Effect of milking time on yield, composition, and antimicrobial components of milk in lactating goats.

Various studies have attempted to improve the milk yield and composition in dairy animals. However, no study has examined the effects of milking at different times on milk yield and composition. Therefore, this study aimed to compare the yield, composition, and antimicrobial components of milk obtained from milking at different times in lactating goats. Eight goats were milked once daily at different times for three consecutive weeks (first week: 06:00 h; second week: 09:00 h; and third week: 12:00 h). The light ranged from 06:30 to 19:00 h. Milk and blood samples were collected once a day during milking time. Milking at 09:00 h resulted in a significantly higher milk yield than that obtained after milking at 06:00 and 12:00 h. Prolactin levels in plasma and the fat, Na+, ß-defensin, and S100A7 (antimicrobial component) levels in milk were the lowest in the 09:00 h milking. These results indicate that milk yield, composition, and antimicrobial components can be affected by milking time, which may be related to the altered concentration of prolactin in the blood. These findings provide a rational basis for achieving maximal milk production with strong immunity by changing to a more effective milking time.

Effect of low milking frequency on the concentration of antimicrobial proteins in goat milk.

This study examined the effects of low frequency milking on the concentrations of antimicrobial components in goat milk. Sixteen goats were divided into two groups of eight each: milking once every 2 d three times (for six days, three times group) or five times (for 10 days, five times group). On other days, milking was performed once daily. Milk was collected, and milk yield, somatic cell count (SCC), and the concentrations of some antimicrobial proteins such as lactoferrin (LF), S100A7, IgA, and sodium ions (Na+) in milk were measured. Milk yield significantly decreased in both the groups during the low-milking frequency period, followed by an increase above the low frequency milking period in both groups. In contrast, SCC and LF concentrations in milk increased in both groups during the low frequency milking period. The concentration of S100A7 in milk temporarily decreased after the low frequency milking period, followed by a significant increase. The S100A7 concentration during this period was higher in the five times group than in the three times group. These results indicated that low frequency milking induced a gradual decrease in milk yield and a concomitant increase in antimicrobial components, such as LF and S100A7, in milk. This increase in the antimicrobial components may be useful in preventing mastitis.

Concentrations of antimicrobial components in milk of Japanese Black cows and their differences from dairy cows.

The components of milk from beef cows remain to be elucidated. This study examined the differences in the antimicrobial components of milk between dairy and beef cows. Quarter milk was collected from both Japanese Black (beef type) and Holstein (dairy type) cows to compare the concentrations of antimicrobial components. The concentration of lingual antimicrobial peptide (LAP) was higher, whereas that of the other antimicrobial components (lactoferrin, S100A7, and S100A8) was lower in beef cows than in dairy cows. Overall, these results indicate that the differences in antimicrobial components between beef and dairy cows may be associated with the difference in the prevalence of mastitis between them.

Innate Immune Training in Chickens for Improved Defense against Pathogens: A Review.

The avian immune system plays a vital role in poultry production to obtain good productibility and products that are safe and of high quality. Historically, adaptive immunity has been the main target of vaccination. However, over the past decade, innate immunity has been reported to be enhanced in different animals through vaccination and feed additives. This enhancement is due to innate immune memory termed "trained immunity," in which epigenetic and metabolic reprogramming play significant roles. Although reports on trained immunity in poultry are limited, several studies have suggested that vaccinations and feed additives affect the innate immunity. This review discusses the possible effects of vaccination and ß-glucan on innate immunity for potential incorporation in advanced strategies to enhance the defense function in poultry while considering the information on trained immunity in mammals.

Effect of mastitis during early-stage pregnancy on the immunity levels and pregnancy function of goats.

In this study, we investigated the effects of mammary inflammation induced by lipopolysaccharide (LPS) and Staphylococcus aureus (SA) infusions on pregnancy function during early pregnancy in goats. In Experiment 1, pregnant goats were subjected to an intramammary LPS infusion for 1 week from Days 60-66 after natural mating (n = 5), and in Experiment 2, they received intramammary infusions of either saline, LPS, or SA for 1 week from Days 45-51 after natural mating (n = 15). Blood was collected to determine the plasma cytokine, cortisol, 13,14-dihydro-15-keto-prostaglandin F2α (PGFM), and progesterone levels. Pregnancy length was significantly longer in the LPS-treated group than that for the saline-treated group of experiment 2. Cytokine levels (IL-1ß, IL-8, Tumor necrosis factor-α: TNF-α) after LPS (in both Experiments 1 and 2) and SA (in Experiment 2) infusion were significantly higher compared with those before infusion. In Experiment 2, the SA-infused group showed significantly higher TNF-α concentrations than those in the saline group. Cortisol levels increased in both experiment 1 and 2 after LPS infusion, but not after saline and SA treatments. Furthermore, PGFM levels increased after LPS infusion in Experiment 1. In Experiment 2, LPS- and SA-infused goats showed significantly higher PGFM levels than those in the saline-infused goats. However, the progesterone levels decreased after LPS treatment in Experiment 1. Our results show that intramammary LPS infusion during the early stage of pregnancy in goats induces inflammatory cytokine and stress hormone production, which prolongs the pregnancy period.

