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Influence of Clinical Mastitis During Early Lactation on Reproductive Performance of Jersey Cows A. R. BARKER,1 F. N. SCHRICK, M. J. LEWIS, H. H. DOWLEN, and S. P. OLIVER2 Department of Animal Science, Institute of Agriculture, The University of Tennessee, Knoxville 37901-1071

ABSTRACT The purpose of this study was to determine the influence of clinical mastitis on reproductive performance of high producing Jersey cows. Cows ( n = 102) with clinical mastitis during the first 150 d of lactation were evaluated. Groups were balanced according to lactation number and days of lactation and subdivided as follows: group 1, clinical mastitis before first artificial insemination ( A I ) ( n = 48); group 2, clinical mastitis between first AI and pregnancy ( n = 14); group 3, clinical mastitis after confirmed pregnancy ( n = 40); and group 4, control cows ( n = 103) with no clinical mastitis. No differences in reproductive performance were detected because of milk production or mastitis caused by Gram-positive or Gram-negative pathogens. The number of days to first AI was significantly greater for cows with clinical mastitis before first AI (93.6 d ) than for all other groups (71.0 d). Artificial inseminations per conception were significantly greater for cows with clinical mastitis after first AI (2.9) than for cows with clinical mastitis before first AI (1.6), cows with no clinical mastitis, or cows with clinical mastitis after confirmed pregnancy (1.7). The number of days to conception for cows with clinical mastitis after first AI (136.6 d ) was significantly greater than that for control cows and that for cows that developed clinical mastitis after confirmed pregnancy (92.1 d). Clinical mastitis during early lactation markedly influenced reproductive performance of Jersey cows. ( Key words: clinical mastitis, reproductive performance) INTRODUCTION Intramammary infections caused by environmental pathogens such as Escherichia coli and Streptococcus

Received August 12, 1997. Accepted December 1, 1997. 1Present address: Division of Animal and Veterinary Science, West Virginia University, Morgantown 26506. 2Corresponding author. 1998 J Dairy Sci 81:1285–1290

uberis occur frequently during early lactation. Many of these infections originate during the nonlactating period and result in clinical mastitis during the first 30 to 60 d after calving. Clinical mastitis during early lactation results in obvious losses such as decreased milk production and alterations in milk composition. However, the influence of clinical mastitis on reproductive performance of dairy cows during early lactation is essentially unknown. Cullor ( 5 ) indicated that endotoxin, a component of the cell wall of Gramnegative bacteria, induced luteolysis and influenced conception and early embryonic survival apparently via the release of inflammatory mediators. Moore et al. ( 1 5 ) indicated that cows from a herd with mastitis caused by predominately Gram-negative pathogens were almost twice as likely to have an altered interestrus interval following clinical mastitis than were herdmates without clinical mastitis. However, in another herd with mastitis caused predominately by Staphylococcus aureus, no significant changes in length of the estrous cycle were detected. Based on this limited information, it would appear that clinical mastitis during early lactation, particularly mastitis caused by Gram-negative pathogens, can affect the reproductive performance of lactating dairy cows. The purpose of the present study was to determine the influence of clinical mastitis during early lactation caused primarily by environmental pathogens on parameters of reproductive performance in a herd of high producing Jersey cows. MATERIALS AND METHODS Jersey cows at The University of Tennessee Dairy Experiment Station research herd at Lewisburg were used. Cows were milked twice daily in a 12-stall trigon milking parlor equipped with automatic milking machine take-offs (Surge; Babson Bros., Oak Brook, IL). Milking machines were backflushed (Surge Backflush II; Babson Bros.) after removal from cows. Milking equipment was evaluated routinely and maintained per the recommendations of the manufacturer. Cows were housed in bedded free stalls; dairy waste solids were separated from a ma-

