Kolhydratsammansättning och in vitro-gasproduktion i gräs vid simulerat hästbete

Hästen är en gräsätare som kan livnära sig på fiberrikt betesgräs som för många hästar utgör en stor del av dieten under sommaren. Betesgräs är i föränderligt tillstånd genom avbetning där faktorer som bland annat gräsets genetiska kapacitet, botaniskt utvecklingsstadium och klimat påverkar den näringsmässiga kvaliteten. Det saknas kunskap om hur avbetning vid upprepade tidpunkter inverkar på kolhydratsamman-sättningen hos olika gräsarter och om eller hur detta inverkar på hästens grovtarms-fermentation. Grovtarmsfermentation är svårt att mäta, men kan estimeras genom vo-lymen producerad gas i in vitro-system där gräsprov inokuleras med träck från häst. I denna studie undersöktes därför kolhydratsammansättning och in vitro-gasprodukt-ion (med hästträck som inokulant) hos sex olika gräsarter som provtagits vid fyra olika tidpunkter som en simulerad betessäsong. De gräsarter som undersöktes var engelskt rajgräs (Lolium perenne), foderlosta (Bromus inermis), hundäxing (Dactylis glomerata), rörsvingel (Festuca arundinacea), timotej (Phleum pratense) och ängs-svingel (Festuca pratensis). Resultatet påvisade att det fanns interaktionseffekter mellan gräsart och avbetningstidpunkt på den kemiska sammansättningen i gräspro-verna. Detta betyder att alla gräsarter inte svarade likadant på upprepad avbetning och foderlosta verkade inte klara den upprepade avbetningen då återväxten var låg. Engelskt rajgräs hade ett högt innehåll av lättlösliga kolhydrater (WSC) och lågt in-nehåll av fiber samtidigt som hundäxing hade ett lågt innehåll av WSC och högt in-nehåll av fiber i relation till de övriga gräsarterna. Innehållet av WSC och fiber hade generellt positiv respektive negativ korrelation med den totala gasproduktionen. Gräsarter med högt innehåll av WSC skulle därmed kunna orsaka större gasbildning, medan arter med högre fiberinnehåll kan ge lägre gasbildning både in vitro och in vivo. Det kan ha betydelse för gräsartens lämplighet i bete för olika kategorier av hästar, då stor och snabb gasproduktion kan innebära grovtarmsstörningar.The horse is a grass eater and can survive on fiber-rich pasture grass which con-tributes to a great part of the diet for many horses during summer. Pasture grass is constantly changing during frequent grazing, as factors like genetic capacity of the grass, stage of plant maturity and climate can affect the nutritional quality. There is lack of knowledge on how repeated grazing influence the carbohydrate composition in various grass species, and whether or how this further affects the fermentation in the hindgut of the horse. The hindgut fermentation is difficult to measure but can be estimated through the volume of produced gas in in vitro systems where grass sam-ples are inoculated with horse faeces. In this study the carbohydrate composition and gas production from in vitro fermentation (with horse faeces as inocula) of six grass species harvested at four different times as a simulated grazing season were studied. The examined grass species were cocksfoot (Dactylis glomerata), bromegrass (Bro-mus inermis), meadow fescue (Festuca pratensis), perennial ryegrass (Lolium perenne), tall fescue (Festuca arundinacea) and timothy (Phleum pratense). The re-sults showed that interaction effects between grass species and grazing time were present for chemical composition in the grass samples. This means that all grass spe-cies did not respond similarly to repeated grazing, and bromegrass did not seem to cope with the repeated grazing due to a low regrowth. Perennial ryegrass had high content of water soluble carbohydrates (WSC) and low concentration of fiber, while cocksfoot hade a low content of WSC and high concentration of fiber in relation to the other grass species. The concentration of WSC had a positive correlation while the fiber content had a negative correlation with the total amount of produced gas. Thus, grass species with a high content of WSC may lead to greater gas formation, while species with higher fiber content may result in lower gas production. This may be of importance for hindgut fermentation disturbances in horses and therefore dif-ferent grass species may have a varied suitability for pasture for different categories of horses

Laktik Asit Bakteri+Enzim Karışımı Silaj İnokulantının Buğday Silajı Üzerine Etkileri

