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Effect of Vitamin E and selenium supplementation on concentrations of plasma cortisol and erythrocyte lipid peroxides and the incidence of retained fetal membranes in crossbred dairy cattle
Theriogenology 64 (2005) 1273–1286 www.journals.elsevierhealth.com/periodicals/the
Effect of Vitamin E and selenium supplementation on concentrations of plasma cortisol and erythrocyte lipid peroxides and the incidence of retained fetal membranes in crossbred dairy cattle§ Sandeep Gupta a,b,*, Harendra Kumar Gupta a, Jyoti Soni b a
Animal Reproduction Division, Indian Veterinary Research Institute, Izatnagar 243122, Bareilly, Uttar Pradesh, India b Faculty of Veterinary Medicine, University College Dublin, Ireland Received 21 July 2004; accepted 14 November 2004
Abstract The objectives were to: (i) determine the effect of prepartum supplementation of Vitamin E (Vit E) and selenium (Se) on plasma cortisol, erythrocyte peroxidation and the incidence of retained fetal membranes (RFM); (ii) estimate myeloperoxidase (MPO), lysozyme, elastase, and acid phosphatase (ACP) enzyme activities in the cotyledons of cows with or without RFM; and (iii) determine the molecular weight (SDS-PAGE) of proteins present in the cotyledons of cows with or without RFM. Fifty dairy (Friesian Sahiwal) cows were equally allocated to one of two treatments, given as an im injection 3 week before calving: 1100 IU of DL a-tocopherol acetate (Vit E) and 30 mg of sodium selenite (Se), or saline (control). Concentrations of plasma cortisol (20 cows) were determined on days 21, 7, 3, 2, 1, and 0 prepartum, and erythrocyte lipid peroxide (all cows) was determined on days 21 and 7 prepartum. Treatment with Vit E and Se did not affect (P = 0.23) the incidence of RFM (12% versus 0%, respectively) but decreased (P < 0.05) erythrocyte lipid peroxide concentrations on day 7 prepartum compared with day 21 prepartum. Plasma cortisol concentration increased (P < 0.05) from day 21 prepartum to the day of parturition in Vit E + Se and control cows. However, on day 0, §
The work was performed at the Animal Reproduction Division, Division of Biochemistry and Food Science, Indian Veterinary Research Institute, Izatnagar 243122, Bareilly, Uttar Pradesh, India and the Military Dairy Farm, Uttar Pradesh, India. * Corresponding author. Present address: Department of Animal Husbandry and Production, Faculty of Veterinary Medicine, University College Dublin, Ireland. Tel.: +353 1 7166231; fax: +353 1 7166253. E-mail address: [email protected] (S. Gupta). 0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.03.008
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plasma cortisol concentrations were lower (P < 0.05) in cows given Vit E + Se than in control cows (with or without RFM). To investigate enzyme activity and peptides in cotyledons, cotyledons were collected (from cows that were not part of the principal experiment), homogenised with PBS, and the supernatant used for the estimation of cationic peptides. Cotyledons of cows with RFM (n = 8) had lower (P < 0.01) MPO and greater (P < 0.05) lysozyme and ACP enzyme activities than those from non-RFM cows (n = 6). A band at < 10 kDa in the SDS-PAGE indicated the presence of cationic peptides. In conclusion, a single treatment of Vit E and Se at 3-week prepartum reduced concentrations of plasma cortisol and erythrocyte peroxide. Altered enzyme activities in the fetal membranes indicated the involvement of leukocytes and trauma at the fetomaternal junction and warrant further investigation. # 2005 Elsevier Inc. All rights reserved. Keywords: Cow; Retained fetal membranes; Cortisol; Placentomes; Oxidative stress
1. Introduction Retention of the fetal membranes (RFM) is a common, albeit poorly understood postpartum disorder that has a detrimental effect on reproductive efficiency and profitability of milk production in dairy cattle [1–3]. The release of fetal membranes postpartum is a physiological process, involving loss of fetomaternal adherence, combined with contraction of uterine musculature [4]. RFM has a multifactorial etiology; it is either due to an abnormal physiological process for the release of fetal membranes or pathological factors that affect the loosening mechanism of the placentomes [5]. The reduced incidence of RFM when Vitamin E (Vit E) and selenium (Se) were administered alone or in combination [6,7] suggested a role of oxidative stress in the etiology of RFM. Vit E prevents oxidative damage to sensitive membrane lipids by destroying hydroperoxide formation [8], acting in conjunction with Se, and protects cellular membranes and lipid containing organelles from peroxidative damage by inhibition and destruction of endogenous peroxides, thus maintaining membrane integrity and reducing oxidative stress [9]. Placental tissues of cows with undisturbed expulsion strongly attract leukocytes [10,11]. Reduced chemotactic activity of leukocytes against bovine cotyledon tissue and altered neutrophil function in the blood of cows has been associated with RFM [12]. Hoedemaker et al. [12] and Tian-Quancai et al. [13] reported decreased myeloperoxidase (MPO) activity in circulating neutrophils of cows with RFM. Furthermore, higher blood lysozyme and acid phosphatase (ACP) activities were reported for cows with acute inflammatory responses to injury [14]. The inflammatory area had increased infiltration of neutrophils [14] that contain antibiotic proteins [12]. The increased cortisol concentrations in cows that developed RFM [15] has immunosuppressive and inhibitory effects on leukocyte migratory activity [16,17]. Vit E and Se have been reported to improve stress-associated immunity in cattle [18], likely through immunomodulatory effects on neutrophil function [19]. The objectives of this study were to: (i) determine the effect of prepartum supplementation of Vit E and Se on plasma cortisol, erythrocyte peroxidation and the incidence of RFM; (ii) estimate the MPO, lysozyme, elastase and ACP enzyme activities in the cotyledons of cows with or without RFM; and (iii) determine the molecular weight of proteins present in cotyledons of cows with or without RFM.
