Levels of lactoferrin, an Fe-binding protein with bacteriostatic properties, were determined in the colostrum and milk of Yorkshire sows during a 21-d lactation. Lactoferrin levels averaged 1,100 to 1,300 μg/ml near the time of farrowing, then declined sharply during the first week of lactation. Concentration of lactoferrin showed considerable variation among sows, but not among teat positions (anterior to posterior). A method for isolating high purity swine lactoferrin is described

Elliot, J. I.

Ernhardt, G.

Fraser, D.

Senft, B.

English

WBI Studies Repository (WellBeing International)

WellBeing International WBI Studies Repository 10-1984 Isolation of Lactoferrin and its Concentration in Sows’ Colostrum and Milk During a 21-Day Lactation J. I. Elliot Agriculture Canada B. Senft Agriculture Canada G. Ernhardt Agriculture Canada D. Fraser Agriculture Canada Follow this and additional works at: https://www.wellbeingintlstudiesrepository.org/nutrit  Part of the Animal Studies Commons, Other Animal Sciences Commons, and the Other Nutrition Commons Recommended Citation Elliot, J. I., Senft, B., Erhardt, G., & Fraser, D. (1984). Isolation of lactoferrin and its concentration in sows' colostrum and milk during a 21-day lactation. Journal of animal science, 59(4), 1080-1084. This material is brought to you for free and open access by WellBeing International. It has been accepted for inclusion by an authorized administrator of the WBI Studies Repository. For more information, please contact wbisr-info@wellbeingintl.org. ISOLATION OF LACTOFERRIN AND ITS CONCENTRATION IN SOWS' COLOSTRUM AND MILK DURING A 21-DAY LACTATION 1'2 J. I. Elliot, B. Senfr 3, G. Erhardt and D. Fraser Animal Research Centre, Agriculture Canada, Ottawa, Ontario K1A 0C6 and University of Giessen, D-6300 Giessen, West Germany Summary Levels of lactoferrin, an Fe-binding protein with bacteriostatic properties, were determined in the colostrum and milk of Yorkshire sows during a 21-d lactation. Lactoferrin levels averaged 1,100 to 1,300/ag/ml near the time of farrowing, then declined sharlSly during the first week of lactation. Concentration of lactoferrin showed considerable variation among sows, but not among teat positions (anterior to posterior). A method for isolating high purity swine lactoferrin is described. (Key Words: Colostrum, Lactation, Lactoferrin, Milk, Sow, Teat Position.) Introduction Lactoferrin is an Fe-binding protein with bacteriostatic properties, found in the whey fraction of milk in humans and domestic livestock species (Oram and Reiter, 1968; Masson and Heremans, 1971 ; Senft and Klobasa, 1973; Schanbacher and Srfiith, 1975). It has been suggested that the bacteriostatic action of lactoferrin is important  in the prevention of udder infections such as mastitis in cattle (Harmon et al., 1975), and in the prevention of intestinal disturbances in the neonate (Bullen et al., 1972). Law and Reiter (1977) suggested that this protein might be used as a bacterio- static agent in milk replacers used for artificial rearing of neonates. However, this has apparently never been a t tempted even though there is suitable methodology for isolating lactoferrin in quant i ty  from the whey fraction of  cows' milk. There is only one report  in the li terature on the concentration of lactoferrin in the mammary secretions of sows throughout  lactation (Masson and Heremans, 1971). The results show high concentrations of lactoferrin in colostrum, with lower levels in normal milk. However, the data were based on pooled milk samples taken at uncertain times throughout  lactation. Roberts and Boursnnell (1975) calculated a concen- tration of .6 mg. lactoferrin/ml whole milk, based