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nure slurry (Alfa-Laval, Inc., Kansas City, MO). Cows were allowed on pasture 4 to 6 h/d. All cows were dried off at approximately 8 wk before expected calving, and all quarters of cows were infused with an approved antibiotic preparation following the last milking of lactation. Cows were observed for estrus for 30 min at least three times daily. In addition, milkers observed cows at milking time, and all farm personnel regularly participated in estrus detection throughout the day. When weather permitted, cows were allowed access to dirt and grass paddocks. Cows in estrus were confined to tie stalls to minimize the chance of injury. Following calving, cows were generally subjected to a voluntary waiting period of 60 d prior to first AI. Postpartum reproductive exams were performed on all cows after the voluntary waiting period. Pregnancy examinations were performed on all cows 50 to 65 d after AI. Cows with clinical mastitis were identified by milking personnel. Samples of foremilk from quarters of cows with clinical mastitis were collected aseptically upon diagnosis as described by Oliver et al. (17). Before sample collection, teats of cows were cleaned thoroughly and dried with individual disposable paper towels, and teat ends were sanitized with swabs containing 70% isopropyl alcohol. After collection, samples were stored frozen until transported to the laboratory. Milk samples were examined following procedures described by Oliver et al. (17). Briefly, foremilk (10 ml ) from each quarter was plated on to one quadrant of a trypticase soy agar plate supplemented with 5% defibrinated sheep blood (Becton Dickinson Microbiology Systems, Cockeysville, MD). Plates were incubated at 37°C, and bacterial growth was recorded at 24-h intervals for 2 d. Bacteria on primary culture medium were identified tentatively according to colony morphologic features, hemolytic characteristics, Gram-stain reaction, and a catalase test. Isolates identified presumptively as staphylococci were tested for coagulase by the tube coagulase method, and all coagulase-positive isolates were plated on mannitol salt agar slants. Isolates identified presumptively as streptococci were evaluated initially for growth in 6.5% NaCl, hydrolysis of esculin and sodium hippurate, and CAMP reaction. Streptococcal organisms were identified to the species level using the API 20 Strep system (bioMerieux Vitek, Inc., Hazelwood, MO). Gram-negative isolates were plated on MacConkey’s agar (Becton Dickinson Microbiology Systems, Bedford, MA) and evaluated initially using the following biochemical tests: triple sugar iron, urea, oxidase, motility, indole, and ornithine decarboxylase. Journal of Dairy Science Vol. 81, No. 5, 1998

TABLE 1. Mastitis pathogens isolated from mammary glands of cows with clinical mastitis during early lactation. Pathogen Staphylococcus aureus Coagulase-negative staphylococci Streptococcus equinus Streptococcus dysgalactiae Streptococcus uberis Enterococcus faecalis Bacillus species Nocardia species Yeast Escherichia coli Serratia marcescens Total

(no.) 1 4 22 12 15 1 3 2 1 39 2 102

Gram-negative isolates were identified to the species level using the API 20E Identification System (bioMerieux Vitek, Inc.). Measures of reproductive performance, including AI per conception, days to conception, breeding period, and days to first AI, were obtained from DHIA records. Reproductive records of 102 cows with clinical mastitis during the first 150 d of lactation were compared with the reproductive records of 103 control cows with no signs of clinical mastitis. The two groups of cows were balanced as closely as possible by lactation number and days of lactation. Cows with clinical mastitis were further subdivided based on the time of occurrence of clinical mastitis as follows: group 1, before first AI ( n = 48); group 2, between first AI and pregnancy ( n = 14); and group 3, after confirmed pregnancy ( n = 40). Data were analyzed according to the general linear models procedure of SAS (23). RESULTS Distribution of clinical mastitis according to the Gram-stain status of mastitis pathogens was as follows: group 1, 31 Gram-positive and 17 Gramnegative; group 2, 5 Gram-positive and 9 Gramnegative; and group 3, 23 Gram-positive and 17 Gram-negative. The majority of infections was caused by environmental mastitis pathogens, primarily E. coli, Streptococcus equinus, Strep. uberis, and Streptococcus dysgalactiae (Table 1). No differences in measures of reproductive performance were detected because of milk production ( P = 0.62) or mastitis caused by Gram-positive or Gram-negative pathogens. Milk production ( X ± SEM) was as follows: group 1, 8482 ± 195 kg; group 2, 8355 ± 360 kg; group 3, 8346 ± 213 kg; and group 4, 8173 ± 133 kg. The number of days to first AI was greater ( P < 0.01) for cows with clinical mastitis before first AI (93.6 ± 5.6

