent of chemical damage induced by heating. The treatment, however, did not eradicate entero- bacteria and was not tested against sporulating species. The aims of the work presented here were to: (i) fine tune this sterilization technique and (ii) validate a method of sterilization–inoculation against an untreated control. Correspondence to: Kamyar Mogodiniyai Kasmaei, Depart- ment of Animal Nutrition and Management, Swedish University of Agricultural Sciences, Box 7024, 750 07 Uppsala, Sweden. E-mail: kamyar.mogodiniyai.kasmaei@slu.se Received 25 August 2014; revised 23 October 2014 668 © 2014 John Wiley & Sons Ltd. Grass and Forage Science, 70, 668–673 doi: 10.1111/gfs.12153 Grass and Forage Science The Journal of the British Grassland Society The Official Journal of the European Grassland Federation