Effects of photoperiod modulation and melatonin feeding around drying-off on bovine mammary gland involution

The risk for a dairy cow to acquire new intramammary infections is high during the transition from lactation to the dry period, because of udder engorgement and altered immune functions. Once the gland is fully involuted, it becomes much more resistant to intramammary infections. Therefore, strategies to depress milk yield before drying-off and accelerate the involution process after drying-off could be beneficial for udder health. The objective of this study was to assess the effect of photoperiod manipulation and melatonin feeding from 14 d before to 14 d after drying-off on the speed of the involution process. Thirty Holstein cows in late lactation were randomly allocated to one of the following treatments: (1) a long-day photoperiod (16 h of light: 8 h of darkness), (2) a short-day photoperiod (8 h of light: 16 h of darkness), and (3) a long-day photoperiod supplemented by melatonin feeding (4 mg/kg of body weight). Milk and blood samples were collected on d ?26, ?19, ?12, ?5, ?1, 1, 3, 5, 7, 10, and 14 relative to the last milking to determine concentrations of mammary gland involution markers and serum prolactin. Additional blood samples were taken around milking on d ?15, before the start of the treatments, and on d ?1, before drying-off, to evaluate the treatment effects on milking-induced prolactin release. The short-day photoperiod slightly decreased milk production and basal prolactin secretion during the dry period. The milking-induced prolactin surge was smaller on d ?1 than on d ?15 regardless of the treatments. Lactoferrin concentration, somatic cell count, and BSA concentration as well as matrix metalloproteinase-2 and -9 activities increased in mammary secretions during the first 2 wk of the dry period, whereas milk citrate concentration and the citrate:lactoferrin molar ratio decreased. The rates of change of these parameters were not significantly affected by the treatments. The long-day photoperiod supplemented by melatonin feeding did not affect milk production, prolactin secretion, or mammary gland involution. Under the conditions in this study, photoperiod modulation and melatonin feeding did not appear to affect the rate of mammary gland involution.     

Inhibiting prolactin by cabergoline accelerates mammary gland remodeling during the early dry period in dairy cows

The inhibition of prolactin release using cabergoline, a dopamine agonist, is an effective strategy to accelerate the changes in mammary secretion composition after drying-off. The objective of this study was to determine how cabergoline may affect mammary tissue remodeling during early involution. Holstein dairy cows were treated with either a single i.m. administration of 5.6 mg of cabergoline (Velactis, Ceva Santé Animale, Libourne, France, n = 7) or placebo (n = 7) at the time of drying-off. Mammary biopsy samples were collected 1 wk before drying-off (d ?6), after 30 h of milk accumulation (d 1), and again 8 d following drying-off (d 8) to determine changes in gene expression, lactoferrin content, and cell turnover. Blood and mammary secretion samples were collected at d ?6 and again at d 1, 2, 3, 4, 8, and 14 following the abrupt cessation of lactation to evaluate indicators of blood-milk barrier integrity and other markers of mammary tissue remodeling. Cabergoline induced less SLC2A1, BAX, CAPN2, and IGFBP5 mRNA expression. In contrast, cabergoline did not modify changes in cell proliferation and apoptosis. Following the cessation of lactation, changes in mammary secretion composition (Na⁺ and K⁺) and blood lactose concentrations were indicative of a loss in the blood-milk barrier function in both treatment groups. Cabergoline treatment affected only Na⁺ and K⁺ concentrations at d 1, suggesting a moderate increase in tight junction permeability. The increase in the activity of MMP9 and in mammary epithelial cell concentration in mammary secretions was greater in cabergoline-treated cows than in control cows, suggesting more mammary tissue remodeling. The increase in lactoferrin immunostaining in the mammary tissue occurred earlier for cabergoline-treated cows than for control cows, and was essentially localized in the stroma. Changes in some key markers of mammary involution suggest that cabergoline accelerates mammary gland remodeling. Thus, a single injection of cabergoline after the last milking would facilitate drying-off by enhancing mammary gland involution.     

