Influence of corn hybrids and type of silage additives on total and γ- zein content during the high moisture corn silage production Silage production is a conservation method used for high moisture crop preservation. It is based on natural fermentation that is under control of epiphytic microflora, optimally-mostly by lactic acid bacteria (McDonald et al., 1991). Protein fractions are important indicators of silage quality (Der Bedrosian et al., 2012; Ferraretto et al., 2014). In particular, corn silage soluble crude protein (sCP) fraction and ammonium (NH3) content are positively associated with proteolysis (Winters et al., 2000; Johnson et al., 2002; Slottnerr and Bertilsson, 2006; Ferraretto et al., 2014) and correlate positively with ruminal in vitro starch digestibility (Ferraretto et al., 2015b). Zein proteins, the most abundant protein fraction in corn grain, surround starch granules and their hydrophobic nature acts as a barrier to starch digestion by rumen microbes (Giuberti et al., 2014). Besides their influence on starch digestibility, zeins also appear to influence the texture and hardness of the grain (Holding and Larkins, 2009). Below et al. (2004) reported that total zein content of corn grain depends primarily on genotypic factors; with environmental factors such as N supply or plant maturity at harvest both causing smaller or larger phenotypic variations. In the corn endosperm zein proteins are located within protein aggregates called protein bodies and are comprised of four distinctive zein classes: alpha (α), beta (β), delta (δ) and gamma (γ) zein. Location of distinctive zein class differs within protein bodies. Gamma zein is thought to be the most important zein fraction. This zein class is located on the surface of protein bodies and in small spots within where they together with beta zein stabilize alpha zein core. Gamma zeins are comprised from three distinctive proteins: 16-kDa gamma zein, 27-kDa gamma zein and 50-kDa gamma zein (Woo et al., 2001; Holding and Larkins, 2006; Boston and Larkins, 2009; Holding and Larkins, 2009). In silage production, lactic acid bacteria (LAB) additives are widely used to optimize fermentation and thereby maximize lactic acid production, quickly reduce pH, and preserve nutrients (McDonald et al., 1991). Lactic acid production during ensiling of forages is well established (McDonald et al., 1991); this most abundant and the strongest acid in silages dissolves zein proteins, leading to a reduction in grain total zein content (Lawton, 2002). In addition to lactic acid, enzymes from plants or microorganisms degrade zein proteins (McDonald et al., 1991; Lawton, 2002; Hoffman et al., 2011). The purpose of the present study was to investigate the additive effect of lactic acid bacteria and lactic acid application on the gamma zeins content, total zein content and grain rumen degradability during high moisture corn fermentation process of corn hybrids that differ in endosperm texture and total zein content. In commercial production ensiling is conducted on yearly basis e.g. between two corn harvests. Main silage conservation characteristics where monitored at different time points during one year ensiling period. The gamma zein proteome analysis e.g. differences between gamma zeins content in silages were detected using 2-D gel electrophoresis followed by densitometry quantification and MALDI-TOF/TOF protein identification. Hybrids (Bc 513 pc, Bc 462 and Bc 344) were grown in 2013 under the same production conditions in split-plot experimental fields at the Faculty of Agriculture of the University of Zagreb. Each hybrid was grown in five replicates. Chopped material from each replicate was divided in three parts. Silages where ensiled at the black layer stage of growth (67.61% – 72.42% DM) in laboratory scale bag silos with one part ensiled with BioSil LAB inoculant (Lactobacillus plantarum DSM 8862 and DSM 8866) in a concentration 3x105 CFU/g of fresh material, second one was left untreated while the third was treated with lactic acid (linear application 5 ‒ 15 g/kg DM). Silages were maintained at 25 ± 2 °C and sampled at the beginning and on 15th, 48th, 98th, 182nd, 274th and 364th day of ensiling. Silage conservation characteristics (DM, lactic acid (LA), titratable acidity (TA), pH, crude protein (CP), sCP, NH3, total zein) where monitored. At the beginning and the end of ensiling period gamma zein content and silage ruminal degradability was evaluated. At the end of ensiling total bacterial count, lactic acid and yeast