Izolacija iz periferne krvi i analiza funkcija heterofila kokoši protočnom citometrijom – metodološko istraživanje

The aim of this study was to modify the flow cytometric method, which is used for analyzing neutrophils, to analyse the functions of avian heterophils. The blood samples used in the experiments were obtained from hens in slaughterhouses. Blood samples were collected from 10 hens for each trial. Within the scope of the present study, trials were carried out regarding the amount of blood, cell suspension, dihydrorodamine-123 (DHR-123), phorbol-12-myristate-13-acetate (PMA), and N-formyl-methionyl-leucyl-phenylalanine (fMLP), as well as the storage duration of blood samples and incubation time. The results showed that 0.5-3 ml of blood could be used to detect heterophil functions, and it would be ideal to conduct analyses using fresh blood samples. In addition, the results showed that blood stored at +4 °C for up to 8 hours may be also used if necessary. In order to isolate the cells, centrifugation for 30 minutes is sufficient, and it is appropriate to use a 30μL cell suspension. 2μL of DHR-123 should be used as a chemical probe to measure heterophil functions. Excessive use of DHR-123 affected the heterophil functions negatively. In addition, it was observed that using 2μL of fMLP, which is used as an oxidative burst stimulant, and 2μL of PMA as a stimulant of chemotaxic activity, were sufficient. It was concluded that incubation at 41 °C for 5 minutes after stimulating the heterophils is also sufficient. We conclude that the methods established in this study could be used to isolate heterophils and to analyze them by flow cytometry. Therefore, this study would contribute to further research and clinical studies in poultry.Cilj je istraživanja bio prilagoditi metodu protočne citometrije, koja se inače primjenjuje u analizi neutrofila, za analizu funkcija ptičjih heterofila. Uzorci krvi 10 kokoši dobiveni su iz klaonica. Uzevši u obzir količinu krvi, kao i trajanje pohrane krvnih uzoraka odnosno vrijeme inkubacije, analizirana su stanična suspenzija, dihidrorodamin-123 (DHR-123), forbol-12-miristat-13-acetat (PMA) i N-formil-metionil-leucil-fenilalanin (fMLP). Rezultati su pokazali da se 0,5 – 3 mL krvi može upotrijebiti za otkrivanje funkcija heterofila te da bi idealno bilo analizirati svježe uzorke krvi. Osim toga rezultati su pokazali da se i krv pohranjena na temperaturi od +4 °C, u vremenu do 8 sati, može upotrijebiti ako je to potrebno. Kako bi se stanice izolirale, dovoljno je centrifugirati 30 minuta uz primjenu stanične suspenzije od 30 μL. Kao kemijsku probu za mjerenje funkcija heterofila trebalo bi upotrijebiti 2 μL DHR-123. Prekomjerna upotreba DHR-123 negativno je utjecala na funkcije heterofila. Također, uočeno je da je bila dovoljna primjena 2 μL fMLP-a, koji služi kao stimulans oksidacijskog izgaranja, kao i primjena 2 μL PMA-a kao stimulansa kemotaktičke aktivnosti. Zaključeno je da je inkubacija na temperaturi od 41 °C, tijekom 5 minuta poslije stimulacije heterofila također dovoljna. Rezultati su pokazali da se metode ustanovljene u ovom radu mogu primijeniti za izolaciju heterofila i njihovu analizu protočnom citometrijom, čime se doprinosi daljnjim istraživanjima i kliničkim studijama u peradi

The alpha amylase activity in plasma crop and pancreas of hens

Changes in a amylase activity of the pancreas plasma crop tissue and crop content related to age and sex were determined in chicks and the correlation among the pancreas plasma crop tissue and crop content were investigated A total of 336 ISA 5 broiler hybrid chicks were used as material The chicks were separated into two groups 224 males and 112 females Pancreas crop content crop tissue and blood samples were taken from male chicks every 3 days The same samples were taken from females every 3 days until the 12th day and from the 12th day to the 42nd day samples were taken every 9 days Some 16 chicks were used in each trial The blood samples were centrifuged instantly and their plasma separated Tissue and content samples were homogenized and centrifuged and their supernatants obtained The a amylase activity was determined in plasma and supernatant samples In addition to changes in the a amylase activity of the pancreas plasma and crop content an increase was determined related to age When the total activities were measured the increase in a amylase activity was observed clearly Low levels of a amylase activity were determined in crop tissue A high correlation was found between a amylase activities of the pancreas and plasma Although there was no difference in a amylase activity between males and females the total pancreatic a amylase activity of males on days 12 21 and 39 was found to be significantly higher than that of the females It was considered that like in mammalians most a amylase in the digestive tract originates from the pancreas in chicks the activity of the crop is very important in starch digestion and the activity of the content may originate from a source outside of the crop Although sex does not affect the level of the enzyme it was concluded that males can digest starch more efficiently than females because of the difference in pancreatic total a amylase activity