Effect of different inflammation states on the antimicrobial components in milk of goat udders after milking cessation.

The aim of this study was to examine the effects of milking cessation under different inflammatory conditions on the changes in antimicrobial components in milk and the process of mammary gland involution. Twenty udder halves were divided into two groups: those with (LPS) and without (control) lipopolysaccharide infusion, followed by cessation of milking for 8 weeks. Milk samples were collected weekly. Udder tissue was collected 4 weeks after milking cessation to measure the area of the lobule and connective tissue. After milking cessation, the somatic cell count (SCC) in the control group increased, whereas that in the LPS group did not. Lactoferrin (LF) and cathelicidin (Cath)-2 concentrations increased in both groups, whereas only LF was significantly lower in the LPS group than in the control group at week 4. The Cath-7 and S100A8 concentrations were significantly lower in the LPS group than in the control group. The lobule area was higher, and the connective tissue area was lower in the LPS group than in the control group. These results indicate that inflammation at milking cessation decreased the concentrations of some antimicrobial components and interfered with mammary gland involution. Therefore, animals with mastitis should recover prior to the onset of the dry period.

Effects of topical application of resveratrol on tight junction barrier and antimicrobial compound production in lactating goat mammary glands.

In mammary glands, the formation of less-permeable tight junctions (TJs) and the production of antimicrobial compounds like lactoferrin and defensins are important for preventing mastitis. Resveratrol, a polyphenol contained in red grapes, is known to protect mammary epithelial cells (MECs) from oxidative stress; however, oral administration of resveratrol causes a decrease in certain biological processes through conjugation and metabolic conversion. In this study, we determined the beneficial effects of resveratrol on TJs and antimicrobial compounds in cultured goat MECs by adding it to the medium, and in lactating goat mammary glands by topical application for percutaneous absorption. TJ barrier function was evaluated by transepithelial resistance and expression or localization pattern of claudins for culture model in vitro and by somatic cell count, Na+, albumin, and IgG in milk for topical application in vivo. Concentrations of antimicrobial compounds and cytokines were measured using ELISA. Activation of STAT3 was evaluated by Western blotting. Resveratrol strengthened TJ barrier function by upregulating claudin-3 in cultured MECs and topical application to udders reduced somatic cell count, Na+, albumin, and IgG in milk. Resveratrol increased ß-defensin and S100A7 levels in cultured MECs and milk. In addition, resveratrol down-regulated cytokine production and STAT3 pathway. These findings suggest that the topical application of resveratrol to udders may be effective in preventing mastitis.

Concentrations of antimicrobial components in milk at dry off and postpartum and their relationships to new high somatic cell counts at quarter level in dairy cows.

We investigated the antimicrobial components in cow milk at dry off and postpartum and their contribution in preventing new high SCC at quarter level. Milk samples from 72 quarters of 19 lactating cows were collected at last milking before dry off and at 7 d after parturition. Milk yield of each cow was recorded and SCC, IgG, IgA, lactoferrin, lingual antimicrobial peptide (LAP), and S100A7 concentrations in each quarter milk sample were measured. The postpartum milk yield was significantly higher than that at dry off. The IgG, IgA and lactoferrin concentrations in milk at dry off were significantly higher than those at postpartum, whereas the LAP concentration was lower. Quarters with SCC < 300 000 cells/ml at both dry off and postpartum were classified as persistent low SCC (PL) whereas those that rose above that same threshold postpartum were classified as new high SCC (NH). At dry off, IgG and LAP concentrations in milk were significantly higher in PL than in NH. These results suggest that high LAP concentrations during the dry period may contribute toward the prevention of new high SCC.

Effect of sodium butyrate treatment at the basolateral membranes on the tight junction barrier function via a monocarboxylate transporter in goat mammary epithelial cells.

