Protective factors in mammary gland secretions during the periparturient period in the mare

I:quine Nutrition and Physiology s

Annual s

Mau 2~'-21, 1 9 9 7 . l::ott Worth, Texas.

PROTECTIVE FACTORS IN MAMMARY GLAND SECRETIONS DURING THE PERIPARTURIENT PERIOD IN THE MARE S. Zou, 1'2 H. A. Brady, 3 W. L. Hurley 1

SUMMARY

Changes in mare mammary secretion composition were characterized prior to and after foaling. Eighteen mares were used for collection of mammary secretions from 23 days prepartum through 44 days postpartum. Concentrations of lactose, total protein, IgG and lysozyme (activity assay) were determined. Considerable variabilty was observed among mares. Mean lactose concentrations by mare were lower (P < .05) in the prepartum period compared with the postpartum period. Mean total protein and immunoglobulin G concentrations by mare were higher (P < .05) in the prepartum period compared with the postpartum period. Mean lysozyme activities by mare tended to be lower (P < .08) during the prepartum period compared with the postpartum period. Mammary secretion protein profiles, characterized by SDS-PAGE, were consistent with changes in concentrations of specific proteins measured in the secretions. Dynamic changes occurring in mammary secretions during the transition from prepartum accumulation of colostrum to postpartum production of milk include factors like IgG and lysozyme which have protective roles in the neonatal foal.

INTRODUCTION

The newborn foal is dependent upon the mare's colostrnm and milk for nutrients and disease protection factors. Many studies have documented the increase in calcium concentrations in mammary secretions of the mare prior to foaling, and this observation has proven valuable as a Authors' addresses: 1Department of Animal Sciences, University of Illinois, Urbana, Illinois 61801; 2Current address, College of Veterinary Medicine, NanjingAgriculturalUniversity,Nanjing,PRC; 3Department of Animal Science and Food Technology, Texas Tech University, Lubbock,

Texas 79409. Address correspondence to H. A. Brady. Acknowledgements:Supportedin partby IllinoisAgricultural Experiment Station Project 35-0315. The authors would like to acknowledge the assistanceof R. D. Ulbricht and P. A. LaCasha in this project.

184

predictive test for time of parturition. 1,2 In addition, concentrations of immunoglobulins, especially IgG, have been studied in colostrum due to their importance in passive transfer of antibodies in the foal. 3,4 Colostrum containing borderline or low levels of IgG poses a significant risk to the health of the neonatal foal. Limited information is available about other components of equine mammary secretions, especially prior to parturition. Of particular interest are other protective factors found in colostrum and milk. Lysozyme is one such nonspecific, disease-resistance factor which is thought to play a role in protection of the newborn by bacteriostatic functions in the gastrointestinal tract. 5 Lysozyme hydrolyses the glycosidic bond between N-acetylmuramic acid and N-acetyl-D-glucosamine in bacterial cell walls. Lysozyme activity has been identified in the milk of a number of species including human, 6 baboon, 7 camel, 8 bovine, 9 feline and canine, 10 and equine.ll Concentrations oflysozyme vary considerably among milks of different species, with human and mare milks containing high concentrations and cow milk containing low concentrations.12 Equine lysozyme has been extensively characterized both biochemically and structurally. 13,14 The dynamics of lysozyme activity in mare mammary secretions and its relationship to other protective factors have not been fully described. This study characterized the compositional changes in mammary secretions of the mare during the peripartum period, with particular emphasis on IgG and lysozyme activity.

MATERIALS AND METHODS A n i m a l s and s a m p l e collection

Standardbred (n = 11), Quarter Horse (n = 3) and Thoroughbred (n =4, including 2 maiden) mares were used in this study. Mammary secretion samples were collected at different time points ranging from 23 days prior to foaling through day 44 of lactation. Numbers of samples collected per mare ranged from 1 to 12. All mares were JOURNAL OF EQUINE VETERINARY SCIENCE

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Figure 1. Content of lactose, total protein, immunoglobulin G, and lysozyme activity in mammary secretions from mares during the prepartum and postpartum periods. Samples were from day 23 prepartum through day 44 postpartum. Each point represents the measured value for one mare. Day 0 (vertical dotted line) indicates day of foaling. maintained under the same management and feeding regimen. Foals of all mares suckled normally. Mammary secretion samples were strained through cheese cloth to remove debris and stored at -20~ until analysis.