This study was carried out to determine the effects of a commercial lactic acid bacteria+enzyme inoculants used as silage additive on the fermentation, crude nutritient contents, cell wall fractions and in vitro dry and organic matter digestibilities wheat (Triticum aestivum L.) harvested and ensiled at milk and dough stages of maturity. Sil-All (Altech, UK) containing water soluble Pediococcus acidilactici, Lactobacillus plantarum and Streptococcus faecium bacteria with cellulase, hemicellulase, pentosonase and amylase was used as bacterial inoculants. The inoculant was applied to the silages at 6.0 log10 cfu/g levels. Wheats were ensiled in 2 liter glass jars and stored at 25 ±2 C in the laboratory. Three jars from each group were sampled for pH, ammonia nitrogen, water soluble carbohydrates, organic acids (acetic, butyric and lactic), crude nutritients, cell wall fractions and microbiological analyses following the 75-day ensiling period. In additions in vitro dry and organic matters digestibility of the silages were determined with enzymatic methods. The inoculant improved fermentation characteristics, decreased neutral and acid detergent fiber contents of wheat silages. However, the in vitro dry and organic matter digestibilities of the silages were not affected by the treatments.Bu çalışma silaj katkı maddesi olarak kullanılan ticari bir laktik asit bakteri+enzim karışımı inokulantın, süt ve hamur olum dönemlerinde hasat edilip, silolanan buğday silajlarının fermantasyon özellikleri ile ham besin maddeleri içerikleri, hücre duvarı kapsamı, in vitro kuru ve organik madde sindirilebilirlikleri üzerindeki etkilerinin saptanması amacıyla düzenlenmiştir. İnokulant olarak Pediococcus acidilactici, Lactobacillus plantarum ve Streptococcus faecium bakterileri ile birlikte sellülaz, hemisellülaz, pentozanaz ve amilaz içeren Sil-All (Altech, UK) kullanılmıştır. İnokulant silajlara 6.0 log10 cfu/g düzeyinde katılmıştır. Silolama 2 litrelik cam kavanozlarda 25±2 °C sıcaklıktaki laboratuar koşullarında gerçekleştirilmiştir. Silolanmadan sonraki 75. günde kavanozlar açılarak, silajlarda pH ölçümleri, amonyak azotu, suda çözünebilir karbonhidrat, organik asit (asetik, bütrik ve laktik), ham besin madde, hücre duvarı fraksiyonlarının belilenmesi ve mikrobiyolojik analizler yapılmıştır. Ayrıca, enzimatik yöntemle silajların in vitro kuru ve organik madde sindirilebilirlikleri saptanmıştır. Laktik asit bakteri+enzim inokulantları buğday silajlarının fermantasyon özelliklerini olumlu yönde etkilemiş, nötr ve asit deterjanda çözünmeyen karbonhidrat kapsamlarını düşürmüş, bunlara karşın in vitro kuru ve organik madde sindirilebilirliklerini etkilememiştir

Vplyv aditív na mykotoxickú kontamináciu kukuričných siláží

Addition of silage additives is commonly concentrated on the improvement of nutritive and fermentative features of silages, but in recent years, mycotoxic contamination is also a feature monitored after the treatment. This study aimed to evaluate the effect of silage additives, in the concrete inoculant consisting of a mixture of Lactobacillus buchneri LN40177, Lactobacillus casei LC329090 and urea, on the concentration of major mycotoxins in maize silages. The ensilage mass was made in three variants, which consisted of maize silage (control variant C), maize silage with the addition of a commercial inoculant (variant A) and maize silage with the addition of urea (variant U). The commercial inoculant was added to the cut mass at a dose of 1 g/t of mass and urea at a dose of 5 kg/t of mass. After application of silage additives, the mass was stored and sealed in silage units. Mycotoxin analysis was performed using Veratox tests with ELISA reader, whereas average samples were prepared according to established protocols. The immuno-enzymatic method revealed that all samples of maize silage showed 100% contamination. The maize silages were characterized by the highest concentration of deoxynivalenol regardless of the treatment. The results confirmed the effect of silage additives on mycotoxic contamination of maize silages. Compared to the control variant, the commercial inoculant had negative increasing effect (P<0.05) on deoxynivalenol, ochratoxins and zearalenone, and a positive decreasing effect (P<0.05) on T-2 toxin and fumonisins. The urea addition resulted in significant reduction of T-2 toxin and increase of zearalenone.Prídavkom silážnych aditív sa bežne sleduje zlepšenie nutričných a fermentačných vlastností siláží, ale v posledných rokoch je mykotoxická kontaminácia tiež sledovaným znakom po ošetrení. Táto štúdia bola zameraná na vyhodnotenie účinku silážnych aditív, konkrétne inokulantu pozostávajúceho zo zmesi Lactobacillus buchneri LN40177, Lactobacillus casei LC329090 a močoviny, na koncentráciu hlavných mykotoxínov v kukuričnej siláži. Silážovaná hmota kukurice bola vyhotovená v troch variantoch, ktoré pozostávali z kukuričnej siláže (kontrolný variant C), kukuričnej siláže s prídavkom komerčného inokulantu (variant A) a kukuričnej siláže s prídavkom močoviny (variant U). Komerčný inokulant bol do narezanej hmoty pridaný v dávke 1g/t silážnej hmoty a močovina v dávke 5 kg/t silážnej hmoty. Po aplikácií silážnych aditív sa hmota uskladnila a zasilážovala do silážnych jednotiek. Analýza mykotoxínov bola vykonaná pomocou Veratox testov na ELISA reader, pričom priemerné vzorky boli upravené podľa stanovených protokolov. Výsledky potvrdili efekt silážnych aditív na mykotoxickú kontamináciu kukuričných siláží. V porovnaní s kontrolným variantom mal komerčný inokulant negatívny stúpajúci efekt (P<0,05) na deoxynivalenol, ochratoxíny a zearalenon, a pozitívny klesajúci efekt (P<0,05) na T-2 toxín a fumonizíny. Výsledkom pridania močoviny bolo preukazné zníženie T-2 toxínu a zvýšenie zearalenonu

