Effects of Feeding Rumen-Protected Choline and Vitamin E on Serum Protein Fractions, Total Thiol Molecules and Total Antioxidant Capacity in Early Lactating Dairy Cows
Mohammadreza Rahmani*, Mehdi Dehghan-banadaky, Romic Kamalyan, Hassan Malekinejad, Fatemeh Rahmani, Mohammad Hossein Hadi Tavatori, Hamed Mohammadi
* Corresponding author: Mohammadreza Rahmani,Email: m.rahmani@live.com
ABSTRACT
Twenty four primiparous and multiparous Holstein cows on early lactation, beginning five weeks postpartum, were used for four weeks to investigate the effects of supplementation of rumen-protected choline (RPC) or vitamin E on blood serum protein fractions, plasma total thiol molecules (TTM), and plasma total antioxidant capacity (TAC). Cows were randomly assigned to one of the following treatments: I - no supplement (control), II - 90 g/d of RPC, and III - 4400 IU/d of vitamin E. Serum protein electrophoresis of samples exhibited four main fractions in the blood serum of the cows including: albumin, α, β, and γ. The electrophoresis was carried out by capillary zone electrophoresis (CZE). In this study, feeding RPC or vitamin E affected the blood serum albumin fraction as well as blood plasma TTM (P<0.05)but the treatments did not affect the different fractions of globulin as well as plasma TAC (P>0.05). The results showed that the increases in serum albumin fractions and TTM which observed in this study, pointed towards a beneficial role of RPC and vitamin E in early lactating dairy cows.
Keywords: Electrophoresis, Serum Proteins, Choline, Vitamin E, Dairy cow.
INTRODUCTION
Normal plasma or serum protein electrophoresis leads to identification of two major protein fractions: albumin and globulin. In humans, sheep, goats, rabbits, dogs, guinea pigs and rats, albumin predominates over globulin while in horses and cows the ratio of albumin and globulin is nearly equal, or globulin predominates (Swenson, 1993). In addition to such species characteristics, there are evidences that some physiological factors, namely: hormones, sexual influences, pregnancy, lactation (Nath et al., 2005; Pourouchottamane et al., 2005; Richard Jagatheesan et al., 2005), nutritional state and many other conditions (dehydration, hemorrhage,
liver and kidney dysfunctions, and inflammatory processes) affect serum protein level (Doxey, 1983; Coles, 1986).
Albumin is essential due to its contribution in the maintenance of osmotic pressure of plasma, because it is carrier of many vital substances like steroid hormones, hemin and fatty acids. The albumin value frequently and markedly declines during different diseases. Globulin fraction contains enzymes, hormones and antibodies, which are synthesized at various places in the body (Nicholson et al., 2000). The α-globulin value increases mainly
in traumas, and some alterations of the lipoprotein metabolism induce changes in the β-globulin fraction. γ-globulin concentration is a reliable indicator of humoral immunity. Its principal component is IgG but other isotypes of antibodies are also present in this fraction (Goldsby et al., 2001). Blood proteins, with the exception of the γ-globulin fraction, are synthesized in liver (Diehl and Delincee., 1986).
To measure classical protein fractions in serum several electrophoretic techniques are available, like separation on cellulose acetate membrane, agarose gel, etc. but capillary zone electrophoresis (CZE) has been suggested as a useful technique for separation and quantification of serum proteins (Henskens et al., 1998).
Total thiol molecules (TTM) are powerful reducing agents capable of acting antioxidants (Ueland et al., 1996). TTM status is important for normal physiological function. Changes in TTM status have been linked to induction of apoptosis (programmed cell death) (Marchetti et al., 1997), and have been observed in a number of diseases including vascular disease and renal failure (Ueland et al., 1996).
Free radicals can be produced during the respiratory oxidation of different cells. Since free radicals can damage various macromolecules as protein, fat, nucleic acids, etc. they are harmful for body (Jamro and Beltwoski, 2002). The natural defense system, which can prevent the damage of free radicals and neutralize them, has been referred to as total antioxidant capacity (TAC) (Kankfer and Lipko, 2006).
Choline is in the structure of lipoproteins which transport lipids in the blood, thus it is an important factor in preventing fatty liver and ketosis in lactating cows (Cooke et al., 2007). Unfortunately most of dietary choline is degraded by microbial populations in the rumen (Sharma and Erdman, 1989), and not much is available for absorption; therefore, choline must be in rumen-protected form when fed. Choline can also be used as an antioxidant (Elsawy et al., 2014) because it has significant antioxidant properties that protects cells (Jansen, 2014). In a research, dietary choline decreased the oxidant damage and regulated the antioxidant system in immune organs of fish (Wua et al., 2014).
