Changes of acute phase protein levels in Saanen goat kids during neonatal period

Accepted Manuscript Title: Changes of Acute Phase Protein Levels in Saanen Goat Kids During Neonatal Period Author: Pinar Alkim Ulutas Bulent Ulutas Funda Kiral Gamze Sevri Ekren Asici Mehmet Gultekin PII: DOI: Reference:

S0921-4488(16)30334-0 http://dx.doi.org/doi:10.1016/j.smallrumres.2016.11.015 RUMIN 5349

To appear in:

Small Ruminant Research

Received date: Revised date: Accepted date:

11-4-2016 17-11-2016 28-11-2016

Please cite this article as: Ulutas, Pinar Alkim, Ulutas, Bulent, Kiral, Funda, Ekren Asici, Gamze Sevri, Gultekin, Mehmet, Changes of Acute Phase Protein Levels in Saanen Goat Kids During Neonatal Period.Small Ruminant Research http://dx.doi.org/10.1016/j.smallrumres.2016.11.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Changes of Acute Phase Protein Levels in Saanen Goat Kids During Neonatal Period Pinar Alkim ULUTAS1, Bulent ULUTAS2, Funda KIRAL1, Gamze Sevri EKREN ASICI1, Mehmet GULTEKIN2 Adnan Menderes University, Faculty of Veterinery Medicine, Department of Biochemistry 1, Department of Internal Medicine2

Corresponding author; Pinar Alkim ULUTAS Address: Adnan Menderes University, Faculty of Veterinery Medicine, Department of Biochemistry, Isikli, Aydin, TURKEY E-mail: [email protected]

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HIGHLIGHTS The developmental changes of plasma acute phase proteins concentrations in healthy Saanen goat kids during neonatal period were investigated. Time-dependent changes were found in concentrations of Serum Amyloid A, ceruloplasmin, fibrinogen and Acid Soluble Glycoprotein during neonatal period. The results will provide new data for scientific research using goat kids as the experimental animals.

Abstract: In the present study, the developmental changes of plasma acute phase proteins concentrations in healthy Saanen goat kids during neonatal period were investigated. Blood samples were collected from eleven goat kids at 0, 1, 3, 7, 14, 21 and 28 days of age. Serum Amyloid A concentrations were higher during first two weeks and significantly decreased at days 21 and 28. There were no statistically differences in the mean haptoglobin concentrations throughout the study. Ceruloplasmin concentrations were lower during first week of life and significantly increased at days 14 and 28. Fibrinogen concentrations were only significantly high at day 28 compared to day 1. Concentrations of Acid Soluble Glycoprotein were higher during first three days and significantly decreased at days 7, 14, 21 and 28. Alterations of these acute phase proteins could be related with birth process and stress and/or factors in colostrum. As a result, the time related changes in acute phase protein levels in Saanen goat kids suggested that these proteins might play an important role in defense and physiological adaptation mechanisms during neonatal period.

Keywords: Acute phase proteins, goat kids, neonatal period.

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1. Introduction Every acute inflammatory process can initiate an acute phase reaction (APR) (Heller and Johns, 2015). The reasons of the APR may be infectious and noninfectious factors such as trauma and strenuous exercise. APR corresponds to changes in the concentration of some plasma proteins, synthesized in the liver, called as Acute Phase Proteins (APPs). APPs play important roles during the APR and known as beneficial to organism with protecting microbial growth, limiting further tissue damage and restoring homeostasis (Tothova et al., 2015). APPs have been proposed as sensitive and rapid indicators of inflammatory processes in ruminants (Eckersal and Bell, 2010; Gonzales et al., 2011). In goats, haptoglobin and Serum amyloid A (SAA) could be thought as major APPs, while ceruloplasmin, fibrinogen and Acid Soluble Glycoprotein (ASG) may be thought as moderate (Gonzales et al., 2008). In all species, the first weeks after birth represents a critical stage; it is a transition period from the sheltered intrauterine to the exposed extrauterine environment. Because of unstable metabolic conditions for newborns; perinatal diseases can result in high mortality rate (Piccione et al., 2008). The immune system of newborns is functional but immature (Morein, 2002). Immaturity of neonatal lymphocytes was documented during the first week of life (Nagahata, 1991). Transfer of immune components such as immunoglobins and cytokines with colostrum starts the effective nonspecific immune response. Host defense properties and production of APPs are very important for newborns to protect themselves against the pathogens. The changes of APPs concentrations in earlier of life could explain the role of immune system adaptation to extrauterine life conditions in newborns (Tothova et al., 2015).

