EVALUATION OF SERUM ACTIVITY OF CPK AND AST IN REPEAT BREEDER COWS WITH CLINICAL AND SUBCLINICAL ENDOMETRITIS

Dept. of Animal Medicine,

Fac. Vet. Med.AssiutUniversity.

Evaluation of serum activity of CPK and AST in repeat breeder cows with clinical and subclinical endometritis  

(With One Table and 2 Figures)

By

Ghada A. Abou El-Ella; H.A. HUssein*

 and D.R.DERAR*

* Dept. Theriogenology, Faculty of Veterinary Medicine, AssiutUniversity

(Received at 23/12/2008)

تقييم نشاط انزيمى ال  CPK و ASTفى مصل الدم فى حالات الشيوع المتکرر المصاحب للاصابات السريرية والتحت السريرية لإلتهاب الجدار المبطن للرحم فى الابقار

غادة احمد محمد ابو العلا ، حسن عبد الصبور على ، ضرار رفعت ابراهيم

يعتبر إلتهاب الجدار المبطن للرحم احد اهم اسباب حالات الشيوع المتکرر في الابقار کما أن الحالات التحت سريرية تلعب دورا هاما في حدوث مثل هذه الحالات. هذا وقد هدفت هذه الدراسة إلى بحث إمکانية استخدام نشاط انزيمي ال CPK  و AST فى مصل الدم کمؤشر للتشخيص المبکر لحالات إلتهاب الرحم بما يسهم في الحد من حالات الشيوع المتکرر وذلک عن طريق التدخل العلاجي المبکر. هذا وقد أجريت هذه الدراسة على عدد إجمالى 23 بقرة من نوع الابقار الخليطة حيث تم تقسيمهم إلى مجموعتين إعتمادا على الأعراض الأکلينيکية والفحص الأکلينيکي. المجموعة الأولى ضمت الأبقار التي تظهر عليها أعراض أکلينيکية متقدمة وتم تصنيفها کالمجموعة السريرية. المجموعة الثانية ضمت الحيوانات التي تظهر عليها أعراض طفيفة والتي تم تصنيفها کالمجموعة التحت سريرية. هذا وقد تم علاج جميع الحيوانات باستخدام العلاج التقليدي والذي يتکون من المطهرات الموضعية داخل الرحم والمضاد الحيوي الجسدي الشامل. تم في هذه الدراسة قياس نشاط انزيمي ال CPK و AST وکذلک بروتينات مصل الدم في کل من المجموعتين قبل وبعد العلاج وتم مقارنتها مع تلک المستخلصة من مجموعة من الابقار السليمة (عدد 20) والتي تم إستخدامها کمجموعة ضابطة. أظهرت نتائج الدراسة وجود زيادة معنوية ( (P<0.001في نشاط انزيمي ال CPK  و AST فى مصل الدم بنسب تصل إلي 10 و3 أضعاف على التوالي وذلک في المجموعة السريرية قبل العلاج مقارنة بالمجموعة الضابطة بينما کان هناک ارتفاع معنوي             ( (P<0.001في نشاط انزيم ال CPK فى مصل الدم فقط في المجموعة التحت سريرية قبل العلاج وصل إلي 3 أضعاف مقارنة بالمجموعة الضابطة. هذا وقد اوضحت الدراسة أنه بعد العلاج وحدوث الحمل تم تسجيل حدوث إنخفاض معنوي  (  (P<0.001في نشاط انزيم    ال CPK  في کل من المجموعة الأولى والثانية مقارنة بنفس المجموعات قبل العلاج بينما تم تسجيل إنخفاض معنوي  ((P<0.001 في نشاط انزيم ال AST وذلک في المجموعة السريرية فقط مقارنة بنفس المجموعة قبل العلاج. بالاضافة إلي ذلک تم تسجيل وجود ارتفاع معنوي ((P<0.001 في ترکيزات البروتين الکلي والجلوبيولينات وکذلک إنخفاض معنوي ((P<0.001 فى نسبة الألبيومين إلي الجلوبيولينات بالمجموعة السريرية فقط وذلک قبل العلاج مقارنة بالمجموعة الضابطة. إلا أنه بعد العلاج تم تسجيل حدوث إنخفاض معنوي ((P<0.001 بترکيز البروتين الکلي والجلوبيولينات وکذلک ارتفاع معنوي ((P<0.001 في نسبة الألبيومين إلي الجلوبيولين وذلک بالمجموعة السريرية مقارنة بنفس المجموعة قبل العلاج. لقد وجد ان نسبة الخصوبة فى الحيوانات التى تم علاجها بالمضاد الحيوى (السيفوکوينوم) فى الحيوانات ذات الالتهابات السريرية وتحت السريرية هى 60%        (3 من 5 ) و 85.7%  و(6 من 7) على التوالى. ووجد ان نسبة الخصوبة فى الحيوانات التى تم علاجها باللوجول فى الحيوانات ذات الالتهابات السريرية وتحت السريرية هى 50% (2 من 4) و %51.15 على التوالى. ووجد ان الفحص بالموجات فوق الصوتية يمکن ان تساعد على التشخيص للالتهابات الرحمية السريرية ولا يمکن تشخيص تحت السريرية. تشير النتائج الأجمالية للبحث إلي إمکانية إستخدام قياس نشاط انزيمي ال CPK  و AST فى مصل الدم کمؤشرات تفيد في التشخيص المبکر لحالات إلتهاب الجدار المبطن للرحم وخصوصا الحالات التحت السريرية منها وذلک بعد الأخذ في الأعتبار استبعاد حالات إلتهاب وتلف العضلات الهيکلية المصاحبة للرقود لفترات طويلة وکذلک حالات نقص الکالسيوم وأمراض الکبد.

