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Department of Animal Science, Agricultural University of Athens, 11855 Athens, Greece
1 Corresponding author: i.politis{at}aua.gr
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ABSTRACT |
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Key Words: periparturient period • urokinase plasminogen activator • superoxide anion • ovine
Sheep farming is a very important animal production activity in Greece, contributing to approximately 40% of the sector’s gross income (Hadjigeorgiou et al., 2002). Two typical indigenous breeds and a synthetic breed were used in this study. The Chios breed is characterized by high milk yield and litter size. On the other hand, the Boutsiko breed is characterized by low milk yield, but it is known for its adaptability under harsh environmental conditions. Furthermore, the Boutsiko breed is less susceptible to mastitis (Hatziminaoglou et al., 1990; Simos et al., 1996; Kominakis et al., 1998; Ploumi et al., 1998). The synthetic breed (50% Boutsiko, 25% Arta, and 25% Chios) has been developed recently in an attempt to upgrade the Boutsiko breed by combining the high productivity of the Chios and Arta breeds with the robustness of the Boutsiko breed.
Several studies have demonstrated a decline in immune function during the periparturient period in dairy cows, but the causes of immunosuppression are not completely understood. It is certain, however, that immunosuppression has been linked to endocrine changes associated with parturition, metabolic stresses associated with lactogenesis, and availability of critical nutrients, including vitamin E and calcium (Kehrli and Goff, 1989; Goff and Horst, 1997; Smith et al., 1997; Kimura et al., 1999). To our knowledge, only one study has dealt partly with immunosuppression during the periparturient period in dairy sheep. Caroprese et al. (2006) reported that cell-mediated and humoral immune responses and plasma IL-6 concentrations underwent marked fluctuations in periparturient ewes. There is a major unfulfilled need for more studies on the suppression of the immune system in periparturient dairy sheep.
Phagocytes (monocytes-macrophages and neutrophils) are critical for a proper immune response, because these cells are capable of migrating from blood circulation to any infected or injured tissue to confront pathogens (Politis et al., 2002). The rapidity of the migratory response depends on the ability of phagocytic cells to overcome the mechanical barriers imposed by the basement membranes. To achieve this objective, monocytes-macrophages and neutrophils use limited proteolytic activity at the regions of contact with the endothelial cells. Ovine monocytes-macrophages and neutrophils express a specific urokinase-plasminogen activator (U-PA) receptor (CD 87; Politis et al., 2002). The U-PA can bind to this receptor and convert the abundant proteolytically inactive proenzyme plasminogen to active plasmin. Plasmin is capable of degrading certain matrix components, in addition to activating other matrix-degrading enzymes such as metalloproteinases.
The objective of the present study was to determine changes in the dynamics of the U-PA system as well as in the production of superoxide anion (SA) by monocytes-macrophages and neutrophils in 3 Greek dairy sheep breeds during the periparturient period.
A total of 33 ewes were used in this study; 11 from the Boutsiko breed, 11 from the synthetic breed (50% Boutsiko, 25% Arta, and 25% Chios), and 11 from the Chios breed. Animals were housed within the premises of the experimental farm of the Agricultural University of Athens. The ewes lambed between November 10, 2005, and January 10, 2006. Furthermore, all ewes lambed within ± 1 d of anticipated lambing. All ewes that lambed outside this period were excluded from the experiment.
Blood samples were collected from all ewes on 18 and 2 d before parturition and on d 15 after parturition. Monocytes-macrophages and neutrophils were isolated as described by Politis et al. (2002). Total cell-associated, membrane-bound U-PA activity and U-PA binding sites in activated monocytes-macrophages and neutrophils were determined by using the methods described by Politis et al. (2002). Activation of phagocytes is achieved following incubation with 81 µM phorbol myristate acetate for 30 min at 37°C. Superoxide anion production by phagocytes, as a direct indicator of respiratory burst activation, was measured by using the superoxide dismutase-inhibitable reduction of ferricytochrome C. All details are as described by Politis et al. (1995).
Significant departures from normal distribution for all variables and possible outliers were initially assessed by using quantile-quartile plots. Calculation of the extreme Studentized deviates for all variables was performed according to Grubbs (1969), and outliers were excluded at a 2-tailed probability of 0.05. Logarithmic transformation for SA production and for free UPA binding sites (by adding the value of 1 because of negative values) was then used in an attempt to meet ANOVA assumptions (i.e., normal distribution and homogeneity of variance within class effects). Transformed values of variables were then analyzed by using a linear mixed model appropriate for repeated measurements per subject (animal) with the compound symmetry for modeling the error covariance structure. The fixed effects part of the model included the effects of breed (Chios, synthetic, and Boutsiko), parturition stage (3 stages: 18 d before, 2 d before, and 15 d after parturition), and the breed by parturition stage interaction, whereas the effect of animal nested within breed was fitted as a random effect. The Satterthwaite method (Steel and Torrie, 1980) was used for computing the denominator degrees of freedom for the tests of fixed effects. The Tukey-Kramer adjustment was used for the P-values when performing multiple comparisons. Results of mixed ANOVA are presented as least squares means with standard errors. All mixed ANOVA analyses were carried out by PROC MIXED in SAS, version 9.0 (SAS Institute, 2004).