on the amount  of protein isolated. Lactoferrin has potential  use as a bacterio- static agent in milk replacers, as part of an ongoing program to develop procedures and diets for the artificial rearing of piglets (Senft and Klobasa, 1971; Elliot et al., 1978). In order to provide a basis for future experiments the following study was undertaken to establish a method for isolating lactoferrin from sows' milk, and to investigate the concentration of  lactoferrin in the mammary secretions of sows throughout lactation. ~Contribution No. 1199, Animal Research Centre. 2The authors are grateful to R. W. Allen for assistance in the collection and analysis of samples, to Dr. B. K. Thompson for discussing the statistical analyses, and to Dr. A. Grunder for assistance and discussion. 3Dr. Senft was a visiting scientist at the Animal Research Centre in 1981. Received November 28, 1983. Accepted April 6, 1984. Materials and Methods All samples were obtained from Yorkshire sows from the Animal Research Centre minimal- disease herd. Farrowings were at tended to determine the exact time of parturit ion. To establish lactoferrin concentration at different stages of  lactation, samples of mam- mary secretion were collected from nine sows on 13 occasions: Within 12 h prefarrowing, 1080 JOURNAL OF ANIMAL SCIENCE, Vol. 59, No. 4, 1984 LACTOFERRIN IN SOWS' COLOSTRUM AND MILK 1081 immediately postfarrowing (within 1 h after the afterbirth was expelled), 12 h postpartum, and on d 1, 2, 3, 4, 5, 6, 7, 11, 14 and 21 of lacta- tion. At least 50 ml of mammary secretion were obtained from each sow at each sampling time by manual expression from randomly selected teats. After the immediate pre- and postfar- rowing periods, milk letdown was induced by an im injection of 40 u oxytocin. Samples were stored frozen at - 2 0  C. To establish teat-to-teat variation in lacto- ferrin content, samples were collected from each teat on one side of  six additional sows. All the samples were collected by hand milking during farrowing (after the expulsion of the first pig and before expulsion of the afterbirth) without injected oxytocin. Preparation and Characterization of Swine Lactoferrin. Sows' colostrum was used for the isolation of lactoferrin. Samples were centri- fuged (17,000 x g, 4 C, 40 min) so that the fat formed a layer at the top of the milk. The fat was then drawn off using an aspirator. The milk was adjusted to pH 7.0 by KHCO3, and the Fe-binding protein lactoferrin was saturated with Fe using ammonium ferrous sulfate (8.6 mg/ml). Six grams of Sephadex CM-504 were added to 500 ml defatted milk at room tem- perature, mixed for 2 h and then allowed to settle. The supernatant milk was carefully decanted and the pink gel that remained was washed first three times on a glass filter funnel (G2, D1) with deionized water, then with a .02 M Na-phosphate buffer pH 7.0, and finally with the same buffer containing .15 M NaC1. About  420 ml of gel was then applied to a column (90 x 2.5 cm) and eluted with a .02 M Na-phosphate buffer containing a linear NaCl-gradient from .2 to .5 M at a flow rate of 20 ml/h. Fractions (3 ml) were collected with an Eldex Universal Fraction Collector Model 4-11, and were read at 280 nm to establish the presence of  protein using a Beckman Model 25 spectrophotometer. Lactoperoxidase was eluted in the first peak of protein (fractions 80 to 110), and the second peak (fractions 120 to 240) contained lacto- ferrin. The lactoferrin containing fractions were pooled, concentrated by ultrafiltration and dialysed against deionized water. 4 Pharmacia Fine Chemicals, Uppsala, Sweden. 