Chapter VI: Results and Discussion Section 1. Parasitological Results

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Chapter VI: Results and Discussion

Section 1. Parasitological Results

1. a) Gastrointestinal strongyles

The following table illustrates the mean values and relevant standard errors of each tested group during the four samplings taken during the experimental period. The first sample reflects the parasitological burden of the animals before receiving treatment. It is evident how the Eggs Per Gram (EPG) values taken from each group in the first sampling are quite homogenous. The homeopathic group is characterized by a lower standard error (87,2) and is therefore the most homogeneous group.

Table 1. Mean EPG and std. errors of the three groups during samplings.

The following graph illustrates the EPG output of strongyles from the three groups during the four samplings taken. In this way, the mean EPG values are clearly illustrated in order to evaluate their trends. The differences between the means of animals tested using the date of sampling as a factor of variability were very significant (p= 0,002) and when the factor of variability was the group nested into the date of sampling the significance was equally valid (p≤ 0,01).

Groups

Samplings Control Drug Homeopathy

Mean Std. error Mean Std. error Mean Std. error

1 587 107,46 646 148,5 698 87,2 2 909 339,63 86 55,5 730 337,28 3 284 111,31 152 124,19 208 44,54 4 509 122,74 427 254,38 344 122,09

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Figure 1. Mean EPG values of the three groups at each sampling taken.

The “Control” group follows a trend which is not influenced by any chemical or homeopathic treatment, therefore reflects the seasonal dynamics of the strongyles present on pasture. The peak noticed in the month of April, is due to the “spring rise” effect (Houdijk, 2008; Ambrosi, 1995). The trend of the “Control” group is similar to those already observed in other studies carried out in the same area (Benvenuti et al., 2004; Benvenuti et al., 2005; Perrucci et al., 2006; Benvenuti et al., 2008).

Mean FECs decreased by the month of July and slightly increased in October. This can be explained by observing the climatic aspects of the area (see Figures 1, 2 and 3 of Chapter 1. Section 1.). During the month of July, maximum temperature did not exceed 24°C, relative humidity was around 70% and average precipitation added up to 15 mm. It is curious how in the month of October, maximum temperature and relative humidity were not so different from those of the month of July (respectively 17°C and 75%). However, the average precipitation registered was relatively much higher (75 mm). It is known that the dispersal of larvae from faeces is highly dependent on sufficient water from dew or rainfall which allows larvae to migrate (Stear et al., 2006). Therefore increased rainfall could indeed be the cause of the higher FECs in the control group in October.

0 100 200 300 400 500 600 700 800 900 1000 1 2 3 4 EPG Samplings

Mean EPG values

Control Drug

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Figure 2. EPG trends of the three groups during the entire research.

The “Drug” group also brings to light an interesting trend. After the chemical treatment with Netobimin, the FECs of these animals dramatically decreased from the month of February to the month of April (see Figure 1). Undoubtedly, the drug used was able to lower parasite burden extremely effectively. Nevertheless, this lowering effect was not permanent because by the month of October, FECs were similar to those previously seen in February (427 versus 646 EPG). This shows how managerial procedures in compliance with anti-parasitic treatments can aid effectiveness in the long run.

The “Homeopathy” group demonstrates a particularly important trend. In the month of April, right after the administration of the treatment, FECs show a slight increase, probably related to the “aggravation” phenomenon, known to indicate that the remedy is appropriate. As spring turns into summer, mean EPG values decrease by 522 EPG among homeopathically treated animals. This is not surprising seeing how it has been stated that homeopathy does not act as an anti-parasitic but contributes to maintain the animal-parasite balance under the threshold of zootechnical risk. Evidently, this is an effect which seems to be beneficial in the long term: in the month of July FECs do not exceed 200 EPG and by October, the means have slightly overtaken those of animals belonging to the “Drug” group (344 versus 427 EPG).

Table 2 (see below) demonstrates the percentages of animals which belong to each level of infestation in the month of February. More than 55% of animals belonging to the three experimental groups, show a FEC which is higher than 600 EPG. Therefore, the majority of animals at the beginning of the study were heavily challenged with nematodes and had a parasitic burden which was well over the threshold of zootechnical damage (≥300 EPG).

0 100 200 300 400 500 600 700 800 900 1000 1 2 3 4 EPG Samplings

EPG output in 3 groups during samplings

Control Drug

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Table 3 (see below) corresponds to Sampling 2 therefore reflects the level of infestation of all animals during the month of April where treatment protocols have been applied. Exactly half of the animals in the control group fall into the interval between 300 and 600 EPG outputs. On the other hand, the animals which received chemical treatment excrete a remarkably lower number of eggs where 57% of the 9 tested animals have no output at all and only 14% continue to excrete between 300 and 600 EPG. Homeopathic animals remain distributed in a higher level of egg output even though 50% show an acceptable parasite burden (≤300 EPG).