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.     

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.     

Infusions of casein hydrolyzates into the mammary gland disrupt tight junction integrity and induce involution in cows

Milk stasis triggers local stimuli, which make the tight junctions leak and trigger involution. The aim of the study was to test the hypothesis that casein hydrolyzates compromise tight junction integrity and dry-off milk secretion in dairy cows. Six repeated doses of casein hydrolyzates after each milking during 3 d caused drastic changes in mammary secretion and composition, which were associated with irreversible cessation of milk secretion. No such changes were recorded in the control glands that had been treated with nonhydrolyzed casein. Treatment with casein hydrolyzates disturbed tight junction integrity within 8 h (as indicated by changes in Na+ and K+ concentrations), reduced the concentrations of lactose precipitously, activated the plasmin activator-plasminogen-plasmin system, and induced the secretion of immunoglobulin type G and lactoferrin. At the end of the 3-d treatments, we stopped milking the experimental and control glands. Milk composition 19 d later was similar in the experimental and control glands and was consistent with the composition expected in fully involuted glands. We conclude that casein hydrolyzates are among the milk-borne factors that cause the disruption of tight junction integrity and induce involution in cows. The process induced by casein hydrolyzate was more rapid and synchronized than the involution induced at drying-off.     

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.     

Invited review: Accelerating mammary gland involution after drying-off in dairy cattle

Bovine mammary gland involution, as a part of the reproductive cycle in dairy cows, is a very important remodeling transformation of the mammary gland for the subsequent lactation. There is considerable incentive to accelerate mammary gland involution to improve udder health, shorten the dry period, and simplify the management process by reducing dietary changes. The complex process of mammary involution is characterized by morphological changes in the epithelial cells and mammary tissue, changes in the composition of mammary secretions, and changes in the integrity of tight junctions. Involution is facilitated by elements of the immune system and several types of proteases and is coordinated by various types of hormones. This review first describes the involution process and then argues for the need to accelerate it. Last, this review focuses on various intervention methods for accelerating involution. Our aim is to provide a comprehensive overview of bovine mammary gland involution as well as potential techniques and new opinions for dry cow management.     

Proteases involved in mammary tissue damage during endotoxin-induced mastitis in dairy cows

During and after diapedesis, milk polymorphonu-clear neutrophils (PMN) release many proteases that have the potential of degrading extracellular matrix proteins and milk proteins. However, the kinetics of milk proteolysis during inflammation and the underlying mechanisms are poorly defined. The enzymes involved in bovine mammary tissue destruction were investigated in this study using an endotoxin-induced mastitis model. Using zymography techniques, the proteolytic activity of milk and mammary tissue during mastitis was examined. Mastitic milk produced 6 caseolysis bands, 4 of which differed from the ones produced by plasmin. Peak proteolytic activity, bovine serum albumin contents, and mammary tissue damage occurred between 6 and 12 h postchallenge. Mastitic milk proteases hydrolyzed casein, gelatin, collagen, hemoglobin, mammary gland membrane proteins, and lactoferrin. These results confirm that mastitic milk proteases have a broad spectrum of activity. The hydrolytic activity of mastitic milk was partially inhibited by aprotinin, EDTA, 1,10-phenanthroline, leupeptin, and pefabloc. When cocultured with normal mammary tissue, mastitic milk, but not normal milk, caused mammary tissue degradation. In situ zymography of mammary gland showed increased proteolytic activity in mastitic tissue compared with normal tissue. The similarity of zymograms of mastitic milk, blood PMN, milk somatic cells, and PMN strongly suggests that proteases in mastitic milk mainly originate from milk PMN. These results suggest that proteases released by PMN are actively involved in udder tissue damage during mastitis.     