Milk component determination Mammary secretions were analyzed for lactose by the method of Teles et a1.15 Total protein content of mammary secretions was determined using a dye binding assay, a Immunoglobulin G was estimated by radial immunodiffusion assay using equine IgG standards.b Lysozyme activity was determined spectrophotometrically with the lysis of Micrococcus lysodeikticus, c as previously described. 16 Briefly, the sample (50 gl) was added to a 1 cm cuvette containing 3 ml of substrate solution (0.25 mg/ml Micrococcus lysodeikticus in 0.1 M sodium phosphate, pH 6.5) and mixed thoroughly. The OD at 600 nm was measured for 3 min (lysis was linear for at least 3 min). One unit of lysozyme activity was defined as a decrease in OD at 600 aBioRad, RockvilleCentre, NY, USA. bVMRD,Inc., Pullman, WA, USA. CSigmaChemicalCo., St. Louis, MO, USA. Volume 18, Number 3, 1998

nm of 0.001 per minute, at 22~

Polyacrylamide gel electrophoresis Proteins in mammary secretions were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli. t7 Gels (15%) were stained with 1% Coomassie blue. Equal volumes of samples were loaded per lane. Molecular weight standards were electrophoresed on each gel. Specific proteins were identified on the basis of migration in the gel and by comigration with known cow milk proteins (not shown). In addition, the identity of lactoferrin, 13-1actogobulin and ~lactalbumin was confirmed by immunoblotting against rabbit antisera specific for those bovine proteins, and lysozyme was confirmed by immunoblotting against antiserum specific for human lysozyme (not shown). Statistical analysis

Statistical analysis was performed using linear regression and the general linear models procedures of SAS. t8 Data were analyzed by mare to derive least squares means 185

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Figure 2. SDS-PAGE of proteins in mammary secretions from mares during the prepartum and postpartum periods. Lanes are samples from different mares collected on various days from parturition (0 = day of foaling). Molecular weight markers (left side) are in kilodattons (kDa). Protein bands are identified (right side) as Lf = lactoferrin, SA = serum albumin, Ig-h = immunoglobulin heavy chain, CN = caseins, Ig-I = immunoglobulin light chains, B-LG = 13lactoglobulin, Ly = lysozyme, c~-La = c~-Iactalbumin, and * = unidentified protein, possible 132-microglobulin. for each variable for the prepartum (-23 to -1 days) and postpartum periods (0 to 44 days). Data were also analyzed by linear regression to derive regression coefficients for each mare for the prepartum and postpartum periods. The general linear models procedure was used to test for differences between the prepartum and postpartum periods. Significance was declared at P < .05.

RESULTS

The period leading up to parturition represents the final stages of mammary structural development and functional differentiation. Samples collected between 23 days and one day prior to foaling represent the late stages of mammary structural development and the period of colostrum formation and early lactogenesis. Samples from day 0 are considered colostrum or the first milk which would be consumed by the foal, and samples from day 0 to day 44 postpartum represent the transition from colostral composition to mature milk. Mean lactose concentrations by mare were lower (P < .05) in the prepartum period compared with the postpartum period (1.98 + .34 vs. 5.48 + .34 %, respectively). Lactose concentrations in prepartum mammary secretions (Figure 1) were highly variable among mares. Despite of this variability, the mean regression coefficient (slope) for mares during the prepartum period was significantly different from zero (P < .05). Comparison of within mare regression coefficients for lactose among the prepartum and postpartum periods was nonsignificant, indicating that differences in concentrations of lactose were more affected by mare than by day from parturition. Much of the change in lactose concentration seemed to occur between one day prepartum and one day postpartum. 186