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2. Materials and methods 2.1. Treatments All animal procedures and protocols were conducted under the approval of the Indian Veterinary Research Institute Ethics Committee. Fifty pregnant crossbred (HolsteinFriesian Sahiwal) cows (sixth parity) in the last quarter of their gestation, were randomly selected from a herd of 170 cows. Cows were equally assigned (using a completely randomised design) to receive one of two treatments as a single im injection (20 mL) 3 week prepartum: 1100 IU of DL a-tocopherol acetate (Vit E) and 30 mg of sodium selenite (Se) in a combined solution (E care Se; Vetcare, Bangalore, India); or saline (control). 2.2. Animal housing and management Before assignment to treatment, cows were maintained on the Military Dairy Farm, Bareilly, Utter Pradesh. Each cow was given access to grass fodder and supplemented with barley plus soybean mix (mean values on DM basis, crude protein = 146 g/kg, crude fibre = 41.9 g/kg, acid hydrolysable oil = 39 g/kg, ash = 58.6 g/kg) with free access to water. Two months before the expected calving date, cows were separated into a free stall barn and those showing signs of approaching parturition were kept in calving boxes with straw bedding, where they were retained for 1 week postpartum. Expected calving date was predicted from the date of AI to be within 1 day of actual calving. Transrectal palpation was performed approximately 30 days before calving to confirm the presence of a viable calf. Cows were free of clinical disease. Cows that calved normally and failed to expel fetal membranes spontaneously within 12 h were considered a clinical case of RFM, as previously defined [20,21]. Conversely, the expulsion of fetal membranes in cows before 12 h was considered clinically normal. Cows were observed every 5 h to determine the time of calving and every 2 h for the expulsion of fetal membranes. This study did not involve assessment of calf health or fertility and health status of cows postpartum. 2.3. Plasma cortisol and erythrocyte lipid peroxide concentrations Blood samples were collected (in heparinised glass tubes) by jugular venipuncture on day 21 (before treatment) and on days 7, 3, 2, 1, and 0 prepartum for plasma cortisol concentrations (n = 20), and on days 21 and 7 for erythrocyte lipid peroxide concentrations (n = 50). The tubes were centrifuged (1600 g for 15 min at 8 8C) and plasma was collected and stored at 20 8C until assayed for cortisol. Commercially available RIA kits were used to determine the plasma cortisol concentrations (Corti-cote; ICN Pharmaceuticals, Orangeburg, NY, USA) within 4 week of sample collection. The concentration of lipid peroxide was estimated in the erythrocyte hemolysate using the method of Placer et al. [22]. The erythrocyte hemolysate from the tubes containing whole blood samples was prepared according to the method of Cohn et al. [23]. Briefly, immediately after collection, blood samples were centrifuged at 1600 g for 10 min. The plasma and buffy coats were removed by aspiration. The sediment containing blood cells was washed three times by resuspending in isotonic saline (0.89 wt.%/vol.% NaCl), followed by re-centrifugation and
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removal of the supernatant fluid. The cells were lysed in nine volumes of ice-cold distilled water (DW) to prepare a 10% erythrocyte hemolysate. An aliquot (0.2 mL) of this hemolysate was added to 1.3 mL of Tris–KCl buffer (0.2 mol Tris in 0.16 mol KCl at pH 7.4), after which 1.5 mL of 0.08% thiobarbituric acid (TBA) reagent (2-TBA in a small amount of NaOH, and neutralised with 7% perchloric acid) was added. The mixture was heated in a boiling water bath for 10 min (using a marble as a condenser). After cooling, 3.0 mL pyridine:n-butanol (3:1, v:v) and 1.0 mL of 1N NaOH (40 g of NaOH pellets in 1 L of DW) was added and mixed by shaking. A blank solution was prepared with 0.2 mLdistilled water in lieu of the erythrocyte hemolysate. The photometric measurement was taken at lmax 548 against the blank, using a spectrophotometer (Electronic Corporation of India Limited, Hydrebad, India). Hemoglobin (Hb) in 10% erythrocyte hemolysate was estimated colorimetrically by the cyanomethemoglobin method [24] expressed in mg/mL of hemolysate; this was converted to nanomoles malonyldialdehyde (nmol MDA)/mL of erythrocyte hemolysate hemoglobin by using 1.56 105 L/mmol/cm as the extinction coefficient [25]. 2.4. Cotyledon tissue To investigate enzyme activity and peptides in cotyledons (without confounding by Vit E + Se treatment or interfering with the primary experiment), 14 additional cows were used. Cotyledonary tissues from cows, with (n = 6) or without (n = 8) RFM were collected immediately postpartum into cold PBS solution. The operator’s arm was covered with sleeve (lubricated within paraffin) that was introduced into the uterus of a cow with RFM. Gentle digital pressure was applied between the maternal caruncle and fetal cotyledon to detach and remove the cotyledon. Cotyledons from non-RFM cows were collected directly from the vagina as the placenta was delivered. Immediately after collection, tissues were transported to the laboratory in an icebox containing PBS. The membranous portions of the cotyledonary tissues were removed with a scissors and forceps. The tissues were washed thoroughly with cold PBS, dried with filter paper and weighed. One part of the tissue was sonicated with two parts of cold PBS and treated with 0.1% triton X-100 to create a homogenate. The homogenate was centrifuged for 20 min at 1200 g and the supernatant of the cotyledon sample was stored in 5 mL aliquots for estimation of cationic peptides. All procedures for homogenate preparation were performed in a cold room at 4 8C. 2.4.1. Enzyme assays in cotyledon tissues Myeloperoxidase (MPO) activity was assayed in the supernatant sample using odianisidine as an electron donor (substrate solution) [26]; the enzyme concentration was calculated by using the molar extinction coefficient for oxidised o-dianisidine. The turbidimetric method [27], using egg white lysozyme for the standard curve, was used to measure lysozyme in the supernatant of the cotyledon samples [28]. Proteolytic activity of elastase was measured with azo-casein as substrate [29]. The total ACP and tartrate resistant ACP activity was assayed [30] by the hydrolysis of p-nitrophenyl phosphate and by measuring the absorbance (at 410 nm) of p-nitrophenyl that was liberated.
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2.4.2. Estimation of total protein content Total protein in the supernatant of the cotyledon samples was estimated using bovine serum albumin as standard, adopting the method described by Lowry et al. [32]. 2.4.3. SDS-PAGE analysis of cotyledon tissue The supernatant of the cotyledon samples from non-RFM and RFM cows were processed by SDS-PAGE using a 5–15% linear gradient gel in a discontinuous buffer system, as described by Laemmli [31]. The protein molecular weight markers used were phosphorylase-b (97.4 kDa), bovine serum albumin (68 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), soyabean trypsin inhibitor (20 kDa) and lysozyme (14.3 kDa). The molecular weights of the sample protein bands were estimated using the references points from the molecular weight markers. 2.5. Statistical analyses Statistical analyses were performed by ANOVA using the General Linear Model (GLM) procedure of the Statistical Analysis System version 8.2 (SAS Institute Inc.; Cary, NC, USA). Probability plots (Shapiro-Wilk test, P < 0.005) using the UNIVARIATE procedure were used to determine the normality of the data [33]. Data for the Vit E and Se response on the occurrence of RFM, erythrocyte lipid peroxidation and enzyme activity were analysed with the student’s t-test. Plasma cortisol concentrations were analysed by repeated measures ANOVA [33]; the model included the effect of group (control with out RFM, control with RFM and Vit E + Se), days and their interaction. If there was a significant main effect or interaction, differences were located using the probability of differences (PDIFF) procedure of SAS on the least square means. Differences in the incidence of RFM were compared with a Fisher’s exact test.
3. Results 3.1. Prepartum administration of Vit E and Se response The incidence of RFM was not different (0% versus 12%) in cows given Vit E + Se and control cows, respectively (P = 0.23). 3.2. Plasma cortisol For plasma cortisol concentrations (Table 1), there were main effects of group, time and their interaction (P < 0.001 for each). There were no significant differences among groups of plasma cortisol concentrations on day 21 prepartum (before treatment). From day 7 prepartum to the day of parturition, plasma cortisol concentrations were greater (P < 0.05) in control cows than those given Vit E + Se. Comparing day of calving with day 21 prepartum, mean plasma cortisol concentrations increased four-fold in cows treated with Vit E and Se (P = 0.05), six-fold in control cows that did not develop RFM (P = 0.01) and 11-fold in control cows that developed RFM (P = 0.001). From days 21 to 7 prepartum,
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Table 1 Mean (S.E.M.) plasma cortisol concentrations (ng/mL) at 21, 7, 3, 2, 1 and 0 day prepartum in cows with or without retained fetal membranes (RFM) Prepartum interval (d)
21 7 3 2 1 0
Treated
Control
Non-RFM (n = 10)
Non-RFM (n = 7)
RFM (n = 3)
3.0 0.23 2.9 0.49 2.5 0.33 4.2 1.36 7.1 1.22 12.5 2.36
4.5 0.84 6.2 1.01 8.1 2.10 7.3 1.74 12.2 4.12 25.6 6.26
5.5 0.89 ax 6.3 1.081 bx 10.4 4.07 by 29.7 6.49 by 40.7 5.34 cy 60.5 4.33 cz
ax ax ax ax ax ay
ax ax by ax by bz
Treated cows were given 1100 IU Vitamin E (DL a-tocopherol acetate) and 30 mg selenium (sodium selenite) 3 week before calving, whereas control cows were given saline. There were effects of group, day and their interaction (P < 0.001 for each). Rows and columns with different letters differ (P < 0.05).
plasma cortisol concentrations were not significantly different between control cows, with or without RFM. In control cows with RFM, plasma cortisol concentrations were lower (P < 0.05) on day 7 prepartum compared with days 3, 2, and 1 prepartum, and the day of parturition. In control cows without RFM, plasma cortisol concentrations were lower (P = 0.01) on day 7 compared with days 3, and 1 prepartum, and the day of parturition. 3.3. Erythrocyte peroxidation The mean levels of MDA in the erythrocyte hemolysate, on day 21 prepartum for the Vit E + Se were not different (P > 0.05) from the control cows (Table 2). However, on the day of calving there was lower (P < 0.05) peroxide formation in the erythrocyte hemolysate of cows given Vit E and Se than control cows (with or without RFM). The administration of Vit E and Se decreased (P < 0.05) peroxide formation in the erythrocyte hemolysate from day 21 prepartum to the day of parturition. However, there was an increase (P < 0.05) in the erythrocyte hemolysate peroxide formation from day 21 prepartum to the day of calving in the control cows that developed RFM. No differences (P > 0.05) in erythrocyte peroxide formation were observed in control cows that did not develop RFM, from day 21 prepartum to the day of calving.
