In vitro ispitivanja uticaja endogenih i egzogenih faktora na funkcionalnu aktivnost neutrofilnih granulocita krava u peripartalnom periodu

Abstract

Hormonske i metaboličke promene, kao i energetska neravnoteža, mogu uticati na zdravlje, proizvodnju i reproduktivne performanse mlečnih krava. Kraj gestacije i početak laktacije kod muznih krava prati niz endokrinih i metaboličkih promena u organizmu i zbog toga je tranzicioni period (3 nedelje pre i 3 nedelje posle teljenja) veoma značajan. Promene koje prate ovaj period dovode do izmena u funkcijama ćelija imunskog sistema (i urođenog i stečenog), tako da mogu biti razlog manje ili više izražene imunosupresije i sve češćih supkliničkih i kliničkih poremećaja zdravlja i reprodukcije. Moguće posledice su: produženi servis period i rano isključenje mlečnih grla iz dalje proizvodnje (usled mastitisa i endometritisa). Ovo se ponekada dešava već u prvoj laktaciji. Neutrofilni granulociti su, kao prva linije odbrane, ključne ćelije u odbrani organizma od bakterija, virusa, gljivica, parazita i drugih stranih ćelija. Od funkcionalne aktivnosti ovih ćelija zavisi opšta otpornost samog organizma, a složena priroda imunskog sistema otežava razumevanje njegove smanjene funkcije u peripartalnom periodu. Iako postoje različite hipoteze o promenama funkcija neutrofilnih granulocita, kao prve linije odbrane organizma u tranzicionom periodu i dalje nije poznato koji faktori dovode do imunosupresije. Tokom tranzicionog perioda uočavaju se promene u vrednostima pojedinih biohemijskih parametara krvi, međutim, njihova koncentracija nije konstantna tokom čitavog perioda da bi, sa III sigurnošću, mogla da objasni nastanak imunosupresije. Cilj ove doktorske disertacije je bio da se utvrdi stepen funkcionalne aktivnosti neutrofilnih granulocita iz pune krvi zdravih krava u periodu pre i posle teljenja, kao i njena korelacija sa metaboličkim i hematološkim promenama koje prate ovaj period. Ispitivana je grupa od 21 krave, koje su praćene u periodu od treće nedelje pre partusa, do treće nedelje nakon partusa, u 7 nedeljnih termina. Poslednje uzorkovanje krvi je izvršeno šest nedelja posle teljenja. Važan deo ovog rada je predstavljalo in vitro ispitivanje mogućnosti modulacije procesa fagocitoze i respiratornog praska neutrofilnih granulocita različitim biljnim ekstraktima i jednim antiseptikom (Armenicum®). Ova laboratorijska ispitivanja su vršena u uzorcima pune krvi 6 nasumično odabranih krava, u nedelji pre i nedelji posle teljenja, odnosno u periodu kada se može očekivati imunosupresija. Stepen fagocitne sposobnosti i respiratornog praska određivan je protočnom citormetrijom. Prosečne vrednosti imunoloških, biohemijskih i parametara krvi, uprkos manjim oscilacijama uglavnom su bile u saglasnosti sa povoljnom kliničkom slikom, proizvodnim i reproduktivnim rezultatima i ukazivale su na dobar tranzicioni menadžment. U ispitivanoj grupi mlečnih krava, najviše prosečne koncentracije NEMK su registrovane u prvoj i drugoj nedelji nakon teljenja (1,05±0,50 mmol/l i 0,81±0,67 mmol/l). Vrednosti BHB takođe rastu nakon teljenja, ali samo do vrednosti od 0,74±0,43 mmol/l, što je ispod donje granice za supkliničku ketozu (> 1,2 mmol/l). Povišene koncentracije NEMK i BHB mogu da utiču na migratornu sposobnost, fagocitozu i respiratorni prasak neutrofilnih granulocita i drugih leukocita. Međutim, u ovom istraživanju koncentracije BHB, NEMK, kao i ostalih indikatora metabolizma jetre i koncentracije minerala u krvi su bile povoljne u posmatranom periodu i pogodovale su aktivaciji neutrofilnih granulocita. Tretman pune krvi različitim koncentracijama etarskih ulja (EU) i antiseptika nije uticao na fagocitnu sposobnost neutrofilnih granulocita. IV Vrednosti dobijene u našem istraživanju ukazuju da nije došlo do stimulacije fagocitoze u uzorcima pune krvi pod dejstvom ispitivanih koncentracija EU i antiseptika, ni pre ni posle teljenja. U uzorcima bez tretmana, neutrofilni granulociti, pobuđeni jakim stimulatorom (PMA), ispoljili su veliku spremnost za fagocitozu, kako pre (87,15%), tako i posle teljenja (90,13%). Dodatna stimulacija egzogenim faktorima nije bila moguća, jer se ovako visok procenat neutrofilnih granulocita, koji su ispoljili aktivnost u smislu fagocitoze u uzorcima pune krvi smatra maksimalnim odgovorom ovih ćelija na stimulans. Statistički značajna odstupanja su ustanovljena između vrednosti respiratornog praska neutrofilnih granulocita između nedelje pre i nedelje nakon teljenja kod istih tretmana. Najbolji efekat u pravcu pojačavanja intenziteta reakcije respiratornog praska neutrofilnih granulocita nakon teljenja, u odnosu na vrednosti pre teljenja, je imao 0,1% ekstrakt mente. Tretmani timijanom (0,0125% ekstrakt) i Armenicum-om® (razređenje 1:800 i razređenje 1:1600) su pozitivno uticali i na intenzitet i na indeks respiratornog praska neutrofilnih granulocita. Na osnovu dobijenih rezultata može da se zaključi da imunosupresija posle teljenja mlečnih krava, koje nisu u negativnom energetskom bilansu nije dominantna pojava. Imunski sistem se "ponaša" racionalno i dozno zavisno u odnosu na pretnje i izazove tranzicionog perioda: porastom neposredno pred teljenja, visokom aktivnošću u prvim nedeljama i brzom stabilizacijom šest nedelja nakon teljenja.

