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* Grup de Recerca en Remugants, Departament de Ciència Animal i dels Aliments, Universitat Autónoma de Barcelona, 08193 Bellaterra, Spain
Sheep and Goat Research Department, Animal Production Research Institute, 4 Nadi El-Said St., 12311 Dokki, Giza, Egypt
1 Corresponding author: gerardo.caja{at}uab.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Key Words: continuous milking • apoptosis • lactogenesis • dairy goat
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Acquisition of passive immunity by the neonate depends on consumption of a sufficient amount of colostral IgG before cessation of macromolecular transport by the intestine (Stott and Fellah, 1983). Continuous milking of 1 half-udder throughout the dry period in dairy cows reduced the massive selective transfer of blood IgG into the colostrum of these glands (Brandon and Lascelles, 1975). In contrast, colostrum formation was normal in the contralateral, nonmilked glands. These results indicate that local regulation of colostrum formation exists and that local signals, such as continuous milking throughout the dry period, can impede colostrogenesis, even during late gestation when hormonal influences favor its establishment. If colostrum contains a small concentration of IgG, the neonate should be fed a large amount of colostrum to obtain a sufficient amount of IgG. However, Stott and Fellah (1983) suggested that large amounts of colostrum containing a small concentration of IgG would not be absorbed adequately; rather, limited amounts of colostrum having a large concentration of IgG may be more beneficial. The effect of omitting the dry period on colostrum quality has not been studied in dairy goats.
Milk yield depends on the activity and number of mammary epithelial cells (Knight, 2000). The number of mammary cells is determined by the rates of cell apoptosis and proliferation. Apoptosis and proliferation occur in the mammary gland throughout lactation, resulting in a considerable turnover of mammary cells (Capuco et al., 2001). Capuco et al. (2001) reported daily rates of proliferation and apoptosis in the bovine mammary gland of 0.3 and 0.56%, respectively. Assuming that these indices are constant throughout lactation and that the cells that die are not those that have proliferated, they calculated that 90% of mammary cells are renewed during lactation. Moreover, they estimated that if mammary cells die after proliferation, then more than half of the cells could renew themselves during a 240-d lactation. Besides stage of lactation, some management factors such as bST injection (Capuco et al., 2001), milking frequency (Hale et al., 2003), and diet energy density (Norgaard et al., 2005) affect the indices of mammary apoptosis and proliferation. It is not known whether omitting the dry period can affect mammary cell turnover in the subsequent lactation.
The objective of this study was to investigate the effects of omitting the dry period on colostrum quality, milk yield, and mammary cell turnover during the subsequent lactation in Murciano-Granadina goats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Animals and Management Conditions
Seventeen pregnant multiparous Murciano-Granadina dairy goats in the herd of the S1GCE (Servei de Granges i Camps Experimentals) of the Universitat Autonoma de Barcelona were used. Goats were mated naturally at wk 29 of lactation (April) after buck-induced estrus. Eight weeks before the expected kidding date (293 ± 0.6 DIM), goats were assigned to 2 experimental dry periods of either 56 d (D56; n = 9) or 0 d (D0; n = 8). Five of the 8 goats assigned to the D0 group reduced milk yield dramatically during pregnancy and dried off spontaneously at 27 ± 4 d before kidding, and were thereafter considered as a separate group (D27). Mean values for age (4.0 ± 0.5, 4.0 ± 0.6, and 3.7 ± 0.7 yr) and parity (2.8 ± 0.4, 3.0 ± 0.6, and 2.7 ± 0.7) were similar for D56, D27, and D0 goats, respectively. Goats grazed for 6 h daily and the diets were supplemented indoors with a commercial concentrate (6.4 MJ of NEL/kg and 160 g of CP/kg; as fed) at a flat rate of 0.5 to 1.0 kg/d according to the stage of lactation, plus 0.5 kg of dehydrated alfalfa hay and 0.5 kg of alfalfa pellets. During the dry period, goats were maintained in pens and fed a dehydrated mixture of 50% whole-plant maize, 50% alfalfa ad libitum, and 0.3 kg/d of concentrate. Moreover, lactating goats received 0.5 kg/d of concentrate in the milking parlor.