Vitamin E (α-tocopherol) is a powerful antioxidant for body defense against oxidative stress (Burton and Traber, 1990; Ibrahim et al., 1997) and is not degraded in the anaerobic ruminal environment (Burton and Traber, 1990; Leedle et al., 1993). In peripartum and early lactating cows, lipid peroxidation increases (Castillo et al., 2005) while serum a-tocopherol decreases (LeBlanc et al., 2004) indicating a higher level of oxidative stress which subsequently can lead to reduced health in dairy cows (Miller et al., 1993). The role of vitamin E in recovering from postpartum-related oxidative stress and decrease in lipid peroxidation in liver has been reported in cattle, mice and rats (Ferre et al., 2001; Bouwstra et al., 2008). In a study, supplemental vitamin E could improve liver antioxidant status in mice with fatty liver (Soltys et al., 2001).
The present study was carried out to compare the oxidative status of dairy cows on early lactation which received either supplemental RPC or vitamin E or those unsupplemented, and also to assess the changes in serum protein fractions.
MATERIALS AND METHODS
Cows, treatments and experimental design
Twenty four early lactating primiparous and multiparous Holstein cows beginning five weeks postpartum (BCS = 2.82 ± 0.12; mean ± S. D. and number of lactation = 2.56; mean) were used for four weeks from October 2011 to November 2011. The cows were free from any diseases, with a normal healthy appearance, and were housed in individual tie stalls. All experimental procedures were in accordance with the guidelines for the use and care of experimental animals and approved by the animal ethical committee of Tehran University. Selection of the cows was based on parity, milk yield of previous lactation (milk yield of dams for the cows in their first lactation) and BCS. In this study, there were 3 blocks based on lactation numbers 1, 2, and 3 or greater. Lactation number was indication of age. Eight cows per treatment were randomly assigned to receive one of the following treatments, using block randomization based on parity: I- no supplement (control), II- 90 g/d of RPC and III- 4400 IU/d of vitamin E. The RPC (Reashure Choline, Balchem, USA; 25%) was a rumen protected source of choline chloride, and the vitamin E was the product of Roche Company (Vitamins Ltd; Switzerland). The cows were fed total mixed rations (TMR) ad libitum. The diet (Table 1) was formulated to meet the nutritional requirements of dairy cows (NRC, 2001). The RPC and vitamin E were top dressed onto the TMR.
Blood Sampling
Blood samples were obtained before morning meal from the coccygeal vein (tail vein) on the last day of the experiment, by using heparinized and non-heparinized Vacutainers tubes (Becton Dickinson, Franklin Lakes, NJ). Blood samples were placed on ice immediately following collection. Then plasma and serum were harvested after centrifugation of the blood at 3000 g for 15 min and were stored at -20 °C until subsequent analyses. The indices of oxidative status including TTM and TAC concentration were analyzed in plasma samples, and the different fractions of blood serum protein were analyzed in serum by CZE.
Capillary Zone Electrophoresis
The Capillary system (Sebia, Issy-les-Moulineaux, France) was operated according to the manufacturer’s instructions under software version 1.4.1. The instrument has eight fused silica capillaries (17 cm in length and 25 μm ID). The alkaline buffer (borate and additives) is pH 10 and sample is diluted 1:10. Detection voltage is 9 kV. Separation is carried out at 35°C and takes 2.5 min. Ultraviolet detection at 200 nm is used for direct quantification of the peptide bonds. When the samples are analyzed in batch, capillary has a throughput of 100 samples/h. A typical electrophoretic pattern for blood serum of a cow is shown in Figure. 1. The proteins were measured at 200 nm wavelength. Protein values were expressed as percentages.
