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Dose of selenium in goat kids and its effect on the antigenic response to Mannheimia haemolytica and oxidative stress
Small Ruminant Research 153 (2017) 171–174
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Dose of selenium in goat kids and its effect on the antigenic response to Mannheimia haemolytica and oxidative stress
MARK
Víctor M. Díaz-Sáncheza, Gabriela Rodríguez-Patiñoa, Patricia Ramírez-Nogueraa, J. Efrén Ramírez-Bribiescab, José F. Morales-Álvarezc, Alma L. Revilla-Vázqueza, ⁎ Raquel López-Arellanoa, a b c
National Autonomous University of Mexico, Cuautitlán, Multidisciplinary Research Unit, Cuautitlán Izcalli, Edo. México, CP 54714 Mexico College of Postgraduate, Livestock Program. Montecillo, Texcoco, Edo. México, CP 56230 Mexico National Institute of Forestry Research, Animal Health, Coyoacán, México, CP 0410 Mexico
A R T I C L E I N F O
A B S T R A C T
Keywords: Selenium Goat kids Antigenic response Oxidative stress
Selenium (Se) prevents oxidative damage and stimulates the immune system. Currently, there are no data available evaluating the Se-induced antigenic response to Mannheimia haemolytica and oxidative stress in goat kids. Twenty-one 6-month-old male Alpine Goat kids (22.9 kg) were immunized against M. haemolytica and divided into 3 groups: Basal diet with no additional Se (CG); Se injected subcutaneously at 0.25 mg Se/kg live weight (LW) (SeSG); and Se administered as intraruminal bolus at 0.46 mg Se/kg LW (SeBG). Blood samples were taken from the animals in all groups at 0, 14, 28, 42 and 56 days post-dosing. Erythrocyte Se levels doubled in SeSG during days 14–28 post-dosing (130.6 ng/g Se) as compared to CG. During days 28–42 post-dosing, Se levels decreased (P < 0.05) in SeSG (106.9 ng/g Se) vs. CG and SeBG (86.2 ng/g Se and 81.7 ng/g Se, respectively). Glutathione (GSH) levels decreased in days 0–28 post-dosing (3.7E–4 nmols/mg of total protein) and then, remained stable up to day 56 (5.6E–4 nmols/mg of total protein). Catalase levels in the SeBG and SeSG (79.9 U) were higher (P < 0.05) than in the CG (56.3 U) by day 14 post-dosing. Malondialdehyde (MDA) levels on day 14 post-dosing increased 7-fold in the SeBG (1.28E–3 ng/mg of total protein). IgG levels increased in the groups treated with Se during days 28–42 post-dosing (1.4 nM) vs. the CG (0.10 nM) (P < 0.05). Treatment with Se improved the immunological response to M. haemolytica.
1. Introduction Infectious diseases and dietary deficiencies are two critical factors causing great losses in goat herds from birth to puberty (RamírezBribiesca et al., 2001). The reported etiologies of pneumonias in goats are Mycoplasma ovipneumoniae, Pasteurella multocida, Klebseilla pneumoniae, Staphylococcus aureus, Shigella spp., E. coli (Saleh and Allam, 2014), and M. haemolytica, which can cause mortality rates in goat kids from 30 to 60% (Mohamed and Abdelsalam, 2008). Specifically, M. haemolytica has been commonly isolated from samples of ruminants with pneumonia in Mexico (Jaramillo-Arango et al., 2009). Consequently, it is necessary to induce a good antigenic response to this microorganism. Selenium (Se) supplementation can stimulate humoral immunity (Hall et al., 2013) through the increase in immunoglobulin G (IgG) levels in bovines immunized against pasteurellosis (Rice et al., 2007). Nevertheless, Se deficiency has been observed in a large part of
the Mexican territory, especially in the Plateau region, which can cause health problems in deficient animals besides the white muscle disease (Ramírez-Bribiesca et al., 2005). Infection by several respiratory pathogens increases production of reactive oxygen species (ROS), damaging cell membranes (Khanal and Knight, 2010). An imbalance between oxidants and antioxidants in favor of the former is called “oxidative stress”. There are numerous lines of defense against ROS, including low molecular weight radicals/ oxidants such as glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) (Rahmanto and Davies, 2012). Oxidative stress and damage to cell membranes produces malondialdehyde (MDA) as degradation product (Grotto et al., 2009). The joint presence of respiratory disease and Se deficiency in the organism induces oxidative stress (Hefnawy and Tórtora-Pérez, 2010). The application of a subcutaneous dose of Se or administration of intraruminal boluses induces an increase in blood Se levels of neonatal ruminants
⁎ Correspondence to: Laboratory of Pharmaceutical Development Tests Faculty of Higher Cuautitlán Studies, Campus 4 UNAM 54714 Cuautitlán Izcalli, State of Mexico Mexico. Tel. +525556231999 extension 39415. E-mail address: [email protected] (R. López-Arellano).
http://dx.doi.org/10.1016/j.smallrumres.2017.06.005 Received 19 February 2017; Received in revised form 2 June 2017; Accepted 4 June 2017 Available online 23 June 2017 0921-4488/ © 2017 Published by Elsevier B.V.