count was determined. At the beginning of ensiling the corn grain hardness was evaluated in green material of hybrid tested with Stenvert hardness test and true density. Effects of time, silage additive and hybrid genotype together with their interactions on silages were tested as a split-plot design using the PROC MIXED procedure in SAS 9.4 (SAS Institute, Cary, NC, USA). Means with different superscript letter groups were obtained with postfitting statistical PROC PLM. The differences of green material endosperm texture and microbial count were performed separately as a split-plot completely randomized design and differences between means were assessed for significance using the Tukey test. The correlation parameters where determined using PROC CORR and linear regression between the dry matter and starch degradability rate with PROC REG in SAS 9.4. Differences and interactions were considered significant when P < 0.05. Grain hardness is important corn characteristic that plays a role in the final product quality and nutritional value of corn. It is highly connected with the ratio of horny and floury endosperm in corn grain and with total zein content (Dombrink-Kurtzman and Bietz, 1993; Pratt et al., 1995). Stenvert hardnes test parameters (time of grinding, volume of grinded sample and coarse to fine particle ratio, Wt C/F) showed the highest hardness in Bc 513 pc and lowest in Bc 344. Hybrids differed significantly in time of grinding and Wt C/F. Bc 513 pc had longer time of grinding (3.94 sec) in contrast with Bc 344 (3.50 sec) and Bc 462 (3.75 sec) and higher Wt C/F than other two hybrids (Bc 513 pc: 1.96; Bc 344: 1.46; Bc 462: 1.48). True density analysis showed similar results to Stenvert hardness test; Bc 513 pc had highest values (1.36 g/ml) in contrast to Bc 344 (1.26 g/ml) and Bc 462 (1.31 g/ml) (P < 0.001). Harder corn grains have higher true density than soft corn grain (Wu, 1992; Radosavljević et al., 2000). Results proved the highest hardness in Bc 513 pc and lowest in Bc 344. Bc 462 is more similar to Bc 513 pc (corn type popcorn) than Bc 344, and is regarded as somewhat harder corn than Bc 344. The total zein content was in accordance with observed differences in hardness between hybrids tested, Bc 513 pc had highest total zein content (59.96 % CP) and Bc 344 lowest (48.75 % CP) total zein content (Bc 462: 55.13 % CP) (P < 0.001). The silage fermentation profiles obtained with these 3 hybrids are in accordance with other high moisture corn silages ensiled at the black layer (Ferraretto et al., 2014). Dry matter value showed significant but numerically low change during ensiling (69.11 ‒ 71.04 %). The hybrid tested had no influence on lactic acid content, pH value or titratable acidity in silages. However, the use of inoculant was associated with higher lactic acid content (12.63 g/kg DM; P < 0.001), faster pH reduction (6.45 ‒ 4.03; P < 0.001) and higher titratable acidity (0.57 meq/g DM; P < 0.05) in tested silages, consistent with the ability of LAB to promote fermentation. Application of lactic acid resulted with similar trend in silages (lactic acid, LA: 8.55 g/kg DM; control, LA: 7.14 g/kg DM; lactic acid, pH: 6.45 ‒ 4.17; control, pH: 6.45 ‒ 4.35; lactic acid, TK: 0.51 meq/g DM; control, TK: 0.51 meq/g DM). Both types of silage additives used in this study improved silage fermentation characteristics when compared to control silages. In all silages regardless of the hybrid or additive tested increase in lactic acid content to 182nd day of ensiling was observed (1.01 ‒ 15.89 g/kg DM; P < 0.001), after which small reduction in LA content up to 364th day was observed (274th: 11.60 g/kg DM; 364th: 9.10 g/kg DM). In substrate deficiency, primarily glucose, lactic acid bacteria (including Lactobacillus plantarum) metabolize lactic acid to acetic acid (Lindgren, 1990). In the case of negative yeast or clostridia activity, parallel to lactic acid reduction the increase in pH should be observed (McDonald et al., 1991; Weinberg and Muck, 1996). However that was not the case in this study. Crude protein content in silages was significantly different between hybrid tested, with highest values observed in Bc 513 pc and lowest in Bc 344 (Bc 513 pc: 133 g/kg DM; Bc 344: 105.6 g/kg DM; Bc 462: 109.8 g/kg DM) and is in accordance with total zein content observed for hybrids tested, with highest values observed for Bc 513. Although the triple interaction influence (corn hybrid×silage additive×ensiling period) observed for ammonium (P < 0.001) and sCP (P < 0.05) content in silages implies different hybrid responses to additive application during ensiling, the same trend was observed. The sCP and ammonium content showed increase during ensiling (sCP: 17.9 ‒ 47.0 % CP; ammonium: 0.38 ‒ 3.73 % CP; P < 0.001) and are evidence of proteolysis in silages (Winters et al., 2000; Johnson et al., 2002; Slottnerr and Bertilsson, 2006). Electrophoretic separation and mass spectrometry identification of gamma zein extract of tested silages showed thirteen distinctive spots of zein proteins. Of three gamma zein types, 16-kDa and 27-kDa gamma zeins were detected. 