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d ) than for all other cows (71.0 ± 2.2 d; Figure 1). The number of AI per conception was greater ( P < 0.01) for cows with clinical mastitis after first AI (2.9 ± 0.3) than for cows with clinical mastitis before first AI (1.6 ± 0.3), cows with no clinical mastitis, or cows with clinical mastitis after confirmed pregnancy (1.7 ± 0.1; Figure 2). The breeding period for cows with clinical mastitis between first AI and pregnancy was longer ( P < 0.01) than that for all other groups of cows (Figure 3). The number of days to conception for cows with clinical mastitis before first AI (113.7 ± 10.8 d ) and for cows with clinical mastitis after first AI (136.6 ± 13.3 d ) was greater ( P < 0.01) than the number of days to conception for control cows and cows that developed clinical mastitis after confirmed pregnancy (92.1 ± 4.6 d; Figure 4). Regardless of clinical mastitis, cows with more than five lactations required more ( P < 0.05) AI per conception (2.5 ± 0.31) than did all other parity groups (1.75 ± 0.15). The number of days to first AI was greater ( P < 0.05) for cows bred from October through December (84.3 d ) than that for cows bred from January through March (70.1 d ) or from April through September (72.3 d). However, analysis of covariance showed no

interaction between season and the presence of mastitis for any of the measures.

Figure 1. Influence of clinical mastitis during early lactation on days to first AI. Group 1, cows with clinical mastitis before first AI; group 2, cows with clinical mastitis between first AI and pregnancy; group 3, cows with clinical mastitis after confirmed pregnancy; and group 4, control cows with no clinical mastitis. Asterisk denotes differences ( P < 0.01) between groups.

Figure 2. Influence of clinical mastitis during early lactation on AI per conception. Group 1, cows with clinical mastitis before first AI; group 2, cows with clinical mastitis between first AI and pregnancy; group 3, cows with clinical mastitis after confirmed pregnancy; and group 4, control cows with no clinical mastitis. Asterisk denotes differences ( P < 0.01) among groups.

DISCUSSION Clinical mastitis during early lactation markedly influenced reproductive performance of lactating Jersey cows. However, in the present study, no differences in reproductive performance were detected between cows with clinical mastitis caused by Gramnegative or Gram-positive pathogens. Moore et al. ( 1 5 ) indicated that cows from a herd with mastitis caused primarily by Gram-negative pathogens were almost twice as likely to have an altered interestrus interval following clinical mastitis than were herdmates without clinical mastitis. However, in another herd with mastitis caused primarily by Staph. aureus, no significant changes in length of the estrous cycle were detected. Gram-positive pathogens associated with clinical mastitis in the present study were primarily environmental Streptococcus species. Thus, it is possible that some Gram-positive pathogens may have more of an impact on reproductive performance than others.

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Past research on the influence of mastitis on reproductive performance focused on experimental coliform mastitis and infusion of endotoxin from Gram-negative pathogens. However, the cell wall of both Gram-negative and Gram-positive bacteria is composed of a mucopeptide, peptidoglycan (22). Although both types of bacteria possess peptidoglycan, Gram-positive bacteria possess many layers of this component (22). The injection of peptidoglycan led to a pyretic response in rabbits that was comparable with the pyretic response elicited by endotoxin (20). The induced fever was eliminated by the administration of antiserum to the peptidoglycan, and peptidoglycan from various bacterial species produced comparable results (21). Therefore, the peptidoglycan fragment of some Gram-positive pathogens, such as Streptococcus species, can possibly produce responses that are similar to endotoxin infusion and coliform mastitis. Increased days to first AI for cows with clinical mastitis prior to first AI could have been due to insufficient follicular development; anovulation, resulting from blockage of the LH surge; or decreased estrogen synthesis, resulting in the loss of behavioral estrus. However, the length of the breeding period for