Table 2 Mean (S.E.M.) lipid peroxide levels (nanomoles MDA/mg of hemoglobin) in erythrocytes at 21 and 7 day prepartum in cows with or without retained fetal membranes (RFM) Prepartum interval (d)
21 7
Treated
Control
Non-RFM (n = 25)
Non-RFM (n = 22)
RFM (n = 3)
2.691 0.064 x 2.457 0.054 ay
2.786 0.067 2.762 0.080 b
2.460 0.072 x 3.677 0.162 cy
Treated cows were given 1100 IU Vitamin E (DL a-tocopherol acetate) and 30 mg selenium (sodium selenite) 3 week before calving, whereas control cows were given saline. Rows and columns with different letters differ (P < 0.05).
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Table 3 Mean S.E.M. enzyme activity in the cotyledons of crossbred dairy cows with or without retained fetal membranes (RFM) Enzymes
Myeloperoxidase, mU/mg protein Lysozyme, KU/mg protein Elastase, U/mg protein Acid phosphatase, U/mg protein Tartate resistant acid phosphatase, U/mg protein Tartrate inhibited acid phosphatase, U/mg protein * **
Enzyme activity Non-RFM (n = 8)
RFM (n = 6)
1.19 0.080 0.06 0.004 1.53 0.096 0.09 0.008 0.06 0.012 0.03 0.005
0.74 0.060** 0.42 0.123** 1.36 0.123 0.12 0.010* 0.07 0.004 0.06 0.008*
Difference between non-RFM and RFM (P < 0.05). Difference between non-RFM and RFM (P < 0.01).
3.4. Cationic peptide enzyme activity in the cotyledon tissue Decreased (P = 0.001) MPO enzyme activity was observed in the cotyledon tissue of cows that developed RFM compared with clinically normal cows (Table 3). Higher (P < 0.05) lysozyme, ACP and tartrate inhibited ACP enzyme activities were observed in cotyledons of the cows that developed RFM, compared with those that did not develop RFM. No differences (P > 0.05) were observed in the enzyme activities of the elastase and tartrate resistant ACP in cows that developed RFM compared with the nonRFM cows. 3.5. SDS-PAGE and cotyledon tissue A sample of the electrophoretic pattern of cotyledons is presented in Fig. 1. There were 20 protein bands in the approximate range of 100–10 kDa. The overall elecotrophoretic pattern of the cotyledon tissue in RFM and non-RFM samples indicated minor differences for different protein bands of varied molecular weight. However, a thick band <10 kDa was observed in the cotyledons of both RFM and non-RFM cows.
4. Discussion In this study, administration of Vit E and Se 3 week prepartum did not alter (P = 0.23) the incidence of RFM (12% versus 0%, respectively). This finding was consistent with earlier observations of Segerson et al. [38], Rosa et al. [39] and Hidiroglou et al. [40] who also reported no decrease in the incidence of RFM when cows were supplemented with Vit E and Se approximately 30–20 days before calving. This is in contrast with the Eger et al. [34], Thomas et al. [35], Kim et al. [36] and Ivandija [37], who reported a decreased incidence of RFM following Vit E and Se supplementations in cattle. In the current study, the incidence of RFM in control cows (12%) was very close to the historical retention rate (14%) for this particular herd. Apart from the Vit E + Se injection schedules employed, there were no changes in nutritional or the herd health management programs.
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Fig. 1. Electrophoretic pattern (SDS-PAGE, 15% linear gradient gel) of cotyledons from cows with (Lanes 1 and 2) or without (Lanes 3, 4 and 5) retained fetal membranes. *Protein molecular weight markers (Lane M) in kilodalton (kDa) are: phosphorylase-b, 97.4 kDa; bovine serum albumin, 68.0 kDa; ovalbumin, 43.0 kDa; carbonic anhydrase, 29.0 kDa; soyabean trypsin inhibitor, 20.0 kDa; and lysozyme, 14.3 kDa.
Relationships among dietary antioxidant, oxidative stress and RFM have been investigated in periparturient dairy cows [41]. Although the pathogenesis of RFM due to Vit E and Se deficiency is not clearly understood, the involvement of oxidative stress in the etiology of RFM syndrome is suggested by a reduction in the incidence of RFM when the antioxidants, Vit E and Se are supplemented [14]. Administration of Vit E and Se, increased the levels of a-tocopherol in RBC, neutrophils and plasma, and increased the
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biochemical activity of glutathione peroxidase (GSH-Px) [42]. Therefore, perhaps supplementation of Vit E in concert with Se reduced oxidative stress and associated changes in the fetomaternal junction. Vitamin E and Se have immunomodulatory effects mediated through improved neutrophil function, by enhancing their random migration and chemotactic response [42]. There is evidence that Se deficiency can affect the function of polymorphonuclear neutrophils (PMN), which is associated with physiological changes in GSH-Px levels [43–45]. The absence of leukocytes at the fetomaternal junction leads to a 100% incidence of RFM [8]. In the present study, Vit E and Se treatment might have increased the leukocyte number and leukocyte chemotaxis at the fetomaternal junction and helped in the normal loosening and expulsion of fetal membranes. Intramuscular administration of Vit E increased serum Vit E concentrations for at least 28 days [46], while injection of Se increased concentration of Se in whole blood and serum for 28 days and increased whole blood GSH-Px activity for at least 84 days [47]. Injection of Vit E and Se, 3-week prepartum, increased erythrocyte GSH-Px in dairy cows during the first 12week of lactation [48]. Lipid peroxidation is a biochemical oxidative degradation of unsaturated fatty acids that causes irreversible denaturation of essential proteins. With respect to the widespread distribution of unsaturated fatty acid in the cellular membranes, the peroxidative damage has the potential to affect many cellular functions and interfere with the regulation of several metabolic pathways [14]. Cytotoxic aldehydes (e.g., MDA), which remain after termination of lipid peroxidation, provide the basis for the thiobarbituric acid test for measuring lipid peroxidation and products in body fluid [22]. The increased levels of the lipid peroxide, from 3 to 0 week prepartum, in cows that failed to expel fetal membranes in this study, were consistent with the Brazezinska-Slebodzinska et al. [49]. The administration of Vit E and Se during the prepartum period reduced erythrocyte substances reactive to thiobarbituric acid. This was in close agreement with Miller et al. [41] and BrazezinskaSlebodzinska et al. [49], who reported a decrease in RBC thiobarbituric acid reactive substances from 375 to 275 nmols MDA/g of Hb in cows supplemented with 1000 IU Vit E for 6 week. In the present study, the combined administration of a-tocopherol acetate and sodium selenite at 3-week prepartum might have reduced the level of lipid peroxidation at 7 day prepartum. It is interesting to note that at parturition, when the clinical features of RFM were not exhibited, MDA (an end product of lipid peroxidation) contents in RBC were increased significantly. Perhaps the biochemical changes leading to elevated levels of MDA could be detected much earlier than the clinical manifestation of inflammatory changes in the infected tissue. Therefore, elevated levels of MDA in erythrocyte hemolysate prepartum may be useful to predict RFM. The prepartum increases in plasma cortisol concentrations observed in this study were similar to those reported previously [15,50]. The signal to initiate parturition comes from the fetal hypothalamic-pituitary-adrenal axis; increased fetal cortisol concentrations [51] cross from the fetal to the maternal circulation [52]. Therefore, increased maternal blood cortisol concentrations 7 day prepartum could be due to increased fetal concentrations. The greater concentrations of plasma cortisol at the prepartum period in RFM compared with non-RFM cows in the present study were consistent with Peter and Bosu [15] and Ras et al. [53]. A sharp rise in cortisol in RFM cows is believed to be in response to stress and inflammation in the pregnant uterus, rather than initiation of parturition [53]. The maturation of placentomes is a hormonally controlled process [54]. The loosening process,
