Abundance and phosphorylation state of translation initiation factors in mammary glands of lactating and nonlactating dairy cows

To test if control of mRNA translation is involved in the increase in protein synthesis by mammary glands during lactation, cellular contents and phosphorylation states of translation factors and their upstream regulators were measured in mammary parenchyma from 12 nonpregnant dairy cows. For a 42-d period, 6 cows in late lactation continued to be milked (L) and 6 at the same stage of lactation were dried off (NL). All cows were then slaughtered and mammary glands and tissue samples obtained. Alveoli and lobules tended to be larger in L cows. Mammary parenchymal mass, cell number, cell size, and RNA, DNA, and protein contents were greater in L cows. Increases (3.1- and 1.8-fold) in the abundance of active, phosphorylated ribosomal protein S6 and its kinase, S6K1, respectively, in L vs. NL parenchyma indicated an ability to sustain greater rates of synthesis of translational machinery, which was also evident in the 102% increase in parenchymal RNA:DNA between the 2 groups. Cellular abundances of the main eukaryotic translation initiation factors (eIF), eIF2 and eIF4E, were 2.6- and 3-fold greater, respectively, in L cows. That these differences were greater than the 102% greater RNA:DNA in L mammary parenchyma suggests an elevated translational efficiency in L glands. Abundance of phosphorylated rpS6 was not different between mammary parenchyma and liver, whereas eIF2α was 50% greater in mammary tissue. In semimembranosus muscle, abundances of phosphorylated rpS6 and eIF2α were 3 to 4 times lower than in mammary parenchyma. In both L and NL mammary glands, 11% of eIF2α was in the inhibitory, phosphorylated form and 48 to 60% of eIF4E was complexed with its binding protein, 4EBP1. It is concluded that up-regulation of initiation of mRNA translation occurs in the fully differentiated milk secretory cell and that, where crucial initiation factors are not present in a maximally active form, the initiation rate might be flexible in response to external stimuli.     

Bovine somatotropin administration to dairy goats in late lactation: effects on mammary gland function,composition and morphology

We investigated the effects of bovine somatotropin (bST) on mammary gland function and composition in the declining phase of lactation in goats. Sixteen Saanen goats, 180 +/- 11 days in milk (DIM), were divided equally into control and treated groups. The treated group received 120 mg/2 wk of slow-release bST for three cycles. Milk yield, milk composition, milk clotting measures, and plasmin-plasminogen activator activities were recorded weekly. Milk Na and K were determined in individual milk samples collected weekly during the third cycle. Blood samples were collected weekly during the second cycle and the plasma analyzed for nonesterified fatty acids (NEFA), glucose, and urea. At the end of the 6 wk, three goats from each group were slaughtered, and the udders were removed. Mammary gland weight, composition, and total DNA content were determined. The histological effects of bST on mammary tissue were investigated. The analyzed parameters included numbers of alveoli, corpora amylacea, apoptotic cells, and laminin fibronectin distribution and localization. An extensive morphological analysis on the epithelial and stromal components was performed. Milk yield was significantly higher in the treated group, fat content was not affected, but protein and nonprotein nitrogen were lower in treated goats milk. Treatment with bST did not influence milk pH but reduced coagulation time. Plasmin and plasminogen activator activities were not affected. Milk K levels were higher and the Na/K ratio was lower in treated animals. Plasma glucose, NEFA, and urea were unaffected. Mammary gland weight and total DNA were higher in treated than control animals, suggesting that with advancing lactation bST treatment maintains cells. Fat, protein, and collagen content of the mammary tissue did not differ between the groups. Treatment with bST significantly increased the number of lactating alveoli (LA) and significantly reduced the number of regressing alveoli (RA) and corpora amylacea, both within and outside the alveolar lumen. Laminin and fibronectin localization were not affected, and very few apoptotic cells were found in both treated and control samples. Our findings suggest that bST administration to dairy goats in late lactation can modulate mammary gland activity and improve lactation persistency; this is associated with maintained total mammary parenchyma weight and lactating alveoli.     