Assessment of plasma total antioxidant capacity (TAC)
TAC of plasma was evaluated by applying the FRAP assay (ferric reducing antioxidant power or ferric reducing ability of plasma) (Benzie and Strain, 1999). The method is based on the reduction of ferric (Fe3+) to ferrous (Fe2+) ion at low pH. This causes a formation of blue colored ferroustripyridyltriazine (Fe2+-TPTZ) complex, which absorbs at 593nm. Absorbance changes are linear over a wide concentration range with antioxidant mixtures, including plasma (Liu et al., 1982; Benzie and Strain, 1999). Results were expressed as mmol/l (mM). The method could be described in brief as the following: the working FRAP reagent was prepared ex tempore by mixing 300 mmol/l acetate buffer, pH 3.6 with 2,4,6-tripyridyl-striazine (TPTZ) solution (10 mM in 40 mM HCl) and 20 mmol/l FeCl3 solution in ratio10:1:1 respectively, and was pre-tempered at 37°C. The reaction was performed by adding of 100 μl plasma, previously diluted 1:1 with distilled water, to 900 μl FRAP working reagent and the mixture was incubated for 25 min at 37°C. The absorbance was measured on 593nm compare to a blank mixture where 100 μl water was added to the working FRAP reagent instead of plasma. Aqueous solutions of known Fe2+ concentration, in range 0.2 to 1 mmol/l (FeSO4. 7H2O) were used for creating of the standard curve. The results were expressed in mmol/l (mM) Fe2+.
Measurement of Plasma Total Thiol Molecules (TTM)
Total sulfhydryl content was determined in plasma by the method of Hu (Hu and Dillared, 1994). A volume of plasma (0.20 ml) was mixed in a 10 ml test tube with 0.6 ml of Tris–EDTA buffer (Tris base 0.25 M, EDTA 20 mM, pH 8.2) followed by the addition of 40 ml of 10 mM of DTNB (Dithiobis-2-nitrobenzoic acid) in methanol. The final volume of the reaction mixture was made up to 4.0 ml by adding 3.16 ml of methanol. The test tube was capped, and the color was developed for 15–20 min, followed by centrifugation at 3000 g for 10 min at ambient temperature. The absorbance of the supernatant was measured at 412 nm. The TTM capacity was expressed as nmol per mg of protein in samples.
Statistical Analyses
Raw data were transformed to their natural logarithm to achieve a normal distribution for analysis. All transformed data were back-transformed for reporting least squares means. Statistical analyses were performed with SAS (SAS, 2002) using GLM procedure in SAS by inspection of standardized residuals plotted against the predicted residuals. Standardized residuals were also inspected graphically to assess fit to a normal distribution. Differences among means were separated with Duncan multiple range test. Each metabolite was considered as an outcome in separate models over the whole experimental period. Significant levels were declared at P<0.05.
RESULTS AND DISCUSSION
Cows undergo a variety of physiological changes during lactation. These occur with respect to the cows’ blood composition due to metabolic changes. In fact, the cows make adjustments to provide an adequate supply of nutrients for producing milk while lactation (Drackley, 1999).
In our study, we fed the experimental cows 90 g/d of RPC, because according to some researches feeding 90 g/d of choline would be optimal dose in lactating dairy cows (Sharma and Erdman, 1989; Xu et al., 2006). In an experiment, oral administration of 800 IU/d of vitamin E resulted in significant improvements in liver function in people with non-alcoholic fatty liver disease (Sanyal et al., 2010), thus we decided to feed 4400 IU/d of vitamin E because a cow’s body weight is approximately 5.5 times heavier than a human’s body weight.
In this research, serum protein electrophoresis of the samples separated into four major fractions: albumin, α-globulin, β-globulin, and γ-globulin (Table 2). The treatments affected albumin fraction (P<0.05), but not different fractions of globulin (P>0.05).
ab different superscripts are indicating significant differences (P<0.05) between the various study groups
Albumin is associated with postpartum diseases and can be used to predict disease risks in early lactation period (Saun, 2004). In spite of concerns about variables confounding albumin interpretation, it seems to be a good disease risk indicator possibly reflecting availability of amino acids from the labile protein pool. In our study, feeding RPC or vitamin E affected albumin fractions (P<0.05). Therefore, due to increases in albumin fractions of the treated cows we could speculate that the treated cows might be more resistant against various diseases compared with the control group.
Generally at the beginning of lactation cycle, the blood level of NEFA, are elevated mainly due to negative energy balance which would result in a reduced performance of the liver (Overton and Waldron, 2004). Choline reduces NEFA in blood stream due to donation of methyl groups which may lead to improving liver function (Cooke et al., 2007; Soltan et al., 2012). Some researchers have reported that the blood metabolites, like total protein, albumin and globulin, in cows and goats were not affected by choline supplementation (Ambrosio et al., 2007; Toghdory et al., 2007; Mohsen et al., 2011).