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which normally occurs between the fetal villi and the maternal crypts, depends upon histological changes in the opposing surface [5] in the presence of estrogens. However, RFM occurs when hormone concentrations and requisite tissue changes are not favorable [54]. Gunnick [10] and Heuwieser and Grunert [55] found a reduction in chemotactic activity and inhibited migratory ability of leukocytes in cows that went on to develop RFM. Cortisol, which inhibits leukocyte activity [17,56] and impairs neutrophil function in bovines, may have suppressed leukocyte activity and contributed to development of RFM. Further studies are required to confirm blood cortisol patterns in cows with RFM. Supplementing Vit E and Se reduced the level of cortisol in the treated group. It is known that a higher level of cortisol is released in response to a variety of acute stressors in cattle, including pregnancy [57]. Stress in pregnancy also increases the oxidative metabolic reaction [58]. Therefore, prepartum supplementation of Vit E and Se might have reduced the reactive oxygen metabolites and/or free radicals, leading to reductions in oxidative stress and cortisol concentrations. That MPO activity was lower in the cotyledons of the RFM cows was consistent with the data of Hoedemaker et al. [12] and Tian-Quancai et al. [13], who reported a depressed activity of MPO in circulating neutrophils of cows with RFM. The MPO activity in cotyledons indicated the presence of neutrophils at the fetomaternal junction. The higher MPO value in the cotyledons of non-RFM cows suggested a greater influx of neutrophils at the fetomaternal junction, thereby promoting the normal release of fetal membranes. Conversely, the lower value of MPO in the cotyledons of RFM cows indicated a transient impairment of neutrophil number, function and migratory ability to the site of the fetomaternal junction, which may have resulted in RFM. However, it was noticed that an increase in the chemotaxis and number of leukocytes in placentomes appeared to be an important part of the detachment process [55]. When both of these requirements were met, the incidence of RFM was approximately 1.4%. When either one was decreased or absent, the incidence was 6.8–9.6% and when both were absent, the incidence approached 100% [10]. Gunnick [10] and Gilbert et al. [59] reported that the normal physiological expulsion of the placenta requires the presence of neutrophils at the fetomaternal junction. Greater tissue and body fluid lysozyme activity was reported for cattle with acute inflammatory responses to injury [14]. Lysozymes are microbicidal (bactericidal against gram-positive bacteria [61]) and are associated with phagocytes and neutrophils [62]. The higher lysozyme activity in the cotyledons of RFM cows in this study may have been associated with the acute inflammatory response at the fetomaternal junction. Moreover, as the postpartum uterus is more prone to infection, the presence of lysozymes in the cotyledons suggests it may be one of the factors in preventing postparturient uterine infection and thereby promoting uterine involution [60]. Elastin is the primary protein constituent of the elastic fibre, the predominant connective tissue of the lung, blood vessels, and skin [63]. Histologically, cotyledons of RFM and nonRFM cows are congested and contain constricted blood vessels, with thickening of tunica media of arterioles and calcification of connective tissues. More focal areas of collagen were also reported in the cotyledons of RFM cows [64]. The proteolytic enzyme elastase is a component of PMN cells. A possible role of elastase is inhibition of bacterial growth [63]. The lack of difference between groups in elastase activity may have been due to a lower degree of bacterial contamination in the placentomes. However, the presence of this
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enzyme in the cotyledons of RFM and non-RFM cows may indicate its role in tissue injury at the fetomaternal junction and could help in the loosening of fetal membranes. The higher activities of ACP and tartrate-inhibited ACP in the cotyledons of RFM than in non-RFM cows in this study indicated trauma to the placental tissues of cows with RFM. Earlier, Mahfooz et al. [65] and Kudlac et al. [66] reported significantly higher values of ACP in the blood of RFM cows at the prepartum stage. The source of this enzyme in the present study could be the fetal membranes themselves or the lysosomes of the PMN cells at the fetomaternal junction. Thus, an increase in ACP levels in the cotyledons of RFM cows may reflect increased enzymatic activity associated with inflammatory changes in placental tissues. It was reported that excess iron in serum could be linked with increased RFM, through increased saturation of serum iron-binding capacity [7]. Certain enzymes of large families of lysosomal tartrate-resistant ACP, like uteroferrin, are the major protein component of uterine secretion [67]. These play important roles in the transportation of iron from maternal to fetal circulation [68]. The presence of tartrate-resistant ACP in the cotyledons indicated the involvement of iron at the fetomaternal junction. That there was no difference between groups in tartrate-resistant ACP activity may have been due to a lack of iron-induced RFM condition. The SDS patterns of non-RFM and RFM cotyledon extracts revealed the presence of >20 bands of peptides. The protein of these bands possessed molecular weights ranging from <10 kDa to >100 kDa. In a series of studies conducted by Kankofer [69,70] and Kankofer and Guz [71] to investigate (using either Western blotting or SDS-PAGE) the activities of various proteins present in bovine placental tissues with or without RFM, it was suggested that the molecular weight of the protein present in both maternal and fetal placental tissues averaged from <35 kDa to >85 kDa. Although Western blotting and SDS-PAGE are semi-quantitative methods, it was possible to detect differences in staining intensity, suggesting differences in the amount of protein present. In general, intensity was lower in fetal than maternal parts of the placenta and patterns of bands were different with respect to the mode of delivery of placenta [71]. In the present study, the lower band in the <10 kDa zone in the SDS-PAGE represented cationic peptides; these concentrations were very high. It has been suggested that these peptides have antibacterial properties (personal communication with Dr. T. More, IVRI, India) and it is known that some cationic peptides are present in neutrophils [61]. This is a preliminary study to use SDS-PAGE in cotyledon tissue and further investigations to develop and understand the potential role of peptides present in cotyledon tissue are clearly warranted. In conclusion, results of this study indicated that the etiopathogenesis of RFM may involve oxidative stress, as the prophylactic prepartum supplementation of antioxidant, Vitamin E (DL a-tocopherol acetate, 1100 IU) and Se (sodium selenite, 30 mg) by single 20 mL im injection, at 3 week prepartum, decreased erythrocyte lipid peroxide concentrations in dairy cows. Elevated prepartum levels of malonyldialdehyde (indicative of oxidative stress) and cortisol could be reliable predictors of retained fetal membranes. Decreased myeloperoxidase activity in the cotyledons of RFM cows, indicated impairment of neutrophil functions at the fetomaternal union. Higher lysozyme and acid phosphatase activity in the cotyledons of cows with RFM indicated an acute inflammatory response at the fetomaternal union. Finally, the involvement of neutrophils and cationic peptides (with natural antibiotic properties) at the fetomaternal junction warrant further investigation.
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Acknowledgements This work was supported by Indian Council of Agricultural Research Fellowship. The authors wish to acknowledge Captain (Dr.) Rajender Singh of the Military Dairy Farm UP, India for his permission to work on the farm, Vet Care Bangalore for the supply of ‘‘E care Se’’ supplement and Mr. Tyagi of C.B.S.H., G.B.P.U.A.T., Pantnagar for providing facilities for the cortisol assay. The authors thank Dr. M.A. Crowe, University College Dublin, Ireland, Mr. Phil Rogers MRCVS, and Mr. Michael Nolan, Grange Research Centre, Teagasc, Dunsany, Co Meath, Ireland for reviewing the manuscript.
