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1 Danish Institute of Agricultural Sciences, Research Centre Foulum, PO Box 50, DK-8830 Tjele, Denmark
2 Hannah Research Institute, Ayr KA6 5HL, United Kingdom
Corresponding author: K. Sejrsen; e-mail: kr.sejrsen{at}agrsci.dk.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Key Words: mammary cell turnover • milking frequency • diet energy • dairy cow
Abbreviation key: ACC = acetyl-CoA carboxylase, FAS = fatty acid synthetase, GT = galactosyl transferase, Nx = N times daily, PCNA = proliferating cell nuclear antigen.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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A restriction in nutrient availability results in decreased milk yield in dairy cows (Friggens et al., 1995; Lacy-Hulbert et al., 1999; Andersen et al., 2003). However, little is known about the cellular events leading to a decrease in milk yield although in rats it has been shown that a decline in the activity of the key enzyme acetyl-CoA carboxylase (ACC), which is involved in milk synthesis, may be affected (Flint and Vernon, 1998).
Increased milking frequency increases milk production (e.g., Erdman and Varner, 1995), and in some cases (Bar-Peled et al., 1995; Hale et al., 2003) but not all (Pearson et al., 1979), milk production remained elevated for a period after the treatment was terminated. On the cellular level, changes in milking frequency can lead to changes in mammary cell turnover as shown for cattle (Hillerton et al., 1990; Hale et al., 2003) and goats (Wilde et al., 1987; Li et al., 1999). The activity of key enzymes involved in milk synthesis has also been shown to change because of changes in milking frequency in goats (Wilde et al., 1986, 1987; Travers and Barber, 1993).
The timing of the onset of the cellular events after stimulation by increased milking frequency is not clear. Recent work of Hale et al. (2003) suggests that mammary cell proliferation may be stimulated after 6 but not 3 d of increased milking frequency whereas apoptosis may be transiently stimulated after 3 d of increased milking frequency.
The main objectives of the present study were to examine the effects of diet energy density and milking frequency on mammary cell turnover and enzyme activity in dairy cows during early lactation. In addition, we examined whether 3 times daily (3x) milking during the first 8 wk postpartum causes a carryover effect on cell turnover and enzyme activity when milking frequency is returned to 2x milking. Finally, it was examined whether the effects of increased milking frequency are established after 1 wk of treatment.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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To examine possible carryover effects of milking frequency on milk yield and mammary cell turnover, milking frequency was reduced from 3x to 2x milking after 8 wk, and the nutritional treatment continued as before. The experiment was terminated after wk 16 postpartum.
Milk yield was measured 3 times per week and milk samples were collected for analysis of milk composition on a weekly basis.
A more thorough description of the experiment design, animals, and diet composition is published in Andersen et al. (2003).
Experiment 2.
Eighteen Holstein-Friesian cows previously used in experiment 1 were included in this experiment. At wk 17 postpartum, milking frequency was increased from 2x to 4x in 2 of the udder quarters (right-front/left-hind). The other udder quarters continued on 2x milking. Treatment continued for 7 d. The nutritional regimen (high- or low-energy density diet) continued as in previous weeks. Milk yield was determined 2 and 1 d before the start of treatment and again on d 6 and 7 of treatment.
All procedures were conducted under the protocols approved by the Danish Animal Experiments Inspectorate and complied with the Danish Ministry of Justice Law no. 382 and Acts 739 concerning animal experimentation and care of experimental animals.
Mammary Biopsies
In experiment 1, mammary biopsies were obtained from the front quarters after approximately 8 wk of either 2x or 3x milking and again after a further approximately 8 wk of 2x milking for all cows, i.e., at approximately wk 16 postpartum. In experiment 2, biopsies were obtained from front quarters milked 2x and 4x for 7 d, within individual cows. All mammary biopsies were preceded by a subcutaneous injection of a local anesthetic (2 mL of xylocaine). Biopsies were obtained through the skin using a biopsy gun (ProMag 2.2, MDTECH, Gainesville, FL) loaded with a 14-g needle (Ultra-Core II, MDTECH). In one sample, up to 20 mg of tissue was collected. A maximum of 3 samples were taken per udder quarter. A portion of the tissue was frozen immediately in liquid nitrogen for enzyme assays and a second portion was placed in 4% neutral buffered formalin for immunohistochemical detection of apoptosis and proliferating cell nuclear antigen (PCNA).