Goats were milked in a double-12 parallel milking parlor (Westfalia-Separator Ibérica, Granollers, Spain) equipped with milk-recording jars (2 L ± 5%), and a low milk pipeline. The milking machine was set at a vacuum pressure of 42 kPa, a pulsation rate of 90 pulses/min, and a pulsation ratio of 66%. The milking routine included machine milking, stripping before cluster removal, and teat dipping in an iodine solution (P3-cide plus; Henkel Hygiene, Barcelona, Spain).
Sample Collection, Analyses, and Measurements
Milk yield was recorded weekly by using the recording jars in the milking parlor until wk 30 of the subsequent lactation (wk 80 of the experiment). After parturition, kids were weighed and separated from their mothers before suckling. Goats were hand milked, and fresh colostrum samples were collected to determine specific gravity (SG) and colostral composition. For IgG concentration, colostral samples were frozen at –20°C until analysis. The SG of colostrum was measured using a density flask (Afora, Madrid, Spain) according to Lewis (1987). Colostrum total solids were calculated after drying in an oven at 103°C overnight. Fat was analyzed by the Gerber method. Noncasein protein was measured by Kjeldahl analysis of the filtrate after precipitation with 10% acetic acid and 1 M sodium acetate, and NPN was measured by Kjeldahl analysis of the filtrate after precipitation with 20% TCA (International Dairy Federation, 1993). Total protein was expressed as N x 6.38, and CN was calculated as the difference between total protein and non-CN protein.
The concentration of IgG in the colostrum was determined by the radial immunodiffusion technique using VET-RID kit plates specific for goat IgG (Bethyl Laboratories, Montgomery, TX). Colostrum samples were diluted (1:10) using PBS (pH = 7.4), and plate wells were then filled with 5 µL of diluted colostrum. Plates were incubated at room temperature for 24 h, after which diameters of the ring-shaped precipitates were measured to the nearest tenth of a millimeter using a scaled ocular of a microscope (Olympus SZH; Olympus Corporation, Tokyo, Japan). A reference curve was constructed by plotting the diameter of the precipitated rings of 3 standard IgG concentrations (250, 1,000, and 2,000 mg/dL) on 2-cycle semilogarithmic graph paper. The concentration of IgG in the samples was determined by interpolation from the reference curve.
Mammary Biopsies and Immunohistochemistry
Seven goats having similar BW (48.3 ± 1.3 kg) and milk yields (0.54 ± 0.19 L/d) at the end of the preceding lactation were chosen for udder biopsy. Mammary biopsies of milked udders were collected after s.c. local anesthesia (2 mL of lidocaine 2%; B. Braun Medical, Barcelona, Spain) and skin incision with a scalpel in a half udder by using a 14-gauge needle (Bard Max·Core, Covington, GA). The biopsy needle was introduced approximately 3 cm into the parenchyma 2 or 3 times to obtain sufficient tissue.
The first biopsy was obtained from 7 goats (3 D56 and 4 D0) at d 284 ± 1.6 of the lactation preceding the dry-period treatment. The second biopsy was programmed 7 d after initiating the dry period to compare lactation and involution in the same D56 goats. No biopsies were planned in the D0 goats because no significant udder changes were expected after 1 wk of lactation. Nevertheless, 3 of the 4 biopsied D0 goats did not maintain lactation and spontaneously dried off, becoming the D27 nonlactating group. The last biopsy was obtained at d 48 ± 1.1 of the lactation subsequent to the dry-period treatment from 9 goats (3 goats in D56 biopsied twice previously, 3 goats in D27 biopsied once previously, 1 goat in D0 biopsied once previously, and 2 goats in D0 biopsied for the first time).
Mammary samples were fixed in 10% neutral-buffered formalin overnight at 4°C before being transferred to 70% ethanol until further processing. Samples were embedded in paraffin, cut at 3 µm, and stained with hematoxylin-eosin, or were processed for detection of cell proliferation by localizing proliferating cell nuclear antigen (PCNA) and cell apoptosis using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL).
PCNA Localization.
Deparaffinized rehydrated tissue slides were incubated in 3% H2O2 for 30 min to inhibit endogenous peroxidase. To unmask the antigens, tissue slides were placed in a covered glass staining dish containing 10 mM citrate buffer (pH 6.0) and were microwave-heated for 10 min. The slides were then incubated with bovine albumin in PBS (60 min) to block nonspecific binding of antibodies. To stain PCNA, slides were then incubated with 100 µL of diluted mouse monoclonal antibody to PCNA (clone PC10, Bio-Genex, San Ramon, CA) overnight at 4°C. The next morning, slides were incubated for 60 min at room temperature with 1:200 goat antimouse Ig biotin (DakoCytomation, Glostrup, Denmark) as a secondary antibody. Slides were then incubated with 1:100 avidin–biotin peroxidase complex (Immuno Pure ABC Peroxidase Staining Kit Standard; Pierce Biotechnology, Rockford, IL) for another 60 min at room temperature. Slides were incubated for 5 to 10 min with the chromagen, 3,3'-diaminobenzidine tetrahydrochloride (Sigma, Madrid, Spain) in PBS containing 0.04% hydrogen peroxide. Finally, slides were counterstained with hematoxylin.