SUMMARY

Endometritis is one of the main causes of repeat breeder syndrome of cows. Sub-clinical endometritis is considered as a major contributor to the syndrome. The goal of the current study was to investigate the possible role of CPK and AST enzymes as early diagnostic parameters for detection of cows with clinical or sub-clinical endometrits and prediction of the fate of the repeat breeder cows. A total of 23 repeat breeder Holstein Friesian cows were subjected to the study. Based on clinical manifestations and the degree of uterine affections, investigated animals were subdivided into 2 groups; the severely affected cows that were assigned as the clinical endometrits group (G1, n=9), and the mildly affected cows that were assigned as the sub-clinical endometritis group (G2, n=14). Five cows from G1 and 7 cows from G2 were treated using local intrauterine Cephquenome 0.5 gm antibiotic and 4 cows from G1 and 7 cow from G2 using Lugol’ Iodine 2% (30 ml) intrauterine.  The activity of CPK and AST enzymes were evaluated in both groups before and after treatment in comparison with healthy control group (n=20). In addition, protein profiles of examined animals were recorded. The results of the current study revealed that, before treatment there were up to 10 folds and 3 folds significant increase (P≤0.001) in the activity of CPK and AST, respectively, in clinical endometritis group as compared to the control healthy group. While there was up to 3 folds significant increase (P≤0.001) only of CPK activity in sub-clinical endometritis group. After treatment and occurrence of conception, significant reduction (P≤0.001) of CPK activity in both clinical and sub-clinical endometritis group and AST activity in clinical endometritis group was recorded as compared to their activities before treatment. In addition, significant elevation (P≤0.001) of total proteins and globulins levels along with significant reduction (P≤0.001) of the A/G ratio were recorded only in clinical endometritis group before treatment as compared to the healthy control group. However, after treatment, significant reduction (P≤0.001) of total proteins and globulins levels and significant elevation (P≤0.001) of A/G ratio were evident as compared to that before treatment. It was found the after using Cephquenome the conception rate in G1 and G2 was 60% (3 from 5 cows) and 85.7%       (6 from 7 cows) respectively. The resulted conception rate after application of Lugol’ Iodine in G1 and G2 was 50% (2 from 4 cows) and 51.14% (4 from 7 cows), respectively. In conclusion, after exclusion of recumbency-based muscular damage, hypocalcemia or liver diseases, the activity of CPK and AST enzymes can be used as a screening parameters for diagnosis of endometrits and early detection of sub-clinical cases.