Udder Volumes
In experiment 1, udder volumes were measured 13.9 ± 1.6 d postpartum, approximately 8 wk postpartum, and again approximately 16 wk postpartum by polyurethane casting as performed by Dewhurst and Knight (1993). To get the best estimate, cows were injected with oxytocin and milked immediately before the volume measurement.
Enzyme and DNA Assays
The activities of ACC, fatty acid synthetase (FAS), and galactosyl transferase (GT) were measured as described by Wilde et al. (1986) under conditions in which activity was related linearly to amount of sample and incubation time. Results were expressed per milligram of DNA. The DNA concentration was measured in tissue homogenates by a fluorometric method using Picogreen dsDNA Quantification Kit (Molecular Probes, Eugene, OR) according to manufacturer’s instructions.
Immunohistology
Mammary tissue was fixed in 4% neutral buffered formalin, processed in paraffin, and sectioned at 4 µm. Mammary apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling of fragmented DNA using the commercial ApopTag Peroxidase in situ Apoptosis Detection Kit (Intergen Company, Purchase, NY). The principle of this assay is an enzymatic addition of digoxigenin-labeled nucleotide triphosphate to the 3'-OH ends of double and single stranded DNA. Labeled DNA fragments then bind to an antidigoxigenin antibody that is conjugated to a peroxidase reporter molecule. The percentage of cells staining positive for PCNA was used as a measure for cell proliferation because PCNA acts as a sliding clamp during DNA replication. For this purpose, a commercial kit was used (DAKO Corporation, Carpinteria, CA). Proliferative cells are labeled by using an antibody against PCNA, which is conjugated to a reporter molecule. Alveolar cells staining positive for apoptosis or PCNA were detected by microscopic examination. The examination was terminated when the fraction of positive cells was stable, although at least 500 alveolar cells were always counted.
Statistical Analyses
To allow for an adaptation period in experiment 1, wk 1 and 2 were not included in the statistical analysis for milk yield and feed intake during the first 8 wk of the experiment. Similarly, wk 9 and 10 were not included in the statistical analysis concerning milk yield and feed intake in wk 9 to 16 postpartum. The experimental design was a 2 x 2 factorial arrangement of treatments within 5 randomized complete blocks. Correlations were calculated as Pearson correlation coefficients (SAS Institute, 1999).
Effects of milking frequency and energy density in the diet within each of the 2 periods (wk 3 to 8 and wk 11 to 16) were analyzed by the MIXED procedure of SAS (SAS Institute, 1999) using the restricted maximum likelihood method and the following statistical model:
 | ([1]) |
where Yijkl is the dependent variable (milk yield, feed intake), µ is the least squares mean, 
i is the fixed effect of block i {i = 1, 2, ... 5}, ßj is the fixed effect of week j {j = 3, ... 8 and 11, ... 16}, 
k is the fixed effect of milking frequency k {k = 2 times daily, 3 times daily}, 
l is the fixed effect of energy density l {l = low, high}, ( delta;)kl, (ß delta;)jkl, (ß)jk and (ßdelta;)jl are the interactions between the 2 experimental factors, Aikl is the random effect of cow i within group kl, Aikl 
N(0, 
A2), Bijkl is the random effect of repeated measurements and Bijkl N(0, B2), 
ijkl is the random residual variation, and {ijkl} N(0, 2). No covariates were included in the statistical model because potential candidates (pretreatment yield and live weight) were included in the blocks by means of previous yield and parity.
Variables such as enzyme activities analyzed within the same week were analyzed using the GLM procedure of SAS (SAS Institute, 1999), where repeated measurements, the variable week, and interaction with week from model [1] were omitted.
In experiment 2, which had a split-plot arrangement, differences in milk yield, DNA concentration, and enzyme activities were evaluated by the MIXED procedure of SAS according to model [1] with the modification that the previous treatment (2x or 3x milking) was included as a class variable. In addition, milk yield measured immediately before start of treatment was used as a covariate to estimate milk yield.
Apoptosis and cell proliferation was analyzed within week with the GENMOD procedure of SAS (SAS Institute, 1999) based on binomial distribution and logit transformation of data and with Pearson as the SCALE option according to the following model:
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where logit(Pijk) is log(Pijk/(1–Pijk)), µ is the least squares mean, i is the fixed effect of block i {i = 1, 2, ... 5}, ßj is the fixed effect of milking frequency j {j = 2 times daily, 3 times daily}, k is the fixed effect of diet energy density k {k = low, high}, (ß)jk is the interaction between milking frequency and energy density and b(500, Pijk). Model [2] used in experiment 2 did not include the variable block.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Feed intake during wk 3 to 8 was 14.9 and 19.4 (P < 0.001) and during wk 11 to 16, it was 16.3 and 21.2 (P <0.001) Scandinavian feed units per day (1 unit = 7.89 MJ of NEL) for the groups fed the low- and high- energy density diets, respectively.
