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,2
* Department of Biology, Faculty of Sciences, University of Sherbrooke, Sherbrooke, Quebec, Canada, J1K 2R1
Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, PO Box 90, STN Lennoxville, Sherbrooke, Quebec, Canada J1M 1Z3
Department of Animal Science, McGill University, Ste. Anne de Bellevue, Quebec, Canada, H9X 3V9
Department of Physiology and Pharmacology, Faculty of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1
# Department of Animal Science, Cornell University, Ithaca, NY 14853
2 Corresponding author: lacassep{at}agr.gc.ca
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ABSTRACT |
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 TOPFOOTNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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, IL-4, and interferon-
). Cows milked 1x produced 31% less milk than cows milked 2x during the first week of lactation. Over the following 13 wk of lactation, the milk production of cows milked 1x during the first week was 8.1% lower than for cows milked 2x. However, because the percentages of fat and protein were greater in the milk from 1x cows, the yields of milk components and energy-corrected milk were similar. Calving induced an increase in the concentrations of NEFA, BHBA, urea, and bilirubin. The increases in levels of NEFA and BHBA were greater in cows milked 2x than in cows milked 1x. During the same period, the serum glucose concentration decreased but remained greater in cows milked 1x. Serum calcium on d 4 and serum phosphorus on d 4 and 5 were greater in cows milked 1x. The differences between the 2 groups persisted beyond treatment until postpartum d 24 for NEFA and glucose and until postpartum d 14 for BHBA. After calving, the concentrations of leptin and stanniocalcin-1 decreased. During the first week postpartum, the decrease of leptin was less marked in cows milked 1x. The immune functions of PBMC and PMNL isolated from experimental cows and incubated using a standard medium did not show clear-cut peripartum immunosuppression. These variables were not significantly affected by the treatments, with the exception of interferon- secretion, which was greater on d 5 and 14 in cows milked 1x. In conclusion, limiting milk production in early lactation had positive effects on metabolite concentration, but larger studies are necessary to establish if this could reduce disease incidence.
Key Words: immune function • leptin • stanniocalcin-1 • transition period
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INTRODUCTION |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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During the periparturient period, the dairy cow experiences a natural state of immunosuppression, which is associated with a high susceptibility to infectious diseases such as mastitis (Kehrli and Harp, 2001). Parturition in dairy cattle is associated with impairment of PMNL phagocytosis and oxidative burst activity (Kehrli et al., 1989; Hoeben et al., 2000) and with a decrease in the ability to fight bacterial infections. This period is also marked by decreased responsiveness of blood lymphocytes to stimulation with mitogenic agents and by decreased immunoglobulin production by B cells (Nonnecke et al., 2003). Various hypotheses have been put forward that attribute the impairment of immune functions around the time of calving to endocrine or metabolic changes. In vitro, the presence of 17β-estradiol (E2), NEFA, and BHBA in high concentrations can adversely affect the respiratory burst and the phagocytic capacity of PMNL (Suriyasathaporn et al., 2000; Lamote et al., 2004, 2006; Scalia et al., 2006). The hormone leptin declines at calving in dairy cows and leptin has been reported in rodents to stimulate cytokine production and immune function (Ingvartsen and Boisclair, 2001). It may therefore be implicated in periparturient immunosuppression. Stanniocalcin-1 (STC-1) is another hormone that has been implicated in regulation of the immune response (Kanellis et al., 2004; Chakraborty et al., 2007). Although the variation of the cow’s systemic STC-1 in early lactation has not been evaluated, it does vary according to lactation cycle in both rodents and cows (Deol et al., 2000; Tremblay et al., 2009).
In the dairy industry, cows are genetically selected for high milk production. Unfortunately, the greater milk output increases health problems in early lactation by increasing the NEB (Goff and Horst, 1997). Milking frequency is a factor that affects both milk yield and metabolic disturbances around calving (Rémond and Pomiès, 2005). Cows milked 3 times daily had 19% greater plasma BHBA concentrations and 6% lower glucose concentrations compared with cows milked twice daily (Andersen et al., 2004). In early lactation, plasma NEFA and BHBA concentrations and milk production are lower in cows milked once daily compared with cows milked 3 times daily (Patton et al., 2006). Therefore, our hypothesis was that reducing the NEB by limiting milk production in early lactation would help to attenuate metabolic disturbances and, at the same time, decrease the magnitude of the periparturient immunosuppression. The objective of this study was to evaluate the effect on immune function and blood metabolite concentration of milking cows once a day during the first week of lactation.