Key words: CPK, AST, Total protein, endometritis, cow

INTRODUCTION

Repeat breeder (RB) is a substantial problem in cattle breeding leading to large economic losses for the dairy producer due to more inseminations, increased calving interval and increased culling rates (de Kruif, 1977 and Bartlett et al., 1986). Repeat breeding has been defined as failure to conceive from 3 or more regularly spaced services in the absence of detectable abnormalities (Zemjanis, 1980). Although there are many causes and factors of repeat breeder in dairy cow, endometritis has been described as the most common cause of the case (Sagartz and Hardenbrook, 1971; LeBlanc et al., 2002; Gilbert et al., 2005 and Ahmadi and Dehghan, 2007). Affected cows were 30% less likely to become pregnant at first service and 70% more likely to be culled for reproductive failure (LeBlanc et al., 2002).

Endometritis is an inflammation of the endometrial lining of the uterus with or without systemic signs. It is usually associated with postpartum bacterial infection of the uterus (Lewis, 1997 and Bondurant, 1999). Clinically, the challenge is to identify those cows that are truly at risk of impaired fertility, in the hope that treatment can be administered to correct the problem.

The incidence and impact of clinical and subclinical endometritis in the postpartum period has been the objective of several studies (LeBlanc et al., 2002; Kasimanickam et al., 2004; Gilbert et al., 2005 and Williams et al., 2007). Clinical endometritis was characterized by a cervical diameter >7.5 cm and the presence of purulent or mucopurulent vaginal discharge after 20 days postpartum. However, in a study by Hartigan et al. (1972) 50% of the genital tracts obtained from an abattoir showed histological evidence of endometritis, yet only 12.5% showed gross lesions. Hence, it is likely that subclinical endometritis is a major contributor to the repeat breeder syndrome of bovine subfertility (Kasimanickam et al., 2004).

Veterinarians must identify and treat uterine diseases efficiently in order to limit their negative effects on fertility. Different treatment regimes, including intrauterine or parenteral antibiotics (e.g. penicillin, dihydrostreptomycin, tetracycline, and cephapirin) and PGF2 have been used to treat postpartum uterine disease (McDougall, 2001 and Ahmadi and Dehghan, 2007). However, it is difficult to determine the true treatment effect on reproductive performance when diagnostic criteria for uterine disease are not validated.

The most common means of diagnosis of endometritis is transrectal palpation of the uterus. However, this method is subjective and often fails to detect subclinical cases (LeBlanc et al., 2002). Therefore, recent studies have focused on a more sophisticated diagnostic tool of endometrial alterations beyond clinical signs of endometritis (Smith et al., 1998 and Sattler and Furll, 2004).

Serum level of some enzymes as creatine phosphokinase (CPK) and aspartate aminotransferase (AST) in relation to endometritis has been subjected to a number of studies. The CK is a muscule-specific enzyme that plays an important role in the energy transfer. It catalyzes the conversion of creatine to creatine phosphate with the conversion of ATP to ADP and release of energy (Galitzer and Oehme, 1985). There are relatively few studies about CK in cows. The CK activity is especially high in tissues with high-energy transfer such as muscle and brain. In the inner organs, there are also high activities of CK, with the CK activities in the uterine tissue being the third highest after skeletal and cardiac muscles (Sattler and Furll, 2004). In some studies, significant CK activity was reported in the serum of pregnant women and guinea pigs before and after delivery. This was explained by the higher metabolic demand in late pregnancy and the mechanical and metabolic stress of the uterine tissue (Clark et al., 1994 and Abramov et al., 1996). Hormonal stimulation of the smooth muscles of bovine uterus had resulted in significant elevation of the CK serum activity (Iyengar et al., 1980).