Effect of Additional of Lactic Acid Bacteria (LAB) Inoculants Pre-harvest and Post-harvest Silage Ferrmantation of Corn Silage

Bu çalışma, hasat öncesi ve hasat sonrası laktik asit bakteri inokulantlarının ilavesinin mısır silajlarının fermantasyon ve aerobik stabiliteleri üzerindeki etkilerini belirlemek amacıyla yürütülmüştür. Çalışmada katkı maddesi olarak homofermantatif ve heterofermantatif laktik asit bakterilerini içeren 2 ticari inokulant kullanılmıştır. İnokulantlar silajlara 6.00 log10 cfu/g düzeyinde katılmıştır. Araştırma materyali hasat öncesi ve hasat sonrası olmak üzere kontrol, homofermantatif (hoLAB) ve heterofermantatif (hetLAB) inokulant uygulaması içeren olmak üzere 3 deneme grubuna bölünmüştür. İnokulantların uygulanmasında firma önerileri dikkate alınmıştır. İnokulantlar hasattan 15 ve 7 gün olmak üzere 2 farklı dönemde tarlada mısırlara el tipi pülverizatör yardımı ile atılmıştır. Hasat öncesi ve sonrasının karşılaştırmak amacıyla, hasat dönemi geldiğinde yine kontrol, homofermantatif (hoLAB) ve heterofermantatif (hetLAB) inokulant uygulamaları yapılmıştır. Hasat öncesi ve hasat sonrası gruplarını içeren uygulamalara ait muameleler CASCVP 260PD marka laboratuar tipi paket silaj makinası ile paketlenmiştir. Her muameleye ait 3’er paket silajın kullanıldığı çalışmada, silajların paketlenmesinden sonra materyaller laboratuvar koşullarında (20-22 oC) depolanmıştır. Fermantasyonun 45. gününde açılan örnekler üzerinden pH, kuru madde kaybı, maya ve küf sayımları için mikrobiyolojik analizlerin yapıldığı çalışmada aerobik stabiliteye ilişkin özellikler ana fermantasyon dönemi sonrası 14 günlük dönemde izlenmiştir. Hasattan öncesi inokulant uygulaması özellikle maya ve küf gelişimini azaltmıştır.This study was conducted in order to investigate the effect of adding inoculated lactic acid bacteria to maize before and after harvest on the fermentation and aerobic stability properties of silages. Two commercial inoculants containing homofermentative and heterofermentative lactic acid bacteria were used as additives. Inoculants were added into the silages at the level of 6.00 log10 cfu/g. Pre and post harvest research materials were divided into three trials groups, namely, control, homofermentative (LAB) and heterofermentative (LAB) inoculants. During the use of inoculants, suggestions by the producers were taken into account. Inoculants were applied to the corn plants in the field by the aid of hand type pulverizator at three different times, 15 and 7 day before the harvest. To compare the pre and post harvest treatments, control, homofermentative and heterofermentative inoculants applications were realised at the time of harvest. The pre and post harvest treated materials were packed using labtype CASCVP 260PD brand named silage machine. After packing, three sample packets from each treatment were stored under laboratory conditions (20-22 oC). pH, dry matter losses, microbiological analyses for yeast and mould count were done on the silage samples opened on the 45th day of fermentation. The same analyses were repeated and compared with the previous results 14 days after the opening in order to assess the aerobic stability

Effect of microbiological inoculants on nutritive value and the aerobic stability of corn silage and alfalfa haylage.