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MATERIALS AND METHODS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Few peripartum diseases were observed: 1 cow experienced metritis (2x group) and 8 experienced mastitis (3 in 1x group and 5 in the 2x group). One case of dystocia (2x group) and 1 case of retained placenta (1x group) were observed.
Isolation of PBMC
Peripheral blood was centrifuged at 540 x g for 30 min, and the plasma and the packed red blood cells were discarded. The PBMC were isolated from the buffy coat by gradient centrifugation in Ficoll Paque Plus (30 min at 540 x g; Amersham Corp., Arlington Heights, IL). The PBMC from the Ficoll gradient were loaded onto a sucrose gradient (7 mL of 10% sucrose and 30 mL of 20% sucrose) and centrifuged at 540 x g for 8 min. The supernatant was discarded and the pellet was washed with Hanks’ Balanced Salt Solution (HBSS, without Ca2+ and Mg2+, Gibco Laboratories, Grand Island, NY) and centrifuged at 300 x g for 8 min. The red blood cells were lysed with Tris-NH4Cl. The PBMC were washed with HBSS and resuspended in 2 mL of RPMI (Gibco) containing 5% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) and antibiotics/antimycotic (A5955, Sigma Chemical Co., St. Louis, MO). The viable cells were counted with a Bright-Line hemacytometer (Hausser Scientific, Horsham, PA) using trypan blue exclusion methodology.
Isolation of Blood PMNL
Polymorphonuclear leukocytes were isolated from peripheral blood by centrifugation at 540 x g for 30 min, and the plasma and buffy coat were discarded. The packed red blood cells were lysed with Tris-NH4Cl twice and centrifuged at 210 x g for 5 min. The supernatant was discarded and the PMNL were washed with 45 mL of HBSS and centrifuged. The PMNL were resuspended in 2 mL of RPMI preheated to 37°C. The cells were counted with a Bright-Line hemacytometer.
Proliferation of PBMC
The proliferation of PBMC was determined by flow cytometric analysis of CFDA-SE (carboxyfluorescein diacetate, succinimidyl ester) staining (Tesfa and Kern, 2003). The PBMC were adjusted to a concentration of 2 x 107 cells/mL in 37°C preheated HBSS with 0.2 mM of the fluorophore CFDA-SE (Molecular Probes, Eugene, OR) and then incubated at 37°C in a water-saturated 5% CO2 atmosphere for 15 min. Then, the cells were centrifuged at 300 x g for 5 min and resuspended in 11 mL of cold RPMI containing 5% FBS and antibiotics/antimycotic; cells were then incubated at room temperature for 15 min in the dark. The PMBC were centrifuged again and resuspended in RPMI (Gibco) containing 5% FBS and antibiotics/antimycotic. The cells were then aliquoted into the wells of a 6-well plate (3.5 mL and 5 x 106 cells per well). The PBMC (2 wells) were incubated with the mitogen concanavalin A (Sigma) at a final concentration of 1 µg/mL at 37°C in a water-saturated 5% CO2 atmosphere for 3.5 d. As a negative control, PBMC were incubated without concanavalin A. The cells were subsequently centrifuged at 300 x g for 5 min and resuspended in PBS containing 0.5% formaldehyde, and the proliferation of PMBC was analyzed by flow cytometry (excitation and emission wavelengths: 492 and 517 nm) on a Coulter Epics XL-MCL flow cytometer (Beckman Coulter, Fullerton, CA).
Cytokine Production
To evaluate tumor necrosis factor- (TNF-) production, PBMC were distributed in 24-well plates at a density of 1 x 107 cells/mL. Phorbol 12-myristate 13-acetate (PMA, Calbiochem, La Jolla, CA) at final concentration of 10 µg/mL and Ca2+ ionophore (ionomycin from Streptomyces conglobatus, Sigma) at final concentration of 100 µg/mL were added, and the PBMC were incubated for 24 h at 37°C. For the determination of IL-4 and IFN-, 1 mL of the supernatant from PBMC stimulated with concanavalin A was recovered 24 h after incubation. Supernatant cytokine content was determined with commercial kits (Endogen, Rockford, IL).