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Very few studies have been carried out on the concentrations of APPs in neonatal period of animals and mainly limited to calves (Itoh et al., 1993; Alsemgeest et al., 1993, 1995; Gentry et al., 1994; Knowles et al., 2000; Tothova et al., 2015) and lambs (Kilpi, 2015). There is only one available report about physiological changes in haptoglobin and ceruloplasmin concentrations in goat kids between 23 and 50 days of age (Magistrelli et al., 2013). The caprine species is one of the least studied among ruminants; however, the economic importance of this species demands a better knowledge of its physiologic characteristics (Greenwood, 1997). Kid mortality rates are generally higher than other farm species, especially during the first days of life (Eales et al., 1983; Roberts and Holland 1993). Newborn disease and mortality are major reasons of economic loss in livestock production. Thus, specific APPs reference ranges could help to promote the ability of clinicians to more accurately interpret clinical pathology data and diagnose neonatal disease. In addition, animal research is currently an important part of medical and veterinary development, and a way of increasing our understanding of the biological processes that underpin these advances. Relevant animal models are critically important for the discovery of disease mechanism targets for medical interventions and for the development of new therapies. Rodents are the most popular, but the use of other, non-rodent mammals is therefore an essential way to develop more relevant and predictable models of human disease. Large animals including sheep, goat, and pig share close similarities with humans in size and in many anatomical, physiological and pathological features. The main objective of the present study was to investigate age dependent changes in some APPs (SAA, haptoglobin, ceruloplasmin, fibrinogen and ASG) in goat kids during neonatal period. The results of this study also will provide a new data to studies that use goat kids.

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2. Materials and Methods 2.1. Animals and sampling: Ten female, pregnant, healthy Saanen goats of similar age were used in this study. Goats were monitored with ultrasonography during all pregnancy period and a total of eleven kids were used for APPs analyses. Goat kids were received colostrum within three hours of birth from their mothers and were fed on milk by suckling during the study period. Water and hay was provided ad libitum. No vaccination or other medications were administered to goats throughout the study. Clinical examination was performed daily for all animals. Blood samples were collected from jugular vein at 0, 1, 3, 7, 14, 21 and 28 days of age at 10:00 am. Serum samples were separated by centrifugation (2000g for 10 min) and stored at -20°C until biochemical analyses. The study protocol, including procedures for animal handling and husbandry, was reviewed and approved by the Animal

Care

and

Use

Committee

of

Adnan

Menderes

University

(Number:

64583101/2013/115). 2.2. Analyses of acute phase proteins: Serum concentrations of SAA were measured by the solid phase sandwich ELISA kit (SAA kit, Tridelta Development Ltd., Greystones, Ireland). Serum concentrations of haptoglobin were determined by the hemoglobin-binding method based on the phase range haptoglobin kit (Haptoglobin kit, Tridelta Development Ltd., Greystones, Ireland) using microtiter plates and an ELISA reader (Optic Ivymen System, Spain). Ceruloplasmin concentrations were estimated in serum described by Sunderman and Nomoto (1970). A colored oxidation product is formed from ceruloplasmin and phenylenediamine and the rate of formation of this product is proportional to the concentration of serum ceruloplasmin. These measurements were made on a UVspectrophotometer (Schimadzu, UV-1601, Japan). Blood fibrinogen concentrations were determined immediately after blood withdrawal with regard to Millars method (Benjamin, 1979). For ASG determinations (Winzler, 1955) serum samples were precipitated with 0.6 M 5

perchloric acid and protein content in supernatant was determined in bicinchoninic acid method using commercial BCA Assay kits (Biovision, USA). 2.3. Statistical Analyses: Arithmetic means and standard errors were calculated using standard descriptive statistic procedures. Data of each APPs were analyzed for homogeneity by using the Shapiro-Wilk tests and all data were normalized with logarithmic transformation before the analysis. Related samples Friedman’s two way-analysis of variance were performed to assess pairwise comparisons between blood sampling days. Software package (SPSS 12.0, SPSS Inc., Chicago, USA) were used for all tests. P value <0.05 was considered statistically significant.