AB Hormonal and metabolical changes, as well as energy disbalans might have an impact on health, productivity and reproductive performances of dairy cows. The end of gestation and the beginning of lactation in dairy cows are accompanied by a series of endocrine and metabolic changes in their body. Therefore the transition period (3 weeks before and 3 weeks after partus) is of great importance. Changes during this period might be the reason for compromited immune system cells functions (both of innate and adaptive arm) resulting in a more or less expressed immunosuppression. They also might lead to the frequent subclinical and clinical disorders of health and reproduction. Possible consequences are: prolonged service period and an early exclusion of dairy cows from further production due to the mastitis and endometritis occurence. In some cases, this happens already in the first lactation. As the first line of defense, neutrophil granulocytes are the key cells in the body protection from bacteria, viruses, fungies, parasites and other foreign cells. General resistance of the organism itself, depends on the functional activity of these cells. Complex nature of the immune system complicates understanding of its lowered function in peripartal period. It is still unknown which factors lead to immunosuppression, although, there are different hypothesis about changed functions of neutrophil granulocytes, as the first line of defense, during the transition period. Moreover, during the transition period, concentrations of some blood biochemical parameters are not stable, so this can not explain the genesis of immunosuppression with acceptable certainty. The aim of this PhD thesis was to estimate functional activity of neutrophil granulocytes in the blood of healthy cows before and after VIII calving, as well to establish possible correlations with metabolic and hematology parameters changes during this period. A group of 21 Simmental cows was monitored during the seven weeks period, starting from the 3 weeks prior to calving until 3 weeks post calving. All blood collections were performed in the regular weekly terms and a final samples were taken at 6th week post calving. An important part of this study was in vitro evaluation of the possible modulation effects of different plant extracts and one antiseptic (Armenicum®) on the phagocytosis and respiratory burst of neutrophil granulocytes. These laboratory tests were performed in samples of whole blood, from 6 randomly choosen cows, one week before and one week after calving, when immunosuppression might be expected. The phagocytosis and respiratory burst activity of neutrophil granulocytes were determined by flow cytometry. The average values of immunological, biochemical and blood parameters, despite small variations were in line with favorable clinical picture, satisfactory production and reproduction results, and pointed to a good transition management. The highest average concentration of NEFA were registered in the 1st and 2nd week after calving (1.05±0.50 mmol/L and 0.81±0.67 mmol/L). Values for BHB also increased after calving but only to 0.74 ± 0.43 mmol/L, being lower than the values indicating subclinical ketosis (higher than 1.2 mmol/L). Higher concentration of NEFA and BHB can influence migratory ability, phagocytosis and respiratory burst activity of neutrophil granulocytes and other leukocytes. However, in this study the concentrations of BHB, NEFA and other indicators of liver metabolism, as well as minerals concentration in the blood, were optimal and favorable for the activation of neutrophil granulocytes. Whole blood treatment with different concentrations of essential oils (EO) and antiseptic did not influence the ability of neutrophil granulocytes for phagocytosis. Values obtained in this study confirm that EO IX and antiseptic did not stimulate phagocytosis in the whole blood samples before calving and neither after it. In the samples with no treatment (control samples), neutrophil granulocytes were stimulated with strong stimulator (PMA) and showed high abilitiy for phagocytosis before calving (87.15%) as well as after it (90.13%). Such a high percentage of neutrophil granulocytes that showed phagocytic activity were considered as the highest response of these cells on stimulants, so no additional stimulation of phagocytosis activity of neutrophil granulocytes was possible. Statistically significant differences were established between respiratory burst activity of neutrophil granulocytes with same treatment, in the week before and week after calving. When we compared effects of treatments on the intensity of neutrophil granulocytes respiratory burst after calving, with respect to pre-calving values, it was possible to conclude that 0.1% extract of the mint had the best effect. Neutrophil granulocytes in the whole blood samples treated with 0.0125% extract of thyme, as well as with Armenicum® (dilutions 1: 800 and 1: 1600), showed stimulation of both intensity and respiratory burst index in neutrophil granulocytes. In accordance with the results obtained in this study, we were able to conclude that in dairy cows with no negative energy balance, immunosuppression after calving is not a dominant phenomenon. The immune system is "behaving" rationally and in a dose-dependent manner on the threats and challenges of the transition period: increasing activity before calving, keeping it at the high level during the first week after calving and followed by stabilization at 6th week after calving.