Results from the third sampling are shown in Table 4 (see below) where it is underlined that only 13 % of all 27 ewes are placed in the higher level, meaning they excrete more than 600 EPG, which is definitely an amelioration as opposed to the month of February. Among the sheep treated with the chemical drug, 60% have stopped excreting all together and only 20% excrete over 600 EPG. The total number of animals treated with the homeopathic remedy at that particular moment excrete between 1 and 300 EPG, which is a remarkably low number of eggs.

Table 5 (see below) demonstrates the distribution of infestation levels which result from the last samples. A percentage of 34% of the total number of animals is placed in the Level 3 interval. In particular, 43% of animals belonging to the control group and 34% of pharmacologically treated animals excrete more than 600 EPG as opposed to 25% of homeopathically treated animals. In the long-run, the homeopathic treatment seems to have kept the majority of animals underneath the critical threshold of 300 EPG, whereas the chemical drug failed from this point of view.

February: levels of infestation among the 3 groups

Groups

FEC level Control (%) Drug (%) Homeopathy (%)

0 9

1 27 29 10

2 9 14 30

3 55 57 60

Table 2. Distribution of the levels of infestation of the three groups in Sampling 1. April: levels of infestation in the 3 groups

Groups

FEC level Control (%) Drug (%) Homeopathy (%)

0 10 57

1 10 29 50

2 50 14 25

3 30 25

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July: levels of infestation of the 3 groups

Groups

FEC level Control (%) Drug (%) Homeopathy (%)

0 10 60

1 60 20 100

2 10

3 20 20

Table 4. Distribution of the levels of infestation of the three groups in Sampling 3.

October: levels of infestation in the 3 groups

Groups

FEC level Control (%) Drug (%) Homeopathy (%)

0 33 12

1 14 38

2 43 33 25

3 43 34 25

Table 5. Distribution of the levels infestation of the three groups in Sampling 4.

When correlating EPG values with PCV, HgB and blood protein values, interesting negative correlations resulted, which were respectively (r= -0,436; r= -0,612 and r= -0,45), therefore denoting that higher EPG values correspond to lower PCV, HgB and blood protein values with high significance (p< 0,01).

On the other hand correlations between EPG with NEFA and triglycerides are not statistically significant.

Table 6 illustrates the EPG mean values of ewes which were in periparturient period (pp) and in non-periparturient period (np) during Sampling 1. Strangely enough, ewes that are in periparturient, posses the lower EPG values.

EPG

Mean Std. error pp 502 130,91 np 676 75,58

Table 6. EPG means and std. errs. of periparturient and non-periparturient ewes during Sampling 1.

1. b) Coccidia oocysts

The following graph (Figure 3) illustrates FECs regarding coccidia oocysts which resulted in being relatively low throughout the study (oocysts mean output is approximately 130 OPG at

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the beginning of the trial). Infact they have almost never constituted a serious/severe problem in farms of this area except in lambs (Perrucci et al., 2006).

Group

Samplings Control Drug Homeopathy

Mean Std. error Mean Std. error Mean Std. error 1 171 58,61 86 25,34 132 76,14 2 304 187,26 217 53,71 565 273,65 3 116 45,59 184 112,85 112 42,71 4 229 138,21 107 53,33 260 226,27

Table 7. Coccidian means and std. err. found in the three groups during Sampling 1.

It must be highlighted that the chemical drug used has no direct effect against coccidia. However, it can be suggested that treatment may have a possible indirect effect by enhancing the whole physiological response of the animal (Perrucci et al., 2006). The significant differences among dates of sampling resulted by the statistical model (p≤0,05). On the contrary, there was no significant difference between the means which result by analyzing treated groups nested into the date of sampling. This is probably due to the fact that values belonging to different groups show small differences between them.

Figure 3. Graph showing coccidia oocysts output from animals belonging to the three experimental groups during the four samplings.

0 100 200 300 400 500 600 1 2 3 4 OPG Samplings

Coccidia oocysts output in the 3 groups

Control Drug

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Coccidia burden was not taken into account in the homeopathic repertory because at this level of infestation it has a low impact on the health status of animals.

Section 2. Blood samples

2.a) Complete blood count

The next table shows all mean values of each parameter evaluated during complete blood count of animals belonging to the three tested groups, during the first sampling. Parameters which fall outside the interval of physiological values are indicated by the coloured arrows.

Table 8. Complete blood count of the three groups from Sample 1. Green arrows indicate values superior to the maximum value of the physiological interval; yellow up-going arrows indicate when values are borderline; yellow horizontal arrows indicate normal results; yellow down-going arrows indicate results which are borderline with the minimum values of the normal interval and red arrows indicate values inferior to the minimum physiological range.