Mean total protein concentrations by mare were lower (P < .05) in the prepartum period compared with the postpartum period (101.8 + 9.7 vs. 40.3 + 9.7 mg/ml, respectively). Comparison of within mare regression coefficients for total protein among the prepartum and postpartum periods was nonsignificant, indicating that differences in concentrations of protein were more affected by mare than by day from parturition. Total protein concentrations were highly variable, especially in the prepartum period (Figure 1), and mean regression coefficients for protein were nonsignificant for the prepartum or postpartum periods. The generally elevated concentrations of protein in secretions prior to parturition were consistent with the increase in immunoglobulins occurring at that time. However, other proteins contributed to the prepartum increase in protein content including serum albumin, casein, B-lactoglobulin, t~-lactalbumin, and lysozyme, as well as other minor proteins (Figure 2). Mean IgG concentrations by mare were higher (P < .05) in the prepartum period compared with the postpartum period (148.2 + 23.6 vs. 9.0 + 23.6 mg/ml, respectively). Comparison of within mare regression coefficients for IgG among the prepartum and postpartum periods was nonsignificant. Mean regression coefficients for IgG were nonsignificant for the prepartum and postpartum periods. Immunoglobulin G concentrations were generally <50 mg/ ml prior to about 10 days prepartum (Figure 1). It appeared that IgG concentrations began declining prior to foaling (Figure 1). By day 1 postpartum and afterward IgG concentration was generally <1 mg/ml, as found in milk (Figure 1). Mean lysozyme activities by mare tended to be lower (P < .08) in the prepartum period compared with the postpartum period (395.5 + 41.7 vs. 508.8 + 41.7 units/ml, respectively). Comparison of within mare regression coefficients for lysozyme activities among the prepartum and postpartum periods was significant (P < .05), indicating that the factors which are responsible for accumulation of lysozyme activity in the secretions differ between the prepartum and postpartum periods. Lysozyme activities were increasing (P < .05) during the early prepartum period (Figure 1). Mean regression coefficient for the postpartum period was nonsignificant. Proteins in representative samples of mammary secretions from 17 days prepartum through 6 days postpartum were separated by SDS-PAGE under denaturing and reducing conditions (Figure 2). Reducing conditions of SDSPAGE caused dissociation of the immunoglobulin heavy and light chains (Ig-h and Ig-1, respectively; Figure 2). Immunoglobulin bands formed the major protein component of prepartum samples. However, immunoglobulin was reduced in colostrum (day 0) and even lower in the postpartum samples, consistent with the measured concentrations in Figure 1. Conversely, casein bands (CN; Figure 2) were typically in low proportion until one day preparJOURNAL OF EQUINE VETERINARY SCIENCE

tuna, but were the major protein bands in milk (day 6 postpartum), f3-Lactoglobulin (13-LG; Figure 2), a major milk whey protein, was present throughout the period illustrated by the gel (Figure 2), but was particularly increased from 5 days prepartum through parturition. In contrast a-lactalbumin ((z-LA; Figure 2) was low in proportion to other proteins until parturition, consistent with its role in lactose synthesis and copious milk secretion of late lactogenesis. Serum albumin (SA; Figure 2) was elevated only in prepartum and colostrum samples. Relatively little of the antimicrobial protein lactoferrin (Lf; Figure 2) was present in the prepartum samples and even less in the postpartum samples. The proportion of lysozyme (Ly; Figure 2) increased during the prepartum period and was greatest in colostrum. Lysozyme remained a major protein in milk postpartum. There was also evidence of a protein band (*; Figure 2) of smaller molecular weight than the c~-lactalbumin band in samples from the prepartum period, but absent in colostrum and milk. The identity of this protein could not be determined from this study, however, the apparent molecular weight (-12 kDa) suggests that it may be ga-microglobulin, as identified in cow milk. 19