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after 20 days post-dosing (Ramírez-Bribiesca et al., 2005; RevillaVázquez et al., 2008). Currently, there are no published papers evaluating the antigenic response against M. haemolytica in goat kids and their association with Se levels. The aim of this study was to evaluate the antigenic response against M. haemolytica through the presence of IgG-specific antibodies and their relationship with oxidative stress markers after administering Se to goat kids as a subcutaneous dose or by oral bolus dosing. 2. Materials and methods 2.1. Standard of ethics and experimental welfare The animals used for this study were handled under the rules of the Internal Committee for the Care and Use of Experimental Animals approved by the Cuautitlán-UNAM, under registration code CICUAE-FESC C15_03.
Fig. 1. Levels of erythrocyte selenium in goat kids with or without selenium supplementation.
completely randomized factorial design (CG, SeSG, and SeBG). The levels of Se, GSH, CAT, MDA and IgG against Mannheimia haemolytica were selected as the response variables. A comparison of means was performed with the Tukey test considering a significant statistical difference (p < 0.05).
2.2. Reception, management, and distribution of the experimental groups Twenty-one 6-month-old male Alpine Goat kids were selected from Cuautitlán Agricultural Farm. They were housed in 3 corrals (each one with an area of 7 m2) with a bed of shavings. The animals were fed as a group with sweet alfalfa [93% dry matter (DM) and 17.12% crude protein (CP)] containing 1.11 μgSe/g of Se. Water was given ad libitum, and the animals were dewormed with Closantel (10 mg/kg LW). On day 15, all the animals were immunized against M. haemolytica by subcutaneously administering 2.5 mL of bacterin-toxoid (Toxo-BacINIFAP®, Mexico, 1 × 109 colony forming units/mL, containing the leukotoxin). On the day after the vaccination, all the goat kids were weighed on a digital scale (Torrey®) and sorted into 3 groups based on their body weight, with each group averaging 22.91 ± 4.15 kg. These experimental groups received the following treatments: 1) Control group (CG), did not receive Se supplementation; 2) Subcutaneous Se dose group (SeSG), 0.25 mg Se/kg LW (as NaSeO3. VALNO, lot: 0347C7); and 3) Intrarruminal Se bolus group (SeBG), 0.46 mg Se/kg (as NaSeO3). Both Se products were manufactured in the pharmacy laboratory of FESC-UNAM. Specifically, the Se bolus were prepared with a densifier (reduced iron), a binder (cutin), and a sliding agent (talc) (Revilla-Vázquez et al., 2008) to be degraded in a maximum time of 4 days. Blood samples were taken before the dosing and on day 14 pst-dosing for 4 periods. Blood extraction was performed by venipuncture of the external jugular vein using 20 G-X 38 mm needles and 6 mL Vacutainer® BD vacuum tubes with EDTA anticoagulant. The blood samples were centrifuged at 4000 rpm for 15′ to separate the plasma from the erythrocyte pack and were stored at −80 °C until their analysis.
3. Results The data are presented as figures showing the means and their standard deviations in the 3 experimental groups. Fig. 1 shows the erythrocyte Se levels. On days 0–14 post-dosing, the average Se levels remained low in the 3 groups (81.12 ± 1.23 ng/g Se and 73.36 ± 7.65 ng/g Se, respectively; P > 0.05). During the days 14–28 post-dosing, the Se levels doubled (P < 0.05) in SeSG and SeBG (130.66 ± 29.77 ng/g Se), while the CG maintained an average Se level below 95 ng/g. During the days 28–42 period post-dosing, the levels of Se decreased (P < 0.05) between SeSG (106.93 ± 15.19 ng/ g Se) vs. CG and SeBG (86.22 ± 12.37 ng/g Se and 81.69 ± 10.19 ng/g Se, respectively). On day 42 post-dosing, an interaction effect (P < 0.05) was observed between the CG and the SeBG. On day 56 post-dosing, an average of 108.87 ± 3.76 ng Se/g (P > 0.05) was obtained among the groups. Fig. 2 shows the GSH content in the erythrocytes. On day 0, the GSH levels in the 3 groups averaged 9.6E–4 ± 1.118E–5 nmols/mg of total protein (P > 0.05). GSH levels decreased during days 0–28 (3.7E−4 ± 4.714E−6 nmols/ mg of total protein) in SeBG and SeSG, and then, they remained stable up to day 56 (5.6E−4 ± 8.49E–5 nmols/mg of total protein). An interaction effect was observed (P < 0.05) between CG and the SeSG and SeBG groups from day 14 up to day 45 post-dosing. Fig. 3 shows the CAT content in the erythrocytes. The mean CAT level at the beginning of the experiment was 73.10 ± 1.85 U. On day 14, the CAT levels in SeBG and SeSG were higher (P > 0.05; 79.90 ± 3.93 U) than in the CG (56.33 ± 1.69 U). Subsequently, from day 14 to day 28 post-
2.3. Laboratory analysis The hematological samples were analyzed with the following tests. Se levels in the erythrocytes: The samples were analyzed according the modified Capelo method (Capelo et al., 2006). Reduced glutathione (GSH) levels in the erythrocytes: GSH was determined according the modified Eyer method, sulfosalicylic (Eyer and Podhradský, 1986). Catalase (CAT) activity in the erythrocytes: A rapid test as described by Iwase and colleagues (Iwase et al., 2013) was used. Measurement of malondialdehyde (MDA) levels: It was conducted according to the modified methods previously described (Lykkesfeldt, 2001). Concentration of immunoglobulin G (IgG) – ELISA test: The IgG concentration was estimated according to the method described by Morales (Morales-Alvarez et al., 1993). 2.4. Statistical analysis The treatments were assigned to each group using a 3-level
Fig. 2. Levels of erythrocyte GSH in goat kids with or without selenium supplementation.