During statistical analysis of data the only parameters which result in being statistically significant when considering the date of sampling or when considering the groups nested into the date of sampling were Red cell Distribution Width (RDW), HgB (g/dL), Eosinophil relative count (%), Lymphocyte relative count (%) and Neutrophil relative count (%).

In observing haematological results it can be noticed that RBC and HgB dosage of animals belonging to the “Control” and “Homeopathy” groups are a little below physiological ranges. Erythrocyte indexes (HCT and HgB) show relatively low values, which could reflect a possible state of anemia. In order to classify the seriousness of an anemic state, HCT represents the main value to be considered. It must be emphasized that the majority of sheep at this moment are either pregnant or lactating; the former of which can deeply influence haematological parameters causing a slight anemia (Lubas, 2005). It can be established that this type of anemia is normocytic and hypercromic, seeing as mean corpuscular volume (MCV) is normal but mean corpuscular haemaglobin concentration (MCHC) is higher than normal. This type of anemia has been associated with in vitro hemolysis; an incorrectly low count of HCT and a falsely high count of HgB (Lubas, 2005).

Mean Std. error Mean Std. error Mean Std. error Physiological range Measuring unit

WBC 5,5 0,74 6,6 0,42 5,7 0,89 5,5-9,5 K/μL RBC 7,2 0,22 7,7 5,68 7,2 0,28 7,5-9,99 M/μ HgB 8,3 0,23 8,8 7,18 8,1 0,41 8,5- 13 g/dL HCT 22,0 0,62 22,6 8,15 21,1 0,75 20-45 % MCV 30,3 0,53 29,5 21,08 29,3 0,16 23-48 fL MCH 11,5 0,10 11,5 29,35 11,3 0,24 8,0-12,0 pg MCHC 37,8 0,60 39,1 11,33 38,6 0,77 31-34 g/dL RDW 20,2 0,52 21,3 38,57 21,1 0,40 18-24.6 %CV

Control Drug Homeopathy

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A slight anemic state could undoubtedly be caused by parasitic diseases. Infact, anemia due to Gastro-Intestinal (GI) strongyle infection should be considered when haematocrit and/or haemoglobin values are inferior to 27% and 9 g/dL respectively, according to Di Loria et al.

(2009). Parasitic diseases, especially if caused by blood-sucking worms such as H. contortus, C. ovina and Bunostomum spp., can be responsible for instating anemia due to loss of plasma or

blood when damaging the inner gut wall.

Anemic conditions can also be classified as regenerative or non-regenerative on the basis of the manner in which the organism responds in compensating the loss of erythrocytes. When the response is regenerative this is indicated by the presence of reticulocytes or other immature red blood cells in the circulatory system, stimulated by the release of erythropoietin. Indeed, this is the case because each and every animal tested in Sampling 1 was found to have reticulocytes in the blood counts which were signaled in the medical reports (data not shown). This is a direct sign of bone marrow reactivity which is initiated starting from 72 hours after a hemorrhage and persists for approximately 2-4 days (Lubas, 2005).

Table 9. Complete blood count of the groups tested in Sampling 2.

Table 9 contains the results obtained from all ewes tested during Sample 2, after treatment protocols have been applied. It is clear how in all animals there seems to have been a profound improvement of health conditions. Anemic states have been defeated. Mean corpuscular heamoglobin concentrations (MCHC) among animals of the three groups are still above maximum range, which at this point could be justified by a probable erroneous laboratory technique since HgB levels are within range.

In considering the values of RDW of each experimental group, the results found indicate that the animals of the “Drug” group possess the highest RDW (22,67 %CV), compared with those of the other groups (p<0,05). RDW can indicate the presence of anisocytosis which occurs when the bone marrow responds to the state of anemia by secreting immature red blood cells into the circulation.

The difference between the two mean values calculated using all the animals tested in February for HgB and again for those in April, was found to be significant (p= 0,05), denoting a mild improvement of this value which increases from 8,4 g/dL to 8,9 g/dL. Infact, in the 2nd Sampling the anemic state does not persist. The animals belonging to the “Drug” group

Mean Std. Error Mean Std. Error Mean Std. error Physiological range Measuring unit

WBC 6,4 0,87 7,0 0,67 6,8 0,66 5,5-9,5 K/μL RBC 7,8 0,20 8,2 0,22 7,6 0,19 7,5-9,99 M/μ HgB 8,8 0,27 9,3 0,25 8,6 0,26 8,5-13 g/dL HCT 23,1 0,85 23,1 0,80 21,5 0,41 20-45 % MCV 29,5 0,62 28,1 0,83 28,7 0,37 23-48 fL MCH 11,4 0,12 11,3 0,23 11,3 0,12 8,0-12,0 pg MCHC 38,5 0,45 40,2 0,52 39,5 0,54 31-34 g/dL RDW 22,4 0,56 22,7 0,61 20,8 0,35 18-24.6 %CV Groups Drug Control Homeopathy

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possess the highest value of HgB, which may be thanks to the fact that they have been treated earlier thus, their parasitic burdens are the lowest of all (see Figure 1). Evidently these specific parasites have a powerful negative effect on the health status of these farm animals.

