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* Department of Animal Science, University of Maryland, College Park 20742
Gene Evaluation and Mapping Laboratory, ANRI, USDA-ARS, Beltsville, MD 20705
Corresponding author:
R. A. Erdman; e-mail:
re13{at}umail.umd.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Key Words: milking frequency • milk yield • mammary growth
Abbreviation key: FIL = feedback inhibitor of lactation, IMF = increased milking frequency, MUN = milk urea nitrogen, MF = milking frequency, MY = milk yield, PP = postpartum, PRL = prolactin, TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labeling, X = times daily
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Experiments using both goats and cattle show that maintenance of peak milk yield and increases in persistency are heavily reliant upon the cellular dynamics of the mammary gland; specifically, the rates of cell proliferation, cell differentiation, apoptosis and the secretory capacity per cell (Tucker, 1981; Wilde et al., 1987; Hillerton et al., 1990). A strong relationship exists between numbers of mammary cells and milk yield (Tucker, 1966; Capuco et al., 2001). During lactation, maintenance of cell number by increasing cell proliferation or decreasing the rate of apoptosis should help maintain milk production. Increasing milking frequency at the beginning of lactation may provide a mechanism for increasing mammary cell numbers therefore increasing lactational MY and providing an explanation for the carryover effect of early lactation IMF on subsequent MY. In goats, IMF treatment corresponds with an increase in mammary cell numbers (Wilde et al., 1987). On the contrary, in cattle, administration of bST during mid or late lactation does not significantly affect mammary cell numbers (Capuco et al., 2001). However, the relationship between IMF during early lactation and mammary cells has not been evaluated in cows.
Finally, in previous studies by Bar Peled et al. (1995) and Sanders et al. (2000) implemented early lactation IMF was initiated on d 1 PP and continued for up to 6 wk. However, since the first few milkings after parturition (up to 72 h) are usually discarded because of antibiotic residues from dry cow therapy, it is of practical importance to determine if the timing of IMF treatment is critical. Specifically, could early lactation IMF be delayed until d 4 PP and still result in the same carryover effects on MY as compared to initiation on d 1 PP?
The objectives of the present investigation were: 1) to determine the effects of early lactation IMF on MY during the early lactation IMF period; 2) to determine the timing at which when IMF treatment must begin in order to stimulate carryover effects on lactation MY; and 3) to investigate potential cellular and hormonal mechanisms responsible for carry-over effects of IMF on lactation MY.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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). Use of animals for these investigations was approved by the Beltsville Agricultural Research Center’s Animal Care and Use Committee.
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Mammary Biopsies
Mammary biopsies were obtained approximately (± 2 d) on d 7 and d 14 of treatment from four cows randomly assigned per treatment group to be biopsied. Biopsies (approximately 1-g wet tissue weight) were obtained on d 7 from the left rear quarter and on d 14 from the right rear quarter from the same 4 cows in each treatment group by the method of Farr et al. (1996). Mammary tissue was used to measure rates of cell proliferation as measured using 3H-thymidine incorporation and Ki-67 nuclear proliferation antigen localization and rates of apoptosis using terminal deoxynucleotidyl transferase nick end labeling (TUNEL). Tissues were placed in Medium 199 (Gibco BRL, Grand Island, NY) and transported to the laboratory for incubation with 3H-thymidine. Mammary tissues for immunohistochemistry were fixed in 10% neutral buffered formalin overnight at 4°C and transferred to 70% ethanol until further processing. Tissues were then dehydrated and embedded in paraffin according to standard techniques and sectioned at 6-µm onto salinated slides.
Immunohistochemistry
Ki-67 localization.