Phagocytosis Assay
The PMNL were centrifuged at 1,700 x g for 5 min and resuspended at a concentration of 1 x 107 cells/mL in Krebs’ Ringer PBS. The PMNL were distributed in 2 tubes, each containing 1 x 106 cells. Before their utilization, Fluoresbrite YO Carboxylate Microspheres (6.0 µm, Polysciences Inc., Warrington, PA) were opsonized with naive cow serum diluted 1:5 in Krebs’ Ringer PBS and incubated for 30 min at 37°C. The opsonized microspheres were added to the PMNL and incubated for 30 min: 1 tube was incubated at 37°C and the other at 4°C (control). The reaction was stopped by adding 1.8 mL of cold PBS. The PMNL were washed 3 times with 1.5 mL of cold PBS and centrifuged at 1,700 x g for 5 min. After the final centrifugation, PMNL were resuspended in 500 µL of cold PBS and fluorescence was analyzed by flow cytometry (excitation and emission wavelengths: 535 and 570 nm) on a Coulter Epics XL-MCL flow cytometer (Beckman Coulter).
Chemotaxis Assay
Zymosan-activated FBS (ZAS) was prepared as described by Lamote et al. (2004). Before use, the ZAS was diluted 1:5 with HBSS and filtered through a 0.22-µm filter.
A micropore Transwell Permeable Support was used to assess the chemotaxis of PMNL (12-mm diameter, 3-µm pore size; Corning Costar 3462, Oneonta, NY). To perform the assay, cells were adjusted to 1 x 107 PMNL/mL in PBS. Two hundred microliters of PMNL suspension was added to the upper chambers and 400 µL of ZAS was placed in the lower chambers. For the negative control, 400 µL of HBSS was used in the lower chamber. After incubation for 90 min at 37°C in a water-saturated 5% CO2 atmosphere, migrated PMNL were counted with a Bright-Line hemacytometer. Results were expressed as the percentage of cells that migrated through the membrane. Each assay was performed in duplicate.
Respiratory Burst Activity Assay
Reactive oxygen species production by PMNL was quantified after stimulation of the cells with PMA. Cell preparation (100 µL of 107 cells/mL), 60 µL of 1 mM luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, stock solution in dimethyl sulfoxide; Sigma), and 20 µL of 0.2 µg/mL PMA (stock solution in dimethyl sulfoxide; EMD, Darmstadt, Germany) were added to 220 µL of preheated (37°C) RPMI without phenol red. The measurement of H2O2 release was monitored using a Modulus Single Tube Luminometer (Turner Biosystems, Sunnyvale, CA) in duplicate over a period of 30 min at 37°C with acquisition every 10 s. A negative control was performed by replacing the PMA solution with an equivalent volume of dimethyl sulfoxide. The area under the curve was calculated over a period of 30 min with SAS software (SAS Institute, Cary, NC).
Ovalbumin Antibody
To evaluate antibody production during the peripartum period, the animals received a subcutaneous injection of 1 mL of ovalbumin antigen (Sigma; final concentration of 1 mg/mL) with 1 mL of incomplete Freund’s adjuvant (Sigma) on d 5 and d 26 after calving. Blood samples were collected from the animals twice a week during the experiment and serum was frozen at –20°C until analyzed. Ovalbumin antibody titer was determined by ELISA. Briefly, 96-well microplates were coated with 5 µg/mL ovalbumin. Serial dilutions (1:2) of the sera were loaded on the microplates. Antibody against ovalbumin was detected with peroxidase-complexed goat antibody against bovine IgG (Jackson Immunoresearch, West Grove, PA, diluted at 1:10,000). Peroxidase activity was detected with tetramethyl benzidine (TMB) reagent (KPL, Gaithersburg, MD) according to the manufacturer’s recommendations.