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during days 14–28, and then, they increased from day 28–42, showing a higher (P < 0.05) level in SeBG (1.43 ± 0.02 nm) vs. CG (0.10 ± 0.09 nm). On day 56, there no difference was observed (P > 0.05) in the IgG absorbances among the 3 groups (0.96 ± 0.12 nm). 4. Discussion The proper level of Se in the serum or blood plasma ranges from 90 to 110 ng/mL (Stowe and Herdt, 1992). If the hematic Se content is as low as 30–80 ng/g, it indicates selenodeficiency (Van Ryssen et al., 2013), which can lead to health disorders and death in newborn goat kids (Ramírez-Bribiesca et al., 2005). In this study, a variability of 25% was initially observed plasma Se levels. To overcome this sample variability, erythrocyte Se content was measured instead. The erythrocytes contain 73% of the Se in the hematic tissue (Maas et al., 1992); therefore, they reflect a more reliable status of this micromineral in the prevention of oxidative damage by GSH activity (Gutzwiller, 1998). In addition, false values are excluded in the determinations of Se in the erythrocytes because none of the samples had hemolyzed, a process that alters the Se content (Qin et al., 2007). Before dosing with Se, all the 6-month old goat kids had an average content of Se of 84.11 ng/g of erythrocytes; a value which indicates a mild selenodeficiency, since none of the animals showed clinical signs of insufficiency. This content of erythrocyte Se was expected due to the lack of Se in the soil and forage located at the area of study (Ramírez-Bribiesca et al., 2005). A high dose of Selenium was considered for the oral bolus administration due to the incorporation of this micromineral in tissues and ruminal microorganisms in the intestinal tract before reaching a measurable blood concentration. Consequently, a lower dose of Selenium was used in the subcutaneous administration due to a quicker incorporation into the Se-dependent enzymes located in the endothelium. This approach was the reason for initially believing in possible differences in the erythrocyte Se levels between both administration routes; however, the results showed similarity. On the other hand, a previous study in bovines demonstrated a correlation between Se levels and the immune response against Pasteurella sp., showing an association between the level of Se in the hematic tissue and the immune response (Reffett Stabel et al., 1985). This hypothesis is more viable in animals that do not lack Se, while selenodeficient animals must first satisfy the need for the microelement in the tissues of the organism and ultimately reflect their status in the hematic tissue (Stefanowicz et al., 2013). In this study, we estimated GSH concentration, thiobarbituric reactive species and Catalase activity in order to know the general redox status after Se supplementation. When levels of oxidative stress increase and the levels of Se decrease, cellular lipoperoxidation occurs and GSH levels increase (Richie et al., 2012). On day 28 post-dosing, a higher level of GSH was found in CG as an acute response. However, this level was lower in the erythrocytes compared with the one found in another study performed on goat blood (Tekeli et al., 2015). Se deficiency influences the concentration of GSH (Celi et al., 2010) and the antioxidant activity of CAT in rats and rabbits (Olisekodiaka et al., 2015). Our study showed a significant difference on day 14: the supplemented groups (SeSG and SeBG) had the highest CAT levels, but after this time point, there were no differences among the groups. MDA is an indicator of oxidative stress, which was measured through the thiobarbituric reactive test, which indicates cellular damage due to lipoperoxidation. The estimation of CAT activity and the levels of MDA are considered good markers of cellular oxidation (Alam et al., 2013). The levels of MDA did not change in the study groups during the progression of the experiment, but on day 14 post-dosing, the goat kids showed a higher content of MDA, indicating a lipoperoxidation effect (Kohen and Nyska, 2002). Evidences show that dietary supplementation of both inorganic and organic Se do not influence MDA level in liver microsomes either (Chung et al., 2007). In this study, the levels of MDA increased at day 14 and then decreased on day 28
Fig. 3. Erythrocyte catalase units in goat kids with or without selenium supplementation.
Fig. 4. Levels of plasma MDA in goat kids with or without selenium supplementation.
Fig. 5. Absorbances of serum IgG in goat kids with or without selenium supplementation.
