| Abstract | Desetljeća korištenja antibiotskih promotora rasta (APR) poljuljala su povjerenje javnosti u sigurnost proizvoda životinjskog podrijetla te pojačali zabrinutost zbog njihovog štetnog djelovanja na imunost i zdravlje ljudi. Učinak APR-a na mikrobiom crijeva pogoduje razvoju korisnih mikroorganizama koji pospješuju probavu i apsorpciju hrane, a posljedično djeluju i na opskrbu hranjivim tvarima potrebnim za proizvodnju. Međutim, dugoročno korištenje antibiotika u subterapijskim dozama dovelo je do pojave rezistentnih sojeva bakterija koje predstavljaju prijetnju zdravlju životinja i ljudi.
Intenzivan uzgoj peradi izlaže životinje različitim stresnim situacijama tijekom njihovog proizvodnog ciklusa. Perad je izložena stresu odmah nakon valjenja kada njen nepotpuno razvijeni probavni sustav prvi puta dolazi u dodir s mikroorganizmima hrane i okoliša. Pile je u tom razdoblju života vrlo podložno invaziji patogenih mikroorganizama, a velika je i mogućnost pojave neke od zaraznih bolesti koje se brzo šire jer su sve životinje u neposrednom kontaktu. Probavni sustav je vjerojatno najvažniji organ u životinji jer predstavlja direktno sučelje s okolinom. Održavanje zdravlja probavnog sustava izuzetno je važno i složeno. Ono se oslanja se na osjetljivu ravnotežu između prehrane, mikropopulacije i sluznice, uključujući probavni epitel i sloj sluzi koji se na njoj nalazi. Epitelne stanice crijeva također su posljednja linija obrane tijela od patogenih bakterija i toksina koji stižu hranom ili vodom. Sastojci hrane, kao i toksini i mikroorganizmi, često oštećuju strukturu gastrointestinalnog trakta. To dovodi do različitih problema s crijevnim bolestima koji se manifestiraju u obliku malapsorpcije hranjivih tvari, proljeva i povećanog rizika od infekcije.
Suvremeni trendovi u proizvodnji životinja nastoje minimizirati ili zabraniti uporabu antibiotika zbog njihovih štetnih nuspojava na životinjama i ljudima. Posljednjih godina sve su češće inicijative koje kao cilj imaju smanjenje korištenja antibiotika u terapijske svrhe kako bi se što duže sačuvali kao učinkovito sredstvo borbe protiv bakterijskih infekcija. Sukladno tome postoji sve veći interes za razvijanje dugoročno održivih pripravaka koji bi bili učinkoviti u održavanju visoke proizvodnosti i zaštiti zdravlja životinja te ne bi imali štetan učinak u vidu bakterijske rezistencije i utjecaja na okoliš.
Đumbir (Zingiber officinale Roscoe) je dobro poznata biljka koja se koristi kao dodatak prehrani za ublažavanje određenih bolesti u tradicionalnoj medicini. Dosadašnja istraživanja o učinkovitosti đumbira kao nutritivnog dodatka za perad variraju ali postoje indikacije da može potaknuti intenzivniji rast, poboljšati funkciju crijeva i oksidativni status peradi. Međutim, doziranje, način aplikacije te ekstrakcijske procese treba standardizirati kako bi mogli biti sigurni u njegovu učinkovitost. Trenutne spoznaje o učinkovitosti đumbira kao nutritivnog dodatka za perad su neujednačena zbog velike varijabilnosti među korištenim pripravcima. Velik broj i različite koncentracije aktivnih spojeva u đumbiru (gingeroli, shogaoli, gingerdioli i gingerdioni) otežavaju objektivnu usporedbu testiranih dodataka i njihovu djelotvornost. U našem istraživanju će se po prvi puta koristiti standardizirani oblik ekstrakta đumbira s kemijski definiranim sastavom koji će omogućiti objektivnije testiranje.