In an experiment on ewes, the data demonstrated that receiving vitamin E, starting two weeks before mating and extending through pregnancy till occurrence of lambing, improved levels of albumin, globulin and total serum protein in treated ewes (El-Shahat and Monem, 2011). Similar finding was obtained by other researches in buffaloes (Helal et al., 2009). In a study on vitamin E deficient rabbits, total plasma protein concentration was not significantly affected by vitamin E deficiency, but albumin levels were lower and globulin levels were higher in deficient animals (Diehl and Delincee., 1986).
In this research, supplementation of RPC or vitamin E affected the concentrations of TTM (P<0.05), but the treatments did not affect TAC (P>0.05; Table 2).
TTM are organic compounds that contain a sulphydryl group. Among all the antioxidants that are available in the body, thiols constitute the major portion of the total body antioxidants and play a significant role in defense against reactive oxygen species. Albumin is exclusively synthesized by the liver, and it is the main source of plasma thiols. Glutathione is mainly synthesized de novo within the liver (Jefferies et al., 2003). The reduction of liver function that is usually observed in the early lactating cows might explain lower plasma thiol levels (Bernabucci et al., 2005).
Considering additional data from literature (Goff and Stabel, 1990; Goff and Horst, 1997), the reduction of vitamins E and A in plasma might help to explain the alteration of the oxidative status after calving. In this regard, some studies have demonstrated that, besides enhancing plasma level of fast-acting antioxidants, the supplementation of vitamin E can be useful against oxidative stress in early lactating dairy cows (Weiss et al., 1990; Brzezinska-Slebodzinka et al., 1994).
CONCLUSION
From the results of the present study, we can conclude that the increases in serum albumin fraction and TTM which observed in both RPC and vitamin E groups pointed towards a beneficial role of RPC and vitamin E.
ACKNOWLEDGMENT
The authors acknowledge the valuable technical assistance of Dr. M.H. Ansari.
REFERENCES
Ambrosio, F.D., A. Campagnoli, F. Susca, E. Fusi, R. Rebucci, A. Agazzi, L. Pinotti and A. Baldi. 2007. Effects of rumen-protected choline supplementation in periparturient dairy goats. Vet. Res. Commun. 31: 393-396.
Benzie, I.F., and J.J. Strain. 1999. Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol. 299: 15-27.
Bernabucci, U., B. Ronchi, N. Lacetera and A. Nardone. 2005. Influence of body condition score on relationships between metabolic status and oxidative stress in priparturient dairy cows. J. Dairy Sci. 88: 2017–2026.
Bouwstra, R.J., R.M.A. Goselink, P. Dobbelaar, M. Nielen, J.R. Newbold and T. Vanwerven. 2008. The Relationship Between Oxidative Damage and Vitamin E Concentration in Blood, Milk, and Liver Tissue from Vitamin E Supplemented and Nonsupplemented Periparturient Heifers. J. Dairy Sci. 91: 977-987.
Brzezinska-Slebodzinka, E., J.K. Miller, J.D. Quigley, J.R. Moore and F.C. Madsen. 1994. Antioxidant status of dairy cows supplemented prepartum with vitamin E and selenium. J. Dairy Sci. 77: 3087–3095.
Burton, G.W., and M.G. Traber. 1990. Vitamin E: antioxidant activity, biokinetics, and bioavailability. Annu. Rev. Nutr. 10: 357-382.
Castillo, C., J. Hemandez, A. Bravo, M. Lopez-Alonso, V. Pereira and J.L. Benedito. 2005. Oxidative status during late pregnancy and early lactation in dairy cows. Vet. J. 169: 286-292.
Coles, E.H. 1986. Veterinary clinical pathology. Saunders, Philadelphia, USA.
Cooke, R.F., R.N. Silva-Del, D.Z. Caraviello, S.J. Bertics, M.H. Ramos and R.R. Grummer. 2007. Supplemental choline for prevention and alleviation of fatty liver in dairy cattle. J. Dairy Sci. 90: 2413-2418.
Diehl, J.F. and H. Delincee. 1986. Vitamin E deficiency in rabbits receiving a high PUFA diet with and without a non-absorbable antioxidant II. Incorporation of 14C-labelled glycine and L-leucine into liver and plasma proteins. Z. Ern & hrungswiss. 25:180-188.
Doxey, D.L. 1983. Clinical pathology and diagnostic procedures, London, UK.