dosing, the CAT levels decreased in all groups (P > 0.05). During days 28–56 post-dosing, the CAT levels fluctuated by amounts of less than 55 units, and there were interactions observed among CG vs. SeBG vs. SeSG (P < 0.05) during days 26–49 post-dosing. Fig. 4 shows the plasma MDA. The mean of the 3 groups on day 0 was 8.20E−5 ± 3.53E−5 ng/mg of total protein (P > 0.05). After day 14 post-dosing, the MDA level in SeBG increased 7-fold (P < 0.05; 1.28E–3 ng/mg of total protein), and a 3-fold increase was observed in SeSG (P < 0.05; 7.00E−4 ng/mg of total protein). In the CG, the MDA level increased by 4-fold, and there was no difference (P > 0.05) with the groups supplemented with Se. On day 28 post-dosing, all the groups showed a decrease of MDA levels to baseline, averaging 1.48E–4 ± 8.52E–5 ng/mg of total protein. Subsequently, from day 28 to day 56, the levels in SeBG and SeSG increased linearly, without exceeding a level of 8.5E–4 ng/mg of total protein. There was an interaction effect observed (P < 0.05) for CG vs. SeBG and SeSG. Fig. 5 shows the absorbance of blood serum IgG. During days 0–14, there was an increase of IgG levels in all groups. The IgG levels were maintained 173
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Selenium effects on glutathione peroxidase and the inmune rsponse of stressed calves challenged with pasteurella hemolytica. J. Anim. Sci. 67, 557–564. Revilla-Vázquez, A., Ramírez-Bribiesca, E., López-Arellano, R., Hernández-Calva, L.M., Tórtora-Pérez, J., García-García, E., Cruz-Monterrosa, R.G., 2008. Suplemento de selenio con bolos intrarruminales de selenito de sodio en ovinos. Agrociencia 42, 629–635. Richie Jr., J.P., Das, A., Calcagnotto, A.M., Aliaga, C.A., El-Bayoumy, K., 2012. Age related changes in selenium and glutathione levels in different lobes of the rat prostate. Exp. Gerontol. 47, 223–228. Rodinova, H., Kroupova, V., Travnicek, J., Stankova, M., Pisek, L., 2008. Dynamics of IgG in the blood serum of sheep with different selenium intake. Vet. Med. (Praha) 53, 260–265. Saini, R.K., Saini, N., Kataria, M., Babu, S., 2007. Effect of selenium and alpha-tocopherol on the antioxidant defense system of goat erythrocytes and the hemic system. Toxicol. Mech. Methods 17, 117–123. Saleh, N.S., Allam, T.S., 2014. Pneumonia in sheep: bacteriological and clinicopathological studies. Am. J. Res. Commun. 2, 73–88. Stefanowicz, F.A., Talwar, D., O’Reilly, D.S.J., Dickinson, N., Atkinson, J., Hursthouse, A.S., Rankin, J., Duncan, A., 2013. Erythrocyte selenium concentration as a marker of selenium status. Clin. Nutr. 32, 837–842. Stowe, H.D., Herdt, T.H., 1992. Clinical assessment of selenium status of livestock. J. Anim. Sci. 70, 3928–3933. Tekeli, H., Kiral, F., Bildik, A., Yilmaz, M., Kaçamakli, Z., 2015. Effect of aging on enzymatic and non-enzymatic antioxidant status in saanen goats. Vet. Zootech. 71, 67–71. Van Ryssen, J.B.J., Coertze, R.J., Smith, M.F., 2013. Time-dependent effect of selenium supplementation on the relationship between selenium concentrations in whole blood and plasma of sheep. Small Rumin. Res. 112, 85–90. Wu, X., Yao, J., Yang, Z., Yue, W., Ren, Y., Zhang, C., Liu, X., Wang, H., Zhao, X., Yuan, S., Wang, Q., Shi, L., Shi, L., 2011. Improved fetal hair follicle development by maternal supplement of selenium at nano size (Nano-Se). Livest. Sci. 142, 270–275. Yue, W., Zhang, C., Shi, L., Ren, Y., Jiang, Y., Kleemann, D.O., 2009. Effect of supplemental selenomethionine on growth performance and serum antioxidant status in taihang black goats. Asian-Aust. J. Anim. Sci. 22, 365–370.
post-dosing, without differences among the 3 groups; this could be related to the general oxidative status in which enzymatic and non-enzymatic antioxidants tend to modulate cellular ROS including Se supplementation. This finding coincides with another study in goats (Chung et al., 2007) but differs from other studies in goats and lambs, where it was shown that Se supplementation decreased the MDA content and improved the activity of antioxidants (Elsheikh et al., 2014). MDA is the main product of lipoperoxidation and is a good indicator to estimate oxidative stress (Wu et al., 2011). This result may be due to the increase in antioxidants directly related to Se and glutathione peroxidase (GPx) (Yue et al., 2009). In another study, Se supplementation in goats showed no difference in lipoperoxidation (Saini et al., 2007), but with levels of 0.3, 0.5, and 1.0 mg Se/kg LW, the MDA content decreased (Yue et al., 2009). Specifically, the ROS that are produced in the body damage the cellular structures, promoting an imbalance in oxidative stress in the organism that can cause disease (Birben et al., 2012). Antioxidants maintain the integrity of the cells that induce the immune response (Chew, 1996), and Se, which is incorporated into selenoproteins, has antioxidant functions. In this case, the Se deficiency that was observed in the non-supplemented Se goat kids had a negative impact on the activation of the immune cell response and increased oxidative stress levels (Huang et al., 2012). The antigenic response of the M. haemolytica-specific IgG increased with time post-dosing. Dosing with Se by both pathways improved IgG levels compared with the CG. Studies in lambs showed an increase in non-specific IgG levels when a dose of Se was given to their mothers before delivery (Rodinova et al., 2008), and in camels, the IgG response also improved when they were supplemented with slow-release intraruminal boluses (Alhidary et al., 2016). 5. Conclusions The immune response to Mannheimia haemolytica was enhanced and cell-modulated responses were promoted in order to improve redox status in Se supplemented goats subcutaneously and orally. The evaluation of oxidative stress levels measured by the amount of GSH, CAT and MDA activity showed significant differences (p < 0.05) with the control group during the first 14 days after dosing with Se. The results suggest that preventive defense systems in immunized goats can be achieved by enzymatic or non-enzymatic mechanisms, including reduced glutathione (GSH) and catalase (CAT). Acknowledgements The authors thank the CONACyT for the doctoral scholarship number349865 and the financial support obtained through the program PAPIIT IN218115 of DGAPA – UNAM. References Alam, M.N., Bristi, N.J., Rafiquzzaman, M., 2013. Review on in vivo and in vitro methods evaluation of antioxidant activity. Saudi Pharm. J. 21, 143–152. Alhidary, I.A., Abdelrahman, M.M., Uallh Khan, R., Harron, R.M., 2016. Antioxidant status and immune responses of growing camels supplemented a long-acting multitrace minerals rumen bolus. Ital. J. Anim. Sci. 15, 343–349. Birben, E., Sahiner, U.M., Sackesen, C., Erzurum, S., Kalayci, O., 2012. Oxidative stress and antioxidant defense. World Allergy Organ. J. 5, 9–19. Capelo, J.L., Fernandez, C., Pedras, B., Santos, P., Gonzalez, P., Vaz, C., 2006. Trends in selenium determination/speciation by hyphenated techniques based on AAS or AFS. Talanta 68, 1442–1447. http://dx.doi.org/10.1016/j.talanta.2005.08.027. Celi, P., Trana, A., Di Claps, S., 2010. Effects of plane of nutrition on oxidative stress in goats during the peripartum period. Vet. J. 184, 95–99. Chew, B.P., 1996. Importance of antioxidant vitamins in immunity and health in animals. Anim. Feed Sci. Technol. 59, 103–114. Chung, J.Y., Kim, J.H., Ko, Y.H., Jang, I.S., 2007. Effects of dietary supplemented inorganic and organic selenium on antioxidant defense systems in the intestine serum, liver and muscle of Korean native goats. Asian-Aust. J. Anim. Sci. 20, 52–59. Elsheikh, A.H., Al-Hassan, M.J., Mohamed, H.E., Abudabos, A.M., 2014. Effect of injectable sodium selenite on the level of stress biomarkers in male aardi goats. Indian J. Anim. Res. 48, 239.
