| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Dept. of Animal Sciences, Auburn University, AL 36849
2 Dept. of Animal Science, Iowa State University, Ames
Corresponding author: K. A. Cummins; e-mail: kcummins{at}acesag.auburn.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT REFERENCES |
|---|
Key Words: diet • calpastatin • muscle • lactation
Abbreviation key: TEMED = N, N, N', N'-tetramethylenediamine
Dairy cattle are capable of mobilizing body protein, largely from skin, muscle and bone, during early lactation (Swick and Benevenga, 1977). This reserve has been estimated from depletion experiments to be as large as 24 kg of protein (Botts et al., 1979; Huber and Kung, 1981). Dairy cows fed 16 or 19% CP diets mobilized 21 kg of body protein between 2 wk prepartum and 5 wk postpartum (Komaragiri and Erdman, 1997). Mobilization of body protein reserves is expected in early lactation and included in dietary nutrient requirements (NRC, 2001), although how the level of dietary CP affects skeletal muscle mobilization is not well established. Vallimont et al. (2001) observed changes in plasma amino acid concentrations consistent with mobilization of skeletal muscle in cows fed a 17.8% CP diet in the immediate postpartum period, and suggested this mobilization may be for carbon skeletons needed to support glucose synthesis, independent of protein requirements.
The calpain/calpastatin system initiates turnover of myofibrillar proteins by making specific cleavages (Goll et al., 1992). Increases in calpastatin activity and resulting decreases in fractional protein degradation appear to be associated with muscle growth in cattle (Morgan et al., 1993), and callipyge lambs (Lorenzen et al., 2000). Various beta-agonists also increase muscle growth and increase calpastatin activity in cattle (Koohmaraie et al., 1991; Wheeler et al., 1992) In steers, changes in calpain and calpastatin mRNA content were proportional to changes in activity when fed a beta-agonist (Parr et al., 1992),
Effects of altering dietary CP on muscle calpain and calpastatin content in lactating dairy cattle have not been investigated. Our hypothesis was that dietary CP restriction and refeeding would alter skeletal muscle content of calpain and calpastatin during peak lactation.
The Auburn University Institutional Animal Care and Use Committee approved all procedures. Animals were housed at the E. V. Smith Research Center Dairy Research Unit at Shorter, AL. Sixteen multiparous Jersey cows were assigned at calving to one of 4 dietary treatments in a completely randomized design. All cows were fed an 18% CP diet from calving to 30 DIM. At 31 DIM, cows were fed a 9, 12, 15, or 18% CP diet until 58 DIM (depletion period). At 59 DIM, each cow was then fed the 18% CP diet until 84 DIM (repletion period). Diets are shown in Table 1
. The diets were fed as TMR.
|
Muscle biopsies were taken at 49 DIM, 19 d after the start of the depletion period, and at 84 DIM, after 26 d of repletion. Biopsies were taken from the semitendinosus muscle of alternate legs using 2% lidocain as a local anesthetic for a regional block. Samples of approximately 5 g wet weight were immediately frozen in liquid nitrogen upon removal.
The procedure used for sarcoplasmic muscle protein extraction was as described by Doumit et al. (1996). Ninety micrograms of protein from each sample was loaded into separate wells of a 10 cm x 12 cm x 1.5 mm Hoefer SE 260B Mighty Small II (Pharmacia Biotech; San Francisco, CA) 15% (for calpastatin) and 10% (for µ-calpain) acrylamide separating gel (acrylamide/bis [100:1 acrylamide: bisacrylamide]), 0.375 M Tris-HCl, pH 8.8, 0.1% SDS, 0.05% ammonium persulfate, and 0.05% N,N,N',N'-Tetramethylenediamine (TEMED). The separating gels had a 5% acrylamide stacking gel (5% acrylamide/bis [100:1 acrylamide: bisacrylamide], 0.125 M Tris-HCl, pH 6.8, 0.1% SDS, 0.125% TEMED, and 0.075% ammonium persulfate). The composition of the running buffer used was 25 mM Tris, 192 mM glycine, and 0.1% SDS, pH 8.3. At completion, the proteins on the gel were transferred immediately to Westran PVDF (polyvinylidene fluoride) membranes (Schleicher & Schuell, Inc.; Keene, NH). Gels were transferred for 90 min at 90 V at 4°C in a TE 22 Transphor Electrophoresis Unit (Hoefer Scientific Instruments; San Francisco, CA). Calpastatin was detected using a mouse anti-calpastatin antibody as described by Lonergan et al. (2001) µ-Calpain was detected with a mouse anti-µ-calpain (cat. # RDI-Ucalpain; Research Diagnostics Inc., Flanders, NJ) diluted 1:1000 in PBS-Tween (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, and 0.1% polyoxyethylene 20-sorbitan monolaurate [Tween 20]). Membranes were incubated for 1 h at room temperature in secondary antibody, goat-anti-mouse IgG conjugated with horseradish peroxidase, (cat. # A2554, Sigma; St. Louis, MO) diluted 1:5,000 in PBS-Tween with 1% nonfat dry milk). Bands were visualized using ECL Western Blotting reagents (Amersham Life Science, Arlington Heights, IL) by exposure to film (Kodak BioMax Light-1 Film, 13 x 18 cm, Kodak No. 8689358) as directed by the manufacturer.
Statistical analysis for milk production, DMI, and milk components was analysis of variance for a completely randomized design with repeated measures. Analysis was done using the PROC GLM procedure of SAS (SAS Institute, 1999), with treatment (diet sequence), period (depletion or repletion) treatment x period, and cow within treatment in the model. Cow within treatment was used to test the main effects of diet and period.
Mean milk production, DMI, and milk protein contents for the depletion and repletion periods are shown in Table 2. Milk production was lower in cows fed low CP during the depletion period (P < 0.05), but not during the repletion period. Cows fed lower dietary CP consumed more DM during the repletion period (P < 0.05). There was a treatment x period interaction for both milk production and DMI (P < 0.05). Milk protein content was reduced in cows fed lower CP diets (P < 0.05). Milk fat content was not altered by period or treatment (P > 0.1).
|
Two Western blots of muscle calpastatin at 84 DIM for 4 cows, one each for the 4 dietary treatments on each blot, are shown in Figure 1. All blots for calpastatin for samples taken during the repletion period (84 DIM) showed the same pattern. A 135-kDa band was observed at 84 DIM only for cows fed the 15 and 18% CP diets from 31 to 58 DIM. Cows fed the 9 and 12% CP diets during this period did not show the 135-kDa band. A band for a 67-kDa form was observed in all cows.
|
Regulation of muscle breakdown and growth during a period of dietary repletion appears to differ in dairy cows from the mechanism regulating growth in young cattle. Calpastatin was altered by periods of protein depletion and repletion but in an opposite manner from that usually observed in growing cattle, decreasing in cattle previously fed lower dietary CP and which would be undergoing muscle protein repletion when fed high CP diets. Dairy cows are mature animals, and, while synthesizing large quantities of protein daily, it is exported in the milk. These factors may explain the different response observed compared to growing cattle.
Received for publication September 9, 2003. Accepted for publication January 30, 2004.
|
REFERENCES |
|---|
|
TOPABSTRACTREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  W. G. Bergen Measuring in vivo intracellular protein degradation rates in animal systems J Anim Sci, April 1, 2008; 86(14_suppl): E3 - E12. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