Detection of Cell Death by TUNEL.
A commercial kit (Apop Tag; Serologicals Corporation, Norcross, GA) was used to visualize apoptotic cells. After deparaffinization and rehydration, slides were incubated with proteinase K (20 µg/mL of PBS). Slides were quenched with 2% H2O2 in PBS for 5 min, incubated in equilibration buffer for 20 min, and then incubated with terminal deoxynucleotidyl transferase for 60 min in a humidified chamber at 37°C. Slides were washed with stop-wash buffer for 20 min at room temperature and then incubated with antidigoxigenin-peroxidase for 30 min at room temperature in a humidified chamber. Tissue sections were incubated for 5 to 10 min with 3,3'-diaminobenzidine tetrahydrochloride (Sigma) in PBS containing 0.04% hydrogen peroxide. Sections were washed with distilled water and then counterstained with hematoxylin.
Cell Counting.
Tissue sections were viewed by light microscopy (Olympus BH2; Olympus Corporation) to quantify PCNA antigen-expressing cells and apoptotic cells. For each tissue section, 5 microscopic fields were quantified. A field was selected under low power and slightly out of focus; the objective was then switched to greater power, a digital image of the microscope field was taken at 100x magnification, and cells were counted on a computer monitor. Photographs of mammary tissue were used to count the numbers of cells per alveolus.
Statistical Analyses
Data were analyzed by using a mixed-models procedure for repeated measurements (PROC MIXED; SAS 8.2; SAS Inst. Inc., Cary, NC). The mixed model contained the random effect of animal within the dry-period duration (treatment); fixed effects of treatment and week of lactation; the interaction between treatment and week of lactation; and residual error. Milk yield during 300 d of the previous lactation was used as a covariate. Milk persistency, colostral composition, and mammary tissue traits were analyzed by using one-way ANOVA (PROC GLM; SAS 8.2; SAS Inst. Inc.) with a model containing the effect of treatment and residual error. Data were tested for the normality of distribution, and a logarithmic transformation (log10) was applied to IgG concentrations. Pearson correlation coefficients between colostral components also were calculated.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Birth Weight of Kids and Colostral Quality
The D56 and D0 goats had similar kidding rates (2.40 kid/goat), but the D27 group had fewer (P < 0.05) kids than the D56 goats (Table 1
). Kids of the D0 goats had smaller (P < 0.05) birth weights than kids from the D56 and D27 goats (Table 1). Decreased birth weights of the D0 kids may be a consequence of nutritive stress resulting from the competition between lactation and pregnancy, especially during the last third of gestation when fetal growth is the greatest. Previously, prepartum energy or protein restriction was shown to reduce birth weight by 6 to 10% in goats (Sahlu et al., 1995). Nevertheless, reducing or eliminating the dry period in dairy cows did not affect calf birth weight (Grummer and Rastani, 2004). The SG of D0 colostrum was smaller (P < 0.01) than those from D56 and D27 colostrum, indicating a poorer quality of colostrum from goats milked throughout pregnancy (Table 1). No values for the SG of colostrum from goats are available to compare with our results. The SG of D0 colostrum (1.032) was slightly greater than values for normal goat milk, which ranged from 1.022 to 1.026 (Guo et al., 2001). Measurement of the colostral SG provides an inexpensive and practical method of estimating the colostral Ig concentration in cows (Fleenor and Stott, 1980). Because kids of D0 goats had smaller birth weights and their mothers produced colostrum having a reduced SG, colostrum from the D27 and D56 goats was used to feed the D0 kids.