5 BioRad Laboratories, Mississauga, Ontario. Hansens Laboratories Inc., Milwaukee, WI. Further purification of the lactoferrin preparation was accomplished on a 40 x 2.5 cm column of  Bio-Rex 70 resin s equilibrated with .01 M Na-acetate pH 7.0. After applying about 5 ml of the equilibrated lactoferrin solution, the column was initially eluted with 150 ml of  .4 M Na-acetate pH 7.0, then a linear gradient was applied by mixing 400 ml .4 M Na-acetate pH 7.0 and 400 ml of 1 M Na-acetate pH 8.0. Fractions of 3 ml were collected and read as described above to determine the presence of protein. The pink solution comprising the third peak (fractions 90 to 130) contained lactofer- tin, which was concentrated by ultrafiltration, dialysed against deionized water and lyo- philized. The protein content in the lactoferrin solutions was determined by the method of Lowry et al. (1951). To estimate its molecular weight, swine lactoferrin was compared with bovine lactoferrin and bovine serum albumin using gel chroma- tography by the method of  Erhardt (1981). Immunoelectrophoresis was performed by the method of  Scheidegger (1955) with anti- serum raised in rabbits against swine colostrum. Quantitative Determination of Swine Lacto- ferrin. The radial immunodiffusion technique (Mancini et al., 1965) was used to determine the lactoferrin concentration of swine whey. The whey was prepared by precipitating the casein using calf rennin 6. Diffusion took place within 24 h at room temperature on glass plates covered with 1.5% agarose in .02 M tris-acetate- buffer pH 7.9 and 1% rabbit anti-swine-lacto- ferrin. The amount of sample applied was 5 vl. Standards were prepared from lactoferrin isolated as previously described. All deter- minations were made in duplicate. The range for the standards was from .03 mg/ml to 4 mg/ml. The antisera were obtained from rabbits after sc injection of 2 mg lyophilized lactoferrin diluted in 1 ml distilled water and 1 ml of complete Freund's adjuvant. The injections were repeated four times at 12-d intervals. Ten days after the last injection, blood was taken from the rabbits by heart puncture. Results and Discussion Preparation and Purification of Lactoferrin. The preparative method used in this investiga- tion makes it possible to use the lactoferrin-free milk for further investigation after having absorbed lactoferrin by Sephadex CM-50. By 1082 ELLIOT ET AL. Figure 1. Immunoelectrophoretic reaction of isolated swine lactoferrin (above) and of swine colostrum (be- low) to antisera produced in rabbits against swine colostrum. mixing the resin into the milk, several time- consuming procedures can be avoided and the economy increased by using CM-50 resin repeatedly. Using the method described herein, a high purity of lactoferrin can be obtained as shown by immunoelectrophoresis (figure 1). The isolated swine lactoferrin formed a strong precipitation line in the ~-area. For comparison the lactoferrin arc is also visible in the separated colostrum sample. This localization of swine lactoferrin is in accordance with the results of Blanc and Isliker (1961) for human lactoferrin. In contrast, bovine lactoferrin is located in the 7-region (Butler, 1973). Gel chromatography showed that swine lactoferrin had an elution pattern similar to that of bovine lactoferrin and bovine serum albumin (figure 2), indicating that swine lactoferrin has a molecular weight of about 70,000. Lactoferrin Content o f  Sows'  Milk. The lactoferrin content of mammary secretions from nine sows during a 21-d lactation is presented in figure 3. Lactoferrin levels remained high (1,100 to 1,300 /~g/ml) near the time of farrowing and for the first 3 d of lactation, and then declined sharply. Two-way analysis of variance, shoWed highly significant variation among sows (Fs,  9s = 12.1, P<.O01) and among sampling times (F12, 9s = 31.3, P<.O01). The two sows with the highest levels of lactoferrin had concentrations about twice as high as the sows with the loWest levels (1,092 and 1,021 E C O oo .1 ,6 g .5 .3 ; ,2  1,0 ,9 ,8 A /Y;, \s.ine I L,~ ", \l~r bovine / I i  ~ \ l  I lactoferr in/  /; \ \~ ] 1 i  \'~ 14 \",, bovine serum .