Sve silaže nezavisno od kvaliteta su podložne aerobnoj degradaciji. Izlaganje silaže vazduhu pri otvaranju silosa a zatim u i hranidbenom prostoru kada je najčešće na farmi sastavni deo TMR neizbežno dovodi do njenog aerobnog kvarenja. Aerobno kvarenje silaža je složen proces koji zavisi od mnogo faktora. Aerobna degradacija rezultira proizvodnjom CO2 i posledično gubicima. Zbog toga, koriste se inokulanti BMK koji mogu da efikasno usmere mlečno kiselinsku fermentaciju u željenom smeru i imaju uticaja na dužu aerobnu stabilnost silaže. Hemijski sastav i epifitna mikroflora isečene mase biljaka u momentu siliranja kao i vrsta primenjenog mikrobiološkog inokulanta utiču na promenu HV vrednosti i dužinu AS silaža. Ciljevi istraživanja bili su: 1. Određivanje razlika u HV, hemijskog i mikrobiološkog sastava između eksperimentalnih silaža 5 različitih hibrida (rani, 2 srednje rana, srednje kasni i 1 kasni) kukuruza siliranim sa tri različita mikrobiološka inokulanta i sa kontrolnim tretmanom tokom 60 dana; 2.Određivanje razlika u HV, hemijskog i mikrobiološkog sastava između eksperimentalnih senaža lucerke siliranim sa tri različita mikrobiološka inokulanta i sa kontrolnim tretmanom u periodu od 40, 90, 120 i 150 dana; 3.Određivanje uticaja 3 različita mikrobiološka inokulanta, kao i epifitne mikroflore u kontrolnom tretmanu na dužinu AS eksperimentalnih silaža kukuruza i senaže lucerke primenom laboratorijske metode Ashbell i Weinberg, (1991). U cilju ispitivanja uticaja mikrobioloških inokulanta na HV i AS silaže kukuruza korišćena su 3 različita inokulanta: inokulant 1 i 2 su sadržali različite heterofermentative i homofermentative BMK ; i inokulant 3 isključivo homofermentativnim BMK. Ukupan broj tretmana bio je 80. U cilju ispitivanja uticaja mikrobioloških inokulanta na HV i AS senaže lucerke korišćena su 3 različita inokulanta: inokulant 1 i 3 su sadržali različite isključivo homofermentativne BMK; i inokulant 2 koji je sadržao heterofermentativne i homofermentativne BMK. Ukupan broj tretmana bio je 64..

Influence of corn hybrids and type of silage additives on total and γ- zein content during the high moisture corn silage production