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The IgG concentration in the colostrum of D0 goats (5.6 mg/mL) was greater than that reported by Levieux et al. (2002) in normal goat milk (0.20 to 2.21 mg/mL). Nevertheless, this concentration seems to be insufficient to achieve the recommended serum concentration of IgG in newborn kids (>12 mg/mL; O’Brien and Sherman, 1993). In addition, a positive correlation (r = 0.86) was detected between concentrations of IgG in the colostrum and in the serum of newborn kids (Argüello et al., 2004). When there is no dry period, a very large amount of colostrum must be fed to supply kids with a sufficient mass of IgG. However, large amounts of colostrum containing reduced concentrations of IgG would not be absorbed adequately (Stott and Fellah, 1983).
Concentrations of IgG did not vary between the D27 and D56 goats (Table 1
), and ranged from 23.5 to 64.0 mg/mL (overall mean = 39.6 mg/mL). In accordance with our results, the immunologic quality of colostrum did not differ between dairy cows dried off for 30 or 70 d (Gulay et al., 2005).
Casein constituted 28.2, 28.1, and 63.1% of total protein in the colostrum of D56, D27, and D0 goats, respectively. The colostrum of D0 goats was close to normal goat milk in the Murciano-Granadina breed, where CN represents 75% of total milk protein. Whey protein, most of which was Ig, constituted over two-thirds of total protein in the colostrum from D56 and D27 goats and was greater (P < 0.01) than in colostrum from D0 goats (Table 1).
To our knowledge, correlations among IgG concentrations, SG, and other components of colostrum have not been reported previously in dairy goats (Table 2). Excluding values for the D0 goats, the concentrations of IgG correlated positively (r = 0.88; P < 0.001) with SG, which were similar to the correlation of 0.84 reported by Fleenor and Stott (1980) in dairy cows but greater than the correlation of 0.53 reported by Morin et al. (2001) in cows. Colostral SG were correlated (P < 0.001) with concentrations of protein (r = 0.96), whey protein (r = 0.95), and IgG (r = 0.88).
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and Table 3.
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).
During the last 3 wk of gestation, milk yield increased for D0 goats (Figure 1). Circulating prolactin in dairy goats remained low up to d 120 of pregnancy and increased during the last part of pregnancy (Kornalijnslijper et al., 1997). Similarly, an increase in milk yield during the days preceding calving was observed in dairy cows (Wheelock et al., 1965), although Remond et al. (1992) did not detect such an increase.
Subsequent Lactation.
Without a dry period, D0 goats produced 16% less (P < 0.01) milk during the subsequent lactation than during the previous lactation. Moreover, the milk yield of D0 goats during the subsequent lactation was reduced (P < 0.05) by 21 and 29% compared with the yields of D56 and D27 goats, respectively (Figure 1 and Table 3). On the other hand, D27 and D56 goats in the subsequent lactation produced 15% (P < 0.01) and 4% (P = 0.122), respectively, more milk than during the equivalent period of the previous lactation. In dairy cows, omitting the dry period also reduced milk production during the subsequent lactation by 15 to 40% (Swanson, 1965; Remond et al., 1992).
Using the half-udder design, Fowler et al. (1991) reported that a dry period between lactations was not necessary in Saanen dairy goats. In their study, half-udders were dried off 2 wk before mating (dry period lasted 23 wk), which is unusual and is 3 times the duration of the dry period in our study. In addition, when one half-udder was dried off, the activity and number of mammary cells increased in the lactating half within the same udder (Capuco and Akers, 1990), and involution was partially inhibited in the nonlactating half (Akers and Keys, 1985). In our study, both udder halves were dried off for a shorter period when goats were in late lactation and advanced pregnancy, which might explain the discrepancy between our results and those reported by Fowler et al. (1991).
Milk persistency was numerically greater (P = 0.25) in D0 than in D56 and D27 goats (Table 3). When comparing D56 and D0 goats, milk yield was 24% less (P < 0.05) in D0 goats during the first 20 wk of lactation and 14% less (P = 0.097) in wk 21 to 30 of lactation (Figure 1 and Table 3). Similarly, milk yield from cows with no dry period (Remond et al., 1997) or with a 30-d dry period (Bachman, 2002) was more persistent than that in cows with a conventional 60-d dry period.
No differences in milk yield were detected between D56 and D27 goats, indicating that shorter dry periods (i.e., 27 d) are sufficient for mammary involution in goats. Consequently, no negative effects were expected when goats spontaneously dried off or were dried off intentionally 1 mo before kidding. Capuco et al. (1997) showed that mammary growth in cows was initiated within the first 25 d of a 60-d dry period. Moreover, cows with a 30-d dry period produced amounts of milk similar to cows with a 60-d dry period (Bachman, 2002).