-~/ " \ k~ I 9 ~ 1 7 6  / \ ~ '  i I , 50 100 150 200 250 ml v o l u m e  Figure 2. Elution pattern comparing swine lactoferrin with bovine lactoferrin and bovine serum albumin, showing absorbance of light at 280 nm in successive 5-ml fractions of eluant taken from a column containing Sephadex G-200. LACTOFERRIN IN SOWS' COLOSTRUM AND MILK 1083 1 4 0 0  . . . . . . . . . . . . .  i~oo 11oo iooo 100 7 9 n r T - ~  T I J T -  r - ~ l e h  l~t 20  3~J 4 0  ~ GO 70  l ~ J  la~F ~l~J l l N [  IN R E L A T I O N  T O  F A R R O W I N G  Figure 3. Mean lactoferrin concentration (#g/ml) in the colostrum and milk of nine sows during a 21-d lactation. Samples were collected before farrowing (PRE), immediately after farrowing (POST), and 12 h to 21 d thereafter. The vertical bar shows twice the standard error of the difference. /ag/ml, vs 532 and 513/~g/ml,  averaged over the 13 sampling times). The  six addi t ional  sows studied at the t ime of  farrowing showed relatively l i t t le variat ion in lactoferr in con ten t  f rom teat  to teat. Mean levels for  individual sows ranged f rom 1,208 to 1,577 gtg/ml, with standard errors of  56 to 150. With teat  posi t ion numbered  f rom 1 (anterior)  to 7 (posterior) ,  two-way analysis of variance showed significant variat ion a t t r ibutable  to  sows (Fs,2s = 11.13, P<.001) ,  bu t  not  to teat  posi t ion (F6,28 = .517). The  rapidly decreasing concent ra t ion  of  lactoferr in during the first week of  lactat ion might  be explained by reduced synthesis of  the protein and(or)  increasing di lu t ion of  lacto- ferrin caused by increased synthesis of  milk. Verdonck  and Paredis (1980) noted  that  the ant imicrobial  act ivi ty of  sows'  colos t rum and milk also declines during lactat ion.  They  at t r ibute  the  ef fec t  to the decrease in the concent ra t ion  o f  immunoglobul ins  in sows'  milk, stating that  lactoferr in is a more constant  const i tuent .  However ,  our  results indicate that  lactoferr in is not  a constant  const i tuent .  This was also demons t ra ted  in human milk by Nagasawa et al. (1972) and in bovine milk by Erhardt  (1981).  In view of its known ant imicrobial  act ivi ty (Bullen et al., 1972), it is qui te  possible that  lactoferr in is an impor t an t  ant ibacterial  con- s t i tuent  of sows'  milk in the early stages o f  lactat ion,  supplement ing the effect  of  immuno-  globulins (Wilson, 1974). It  is interesting to note  that  the decrease in lactoferr in con ten t  corresponds with the deve lopment  of  the pig's endogenous  capabili ty to produce  and secrete immunoglobul ins  into the  intestinal lumen as described by Wilson (1974).  There  may  be an impor tan t  role for lacto- ferrin in the deve lopment  of  milk replacers to be used in the artificial rearing of  piglets. The nonspecif ic  bacter iosta t ic  activity of  lactoferr in has been demons t ra ted  in numerous  investi- gations (Oram and Reiter ,  1968; Bullen et al., 1972), and the mechanism of  act ion shown to be based on the binding of  Fe  to the pro- tein, thereby rendering it unavailable for  bacterial  growth.  