U okviru ovog istraživanja procijenjen je učinak tri doze ekstrakta đumbira (2.5, 5 i 10 g/kg hrane) na ukupno dvije stotine Ross 308 tovnih pilića u dobi od 1 do 42 dana starosti. Tijekom pokusa praćeni su proizvodni rezultati, biokemijski i hematološki parametri, antioksidacijski status te mikrobiom i morfometrija crijeva. Dodatak ekstrakta đumbira u najnižoj i najvišoj dozi djelovao je negativno na završne mase dok je u najvišoj dozi djelovao negativno i na intenzitet rasta pilića u tovu. Povećanje doze pripravka imalo je negativan učinak na konzumaciju hrane tovnih pilića, a skupina koja je dobivala najvišu dozu je ostvarila i najviši mortalitet. S druge strane, dodatak ekstrakta đumbira u dozi od 5g/kg hrane pozitivno je djelovao na iskorištavanje hrane. Testirani pripravak nije negativno utjecao na raznolikosti i bogatstvo bakterijskih rodova u ileumu brojlera. Sve pokusne skupine, a pogotovo u dozi od 5 g/kg hrane, značajno su doprinijele održavanju poželjne ravnoteže crijevnih bakterija na razini koljena i roda. Dodatak ekstrakta đumbira u dozi od 5 g/kg hrane pozitivno je utjecao na morfometrijske parametre segmenata tankog crijeva te nije detektiran negativan učinak na pojavnost patoloških promjena na tkivu jetre. Pripravak đumbira je većim dijelom poboljšao antioksidacijski status tovnih pilića u intenzivnom uzgoju. Uzevši u obzir sve promatrane parametre ovog istraživanja skloni smo zaključiti da je doza ekstrakta đumbira od 5 g/kg hrane ostvarila najbolje rezultate te bi stoga mogla biti od koristi peradarskoj industriji u poboljšanju učinkovitosti i održivosti proizvodnje pilećeg mesa. |
| Abstract (english) | Objectives: Decades of use of antibiotic growth promoters (AGP) have shaken public confidence in the safety of products of animal origin and heightened concerns about their harmful effects on immunity and human health. The effect of AGP on the microbiome of the intestine favors the development of beneficial microorganisms that promote digestion and absorption of feed, and consequently act on the supply of nutrients necessary for production. However, the long-term use of antibiotics in subtherapeutic doses has led to the emergence of resistant strains of bacteria that pose a threat to animal and human health. Intensive poultry production exposes animals to various stressful situations during their life cycle. Poultry is exposed to stress immediately after hatching when its immature digestive system first comes into contact with feed and microorganisms. Chickens are very susceptible to the infection from pathogenic microorganisms during this period of life so the possibility of rapidly spreading infectious diseases is high. The digestive system is probably the most important organ in an animal because it represents a direct interface with the environment. Maintaining the health of the digestive system is extremely important and complex. It depends on the delicate balance between diet, micropopulation and mucous membranes, including the digestive epithelium and the mucus layer contained on it. The epithelial cells of the intestine are also the body's last line of defense against pathogenic bacteria and toxins that arrive by feed or water. Feed ingredients, as well as toxins and microorganisms, often damage the structure of the gastrointestinal tract. This leads to various problems with intestinal diseases that manifest themselves in the form of malabsorption of nutrients, diarrhea, and an increased risk of infection. Modern trends in animal production seek to minimize or prohibit the use of antibiotics due to their harmful side effects on animals and humans. In recent years, initiatives aimed at reducing the use of antibiotics for therapeutic purposes have been increasing to preserve them as an effective means of combating bacterial infections for as long as possible. Accordingly, there is a growing interest in developing long-term sustainable preparations that would be effective in maintaining high productivity and protecting animal health without a detrimental effect in the form of bacterial resistance and environmental impact. Ginger (Zingiber officinale Roscoe) is a well-known herb used as a dietary supplement to relieve certain diseases in traditional medicine. Previous
research on the effectiveness of ginger as a nutritional supplement for poultry is highly variable, but there are indications that it can promote growth, improve digestion and oxidative status of poultry. However, dosing, application mode and extraction processes need to be standardized to be certain of its effectiveness. Current knowledge about the efficacy of ginger as a nutritional supplement for poultry is uneven due to the high variability among the preparations used. The large number and different concentrations of active compounds in ginger (gingerols, shogaols, gingerdiols and gingerdions) make it difficult to objectively compare the tested supplements and their effectiveness. Our research will for the first time use a standardized form of ginger extract with a chemically defined composition that will allow more objective testing.