Role of prolactin, growth hormone and insulin-like growth factor 1 in mammary gland involution in the dairy cow

Bovine mammary involution, an important process for subsequent lactations, is characterized by loss of epithelial cells by apoptosis, but its hormonal regulation is still not well defined. Prolactin (PRL) and growth hormone (GH) play a specific role on rat mammary gland apoptosis, through insulin-like growth factor 1 (IGF-1) and the IGF binding protein (IGFBP) system. The purpose of our investigation was to determine the possible role of PRL, GH, and IGF-1 on cell survival and on IGFBP-5 expression in the bovine mammary gland. Mammary gland explants were cultured in the presence of cortisol, 17beta-estradiol, progesterone, insulin, PRL, GH, and IGF-1 and with the same treatment but without PRL, GH or IGF-1, respectively. After 24 h of culture, we determined the level of apoptosis through evaluation of DNA laddering in the oligonucleosomal fraction and examined IGFBP-5 messenger RNA (mRNA) expression. The results show a high level of DNA laddering and an increase in IGFBP-5 mRNA content in mammary explants cultured in the absence of PRL, GH, or IGF-I with respect to explants treated with all hormones. Moreover, explants cultured in presence of PRL, GH, or IGF-I show a low level of DNA laddering and IGFBP-5 expression with respect to explants cultured without any hormones. These data demonstrate a relationship between levels of apoptosis and IGFBP-5 mRNA expression in the bovine mammary gland and confirm the involvement of this binding protein programmed cell death and its relationship with the main lactogenic hormones.     

Cell turnover and activity in mammary tissue during lactation and the dry period in dairy cows

Milk yield of the dairy cow follows a pattern termed the lactation curve. We have investigated the cellular background for this pattern. Seven mammary biopsies were obtained from each of 10 cows: at the end of lactation (d 347, equal to d 77 before next parturition); during the dry period at d 48 (4 d after dry off); 16 d before parturition; and during lactation at d 14, 42, 88, and 172. The fraction of proliferating (staining positive for Ki-67) alveolar cells was higher during the dry period (8.6%) than during lactation (0.5%). The fraction of apoptotic (staining positive by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) alveolar cells was higher immediately after dry off (0.37%) and in early lactation (0.76%) than during other periods (0.15%). The enzyme activities of fatty acid synthetase, acetyl CoA-carboxylase, and galactosyl transferase were approximately 12-, 11-, and 4-fold higher, respectively, during lactation than during the dry period. In conclusion, mammary cell proliferation is substantial in a period near parturition but otherwise low, and apoptosis is elevated at dry off and in early lactation. The increase in apoptosis in early lactation may be due to discarding nonfunctional or senescent cells or to removal of a surplus of newly synthesized cells. The activity of selected enzymes central for milk synthesis is probably not limiting for milk production.     

王不留行促进奶牛乳腺泌乳的信号转导途径

应用Western blot检测王不留行水浸提液作用后,乳腺上皮细胞泌乳信号转导基因P-AKT1、cyclinD1、PRLR、P-STAT5a和AMPK、GULT1的变化,结果表明:中药王不留行对P-AKT1、cyclinD1、PRLR、P-STAT5a和AMPK、GULT1基因表达具有一定作用,可能通过mTOR信号转导途径促进乳腺上皮细胞的增殖;通过激活PRLR从而激活Stat5a泌乳信号转导途径激活其下游β-酪蛋白基因的表达;且可能通过激活AMPK而促进GLUT1表达的增强,从而增加细胞对葡萄糖的摄取能力促进乳糖的合成。     

Effect of photoperiod in the third trimester of gestation on milk production and circulating hormones in dairy goats