Immunohistochemical localization of Ki-67 cell proliferation antigen was performed as described by Capuco et al. (2001). Briefly, slides were deparaffinized in xylene and hydrated in a graded series of ethanol. Tissue sections were quenched with 3% H2O2 in PBS for 10 min and then washed in PBS (3 x 2 min). Slides were microwaved at high power (650 W) in 400 ml of 10 mM citrate buffer (pH 6.0) in a covered glass staining dish for 5 min, remained undisturbed for 5 min, and then were microwaved for an additional 5 min. Slides remained in the buffer for a 30-min cooling period. They were then washed in PBS (3 x 2 min) and blocked with 5% non-immune goat serum in PBS (30 min) prior to histochemical localization of Ki-67 antigen. Staining Ki-67 utilized the MIB-1 monoclonal antibody (prediluted MIB-1, Zymed Laboratories, San Francisco, CA) and Histostain SP kit (Zymed). The slides were incubated with primary antibody for 60 min at room temperature and then washed with PBS (3 x 5 min). Slides were then incubated for 10 min at room temperature with biotinylated secondary antibody and washed in PBS (3 x 2 min). Slides were then incubated with streptavidin-peroxidase-conjugate for 10 min at room temperature. After washing in PBS (3 x 5 min), sections were incubated with diaminobenzidine. Slides were washed with distilled H2O, counterstained with hematoxylin, dehydrated, and then mounted with Permaslip.
Terminal deoxynucleotidyl transferase dUTP nick end labeling.
As described by Capuco et al. (2001), in situ detection of apoptotic cells utilized TUNEL to visualize cells that exhibited endonucleotidyl degradation of DNA, a key feature of apoptotic cells. A commercial kit (Apoptag kit, Oncor, Gaithersburg, MD) was used. After deparaffinization and hydration, slides were incubated with proteinase K (20 µg/ml of PBS, Oncor). Tissue sections were quenched with 2% H2O2 in PBS for 10 min and then washed in PBS (2 x 5 min). Slides were incubated in equilibration buffer for 10 min and then incubated with terminal deoxynucleotidyl transferase for 60 min in a humidified chamber at room temperature. Tissue sections were washed with stop/wash buffer for 10 min at room temperature and then incubated with anti-digoxigenin-peroxidase for 30 min at room temperature in a humidified chamber. Tissue sections were washed in PBS (4 x 2 min) and then incubated with diaminobenzidine until development of color, approximately 4 min. Sections were washed with distilled H2O and then counterstained with 0.1% methyl green in acetate buffer, pH 4, washed in three changes of distilled H2O, dehydrated in butanol and xylene and then mounted with Permaslip.
Quantitation of immunohistochemistry.
Tissue sections were viewed by light microscopy to quantify Ki-67 antigen expressing cells, and apoptotic cells. For each tissue section, five microscopic fields were quantified. A field was selected under low power and slightly out of focus, then the objective was switched to higher power, a digital image of the microscope field was taken at 100x magnification and cells were counted on a computer monitor.
3H-thymidine Incorporation
Mammary tissue from cows was sliced with a Stadie-Riggs hand microtome (Arthur H. Thomas Co., Philadelphia, PA). Triplicate slices (approximately 60 mg) were each incubated in 3 ml of Medium 199 containing 1 µCi of 3H-thymidine/ml (Amersham Biosciences, Piscataway, NJ). Incubation was for 2 h at 37°C in a shaking water bath, under an atmosphere of 5% CO2 and 95% O2. After incubation, tissues were rinsed in 0.9% saline and frozen at -20°C until quantification of incorporation. Tissue slices were homogenized in saline, precipitated with trichloroacetic acid and radioactivity determined by liquid scintillation spectroscopy, as described previously (Capuco and Akers, 1990).
DNA Analyses
To quantify nucleic acids, mammary tissue was homogenized (1:15 wt/vol) in DNA assay buffer (50 mM Na2PO4, 2 M NaCl, 2 mM Na2EDTA) using a Tekmar Homogenizer (Tekmar, Cincinnati, OH). DNA was quantified using Hoechst 33258 dye binding (Labarca and Paigen, 1980). Five-micrometer aliquots of sample homogenates were transferred to a 96-well microplate. Then 195 µl of DNA assay buffer containing Hoechst dye [99.8 ml of DNA assay buffer + 200 µl dye solution (1 µg of Hoechst 33258 dye/µl distilled water)] was added to each sample. A standard curve containing 0 to 2 µg of calf thymus DNA per well was prepared. Fluorescence was read using a Bio-Tek FL600 plate reader with a 360/460 nm filter set (Bio-Tek Instruments, Inc., Winooski, VT).