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Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Dose of selenium in goat kids and its effect on the antigenic response to Mannheimia haemolytica and oxidative stress
MARK
Víctor M. Díaz-Sáncheza, Gabriela Rodríguez-Patiñoa, Patricia Ramírez-Nogueraa, J. Efrén Ramírez-Bribiescab, José F. Morales-Álvarezc, Alma L. Revilla-Vázqueza, ⁎ Raquel López-Arellanoa, a b c
National Autonomous University of Mexico, Cuautitlán, Multidisciplinary Research Unit, Cuautitlán Izcalli, Edo. México, CP 54714 Mexico College of Postgraduate, Livestock Program. Montecillo, Texcoco, Edo. México, CP 56230 Mexico National Institute of Forestry Research, Animal Health, Coyoacán, México, CP 0410 Mexico
A R T I C L E I N F O
A B S T R A C T
Keywords: Selenium Goat kids Antigenic response Oxidative stress
Selenium (Se) prevents oxidative damage and stimulates the immune system. Currently, there are no data available evaluating the Se-induced antigenic response to Mannheimia haemolytica and oxidative stress in goat kids. Twenty-one 6-month-old male Alpine Goat kids (22.9 kg) were immunized against M. haemolytica and divided into 3 groups: Basal diet with no additional Se (CG); Se injected subcutaneously at 0.25 mg Se/kg live weight (LW) (SeSG); and Se administered as intraruminal bolus at 0.46 mg Se/kg LW (SeBG). Blood samples were taken from the animals in all groups at 0, 14, 28, 42 and 56 days post-dosing. Erythrocyte Se levels doubled in SeSG during days 14–28 post-dosing (130.6 ng/g Se) as compared to CG. During days 28–42 post-dosing, Se levels decreased (P < 0.05) in SeSG (106.9 ng/g Se) vs. CG and SeBG (86.2 ng/g Se and 81.7 ng/g Se, respectively). Glutathione (GSH) levels decreased in days 0–28 post-dosing (3.7E–4 nmols/mg of total protein) and then, remained stable up to day 56 (5.6E–4 nmols/mg of total protein). Catalase levels in the SeBG and SeSG (79.9 U) were higher (P < 0.05) than in the CG (56.3 U) by day 14 post-dosing. Malondialdehyde (MDA) levels on day 14 post-dosing increased 7-fold in the SeBG (1.28E–3 ng/mg of total protein). IgG levels increased in the groups treated with Se during days 28–42 post-dosing (1.4 nM) vs. the CG (0.10 nM) (P < 0.05). Treatment with Se improved the immunological response to M. haemolytica.
1. Introduction Infectious diseases and dietary deficiencies are two critical factors causing great losses in goat herds from birth to puberty (RamírezBribiesca et al., 2001). The reported etiologies of pneumonias in goats are Mycoplasma ovipneumoniae, Pasteurella multocida, Klebseilla pneumoniae, Staphylococcus aureus, Shigella spp., E. coli (Saleh and Allam, 2014), and M. haemolytica, which can cause mortality rates in goat kids from 30 to 60% (Mohamed and Abdelsalam, 2008). Specifically, M. haemolytica has been commonly isolated from samples of ruminants with pneumonia in Mexico (Jaramillo-Arango et al., 2009). Consequently, it is necessary to induce a good antigenic response to this microorganism. Selenium (Se) supplementation can stimulate humoral immunity (Hall et al., 2013) through the increase in immunoglobulin G (IgG) levels in bovines immunized against pasteurellosis (Rice et al., 2007). Nevertheless, Se deficiency has been observed in a large part of
the Mexican territory, especially in the Plateau region, which can cause health problems in deficient animals besides the white muscle disease (Ramírez-Bribiesca et al., 2005). Infection by several respiratory pathogens increases production of reactive oxygen species (ROS), damaging cell membranes (Khanal and Knight, 2010). An imbalance between oxidants and antioxidants in favor of the former is called “oxidative stress”. There are numerous lines of defense against ROS, including low molecular weight radicals/ oxidants such as glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) (Rahmanto and Davies, 2012). Oxidative stress and damage to cell membranes produces malondialdehyde (MDA) as degradation product (Grotto et al., 2009). The joint presence of respiratory disease and Se deficiency in the organism induces oxidative stress (Hefnawy and Tórtora-Pérez, 2010). The application of a subcutaneous dose of Se or administration of intraruminal boluses induces an increase in blood Se levels of neonatal ruminants
⁎ Correspondence to: Laboratory of Pharmaceutical Development Tests Faculty of Higher Cuautitlán Studies, Campus 4 UNAM 54714 Cuautitlán Izcalli, State of Mexico Mexico. Tel. +525556231999 extension 39415. E-mail address: [email protected] (R. López-Arellano).