Drackley, J.K. 1999. Biology of dairy cows during the transition period: the final frontier? J. Dairy Sci. 82: 2259-2273.
El-Shahat, K.H. and U.M.A. Monem. 2011. Effects of Dietary Supplementation with Vitamin E and /or Selenium on Metabolic and Reproductive Performance of Egyptian Baladi Ewes under Subtropical Conditions. WASJ. 12: 1492-1499.
Elsawy, G., O. Abdelrahman and A. Hamza. 2014. Effect of choline supplementation on rapid weight loss and biochemical variables among female Taekwondo and Judo athletes. JHK. 40: 77-82.
Ferre, N., J. Camps, A. Paul, M. Cabre, L. Calleja, J. Osada and J. Joven. 2001. Effects of high-fat, low-cholesterol diets on hepatic lipid peroxidation and antioxidants in apolipoprotein E-deficient mice. Mol. Cell. Biochem. 218: 165–169.
Goff, J.P. and J.R. Stabel. 1990. Decreased plasma retinol, alphatocopherol,and zinc concentration during the periparturient period: Effect of milk fever. J. Dairy Sci. 73: 3195–3199.
Goff, J.P., and R.L. Horst. 1997. Physiological changes at parturition and their relationship to metabolic disorders. J. Dairy Sci. 80: 1260–1268.
Goldsby, R.A., T.J. Kindt and B.A. Osborne. 2001. Kuby Immunology, New York, USA.
Helal, T.S., F.A. Ali, O. Ezzo and M.A. El-Ashry. 2009. Effect of supplement ting some vitamins and selenium during the last stage of pregnancy on some reproductive aspects of Egyptian dairy buffaloes. J. Agri. Sci. 19: 4289-4299.
Henskens, Y., J. De-Winter, M. Pekelharing and G. Ponjee. 1998. Detection and identification of monoclonal gammopathies by capillary electrophoresis. Clin Chem. 44: 1184-90.
Hu, M.L. and C.J. Dillared. 1994. Plasma SH and GSH measurement. Method Enzymol. 233: 385–387.
Ibrahim, W., U.S. Lee, C.C. Yeh, J. Szabo, G. Bruckner and C.K. Chow. 1997. Oxidative stress and antioxidant status in mouse liver: effects of dietary lipid, vitamin E and iron. J. Nutr. 127: 1401-1406.
Jamro, A. and J. Beltwoski. 2002. Cervastatin modulates plasma paraoxonase/arylesterase activity and oxidant/antioxidant balance in rat. Pharmcol. 54: 143-150.
Jansen, S. 2014. WebMD. http://www.brain-smart.net/brain-vitamins-101-what-is-choline-bitartrate/#axzz33qVfnOap.htm, Accessed. 2014.
Jefferies, H., J. Coster, A. Kahlil, J. Bot, R.D. McCauley and J.C. Hall. 2003. Glutathione. ANZ J. Surg. 73: 517–522.
Kankfer, M. and J. Lipko. 2006. The relationship between lipid peroxidation in tensity and total antioxidant capacity in cases of spontanously released and retained bovine placenta. Vet. Med. 157: 405-409.
LeBlanc, S.J., T.H. Herdt, W.M. Seymour, T.F. Duffield and K.E. Leslie. 2004. Peripartum serum vitamin E, retinol, and beta-carotene in dairy cattle and their associations with disease. J. Dairy Sci. 87: 609-619.
Leedle, R.A., J.A. Leedle and M.D. Butine. 1993. Vitamin E is not degraded by ruminal microorganisms: assessment with ruminal contents from a steer fed a high-concentrate diet. J. Anim Sci. 71: 3442-3450.
Liu, T.Z., N. Chin, M.D. Kiser and W.N. Bigler. 1982. Specific spectrophotometry of ascorbic acid in serum or plasma by use of ascorbate oxidase. Clin Chem. 28: 2225-2228.
Marchetti, P., D. Decaudin, A. Macho, N. Zamzami, T. Hirsch, S.A. Susin and G. Kroemer. 1997. Redox regulation of apoptosis: Impact of thiol oxidation status on mitochondrial function. Eur. J. Immunol. 27: 289-296.
Miller, J.K., E. Brzezinska-Slebodzinska and F.C. Madsen. 1993. Oxidative stress, antioxidants, and animal function. J. Dairy Sci. 76: 2812-2823.