Table 10. Leukocyte differential count resulted from microscopic analysis of animals tested in Sampling 1.

In the above table, the only alteration which can be observed is the increase of eosinophil mean relative percentages in untreated animals of all three groups. The animals of the homeopathic group are characterized by a smaller standard error (1,45) therefore differences among singular values are limited.

Parasitism is notoriously one of the main causes of hypereosinophilia because one of the main tasks these cells have is to phagocitate non-self substances (Archetti and Ravarotto, 2002; Lubas, 2005; Meeusen et al., 2005; ). Stress is also a causal agent which could justify the increase of eosinophil count which results from the microscopic examination of the blood samples (Lubas, 2005). Infact, granulocytes are very responsive to elevated levels of glucocorticoid hormones which are responsible for the inhibitory effect on the immune system during periods of stress (Carroll and Forsberg, 2007).

Table 11. Leukocyte differential count resulted from microscopic analysis of animals tested in Sampling 2.

From the second sampling carried out in the month of April, mainly three alterations of the leukocyte differential count can be observed: lymphopenia; hypereosinophilia and neutrophilia.

Mean Std. error Mean Std. error Mean Std. error Physiological range Measuring unit

LYMPH 53,0 2,31 50,0 4,65 51,3 1,76 45,0-65,0 %L

BASO 0,0 0,00 0,0 0,00 0,0 0,00 0,00-1,00 %B

EOS 8,7 2,58 8,0 1,90 7,5 1,45 0,00-4,00 %E

MONO 0,0 0,00 1,7 1,05 1,7 1,17 0,00-6,00 %M

NEUTR 38,3 2,95 40,3 4,29 39,5 2,68 30,0-45,0 %N

BAND NEUTR 0,0 0,00 0,0 0,00 0,0 0,00 0-3 (rare) %B

Sampling 1 (manual count) Homeopathy Drug

Control

Mean Std. error Mean Std. error Mean Std. error Physiological range Measuring unit

LYMPH 40,3 1,91 44,5 3,40 34,7 3,61 45,0-65,0 %L

BASO 0,0 0,00 0,0 0,00 0,0 0,00 0,00-1,00 %B

ESO 10,8 2,76 12,0 3,61 12,8 2,09 0,00-4,00 %E

MONO 2,0 1,00 0,0 0,00 2,0 1,00 0,00-6,00 %M

NEUTR 46,8 2,51 43,5 2,23 50,5 4,82 30,0-45,0 %N

BAND NEUTR 0,0 0,00 0,0 0,00 0,0 0,00 0-3 (rare) %B

Sampling 2 (manual count)

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In combination, these three results could be an indication of prolonged stress which weighs on the organism and causes alteration of these parameters. It has been observed that stressful events in sheep, such as transportation even if brief, can cause the lowering of the percentages of lymphocytes and increase those of granulocytes (Fagiolo et al., 2004), providing us with a probable explanation. Indeed, lymphocytes diminish in response to conditions of distress which undoubtedly have consequential effects upon certain physiological parameters in the blood (Moro et al., 2008). Lymphocyte mean values decreased from Sampling 1 to Sampling 2 (p> 0,01).

Eosinophils are still quite high, even though these animals have been treated, thus parasitic burden has been lowered. It is also true that parasitic burden in April is lowered only in animals belonging to the “Drug” group and all the other animals continue to excrete large amounts of eggs (see Figure 1). Nevertheless, there was a slight decrease of mean values calculated from Sampling 1 to Sampling 2 which is seen to be significant (p= 0,05).

The increase of neutrophils is generally caused by three events: inflammation, stress and in response to catecholamines (Lubas, 2005). However, during inflammation the total number of WBC should be superior to the physiological interval which is not the case. Stress, on the other hand, notably diminishes WBC especially due to glucocorticoid levels, and increase neutrophils by mobilizing the portion of these which reside in small vessels and move at a slower rate. Infact, there is a gradual increase of neutrophil counts in patients following a stressful event (Zahorec, 2001). This effect is also produced when catecholamine hormones are secreted except catecholamines also induce an increase of lymphocytes which is not the case (Lubas, 2005). Therefore, it can be stated that the variation of these haematological parameters could reflect a condition of stress or distress in these tested animals. Infact, neutrophil mean values from Sampling 1 to Sampling 2 have increased (p< 0,01).