Impaired autophagy aggravates oxidative stress in mammary gland of dairy cows with clinical ketosis

When ketosis occurs, supraphysiological levels of free fatty acids (FFA) can cause oxidative injury to the mammary gland and autophagy can regulate the cellular oxidative status. The aim of this study was to investigate the autophagy status of mammary tissue and its associations with oxidative stress in healthy and clinically ketotic dairy cows. Mammary tissue and blood samples were collected from healthy cows [n = 15, β-hydroxybutyrate (BHB) <0.6 mM] and clinically ketotic cows (n = 15, BHB >3.0 mM) at 3 to 15 (average = 7) days in milk. For in vitro study, bovine mammary epithelial cells (BMEC) isolated from healthy cows were treated with 0, 0.3, 0.6, or 1.2 mM FFA for 24 h. Furthermore, BMEC were pretreated with 100 nM rapamycin, an autophagy activator, for 4 h or 50 mM 3-methyladenine (3-MA), an autophagy inhibitor, for 1 h, followed by treatment with or without FFA (1.2 mM) for another 24 h. Oxidation indicators and autophagy-related protein abundance were measured. Compared with healthy cows, serum concentrations of FFA, BHB, and malondialdehyde were greater in clinically ketotic cows, but milk production (kg/d), milk protein (kg/d), activities of superoxide dismutase, catalase, and glutathione peroxidase were lower. Abundances of mRNA and protein of autophagy-related gene 5 (ATG5) and 7 (ATG7) were lower, but sequestosome-1 (SQSTM1, also called p62) greater in mammary tissue of clinically ketotic cows. The mRNA abundance of microtubule-associated protein 1 light chain 3 (MAP1LC3, also called LC3) and protein abundance of LC3-II were lower in mammary tissue of clinically ketotic cows. In vitro, exogenous FFA increased the content of malondialdehyde and reactive oxygen species, but decreased the activities of superoxide dismutase, catalase, and plasma glutathione peroxidase. Compared with the 0 mM FFA group, abundance of ATG5, ATG7, LC3-II was greater, but p62 was lower in the 0.6 mM FFA-treated cells. Similarly, abundance of ATG5, ATG7, and LC3-II was lower, but p62 greater in the 1.2 mM FFA-treated cells relative to 0 mM FFA group. Culture with rapamycin alleviated oxidative stress induced by 1.2 mM FFA, whereas 3-MA aggravated it. Overall, results indicated that a low concentration (0.6 mM) of FFA can induce oxidative stress and activate autophagy in BMEC. At higher concentrations of FFA (1.2 mM), autophagy is impaired and oxidative stress is aggravated. Autophagy is a mechanism for BMEC to counteract FFA-induced stress. As such, it could serve as a potential target for further development of novel strategies against oxidative stress.     

Lipopolysaccharide and lipoteichoic acid induce different immune responses in the bovine mammary gland

Different pathogens, such as Escherichia coli and Staphylococcus aureus, can be responsible for different outcomes of mastitis; that is, acute and severe or chronic and subclinical. These differences in the disease could be related to different mammary responses to the pathogens. The objective of this study was to determine if intramammary challenge with the endotoxins lipopolysaccharide (LPS), from E. coli, and lipoteichoic acid (LTA), from Staph. aureus, induce different immune responses in vivo in milk cells and mammary tissue. To provide a reference level for comparing the challenge and to show the different stimulation of the mammary immune system on a quantitatively similar level, dosages of LPS and LTA were chosen that induced an increase of somatic cells in milk to similar maxima. One udder quarter in each of 21 lactating dairy cows was challenged with 0.2 μg of LPS or 20 μg of LTA. From these quarters and from respective control quarters, milk cells or tissue biopsies were obtained at 0, 6, and 12 h relative to the challenge to measure mRNA expression of tumor necrosis factor-α (TNFα), IL-1β, IL-8, lactoferrin, and RANTES (regulated upon activation, normal T-cell expressed and secreted). Furthermore, if no biopsies were performed, hourly milk samples were taken for measurement of somatic cell count, lactate dehydrogenase (LDH), and TNFα. Somatic cell count increased in all treatments to similar maxima with LPS and LTA treatments. Concentrations of TNFα in milk increased with LPS but not with LTA. The activity of LDH in milk increased in both treatments and was more pronounced with LPS than with LTA. The mRNA expression of TNFα, IL-1β, IL-8, and RANTES showed increases in milk cells, and LPS was a stronger inducer than LTA. Lactoferrin mRNA expression decreased in milk cells with LPS and LTA treatments. The measured factors did not change in either treatment in mammary tissue. Challenge of udder quarters with dosages of LPS and LTA that induce similar increases in SCC stimulate the appearance of different immune factor patterns. This dissimilar response to LPS and LTA may partly explain the different course and intensity of mastitis after infection with E. coli and Staph. aureus, respectively.     