Multiparous Israeli Saanen goats (n = 8) were blocked at dry off (approximately 45 d prepartum) into 2 treatments of 4 goats each based on body weight (BW), previous milk production, and the number of detected embryos in utero. Treatments consisted of long-day (16 h light:8 h dark) and short-day (8 h light:16 h dark) photoperiods at normothermic ambient temperature (22°C, 72% relative humidity). All goats were returned to ambient photoperiod after kidding, milked twice daily, and milk yield was automatically recorded. Dry matter intake was similar between treatments and averaged 980 g/d. Milk production was greater in the short-day than in the long-day treatment (2,932 vs. 2,320 g/d) during the 12-wk experimental period. Milk protein and lactose contents were similar in both treatments and averaged 3.61 and 4.88%, respectively, whereas milk fat was greater in the long-day treatment (4.80 vs. 4.22%). Plasma insulin-like growth factor 1 was greater in the long-day treatment (149 vs. 73 ng/mL) during the dry period than in the short-day treatment, but was similar postkidding, averaging 76 ng/mL. Concentrations of triiodothyronine in plasma were similar in both treatments during the dry period, but greater during lactation in the short-day treatment (122.1 vs. 94.1 ng/mL). Plasma prolactin was greater in the long-day than in the short-day treatment during the dry period (28.0 vs. 17.5 ng/mL), whereas it was similar throughout lactation (11.7 ng/mL). These data support the idea that greater milk production in goats exposed to short days during the dry period is not explained by differences in feed intake or increased secretion of insulin-like growth factor 1.     

Characterization of mammary stromal remodeling during the dry period

During the dry period between successive lactations, the mammary gland of dairy cows undergoes extensive remodeling that is marked by phases of involution and mammogenesis. Changes in the mammary epithelium during the dry period have been well characterized; however, few studies have examined the changes that occur in stromal tissue. The objective of this study was to characterize changes that occur in mammary stroma during the dry period. Mammary biopsies were taken from 9 multigravid Holstein cows in late lactation, at 1 wk after dry-off, 3 wk before expected calving date, and 1 wk before expected calving date. Tissue was fixed in formalin, embedded in paraffin, and cut into 5-μm sections. Sections were stained with hematoxylin and eosin or with immunohistochemistry for expression of smooth muscle α actin (SMA), fibronectin, stromelysin-1 (MMP-3), transforming growth factor-β1 (TGF-β1), and TGF-β receptor 2 (TGF-βR2). Images of tissues were captured with light microscopy, and imaging software was used to measure intralobular stromal area, number of activated fibroblasts, as identified by expression of SMA, and percentage of intralobular stromal area expressing fibronectin, MMP3, TGF-β1, and TGF-βR2. Analyses of variance were conducted and statistical differences were based on the least squares means of biopsy stage. Number of activated fibroblasts was greater at 1 wk dry than at 1 wk before calving (2,720 vs. 1,800 cells/mm²), percentage intralobular stromal area was greater at 1 wk dry (32%) and 3 wk before calving (37%) than at 1 wk before calving (25%), and TGF-β1 expression decreased 15% from late lactation to the dry period. The percentages of stromal area expressing fibronectin, MMP-3, and TGF-βR2 and the percentage of myofibroblasts were not different across biopsy stages. These results support the concept that stromal expression of transforming growth factor-β1 and fibroblast proliferation may be important for remodeling during the dry period.     

Invited review: Metabolic challenges and adaptation during different functional stages of the mammary gland in dairy cows: Perspectives for sustainable milk production