http://dx.doi.org/10.1016/j.smallrumres.2017.06.005 Received 19 February 2017; Received in revised form 2 June 2017; Accepted 4 June 2017 Available online 23 June 2017 0921-4488/ © 2017 Published by Elsevier B.V.
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after 20 days post-dosing (Ramírez-Bribiesca et al., 2005; RevillaVázquez et al., 2008). Currently, there are no published papers evaluating the antigenic response against M. haemolytica in goat kids and their association with Se levels. The aim of this study was to evaluate the antigenic response against M. haemolytica through the presence of IgG-specific antibodies and their relationship with oxidative stress markers after administering Se to goat kids as a subcutaneous dose or by oral bolus dosing. 2. Materials and methods 2.1. Standard of ethics and experimental welfare The animals used for this study were handled under the rules of the Internal Committee for the Care and Use of Experimental Animals approved by the Cuautitlán-UNAM, under registration code CICUAE-FESC C15_03.
Fig. 1. Levels of erythrocyte selenium in goat kids with or without selenium supplementation.
completely randomized factorial design (CG, SeSG, and SeBG). The levels of Se, GSH, CAT, MDA and IgG against Mannheimia haemolytica were selected as the response variables. A comparison of means was performed with the Tukey test considering a significant statistical difference (p < 0.05).
2.2. Reception, management, and distribution of the experimental groups Twenty-one 6-month-old male Alpine Goat kids were selected from Cuautitlán Agricultural Farm. They were housed in 3 corrals (each one with an area of 7 m2) with a bed of shavings. The animals were fed as a group with sweet alfalfa [93% dry matter (DM) and 17.12% crude protein (CP)] containing 1.11 μgSe/g of Se. Water was given ad libitum, and the animals were dewormed with Closantel (10 mg/kg LW). On day 15, all the animals were immunized against M. haemolytica by subcutaneously administering 2.5 mL of bacterin-toxoid (Toxo-BacINIFAP®, Mexico, 1 × 109 colony forming units/mL, containing the leukotoxin). On the day after the vaccination, all the goat kids were weighed on a digital scale (Torrey®) and sorted into 3 groups based on their body weight, with each group averaging 22.91 ± 4.15 kg. These experimental groups received the following treatments: 1) Control group (CG), did not receive Se supplementation; 2) Subcutaneous Se dose group (SeSG), 0.25 mg Se/kg LW (as NaSeO3. VALNO, lot: 0347C7); and 3) Intrarruminal Se bolus group (SeBG), 0.46 mg Se/kg (as NaSeO3). Both Se products were manufactured in the pharmacy laboratory of FESC-UNAM. Specifically, the Se bolus were prepared with a densifier (reduced iron), a binder (cutin), and a sliding agent (talc) (Revilla-Vázquez et al., 2008) to be degraded in a maximum time of 4 days. Blood samples were taken before the dosing and on day 14 pst-dosing for 4 periods. Blood extraction was performed by venipuncture of the external jugular vein using 20 G-X 38 mm needles and 6 mL Vacutainer® BD vacuum tubes with EDTA anticoagulant. The blood samples were centrifuged at 4000 rpm for 15′ to separate the plasma from the erythrocyte pack and were stored at −80 °C until their analysis.
3. Results The data are presented as figures showing the means and their standard deviations in the 3 experimental groups. Fig. 1 shows the erythrocyte Se levels. On days 0–14 post-dosing, the average Se levels remained low in the 3 groups (81.12 ± 1.23 ng/g Se and 73.36 ± 7.65 ng/g Se, respectively; P > 0.05). During the days 14–28 post-dosing, the Se levels doubled (P < 0.05) in SeSG and SeBG (130.66 ± 29.77 ng/g Se), while the CG maintained an average Se level below 95 ng/g. During the days 28–42 period post-dosing, the levels of Se decreased (P < 0.05) between SeSG (106.93 ± 15.19 ng/ g Se) vs. CG and SeBG (86.22 ± 12.37 ng/g Se and 81.69 ± 10.19 ng/g Se, respectively). On day 42 post-dosing, an interaction effect (P < 0.05) was observed between the CG and the SeBG. On day 56 post-dosing, an average of 108.87 ± 3.76 ng Se/g (P > 0.05) was obtained among the groups. Fig. 2 shows the GSH content in the erythrocytes. On day 0, the GSH levels in the 3 groups averaged 9.6E–4 ± 1.118E–5 nmols/mg of total protein (P > 0.05). GSH levels decreased during days 0–28 (3.7E−4 ± 4.714E−6 nmols/ mg of total protein) in SeBG and SeSG, and then, they remained stable up to day 56 (5.6E−4 ± 8.49E–5 nmols/mg of total protein). An interaction effect was observed (P < 0.05) between CG and the SeSG and SeBG groups from day 14 up to day 45 post-dosing. Fig. 3 shows the CAT content in the erythrocytes. The mean CAT level at the beginning of the experiment was 73.10 ± 1.85 U. On day 14, the CAT levels in SeBG and SeSG were higher (P > 0.05; 79.90 ± 3.93 U) than in the CG (56.33 ± 1.69 U). Subsequently, from day 14 to day 28 post-
2.3. Laboratory analysis The hematological samples were analyzed with the following tests. Se levels in the erythrocytes: The samples were analyzed according the modified Capelo method (Capelo et al., 2006). Reduced glutathione (GSH) levels in the erythrocytes: GSH was determined according the modified Eyer method, sulfosalicylic (Eyer and Podhradský, 1986). Catalase (CAT) activity in the erythrocytes: A rapid test as described by Iwase and colleagues (Iwase et al., 2013) was used. Measurement of malondialdehyde (MDA) levels: It was conducted according to the modified methods previously described (Lykkesfeldt, 2001). Concentration of immunoglobulin G (IgG) – ELISA test: The IgG concentration was estimated according to the method described by Morales (Morales-Alvarez et al., 1993). 2.4. Statistical analysis The treatments were assigned to each group using a 3-level
Fig. 2. Levels of erythrocyte GSH in goat kids with or without selenium supplementation.