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Effect of Vitamin E and selenium supplementation on concentrations of plasma cortisol and erythrocyte lipid peroxides and the incidence of retained fetal membranes in crossbred dairy cattle§ Sandeep Gupta a,b,*, Harendra Kumar Gupta a, Jyoti Soni b a
Animal Reproduction Division, Indian Veterinary Research Institute, Izatnagar 243122, Bareilly, Uttar Pradesh, India b Faculty of Veterinary Medicine, University College Dublin, Ireland Received 21 July 2004; accepted 14 November 2004
Abstract The objectives were to: (i) determine the effect of prepartum supplementation of Vitamin E (Vit E) and selenium (Se) on plasma cortisol, erythrocyte peroxidation and the incidence of retained fetal membranes (RFM); (ii) estimate myeloperoxidase (MPO), lysozyme, elastase, and acid phosphatase (ACP) enzyme activities in the cotyledons of cows with or without RFM; and (iii) determine the molecular weight (SDS-PAGE) of proteins present in the cotyledons of cows with or without RFM. Fifty dairy (Friesian Sahiwal) cows were equally allocated to one of two treatments, given as an im injection 3 week before calving: 1100 IU of DL a-tocopherol acetate (Vit E) and 30 mg of sodium selenite (Se), or saline (control). Concentrations of plasma cortisol (20 cows) were determined on days 21, 7, 3, 2, 1, and 0 prepartum, and erythrocyte lipid peroxide (all cows) was determined on days 21 and 7 prepartum. Treatment with Vit E and Se did not affect (P = 0.23) the incidence of RFM (12% versus 0%, respectively) but decreased (P < 0.05) erythrocyte lipid peroxide concentrations on day 7 prepartum compared with day 21 prepartum. Plasma cortisol concentration increased (P < 0.05) from day 21 prepartum to the day of parturition in Vit E + Se and control cows. However, on day 0, §
The work was performed at the Animal Reproduction Division, Division of Biochemistry and Food Science, Indian Veterinary Research Institute, Izatnagar 243122, Bareilly, Uttar Pradesh, India and the Military Dairy Farm, Uttar Pradesh, India. * Corresponding author. Present address: Department of Animal Husbandry and Production, Faculty of Veterinary Medicine, University College Dublin, Ireland. Tel.: +353 1 7166231; fax: +353 1 7166253. E-mail address: [email protected] (S. Gupta). 0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.03.008
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plasma cortisol concentrations were lower (P < 0.05) in cows given Vit E + Se than in control cows (with or without RFM). To investigate enzyme activity and peptides in cotyledons, cotyledons were collected (from cows that were not part of the principal experiment), homogenised with PBS, and the supernatant used for the estimation of cationic peptides. Cotyledons of cows with RFM (n = 8) had lower (P < 0.01) MPO and greater (P < 0.05) lysozyme and ACP enzyme activities than those from non-RFM cows (n = 6). A band at < 10 kDa in the SDS-PAGE indicated the presence of cationic peptides. In conclusion, a single treatment of Vit E and Se at 3-week prepartum reduced concentrations of plasma cortisol and erythrocyte peroxide. Altered enzyme activities in the fetal membranes indicated the involvement of leukocytes and trauma at the fetomaternal junction and warrant further investigation. # 2005 Elsevier Inc. All rights reserved. Keywords: Cow; Retained fetal membranes; Cortisol; Placentomes; Oxidative stress
1. Introduction Retention of the fetal membranes (RFM) is a common, albeit poorly understood postpartum disorder that has a detrimental effect on reproductive efficiency and profitability of milk production in dairy cattle [1–3]. The release of fetal membranes postpartum is a physiological process, involving loss of fetomaternal adherence, combined with contraction of uterine musculature [4]. RFM has a multifactorial etiology; it is either due to an abnormal physiological process for the release of fetal membranes or pathological factors that affect the loosening mechanism of the placentomes [5]. The reduced incidence of RFM when Vitamin E (Vit E) and selenium (Se) were administered alone or in combination [6,7] suggested a role of oxidative stress in the etiology of RFM. Vit E prevents oxidative damage to sensitive membrane lipids by destroying hydroperoxide formation [8], acting in conjunction with Se, and protects cellular membranes and lipid containing organelles from peroxidative damage by inhibition and destruction of endogenous peroxides, thus maintaining membrane integrity and reducing oxidative stress [9]. Placental tissues of cows with undisturbed expulsion strongly attract leukocytes [10,11]. Reduced chemotactic activity of leukocytes against bovine cotyledon tissue and altered neutrophil function in the blood of cows has been associated with RFM [12]. Hoedemaker et al. [12] and Tian-Quancai et al. [13] reported decreased myeloperoxidase (MPO) activity in circulating neutrophils of cows with RFM. Furthermore, higher blood lysozyme and acid phosphatase (ACP) activities were reported for cows with acute inflammatory responses to injury [14]. The inflammatory area had increased infiltration of neutrophils [14] that contain antibiotic proteins [12]. The increased cortisol concentrations in cows that developed RFM [15] has immunosuppressive and inhibitory effects on leukocyte migratory activity [16,17]. Vit E and Se have been reported to improve stress-associated immunity in cattle [18], likely through immunomodulatory effects on neutrophil function [19]. The objectives of this study were to: (i) determine the effect of prepartum supplementation of Vit E and Se on plasma cortisol, erythrocyte peroxidation and the incidence of RFM; (ii) estimate the MPO, lysozyme, elastase and ACP enzyme activities in the cotyledons of cows with or without RFM; and (iii) determine the molecular weight of proteins present in cotyledons of cows with or without RFM.