Milk yield followed the normal lactation curve with yield reaching a peak between wk 4 and 6 postpartum (Figure 1
). During wk 3 to 8 postpartum, cows fed the low-energy density diet had 17% lower milk yield (P = 0.003) than cows fed the high-energy density diet. The milk yield of cows fed the low- energy density diet during wk 11 to 16 postpartum was 24% lower (P < 0.001) than that of cows fed the high-energy density diet (Table 1).
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. For a thorough description of performance, refer to a companion paper by Andersen et al. (2003).
The first measurement of udder volume was not performed until d 14 postpartum due to udder edema. The udder volumes are given in Table 2. Feeding a low-energy density diet did not result in smaller udders in wk 2, 8, or 16. In wk 2 and 8 postpartum, udders were larger (P < 0.05) for cows milked 3x compared with those milked 2x. This difference also tended (P = 0.06) to exist at wk 16. In spite of the larger udder volumes of the cows milked 3x, there were no significant correlations between udder volume and milk yield (r = 0.16 and 0.26 for wk 8 and 16, respectively). Table 2 shows the concentration of DNA in mammary tissue. Neither energy density in the diet nor milking frequency had any significant effect on mammary DNA concentration. The DNA concentrations determined in this study were lower than previously reported for lactating cows (Capuco et al., 2001) and goats (Knight et al., 1990). The reason for this discrepancy is that DNA was measured in tissue homogenates suspended in a buffer with high salt (NaCl) concentration, because subsequent measurements in some of the samples suspended in a buffer with one-hundredth the concentration of salt yielded twice as much DNA. There was not sufficient material in all samples for DNA measurement in a low- salt concentration buffer, and therefore the DNA measures from the high salt concentration buffer are used. However, because there was a significant (P < 0.05) correlation between the DNA measures obtained in the high-and low-salt buffers, the DNA and the enzyme activity data (which are expressed per unit of DNA) are valid for comparisons between treatment groups.
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), resulting in only 8.6% of the amount of cell proliferation (P = 0.001) in cows on the low-energy density diet compared with cows on the high-energy density diet. Diet energy density had no significant effects on mammary cell apoptosis at this time point. At wk 16 postpartum, neither cells staining positive for PCNA nor apoptosis were significantly different between cows fed the low or the high-energy density diet.
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There were no interactions between the 2 experimental factors except for cell proliferation at wk 16 postpartum.
In general, there were no significant differences between treatment groups for enzyme activities per milligram of DNA measured in mammary biopsies taken at wk 8 and 16 postpartum (Table 4). However, at wk 8 there was a tendency (P = 0.14 and 0.08, respectively) toward greater GT activity in cows milked 3x and in cows fed the high-energy density diet than in cows from the 2 opposing treatments. Acetyl- CoA carboxylase and FAS showed increased activity in biopsies obtained in wk 8 compared with wk 16 (Pwk = 0.14 and 0.06 for ACC and FAS, respectively). No interaction was observed between the 2 experimental factors. Correlations between lactose yield and GT activity were significant in wk 8 (0.51, P = 0.03), and correlations between fat yield and ACC and FAS were strongest in wk 16 (0.37, P = 0.12 and 0.41, P = 0.08, respectively).
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There was no effect of diet energy density and no interaction between milking frequency and diet energy density for any of the measured variables.
Increasing milking frequency to 4x increased milk yield by 18% (P = 0.005) after the 7 d of treatment. However, 7 d after initiation of this treatment neither enzyme activities nor cell turnover were affected (Table 5 and 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Previously, a significant relationship between milk yield and udder volume has been reported (Knight, 2000). This relationship could not be confirmed in the present experiment. However, the majority of animals had udder edema at the time of the first measurement of udder volume. At wk 8, when the second measurement was taken, udder edema was still present in some of the cows. The measures of udder volume, therefore, may be unreliable due to edema.