IGFBP-5在乳腺发育过程中的生物学作用

细胞生长的调节主要由IGFs起作用,IGFBPs能够通过蛋白水解、翻译后修饰以及同细胞表面和细胞外基质的相互作用来加强或抑制IGFs的作用,从而调节细胞的增殖、存活和分化.IGFBPs也具有独立的作用方式.IGFBP-5与乳腺发育特别是乳腺退化关系密切,IGFBP-5的表达可能是细胞凋亡发生时的普遍事件,尤其是在乳腺退化的初始阶段.     

Effect of casein and propionate supply on mammary protein metabolism in lactating dairy cows

The effects of casein (CN) and propionate (C3) on mammary AA metabolism were determined in 3 multiparous Holstein cows fitted with both duodenal and ruminal cannulas and used in a replicated Youden square with six 14-d periods. Casein (743 g/d in the duodenum) and C3 (1,041 g/d in the rumen) infusions were tested in a factorial arrangement. For each period, l-[1-¹³C]Leu (d 11) and NaH[¹³C]O₃ (d 13) were infused into a jugular vein, and blood samples were taken from the carotid artery and the mammary vein to determine Leu kinetics and net uptake of AA. Both CN and C3 treatments separately increased milk protein concentration and yield. With CN there was a general response in mammary protein metabolism, involving increases in Leu net uptake (30%), the uptake:output ratio (8%), protein synthesis (11%), secretion in milk protein (21%), and oxidation (259%). In contrast, C3 treatments tended to increase only Leu in milk protein (7%) and, when in combination with CN, to reduce Leu used for protein synthesis (5%). Across all treatments, most Leu uptake by the mammary gland was accounted for as Leu in milk or oxidized, and the Leu balance was therefore achieved without involvement of either net peptide use or production. Mammary uptake of group 1 AA increased to match milk output with all infusions. In contrast, mammary uptake of group 2 AA exceeded output to a greater extent with CN than with C3 infusions, whereas the increment in uptake of group 3 AA increased with C3 treatments. Overall, these data suggest that different mechanisms operate to improve milk protein production when either protein or energy is supplied.     

A high-concentrate diet induces mitochondrial dysfunction by activating the MAPK signaling pathway in the mammary gland of dairy cows