Siliranje visoko vlažnog zrna kukuruza način je da se očuva zrno kukuruza od berbe do berbe. U klasičnoj teoriji siliranje je metoda konzerviranja uz pomoć anaerobnih uvjeta i bakterija mliječne kiseline. Proizvodnja organskih kiselina, u najvećem dijelu mliječne kiseline, snižava pH koji inhibira nepoželjne mikroorganizame i sprječava daljnje promjene u siliranoj masi 1−2 mjeseca nakon početka siliranja. Suvremena istraživanja ukazuju na to da silaža nastavlja fermentirati i nakon 1−2 mjeseca, što bi za posljedicu moglo imati i kontinuirane promjene sastava i probavljivosti zrna kukuruza. Cilj istraživanja bio je utvrditi kako dodatak inokulanta i mliječne kiseline prilikom siliranja vlažnog zrna u periodu od godine dana utječe na intenzitet fermentacije i kvalitetu silaža te na razgradnju 16 kDa i 27 kDa γ-zeina hibrida različite tvrdoće zrna i udjela ukupnih zeina. Dodatno se ispitala i buražna razgradivost suhe tvari i škroba ispitivanih silaža. Tri hibrida (Bc 513 pc, Bc 462 i Bc 344) uzgojena u split-plot dizajnu u pet ponavljanja pri istim agroklimatskim uvjetima razlikovala su se u tvrdoći zrna, što su pokazali rezultati Stenvertove analize tvrdoće zrna i prava gustoća. Udio ukupnih zeina razlikovao se signifikantno između tri ispitivana hibrida kukruza (Bc 513 pc: 59,96% SP; Bc 462: 55,13% SP; Bc 344: 48,75% SP). Silaže sva tri hibrida silirane su s dodatkom inokulanta (Bio-Sil; 3x105 CFU/g), dodatkom mliječne kiseline (linearni porast 5 ‒ 15 g/kg ST) i bez dodataka (kontrola) u periodu od godine dana. Silaže su imale optimalne fermentacijske karakteristike tijekom godine dana siliranja. Suha tvar (ST) silaža brojčano se minimalno mijenjala tijekom siliranja 69,11 ‒ 71,04% i nije se razlikovala između tretmana i hibrida. Sadržaj mliječne kiseline signifikantno je rastao do 182. dana stajanja silaža (1,01 ‒ 15,38 g/kg ST), iza čega je opadao do zadnjeg uzorkovanja (9,10 g/kg ST). Nije utvrđena razlika između silaža pojedinih hibrida u sadržaju mliječne kiseline. Skoro dvostruko viši sadržaj mliječne kiseline imale su silaže s inokulantom, manje s mliječnom kiselinom i najmanje silaže bez dodataka. Titracijska kiselost rasla je tijekom stajanja silaža (0,02 ‒ 0,74 meq/g ST) s najvišim vrijednostima u silažama s dodatkom inokulanta. Vrijednost pH bilježila je očekivan signifikantan pad tijekom siliranja (6,45 ‒ 4,18), s najnižim vrijednostima u silažama s inokulantom i sukladno porastu mliječne kiseline te titracijske kiselosti u silažama s dodatkom inokulanta. Tijekom godine dana stajanja silaža rastao je i sadržaj produkata proteolize: topljivi protein (17,9 ‒ 47,0% SP) te amonijak (0,38 ‒ 3,73% SP). Silaže hibrida Bc 513 pc imale su viši sadržaj sirovog proteina i ukupnih zeina od silaža ostalih dvaju hibrida u svim vremenskim točkama. Na kraju siliranja zabilježena je redukcija količine 16 kDa i 27 kDa γ-zeina, što ukazuje na to da siliranje uzrokuje razgradnju ovih proteina. U svim silažama na kraju siliranja nije detektiran 27 kDa γ-zein. S druge strane razgradnja 16 kDa γ-zeina bila je različita u silažama ispitivanih hibrida siliranim s različitim dodacima. Dodatak inokulanta imao je veći utjecaj na razgradnju γzeina silaža tvrđih hibrida (Bc 513 pc i Bc 462), a dodatak mliječne kiseline na silaže mekšeg hibrida (Bc 344). Silaže s dodacima imale su veću razgradnju 16 kDa γ-zeina od kontrolnih silaža kod svih ispitivanih hibrida. Na kraju siliranja zabilježen je signifikantan porast buražne razgradivosti suhe tvari (od 0,59 na početku do 0,77 na kraju) i škroba (od 0,53 do 0,78), što je potvrda da siliranje (i razgradnja γ-zeina) povisuju razgradnju škroba i suhe tvari. Istraživanjem je potvrđena hipoteza o većoj razgradnji 16 kDa i 27 kDa γ-zeina u starijim silažama te hibridima tvrđeg, nego mekšeg endosperma te u silažama s dodacima, nego bez dodataka. Dodatno, istraživanje ukazuje da uz slične vrijednosti razgradivosti suhe tvari i škroba na kraju siliranja, kao i hibridi mekšeg zrna, hibridi tvrđeg zrna (Bc 462) imaju bolje proizvodne karakteristike.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

Bakteriyel İnokulant ve Organik Asit İlavesi ile Yeniden Silolamanın Mısır Silajının Aerobik Stabilitesi ve In Vitro Gaz Üretim Parametreleri Üzerine Olan Etkileri