3. Results The results of this study were shown in Table 1 and Figure 1. Serum Amyloid A concentrations were higher during first two weeks of life and significantly decreased at days 21 and 28 (Figure 1a). There were no statistically differences in the mean haptoglobin concentrations throughout the observation (Figure 1b). Ceruloplasmin concentrations were lower during first week of life and significantly increased at days 14 and 28 (Figure 1c). Fibrinogen concentrations were only significantly high at day 28 compared to day 1 (Figure 1d). Concentrations of ASG were higher during first three days and significantly decreased at days 7, 14, 21 and 28 (Figure 1e).

4. Discussion

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In the present study, the developmental changes of plasma acute phase proteins concentrations in healthy Saanen goat kids during neonatal period were investigated. Significant temporal changes were found in most of APPs. The most significant changes occurred in the concentrations of SAA and ASG. Adaptation to extrauterine life is a complicated physiological process involving many different mechanisms. APR is one of the important mechanisms to create homeostasis. Colostrum intake is the external factor most likely to cause the temporal changes in APP concentrations in newborn animals (Orro et al., 2008). Colostrum containing high quantities of proinflammatory cytokines and transfer of colostral cytokines to blood has been reported before (Yamanaka et al., 2003). Transfer of some APPs such as CRP from colostrum to newborns may potentially happen (Schroedl et al., 2003). Physical stress or trauma during parturition may also start the synthesis of APPs (Marchini et al., 2000). Furthermore, immaturity of liver in neonatal animals could affect the synthesis of APP (Orro, 2008). In the present study, the most obvious change occurred in concentrations of SAA, which started to increase after birth and reached the highest level at day 7, gradually decreased after first week, significantly lower at days 21 and 28. Although there was no evidence for transfer of SAA directly from colostrum to newborn ruminants, proinflammatory cytokines that present in colostrum could have crossed the neonatal circulation and stimulated the synthesis of hepatic APPs (Yamanaka et al., 2003). Colostrum that contains high quantities of cytokines is the main inducer of production of APPs by the newborn liver (Tothova et al., 2015). Human mammary-associated SAA was shown to have a primarily protective effect on the gastrointestinal tract of neonate by stimulating mucin synthesis and reducing the connection of pathogens (McDonald et al., 2001). Earlier studies indicated that birth stress and trauma during the parturition may stimulate the rise in the concentrations of APPs (Orro et al., 2008; Marchini et al., 2000). This can explain the reason of the high 7

concentrations of SAA for the first week of neonatal period. Similar to our results, Orro et al. (2006; 2008) have reported increased SAA levels in newborn dairy calves until days 8-14 and day 7 after birth, which was followed by a decrease. Correspondingly, Tothova et al. (2015) demonstrated that SAA concentrations were lower in few hours at birth and significantly increased on third day of life. Mean haptoglobin concentrations were not changed significantly in goat kids during this study. It may be explained with the different kinetic characterization of haptoglobin compared to other APPs (Tuna and Ulutas, 2015) and hemolysis of fetal red blood cells which can affect the biosynthesis and catabolism of haptoglobin (Orro et al., 2008). In the present study, ceruloplasmin concentrations were lower during first week of life and significantly increased at days 14 and 28. These findings may be related with response to birth or activity of the liver which has been described by Tothova et al. (2015). In addition, ceruloplasmin carries about 70% of the total copper in plasma and may play an important role in Cu homeostasis (Tothova et al., 2014). Despite the serum copper concentrations were not analyzed in this study, colostrum intake would also have a role in high ceruloplasmin concentrations after the first week of life (Tabrizi et al., 2011). ASG is a moderate APPs in goats and showed an extended response compared with SAA. Gonzales et al. (2008) have reported that a two fold increase in ASG concentrations after turpentine induced inflammation in goats. Changes of ASG concentrations were similar in size and time for appearance that previously described in cattle (Conner et al., 1989; Nagahata et al., 1991). Although ASG is more likely to be associated with chronic conditions, concentrations of ASG were higher during first three days and were significantly decreased at days 7, 14, 21 and 28 in the present study. We thought that high concentrations of ASG during the first three days may not be related with stimulation of APR by external stimulus