The aspartate amino transferase (AST) is considered a muscle and liver-specific enzyme in cattle. The highest AST activities were recorded in case of damage of liver followed by skeletal muscles and heart muscles (Medway et al., 1974). However, AST is not liver-specific enzyme as it found in most tissues. Previous studies had recorded significant AST activity in many clinical cases that involve the bovine uterine tissues as puerperal disorders in dairy cows, uterine torsion and endometritis (El-Din Zain et al., 1997, Oliveira et al., 1998 and Amer et al., 2008). It was found that cows with highly abnormal puerperium 3 weeks postpartum had higher AST activities than healthy cows at the same postpartum period (Oliveira et al., 1998).

Obviously, there is an apparent connection between the activity of CPK and AST enzymes and uterine affections. Therefore, the current study aimed to evaluate the variation of CPK and AST activities in relation to clinical and sub-clinical endometritis. Moreover, the influence of conventional treatment of endometritis with the subsequent conception on the activity of CPK and AST activities was also investigated. The ultimate goal is to assess the possibility of using these enzymes as sensitive parameter for evaluation of the status of uterine wall and the extent of its affection.

MATERIALS and METHODS

Animals: The current study was conducted between December 2007 and July 2008 on suspected repeat breeder mixed breed cows that were presented to the Veterinary Clinic, Department of Theriogenology, faculty of Veterinary Medicine, AssiutUniversity. All presented cows failed to conceive and came into heat regularly every 20-21 day for at least 3 successive times. Thorough clinical examination of tested cows revealed no any visible abnormal clinical manifestations related to liver disease.

Clinical examination: All suspected cows were subjected to thorough clinical examination. During which, suspected cows were first inspected for the presence offresh discharge on the vulva, perineum, or tail. If dischargewas not visible externally, cows were examined vaginoscopically. The vaginoscope was inserted into the vagina up to the levelof the external os of the cervix. Inspection of the cervix andvagina was performed with illumination from a penlight. The nature of dischargewas recorded (clear mucus with or without flecks of pus; muco-purulent or purulent discharge). Following inspection and vaginoscopy, transrectal palpationof the reproductive tract was performed. Moreover, all suspected cows were ultrasonographically examined by using a 6/8 MHz linear array transducer connected to B-mode ultrasound scanner (100 LC, Pie Medical, Masstricht, Netherland) for assessment of uterine echogenicity, the uterine thickness and uterine fluid.

Study design: Animals with pyometra at admission(uterine horn distended with fluid in the presence of a CL,diagnosed by palpation) or those with adhesions involving the internalgenitalia were all excluded from the study. For the current study, 23 cows with endometritis were selected and sub-divided into 2 group based on the clinical findings according to Kasimanickam et al. (2006). Group 1 (G1, n=9 cows), which included severely affected cows and with external and/or internal mucopurulent uterine discharge, thick uterine wall and enlarged cervix (<7 cm diameter). Group 2 (G2, n=14 cows), which included mildly affected cows. By examination of their genitalia revealed the absence of external discharge, normal uterine wall, normal cervix diameter (>5 cm). In addition, 20 mixed breed cows were selected from local private farms of surrounding villages and subjected to clinical examination and proved healthy and free from endometritis and were used as a control group.

Treatment of affected cows: The suspected clinically and sub-clinically affected cows were subjected to one of intrauterine infusion of the following treatments: 1) Cephquenome 0.5 gm antibiotic (Cobactan®, Hoeschst Russel Vet., Turkey). The treatment was applied for 5 cows from G1 and 7 cows from G2. 2). For 4 cows from G1 and 7 cows from G2 intrauterine infusion using 30 ml of Lugol’s Iodine 2% was used (prepared by dissolving 40gm potassium iodide in 1000 distilled water then dissolved 20 gm inorganic iodine). Treatments were repeated in some cases twice or three times at one week interval. The control healthy animals were left without treatment. Post treatment; all cows were observed for heat and were artificially inseminated by experienced inseminators. Pregnancy was diagnosed using trans-rectal Ultrasonography (US) between 27 and 34 days and by rectal palpation between 40 and 90 days post-insemination for all treated and control cows.