Because of  the nonspecif ic  act ivi ty of  the lactoferr in f rom di f ferent  species and the dif f icul ty  in obtaining large amounts  o f  sows' milk,  it is p roposed  to investigate the use of  bovine lactoferr in in milk replacers for piglets. In view of  the large variat ion among sows noted here, it would  be useful to de te rmine  whether  piglet survival is related to the lacto- ferrin concent ra t ion  o f  the dam's  milk. If so, this variable might  be useful in genetic selection.  Literature Cited Blanc, B. and H. Isliker. 1961. Isolement et carac- t6risation de la proteine rouge sld~rophile du lait materneh la lact0ferrine. Helv. Physiol. Pharma- col. Acta 19:C13. Bullen, J. J, H. J. Rogers and L. Leigh. 1972. Iron- binding proteins in milk and resistance to Escher- ichia coli infection in infants. Brit. Med. J. 1:69. Butler, J. E. 1973. The occurrence of immunoglobulin fragments, two types of lactoferrin and a lacto- ferrin IgG2-complex in bovine colostral and milk-whey. Biochim. Biophys. Acta 295:341. Elliot, J. I., G. A. Lodge and T. G. Hartsock. 1978. Practical artificial rearing of neonatal piglets under non-isolated conditions. Can. J. Anim. Sci. 58:799. Erhardt, G. 1981. Zusammenhange zwischen Zitrat und Lactoferrin in der Milch laktierender und nicht laktierender Kuhe sowie nach experi- menteller Infektion der Milchdruse mit Staphy- lococcus aureus. Giessner Schriftenreihe Tierzucht und Haustiergenetik, Verlag Paul Parey, Hamburg und Berlin. Itarmon, J., F. L. Schanbacker, L. C. Ferguson and K. L. Smith. 1975. Concentration of lactoferrin in milk of normal lactating cows and changes occurring during lactation. Amer. J. Vet. Res. 36:1001. Law, B. A. and B. Reiter. 1977. The isolation and bacteriostatic properties of lactoferrin from bovine milk whey. J. Dairy Res. 44:595. Lowry, O. J., N. J. Rosebrough, A. L. Farr and R. J. Randall. 1951. Protein measurement with the folin-phenol reagent. J. Biol. Chem. 193:265. Mancini, G., A. O. Carbonara and J. F. Heremans. 1965. Immunochemical quantitations of antigene by single radial immunodiffision. Immuno- 1084 ELLIOT ET AL. chemistry 2:235. Masson, P. L. and J. F. Heremans. 1971. Lactoferrin in milk from different species. Comp. Biochem. Physiol. 39B: 119. Nagasawa, T., I. Kiyosawa and K. Kuwahara. 1972. Amounts of lactoferrin in human colostrum and milk. J. Dairy Sci. 55:1651. Oram, J. D. and B. Reiter. 1968. Inhibition of bacteria by laetoferrin and other iron-chelating agents. Biochim. Biophys. Acta 170: 351. Roberts, T. K. and J. C. Boursnell. 1975. The isolation and characterization of lactoferrin from sow milk and boar seminal plasma. J. Reprod. Fertil. 42:579. Schanbacher, F. L. and K. L. Smith. 1975. Formation and role of unusual whey proteins and enzymes: Relation to mammary function. J. Dairy Sci. 58:1048. Scheidegger, J. J. 1955. Une micromethode de l'im- muno-electrophor~se. Int. Arch. Allergy 7:103. Senft, B. and F. Klobasa. 1971. Untersuchungen uber die kunstliche Aufzucht von Ferkeln. I. Der Einstaz yon Rinderkolostralmilch und reifer Kuhmilch. Zuchtungskunde 43: 371. Senft, B. and F. Klobasa. 1973. Untersuchungen uber die Konzentration an Lactoferrin in Kolostral- milch und reifer Milch von Kuhen. Milchwissen- schaft 28:750. Verdonck, M. and F. Paredis. 1980. A comparative study of the in vitro antimicrobial activity of colostrum and milk in the pig. IRCS Med. Sci. 8:287. Wilson, M. R. 1974. Immunologic development of the neonatal pig. J. Anita. Sci. 38:1018. 

Isolation of Lactoferrin and its Concentration in Sows’ Colostrum and Milk During a 21-Day Lactation

https://www.wellbeingintlstudiesrepository.org/cgi/viewcontent.cgi?article=1002&context=nutrit

Isolation of Lactoferrin and its Concentration in Sows’ Colostrum and Milk During a 21-Day Lactation

Abstract

Similar works

Full text

Available Versions

WBI Studies Repository (WellBeing International)