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such as colostrum or birth process. Because, ASG concentrations are differently regulating in newborns compared to adults and probably regulated by fetus as reported in calves and piglets (Stone and Maurer, 1987; Itoh et al., 1993). Therefore high concentration of this protein might be related with prenatal period. This information would be beneficial for scientific research using newborn goat kids as the experimental animals. 5. Conclusion APR is not always related with the disease, it might be a part of physiological period. Thus, changes observed in this study may help us to understand the adaptation of extrauterine life in newborn goat kids. The present study includes normal reference intervals of SAA, haptoglobin, ceruloplasmin, fibrinogen and ASG in goat kids during neonatal period. In conclusion, the time related changes in acute phase protein levels in healthy Saanen goat kids suggested that these proteins might play an important role in defense and physiological adaptation mechanisms during neonatal period. The results also will provide new data for scientific research using goat kids as the experimental animals.

Acknowledgement: This study has been supported by the Adnan Menderes University Research Foundation (VTF-14006).

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Itoh, H., Takamura, K., Izumi, M., Motoi, Y., Funayama, Y., 1993. Characterization of serum alpha-1 acid glycoprotein in fetal and newborn calves during development. Am. J. Vet. Res. 54, 591-595. Kilpi, A.J. 2015, Serum concentrations of globulins, albumin and serum amyloid a of neonatal lambs and associations with weight gain during summer rearing period, Estonian University of Life Sciences Institute of Veterinary Medicine and Animal Sciences Graduation, Thesis in Veterinary Medicine, Tartu, Estonia. Knowles, T.G., Edwards, J.E., Bazeley, K.J., Brown, S.W., Butterworth, A., Warris, PD., 2000. Changes in the blood biochemical and haematological profile of neonatal calves with age. Vet. Rec. 147, 593-598. Magistrelli, D., Aufy, A.A., Pinotti, L., Rosi, F., 2013. Analysis of weaning-induced stres in Saanen goat kids. J. Anim. Physiol. Anim. Nutr. 97, 732-739. Marchini, G, Berggren, V., Djilali-Merzoug, R., Hansson, L.O., 2000. The birth process initiates an acute phase reaction in the fetus-newborn infant. Acta. paediatr. 89, 1082– 1086. McDonald, T.L., Larson, M.A., Mack, D.R., Weber, A., 2001. Elevated extrahepatic expression and secretion of mammary-associated serum amyloid A 3 (M-SAA3) into colostrum. Vet. Immunol. Immunopathol. 83, 203–211. Morein, B., Abusurga, I., Blomqvist, G., 2002. Immunity in neonates. Vet. Immunol. Immunopathol. 87, 207-213. Nagahata, H., Kojima, N., Higashianti, I., Ogawa, H., Noda, H., 1991. Postnatal changes in lymphocyte function of dairy calves. Zentralbl.Veterinarmed. B. 38, 49-54. Orro, T., 2008. Acute phase proteins in dairy calves and reindeer. Changes after birth and in respiratory infections. Department of Production Animal Medicine Faculty of Veterinary Medicine University of Helsinki, Finland. Orro, T., Jacobsen, S., LePage, F.P., Niewold, T., Alasuutari, S., Soveri, T., 2008. Temporal changes in serum concentrations of acute phase proteins in newborn dairy calves. Vet. J. 176, 182-187.