Subacute rumen acidosis can lead to mastitis in dairy cows. Mitochondrial dysfunction is closely related to the inflammatory response. This experiment was conducted to investigate the effects of a high-concentrate diet on mammary gland inflammation and mitochondrial damage in dairy cows. Twelve Holstein dairy cows in mid-lactation were randomly divided into 2 groups and fed a 40% concentrate (low concentrate, LC) diet or a 60% concentrate (high concentrate, HC) diet. Cows were fed individually, and the experiment lasted for 3 wk. After the experiment, mammary gland tissue, blood, and rumen fluid were collected. Compared with the LC diet, the HC diet significantly decreased rumen pH; the pH was <5.6 for more than 3 h. The HC diet also increased the concentration of LPS in the blood (7.17 ± 1.25 µg/mL vs. 12.12 ± 1.26 µg/mL), which indicated that feeding the HC diet successfully induced subacute rumen acidosis. The HC diet also increased the concentration of Ca²⁺ (34.80 ± 4.23 µg/g vs. 46.87 ± 7.24 µg/g) in the mammary gland and upregulated the expression of inflammatory factors IL-6 (1,128.31 ± 147.53 pg/g vs. 1,538.42 ± 241.38 pg/g), IL-1β (69.67 ± 5.86 pg/g vs. 90.13 ± 4.78 pg/g), and tumor necrosis factor-α (91.99 ± 10.43 pg/g vs. 131.75 ± 17.89 pg/g) in mammary venous blood. The HC diet also increased the activity of myeloperoxidase (0.41 ± 0.05 U/g vs. 0.71 ± 0.11 U/g) and decreased the content of ATP (0.47 ± 0.10 µg/mL vs. 0.32 ± 0.11 µg/mL) in the mammary gland. In addition, phosphorylation of JNK (1.00 ± 0.21 vs. 2.84 ± 0.75), ERK (1.00 ± 0.20 vs. 1.53 ± 0.31), and p38 (1.00 ± 0.13 vs. 1.47 ± 0.41) and protein expression of IL-6 (1.00 ± 0.22 vs. 2.21 ± 0.27) and IL-8 (1.00 ± 0.17 vs. 1.96 ± 0.26) were enhanced in cows of the HC group, indicating that the mitogen-activated protein kinase (MAPK) signaling pathway was activated. Compared with the LC diet, the HC diet reduced the protein expression of mitochondrial biogenesis–related proteins PGC-1α (1.00 ± 0.17 vs. 0.55 ± 0.12), NRF1 (1.00 ± 0.17 vs. 0.60 ± 0.10), TFAM (1.00 ± 0.10 vs. 0.73 ± 0.09), and SIRTI (1.00 ± 0.44 vs. 0.40 ± 0.10). The HC diet promoted mitochondrial fission and inhibited mitochondrial fusion by reducing protein expression of MFN1 (1.00 ± 0.31 vs. 0.49 ± 0.09), MFN2 (1.00 ± 0.19 vs. 0.69 ± 0.13), and OPA1 (1.00 ± 0.08 vs. 0.72 ± 0.07), and by increasing that of DRP1 (1.00 ± 0.09 vs. 1.39 ± 0.10), MFF (1.00 ± 0.15 vs. 1.89 ± 0.12), and TTC1/FIS1 (1.00 ± 0.08 vs. 1.76 ± 0.14), leading to mitochondrial dysfunction. The HC diet increased mitochondrial permeability by upregulating the protein expression of VDAC1 (1.00 ± 0.42 vs. 1.90 ± 0.44), ANT (1.00 ± 0.22 vs. 1.27 ± 0.17), and CYPD (1.00 ± 0.41 vs. 1.82 ± 0.43). Taken together, these results indicated that feeding the HC diet induced mitochondrial damage via the MAPK signaling pathway in the mammary gland of dairy cows.     

Regulation of protein synthesis in mammary glands of lactating dairy cows by starch and amino acids

The objective of this study was to evaluate local molecular adaptations proposed to regulate protein synthesis in the mammary glands. It was hypothesized that AA and energy-yielding substrates independently regulate AA metabolism and protein synthesis in mammary glands by a combination of systemic and local mechanisms. Six primiparous mid-lactation Holstein cows with ruminal cannulas were randomly assigned to 4 treatment sequences in a replicated incomplete 4 × 4 Latin square design experiment. Treatments were abomasal infusions of casein and starch in a 2 × 2 factorial arrangement. All animals received the same basal diet (17.6% crude protein and 6.61 MJ of net energy for lactation/kg of DM) throughout the study. Cows were restricted to 70% of ad libitum intake and abomasally infused for 36 h with water, casein (0.86 kg/d), starch (2 kg/d), or a combination (2 kg/d starch + 0.86 kg/d casein) using peristaltic pumps. Milk yields and composition were assessed throughout the study. Arterial and venous plasma samples were collected every 20 min during the last 8 h of infusion to assess mammary uptake. Mammary biopsy samples were collected at the end of each infusion and assessed for the phosphorylation state of selected intracellular signaling molecules that regulate protein synthesis. Animals infused with casein had increased arterial concentrations of AA, increased mammary extraction of AA from plasma, either no change or a trend for reduced mammary AA clearance rates, and no change in milk protein yield. Animals infused with starch had increased milk and milk protein yields, increased mammary plasma flow, reduced arterial concentrations of AA, and increased mammary clearance rates and net uptake of some AA. Infusions of starch increased plasma concentrations of glucose, insulin, and insulin-like growth factor-I. Starch infusions increased phosphorylation of ribosomal protein S6 and endothelial nitric oxide synthase, consistent with changes in milk protein yields and plasma flow, respectively. Phosphorylation of the mammalian target of rapamycin was increased in response to starch only when casein was also infused. Thus, cell signaling molecules involved in the regulation of protein synthesis differentially responded to these nutritional stimuli. The hypothesized independent effects of casein and starch on animal metabolism and cell signaling were not observed, presumably because of the lack of a milk protein response to infused casein.     