Mammary Cell Turnover
Indices of apoptosis and proliferation in D56 goats at 284 DIM of the preceding lactation, 7 d after drying off, and 48 DIM of the subsequent lactation are shown in Figure 2. Cellular indices of apoptosis (0.61%) and proliferation (2.52%) did not vary between mammary biopsies on d 284 and 48 of the preceding and subsequent lactations, respectively. These apoptosis values are in the range (0.2 to 3.0%) reported in lactating cows (Capuco et al., 2001; Norgaard et al., 2005) and lactating goats (Li et al., 1999). Similar to our proliferation values, cells expressing PCNA accounted for 0.17 to 2.99% of mammary cells during lactation in dairy cows (Norgaard et al., 2005). In our results, cell proliferation exceeded cell death, which suggests that the mammary gland grows throughout lactation rather than regressing. The TUNEL test detects the proportion of cells dying at a given moment before being phagocytosed by macrophages and adjacent alveolar cells, whereas PCNA indicates proliferating cells for approximately 24 h (Knight, 2000). In addition, milk may be a vehicle for eliminating mammary apoptotic cells as well as for phagocytosis. We observed some apoptotic cells in the alveolar lumen, and most of these cells were localized in the alveolar epithelium (Figure 3).
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). Similarly, Li et al. (1999) observed that the apoptosis index was less than 1% of cells in lactating tissues and increased at wk 1 (2 to 3%) and 2 (5%) after drying off nonpregnant goats. The peak apoptosis probably occurred before d 7 of involution in our goats because they were in late pregnancy at drying off. Annen et al. (2003) detected the greatest incidence of apoptosis in pregnant cows at d 2 after drying off, and by d 8 the number of apoptotic cells did not differ from those in lactating cows. Elevated apoptosis and proliferation indices during dry-off in our study indicate significant mammary cell turnover, as previously observed by Capuco et al. (1997) in dairy cows, in which proliferation of mammary epithelial cells in dry cows was greater than in cows without dry periods to replace senescent and damaged cells.
In the subsequent lactation (48 DIM), the numbers of cells per alveolus in D56 (P = 0.14) and D27 (P = 0.19) goats were greater than those in D0 goats (Table 4), in accordance with milk yield values. In our results, milk yield correlated positively (r = 0.81; P < 0.05) with cell number per alveolus. No differences were detected between treatments in apoptosis or proliferation indices for D56, D27, and D0 goats, respectively (Table 4). Our results indicate that the length of the dry-off period did not affect mammary cell turnover in the following lactation.
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). Fitzgerald et al. (2004) observed that the proliferation of mammary epithelial cells in the glands of primiparous dairy cows dried off for 60 d was greater than in the glands without dry-off at 7 d before calving. These authors reported no differences after parturition in cell proliferation between glands with or without dry periods, suggesting that reduced cell proliferation before parturition caused milk yield losses in the subsequent lactation in glands without a dry period. |
CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Received for publication March 1, 2006. Accepted for publication June 22, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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-lactalbumin and serum albumin in colostrum and milk during early post partum period. J. Dairy Res. 69:391–399.[Medline]This article has been cited by other articles:
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  B. R. Andrade, A. A. K. Salama, G. Caja, V. Castillo, E. Albanell, and X. Such Response to Lactation Induction Differs by Season of Year and Breed of Dairy Ewes J Dairy Sci, June 1, 2008; 91(6): 2299 - 2306. [Abstract] [Full Text] [PDF]
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A. A. K. Salama, G. Caja, E. Albanell, S. Carne, R. Casals, and X. Such Mammogenesis and Induced Lactation With or Without Reserpine in Nulliparous Dairy Goats J Dairy Sci, August 1, 2007; 90(8): 3751 - 3757. [Abstract] [Full Text] [PDF]
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; n = 17). At wk 42 of lactation, goats were allocated into 2 treatments: a dry period of 56 d (
; n = 9) or no dry period (n = 8). At wk 47 of lactation, 5 of the 8 goats assigned to have no dry period dried off spontaneously at 27 d (
; n = 5) before parturition, and the remainder were milked continuously until parturition (
; n = 3).
) indices in dairy goats milked for 300 d and dried off for 56 d before kidding (n = 3). Biopsies were collected during late lactation (284 DIM), involution (7 d after drying off), and early lactation (48 DIM in the subsequent lactation).