Bu çalışmada, mısır silajının, inokulant ve organik asit ilave edilerek farklı sürelerde yeniden silolanmasının silaj kalitesi ve aerobik stabilite açısından değerlendirilmesi amaçlanmıştır. Çalışmanın bitkisel materyalini II. ürün mısır silajı oluşturmuştur. Fermantasyon döneminin 150. gününde açılan mısır silajı 3 muamele grubuna bölünmüştür. Muamele grupları 1- Kontrol, 2- Lactobacillus buchneri (LB) 3- Organik Asit (OA)’ten oluşturulmuştur. Yeniden silolama süresinin etkisini ortaya koyabilmek amacı ile her bir muamele grubu kendi içerisinde alt muamele gruplarına ayrılarak oksijene maruz kaldığı sürenin 6., 12. ve 24. saatinde yeniden silolanarak fermantasyona tabi tutulmuştur. Yeniden silolamanın 60. günü açılan silajlarda kimyasal ve mikrobiyolojik analizler yapılmıştır. Ayrıca, silajların in vitro gaz üretim değerleri saptanmıştır. Silolama döneminde (60. gün) açılan tüm silajlara 7 gün süre ile aerobik stabilite testi uygulanmıştır. Araştırma sonuçları, mısır silajlarının katkı maddesi ilave etmeden yeniden silolanabileceğini, ancak yeniden silolamada organik asit kullanımının aerobik stabiliteyi geliştirdiği yönündedir. Yemlerin in vitro gaz değerlerinden elde edilen verilere göre, inkübasyon süresinin 24. saatinden 96. saatine kadar silolama süresinin 6. saatinde LB ve OA silajların metan üretimi, kontrol grubuna göre yüksek bulunmuştur (P&lt;0.05)

Farklı Olgunluk Dönemlerinde Hasat Edilen Yonca Bitkisinden Hazırlanan Silajlarda Laktik Asit Bakterisi ve Enzim Karışım İnokulant İlavesinin Silaj Fermantasyon Özellikleri ve Yem Değeri Üzerindeki Etkileri

This study was carried out to determine the effects of lactic acid bacteria+ enzyme (LAB+E)inoculants on the fermentation characteristics and feed values of silages prepared from alfalfaharvested at three maturity stages. Alfalfa was harvested at the early, middle and late floweringstages. Sil-All (Alltech, UK) were used as LAB+E inoculants. Inoculants were applied to thesilages at the rates of 1×105, 5×105and 1×106cfu/g levels in 1 liter capacity plastic bags. Thebags were stored at 20±2°C under the laboratory conditions. Three bags from each group weresampled for chemical and microbiological analyses on the 45th day after ensiling. The resultsshowed that LAB+E inoculants reduced pH values and ammonia-nitrogen content, whereasincreased lactic acid contents and lactobacillus count of alfalfa silages. High doses LAB+Einoculant decreased neutral detergent fiber and acid detergent fiber content, increased in vitroorganic matter digestibility and metabolic energy of alfalfa silages. It has been demonstratedthat the most effective application dose of LAB+E inoculant to improve fermentation and feedvalue of alfalfa silage was 1×106cfu/g, but 1x105and 5×105cfu/g level can also be consideredas effective doseBu çalışma, üç ayrı vejetasyon döneminde hasat edilen yonca bitkisine farklı düzeylerde laktik asit bakteri+enzim (LAB+E) inokulantı ilavesinin silaj fermantasyon özellikleri ve yem değeri üzerindeki etkilerinin saptanması amacıyla yürütülmüştür. Yonca bitkisi çiçeklenme başlangıcı, çiçeklenme ortası ve çiçeklenme sonu döneminde hasat edilmiştir. Laktik asit bakteri+enzim karışımı inokulant kaynağı olarak Sil-All (Alltech, UK) kullanılmıştır. İnokulant, yonca hasıllarına 1×105 , 5×105 ve 1×106 kob/g düzeyinde katılmıştır. Kontrol ve katkı maddeleri ile muamele edilen yonca 1 litre hacimli polietilen torbalarda silolanmıştır. Torbalar laboratuvar koşullarında 20±2°C sıcaklıkta depolanmışlardır. Silolamadan sonraki 45. günde her gruptan 3'er torba açılarak silajlarda kimyasal ve mikrobiyolojik analizler yapılmıştır. Sonuç olarak, LAB+E inokulantı silajların pH ve amonyak azotu içeriklerini azaltırken; laktik asit, asetik asit içerikleri ve lactobacilli sayısını artırmıştır. Yüksek dozda LAB+E ilavesi silajların nötr deterjanda çözünmeyen lif ve asit deterjanda çözünmeyen lif içeriğini azaltmış, in vitro organik madde sindirilebilirliğini ve metabolik enerji değerlerini artırmıştır. Yoncanın LAB+E inokulantı ilave edilerek silolanmasının fermantasyon özellikleri ve yem değerini iyileştirdiği, en etkili dozun 1×106 kob g/kg olmakla birlikte, 1×105 kob g/kg ve 5×105 kob/g dozlarında da uygulanabileceği belirlenmişti