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2. Materials and methods 2.1. Treatments All animal procedures and protocols were conducted under the approval of the Indian Veterinary Research Institute Ethics Committee. Fifty pregnant crossbred (HolsteinFriesian Sahiwal) cows (sixth parity) in the last quarter of their gestation, were randomly selected from a herd of 170 cows. Cows were equally assigned (using a completely randomised design) to receive one of two treatments as a single im injection (20 mL) 3 week prepartum: 1100 IU of DL a-tocopherol acetate (Vit E) and 30 mg of sodium selenite (Se) in a combined solution (E care Se; Vetcare, Bangalore, India); or saline (control). 2.2. Animal housing and management Before assignment to treatment, cows were maintained on the Military Dairy Farm, Bareilly, Utter Pradesh. Each cow was given access to grass fodder and supplemented with barley plus soybean mix (mean values on DM basis, crude protein = 146 g/kg, crude fibre = 41.9 g/kg, acid hydrolysable oil = 39 g/kg, ash = 58.6 g/kg) with free access to water. Two months before the expected calving date, cows were separated into a free stall barn and those showing signs of approaching parturition were kept in calving boxes with straw bedding, where they were retained for 1 week postpartum. Expected calving date was predicted from the date of AI to be within 1 day of actual calving. Transrectal palpation was performed approximately 30 days before calving to confirm the presence of a viable calf. Cows were free of clinical disease. Cows that calved normally and failed to expel fetal membranes spontaneously within 12 h were considered a clinical case of RFM, as previously defined [20,21]. Conversely, the expulsion of fetal membranes in cows before 12 h was considered clinically normal. Cows were observed every 5 h to determine the time of calving and every 2 h for the expulsion of fetal membranes. This study did not involve assessment of calf health or fertility and health status of cows postpartum. 2.3. Plasma cortisol and erythrocyte lipid peroxide concentrations Blood samples were collected (in heparinised glass tubes) by jugular venipuncture on day 21 (before treatment) and on days 7, 3, 2, 1, and 0 prepartum for plasma cortisol concentrations (n = 20), and on days 21 and 7 for erythrocyte lipid peroxide concentrations (n = 50). The tubes were centrifuged (1600 g for 15 min at 8 8C) and plasma was collected and stored at 20 8C until assayed for cortisol. Commercially available RIA kits were used to determine the plasma cortisol concentrations (Corti-cote; ICN Pharmaceuticals, Orangeburg, NY, USA) within 4 week of sample collection. The concentration of lipid peroxide was estimated in the erythrocyte hemolysate using the method of Placer et al. [22]. The erythrocyte hemolysate from the tubes containing whole blood samples was prepared according to the method of Cohn et al. [23]. Briefly, immediately after collection, blood samples were centrifuged at 1600 g for 10 min. The plasma and buffy coats were removed by aspiration. The sediment containing blood cells was washed three times by resuspending in isotonic saline (0.89 wt.%/vol.% NaCl), followed by re-centrifugation and
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removal of the supernatant fluid. The cells were lysed in nine volumes of ice-cold distilled water (DW) to prepare a 10% erythrocyte hemolysate. An aliquot (0.2 mL) of this hemolysate was added to 1.3 mL of Tris–KCl buffer (0.2 mol Tris in 0.16 mol KCl at pH 7.4), after which 1.5 mL of 0.08% thiobarbituric acid (TBA) reagent (2-TBA in a small amount of NaOH, and neutralised with 7% perchloric acid) was added. The mixture was heated in a boiling water bath for 10 min (using a marble as a condenser). After cooling, 3.0 mL pyridine:n-butanol (3:1, v:v) and 1.0 mL of 1N NaOH (40 g of NaOH pellets in 1 L of DW) was added and mixed by shaking. A blank solution was prepared with 0.2 mLdistilled water in lieu of the erythrocyte hemolysate. The photometric measurement was taken at lmax 548 against the blank, using a spectrophotometer (Electronic Corporation of India Limited, Hydrebad, India). Hemoglobin (Hb) in 10% erythrocyte hemolysate was estimated colorimetrically by the cyanomethemoglobin method [24] expressed in mg/mL of hemolysate; this was converted to nanomoles malonyldialdehyde (nmol MDA)/mL of erythrocyte hemolysate hemoglobin by using 1.56 105 L/mmol/cm as the extinction coefficient [25]. 2.4. Cotyledon tissue To investigate enzyme activity and peptides in cotyledons (without confounding by Vit E + Se treatment or interfering with the primary experiment), 14 additional cows were used. Cotyledonary tissues from cows, with (n = 6) or without (n = 8) RFM were collected immediately postpartum into cold PBS solution. The operator’s arm was covered with sleeve (lubricated within paraffin) that was introduced into the uterus of a cow with RFM. Gentle digital pressure was applied between the maternal caruncle and fetal cotyledon to detach and remove the cotyledon. Cotyledons from non-RFM cows were collected directly from the vagina as the placenta was delivered. Immediately after collection, tissues were transported to the laboratory in an icebox containing PBS. The membranous portions of the cotyledonary tissues were removed with a scissors and forceps. The tissues were washed thoroughly with cold PBS, dried with filter paper and weighed. One part of the tissue was sonicated with two parts of cold PBS and treated with 0.1% triton X-100 to create a homogenate. The homogenate was centrifuged for 20 min at 1200 g and the supernatant of the cotyledon sample was stored in 5 mL aliquots for estimation of cationic peptides. All procedures for homogenate preparation were performed in a cold room at 4 8C. 2.4.1. Enzyme assays in cotyledon tissues Myeloperoxidase (MPO) activity was assayed in the supernatant sample using odianisidine as an electron donor (substrate solution) [26]; the enzyme concentration was calculated by using the molar extinction coefficient for oxidised o-dianisidine. The turbidimetric method [27], using egg white lysozyme for the standard curve, was used to measure lysozyme in the supernatant of the cotyledon samples [28]. Proteolytic activity of elastase was measured with azo-casein as substrate [29]. The total ACP and tartrate resistant ACP activity was assayed [30] by the hydrolysis of p-nitrophenyl phosphate and by measuring the absorbance (at 410 nm) of p-nitrophenyl that was liberated.
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2.4.2. Estimation of total protein content Total protein in the supernatant of the cotyledon samples was estimated using bovine serum albumin as standard, adopting the method described by Lowry et al. [32]. 2.4.3. SDS-PAGE analysis of cotyledon tissue The supernatant of the cotyledon samples from non-RFM and RFM cows were processed by SDS-PAGE using a 5–15% linear gradient gel in a discontinuous buffer system, as described by Laemmli [31]. The protein molecular weight markers used were phosphorylase-b (97.4 kDa), bovine serum albumin (68 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), soyabean trypsin inhibitor (20 kDa) and lysozyme (14.3 kDa). The molecular weights of the sample protein bands were estimated using the references points from the molecular weight markers. 2.5. Statistical analyses Statistical analyses were performed by ANOVA using the General Linear Model (GLM) procedure of the Statistical Analysis System version 8.2 (SAS Institute Inc.; Cary, NC, USA). Probability plots (Shapiro-Wilk test, P < 0.005) using the UNIVARIATE procedure were used to determine the normality of the data [33]. Data for the Vit E and Se response on the occurrence of RFM, erythrocyte lipid peroxidation and enzyme activity were analysed with the student’s t-test. Plasma cortisol concentrations were analysed by repeated measures ANOVA [33]; the model included the effect of group (control with out RFM, control with RFM and Vit E + Se), days and their interaction. If there was a significant main effect or interaction, differences were located using the probability of differences (PDIFF) procedure of SAS on the least square means. Differences in the incidence of RFM were compared with a Fisher’s exact test.
3. Results 3.1. Prepartum administration of Vit E and Se response The incidence of RFM was not different (0% versus 12%) in cows given Vit E + Se and control cows, respectively (P = 0.23). 3.2. Plasma cortisol For plasma cortisol concentrations (Table 1), there were main effects of group, time and their interaction (P < 0.001 for each). There were no significant differences among groups of plasma cortisol concentrations on day 21 prepartum (before treatment). From day 7 prepartum to the day of parturition, plasma cortisol concentrations were greater (P < 0.05) in control cows than those given Vit E + Se. Comparing day of calving with day 21 prepartum, mean plasma cortisol concentrations increased four-fold in cows treated with Vit E and Se (P = 0.05), six-fold in control cows that did not develop RFM (P = 0.01) and 11-fold in control cows that developed RFM (P = 0.001). From days 21 to 7 prepartum,
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Table 1 Mean (S.E.M.) plasma cortisol concentrations (ng/mL) at 21, 7, 3, 2, 1 and 0 day prepartum in cows with or without retained fetal membranes (RFM) Prepartum interval (d)
21 7 3 2 1 0
Treated
Control
Non-RFM (n = 10)
Non-RFM (n = 7)
RFM (n = 3)
3.0 0.23 2.9 0.49 2.5 0.33 4.2 1.36 7.1 1.22 12.5 2.36
4.5 0.84 6.2 1.01 8.1 2.10 7.3 1.74 12.2 4.12 25.6 6.26
5.5 0.89 ax 6.3 1.081 bx 10.4 4.07 by 29.7 6.49 by 40.7 5.34 cy 60.5 4.33 cz
ax ax ax ax ax ay
ax ax by ax by bz
Treated cows were given 1100 IU Vitamin E (DL a-tocopherol acetate) and 30 mg selenium (sodium selenite) 3 week before calving, whereas control cows were given saline. There were effects of group, day and their interaction (P < 0.001 for each). Rows and columns with different letters differ (P < 0.05).
plasma cortisol concentrations were not significantly different between control cows, with or without RFM. In control cows with RFM, plasma cortisol concentrations were lower (P < 0.05) on day 7 prepartum compared with days 3, 2, and 1 prepartum, and the day of parturition. In control cows without RFM, plasma cortisol concentrations were lower (P = 0.01) on day 7 compared with days 3, and 1 prepartum, and the day of parturition. 3.3. Erythrocyte peroxidation The mean levels of MDA in the erythrocyte hemolysate, on day 21 prepartum for the Vit E + Se were not different (P > 0.05) from the control cows (Table 2). However, on the day of calving there was lower (P < 0.05) peroxide formation in the erythrocyte hemolysate of cows given Vit E and Se than control cows (with or without RFM). The administration of Vit E and Se decreased (P < 0.05) peroxide formation in the erythrocyte hemolysate from day 21 prepartum to the day of parturition. However, there was an increase (P < 0.05) in the erythrocyte hemolysate peroxide formation from day 21 prepartum to the day of calving in the control cows that developed RFM. No differences (P > 0.05) in erythrocyte peroxide formation were observed in control cows that did not develop RFM, from day 21 prepartum to the day of calving.