Milk production of dairy cows has increased markedly during recent decades and continues to increase further. The evolutionarily conserved direction of nutrients to the mammary gland immediately after calving provided the basis for successful selective breeding toward higher performance. Considerable variation in adaptive responses toward energy and nutrient shortages exists; however, this variation in adaptability recently gained interest for identifying more metabolically robust dairy cows. Metabolic challenges during periods of high milk production considerably affect the immune system, reproductive performance, and product quality as well as animal welfare. Moreover, growing consumer concerns need to be taken into consideration because the public perception of industrialized dairy cow farming, the high dependency on feed sources suitable for human nutrition, and the apparently abundant use of antibiotics may affect the sales of dairy products. Breeding for high yield continues, but the metabolic challenges increasingly come close to the adaptational limits of meeting the mammary gland's requirements. The aim of the present review is to elucidate metabolic challenges and adaptational limitations at different functional stages of the mammary gland in dairy cows. From the challenges and adaptational limitations, we derive perspectives for sustainable milk production. Based on previous research, we highlight the importance of metabolic plasticity in adaptation mechanisms at different functional stages of the mammary gland. Metabolic adaptation and plasticity change among developing, nonlactating, remodeling, and lactational stages of the mammary gland. A higher metabolic plasticity in early-lactating dairy cows could be indicative of resilience, and a high performance level without an extraordinary occurrence of health disorders can be achieved.     

奶山羊泌乳激素及其受体在乳腺发育过程中的变化规律及作用

探讨奶山羊乳腺发育过程中泌乳激素(E2,P和PRL)及其受体(ERα,PR和PRLR)的变化规律及作用.分别在关中奶山羊乳腺发育的青春期、妊娠期、泌乳期及退化期,采集血清并用放射性免疫学方法测定各激素浓度,采集乳腺组织并用免疫荧光和real-time PCR检测各激素受体表达量.青春期和妊娠早期E2,P和PRL缓慢上升,妊娠晚期E2和P达峰值,泌乳启动PRL高峰,泌乳高峰过后各激素均回复正常水平.ERα和PRLR青春期开始缓慢上升,泌乳启动后ERα达到高峰,而泌乳期PRLR维持在高水平,并在退化期达到表达高峰.青春期PR较妊娠期高,妊娠后期又开始升高,退化期下降.E2与ERα,E2与P及与ERα之间正相关,其余的激素和受体之间相关性不显著.     

Milk production of dairy cows treated with estrogen at the onset of a short dry period

The objective was to determine whether the use of estradiol-17beta (E2) at the initiation of short dry periods prevented an anticipated decline in milk production in the subsequent lactation. Lactating Holstein cows (n = 66) were dried at either 60 or 30 d before expected calving. Treatments in a 2 x 2 factorial arrangement included: D60 (n = 19, 60-d dry, no E2), D60 + E2 (n = 18, 60-d dry, E2), D30 (n = 15, 30-d dry, no E2), and D30 + E2 (n = 14, 30-d dry, E2). To accelerate mammary involution, estradiol-17beta (15 mg in 4 ml of ethanol) was injected subcutaneously daily for 4 d beginning 30 d before expected calving. Parturitions occurred between November 1995, and March 1996. Actual days dry for respective treatments were 57.3, 60.6, 33.9, and 33.8 +/- 1.7 d. Onset of parturition, calving difficulty, and cow health were not affected by E2. Actual 305-d milk yields for the lactation completed immediately before the experimental dry period were 10,318, 10,635, 10,127, and 10,447 +/- 334 kg, respectively; and were 9942, 9887, 9669, and 10,172 +/- 387 kg, respectively, for the lactation immediately following treatment. Respective pre- and posttreatment mature equivalent 305-d yields were 9574, 9861, 9812, and 9724 +/- 297 kg; 8987, 8843, 9126, and 9008 +/- 294 kg. Milk yields did not differ across treatments. Cows with a 34-d dry period were as productive as cows with a 59-d dry period. Estradiol-17beta had no effect, but perhaps should be evaluated with dry periods shorter than 34 d.     