奶牛乳腺中瘦素及其受体的表达与定位

为探讨瘦素在奶牛乳腺发育、泌乳及退化各时期的表达变化规律及在乳腺组织中的具体位置,采用免疫印迹(western blot-ting)技术及激光共聚焦技术检测奶牛乳腺组织中瘦素及瘦素受体(OB-Rb)的表达变化及其定位。结果表明,瘦素在青春期表达量较高,在泌乳期表达量最低。瘦素受体在青春期和妊娠期表达量较高,泌乳期表达量相对较低,退化期逐渐恢复到妊娠期水平。     

Induced hyperketonemia affects the mammary immune response during lipopolysaccharide challenge in dairy cows

Metabolic adaptations during negative energy and nutrient balance in dairy cows are thought to cause impaired immune function and hence increased risk of infectious diseases, including mastitis. Characteristic adaptations mostly occurring in early lactation are an elevation of plasma ketone bodies and free fatty acids (nonesterified fatty acids, NEFA) and diminished glucose concentration. The aim of this study was to investigate effects of elevated plasma β-hydroxybutyrate (BHBA) at simultaneously even or positive energy balance and thus normal plasma NEFA and glucose on factors related to the immune system in liver and mammary gland of dairy cows. In addition, we investigated the effect of elevated plasma BHBA and intramammary lipopolysaccharide (LPS) challenge on the mammary immune response. Thirteen dairy cows were infused either with BHBA (HyperB, n = 5) to induce hyperketonemia (1.7 mmol/L) or with a 0.9% saline solution (NaCl, n = 8) for 56 h. Two udder quarters were injected with 200 μg of LPS after 48 h of infusion. Rectal temperature (RT) and somatic cell counts (SCC) were measured before, at 48 h after the start of infusions, and hourly during the LPS challenge. The mRNA abundance of factors related to the immune system was measured in hepatic and mammary tissue biopsies 1 wk before and 48 h after the start of the infusion, and additionally in mammary tissue at 56 h of infusion (8 h after LPS administration). At 48 h of infusion in HyperB, the mRNA abundance of serum amyloid A (SAA) in the mammary gland was increased and that of haptoglobin (Hp) tended to be increased. Rectal temperature, SCC, and mRNA abundance of candidate genes in the liver were not affected by the BHBA infusion until 48 h. During the following LPS challenge, RT and SCC increased in both groups. However, SCC increased less in HyperB than in NaCl. Quarters infused with LPS showed a more pronounced increase of mRNA abundance of IL-8 and IL-10 in HyperB than in NaCl. The results demonstrate that an increase of plasma BHBA upregulates acute phase proteins in the mammary gland. In response to intramammary LPS challenge, elevated BHBA diminishes the influx of leukocytes from blood into milk, perhaps by via modified cytokine synthesis. Results indicate that increased ketone body plasma concentrations may play a crucial role in the higher mastitis susceptibility in early lactation.