these cows was normal compared with that of control cows and did not require an additional AI as was necessary for cows with clinical mastitis between first AI and conception, suggesting that the ovulated follicle in these cows was viable. Infusion of bovine mammary glands with E. coli led to coliform mastitis and endotoxemia (16). Endotoxin, the lipopolysaccharide component of the Gram-negative bacterial cell wall (12, 16), is released upon lysis of the organism. Cullor ( 5 ) suggested that endotoxin may induce luteolysis and influence conception and early embryonic survival by release of inflammatory mediators. Specifically, endotoxin stimulates synthesis of PGF2a (4, 6), glucocorticoids (2, 12, 18), ACTH ( 3 ) , and interleukin-1 ( 1 2 ) and decreases GnRH release ( 2 ) . In addition, infusion of endotoxin resulted in a pyretic response in gilts ( 4 ) and dairy cows (11). Battaglia et al. ( 2 ) reported significant inhibition of GnRH pulse amplitude and total GnRH following intravenous endotoxin infusion. Consequently, insufficient follicular development could lead to insufficient estrogen production and subsequent anovulation. Blockage of the LH surge and lack of behavioral estrus could occur via various mechanisms. Increased corticosteroids were detected following experimentally

Figure 3. Influence of clinical mastitis during early lactation on length of breeding period. Group 1, cows with clinical mastitis before first AI; group 2, cows with clinical mastitis between first AI and pregnancy; group 3, cows with clinical mastitis after confirmed pregnancy; and group 4, control cows with no clinical mastitis. Asterisk denotes differences ( P < 0.01) among groups.

Figure 4. Influence of clinical mastitis during early lactation on days to conception. Group 1, cows with clinical mastitis before first AI; group 2, cows with clinical mastitis between first AI and pregnancy; group 3, cows with clinical mastitis after confirmed pregnancy; and group 4, control cows with no clinical mastitis. Asterisks denote differences ( P < 0.01) among groups.


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induced coliform mastitis (12, 18). Paape et al. ( 1 8 ) suggested that intramammary infusion of endotoxin may release interleukin-1 and other factors that can then be absorbed systemically. Increased concentrations of interleukin-1 reduced LH receptor concentration (16), stimulated release of corticotropinreleasing factor and ACTH, and increased steroidogenesis in the adrenal gland (12). Intravenous infusion of endotoxin increased cortisol ( 5 ) . Increased cortisol, in combination with a reduced number of LH receptors, blocked the LH surge and led to the formation of cystic ovaries (12, 25) and resulted in the absence of behavioral estrus (25). Cortisol inhibited aromatase activity in vitro (21), which could lead to blockage of the LH surge by inhibiting estrogen synthesis by the ovary (19). Cows with clinical mastitis between first AI and conception required an additional AI, had a greatly lengthened breeding period, and had the greatest number of days to conception compared with all other cows. These results could have been due to luteolysis, subsequent loss in progesterone, and early embryonic death. The bovine mammary gland synthesizes PGF2a (10, 13, 14), and increased PGF2a was found in milk from cows with clinical mastitis ( 1 ) . Intramammary infusion of Klebsiella pneumoniae led to increased plasma and milk PGF2a ( 5 ) . Recent evidence suggests that subluteolytic concentrations of PGF2a may compromise early embryonic development (24). Giri et al. (8, 9 ) infused E. coli endotoxin and caused a dose-dependent increase in serum PGF2a ( 9 ) and milk PGF2a ( 8 ) . Although this increase in plasma concentrations of PGF2a could induce luteolysis, others (1, 11) found no increase in the hormone during mastitis induced by endotoxin. Intravenous infusion of E. coli endotoxin led to a shortening of the estrous cycle in heifers ( 7 ) and increased plasma concentrations of PGF2a and cortisol, followed by a decrease in concentrations of progesterone in pregnant cows ( 5 ) , which led to abortion when infused in the first trimester but not when infused in the second or third trimester. These results suggested that luteolysis induced by endotoxin was more harmful when placental progesterone was not available ( 5 ) . In contrast, Lopez-Diaz and Bosu ( 1 2 ) reported that abortion was not induced after intramammary infusion of endotoxin in ruminants. Intravenous injection of E. coli endotoxin increased PGF2a metabolite, decreased progesterone, and resulted in abortion in goats ( 6 ) and gilts ( 4 ) . Treatment of gilts ( 4 ) and cows ( 9 ) with the cyclooxygenase inhibitor flunixin meglumine prevented PGF2a synthesis following endotoxin injection, thus