DISCUSSION

Composition of mammary secretions changed dramatically in the peripartum period. The increase in lactose at the time of parturition occurred coincident with the rapid increase in milk secretion which accompanies lactogenesis. Changes in total protein content in secretions reflected the accumulation of immunoglobulins and other milk proteins during the prepartum period. Conversely, the decrease in protein content at or immediately after foaling was consistent with the decline in IgG concentration immediately after foaling, combined with the dilution effect of increased secretion volume. Lysozyme activity increased during the perpartum period and then remained elevated. Changes in the profiles of specific mammary secretion proteins were consistent with the quantitative estimates of these protein components. The period immediately prior to and after parturition is a time of rapid and complex change in the mammary gland. The processes involved in lactogenesis include functional differentiation of mammary cells toward the synthesis of milk components and these functional changes result in changes in composition of the secretions. 2~ The early stages of lactogenesis also occur concurrent with the formation of colostrum, especially the accumulation ofimmunoglobulins? 9 Once extensive lactose synthesis begins, water is osmotically drawn into the mammary cells resulting in a dilution of secretory components. Prepartum immunoglobulin concentrations probably reflect the accumulation of those proteins during the period Volume 18, Number 3, 1998

when secretions are not being removed by the foal. Immunoglobulin transport across the mammary epithelial barrier occurs by a receptor mediated mechanism 19 as part of the process ofcolostrum formation. Immunoglobulin concentration then declines rapidly after parturition in the mare. el In the present study, IgG concentration seemed to decline immediately prior to the day of parturition, suggesting that the rapid increase in secretion volume associated with lactogenesis was having a dilution effect. This has been observed in the cow.19 However, it also may suggest that the IgG transport mechanism in the mammary cells declines prior to parturition, coincident with the rapid increase in lactose synthesis. Lysozyme activity increased in the prepartum period in mare mammary secretions. This is in contrast to observations in prepartum human breast secretions where lysozyme content was already near preterm levels at 3-5 weeks prepartum. 22'2a In the present study, postparturient lysozyme activities in mare milk at 5-6 weeks of lactation were still about 50% of the parturient levels. Human milk also has lower concentrations of lysozyme at that stage of lactation compared with the colostral concentrations. 22,23 In human milk, lysozyme concentration increases once again after about 10 weeks of lactation. 22 It is not known whether lysozyme activity continues to change during later lactation in the mare. Immunoglobulin G ingested by the newborn foal is absorbed into the blood to provide passive immunity, la In contrast, human breast milk lysozyme is thought to have protective value to the neonate by acting as an antimicrobial agent in the intestine. 5 In addition to its antimicrobial activity, milk lysozyme is generally resistant to digestive enzymes, acts synergistically with other antimicrobial agents, especially IgA and lactoferrin, and acts without triggering an inflammatory response. 5 Human and mare milks have some of the highest activities of lysozyme identified in milk. 12 The protective role of lysozyme in the neonate foal remains to be demonstrated, but this factor presumably continues to act in the intestine even after closure to immunoglobulin absorption. Lysozyme also may contribute to the host defense mechanisms of the mare mammary gland in preventing mastitis. Lysozyme concentrations are increased during mastitis in the cow, e4 although activities of this enzyme are normally quite low in bovine milk. Mastitis incidence is lower in the mare than in the cow. 25 The high activity of lysozyme in mare milk may be one factor contributing to this low incidence of equine mastitis. Further study of the function of lysozyme in the foal and in the mare mammary gland is warranted.

CONCLUSIONS

The concentration of components in mammary secre187

tions in constant change during the prepartum and the immediate postpartum periods. Immunoglobulin content accumulates in the prepartum gland in preparation for transfer of passive immunity to the newborn foal. Other protective factors such as lysozyme remain at elevated levels in milk well after parturition and may continue acting in the intestine after closure to absorption of macromolecules. The high activity of lysozyme in mare milk may contribute to the well-being of the foal by protecting the intestine, in concert with other protective agents. Lysozyme may also contribute to the protection of the mammary gland against infection.