Mohsen, M.K., H.M.A. Gaffar, M.M. Khalafalla, A.A. Shitta and A.M. Yousif. 2011. Effect of Rumen Protected Choline Supplementation on Digestibility, Rumen Activity and Milk Yield in Lactating Friesian Cows. J. Anim. Sci. 44: 2011.
Nath, H.C., K.K. Baruah and A. Baruah. 2005. Serum cholesterol and protein in pre, peri and postpartum cows. Indian Vet. J. 82: 519–521.
Nicholson, J.P., M.R. Wolmarans and G.R. Park. 2000. The role of albumin in critical illness. Br. J. Anaesth. 85: 599-610.
NRC. 2001. National Research Council. Nutrient Requirements of Dairy Cattle, Washington, DC. National Academies Press.
Overton, T.R. and M.R. Waldron. 2004. Nutritional management of transition dairy cows: strategies to optimize metabolic health. J. Dairy Sci. 87 (E. Suppl): E105-E119.
Pourouchottamane, R., A. Chatterjee and I.U. Shikh. 2005. Blood biochemical constituents of female yaks in different physiological status. Indian Vet J. 82: 1108–1109.
Richard Jagatheesan, P.N., M. Selvaraju and R.V. Sarvana Kumar. 2005. Effect of advanced pregnancy and early lactation on blood biochemical constituents in Murrah buffaloes. Indian Vet J. 82: 401–403.
Sanyal, A.J., N. Chalasani, K.V. Kowdley, A. McCullough, A.M. Diehl, N.M. Bass, B.A. Neuschwander-Tetri, J.E. Lavine, J. Tonascia, A. Unalp, M.V. Natta, J. Clark, E.M. Brunt, D.E. Kleiner, J.H. Hoofnagle and P.R. Robuck. 2010. Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. N. Engl. J. Med. 362: 1675-1685.
SAS. 2002. SAS User’s Guide v. 9.1: Statistics., Cary, NC: SAS Institute, Inc.
Saun R.J.V. (2004). Metabolic profiling and health risk in transition cows. Proc. Am. Assoc. Bov. Pract. 37: 212-213.
Sharma, B.K. and R.A. Erdman. 1989. Effects of dietary and abomasally infused choline on milk production responses of lactating dairy cows. J. Nutr. 119: 248-254.
Soltan, M.A., A.M. Mujalli, M.A. Mandour and M. El-Shinway Abeer. 2012. Effect of Dietary Rumen Protected Methionine and/or Choline Supplementation on Rumen Fermentation Characteristics and Productive Performance of Early Lactating Cows. Pak J Nutr. 11: 221-230.
Soltys, K., G. Dikdan and B. Koneru. 2001. Oxidative stress in fatty livers of obese Zucker rats: Rapid amelioration and improved tolerance to warm ischemia with tocopherol. Hepatology. 34: 13-18.
Swenson, M.J. 1993. Dukes physiology of domestic animals, Ithaca, NY.
Toghdory, A., S. Emanuele, T. Choorchi and A. Naerian. 2007. Effect of choline and rumen protected choline on milk production, milk composition and blood metabolites of lactating dairy cows. J. Dairy Sci. 90: 353-364.
Ueland P.M., M.A. Mansoor, A.B. Guttormsen, F. Mullerm, P. Aukrust, H. Refsum and A.M. Svardal. 1996. Reduced, oxidized and protein-bound forms of homo-cysteine and other aminothiols in plasma comprise the redox thiol status - a possible element of the extracellular antioxidant defense system. J. Nutr. 126: 1281-1284.
Weiss, W.P., D.A. Todhunter, J.S. Hogan and K.L. Smith. 1990. Effect of duration of supplementation of selenium and vitamin E on periparturient dairy cows. J. Dairy Sci. 73: 3187–3195.
Wua, P., W. Jianga, Y. Liua, G. Chena, J. Jiangb, S. Lib, L. Fenga and X. Zhoua. 2014. Effect of choline on antioxidant defenses and gene expressions of Nrf2 signaling molecule in the spleen and head kidney of juvenile Jian carp (Cyprinus carpio var. Jian). Fish Shellfish Immun. 38: 374–382.
Xu, G., J. An Ye, J. Liu and Y. Yu. 2006. Effect of Rumen-protected Choline Addition on Milk Performance and Blood Metabolic Parameters in Transition Dairy Cows. Asian-Aust. J. Anim. Sci. 19: 390-395.
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