Effects of Diet Energy Density on Mammary Cells
To our knowledge, there is no previous research into the way a constraint on nutrient availability will affect mammary epithelial cells in ruminants. With regard to cell turnover, our results on PCNA at wk 8 postpartum indicate that epithelial cell proliferation was considerably lower in cows fed the low-energy density diet compared with cows fed the high-energy density diet, whereas epithelial cell apoptosis did not differ. Thus, our data indicate that the cell number of the mammary gland accommodates to nutrient availability, i.e., a decrease in nutrient availability will lead to a decrease in the number of mammary cells. Insulin-like growth factor-I, which is known to stimulate cell proliferation (Shamay et al., 1988), may at least partly be mediating this effect because IGF-I plasma concentration was 38% lower in cows fed the low-energy density diet compared with those fed the high-energy density diet (Andersen et al., 2004). However, it should be noted that IGF-I interacts with IGF binding proteins and that there is a local mammary IGF-I synthesis (Flint et al., 2000).
Enzyme activities were numerically lower in mammary tissue from cows fed the low-energy density diet compared with the cows fed the high-energy density diet in experiment 1, but only the activity for GT measured 8 wk postpartum approached statistical significance. Taken together with the work of Flint and Vernon (1998), who observed a lower mammary ACC activity in rodents with feed intake restricted to 50 or 25% of ad libitum intake, the results suggest that the activity of some mammary enzymes may accommodate to the nutrient availability. However, because enzyme activity is expressed per unit of DNA originating from all cell types, it cannot be determined whether the change in activity originates from differentiated epithelial cells or is due to a change in the ratio between differentiated and undifferentiated cells or in the ratio between epithelial and stromal cells. The correlations observed between the yield of milk fat and the ACC and FAS enzymes and between the yield of lactose and the GT enzyme suggested that the enzyme activity of differentiated epithelial cells is accommodated to the nutrient availability.
The significantly higher enzyme activities observed for ACC and FAS in wk 16 compared with wk 8 postpartum are indicators of a change in ratio between differentiated epithelial cells and the other mammary cells or it may be an indicator of decreased epithelial cell efficiency with advancing lactation. Comparable results on enzyme activity with advancing lactation have been reported for well-fed goats (Wilde et al., 1986).
Effects of Milking Frequency on Mammary Cells
Udder volumes for cows milked 3x were significantly bigger than for cows milked only 2x. This is in agreement with the work of Wilde et al. (1987) and Knight et al. (1990) in goats, whereas it is not consistent with the work on cows by Stelwagen and Knight (1997). The inconsistency among the results on udder volume may be due to inaccuracies related to udder edema in our data.
The number of cells undergoing apoptosis or staining positive for PCNA in wk 8 did not differ significantly between milking frequencies although apoptosis was numerically lowest and PCNA staining was numerically highest when cows were milked 3x. In agreement (although not significant), Hillerton et al. (1990) found a higher number of cells per alveolus after 4 wk of 4x milking starting at wk 11 postpartum. Similarly, Hale et al. (2003) observed a tendency of numerically higher percentage of epithelial cells undergoing proliferation at d 7 and 14 postpartum when cows were milked 4x compared with 2x. Thus, although the experimental foundation is weak, the present information indicates that increased milking frequency may increase cell proliferation and decrease apoptosis thus giving rise to relatively more mammary epithelial cells. In agreement with the work of Hillerton et al. (1990), there are indications that the activity of mammary cell enzymes may increase with the increase in milking frequency.
In experiment 2, there were no clear short-term effects of milking 4x for 7 d during wk 17 postpartum on mammary cell turnover or enzyme activity, even though milk yield increased in this period. However, in the work of Hale et al. (2003) there were indications that apoptosis may be altered within 3 d and cell proliferation within 6 d after initiation of increased milking frequency.
The absence of clear effects on cell turnover and enzyme activity after a few days as well as after several weeks of altered milking frequency indicate that changes in milking frequency may affect other mechanisms. Thus, we suggest that negative feedback loops in response to filling of the gland are less activated because mammary glands are emptied more often with increased milking frequency. Candidates for having roles in these mechanisms are "feedback inhibitor of lactation," (Peaker et al., 1998) and serotonin (Matsuda et al., 2004).
In accordance with the lack of a long-term carryover effect of milking frequency on milk yield, there was also no significant long-term carryover effect on cell turnover or enzyme activity.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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The increase in milk yield that was the consequence of the increase in milking frequency for 1 or 8 wk was not accompanied by significant changes in mammary cell turnover or enzyme activity. We therefore suggest that tuning of negative feedback loops in response to filling of the gland may be the dominating effects of changes in milking frequency. In accordance with the lack of a long-term carryover effect of milking frequency on milk yield, mammary cell turnover and enzyme activity were not affected in the long term.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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FOOTNOTES |
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JN and AS contributed equally to this work.
Received for publication July 1, 2004. Accepted for publication November 10, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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