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Orro, T., Nieminen, M., Tamminen, T., Sukura, A., Sankari, S., Soveri, T., 2006. Temporal changes in concentrations of serum amyloid A and haptoglobin in their association with weight gain in neonatal reindeer calves. Comp. Immunol. 29, 79-88. Piccione, G., Bertolucci, C., Giannetto, C., Giudice, E., 2008. Clotting profiles in newborn Maltese kids during the first week of life. J. Vet. Diagn. Invest. 20(1), 114-118. Roberts L.R., Holland L.J., 1993. Coordinate transcriptional regulation of three fibrinogen subunit genes by glucocorticoids in cultured primary liver cells from Xenopus laevis. Endocrinology. 132,2563-2570. Schroedl, W., Jaekel, L., Krueger, M., 2003. C-reactive protein and antibacterial activity in blood plasma of colostrum-fed calves and the effect of lactulose. J. Dairy Sci. 86, 33133320. Stone, R.T., Maurer, R.A., 1987. Cloning and developmental regulation of alpha 1 acid glycoprotein in swine. Developmental. Genetics. 8. 295– 304. Sunderman, F.W., Nomoto, S., 1970. Measurement of human serum ceruloplasmin by its p-phenylenediamine oxidase activity. Clin. Chem. 16, 903-910. Tabrizi, B.A., Hasanpour, A., Mousavi, G., Hajialilou, S., 2011. Evaluation of serum levels of copper in holstein cows and their calves during colostrum nourishing. Middle East J. Sci. Res. 7 (5), 712-714 Tothova, C., Nagy,O., Nagyova, V., Kovac, G., 2015. Changes in the concentration of acute phase proteins in calves during the first month of life. Acta. Vet. Boegrad. 65(2), 260-270. Tothova, C., Nagy, O., Kovac, G., 2014. Acute phase proteins and their use in the diagnosis of diseases in ruminants: a review. Vet Med-Czech. 59(4), 163-180. Tuna, G.E., Ulutas, B., 2015. Acute Phase Proteins as Biomarkers of Diseases. Turkiye Klinikleri J. Vet. Sci. Intern. Med-Special. 1(1), 8-19. Winzler, R.J., 1955. The determination of serum glycoproteins. Methods. Biochem. Anal. 2, 279-314.

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Yamanaka, H., Hagivara, K., Kirisawa, R., Iwai, H., 2003. Transient detection of proinflammatory cytokines in sera of colostrum-fed newborn calves. J. Vet. Med. Sci. 65, 813-816.

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a)

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H a p t o g lo b in ( g /L )

S A A ( µ g /m l)

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Figure 1a-e. Distribution of the concentrations of SAA (a), haptoglobin (b), ceruloplasmin (c), fibrinogen (d) and ASG (e) in goat kids during the first 28 days after birth.

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Table 1. Changes of the mean concentrations of acute phase proteins (SAA, haptoglobin, ceruloplasmin, fibrinogen and ASG) in goat kids during the first 28 days after birth

SAA (µg/ml) Haptoglobin (g/L) Ceruloplasm in (mg/dL) Fibrinogen (g/L) ASG (g/L)

Day 0

Day 1

Day 3

Day 7

Day 14

Day 21

Day 28

p

18,23±6,6

29,11±5,3

40,03±8,3

51,64±17,1

10,81±2,4

5,10±0,8

5,68±0,8

***

0,269±0,0 4

0,292±0,0 4

0,269±0,06

0,324±0,12

0,273±0,05

0,167±0,04

0,223±0,07

N.S.

5,24±0,9

5,86±0,8

7,48±1,2

10,48±2,2

15,08±2,9

9,45±1,8

14,09±1,9

**

3,12±0,3

2,90±0,4

3,16±0,2

3,90±0,3

3,65±0,1

3,26±0,2

4,33±0,2

*

3,92±0,4

3,02±0,3

2,38±0,2

1,64±0,1

1,53±0,1

1,57±0,1

1,67±0,1

***

*: p<0.05; **: p<0.01, ***: p<0,001, N.S. Not Significant

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