Effects of dry period length and concentrate protein content in late lactation on body condition score change and subsequent lactation performance of thin high genetic merit dairy cows

Improving body condition score of thin cows in late lactation is necessary, because cows that are thin at drying off exhibit decreased fertility postpartum and are at increased risk of disease and of being culled in the subsequent lactation. Offering a diet low in crude protein (CP) content in late lactation may help to improve body condition score (BCS) at drying off, whereas imposing an extended dry period (EDP) has been advocated as another way to increase BCS at calving. To test these hypotheses, 65 thin cows (mean BCS 2.25 at 14 wk precalving) were managed on 1 of 3 treatments between 13 and 9 wk prepartum: normal protein control {NP; grass silage + 5 kg/d of a normal protein concentrate [228 g of CP/kg of dry matter (DM)]}, low protein [LP; grass silage + 5 kg/d of a low-protein concentrate (153 g of CP/kg of DM)], or EDP (cows dried off at 13 wk precalving and offered a grass silage-only diet). Both NP and LP cows were dried off at wk 8 prepartum, after which all cows were offered a grass silage-only diet until calving. After calving, all cows were offered a common diet (supplying 11.1 kg of concentrate DM/cow per day) for 19 wk. Between 13 and 9 wk prepartum, LP cows had lower DM intake, milk yield, and body weight than NP cows. Whereas EDP cows had lower serum β-hydroxybutyrate and fatty acid concentrations than those of NP cows, BCS at wk 9 prepartum did not differ between treatments. Cows on the LP treatment continued to have lower DMI and BW than those of NP and EDP cows between 8 wk prepartum and calving, but only EDP cows had a higher BCS at calving. Treatment did not affect calving difficulty score or calf birth weight. Although all cows were offered a common diet postpartum, cows on the LP treatment had lower DM intake and milk fat + plus protein yield than cows on any other treatment during the 19-wk period postpartum, but we found no differences in any postpartum indicator of body tissue reserves. The treatments imposed from wk 13 to 9 prepartum had no effect on any fertility or health parameters examined postpartum. Extending the dry period for thin cows improved their BCS at calving but did not allow these cows to achieve the target BCS of 2.75, and we found no beneficial effects of this treatment on cow performance postpartum. Offering a lower-protein diet to thin cows in late lactation did not improve BCS at calving above that of cows on a normal protein diet, but had unexplained long-term negative effects on cow performance.     

Effect of heat stress during the dry period on mammary gland development

Heat stress during the dry period negatively affects hepatic metabolism and cellular immune function during the transition period, and milk production in the subsequent lactation. However, the cellular mechanisms involved in the depressed mammary gland function remain unknown. The objective of the present study was to determine the effect of heat stress during the dry period on various indices of mammary gland development of multiparous cows. Cows were dried off approximately 46 d before expected calving and randomly assigned to 2 treatments, heat stress (HT, n = 15) or cooling (CL, n = 14), based on mature equivalent milk production. Cows in the CL treatment were provided with sprinklers and fans that came on when ambient temperatures reached 21.1°C, whereas HT cows were housed in the same barn without fans and sprinklers. After parturition, all cows were housed in a freestall barn with cooling. Rectal temperatures were measured twice daily (0730 and 1430 h) and respiration rates recorded at 1500 h on a Monday-Wednesday-Friday schedule from dry off to calving. Milk yield and composition were recorded daily up to 280 d in milk. Daily dry matter intake was measured from dry off to 42 d relative to calving. Mammary biopsies were collected at dry off, −20, 2, and 20 d relative to calving from a subset of cows (HT, n = 7; CL, n = 7). Labeling with Ki67 antigen and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling were used to evaluate mammary cell proliferation and apoptosis, respectively. The average temperature-humidity index during the dry period was 76.6 and not different between treatments. Heat-stressed cows had higher rectal temperatures in the morning (38.8 vs. 38.6°C) and afternoon (39.4 vs. 39.0°C), greater respiration rates (78.4 vs. 45.6 breath/min), and decreased dry matter intake (8.9 vs. 10.6 kg/d) when dry compared with CL cows. Relative to HT cows, CL cows had greater milk production (28.9 vs. 33.9 kg/d), lower milk protein concentration (3.01 vs. 2.87%), and tended to have lower somatic cell score (3.35 vs. 2.94) through 280 d in milk. Heat stress during the dry period decreased mammary cell proliferation rate (1.0 vs. 3.3%) at −20 d relative to calving compared with CL cows. Mammary cell apoptosis was not affected by prepartum heat stress. We conclude that heat stress during the dry period compromises mammary gland development before parturition, which decreases milk yield in the next lactation.     