In lactating mammary glands, tight junctions (TJs) prevent blood from mixing with milk and maintain epithelial cell polarity, which is important for milk production. This study aimed to investigate the effect of sodium acetate and sodium butyrate (SB) stimulation direction on the TJ barrier function, which is measured with regard to transepithelial electrical resistance and fluorescein flux, in goat mammary epithelial cells. The expression and localization of the TJ proteins claudin-3 and claudin-4 were examined using Western blotting and immunofluorescence. SB treatment in the lower chamber of cell culture inserts adversely affected the TJ barrier function, whereas sodium acetate barely had any effect, regardless of stimulation direction. In addition, SB treatment in the lower chamber significantly upregulated claudin-3 and claudin-4, whereas TJ proteins showed intermittent localization. Moreover, SB induced endoplasmic reticulum (ER) stress. ARC155858, a monocarboxylate transporter-1 inhibitor, alleviated the adverse impact of SB on TJs and the associated ER stress. Interestingly, sodium ß-hydroxybutyrate, a butyrate metabolite, did not affect the TJ barrier function. Our findings indicate that sodium acetate and SB influence the TJ barrier function differently, and excessive cellular uptake of SB can disrupt TJs and induce ER stress.

Effects of oral administration of lipopolysaccharide derived from Pantoea agglomerans on innate immunity of mammary glands in dairy goats.

This study aimed to evaluate the effect of orally administered lipopolysaccharide (LPS) derived from Pantoea agglomerans (LPSpa) on innate immune functions, including the concentrations of antimicrobial components and interleukin (IL)-10 in goat milk, for the prevention of goat mastitis. Twelve Tokara goats were divided into two groups of six goats. Goats in the LPSpa and control groups were orally administrated with 0.4 g/kg dextrin with or without 0.02 mg/kg LPSpa for 7 days (day 0-6), respectively. After treatment (i.e., day 7), 1 µg LPS from Escherichia coli O111 (LPSec) was infused into one side of the udder in both groups to induce mastitis. Milk from all sides of the udder, saliva, and feces were collected on days 0 and 7. After LPSec infusion into the udders, milk was collected from the infused side of the udder on days 8, 10, and 12. Milk yields and somatic cell counts were recorded during the examination period. The concentrations of immunoglobulin (Ig) A in saliva, feces, and milk and the concentrations of lactoferrin, goat ß defensin-1 (GBD1), S100A7, and IL-10 in milk were measured. After LPSpa oral administration, the concentrations of GBD-1 and IL-10 in the milk of the LPSpa group were significantly higher on day 7 than those in the control group, and the concentration of IgA in the feces tended to be higher than that in the control group. After LPSec intramammary infusion, S100A7 concentration on day 12 was significantly lower in the LPSpa group than in the control group. These findings suggest that the oral administration of LPSpa may prevent mastitis by increasing the concentration of GBD1 in milk.

Effect of intramammary lipopolysaccharide challenge after repeated intrauterine infusion of lipopolysaccharide on the inflammation status of goat mammary glands.

Previous studies have shown that a single infusion of lipopolysaccharide (LPS) into the uterus induces mammary gland inflammation. However, repeated LPS infusions return the mammary glands to their basal state of inflammation. To confirm that this is a state of tolerance to LPS, we examined whether tolerance induced by repeated intrauterine LPS infusions limits mammary gland inflammation following subsequent intramammary LPS infusions. In the first experiment, three goats were treated with repeated intrauterine infusions of LPS dissolved in black ink for 5 consecutive days. Blood and milk samples were collected at 2, 4, 6, 12, 24, 48, 72, 96, and 120 h and smeared on glass slides to confirm the translocation of LPS from the uterus to the mammary gland. Black particles were detected in the blood and milk samples more than 2 h after the first infusion and in the connective tissue of the mammary gland after day 5. In the second experiment, goats were divided into two groups: an intrauterine infusion group (IU; n = 7) and a control group (CON; n = 6). The IU group received an intrauterine infusion of 100 µg of LPS in saline for 5 days. Subsequently, LPS was infused into the mammary glands of both groups to examine the effect of intrauterine treatment on the mammary inflammatory response after intramammary LPS infusion. Blood and milk samples were collected at 6, 12, and 24 h, and then daily until 7 d after the intramammary LPS challenge. Interestingly, a significant increase in the milk somatic cell count (SCC), IL-8, IL-1ß, and TNF-α concentrations were observed in the CON group compared to the IU group. This suggests that pretreatment with repeated intrauterine infusions of LPS suppresses the inflammatory responses to subsequent intramammary LPS challenges.

Potential effects of gingerol topical application on components of the innate immunity in lactating goat mammary glands.

In the mammary glands, production of antimicrobial components and formation of less-permeable tight junctions (TJs) are important for safe milk production. Previously, we reported that local heat treatment of udders using disposable heating pad enhances the components of innate immunity in lactating goat mammary glands. Gingerol is a polyphenol present in ginger that can induce heat-like effects. However, oral administration of polyphenols causes a decrease in biological activity through conjugation and metabolic conversion. Here, we investigated the effects of gingerol on antimicrobial components and TJs by topically applying it to lactating goat udders. Gingerol application increased the somatic cell count, cathelicidin-2 concentration, and proportion of polymorphonuclear cells in the milk and interleukin-8 production. Moreover, gingerol treatment enhanced ß-defensin-1 production in milk, cultured mammary epithelial cells, and cultured somatic cells. Contrastingly, gingerol treatment did not affect the concentrations of blood-derived components (Na+, albumin, and IgG) in the milk or the TJ barrier function of cultured mammary epithelial cells. These findings suggest that the topical application of gingerol, similar to local heat treatment, to udders enhances the components of innate immunity in mammary glands. These findings may be useful for the prevention of mastitis in milk-producing animals and, hence, safe and stable dairy production.