Hormone Assays
The serum concentration of E2 was determined by RIA using the approach described by Delbecchi and Lacasse (2006). The serum concentration of STC-1 was determined by RIA as described by Niu et al. (2000). The plasma leptin concentrations were determined by RIA as described by Ehrhardt et al. (2000).
Metabolite Assays
Plasma urea was determined using a BUN commercial kit (Diagnostic Chemicals Ltd., Charlottetown, Prince Edward Island, Canada), and absorption was monitored using a Pharmacia Biotech Ultrospec-3000 spectrophotometer (Pharmacia Biotech, London, UK). Bilirubin was determined in a commercial laboratory (BioVet, St-Hyacinthe, Quebec, Canada). Serum calcium was determined by using the QuantiChrom Calcium Assay Kit (BioAssay Systems, Hayward, CA). Serum BHBA was evaluated with the β-hydroxybutyrate reagent kit (Pointe Scientific Inc., Canton, MI). Serum phosphorus was determined with the Phosphorus-SL assay (Diagnostic Chemicals Ltd.).
Plasma NEFA was determined with the NEFA-HR-2 kit (Wako, Richmond, VA), following the manufacturer’s protocol modified to accommodate a 96-well microplate. Briefly, reagents A and B were prepared as described in the protocol. Five standards were prepared from the supplied stock. The samples, standards, and blanks were added in duplicate at 10 µL per well. Then, 100 µL of reagent A was added and incubated for 30 min. Finally, 200 µL of reagent B was added and incubated for an additional 30 min and then the plate was read at 550 nm using a SpectraMAX 250 microplate reader (Molecular Devices Co., Sunnydale, CA).
Plasma glucose was determined with the GLU Roche/Hitachi kit by following a modified version of the manufacturer’s protocol to accommodate a 96-well microplate (Roche Diagnostics, Indianapolis, IN). For the glucose assay, 200 µL of GOD-PAP reagent from the kit was added to 10 µL of plasma sample and read at 510 nm on a Spectra Max 250 microplate reader (Molecular Devices Co.).
Statistical Analysis
Data were analyzed separately for the prepartum period, the treatment period (the week following calving), and wk 2 to 14 of lactation. Because measurements made before calving were not made at fixed times (calving time is not precisely predictable), data were analyzed using time as a regression variable. Postcalving data were analyzed as repeated measurements using the MIXED procedure of SAS with spatial power as the covariance structure. For DMI, a global analysis was performed over the 3 periods using the MIXED procedure of SAS with spatial power as the covariance structure. The weekly increase in DMI in early lactation was calculated and analyzed with PROC MIXED. Evolution of immune parameters across time was determined by averaging the data by periods (precalving, treatment, and posttreatment) and analyzed using the MIXED procedure of SAS. Cytokine production was analyzed using the GLIMMIX procedure with the Gamma distribution function (SAS Institute).
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RESULTS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Blood Metabolites
Following calving, concentrations of both NEFA and BHBA (Figure 2, panels A and B) were significantly greater in cows milked 2x compared with 1x (P < 0.05 and <0.01 for NEFA and BHBA, respectively). After parturition, the glucose concentration decreased (P < 0.001) in the 2 groups but remained greater (P < 0.001) in cows milked 1x (Figure 2, panel C). The difference between the 2 groups persisted beyond the treatment period until postpartum d 24 for NEFA and glucose and until postpartum d 14 for BHBA. Serum calcium on d 4 (P < 0.05) and serum phosphorus on d 4 (P = 0.07) and d 5 (P < 0.02) were greater in cows milked 1x (Figure 3, panels A and B). At calving, bilirubin increased (P < 0.001) but there was no difference between the 2 experimental groups (data not shown). Plasma urea was greater in cows milked 2x on d 3 (4.53 vs. 5.66 mM; P < 0.05; data not shown).
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was greater in cows milked 1x on d 5 (P < 0.02) and d 14 (P < 0.01; Figure 6, panel A). Although the release of IL-4 increased in the posttreatment period (P < 0.01), no treatment effect was found (Figure 6, panel B). At calving, the production of TNF- by stimulated PBMC increased (P < 0.05) and then declined afterward (Figure 6, panel C). No treatment effect on TNF- production was noted.