Measurements
Milk production was electronically recorded at each milking for the 305-d lactation period. Milk samples were collected weekly from two consecutive milkings and analyzed for fat, protein, solids, lactose, and SCC using an infrared analyzer (Bentley Instruments, St. Paul, MN) and milk urea nitrogen (MUN; Bentley Chemspec, Chaska, MN) by Lancaster DHIA (Manheim, PA) for the first 10 wk PP.
Cow body weight and body condition scores were recorded on the same day weekly for the first 10 wk PP. Body condition scores were based on a 5-point scale (Edmonson et al., 1989) and were assessed by a single individual (S. A. Hale).
Blood Sampling and Analysis
Blood was sampled at 1300 h three times per week during the first 2 wk and then once per week during wk 3, 4, 5, 6, 8, and 10. Blood was collected by tail venipuncture with 9 ml K3 EDTA Vacutainer tubes and 20-gauge needles (Becton Dickinson Vacutainer Systems, Rutherford, NJ). Samples were centrifuged at 2000 x g for 30 min, and plasma was decanted and stored at -20°C until concentrations of prolactin (PRL), bST and IGF-1 were determined using previously validated radioimmunoassays (Elsasser et al., 1989; Miller et al., 1999). Bovine PRL AFP-4835B; anti-bPRL AFP-753180Rb; anti-hIGF-1 AFP-4892898; were provided by Dr. Parlow, NHPP, and NIDDK, Torrance, CA. Bovine somatotropin (USDA-bST-b1) was provided by the USDA Animal Hormone Program (Dr. Proudman, Building 200, BARC-East, Beltsville, MD). Rabbit-anti-bovine somatotropin (R1-1-3) was kindly donated by Dr. Elsasser (USDA-ARS Bldg. 200, BARC-East, Beltsville, MD). Intraassay and interassay coefficients of variation averaged, respectively, 12 and 9% for IGF-1 (two assays). Intraassay coefficients of variation were 5% for PRL (one assay) and 9% for bST (one assay).
Statistical Analyses
Milk production data and milk component data was analyzed using ANOVA with repeated measures using the Mixed Procedure in SAS (version 8; SAS Institute, Inc., Cary, NC). The statistical model included: Covariant, Block, Treatment, Cow (treatment), day PP, and day PP by treatment interaction. Previous 305-d adjusted milk production was used as a covariate in the ANOVA and milk production data is reported as covariance adjusted least squares means. This model was tested separately between treatment groups for the wk 1 to 3, wk 4 to 10, wk 4 until 305-d and the complete lactation. Mammary tissue parameters and blood hormonal data were analyzed by ANOVA with repeated measures using the Mixed Procedure in SAS. The statistical model included: Block, Treatment, Cow (treatment), day postpartum, and day postpartum x treatment interaction.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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and Table 2. During wk 1 to 3 daily, milk yield in the IMF1 and IMF4 groups were 8.8 and 4.8 kg higher than the 2X cows. Although statistically not different, milk production in the IMF1 group was consistently greater than that in the IMF4 group during the treatment period (Figure 1). In proportion to the short milking interval, milk production for the second 4X morning and evening milkings averaged 6.0 kg (SE ± 0.28) and 4.9 kg (SE ± 0.24), respectively across the IMF groups. Peak milk production occurred during wk 7 for the 2X cows, and during wk 8 for IMF1 and IMF4 cows (Figure 1). Milk production was greater in the 4X groups than the 2X group during the majority of lactation until all groups converged during late lactation (P < 0.05) demonstrating a carryover effect of IMF treatment.
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) and were similar for the remainder of lactation. For the complete lactation, milk production for the IMF1 and IMF4 groups were similar and 3.0 kg/d greater than for 2X group (P < 0.05).
Concentrations of milk components are shown in Table 2. During the 3 wk treatment period, there was decreased fat percentage between the 4X and 2X groups (P < 0.05). After treatment, the 2X group maintained the higher fat percentage (P < 0.05). The IMF1 group had higher fat percentage from wk 4 to 44 as compared to IMF4 (P < 0.05) (Table 2). The 4X groups had tended to have lower milk protein percentage during wk 4 to 44 (P < 0.10). Increased milking frequency also tended to decrease the lactose percentage during the treatment period and the posttreatment period, wk 4 to 10 (P < 0.10). There were no treatment effects on milk urea nitrogen. Milk SCC was low for all the treatments and did not differ between the 4X and 2X groups (P < 0.05). During wk 4 to 10, milk SCC was significantly lower in the IMF4 group as compared to the IMF1 group (P < 0.05).