Table 2 Mean (S.E.M.) lipid peroxide levels (nanomoles MDA/mg of hemoglobin) in erythrocytes at 21 and 7 day prepartum in cows with or without retained fetal membranes (RFM) Prepartum interval (d)
21 7
Treated
Control
Non-RFM (n = 25)
Non-RFM (n = 22)
RFM (n = 3)
2.691 0.064 x 2.457 0.054 ay
2.786 0.067 2.762 0.080 b
2.460 0.072 x 3.677 0.162 cy
Treated cows were given 1100 IU Vitamin E (DL a-tocopherol acetate) and 30 mg selenium (sodium selenite) 3 week before calving, whereas control cows were given saline. Rows and columns with different letters differ (P < 0.05).
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Table 3 Mean S.E.M. enzyme activity in the cotyledons of crossbred dairy cows with or without retained fetal membranes (RFM) Enzymes
Myeloperoxidase, mU/mg protein Lysozyme, KU/mg protein Elastase, U/mg protein Acid phosphatase, U/mg protein Tartate resistant acid phosphatase, U/mg protein Tartrate inhibited acid phosphatase, U/mg protein * **
Enzyme activity Non-RFM (n = 8)
RFM (n = 6)
1.19 0.080 0.06 0.004 1.53 0.096 0.09 0.008 0.06 0.012 0.03 0.005
0.74 0.060** 0.42 0.123** 1.36 0.123 0.12 0.010* 0.07 0.004 0.06 0.008*
Difference between non-RFM and RFM (P < 0.05). Difference between non-RFM and RFM (P < 0.01).
3.4. Cationic peptide enzyme activity in the cotyledon tissue Decreased (P = 0.001) MPO enzyme activity was observed in the cotyledon tissue of cows that developed RFM compared with clinically normal cows (Table 3). Higher (P < 0.05) lysozyme, ACP and tartrate inhibited ACP enzyme activities were observed in cotyledons of the cows that developed RFM, compared with those that did not develop RFM. No differences (P > 0.05) were observed in the enzyme activities of the elastase and tartrate resistant ACP in cows that developed RFM compared with the nonRFM cows. 3.5. SDS-PAGE and cotyledon tissue A sample of the electrophoretic pattern of cotyledons is presented in Fig. 1. There were 20 protein bands in the approximate range of 100–10 kDa. The overall elecotrophoretic pattern of the cotyledon tissue in RFM and non-RFM samples indicated minor differences for different protein bands of varied molecular weight. However, a thick band <10 kDa was observed in the cotyledons of both RFM and non-RFM cows.
4. Discussion In this study, administration of Vit E and Se 3 week prepartum did not alter (P = 0.23) the incidence of RFM (12% versus 0%, respectively). This finding was consistent with earlier observations of Segerson et al. [38], Rosa et al. [39] and Hidiroglou et al. [40] who also reported no decrease in the incidence of RFM when cows were supplemented with Vit E and Se approximately 30–20 days before calving. This is in contrast with the Eger et al. [34], Thomas et al. [35], Kim et al. [36] and Ivandija [37], who reported a decreased incidence of RFM following Vit E and Se supplementations in cattle. In the current study, the incidence of RFM in control cows (12%) was very close to the historical retention rate (14%) for this particular herd. Apart from the Vit E + Se injection schedules employed, there were no changes in nutritional or the herd health management programs.
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Fig. 1. Electrophoretic pattern (SDS-PAGE, 15% linear gradient gel) of cotyledons from cows with (Lanes 1 and 2) or without (Lanes 3, 4 and 5) retained fetal membranes. *Protein molecular weight markers (Lane M) in kilodalton (kDa) are: phosphorylase-b, 97.4 kDa; bovine serum albumin, 68.0 kDa; ovalbumin, 43.0 kDa; carbonic anhydrase, 29.0 kDa; soyabean trypsin inhibitor, 20.0 kDa; and lysozyme, 14.3 kDa.
Relationships among dietary antioxidant, oxidative stress and RFM have been investigated in periparturient dairy cows [41]. Although the pathogenesis of RFM due to Vit E and Se deficiency is not clearly understood, the involvement of oxidative stress in the etiology of RFM syndrome is suggested by a reduction in the incidence of RFM when the antioxidants, Vit E and Se are supplemented [14]. Administration of Vit E and Se, increased the levels of a-tocopherol in RBC, neutrophils and plasma, and increased the
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biochemical activity of glutathione peroxidase (GSH-Px) [42]. Therefore, perhaps supplementation of Vit E in concert with Se reduced oxidative stress and associated changes in the fetomaternal junction. Vitamin E and Se have immunomodulatory effects mediated through improved neutrophil function, by enhancing their random migration and chemotactic response [42]. There is evidence that Se deficiency can affect the function of polymorphonuclear neutrophils (PMN), which is associated with physiological changes in GSH-Px levels [43–45]. The absence of leukocytes at the fetomaternal junction leads to a 100% incidence of RFM [8]. In the present study, Vit E and Se treatment might have increased the leukocyte number and leukocyte chemotaxis at the fetomaternal junction and helped in the normal loosening and expulsion of fetal membranes. Intramuscular administration of Vit E increased serum Vit E concentrations for at least 28 days [46], while injection of Se increased concentration of Se in whole blood and serum for 28 days and increased whole blood GSH-Px activity for at least 84 days [47]. Injection of Vit E and Se, 3-week prepartum, increased erythrocyte GSH-Px in dairy cows during the first 12week of lactation [48]. Lipid peroxidation is a biochemical oxidative degradation of unsaturated fatty acids that causes irreversible denaturation of essential proteins. With respect to the widespread distribution of unsaturated fatty acid in the cellular membranes, the peroxidative damage has the potential to affect many cellular functions and interfere with the regulation of several metabolic pathways [14]. Cytotoxic aldehydes (e.g., MDA), which remain after termination of lipid peroxidation, provide the basis for the thiobarbituric acid test for measuring lipid peroxidation and products in body fluid [22]. The increased levels of the lipid peroxide, from 3 to 0 week prepartum, in cows that failed to expel fetal membranes in this study, were consistent with the Brazezinska-Slebodzinska et al. [49]. The administration of Vit E and Se during the prepartum period reduced erythrocyte substances reactive to thiobarbituric acid. This was in close agreement with Miller et al. [41] and BrazezinskaSlebodzinska et al. [49], who reported a decrease in RBC thiobarbituric acid reactive substances from 375 to 275 nmols MDA/g of Hb in cows supplemented with 1000 IU Vit E for 6 week. In the present study, the combined administration of a-tocopherol acetate and sodium selenite at 3-week prepartum might have reduced the level of lipid peroxidation at 7 day prepartum. It is interesting to note that at parturition, when the clinical features of RFM were not exhibited, MDA (an end product of lipid peroxidation) contents in RBC were increased significantly. Perhaps the biochemical changes leading to elevated levels of MDA could be detected much earlier than the clinical manifestation of inflammatory changes in the infected tissue. Therefore, elevated levels of MDA in erythrocyte hemolysate prepartum may be useful to predict RFM. The prepartum increases in plasma cortisol concentrations observed in this study were similar to those reported previously [15,50]. The signal to initiate parturition comes from the fetal hypothalamic-pituitary-adrenal axis; increased fetal cortisol concentrations [51] cross from the fetal to the maternal circulation [52]. Therefore, increased maternal blood cortisol concentrations 7 day prepartum could be due to increased fetal concentrations. The greater concentrations of plasma cortisol at the prepartum period in RFM compared with non-RFM cows in the present study were consistent with Peter and Bosu [15] and Ras et al. [53]. A sharp rise in cortisol in RFM cows is believed to be in response to stress and inflammation in the pregnant uterus, rather than initiation of parturition [53]. The maturation of placentomes is a hormonally controlled process [54]. The loosening process,