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DISCUSSION |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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In our experiment, we found that milk production tended to be less even after a return to twice-daily milking. Hale et al. (2003) reported that increasing milking to 4 times a day during the first 3 wk of lactation increases milk production for the first 6 mo of lactation. However, the reduction in milk production was completely compensated by an increase in the concentration of milk components, and the yields of protein, fat, lactose, and ECM were similar. Accordingly, Hale et al. (2003) and Fernandez et al. (2004) reported that milking 4 times a day in early lactation reduced subsequent milk fat and protein content, so that the yields of milk fat and protein did not differ.
In our study, reduced milking frequency in early lactation led to a decrease in metabolic imbalances as reflected in findings similar to those described previously (Patton et al., 2006). Accordingly, an increase in milking frequency in early lactation has been reported to accentuate metabolic imbalances (Andersen et al., 2004; Fernandez et al., 2004). As expected, NEFA and BHBA blood concentrations increased after calving. The concentrations of NEFA and BHBA provide an indication of fat mobilization, and NEFA is often used as an indicator of NEB. The NEFA and BHBA profiles differed between cows milked 1x and those milked 2x. The blood concentrations of NEFA and BHBA were lower in cows milked 1x and stayed lower even after the switch to twice-daily milking was made in the second week of lactation. The fact that the urea level was transiently lower in 1x cows is also indicative of a lower need to use amino acid for gluconeogenesis in these animals. These results show that reducing milking frequency during the transition period contributes to a better energy balance that persists beyond the treatment period.
In ruminants, plasma glucose is derived almost exclusively from gluconeogenesis. Around calving, plasma glucose concentrations decrease because of the high energy demands associated with milking and the harnessing of glucose for the production of lactose. Low glucose levels can also be a consequence of decreased appetite and impaired liver function (Drackley et al., 2001). In our study, the plasma glucose concentration was greater in cows milked 1x, a result that is consistent with the positive effects observed on energy status and metabolite profiles. The glucose concentration in cows milked 1x remained higher even after the treatment period. Ketone bodies are known to impair gluconeogenesis (Goff and Horst, 1997). Therefore, these cows have greater glucose levels, probably because of their lower milk production, lower fat mobilization, and increased gluconeogenesis.
Serum concentrations of phosphorus and calcium were slightly greater in 1x cows during the first week of lactation. Calcium and phosphorus are both present in milk. During the first week of lactation, the calcium and the phosphorus concentrations decreased for a few days because of the adaptation required to meet the high demand for milk production. None of our cows experienced clinical hypocalcemia. A larger study would be needed to determine if this gain in calcium and phosphorus is sufficient to reduce the incidence of milk fever.
There was no difference between the DMI of cows milked 1x and cows milked 2x. However, between wk 1 and 2 of lactation, the increase in DMI was significantly greater for cows milked 1x. High NEFA and BHBA levels cause a decrease in appetite (Ingvartsen and Andersen, 2000) and may have delayed the increase in DMI in 2x cows. In early lactation, cows milked 1x have lower blood concentrations of NEFA and BHBA, which can lead to an increase in appetite and contribute to a less negative energy balance. Similarly, the BW loss was reduced in cows milked 1x, reflecting a less negative energy balance.
Plasma leptin has been shown to be negatively correlated with NEFA concentration, and the decrease in leptin around parturition is caused by the NEB (Block et al., 2001). The present study clearly showed that leptin decreased at calving, which is in agreement with the findings of previous studies. During the treatment period, the plasma leptin concentration was greater in cows milked 1x, probably because of their less negative energy balance. In mice, starvation-induced immunosuppression was reversed by exogenous leptin (Lord et al., 1998). The role of leptin in regulation of bovine immune function has not been established, but its implication in the immunosuppression observed in postpartum cows cannot be ruled out.
As previously observed, estrogen levels increased just before calving and then declined (Goff and Horst, 1997). In an earlier study in our laboratory, E2 injections were found to increase the risk of mastitis (Delbecchi et al., 2005). Polymorphonuclear leukocytes have estrogen receptors and in vitro studies reported that E2 decreased their functional properties (Lamote et al., 2004, 2006). We therefore hypothesized that estrogens may play a role in suppressed immunity observed at calving. In our study, the E2 level peaked a few days before calving and E2 concentrations did not vary significantly among the cows. After calving, the E2 level declined sharply. Cows milked 1x had greater blood concentrations of E2 during the treatment period; however, the level was very low and the difference would probably not affect the immune system.