Table 12 (see below) illustrates results obtained by calculating the ratio between neutrophil relative counts (%) and lymphocyte relative counts (%) of adult females tested during Sampling 1. All values remain below the maximum value indicating the absence of major stressful events. Indeed, the neutrophil/lymphocyte stress factor remains beneath the maximum interval and never indicates a highly intense stressful event in both samples. However, there is a mild increase in all three groups during the second sampling (Table 13). It is also true that this reflects the same trend noticed when observing the leukocyte differential counts of the three groups from February to April (see Tables 10 and 11).

Neutrophil/Lymphocyte ratio Sampling 1

Control Drug Homeopathy Normal ratio NLSF 0,73 0,81 0,77 <5

Table 12. Neutrophil/Lymphocyte Stress Factors of the three groups from Sampling 1.

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Neutrophil/Lymphocyte ratio Sampling 2

Control Drug Homeopathy Normal ratio NLSF 1,16 0,98 1,45 <5

Table 13. Neutrophil/Lymphocyte Stress Factors of the three groups from Sampling 1.

2. b) Blood chemistry results

The following table illustrates the results obtained from all adult sheep during Sampling 1.

Table 14. Blood chemistry from Sampling 1.

Alanine transaminase is known to be a highly reliable indicator of liver dysfunction. Infact, the ratio calculated between AST/ALT should normally be superior to 1; otherwise it would indicate liver suffering or cellular damage, which fortunately is not the case here (ratios are superior to 4 in all three cases). Nevertheless, this specific result allows us to state that a slight metabolic distress could be causing increments of cellular enzymes in all tested animals.

Blood urea nitrogen resulted in being superior to physiological values. Considering that blood was always retrieved from animals when on an empty stomach, increased values of BUN in this case could be ascribed to the fact that the majority of these animals were in lactation. Therefore, gluconeogenesis is profoundly stimulated and amino-acidic deposits are being mobilized.

Prolonged stress often due to pre-pathological conditions when severe inflammation is advancing/proceeding, can indeed is responsible for this metabolic state (Bertoni, 1999). Possibly, grasslands and pastures offered to these sheep do not contain enough metabolizable energy to suffice the organism’s increased requirements during certain physiological stages such as pregnancy and lactation (Moro et al., 2008).

ALT 24 1,32 24,3 0,81 24,3 2,01 <18 U/L AST 101,8 3,72 112,1 8,81 99,8 2,94 40 - 123 U/L BUN 26,9 0,82 28,1 1,75 27 1,42 8 - 20 mg/dL CHO 57,5 2,07 58,4 3,39 58,6 3,46 60 - 150 mg/dL COR 2,75 0,43 1,7 0,45 2,3 0,52 1,88 - 2,6 μg/dL Creat 0,77 0,02 0,8 0,02 0,7 0,03 0,7 - 3 mg/dL Gluc 52,7 1,45 53,4 2,3 51,2 2,11 50 - 80 mg/dL NEFA 180,2 27,24 199,1 29,24 185,1 19,78 102-450 μmol/l Prot 7,12 0,13 7,1 0,21 7 0,15 6-7,9 g/dL Trig 20,6 1,89 21,1 2,41 18,6 2,72 13 - 23 mg/dL

Control Drug Homeopathy Sampling 1

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Low cholesterol levels can be observed in farm animals right at the beginning of lactation. The majority of the tested animals in February had given birth 25-35 days beforehand. The lactation period in these animals has a duration of about 2-3 months.

Cholesterol is synthesized in the liver therefore is also an indicator of hepatocyte failure or dysfunction. Liver dysfunction can be confirmed by reduced levels of cholesterol and HCT (Hematocrit) which are sensitive to modifications of the liver’s functionality. Low cholesterol values observed in Sampling 1 can be justified in this way.

Cortisol results differ between the three groups: in the “Control” group the mean is higher than the normal range; in the “Drug” group, the mean is lower and in the “Homeopathy” group the value is within the normal interval.

Cortisol measurement is frequently used to indicate distress in farm animals (Fagiolo et

al., 2002; Fagiolo et al., 2004; Moro et al., 2008). It causes lymphocytopenia and neutrophilia

(Lubas, 2005).

The animals of the “Control” group seem to be the only ones in a state of distress and curiously enough, the WBC was slightly lower in this group.

Table 15. Blood chemistry from Sampling 2.

The only altered parameters in the second sampling taken in April regard creatinine and NEFA values. The remaining parameters are in accord with those which result from Sampling 1, denoting consistency of physiological values studied in these animals. Cholesterol, creatinine, total blood proteins, NEFA and glucose values demonstrate a statistical significance when analyzed using the date of sampling as the variability factor. Unfortunately, except for triglycerides, no statistical significance was found when analyzing means among the three groups.