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during days 14–28, and then, they increased from day 28–42, showing a higher (P < 0.05) level in SeBG (1.43 ± 0.02 nm) vs. CG (0.10 ± 0.09 nm). On day 56, there no difference was observed (P > 0.05) in the IgG absorbances among the 3 groups (0.96 ± 0.12 nm). 4. Discussion The proper level of Se in the serum or blood plasma ranges from 90 to 110 ng/mL (Stowe and Herdt, 1992). If the hematic Se content is as low as 30–80 ng/g, it indicates selenodeficiency (Van Ryssen et al., 2013), which can lead to health disorders and death in newborn goat kids (Ramírez-Bribiesca et al., 2005). In this study, a variability of 25% was initially observed plasma Se levels. To overcome this sample variability, erythrocyte Se content was measured instead. The erythrocytes contain 73% of the Se in the hematic tissue (Maas et al., 1992); therefore, they reflect a more reliable status of this micromineral in the prevention of oxidative damage by GSH activity (Gutzwiller, 1998). In addition, false values are excluded in the determinations of Se in the erythrocytes because none of the samples had hemolyzed, a process that alters the Se content (Qin et al., 2007). Before dosing with Se, all the 6-month old goat kids had an average content of Se of 84.11 ng/g of erythrocytes; a value which indicates a mild selenodeficiency, since none of the animals showed clinical signs of insufficiency. This content of erythrocyte Se was expected due to the lack of Se in the soil and forage located at the area of study (Ramírez-Bribiesca et al., 2005). A high dose of Selenium was considered for the oral bolus administration due to the incorporation of this micromineral in tissues and ruminal microorganisms in the intestinal tract before reaching a measurable blood concentration. Consequently, a lower dose of Selenium was used in the subcutaneous administration due to a quicker incorporation into the Se-dependent enzymes located in the endothelium. This approach was the reason for initially believing in possible differences in the erythrocyte Se levels between both administration routes; however, the results showed similarity. On the other hand, a previous study in bovines demonstrated a correlation between Se levels and the immune response against Pasteurella sp., showing an association between the level of Se in the hematic tissue and the immune response (Reffett Stabel et al., 1985). This hypothesis is more viable in animals that do not lack Se, while selenodeficient animals must first satisfy the need for the microelement in the tissues of the organism and ultimately reflect their status in the hematic tissue (Stefanowicz et al., 2013). In this study, we estimated GSH concentration, thiobarbituric reactive species and Catalase activity in order to know the general redox status after Se supplementation. When levels of oxidative stress increase and the levels of Se decrease, cellular lipoperoxidation occurs and GSH levels increase (Richie et al., 2012). On day 28 post-dosing, a higher level of GSH was found in CG as an acute response. However, this level was lower in the erythrocytes compared with the one found in another study performed on goat blood (Tekeli et al., 2015). Se deficiency influences the concentration of GSH (Celi et al., 2010) and the antioxidant activity of CAT in rats and rabbits (Olisekodiaka et al., 2015). Our study showed a significant difference on day 14: the supplemented groups (SeSG and SeBG) had the highest CAT levels, but after this time point, there were no differences among the groups. MDA is an indicator of oxidative stress, which was measured through the thiobarbituric reactive test, which indicates cellular damage due to lipoperoxidation. The estimation of CAT activity and the levels of MDA are considered good markers of cellular oxidation (Alam et al., 2013). The levels of MDA did not change in the study groups during the progression of the experiment, but on day 14 post-dosing, the goat kids showed a higher content of MDA, indicating a lipoperoxidation effect (Kohen and Nyska, 2002). Evidences show that dietary supplementation of both inorganic and organic Se do not influence MDA level in liver microsomes either (Chung et al., 2007). In this study, the levels of MDA increased at day 14 and then decreased on day 28
Fig. 3. Erythrocyte catalase units in goat kids with or without selenium supplementation.
Fig. 4. Levels of plasma MDA in goat kids with or without selenium supplementation.