preventing destruction of the corpus luteum, decrease of progesterone, and abortion. CONCLUSIONS Results of this study suggest that clinical mastitis during early lactation can have a markedly negative impact on the reproductive performance of dairy cows. This phenomenon was not restricted only to Gramnegative pathogens as observed previously (5, 15). Therefore, both Gram-negative and Gram-positive pathogens may act through similar mechanisms to increase inflammatory mediators, leading to reproductive failure during early lactation. However, additional studies delineating mechanisms by which IMI influence reproductive performance are necessary to substantiate this hypothesis. ACKNOWLEDGMENTS This work was supported by the Tennessee Agricultural Experiment Station and The University of Tennessee, College of Veterinary Medicine, Center of Excellence Research Program in Livestock Diseases and Human Health. We thank Arnold Saxton and Edwin Townsend for advice on statistical analysis. REFERENCES 1 Anderson, L. K., H. Kindahl, R. A. Smith, L. E. Davis, and B. K. Gustafsson. 1986. Endotoxin-induced bovine mastitis: arachidonic acid metabolites in milk and plasma and effect of flunixin meglumine. Am. J. Vet. Res. 47:1373–1377. 2 Battaglia, D. F., J. M. Bowen, H. B. Krasa, L. A. Thrun, C. Viguie, and F. J. Karsch. 1996. Immune stress and reproductive neuroendocrine function: physiologic evidence for profound inhibition of GnRH secretion. Biol. Reprod. 54(Suppl. 1): 93.(Abstr.) 3 Besedosbky, H., A. DelRey, E. Sorkin, and C. A. Dinarello. 1986. Immunoregulatory feedback between interleukin-1 and glucocorticoid hormones. Science (Washington, DC) 233: 652–654. 4 Cort, N., and H. Kindahl. 1990. Endotoxin-induced abortion in early pregnant gilts and its prevention by flunixin meglumine. Acta Vet. Scand. 31:347–358. 5 Cullor, J. S. 1990. Mastitis and its influence upon reproductive performance in dairy cattle. Pages 176–180 in Proc. Int. Symp. Bovine Mastitis, Indianapolis, IN. Natl. Mastitis Counc., Inc. and Am. Assoc. Bovine Pract., Arlington, VA. 6 Fredricksson, G., J. Kindahl, and L. E. Edquist. 1985. Endotoxin-induced prostaglandin release and corpus luteum function in goats. Anim. Reprod. Sci. 8:109–121. 7 Gilbert, R. O., W.T.K. Bosu, and A. T. Peter. 1990. The effect of Escherichia coli endotoxin on luteal function in Holstein heifers. Theriogenology 33:645–651. 8 Giri, S. N., Z. Chen, E. J. Carroll, R. Mueller, M. J. Schiedt, and L. Panico. 1984. Role of prostaglandins in pathogenesis of bovine mastitis induced by Escherichia coli endotoxin. Am. J. Vet. Res. 45:586–591. 9 Giri, S. N., G. H. Stabenfeldt, T. A. Mosley, T. W. Graham, M. L. Bruss, R. H. BonDurant, J. S. Cullor, and B. I. Osburn. 1991. Role of eicosanoids in abortion and its prevention by Journal of Dairy Science Vol. 81, No. 5, 1998

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