The mean of all three groups during Sampling 1 regarding cholesterol levels results in being 58,2 mg/dL, whereas the mean resulting from Sampling 2 is much lower - 50 mg/dL (p< 0,01). This clearly indicates that gradually ewes are entering a predominantly anabolic phase of the metabolism associated with a diminished lipid mobilization, thus resulting in lower values

ALT 17,4 1,08 17,9 1,01 18,9 1,38 <18 U/L AST 105,3 4,88 109,9 9,87 101,8 3,64 40 - 123 U/L BUN 32,5 1,35 30,9 1,67 31,1 1,66 8 - 20 mg/dL CHO 49,4 1,67 51,7 2,06 48,9 2,95 60 - 150 mg/dL COR 2,7 0,27 1,7 0,41 2,8 0,39 1,88 - 2,6 μg/dL Creat 0,6 0,03 0,6 0,01 0,6 0,02 0,7 - 3 mg/dL Gluc 64,4 0,97 66,1 1,44 65,1 2,46 50 - 80 mg/dL NEFA 48,9 3,97 59,4 7,19 44,1 4,36 102-450 μmol/l Prot 7,4 0,20 7,7 0,22 7,2 0,13 6-7,9 g/dL Trig 22,4 2,14 18,7 1,80 20,8 1,50 13 - 23 mg/dL

Control Drug Homeopathy Sampling 2

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of this lipoprotein. Cholesterol has been found to follow the trend of milk productivity (Bertoni,1999). Infact, in Sampling 2 all ewes were approaching the end of lactation thus manifesting a lower cholesterol value.

Interestingly enough, the mean value of the “Drug” group is higher (51,7 mg/dL) than the other animals although this is not statistically significant.

Differences between the two mean values of creatinine calculated from 1st and 2nd Samplings are highly significant (p<0,01), whereas creatinine mean values of each group nested into date of sampling is not significant. However, creatinine levels did undergo a decrease from February to April, but this parameter has a tendency to decrease once lactation has come to an end (Bertoni, 1999).

Creatinine levels can also decrease due to a delayed and prolonged mobilization of muscle tissue proteins, in order to compensate for metabolic deficiency which is often the case in the first stages of lactation. It is also interesting to note that low creatinine levels indicate that the muscular mass could be relatively lower than body mass (Bertoni, 1999). Animals analyzed in April may have a worst body condition than they did in February, since creatinine levels have dropped from a mean of 0,75 to 0,62.

The two mean values calculated using glucose levels which resulted from Sampling 1 and Sampling 2 indicate a slight increase from 52,4 to 65,2 mg/dL (p< 0,01). Even though the mean levels among groups are constantly within normal intervals, there seems to have been an improvement regarding glucose levels. These can be influenced by many factors but in ruminants mainly by the fermentations which occur in the rumen and the amount of proprionic acid which is produced, since it is the direct precursor of glucose. The quantity of glucose supplied by the diet is also influenced by the amount of starch present in fodder (Bertoni, 1999). Among the three groups, those in April with higher glucose means were the ones belonging to the “Drug” group (66,14 mg/dL), however this is not significant and the differences with animals of the other two groups is negligible.

In considering NEFA, statistical analysis demonstrates an important decrease in the mean values of all animals tested in February (188,1 mg/dL) and in April (50,8 mg/dL) which was highly significant (p<0,01). This drastic decrease could be caused by cessation of lactation which evidently represented a state of excessive catabolism for these sheep. As lactation proceeds, normally NEFA decreases and glucose increases (Bertoni, 1999). However, this drastic decrease could also be ascribed to a slight technical problem , since NEFA are very sensitive to stocking conditions and tend to diminish in time if samples are not analyzed immediately (Cavallina, personal communication).

In considering total blood proteins, there is a statistically significant difference between the two mean values calculated of all animals tested in Sampling 1 and Sampling 2 (p= 0,01), where values seem to increase mildly from 7,1 to 7,4. This denotes a slight improvement which could be ascribed either to the reduction in FECs or to the cessation of the lactation period in all ewes. In April, the group which possessed higher values of blood proteins are those belonging to the “Drug” group (7,7 g/dL), however it is not statistically significant.

Differences between ALT, AST, NEFA and BUN mean values of the three groups tested during 2nd Sampling did not result in being statistically significant.

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In considering the BUN values singularly, it is evident that animals of the “Control” group possess the highest value (31,9 mg/dL) which is superior to the normal interval. Lactation is known to be a physiological event which increases BUN values (Bertoni, 1999).

With regard to cortisol levels, animals belonging to the “Homeopathy” group result in having a slightly higher mean value, even though these differences were found not to be statistically significant. Nevertheless, this could be explained by the fact that animals of this group, in April showed the highest EPG and OPG mean values (730 and 565 respectively), as opposed to animals of the other 2 groups. On the contrary, the animals who had just received the chemical treatment had the lowest mean value (1,7 μg/dL). On the whole, from Sampling 1 to Sampling 2 cortisol levels hardly vary (from an average of 2,25 μg/dL to 2,4 μg/dL).