50-kDa gamma zein is expressed usually in undetectable quantities on molecular level (Woo et al., 2001). At the end of ensiling, densitometry of electrophoretically separated proteins showed reduction of both gamma zein proteins. 27-kDa zein was reduced at undetectable level in all silages. Inoculant application resulted with higher degradability of 16-kDa gamma zein proteins in silages of corn hybrids with harder endosperm type. Lactic acid application resulted with higher degradability of 16-kDa gamma zein proteins in softer corn hybrid (Bc 344) silages. In all silages degradation of 16-kDa zein was lowest in control silages regardless of the hybrid used. Other zein detected were alpha zeins, which showed reduction at the end of ensiling. However the reduction was less expressive than in the case of gamma zeins. Gamma zeins are located on the surface of protein bodies and in small spots at the core of protein bodies (Holding and Larkins, 2006) which can explain higher degradability of gamma zeins compared to alpha zeins. Interestingly, increase in total zein content was observed during ensiling regardless of the hybrid tested or additive used and is opposite to proteolytic activity observed in silages (increase in ammonium and sCP contents) and observed reduction in gamma zein and alpha zein. Analytical method used for zein quantification proved to be efficient for defining difference between hybrids tested, but inefficient for defining zein degradation rate during ensiling (Hoffman et al., 2011). High moisture corn is highly used feed in ruminant and monogastic animals nutrition as it reduces costs of drying during corn grain storage and it allows earlier harvest of corn grain during unfavourable weather. Additionally, high moisture corn silage preparation allows corn utilization from harvest to harvest, during whole year and it has positive effect on corn grain nutritional value. Ensiling boosts ruminal starch degradability (Philippeau et Michalet-Doreau, 1998; Firkins, 2001; Ferraretto et al., 2013). High moisture corn silages had higher starch and dry matter degradation parameters at the end of ensiling when compared with green material (effective degradability of dry matter: from 0.59 to 0.77; effective degradability of starch: from 0.53 to 0.78; P < 0.001). All the starch degradation parameters showed improvement at the end of ensiling; Lag phase was shorter (from 7.37 to 5.43 hours; P < 0.001), rapidly degradation fraction was higher (from 0.28 to 0.54; P < 0.001) as well as the rate of starch degradation (from 0.051 h-1 to 0.098 h-1; P < 0.001). Similar trend was observed in dry matter degradation. Strong linear regression of rate of dry matter degradation and starch degradation observed by us implies that dry matter degradation is highly related to starch degradation in high moisture corn silages (Philippeau et al., 2000; Correa et al., 2002; Ngonyamo-Majee et al., 2008). Inoculant application had positive effect on degradation of dry matter and starch, but overall this effect was minimal. The hybrid effect on dry matter degradability or starch degradability showed somewhat better parameters for softer corn hybrid (Bc 344) although the difference was minimal compared to harder corn hybrid (Bc 462). The study confirmed the hypothesis of a greater degradation of 16-kDa gamma zein in silages at the end of one year ensiling and in hybrids with harder than softer grain, as well in silages with additives. Inoculant application had more significant influence on harder grain hybrids; and lactic acid on softer hybrid. The greater 16-kDa gamma zein degradation was connected with higher ruminal degradation of starch and dry matter. Results showed that harder hybrid (Bc 462) had similar ruminal degradability as softer hybrid (Bc 344) with the somewhat better fermentation characteristics and better 16-kDa gamma zein degradation thus implying better production characteristics for more vitreous corn.