Influence of estrus of dairy goats on somatic cell count, milk traits, and sex steroid receptors in the mammary gland

Two experiments were conducted to study the effect of the stage of a spontaneous estrus cycle on milk yield and constituents [somatic cell count (SCC), fat, protein, caseins, lactose, and urea content] and on estrogen receptor-α (ERα) and progesterone receptor (PR) immunostaining in the mammary gland. In experiment I, the major components of milk and SCC were monitored weekly in 80 lactating Saanen goats for 6 wk, whereas detection of estrus was daily. In experiment II, milk samples were collected daily for SCC determination during 1 spontaneous estrus (d 0) until the second spontaneous estrus in 14 Saanen goats. The day of the estrous cycle was confirmed by plasma progesterone and 17β-estradiol levels. Immunoreactivity of ERα and PR was analyzed in mammary gland samples of 8 Saanen goats (d 0, n = 4; d 10, n = 4) and the number of positive nuclei and intensity of the staining were evaluated in 1,000 cells. In experiment I, milk casein and protein percentages were significantly affected by the stage of estrous cycle; during proestrus and estrus, these variables were higher (3.32 ± 0.06 and 4.44 ± 0.08) than during metestrus (3.03 ± 0.07 and 4.07 ± 0.10), but not higher than during diestrus (3.23 ± 0.06 and 4.35 ± 0.09, respectively). In experiment II, daily measurement of SCC revealed higher levels at estrus (7,195 ± 672 × 10³ cells/mL) and a decline toward the luteal phase (1,694 ± 672 ± 10³ cells/mL). Estrogen receptor-α and PR immunostaining were exclusively detected on epithelial cells. The percentage of positive nuclei to ERα was higher on d 0 than on d 10 (75.4 ± 8.8 vs. 68.3 ± 8.8%), but no change was observed for PR (4.0 ± 0.3 vs. 3.5 ± 0.4%). The average immunostaining intensity for both receptors was greater on d 0 than on d 10 (ERα : 1.44 ± 0.02 vs. 1.35 ± 0.02; PR: 0.079 ± 0.008 vs. 0.057 ± 0.008). The high SCC at estrus in experiment II was associated with high plasma estradiol and low progesterone, suggesting that the increased SCC could be brought about by the estrogen-induced proliferation and exfoliation of epithelial cells. In addition, this action may be supported by the higher sensitivity to estrogens (ERα content) found at d 0.     

Short communication: Dietary conjugated linoleic acid down-regulates fatty acid transporters in the mammary glands of lactating rats

Recent studies indicated that reduction of milk triacylglycerol concentrations by dietary conjugated linoleic acid (CLA) involves an impairment of both de novo fatty acid synthesis and uptake of fatty acids from circulating triacylglycerol-rich lipoproteins into the mammary gland. However, nonesterified fatty acids (NEFA) in the plasma released from adipose tissue and taken up into the mammary gland by fatty acid transporters are a further important source of fatty acids available for milk triacylglycerol synthesis. Therefore, the aim of the present study was to investigate the effect of dietary CLA on plasma concentrations of NEFA and the expression of fatty acid transporters in the mammary glands of lactating rats fed either a CLA diet or a control diet. Dams fed diets with CLA had a greater concentration of NEFA in plasma than those fed the control diet. In addition, relative mRNA concentrations of fatty acid transporters (fatty acid translocase/CD36, fatty acid transport protein, and plasma membrane fatty acid binding protein) were about 45, 75, and 70% lower, respectively, in the mammary gland of dams fed diets with CLA compared with those fed the control diet. In conclusion, the present findings indicate that reduced uptake of circulating NEFA released from white adipose tissue into the mammary gland could also contribute to the reduction of milk triacylglycerol concentrations by dietary CLA in rats. The mechanism through which CLA inhibits expression of fatty acid transporters deserves further study.