The differences between the two dates of sampling and the groups regarding triglycerides have no statistical significance. However, evaluation of each group showed that animals of the “Control” group have higher values (22,3 mg/dL) which could signify that catabolic processes are predominant in these ewes, as opposed to those who have received chemical treatment and have the lowest value (18,7 mg/dL). None of these ewes are in periparturient period during the second sampling.

2. c) Immunological results

Table 16. Immunological parameters tests during Sampling 1.

BPI is indicated as the percentage of bactericidal capacity of serum and should be superior to 90%. Only animals belonging to the “Drug” group possess this capacity at a physiological value. “Control” and “Homeopathy” animals, on the contrary, seem to be deficient of antibacterial proteins which could indicate the presence of suppressive factors towards the immune system and its components (complement and certain antibodies). Nevertheless, values are strictly borderline. Thus the innate immune response seems to be slightly depressed.

Mean lysozyme levels are high in all three groups which indicate that these animals in Sampling 1 were indeed struck by an inflammatory event. Infact, in the month of February all animals were parasitized as previously described (see Figure 1). Lysozyme is an important substance contained in neutrophils and belongs to the innate immune response. Thus, lysozyme activity can provide information about the functionality of neutrophils in the blood,

mean err. Std. mean err. Std. mean err. Std. Physiological range Measuring unit

BPI 89,7 0,33 90,9 0,46 89,7 0,47 >90 % Lysoz 4,8 0,81 4,6 1,04 3,4 0,49 1 - 3 μg/ml CD4+ 35,3 1,75 32,1 1,52 33,9 2,27 8 - 22 % CD8+ 22,4 1,82 22,5 2,43 17,7 1,37 4 - 22 % CD4/CD8 1,6 0,15 1,5 0,14 2,0 0,13 >1, 55 Sampling 1 Groups

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indicating the possible presence of inflammation. Neutrophils were not found to be particularly high among the three groups in Sampling 1, nevertheless it can be stated that all ewes are characterized by having an activated innate immune response particularly focused against Gram positive bacteria (Moscati and Beghelli, 2008).

High CD4+ values reflect a stimulated activity of regulatory lymphocytes in a positive manner. High CD8+ levels, on the other hand, indicate a poor reactivity of the acquired immune response (Tizard, 2004).

Table 17. Immunological parameters tests during Sampling 2.

Statistical analysis proves that lysozyme values and CD4+ percentages are the only parameters showing a significant difference when analyzed from Sampling 1 to Sampling 2. In addition, a very interesting and significant correlation was found between the CD4+/CD8+ ratio and Cortisol levels of ewes tested at Sampling 2.

Lysozyme levels have been rectified in all three groups by the 2nd date of sampling and all fall within normal range. In considering the difference of lysozyme values from February to April, it follows that with a highly statistical significance (p< 0,01) the mean values of the 1st Sample are more than four times those of the 2nd Sampling (from 4,3 to 1,) which could be influenced by the important decrease of parasite challenge.

CD4+ values were evaluated in each of the three groups using the percentages collected from Sampling 2 and significant results (p≤ 0,05) underline that animals of the “Control” group have a higher percentage (33,5%).

On the other hand, other immunological parameters do not result in any statistical significance.

BPI values are found to be low in all three groups. Moreover, mean values of all three groups were calculated for the 1st Sample and for the 2nd and are 90,1 and 85,4 % respectively, even though differences are not statistically significant. We can notice a worsening in all three groups from February to April.

Animals belonging to the “Control” group possess the lowest BPI capacity (80,5%), however, they also have a larger standard error which could indicate less homogeneity among animals. Low BPI percentages could indicate an unfavourable innate immune response.

In considering the three groups singularly, animals treated homeopathically resulted in having a higher BPI capacity (88,8%) than animals of the other two groups.

mean err. Std. mean err. Std. mean err. Std. Physiological range Measuring unit

BPI 80,5 5,69 87,0 2,42 88,8 1,66 >90 %

Lysoz 1,2 0,14 1,0 0,06 1,1 0,14 1 - 3 μg/ml

CD4+ 33,5 1,95 28,6 2,03 28,4 1,45 8 - 22 %

CD8+ 17,4 1,19 18,6 1,68 15,1 1,74 4 - 22 %

CD4/CD8 2,0 0,19 1,6 0,11 2,0 0,16 1, 55

Control Drug Homeopathy

Sampling 2 Groups

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The “Drug” group has the highest CD8+ percentage (18,6%) even though this result is not significant and within normal range; curiously these same animals possess the lowest cortisol levels in the same month.

On the contrary, animals belonging to the “Homeopathy” group in April possess the highest cortisol values (2,8 μg/dL) while CD8+ percentage is the lowest of all groups (15,1%).