Biochemical analysis: Blood samples without anti-coagulant were collected from studied cows for assessment of the serum activity of CPK (U/l) and AST (U/l) enzyme as well as total proteins (g/l), albumin (g/l), globulins (g/l) and A/G ratio in both clinical and sub-clinical endometritis groups (G1, G2) before treatment as comparison to that of healthy control group.In addition, blood samples were collected from conceived cows of G1 and G2 after treatment for assessment of the same parameters in comparison to that before treatment.  The serum activity of CPK and AST enzymes were measured by kinetic method using standard testkits supplied by STANBIO Laboratory, Berne, TX. The serum levels of total proteins and albumin were determined colorometerically using commercial testkits supplied by QUIMICA CLINICA APLICADA S. A. (QCA), Amposta, Spain,  while globulins level were calculated as the difference between total proteins and albumin (Latimer et al., 2003). All measurements were carried out using spectro UV-Vis RS digital spectrophotometer UV 2500, Labo Med, Inc., CA, USA.

Statistical analysis: The findings were expressed as the means ± SD. Statistical analyses were performed by one-way ANOVA using the Statistical Package for the Social Sciences for Windows (SPSS, version 10.0, Chicago, IL, USA).

RESULTS

Biochemical analysis findings

A) Enzymatic activity of serum CPK and AST: The results of the current study revealed that, before treatment there were up to 10 folds and 3 folds significant increase (P≤0.001) in the activity of serum CPK and AST, respectively, in clinical endometritis group (G1) as compared to the control healthy group (Table 1, Figure 1). On the other hand, regarding the sub-clinical endometritis group (G2), up to 3 folds significant increase (P≤0.001) of CPK activity in sub-clinical endometritis group was recorded as compared to control healthy group. While non-significant change of the AST activity was recorded (Table 1, Figure 2).

After treatment and occurrence of conception, biochemical analysis revealed significant reduction (P≤0.001) of CPK activity in both clinical and sub-clinical endometritis group and AST activity in clinical endometritis group as compared to their activities before treatment (Table 1, Figure 2).

B) Proteingram: Significant elevations (P≤0.001) of serum total proteins and globulins concentration along with significant reduction (P≤0.001) of the A/G ratio were recorded in the clinical endometritis group (G1) before treatment as compared to the healthy control group (Table 1, Figure 1). However, after treatment, significant reduction (P≤0.001) of total proteins and globulins and significant elevation (P≤0.001) of A/G ratio were evidence as compared to that before treatment (Table 1, Figure 2). On the other hand, and regarding the sub-clinical endometritis group (G2), non-significant or hardly significant (P≤0.05) changes were recorded in the concentrations of serum total proteins, albumin, globulins and A/G ratio in this group before treatment as compared to control healthy group (Table 1, Figure 1).

C) Conception rate: It was found that after using Cephquenome the conception rate in G1 and G2 was 60% (3 from 5 cows) and 85.7% (6 from 7 cows) respectively. The resulted conception rate after application of Lugol’ Iodine in G1 and G2 was 50% (2 from 4 cows) and 51.14% (4 from 7 cows), respectively. In control, all animals were conceived.

D) Ultrasonographic examination: Ultrasonographic diagnosis of endometritis using echogenicity of the sonographic image was useful for the G1 animals but had no diagnostic value for G2 cows.  The image appeared hyperechogenic with hyperechogenic flakes in the endometrium. The endometrial thickness in G1 was 12.8 ± 0.8 mm and in G2 was 6.7 ± 0.9 mm and the difference was highly significant. (P≤0.001). There were no significant differences between the endometrial thickness in G2 and control group (6.7 ± 0.9 vs. 6.2 ± 1.1 mm) (Figure2). Five cows in G1 showed endometrial fluid with hyperechogenic flakes, the diameter of the uterine lumen was 3.6 ± 0.8 mm and it was recorded no endometrial fluid inside the uterus in G2 and control animals except if the cow was examined during estrus period (2 cows).

          Table 1: Mean and SD of biochemical parameters in clinical and sub-clinical endometritis groups before treatment as compared to healthy control and same groups after treatment.

                        ^*** (P≤0.001), ^* (P≤0.05).

                        ^a Significant changes in studied parameters in investigated group as compared to that of control group.

                        ^b Significant changes in investigated group after treatment as compared to that before treatment.