CE provide an ideal model for investigating the development of pathogens such as viruses and testing drug effects, evaluating both toxicity and pharmacokinetics. Thus, CE as an animal model can be an important ally for carrying out experiments that require quick and less complex execution and with limited resources.
The CE and its annexes constitute a favourable environment for replicating several viruses, being widely used for the isolation or production of vaccines9. IBV causes dwarfism, haemorrhage and death when inoculated into embryonated eggs. Some strains of the gammacoronavirus can cause nephropathies and others haemagglutination14. However, IBV has a minimal agglutination capacity for erythrocytes in chickens15. Our study showed a decrease in haemoglobin concentration, haematocrit value and the number of erythrocytes in infected embryos compared to the NC group, indicating anaemia. Haematimetric indices did not differ in relation to the NC group. Thus, we can classify anaemia as normocytic and normochromic. This means that the cells were of standard size and haemoglobin concentration, so there was a decrease in RBC, but there was no response from the bone marrow to release cells into the bloodstream. Normocytic and normochromic anaemia can indicate decreased erythrocyte production, which can develop rapidly in birds with diseases involving infectious agents16.
Although there was no statistical difference in the number of leucocytes in the infected animals compared to embryos in the NC group, some animals had a high leucocyte count (> 30,000/mm3). In contrast, others had a low count (< 4,000/mm3) (Supplementary Table 1). These results contributed to the increase in the standard deviation in this analysis. The response of the white blood cell count during an infection can vary. In the initial moment of disease, leucocytosis may occur as a response by the body. However, as the leucocytes are consumed, leucopenia may occur when the demand for leucocytes is above the production capacity of these cells by the bone marrow16. From 12 EID, the haematopoiesis in CE is more active and occurs mainly in bone marrow 17. Therefore, in this work, haematopoiesis in the CE was intense since we used animals between 10–17 EID. However, although the CE used in this work were from the same flock and the mother had high consanguinity, there may be an inherent variation in the model itself.
There was no change in the Creat and UA levels in serum and allantois samples from infected CE compared to the NC group, and macroscopic kidney lesions were not observed either. On the other hand, the liver of infected animals was macroscopically altered, with an increase in volume and a greenish colour. In addition, we observed a moderate inflammatory reaction by histopathologic analyses (Figure 2), although liver enzymes were not increased in the serum or allantois compared to the control group18. There was also a decrease in lipid peroxidation rather than an increase. The absence of a rise in GGT, ALT, AST and the reduction in lipid peroxidation indicate that the stress was very intense, reducing liver responsiveness.
Serum calcium levels from virus-infected CE decreased compared to the NC group. Possibly the viruses can hijack the host cell’s machinery and utilise the host cell’s calcium to create an environment adapted to meet their own demands for replication19. This hypothesis is supported in the work of Cao et al. (2011)20. These authors observed that the expression level of some calcium-binding proteins was increased after in ovo IBV infection. These proteins facilitate the transcellular transport of calcium, suggesting that IBV may disrupt cellular Ca2 homeostasis for its own benefit.
In addition to virus experiments, drug studies using CE as a model provide a technically simple way to study complex biological systems for in vivo drug toxicity and pharmacokinetic assessment. For example, DX is a widely used glucocorticoid. Its administration in CE can cause immunosuppressive effects increasing embryonic catecholamines that alter development and cause death21.
There was high macroscopic lesion and mortality rate after the DX inoculation, similar to other studies21–23. As expected, the mortality was age-dependent. In addition, in our study, CE treated with DX reduced the weight in all CE that lived. The reduced weight in CE treated with DX can be related to muscle and bone development inhibition22,23. Furthermore, high doses of glucocorticoids can generate suppression of growth hormone activity in the pituitary gland which is fully established at the beginning of the last week of the embryonic development of CE23. Thus, we can suggest that DX promotes a delay in embryonic development, negatively influencing embryo weight gain, which may explain the delay in the complete formation of CAM found by us.
Unlike DX, FG did not cause significant changes and deaths. The only death observed in an embryo inoculated at zero EID was not accompanied by changes and occurred in an embryo from the NC group, probably caused by a natural process. Similarly, the injury in the CE inoculated via CAM possibly resulted from the inoculation process since haemorrhage may occur using this route.
CE treated with DX at 10 and 12 EID were anaemic. The CE treated with DX showed a decreased erythrocyte count and decreased haematocrit and at 10 EID, haemoglobin concentration compared to CE in the NC group. There were also decreases in haematocrit, haemoglobin and erythrocyte values in animals treated with DX at 12 EID, but only erythrocytes showed a statistical difference for the NC group. Despite that, there was a high correlation between haemoglobin and haematocrit (r value = 0.78), haematocrit and erythrocyte (r value = 0.77) and haemoglobin and erythrocytes (r value = 0.81). Therefore, the erythrocyte reduction accompanied the reduction in haemoglobin and haematocrit. Although the CE treated with DX had anaemia, the haematimetric indices were not statistically different between the groups treated and the NC, defining anaemia as normocytic and normochromic. Chickens treated with corticosteroids show increased energy expenditure24. Embryos treated with DX at 10 EID had higher yolk consumption, indicated by the lower yolk weight of these embryos compared to animals in the NC group. The nutritional deficit associated with liver damage caused by the drug may have impaired the production of erythrocytes, causing normocytic normochromic anaemia.