According to Moro et al. (2008), stress seems to have a negative effect on CD8+ cells which is in accordance with the mentioned results found in the homeopathic group.

CD4+/CD8+ ratio is constantly superior to the normal range mainly because of the high CD4+ values which are increased during the 1st and 2nd Samplings in all three groups. This indicates lymphocyte reactivity and in addition, has determined a significant and important correlation (r= 0,503) between the CD4+/CD8+ ratio and cortisol levels (p=0,03). Therefore, it can be stated that as cortisol values increase the ratio of these two T cell subsets behave in the same way manifesting a positive relationship. This intriguing result is in accordance with Moro

et al. (2008).

Lastly, during Sampling 1, it was observed that more than half of adult ewes tested were in periparturient period. Certain correlations were performed in order to verify if immunological parameters could have undergone a negative influence related to this critical physiological phase, which would naturally weigh upon the organism’s functionalities. In the end, three very interesting and statistically significant results were noticed which are here-below displayed.

Table 18. Parameters correlated with the period of periparturient in ewes.

Periparturient ewes show a lower cortisol level (p= 0,05). This could be attributed to the inhibitory tone on hypothalamic-pituitary-adrenal (HPA) axis activity that prolactin has been found to exert. Indeed, in preparation of lactation neuro-endocrine and behavioural responses to acute stress become attenuated in female mammals (Torner et al., 2002).

Lysozyme mean values are high in all adult ewes, whether in periparturient period or not. This could indicate that all animals before receiving treatment were indeed combating against parasites. Ewes in periparturient time have a mean value which is almost double the mean of non-periparturient ewes (p≤ 0,02). Evidently, periparturient ewes are characterized by the momentarily expression of an enhanced innate immunity. Lysozyme levels are regulated by degenerated neutrophils in the blood (Moscati and Beghelli, 2008). Oxytocin is produced during mammalian nursing of young. It is known that oxytocin stimulates T-helper cells to

Mean Std. error Mean Std. error Physiological range Measuring unit

Cortisol 1,4 0,54 2,6 0,29 1,88-2,6 μg/dL

Lysozyme 6,1 0,85 3,7 0,47 1 - 3 μg/ml

CD4+/CD8+ 1,4 0,15 1,8 0,09 1, 55

pp np

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produce IFN-γ which, in this circumstance, activates components of the innate immune system (Broom, 2006).

Periparturient ewes also show a lowering of the ratio between the two T-cell subsets as opposed to non-periparturient ewes which show a higher value of this ratio (p= 0,05). When the ratio falls below 1 there is a good possibility that immunosuppression is in action (Zahorec, 2001). This is not yet the case but evidently periparturient ewes are tending towards this direction. Lymphocytopenia can be induced by high serum concentrations of prolactin (Zahorec, 2001).

It can be concluded that periparturient ewes seem to have a less effective immune response as opposed to non-periparturient ewes which show a higher CD4+/CD8+ ratio, high lysozyme values and a physiological cortisol level. Evidently, there is a stronger statistical correspondence between immunological parameters and the periparturient period in sheep than there is with homeopathic medication.

2. d) Zootechnical results

Bodily condition is known to reflect the health status of animals of a previous period. Table 19 (see below) illustrates BCS estimated during the 1st Sampling. It results that 78% of all animals are attributed score 3 which corresponds to a medium fatty subcutaneous deposit. All animals are above the score 2,5.

On the other hand, in October, 47% of animals were attributed score 3; 22% had a score of 2,5 and 5% had a score of 2. This could indicate that catabolic mechanisms were predominant in the previous period which would reflect a nutritional deficiency since quality of pastures is scarce during the summer.

Striking changes of BCS may be more frequently noticed in environments where

Teladorsagia or Trichostrongylus are predominant (Burke et al., 2007), which were indeed seen

to be among the most prevalent parasitic species in former studies completed on Zeri territory (Perrucci et al., 2006).

Table 21 (see below) illustrates FAMACHA scores registered during the first Sampling. As a rule, the eye membrane tends to be of a whitish colour at higher FAMACHA scores. By summarizing our data, it was found that the eleven animals with a FAMACHA score of 3 have a PCV of 22,6%. Animals with FAMACHA scores of 4 and a PCV of 21,1% were 31. This is in contrast with official FAMACHA scores which dictate that score 4 corresponds to the value of PCV of 13-17% indicating that we have over-estimated FAMACHA scores.

The second collection of FAMACHA scores was carried out in the month of October and corresponds to the moment in which the fourth sampling of faeces was effectuated. In analyzing data, 57,7% of all animals in Sampling 1 are appointed FAMACHA score 4 and 39% manifest a score of 3. In sampling 2, 88% of animals are appointed score 4 which is in agreement with the haematological parameters analyzed previously. Together with RBC and HgB, this confirms that the anemic conditions have been indeed defeated after treatment administered previously.

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