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Periparturient Diseases of Cattle Research Unit, USDA–ARS, National Animal Disease Center, Ames, IA 50010
Corresponding author: T. A. Reinhardt; e-mail: treinhar{at}nadc.ars.usda.gov.
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ABSTRACT |
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Key Words: Ca2+-adenosine triphosphatase • milk fever • milk fat globule membrane • mammary gland
Abbreviation key: MF = milk fever, MFGM = milk fat globule membrane, MG = mammary gland, PMCA = plasma membrane Ca2+-ATPase (The number following PMCA and SPCA refers to the specific isoform. The first lowercase letter following the number in PMCA refers to the splice site C splice form, and the next lowercase letter refers to the splice site A splice form). SPCA = secretory pathway Ca2+-ATPase
Research on milk fever (MF) has historically focused on methods to control intestinal Ca absorption and bone Ca resorption in the periparturient cow (Goff and Horst, 2003; Horst et al., 2003). The potential role that mammary gland (MG) Ca storage and transport has on the development of MF has been ignored despite the primary role of the MG in MF development (Goff et al., 2002). The objectives of this study were to measure MG and milk fat globule membrane (MFGM) expression of the major mammary Ca2+-ATPase proteins in the periparturient period and examine Ca2+-ATPases protein expression in normal and MF cows.
Three weeks prepartum, 15 Jersey cows were fed a diet that promoted MF (Goff et al., 2002). Mammary gland biopsies were collected from 12 cows on d –7, 0, 7, 14, with d 0 representing calving day. Because of the uncertainty of the time of calving, the d –7 samples ranged from –4 to –12 d. After calving, cows were fed a normal lactation diet (Goff et al., 2002). Blood samples were collected to monitor plasma Ca and plasma 1,25(OH)2D3 concentrations (Reinhardt et al., 1984). Cows were classified MF cows according to their clinical signs and plasma Ca levels. Mammary gland, MFGM, and bovine brain membranes were prepared as previously described (Reinhardt et al., 2000). Membrane preparations were stored at –70°C. Proteins were determined using the BioRad Protein Assay Kit. The Ca2+-ATPase protein expression in the MG and MFGM was determined by Western blotting as described by Reinhardt et al. (2000). Antiplasma membrane Ca2+-ATPase 2 and 4 (PMCA2, PMCA4) and secretory pathway Ca2+-ATPase 1 (SPCA1) antibodies were described previously (Reinhardt et al., 2000, 2004a,Reinhardt et al., b). One lane in every gel was loaded with a constant amount of brain membrane to serve as a standard. The results from image analysis for the blots were normalized to the brain microsomal membrane lane and expressed in arbitrary units.
Data were analyzed by repeated measures using the PROC MIXED procedure of SAS (SAS Inst., Inc., Cary, NC). The model included the fixed effects of time and MF status, time by MF interaction, the random effect of cows nested within MF status, and the residual error. For each variable analyzed, cow nested within MF status was subjected to 3 covariance structures; compound symmetry, autoregressive order 1, and unstructured covariance. The covariance that resulted in the Akaike’s information criterion closest to zero was used, which was autoregressive order 1. Means and standard errors of the means are reported for all data. Means separation was conducted by the Tukey-Kramer option in SAS.
Seven of 15 cows developed MF. Plasma Ca concentration decreased in MF cows just before (P < 0.05) and at parturition (P < 0.001) (Figure 1A
) compared with non-MF cows. Plasma 1,25-(OH)2D3 concentrations were elevated on d 1 prepartum (P < 0.05) through d 2 (P < 0.001) of lactation in MF cows (Figure 2B) compared with non-MF cows. The expression of Ca2+-ATPase proteins in the MG was measured in MG biopsies (Figure 2). There was an effect of MF status on SPCA1 expression (P < 0.05), and the MF by time interaction was significant (P < 0.001). Milk fever cows expressed more SPCA1 (P < 0.001) prepartum than non-MF cows (Figure 2A). The SPCA1 expression was not different between MF and non-MF cows from parturition through d 14 of lactation and declined from prepartum levels (P < 0.001). The PMCA2bw expression (Figure 2B) was not different between MF and non-MF cows. The MF by time interaction was not significant. Expression of PMCA2bw did increase with time and was elevated (P < 0.05) on d 14 of lactation compared with d –7. Expression of PMCA4b (Figure 2C) remained constant and was not different between MF and non-MF cows. The interaction was not significant. Expression of PMCA2bw in MFGM (Figure 3A) was not affected by MF status. The MF by time interaction was not significant. Expression of PMCA2bw in MFGM, however, increased (P < 0.001) in all cows by d 7 and 14 of lactation. Expression of PMCA4b in MFGM was lower (P < 0.05) in cows that developed MF (Figure 3A), but the interaction was not significant. In all cows, PMCA4b expression (Figure 3B) in MFGM declined as lactation proceeded (P < 0.05).
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). At parturition, SPCA1 expression in the MF cows was equal to that of the non-MF cows, but its higher expression prepartum may be just enough Ca stress to upset Ca homeostasis, thus leading to MF. In the MFGM, PMCA4b was significantly lower in MF cows (Figure 3B). For this, we have no explanation, as the role of PMCA4b in the MG has yet to be explored. It has been speculated the PMCA4b may be important to mammary development (Reinhardt et al., 2000), but PMCA4 has been shown to have few critical functions (Okunade et al., 2004). The precise metabolic lesions responsible for MF development are still being debated and are likely a complex interacting network of Ca stresses that, when added together, result in the development of this disease. This study provides data that indirectly suggest that increased prepartum MG Ca storage may contribute to the development of MF. Cows that develop MF express significantly more SPCA1 in their MG prepartum. The SPCA1 is a Golgi Ca pump responsible for loading Ca into the Golgi. Therefore, these data provide new and novel insights into potential additional mechanisms associated with MF development. In addition, data on the protein expression of the Ca pump (PMCA2bw) responsible for Ca transport into milk (Reinhardt et al., 2004b), SPCA1, and PMCA4b are presented for the first time in the cow.
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FOOTNOTES |
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Received for publication August 26, 2004. Accepted for publication November 29, 2004.
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) and non-MF cows (n = 8; •) during the periparturient period. Significantly different from non-MF cows: *P < 0.05; **P < 0.001.