In our study, the STC-1 concentration decreased after calving. This is the first time that peripartum STC-1 concentrations are reported in cows but the profile is similar to that in mice (Deol et al., 2000). In the Delbecchi et al. (2005) study, we observed that estradiol injections increased both milk and serum level of STC-1, as well as increasing the risk of mastitis. In humans, STC-1 inhibits migration of macrophages and T lymphocytes (Kanellis et al., 2004; Chakraborty et al., 2007). As the postpartum level of STC-1 was low and unaffected by treatment, it is unlikely a major component of postcalving immunosuppression. Nevertheless, an effect in late lactation cannot be excluded.
The immunosuppression phenomenon observed at calving appears to be linked to the production of milk. In mastectomized cows, the number of immune cells and their associated functions did not provide clear evidence that calving itself causes immunosuppression (Kimura et al., 1999, 2002; Nonnecke et al., 2003). We therefore hypothesized that reducing milking frequency immediately after parturition would counter immunosuppression. In our study, the immune functions evaluated for PBMC (proliferation and cytokine production) are indicative of a mild immunosuppression around calving. Lymphocyte proliferation under concanavalin A stimulation indicates that there is a slight reduction in lymphocyte activation during the week after calving, but the functions were not significantly affected by the treatment. The production of IL-4 and TNF- also provided evidence of immunosuppression but these cytokines were not affected by the treatment. In addition, the PMNL functions that were evaluated did not show a clear-cut immunosuppressive state. The causes of immunosuppression in early lactation are not known. In vitro studies have shown that the addition of NEFA, BHBA, and E2 have a negative effect on the cows’ immune cells (Suriyasathaporn et al., 2000; Lamote et al., 2004, 2006; Scalia et al., 2006). In our study, the immune cells isolated from the cows’ blood were incubated in FBS-supplemented medium. Conversely, when PBMC from mid-lactation cows were incubated with serum from cows of this experiment, the immunosuppressive effects were observed (C. Ster, M. C. Loiselle, and P. Lacasse; unpublished data). These data indicate that the immunosuppression relates to the metabolic milieu of early lactation rather than autonomous immune cell defects.
The production of IFN- by activated lymphocytes was greater in the lymphocytes of cows milked 1x after calving. Interferon- induces an antiviral state that promotes the expression of major histocompatibility complex antigens and enhances the activation of macrophages. In addition, it is important for the development of an effective cell-mediated immune response. Some studies show a decrease in IFN- production during the postpartum period (Ametaj et al., 2000). In the study by Nonnecke et al. (2003), the IFN- response was maintained in cows where the mammary gland was removed. This supports our finding that a reduction in milk production attenuates the decrease of IFN- around calving. In vitro, the presence of NEFA was shown to decrease the production of IFN- (Lacetera et al., 2003). In our study conducted ex vivo, the blood concentration of NEFA was lower in cows milked 1x, which suggests a persisting effect of NEFA on the ex vivo ability of lymphocytes to produce IFN-.
The transition from pregnancy to lactation is marked by metabolic changes that have an effect on animal health. These metabolic changes relate to the mobilization of reserves to enable milk production. In nature, the quantity of milk needed for offspring increases over time but is relatively low during the newborn’s first days of life. This explains the gradual transition from the dry period to a situation of high milk demand, which helps to reduce the magnitude of the NEB. In this experiment we tested an approach that limits milk production by reducing milking frequency. The results indicate that this approach was successful in limiting metabolite imbalances in early lactation. Larger studies are needed to determine whether these positive effects lead to better animal health.
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ACKNOWLEDGMENTS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Received for publication May 27, 2008. Accepted for publication December 23, 2008.
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= cows milked once daily during the first week of lactation and then twice daily for the rest of lactation; 
= cows milked twice daily throughout lactation. Milk production was lower (P < 0.002) during wk 1 and tended to be lower (P = 0.06) during wk 2 to 14 in cows milked once daily. Data are presented as least squares means ± SEM.