Influence of corn hybrids and type of silage additives on total and γ- zein content during the high moisture corn silage production

Siliranje visoko vlažnog zrna kukuruza način je da se očuva zrno kukuruza od berbe do berbe. U klasičnoj teoriji siliranje je metoda konzerviranja uz pomoć anaerobnih uvjeta i bakterija mliječne kiseline. Proizvodnja organskih kiselina, u najvećem dijelu mliječne kiseline, snižava pH koji inhibira nepoželjne mikroorganizame i sprječava daljnje promjene u siliranoj masi 1−2 mjeseca nakon početka siliranja. Suvremena istraživanja ukazuju na to da silaža nastavlja fermentirati i nakon 1−2 mjeseca, što bi za posljedicu moglo imati i kontinuirane promjene sastava i probavljivosti zrna kukuruza. Cilj istraživanja bio je utvrditi kako dodatak inokulanta i mliječne kiseline prilikom siliranja vlažnog zrna u periodu od godine dana utječe na intenzitet fermentacije i kvalitetu silaža te na razgradnju 16 kDa i 27 kDa γ-zeina hibrida različite tvrdoće zrna i udjela ukupnih zeina. Dodatno se ispitala i buražna razgradivost suhe tvari i škroba ispitivanih silaža. Tri hibrida (Bc 513 pc, Bc 462 i Bc 344) uzgojena u split-plot dizajnu u pet ponavljanja pri istim agroklimatskim uvjetima razlikovala su se u tvrdoći zrna, što su pokazali rezultati Stenvertove analize tvrdoće zrna i prava gustoća. Udio ukupnih zeina razlikovao se signifikantno između tri ispitivana hibrida kukruza (Bc 513 pc: 59,96% SP; Bc 462: 55,13% SP; Bc 344: 48,75% SP). Silaže sva tri hibrida silirane su s dodatkom inokulanta (Bio-Sil; 3x105 CFU/g), dodatkom mliječne kiseline (linearni porast 5 ‒ 15 g/kg ST) i bez dodataka (kontrola) u periodu od godine dana. Silaže su imale optimalne fermentacijske karakteristike tijekom godine dana siliranja. Suha tvar (ST) silaža brojčano se minimalno mijenjala tijekom siliranja 69,11 ‒ 71,04% i nije se razlikovala između tretmana i hibrida. Sadržaj mliječne kiseline signifikantno je rastao do 182. dana stajanja silaža (1,01 ‒ 15,38 g/kg ST), iza čega je opadao do zadnjeg uzorkovanja (9,10 g/kg ST). Nije utvrđena razlika između silaža pojedinih hibrida u sadržaju mliječne kiseline. Skoro dvostruko viši sadržaj mliječne kiseline imale su silaže s inokulantom, manje s mliječnom kiselinom i najmanje silaže bez dodataka. Titracijska kiselost rasla je tijekom stajanja silaža (0,02 ‒ 0,74 meq/g ST) s najvišim vrijednostima u silažama s dodatkom inokulanta. Vrijednost pH bilježila je očekivan signifikantan pad tijekom siliranja (6,45 ‒ 4,18), s najnižim vrijednostima u silažama s inokulantom i sukladno porastu mliječne kiseline te titracijske kiselosti u silažama s dodatkom inokulanta. Tijekom godine dana stajanja silaža rastao je i sadržaj produkata proteolize: topljivi protein (17,9 ‒ 47,0% SP) te amonijak (0,38 ‒ 3,73% SP). Silaže hibrida Bc 513 pc imale su viši sadržaj sirovog proteina i ukupnih zeina od silaža ostalih dvaju hibrida u svim vremenskim točkama. Na kraju siliranja zabilježena je redukcija količine 16 kDa i 27 kDa γ-zeina, što ukazuje na to da siliranje uzrokuje razgradnju ovih proteina. U svim silažama na kraju siliranja nije detektiran 27 kDa γ-zein. S druge strane razgradnja 16 kDa γ-zeina bila je različita u silažama ispitivanih hibrida siliranim s različitim dodacima. Dodatak inokulanta imao je veći utjecaj na razgradnju γzeina silaža tvrđih hibrida (Bc 513 pc i Bc 462), a dodatak mliječne kiseline na silaže mekšeg hibrida (Bc 344). Silaže s dodacima imale su veću razgradnju 16 kDa γ-zeina od kontrolnih silaža kod svih ispitivanih hibrida. Na kraju siliranja zabilježen je signifikantan porast buražne razgradivosti suhe tvari (od 0,59 na početku do 0,77 na kraju) i škroba (od 0,53 do 0,78), što je potvrda da siliranje (i razgradnja γ-zeina) povisuju razgradnju škroba i suhe tvari. Istraživanjem je potvrđena hipoteza o većoj razgradnji 16 kDa i 27 kDa γ-zeina u starijim silažama te hibridima tvrđeg, nego mekšeg endosperma te u silažama s dodacima, nego bez dodataka. Dodatno, istraživanje ukazuje da uz slične vrijednosti razgradivosti suhe tvari i škroba na kraju siliranja, kao i hibridi mekšeg zrna, hibridi tvrđeg zrna (Bc 462) imaju bolje proizvodne karakteristike.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