Materials and Methods: Two hundred one-day-old Ross 308 chickens (mixed sex) were acquired from a local hatchery. Chickens were randomly distributed into 20 pens (10 birds per pen) which were then randomly divided into 4 groups (treatments, 5 pens per treatment). One group was given only basic feed (control), and the others were fed with basic feed in which a standardized ginger extract was added at the level of 2.5, 5 and 10 g/kg feed. The experiment was set up as a complete randomized block design, and the pens were used as replication units. Broilers were fed starter feed from day 1 to 21 and grower feed from day 22 to 42. The birds were kept in a production system that resembles the modern intensive broiler production farm. The lights were turned on 24 hour per day and the birds were fed ad libitum and had free access to water throughout the experiment. During the experiment, production results, biochemical and hematological parameters, antioxidant status, microbiome and intestinal morphometry were assessed. Body weight and feed intake were measured weekly at pen level to determine average daily weight gain, average daily feed intake and feed conversion ratio. Mortality and health status were visually observed and recorded daily throughout the experimental period. On the morning of the 21st and 42nd day of the experiment, 40 birds (2 birds per pen and 10 per treatment) were randomly selected after 12 hours of fasting to collect peripheral blood needed to determine hematological and biochemical parameters. At day 42. two birds per cage (a total of 40 animals) were randomly selected for slaughter to collect liver tissue and segments of the small intestine for histological and morphometric examination. A 3 cm long segment of the ileum for determining the microbiome was taken in the middle between the Meckel diverticulum and the ileo-cecal junction. Those samples with content were stored in a cooled container with dry ice and transported to a laboratory where they are stored at a temperature of – 80° C until DNA extraction. For the extraction of DNK from small intestine content samples of fattening chickens, GenElute™ Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich,
USA) was used in this study. Due to its complex composition, the contents of the small intestine are classified as mammalian tissue. Isolated DNA from a sample of the contents of the small intestine was sent for sequencing to the laboratory of Novogene Co (Novogene Co, Cambridge Science Park, UK). Histological preparations of the intestinal segments were examined with a light microscope Nikon-Microphot-FXA (Nikon, Tokyo, Japan), which is equipped with a digital microscope camera GXCAM-U3-18 (GT Vision, Wickhambrook, UK) with which representative fields were photographed. The GXCapture-T computer program (GT Vision, Wickhambrook, UK) was used for capturing images. Quantitative computational morphometric analysis was performed on the images of the prepared cross-sections. The images were analyzed using ImageJ software (Bethesda, MD, USA) to measure the height of the villi, the depth of the crypt and the width of the villi at the crypt/villi junction, as well as the tip. The measurement was based on the mean value of 15 villi per sample (x10). The level of metabolites, enzymes and electrolytes in the serum of fattening chickens were determined by using standard methods on the Abbott Architect C4000 (Abbott, USA) automatic biochemical analyzer. All parameters were determined using Abbott reagents (Abbott, USA) except globulin and GPx. Globulin content was determined by calculation as the difference between total protein and albumin, and GPx was determined by Randox reagents (Randox Laboratories, Crumlin, UK). Malonaldehyde content (MDA) was measured using the HPLC method. Aliquot of 20 μL is injected into Shimadzu LC-2010HT with inert-Sustain C18 column (4.6 mm 150 mm, particle size 5 μm; GL Sciences, Tokyo, Japan). The standard curve prepared with 1,1,3,3-tetraetoxypropane is used. Substances that react with thiobarbituric acid (TBARS) are expressed as nmol per gram of wet tissue.
Results: The addition of ginger extract in the lowest and highest dose had a negative effect on the final masses, while the highest dose had also a negative effect on the growth rate of fattening chickens. An increase in the dose of the preparation had a negative effect on feed consumption of fattening chickens, and the group that received the highest dose had the highest mortality. On the other hand, the addition of ginger extract at a dose of 5g/kg of food had a positive effect on feed efficiency. The tested preparation did not adversely affect the diversity and richness of bacterial genera in broiler ileum. All experimental groups showed an increase in the proportion of beneficial Firmicutes, which was proportional to the increase in the dose of the tested preparation, while at the same time there was a decrease in the proportion of Proteobacteria which are considered a marker of intestinal microbiota imbalance. An increase in the relative abundance of bacteria from the genus Candidatus Arthromitus and Romboutsia and a decrease
in the genus Pseudomonas and Termoanaerobakteria were observed in the groups of chickens supplemented with ginger extract. The group supplemented with the highest dose of ginger extract recorded a significantly higher proportion of Lactobacillus bacteria compared to other groups. In the same group, a significantly higher proportion of Enterococcus bacteria belonging to probiotic species such as Lactobacillus, Bacillus, Bifidobacterium, Streptococcus and Faecalibacterium was recorded, which have a positive effect on the performance and health status of broilers. All the experimental groups, especially the one receiving 5 g/kg of feed, significantly contributed to maintaining the desirable balance of intestinal bacteria at the phylum and genus level. The addition of ginger extract at a dose of 5 g/kg of feed had a positive effect on the morphometric parameters of the small intestine segments and no negative effect was detected on the occurrence of pathological changes in liver tissue. A significant increase in GPx activity in serum of fattening chickens was observed on the 42nd day of the study while there were no differences on day 21. The increase was recorded in groups supplemented with 2.5 and 5 g/kg feed, while in 10 g/kg group there were no differences compared to the control group. Different trend was observed in MDA concentration, with the lower concentration recorded in groups supplemented with 2.5 and 5 g/kg feed on the 21st day of the experiment. At the end of the study there were no differences in MDA between the groups, but the level of MDA was significantly lower compared to the day 21. Overall, the ginger preparation has improved the antioxidative status of fattening chickens in intensive breeding.
Conclusion: The research demonstrates that the positive effects of ginger extract are clearly dose dependent but to pinpoint the optimal dosage and application method of this feed additive for broiler chickens further research is needed. Considering all the observed parameters of this study, we tend to conclude that a dose of ginger extract of 5 g/kg of feed achieved the best results and could therefore be useful to the poultry industry in improving the efficiency and sustainability of chicken meat production. |