7. Buss DH: Lysozyme activity in baboon milk. Comp Biochem Physiof 1969;31:783-787. 8. Duhaiman AS: Purification of camel milk lysozyme and its lytic effect on Escherichia coil and Micrococcus lysodeikticus. Comp Biochem Physiol 1988;91 B:793-796. 9. Chandan RC, Parry RM, Jr, Shahani KM: Lysozyme, lipase, and ribonuclease in milk of various species. J Dairy Sci 1968;51:606-607. 10. Halliday JA, Bell K, Shaw DC: Feline and canine milk lysozymes. Comp Biochem Physiol 1993; 106B:859-865. 11. Jauregui-Adell J, Cladel G, Ferraz-Pina C, Rech J: Isolation and partial characterization of mare milk lysozyme. Arch Biochem Biophys 1972;151:353-355. 12. Farkye NY: Indigenous enzymes in milk. Other enzymes. In: Fox PE (ed.), Advanced Dairy Chemistry. Vol. 1. Proteins. Elsevier Applied Science: New York, pp 348-350, 1992. 13. Jauregui-Adell J: Heat stability and reactivation of mare milk lysozyme. J Dairy Sci 1975;58:835-838. 14. McKenzie HA, Shaw DC: The amino acid sequence of REFERENCES equine milk lysozyme. Biochem Int 1985;10:23-31. 15. Teles FF, Young CK, Stull JW: A method of rapid determination of lactose. J Dairy Sci 1978;61:506-508. 1. Peaker M, Rossdale PD, Forsyth IA, Falk M: Changes 16. Shugar D:The measurement of lysozyme activity and the in mammary developmentand the composition of secretion during ultra-violet inactivation of lysozyme. Biochim Biophys Acta late pregnancy in the mare. J Reprod Fertil Supp11979;27:5551952;8:302-309. 561. 17. Laemmli UK: Cleavage of structural proteins during 2. Ousey JC, Dudan FE, Rossdale PD: Preliminary assembly of the head of bacteriophage T4. Nature (Lond) studies of mammary secretions in the mare to assess readiness 1970;227:680-685. for birth. Equine Vet J 1984;16:259-263. 18. SAS/STA'I'~: User's Guide, Version 6, 4th Edition. Cary, 3. LaBlanc MM, McLauren BI, Boswell R: Relationships NC: SAS Inst., Inc., 1990. among serum immunoglobulin concentrations in foals, colostral 19. Larson BL: Immunoglobulinsof the mammary secretions. specific gravity, and colostral immunoglobulinconcentration. JAm In: Fox PE (ed.), Advanced Dairy Chemistry. Vol. 1. Proteins. New Vet Med Assoc 1986; 189:57-60. York: Elsevier Applied Science, pp 231-254, 1992. 4. LaBlanc MM, Tran T, Baldwin JL, Pritchard EL: Factors 20. Hartmann PE: Changes in the composition and yield of that influence passive transfer of immunoglobulins in foals. J Am the mammary secretion of cows during the initiation of lactation. Vet Med Assoc 192;200:179-183. J Endocrin 1973;59:231-247. 5. Goldman AS: The immune system of human milk: 21. Rouse BT, Ingram DG: The total protein and antimicrobial, antiinflammatory and immunomodulating immunoglobulin profile of equine colostrum and milk. Immunology properties. Pediatr Infect Dis J 1993; 12:664-671. 1970; 19:901-907. 6. Parry RM, Jr, Chandan RC, Shahani KM: Isolation and 22. Hyslop NE, Jr, Kern KC, Walker WA: Lysozyme in human characterization of human milk lysozyme. Arch Biochem Biophys colostrum and breast milk. In: Osserman EF, Canfield RE, 1969; 103:59-65. Beychok S. (ed.), Lysozyme. NewYork: Academic Press, pp 449462, 1974. 23. Lewis-Jones DI, Reynolds G J: A suggested role for precolostrum in preterm and sick newborn infants. Acta Paediatr Scand 1983;72:1317. 24. Carlsson A, Bjorck L, Persson K: Lactoferrin and lysozyme in milk during acute mastitis and their 'M 9 I m p r o v e s f e e d e f f i c i e n c y inhibitory effect in Delvost P. J Dairy Sci 1989;72:3166-3175. 9 Reduces need for additional 25. Jackson PGG: Equine mastitis: comparative lessons. Equine Vet supplements J, 1986;18:88-89.

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