In CE inoculated with DX at 10 EID, there was a decrease in the number of thrombocytes compared to embryos from the NC group. Perhaps the production of thrombocytes in CE treated with DX at 10 EID was impaired by the damage caused in the liver. Thrombopoietin (TPO) is the regulator of megakaryocyte development and thrombocyte production and its expression in chickens occurs mainly in the liver25. Hemopoietic activity in the liver starts at seven EID with a peak at 14 EID26, but is more active from 12 EID17. In animals treated with DX at 12 EID, the opposite was observed. There was an increase in the number of thrombocytes compared to CE from the NC group. Thrombocytosis may reflect a rebound response after recovery from other conditions associated with excessive use of thrombocytes. Considering that CE from 12 EID had a more mature liver, active bone marrow and hemopoietic activity17, thrombocytosis can be explained in CE treated at 12 EID. Additionally, it should also be considered that in birds, thrombocytes have a phagocytic function27and the influence of glucocorticoids on these cells is unknown28.
In embryos treated with DX at 12 EID, there was an increase in the number of leucocytes, different to that observed in CE inoculated at 10 EID. This can be explained by the onset of lymphoid activity in the Bursa of Fabricius at 12 EID29, with a greater capacity to respond to the stimulus caused by the drug. In mammals treated with DX, an initial leucocytosis may occur, mainly due to neutrophilia15. In fact, CE inoculated at 12 EID showed an increase of H/L ratio due to the increase in heterophils and a decrease in lymphocytes. This corroborates several studies in born animals30–32. During CE development, granulopoiesis is more predominant; however, at hatching, the granulocytes begin to be replaced by lymphocytes by the first three days16.
Taken together, the results show that embryos with a small difference in embryo development stage can completely alter the cell count response. We do not know if this occurs with other drugs. However, knowing that the embryo is an ascending animal model, further work must be carried out to consider the best age for using the model depending on the expected objective.
As a granulocyte colony-stimulating factor (G-CSF), FG is used in human medicine to increase levels of neutrophils in the bloodstream. Therefore, we expected that the same effect would be observed in the CE in this experiment, increasing heterophils that have characteristics and performance corresponding to human neutrophils. However, no increase in granulocytes was observed in animals treated with FG at 10 EID or 12 EID either by inoculation in CAM or SM. Perhaps this did not occur because FG is a synthetic compound for human use and may not have the same results in other species.
The analysis of biochemical parameters provides essential data for the assessment of the clinical status of the animal. However, blood samples collected in research with CE do not always allow this analysis because it is not easy and takes time. Therefore, alternatives samples have been used to assess these parameters, such as amniotic fluid and allantoic fluid5,7,33. In this work, we compared the biochemical analyses of serum and allantoic fluid and observed that the values found for the two samples were not always similar. However, the differences observed between groups in serum samples were also observed in allantoic samples, except for AST, which may indicate that this enzyme does not have a good analysis from allantois.
It is known that proteins found in serum have different physical and biochemical properties and change in various physiological and pathological conditions. One of the problems with enzymatic analysis methods is that the reagents were designed to provide the substrate and its optimal concentrations for human plasma, but these variables can change depending on the species. For example, birds have deficient levels of activity of the enzyme ALT in the liver tissue, so in cases of severe liver damage, this enzyme may present normal values. Aspartate aminotransferase (AST) activity occurs in multiple tissues, but the main ones are liver and muscle, being considered sensitive but not very specific in cases of liver problems. GGT activity is increased in all conditions in which hepatocellular damage is present. Alkaline phosphatase (ALP) is associated with the regulation of bird growth, participating in chondrogenic and osteoblastic activities. Thus, physiological variations can be observed, with higher activity levels resulting from bone growth in young birds. Elevations in ALP may be associated with liver disease even if its activity in this organ is minimal26.
In our study, only the AST enzyme of CE treated with DX at 10 EID showed a statistical difference compared to the CN group. However, histopathological findings and results of oxidative stress biomarkers indicate that there was liver damage. At 12 EID, there was no increase of AST in CE treated with DX. At this age, just 50% of the CE had a macroscopic injury (Table 2) and maybe because of this, the AST level did not increase. However, the AST maximum value of the group treated of DX at 19 EID was greater than the NC (Supplementary Table 2). Thus, AST may be the best parameter for analysis of liver function in embryo serum.
High concentrations (up to five times) of UA in plasma can lead to precipitation of this acid in the form of crystals, which accumulate in tissues. Situations of hypouricemia are rare and may be related to severe liver damage with a consequent decrease in UA production34. The excretion of Crea occurs via the kidneys, but in birds, most creatine is excreted before being converted to creatinine16. Thus, increased Crea concentrations are rare and may occur in severe renal impairment, significantly if filtration is affected34. In our study, no changes in urea or Creat were observed in any of the treatments. Macroscopic lesions were also not observed. This can mean that there was no damage to the CE kidneys. However, as we did not perform histopathological analysis of the kidneys, we cannot rule out the possibility that the high damage caused by the treatment does not lead to changes in biochemical parameters.