Fat corrected milk (3.5%) and milk component yields during the first 10 wk PP and monthly thereafter are shown in Table 3. There were no differences due to IMF treatment in FCM, or protein yield. After treatment, fat yield tended to be lower in the IMF4 group as compared to the IMF1 group (P < 0.10). Fat yield in the 2X group compared to the IMF groups did not differ (P > 0.10). Lactose yield tended to be higher in the 4X groups as compared to the 2X groups during the treatment period (P < 0.01). Lactose yield did not differ between groups during wk 4 to 10.
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. Mean initial body weight (Figure 2; left panel) was greater in the IMF1 and IMF4 cows than the 2X cows (P > 0.05). Body weights of the IMF1 and IMF4 groups declined until wk 5. Body condition score declined by 0.1 to 0.2 units from wk 1 to 5 PP. There were no significant effects of IMF treatment on body condition (Figure 2; right panel; P > 0.05).
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), were greater in the IMF1 group (P < 0.05) than in the 2X and IMF4 groups when measured on d 7 PP. On d 14, cell proliferation in the IMF1 group tended to be higher than 2X and IMF4 groups (P < 0.16 and 0.06, respectively). There were no other differences between groups or biopsy days.
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). The percentage of mammary epithelial and stromal cells expressing Ki-67 in IMF and 2X cows did not differ due to treatment or biopsy day (P > 0.05). However, the epithelial labeling indices were numerically greatest for IMF1 on d 7 and IMF4 on d 14. Although not compared statistically, rates of stromal cell proliferation were approximately twice that of epithelial cell proliferation using Ki-67 (Figure 4).
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. There was a higher percentage apoptotic mammary epithelial cells on d 7 in the IMF4 group (Fig. 5; left panel). The IMF4 d-7 apoptotic percentage was significantly higher than the d 7 biopsy in the 2X group and the d-14 biopsy in the IMF1 group (P < 0.05). Percentages of apoptotic mammary epithelial cells tended to be higher for IMF4 tissue on d 7 than all other treatments and biopsy days (P < 0.10).
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; right panel) was numerically greater (P < 0.05) in the IMF1 treatment on d 7 as compared to the 2X d-7 biopsy. On d 14, IMF4 stromal apoptosis was greater than 2X (P < 0.05). Mammary stromal cells in IMF1 tissue tended to have a higher percentage of apoptotic cells on d 7 than 14 (P < 0.10).
Concentrations of serum bST, IGF-1 and PRL by treatment and days PP are shown in Figures 6 through 8. Serum bST declined with time PP but was not affected by treatment (P > 0.05). Serum IGF-1 increased as lactation progressed and tended to be lower in the IMF groups than the 2X group, especially during the post treatment period (Figure 7). During the wk 1 to 3, there were no effects of IMF treatment or day PP on serum PRL (Figure 8). During wk 4 to 10, serum PRL was not affected by treatment but was numerically higher in the IMF4 group as compared to the 2X and IMF1 group (P > 0.10).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Consistent with other early lactation IMF studies (Bar Peled et al., 1995; Sanders, 2000), MY remained higher in 4X cows than 2X cows after the treatment period. In previous studies (Bar Peled et al., 1995; Sanders, 2000), the treatment period was 6 wk and IMF cows were milked 6X beginning d 1 PP. The current study employed a 3-wk treatment period during which IMF cows were milked 4X beginning d 1 PP or d 4 PP and daily milking intervals were not evenly distributed. Even with shorter and uneven milking intervals of approximately 9, 3, 9, and 3h, MY responses were almost equal to IMF treatment where the milking interval was equally spaced throughout 24 h (Bar-Peled et al., 1995; Sanders, 2000).