Fig. 5. Absorbances of serum IgG in goat kids with or without selenium supplementation.
dosing, the CAT levels decreased in all groups (P > 0.05). During days 28–56 post-dosing, the CAT levels fluctuated by amounts of less than 55 units, and there were interactions observed among CG vs. SeBG vs. SeSG (P < 0.05) during days 26–49 post-dosing. Fig. 4 shows the plasma MDA. The mean of the 3 groups on day 0 was 8.20E−5 ± 3.53E−5 ng/mg of total protein (P > 0.05). After day 14 post-dosing, the MDA level in SeBG increased 7-fold (P < 0.05; 1.28E–3 ng/mg of total protein), and a 3-fold increase was observed in SeSG (P < 0.05; 7.00E−4 ng/mg of total protein). In the CG, the MDA level increased by 4-fold, and there was no difference (P > 0.05) with the groups supplemented with Se. On day 28 post-dosing, all the groups showed a decrease of MDA levels to baseline, averaging 1.48E–4 ± 8.52E–5 ng/mg of total protein. Subsequently, from day 28 to day 56, the levels in SeBG and SeSG increased linearly, without exceeding a level of 8.5E–4 ng/mg of total protein. There was an interaction effect observed (P < 0.05) for CG vs. SeBG and SeSG. Fig. 5 shows the absorbance of blood serum IgG. During days 0–14, there was an increase of IgG levels in all groups. The IgG levels were maintained 173
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post-dosing, without differences among the 3 groups; this could be related to the general oxidative status in which enzymatic and non-enzymatic antioxidants tend to modulate cellular ROS including Se supplementation. This finding coincides with another study in goats (Chung et al., 2007) but differs from other studies in goats and lambs, where it was shown that Se supplementation decreased the MDA content and improved the activity of antioxidants (Elsheikh et al., 2014). MDA is the main product of lipoperoxidation and is a good indicator to estimate oxidative stress (Wu et al., 2011). This result may be due to the increase in antioxidants directly related to Se and glutathione peroxidase (GPx) (Yue et al., 2009). In another study, Se supplementation in goats showed no difference in lipoperoxidation (Saini et al., 2007), but with levels of 0.3, 0.5, and 1.0 mg Se/kg LW, the MDA content decreased (Yue et al., 2009). Specifically, the ROS that are produced in the body damage the cellular structures, promoting an imbalance in oxidative stress in the organism that can cause disease (Birben et al., 2012). Antioxidants maintain the integrity of the cells that induce the immune response (Chew, 1996), and Se, which is incorporated into selenoproteins, has antioxidant functions. In this case, the Se deficiency that was observed in the non-supplemented Se goat kids had a negative impact on the activation of the immune cell response and increased oxidative stress levels (Huang et al., 2012). The antigenic response of the M. haemolytica-specific IgG increased with time post-dosing. Dosing with Se by both pathways improved IgG levels compared with the CG. Studies in lambs showed an increase in non-specific IgG levels when a dose of Se was given to their mothers before delivery (Rodinova et al., 2008), and in camels, the IgG response also improved when they were supplemented with slow-release intraruminal boluses (Alhidary et al., 2016). 5. Conclusions The immune response to Mannheimia haemolytica was enhanced and cell-modulated responses were promoted in order to improve redox status in Se supplemented goats subcutaneously and orally. The evaluation of oxidative stress levels measured by the amount of GSH, CAT and MDA activity showed significant differences (p < 0.05) with the control group during the first 14 days after dosing with Se. The results suggest that preventive defense systems in immunized goats can be achieved by enzymatic or non-enzymatic mechanisms, including reduced glutathione (GSH) and catalase (CAT). Acknowledgements The authors thank the CONACyT for the doctoral scholarship number349865 and the financial support obtained through the program PAPIIT IN218115 of DGAPA – UNAM. References Alam, M.N., Bristi, N.J., Rafiquzzaman, M., 2013. Review on in vivo and in vitro methods evaluation of antioxidant activity. Saudi Pharm. J. 21, 143–152. Alhidary, I.A., Abdelrahman, M.M., Uallh Khan, R., Harron, R.M., 2016. Antioxidant status and immune responses of growing camels supplemented a long-acting multitrace minerals rumen bolus. Ital. J. Anim. Sci. 15, 343–349. Birben, E., Sahiner, U.M., Sackesen, C., Erzurum, S., Kalayci, O., 2012. Oxidative stress and antioxidant defense. World Allergy Organ. J. 5, 9–19. Capelo, J.L., Fernandez, C., Pedras, B., Santos, P., Gonzalez, P., Vaz, C., 2006. Trends in selenium determination/speciation by hyphenated techniques based on AAS or AFS. Talanta 68, 1442–1447. http://dx.doi.org/10.1016/j.talanta.2005.08.027. Celi, P., Trana, A., Di Claps, S., 2010. Effects of plane of nutrition on oxidative stress in goats during the peripartum period. Vet. J. 184, 95–99. Chew, B.P., 1996. Importance of antioxidant vitamins in immunity and health in animals. Anim. Feed Sci. Technol. 59, 103–114. Chung, J.Y., Kim, J.H., Ko, Y.H., Jang, I.S., 2007. Effects of dietary supplemented inorganic and organic selenium on antioxidant defense systems in the intestine serum, liver and muscle of Korean native goats. Asian-Aust. J. Anim. Sci. 20, 52–59. Elsheikh, A.H., Al-Hassan, M.J., Mohamed, H.E., Abudabos, A.M., 2014. Effect of injectable sodium selenite on the level of stress biomarkers in male aardi goats. Indian J. Anim. Res. 48, 239.
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