Hasat Öncesi Ve Hasat Sonrasi Laktik Asit Bakteri (Lab) İnokulantlarinin İlavesinin, Misir Silaj Fermantasyonu Ve Aerobik Stabilite Üzerine Etkileri

Bu çalışma, hasat öncesi ve hasat sonrası laktik asit bakteri inokulantlarının ilavesinin mısır silajlarının fermantasyon ve aerobik stabiliteleri üzerindeki etkilerini belirlemek amacıyla yürütülmüştür. Çalışmada katkı maddesi olarak homofermantatif ve heterofermantatif laktik asit bakterilerinin içeren 2 ticari inokulant kullanılmıştır. İnokulantlar silajlara 6.00 log10 cfu/g düzeyinde katılmıştır. Araştırma materyali hasat öncesi ve hasat sonrası olmak üzere kontrol, homofermantatif (LAB) ve heterofermantatif (LAB) inokulant uygulaması içeren olmak üzere 3 deneme grubuna bölünmüştür. İnokulantların uygulanmasında firma önerileri dikkate alınmıştır. İnokulantlar hasattan 15 ve 7 gün olmak üzere 2 farklı dönemde tarlada mısırlara el tipi pülverizatör yardımı ile atılmıştır. Hasat öncesi ve sonrasının karşılaş- tırmak amacıyla, hasat dönemi geldiğinde yine kontrol, homofermantatif (LAB) ve heterofermantatif (LAB) inokulant uygulamaları yapılmıştır. Hasat öncesi ve hasat sonrası gruplarını içeren uygulamalara ait muameleler CASCVP 260PD marka laboratuar tipi paket silaj makinası ile paketlenmiştir. Her muameleye ait 3?er paket silajın kullanıldığı çalışmada, silajların paketlenmesinden sonra materyaller laboratuvar koşullarında (20-22 oC) depolanmıştır. Fermantasyonun 45. gününde açılan örnekler üzerinden pH, kuru madde kaybı, maya ve küf sayımları için mikrobiyolojik analizlerin yapıldığı çalışmada aerobik stabiliteye ilişkin özellikler ana fermantasyon dönemi sonrası 14 günlük dönemde izlenmiştir. Araştırma sonucunda, hasattan öncesi inokulant uygulamasının özellikle maya ve küf gelişimi üzerinde olumlu etkileri olduğu ve özellikle maya ve küf gelişiminin azalttığı yö- nündedir.This study was conducted in order to investigate the effect of adding inoculated lactic acid bacteria to maize before and after harvest on the fermentation and aerobic stability properties of silages. Two commercial inoculants containing homofermentative and heterofermentative lactic acid bacteria were used as additives. Inoculants were added into the silages at the level of 6.00 log10 cfu/g. Pre and post harvest research materials were divided into three trials groups, namely, control, homofermentative (LAB) and heterofermentative (LAB) inoculants. During the use of inoculants, suggestions by the producers were taken into account. Inoculants were applied to the corn plants in the field by the aid of hand type pulverizator at three different times, 15 and 7 day before the harvest. To compare the pre and post harvest treatments, control, homofermentative and heterofermentative inoculants applications were realised at the time of harvest. The pre and post harvest treated materials were packed using lab-type CASCVP 260PD brand named silage machine. After packing, three sample packets from each treatment were stored under laboratory conditions (20-22 oC). pH, dry matter losses, microbiological analyses for yeast and mould count were done on the silage samples opened on the 45th day of fermentation. The same analyses were repeated and compared with the previous results 14 days after the opening in order to assess the aerobic stability. The results showed that inoculants applications before the harvest had a positive effects on silage quality by decreasing yeast and mould growth