Drugs can cause increased production of oxidants and the formation of free radicals, which, by exceeding the body’s ability to neutralise and scavenge these radicals, can cause organ damage35. Our study showed an increase in oxidative stress biomarkers ROS in the livers of embryos treated with DX at 10 EID and 12 EID and an increase in lipid peroxidation in embryos treated at 12 EID. In adult animals, corticosteroids can increase oxidative stress36, but a similar approach was never studied in CE. The increased energy expenditure triggered by high circulating corticosteroid levels may be responsible for the augmented formation of ROS as reflected by increased lipid peroxidation36. The body uses enzymatic and non-enzymatic antioxidants to neutralise damage caused by free radicals and minimise excessive oxidative stress. Sulphydryl groups are an example of essential antioxidants in controlling these processes and protecting against damage. Embryos that were treated at 12 EID showed decreased sulphydryl groups and FRAP values, indicating that these embryos had difficulty matching the damage caused by free radicals in the induction of oxidative stress by DX.
Embryos treated with FG at 12 EID did not show changes in oxidative stress biomarkers compared to CE from the NC group. However, in CE inoculated at 10 EID via CAM, FG increased the sulphydryl group. Filgrastim is a granulocytic colony-stimulating factor (G-CSF). It has biological activity identical to that of endogenous human G-CSF with a free cysteine at position 17 with an ionised sulphydryl group that is very reactive to free radical oxidation37. From this, we can conclude that FG may have shown an antioxidant effect, with possible protection of the embryo against ROS.
To measure haemoglobin in this experiment, we used Drabkin’s solution and compared it with the values obtained using the Drabkin solution method. Our study showed a moderate correlation in embryos treated at 12 EID and a strong correlation in embryos treated at 10 EID13. Thus, we can conclude that the calculation based on haematocrit can be used to approximate the haemoglobin value in situations where measurement by spectrophotometry is not possible. However, this replacement is only possible if there is no suspicion of haemolysis, since haemolysis due to collection or pathological problems, promotes a decrease in haematocrit without a proportional reduction in haemoglobin.
To perform the differential count of leucocytes in birds, there is a great difficulty is differentiating between thrombocytes and lymphocytes. Though not identical, the nuclei of thrombocytes and small lymphocytes are too similar to serve as a basis for distinguishing between these two cell types. In the present study, cells in fast panoptic stained CE blood smears with small, round or oval nuclei with dense chromatin clumps were categorised as thrombocytes if they had cytoplasmic vacuoles and colourless cytoplasm. Cells classified as small lymphocytes had similar nuclei but scant amounts of blue or dark blue cytoplasm without vacuoles. To establish a basis for the categorisation of these cells, some cytochemical properties of these cells were compared. The typical thrombocytes were visualised by UV after being exposed to gaseous formaldehyde. Swayne et al. (1986)38 observed that most of the thrombocytes (99%) had been fluorescent after gaseous formaldehyde treatment and all of the small lymphocytes had been non-fluorescent. This fluorescence resulted from serotonin condensation products. We measured the thrombocytes found in the slides of animals inoculated in this study and compared them with cells found in the blood smear slides of adult chickens. Thrombocytes from adult chickens had a larger size compared to the NC group, which averaged 8.5 µm. We could not determine why FG increased the size of thrombocytes.
Using the PAS, we characterised the thrombocytes as PAS positive while lymphocytes and erythroblasts were PAS negative (Figure 6). The eosinophils were SBB positive, while the heterophils were SBB negative (Figure 6). The use of PAS and SBB is important for better cell classification.
Despite the different forms of differentiation used to identify cells in the blood smears of embryos in this experiment, some cells remained without conclusive identification. The authors identified these cells as being a type of granulocyte, but they could be confused with eosinophils, basophils or granulocyte precursors16,27,39. These cells were found in samples from embryos treated with FG (20%) and DX (25%) inoculated at 10 EID, animals infected with virus (100%), and in animals from the NC group (23.07%) inoculated at 10 EID.
Another response observed in the use of FG in humans is the stimulation of endothelial cells with consequent angiogenesis40–42. In this study, we analysed the blood vessels of the CAM of animals inoculated with FG to identify a possible increase in vessel density characterising the occurrence of angiogenesis. However, there was no statistical difference between CE inoculated with FG and the NC group.
Overall, the results of this work provide vital information on the use of CE as an experimental model. The response of CE to challenges from viruses and drugs does not always go as expected. Although macro and microscopic damage was visible in viruses, white blood cell counts and inflammation biomarkers such as CRP did not change. It is important to mention that some drugs can be innocuous and not result in expected effects on CE, which was the case with FG. In the case of DX, changes in blood parameters and biomarkers seem to be inherent to the model and are highly dependent on the developmental stage of the CE.
This article reiterates the wonderful value of the CE as an animal model. However, our work sheds light on the importance of standardisation and the correct use of the model (considering the laboratory analysis, drug, age, route) so that the infection, toxicity and pharmacokinetic results are reliable.