Erdman and Varner (1995) reported a fixed yield increase of 3.5 kg/d in cows milked 3X for an entire lactation. Other studies reported a 3.0 kg/d (Pearson et al., 1979) and 4.0 kg/d (Poole, 1982) increase of 3X over 2X milking during the 3X treatment period that continued until mid lactation with small and nonsignificant carryover effects. In these experiments (Pearson et al., 1979; Poole 1982), carryover effects on MY were lower than in the current study where 4X cows continued to produce 2.8 kg/d more than 2X cows until wk 36 when MY of all cows converged for the remainder of lactation. In contrast, Bar Peled et al. (1995) saw posttreatment (wk 7 to 18) increases in MY of 5.1 kg/d. In the present study, MY of 4X cows was 4.0 kg/d higher than 2X cows for a 14 wk period posttreatment. These data suggest that, there is a window during early lactation when IMF treatment may positively affect the mammary gland, resulting in increased MY even after IMF treatment has ceased.
IMF treatment generally alters the milk composition, specifically, fat and protein. Usually, as milking frequency increases, fat and protein decrease (Erdman and Varner, 1995). In the present study, there was a significant decrease in protein percentage during wk 4 to 10, a decreased fat percentage (P < 0.05) during wk 1 to 3 and 4 to 44, and a tendency for decreased lactose percentage during wk 1 to 3 and 4 to 10. The majority of the reduction in the 4X treatments could be attributed to the IMF4 treatment where fat percentage was 0.4 to 0.5 percentage units lower than 2X throughout the experiment. Because milk samples were obtained only at the first AM and PM milkings rather than all four milkings for the 4X groups, this may account for the lack of differences in FCM between the 2X and the 4X cows during the treatment period since milk fat content generally increases as a milking continues. However, this would not explain differences in milk fat during the posttreatment period.
Previously work by Wilde et al. (1995) proposed the hypothesis that increased MY as a result of IMF was due to the removal of a whey protein referred to as the feedback inhibitor of lactation (FIL). High concentrations of FIL have been associated with decreased milk secretion (Wilde et al., 1995) and more frequent removal of milk may rid the mammary gland of FIL therefore maintaining higher rates of milk secretion (Henderson and Peaker, 1984). Although this concept would explain MY responses during a period of IMF, it cannot explain the carryover effect of increased early lactation IMF on MY seen after IMF treatment ceased that was observed in the present study and others (Bar Peled et al., 1995; Henshaw et al., 2000). This would seem to preclude the possibility of an operative FIL mechanism posttreatment since milk removal in 2X cows was as frequent as 4X cows during this time and according to the FIL hypothesis, milk production should be similar between treatment groups.
In addition, though FIL has been shown to inhibit differentiation in vitro (Wilde et al., 1990), this effect of FIL on mammary cells is not consistent with carryover effects on MY since other studies have suggested the increase in MY until peak may be due to increased differentiation since there is no appreciable increase in mammary cells during early lactation (Capuco et al., 2001). Though mammary cell differentiation was not evaluated in this experiment, it is possible that IMF treatment may stimulate mammary cell differentiation.
To our knowledge, this is the first early lactation IMF study that also evaluated mammary growth effects in dairy cattle. Capuco et al. (2001) showed no net growth of mammary cells during early lactation in cows milked twice daily. However, small increases at the beginning of lactation may have been missed since the earliest mammary tissue sampling was 14 d PP and it remained to be determined if IMF could induce mammary growth during early lactation in cows. Wilde et al. (1987) suggested that in goats, under a short, 2-wk IMF regimen, increased MY is due to an increase in mammary epithelial cell activity, whereas a longer IMF treatment period elicited an increase in mammary cell proliferation. In the current study, IMF1 treatment increased 3H-thymidine incorporation in the d 7 mammary tissue. Though the Ki-67 localization results were not statistically significant, there was a quantitatively higher percentage of epithelial cells expressing the Ki-67 antigen in the d 7 tissue of IMF1 cows and d 14 tissue of IMF4 cows. These results are consistent with our hypothesis that IMF treatment increases mammary cell proliferation, therefore providing a mechanism for increased MY and carryover effects.