Total and membrane bound U-PA activities in monocytes-macrophages and neutrophils during the periparturient period for the 3 Greek dairy sheep breeds are presented in Tables 1
and 2, respectively. Total and membrane-bound U-PA activities remained constant during the periparturient period for the Boutsiko breed for both monocytes-macrophages and neutrophils. In contrast, there was a decline in total and membrane-bound U-PA activities of both monocytes-macrophages and neutrophils at d 2 prior to parturition in the Chios breed. In the synthetic breed, the lowest values for total and membrane-bound U-PA activities of monocytes-macrophages were observed at d 15 after parturition, and the highest were observed at d 18 prior to parturition. There were no significant differences in total and membrane-bound U-PA activities of neutrophils isolated from the synthetic breed during the periparturient period.
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and 2 also allow comparisons to be made among breeds. There were no significant differences (P > 0.05) in total and membrane-bound U-PA activities of monocytes-macrophages among breeds at d 15 after parturition and at d 18 before parturition. Furthermore, no significant differences were observed among breeds regarding total U-PA activity of neutrophils at d 15 after parturition and at d 18 before parturition and in membrane-bound U-PA activity of neutrophils at d 15 after parturition. In contrast, total and membrane-bound U-PA activities were lower in the Chios breed compared with the other 2 breeds for both monocytes-macrophages (P < 0.01) and neutrophils (P < 0.001) at d 2 before parturition and in neutrophils (P < 0.01) at d 18 before parturition.
Total U-PA activity was greater by 77 to 130% in neutrophils compared with the corresponding values in monocytes-macrophages. In a similar manner, membrane bound U-PA activity was greater by 73 to 122% in neutrophils than in monocyte-macrophages. The majority of the U-PA was detected in the cell membrane (Tables 1 and 2).
There were no significant differences (P > 0.05) in free U-PA binding sites of monocytes-macrophages and neutrophils during the periparturient period in all 3 Greek dairy sheep breeds used in our study. Furthermore, there were no significant differences (P > 0.05) between breeds in free U-PA binding sites on the membrane of monocytes-macrophages and neutrophils at all sampling points (d 18 and d 2 before and d 15 after parturition; data not shown).
Superoxide anion production of monocytes-macrophages and neutrophils during the periparturient period for the 3 Greek dairy sheep breeds is presented in Table 3. Superoxide anion production remained constant during the periparturient period for the Boutsiko breed for both monocytes-macrophages and neutrophils. In the Chios breed, the lowest value for SA production by monocytes-macrophages was observed at d 2 prior to parturition and the highest at d 15 after parturition. In contrast, there were no significant differences in SA production by neutrophils isolated from the Chios breed during the periparturient period. In the synthetic breed, there was a decline in SA production by monocytes-macrophages and neutrophils at d 15 after parturition. There were no significant differences (P > 0.05) among breeds in SA production by monocytes-macrophages and neutrophils at d 18 before parturition and at d 15 after parturition. Superoxide anion production by monocytes-macrophages and neutrophils was lower in the Chios breed compared with the other 2 breeds at d 2 before parturition.
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The second finding emerging from this study was that immunosuppression was more pronounced in the Chios breed, because both monocytes-macrophages and neutrophils were affected. In the synthetic breed, immunosuppression was mainly observed in monocytes-macrophages (total and membrane-bound U-PA, SA), whereas in neutrophils only SA production was affected. The extent of immunosuppression observed in the 2 Greek dairy sheep breeds was less pronounced than that observed in dairy cows for the same time period. Politis et al. (2004) reported that SA production at parturition was less than 50% of that observed 4 wk before and 4 wk after parturition. Similarly, Politis et al. (2001) reported that membrane-bound U-PA activity of neutrophils was in the range of 0.1 to 0.12 
A/h per 106 cells around parturition and 0.3 A/h per 106 cells at 4 wk after parturition. In contrast, reductions in the same parameters in our study never exceeded the level of 24%. Mild immunosuppression occurred in the prepartum period in the Chios breed and in the postpartum period in the synthetic breed.
The nature of the factors inducing the mild immunosuppression observed in the 2 highest milk-producing Greek dairy sheep breeds is not known with certainty. The hormonal environment of the blood and extracellular tissue fluids can probably influence phagocyte function and activity. It is known that major hormonal changes occur around parturition in dairy sheep. Plasma concentrations of sex hormones in sheep change spectacularly as the animal approaches parturition; progesterone concentration falls dramatically, whereas cortisol and estradiol concentrations rise in a similar manner leading up to parturition (El-Belely et al., 2000). Fluctuation of these hormones negatively affects the function on the immune system, at least in the case of the dairy cow.
In conclusion, mild immunosuppression occurred in the 2 highest milk-producing breeds (Chios and synthetic) during the periparturient period, but not in the lower milk-producing but environmentally tolerant Boutsiko breed. The patterns of mild immunosuppression were different for these 2 breeds; for the Chios breed it occurred in the prepartum period, whereas for the synthetic breed it occurred in the postpartum period. Interactions in the physiology among reproduction, lactation, and immune function in the dairy sheep require further research.
Received for publication March 30, 2007. Accepted for publication July 31, 2007.
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