Creatine kinase and C reactive protein as an indicator for tissue damage in the retained placenta in cows

Introduction

C reactive protein (CRP) is a ring shape protein that synthesis and release from the liver with fibrinogen in response to the production of interleukin six released from stimulated macrophages, T lymphocytes and adipocytes by dying or dead cell due to a wide range of inflammatory process either acute or even chronic reaction, also due to rheumatic arthritis, pathogenic microorganisms, lethal or sublethal cellular injury (1). CRP is a member of patterns recognition receptor (PRR) system that responsible for the detection of necrotic, apoptotic cells as well as pathogenic microorganisms (especially bacterial pathogens), in which CRP where bind to lysophosphatidylcholine (lysolecithins) on the cell membrane of dead and dying cells, this lysolecithins were synthesis by hydrolysis of lipids present on the cell membrane by the action of phospholipase (especially phospholipase A2 enzyme) that released from affected cells, these complex of CRP with lysolecithins will cause activation 400 kDa complement compound known as C1q that lead to initiation and ignition of the complement system to promote and activate phagocytosis conducted by macrophages to necrotic and apoptotic cells (2). Creatine kinase (CK) is a crystal shape enzyme synthesis in a wide range of cells especially those of high consuming ATP as a source for energy in muscle, brain, retina, ear, spermatogenesis process and graved placenta (3). CK function is to catalyzes a reversible conversion of creatine to phosphocreatine and ADP using ATP as a source of energy in response to high energy-consuming in situ within cells these can be attributed to fast intracellular transportation to phosphocreatine as a source of energy in which phosphocreatine can react with ADP to produce ATP and creatine in the presence of CK enzyme for fast intracellular energy transportation, also, CK has main three subtypes, brain CK (CKb), muscle CK (CKm) and mitochondrial CK (CKmt) (4). CK concentration increases during cellular damage occur in tissue rich with CK especially muscle as in cases of myocardial infarction, rhabdomyolysis and myositis, in which destruction of these cells for any cellular injuries lead to releasing of CK into the bloodstream and increase its level in blood serum (5). Retention of fetal membranes in cows occurs when the uterus fails to expel the fetus covering membranes after one day of calving, which usually occurs within 4-6 hours after parturition. The occurrence of such case in cows could be affected by many factors; mainly types of parturition (normal or dystocia), sex and size of the fetus, twin’s deliveries, number of previous parturitions, abortion and nutritional status of cows (6). Naturally, the retained placenta will be expelled with four to six hours after parturition (7).

The objective of this study was to investigate the serum concentration of CRP and CK in cows that suffered from retention of the placenta as an indicator of tissue damage and the relationship between CRP and CK with the type of parturition process, sex of the fetus, previous calve delivery and days required to expel the placenta.

Materials and methods

Animals and blood samples

A total of sixty-three cows aged from two to seven years, suffering from retention of placenta. Another ten cows (control group) with normal parturition and expulsion of the placenta within three to six hours were also used in the current study during the period from December 1, 2018, till September 1, 2019. Blood samples were collected from jugular veins (10 ml) using a round gel tube with clot activator. The blood samples were left for 30 minutes to allow the blood clot to be formed and to separate serum, later blood clot removed by centrifuge at 2500 rpm for 20 minutes. Using Pasteur pipette, the serum was collected, transported to Eppendorf tubes and stored at - 20 ºC in the freezer.

The serum concentration of CRP and CK in cows

The concentrations of CRP and CK in blood's serum were estimated by direct ELIZA kits obtained from Monobind Inc, USA. Estimation of the CRP concentration (µg/ml) was measured following methods described by Kimberly et al. (8). In contrast, the concentration of CK (ng/ml) was determined by using the procedure described by Panteghini et al. (9).

Statistical analysis

The mean concentration of CRP and CK were statistically analyzed using SPSS software version 19.0. A one-way analysis of variance was used to explain the signification differences according to experiment design. Duncan's test used as a post hock test to identify the significant differences in serum concentration of CRP and CK in relation to previous parturition and time to retained placenta at P

Results

Type of parturition

Significant differences were detected for CRP and CK serum levels between normal parturition and retained placenta. CRP and CK levels were higher in cases of parturition associated with the retained placenta in comparison to those of normal parturition in cows (Table 1).

Table 1: Effect of parturition type on serum concentration of CRP and CK

* there are significant differences within the groups in the same parameter at P<0.05.

Gender of fetus

The result represented in table 2 showed that there were no significant differences in serum concentrations of CRP and CK in cows whatever the born calf sex was (male or female). Still, the serum concentrations of CRP and CK in cows that have male or female calves with retained placenta were significantly higher concentration in comparison with cows that have male or female calves without retention of placenta.

Previous parturitions

The result present in table 3 showed that the serum concentration of CRP and CK did not significantly differ in relation to previous parturition status of cows with retained placenta.

Time of retained placenta

The result of table 4 showed that the serum concentration of CRP and CK were increased significantly when there was an increase in time that required till placenta expel, in which the highest serum concentration of CRP and CK were recorded in cows with the retained placenta after four days. The serum concentration of CRP and CK were decreased significantly when the time to expel placenta has been reduced.

Table 2: Effect of fetal sex on serum concentration of CRP and CK

* there is a significant difference within the groups in same-sex at P<0.05.