Manipulating milking frequency not only alters the proliferative state of the mammary gland, but also might alter the apoptotic state. Studies by Li et al. (1999) showed differences in the apoptotic processes as evidenced by morphologic variation between 2X/unmilked glands and 3X/1X glands. This suggested a possible regulation of mammary apoptosis through local mechanisms sensitive to the frequency of milk removal (Li et al., 1999). In cattle, the proportion of apoptotic mammary cells during mid lactation has been estimated at 2.4% (Wilde et al., 1987) while other estimates of apoptotic rate are much lower, 0.07% during mid to late lactation and 0.27% during early lactation (Capuco et al., 2001). The apoptotic index estimated by Wilde et al. (1987) during mid lactation is similar to the apoptotic index seen in mammary epithelial cells of IMF4 cows at d 7 of lactation of 2%. The quantification of apoptotic mammary epithelial cells this early in lactation may be misleading since increased migration of leukocytes into the mammary gland and milk is characteristic of this lactation stage (Concha, 1986; Sordillo et al., 1997) and morphological changes that occur during the apoptotic process make it difficult to distinguish between an epithelial cell and an apoptotic leukocyte that has migrated into the epithelial compartment. Therefore, the incidence of apoptosis in mammary epithelial cells in early lactation may be lower than that estimated by TUNEL, with a higher rate of apoptosis may be due to migration of apoptotic leukocytes and increased tissue edema.
Implementing an IMF program might not only affect mammary cell turnover, but also endocrine profiles. Blood samples were collected approximately 1 to 2 h after milking to obtain basal concentrations of prolactin rather than milking-induced concentrations in the circulation. Milking frequency did not alter basal concentrations of PRL during or after the treatment period. However, increased milking frequency should have increased exposure of mammary epithelial cells to circulating prolactin due to milking-induced secretion of PRL at four milkings rather than two. Furthermore, Bar-Peled et al. (1995) observed more PRL released per milking when milking frequency was increased (6X versus 3X). Although basal concentrations of PRL were not altered by milking frequency, we cannot rule out PRL as a stimulator of mammary growth or differentiation during the treatment period.
Studies investigating bST effects on mammary growth appear contradictory. Whereas some studies (Capuco et al., 1989; Binelli et al., 1995) show no mammogenic effect of bST, other studies (Knight et al., 1992; Capuco et al., 2001) show a possible mammogenic affect when cattle are administered bST. Moreover, in cows that are administered bST under an IMF routine, Knight et al. (1992) suggested that increased mammary cell proliferation may contribute to the increase in MY. Work by Bar-Peled et al. (1995) showed higher endogenous bST levels in cows milked 6X as compared to 3X, though these results were not significantly different, and may have reflected a change in energy status rather than an increase in MY (Bar Peled et al., 1995). Serum bST concentrations in the current study did not differ between IMF groups and 2X and decreased with time, suggesting that the possible proliferative effects on the mammary gland by IMF treatment were not mediated by systemic bST.
Insulin-like growth factor-1 has been shown to stimulate mammary DNA synthesis in vitro and is presumed to be the primary mediator of the galactopoietic action of bST in the ruminant (Prosser et al., 1990; Forsyth, 1996). Lower concentrations of IGF-1 in IMF cows argue against its involvement in eliciting a mammogenic response to IMF. Although we did not measure IGF-1 binding proteins, changes in IGF-1 binding proteins could have played an important role in regulating IGF-1 levels both in the mammary gland and systemically. Furthermore, IGF-1 and IGF-1 binding proteins are also synthesized locally in the mammary gland and may act in a paracrine manner to regulate mammary growth (Hauser et al., 1990; McGrath et al., 1991).
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Received for publication September 7, 2002. Accepted for publication December 6, 2002.
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G. E. Dahl, R. L. Wallace, R. D. Shanks, and D. Lueking Hot Topic: Effects of Frequent Milking in Early Lactation on Milk Yield and Udder Health J Dairy Sci, April 1, 2004; 87(4): 882 - 885. [Abstract] [Full Text] [PDF]
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; IMF1) and 4X starting at d 4 PP until d 21 PP (
; IMF4) for the complete 305-d lactation. SEM ± 1.91 kg/d.