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which normally occurs between the fetal villi and the maternal crypts, depends upon histological changes in the opposing surface [5] in the presence of estrogens. However, RFM occurs when hormone concentrations and requisite tissue changes are not favorable [54]. Gunnick [10] and Heuwieser and Grunert [55] found a reduction in chemotactic activity and inhibited migratory ability of leukocytes in cows that went on to develop RFM. Cortisol, which inhibits leukocyte activity [17,56] and impairs neutrophil function in bovines, may have suppressed leukocyte activity and contributed to development of RFM. Further studies are required to confirm blood cortisol patterns in cows with RFM. Supplementing Vit E and Se reduced the level of cortisol in the treated group. It is known that a higher level of cortisol is released in response to a variety of acute stressors in cattle, including pregnancy [57]. Stress in pregnancy also increases the oxidative metabolic reaction [58]. Therefore, prepartum supplementation of Vit E and Se might have reduced the reactive oxygen metabolites and/or free radicals, leading to reductions in oxidative stress and cortisol concentrations. That MPO activity was lower in the cotyledons of the RFM cows was consistent with the data of Hoedemaker et al. [12] and Tian-Quancai et al. [13], who reported a depressed activity of MPO in circulating neutrophils of cows with RFM. The MPO activity in cotyledons indicated the presence of neutrophils at the fetomaternal junction. The higher MPO value in the cotyledons of non-RFM cows suggested a greater influx of neutrophils at the fetomaternal junction, thereby promoting the normal release of fetal membranes. Conversely, the lower value of MPO in the cotyledons of RFM cows indicated a transient impairment of neutrophil number, function and migratory ability to the site of the fetomaternal junction, which may have resulted in RFM. However, it was noticed that an increase in the chemotaxis and number of leukocytes in placentomes appeared to be an important part of the detachment process [55]. When both of these requirements were met, the incidence of RFM was approximately 1.4%. When either one was decreased or absent, the incidence was 6.8–9.6% and when both were absent, the incidence approached 100% [10]. Gunnick [10] and Gilbert et al. [59] reported that the normal physiological expulsion of the placenta requires the presence of neutrophils at the fetomaternal junction. Greater tissue and body fluid lysozyme activity was reported for cattle with acute inflammatory responses to injury [14]. Lysozymes are microbicidal (bactericidal against gram-positive bacteria [61]) and are associated with phagocytes and neutrophils [62]. The higher lysozyme activity in the cotyledons of RFM cows in this study may have been associated with the acute inflammatory response at the fetomaternal junction. Moreover, as the postpartum uterus is more prone to infection, the presence of lysozymes in the cotyledons suggests it may be one of the factors in preventing postparturient uterine infection and thereby promoting uterine involution [60]. Elastin is the primary protein constituent of the elastic fibre, the predominant connective tissue of the lung, blood vessels, and skin [63]. Histologically, cotyledons of RFM and nonRFM cows are congested and contain constricted blood vessels, with thickening of tunica media of arterioles and calcification of connective tissues. More focal areas of collagen were also reported in the cotyledons of RFM cows [64]. The proteolytic enzyme elastase is a component of PMN cells. A possible role of elastase is inhibition of bacterial growth [63]. The lack of difference between groups in elastase activity may have been due to a lower degree of bacterial contamination in the placentomes. However, the presence of this
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enzyme in the cotyledons of RFM and non-RFM cows may indicate its role in tissue injury at the fetomaternal junction and could help in the loosening of fetal membranes. The higher activities of ACP and tartrate-inhibited ACP in the cotyledons of RFM than in non-RFM cows in this study indicated trauma to the placental tissues of cows with RFM. Earlier, Mahfooz et al. [65] and Kudlac et al. [66] reported significantly higher values of ACP in the blood of RFM cows at the prepartum stage. The source of this enzyme in the present study could be the fetal membranes themselves or the lysosomes of the PMN cells at the fetomaternal junction. Thus, an increase in ACP levels in the cotyledons of RFM cows may reflect increased enzymatic activity associated with inflammatory changes in placental tissues. It was reported that excess iron in serum could be linked with increased RFM, through increased saturation of serum iron-binding capacity [7]. Certain enzymes of large families of lysosomal tartrate-resistant ACP, like uteroferrin, are the major protein component of uterine secretion [67]. These play important roles in the transportation of iron from maternal to fetal circulation [68]. The presence of tartrate-resistant ACP in the cotyledons indicated the involvement of iron at the fetomaternal junction. That there was no difference between groups in tartrate-resistant ACP activity may have been due to a lack of iron-induced RFM condition. The SDS patterns of non-RFM and RFM cotyledon extracts revealed the presence of >20 bands of peptides. The protein of these bands possessed molecular weights ranging from <10 kDa to >100 kDa. In a series of studies conducted by Kankofer [69,70] and Kankofer and Guz [71] to investigate (using either Western blotting or SDS-PAGE) the activities of various proteins present in bovine placental tissues with or without RFM, it was suggested that the molecular weight of the protein present in both maternal and fetal placental tissues averaged from <35 kDa to >85 kDa. Although Western blotting and SDS-PAGE are semi-quantitative methods, it was possible to detect differences in staining intensity, suggesting differences in the amount of protein present. In general, intensity was lower in fetal than maternal parts of the placenta and patterns of bands were different with respect to the mode of delivery of placenta [71]. In the present study, the lower band in the <10 kDa zone in the SDS-PAGE represented cationic peptides; these concentrations were very high. It has been suggested that these peptides have antibacterial properties (personal communication with Dr. T. More, IVRI, India) and it is known that some cationic peptides are present in neutrophils [61]. This is a preliminary study to use SDS-PAGE in cotyledon tissue and further investigations to develop and understand the potential role of peptides present in cotyledon tissue are clearly warranted. In conclusion, results of this study indicated that the etiopathogenesis of RFM may involve oxidative stress, as the prophylactic prepartum supplementation of antioxidant, Vitamin E (DL a-tocopherol acetate, 1100 IU) and Se (sodium selenite, 30 mg) by single 20 mL im injection, at 3 week prepartum, decreased erythrocyte lipid peroxide concentrations in dairy cows. Elevated prepartum levels of malonyldialdehyde (indicative of oxidative stress) and cortisol could be reliable predictors of retained fetal membranes. Decreased myeloperoxidase activity in the cotyledons of RFM cows, indicated impairment of neutrophil functions at the fetomaternal union. Higher lysozyme and acid phosphatase activity in the cotyledons of cows with RFM indicated an acute inflammatory response at the fetomaternal union. Finally, the involvement of neutrophils and cationic peptides (with natural antibiotic properties) at the fetomaternal junction warrant further investigation.
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Acknowledgements This work was supported by Indian Council of Agricultural Research Fellowship. The authors wish to acknowledge Captain (Dr.) Rajender Singh of the Military Dairy Farm UP, India for his permission to work on the farm, Vet Care Bangalore for the supply of ‘‘E care Se’’ supplement and Mr. Tyagi of C.B.S.H., G.B.P.U.A.T., Pantnagar for providing facilities for the cortisol assay. The authors thank Dr. M.A. Crowe, University College Dublin, Ireland, Mr. Phil Rogers MRCVS, and Mr. Michael Nolan, Grange Research Centre, Teagasc, Dunsany, Co Meath, Ireland for reviewing the manuscript.
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