Effects of hydrostatic compression on milk production-related signaling pathways in mouse mammary epithelial cells.

Mammary epithelial cells (MECs) secrete milk into the mammary alveolar lumen during lactation. The secreted milk accumulates in the alveolar lumen until milk ejection occurs, and excess milk accumulation downregulates milk production in alveolar MECs. Intramammary hydrostatic pressure also increases in the alveolar lumen in a manner dependent on milk accumulation. In this study, we investigated whether high hydrostatic compression directly affects lactating MECs, using a commercial compression device and a lactation culture model of MECs, which have milk production ability and less permeable tight junctions. High hydrostatic compression at 100 kPa for 8 h decreased ß-casein and increased claudin-4 levels concurrently with inactivation of STAT5 and glucocorticoid receptor signaling pathways. In addition, high hydrostatic compression for 1 h inactivated STAT5 and activated p38 MAPK signaling. Furthermore, repeated rises and falls of the hourly hydrostatic compression induced activation of positive (Akt, mTOR) and negative (STAT3, NF-κB) signaling pathways for milk production concurrently with stimulation of casein and lactoferrin production in MECs. These results indicate that milk production-related signaling pathways in MECs change in response to hydrostatic compression. Hydrostatic compression of the alveolar lumen may directly regulate milk production in the alveolar MECs of lactating mammary glands.

Menthol application on healthy and inflamed goat udders changes antimicrobial components in milk.

Mammary glands with mastitis are usually treated with antibiotics in combination with anti-inflammatory drug application on the udder skin. Menthol is an anti-inflammatory drug. The aim of the present study was to investigate the effect of surface application of menthol on goat udders on the production of antimicrobial components in milk. Goats (5 Shiba and 11 Tokara goats) were subjected to menthol application to the udder under both healthy and inflammatory conditions. An intramammary infusion of lipopolysaccharides was carried out to induce inflammatory conditions in the udder. Milk samples were collected to determine somatic cell count (SCC) and sodium ion (Na+ ), antimicrobial component and cytokine concentrations. In healthy udders, menthol application increased the concentration of antimicrobial components (S100A7 and S100A8), but not in the control. In the inflamed udder, antimicrobial component (lactoferrin, S100A7, and S100A8) and inflammatory cytokine (IL-1ß) concentrations were higher in the menthol group than in the control group. These results suggest that menthol application on udders augments the antimicrobial component concentration in the mammary gland under both healthy and inflammatory conditions.

Effects of Newcastle Disease/Infectious Bronchitis Vaccine and Feeding Yeast Products on the Innate Immune System in the Proventriculus and Ileum of Broiler Chicks.

The aim of this study was to determine whether Newcastle disease/infectious bronchitis (ND/IB) vaccination and yeast product diet supplementation modulate the expression of innate immune molecules in the proventriculus and ileum of broiler chicks. One-day-old male broiler chicks were divided into four groups (V-Y- (control), V-Y+, V+Y-, and V+Y+ groups, where V and Y represent vaccination and yeast product supplementation, respectively). Chicks in the V+Y- and V+Y+ groups were immunized with the live ND/IB vaccine, whereas chicks in the V-Y- and V-Y+ groups were not. Chicks in the V-Y+ and V+Y+ groups received feed containing yeast products from day 4, whereas chicks in the V-Y- and V+Y- groups did not. The proventriculus and ileum were collected on day 7 to analyze the expression of seven Toll-like receptors (TLRs) and Dectin-1. In the proventriculus, compared with those of the V-Y- control group, the TLR7 and TLR21 expression levels were higher in the V+Y- group; however, there were no differences in the expression levels of any TLR or Dectin-1 in the ileum. There were also no differences in the expression of avian ß-defensins and cathelicidin-1 in the proventriculus and ileum between the control and treatment groups. The expression of granzyme in cytotoxic cells and interleukin (IL)-1B was upregulated by ND/IB vaccination in the proventriculus. Supplementation with yeast products upregulated only granzyme expression in the ileum and downregulated IL-6 expression in the proventriculus in chicks immunized with the ND/IB vaccine. Thus, we concluded that ND/IB vaccination is effective at enhancing the innate immune system in the proventriculus of chicks, at least until day 7 post-hatching, whereas the effects of diet supplementation with yeast products may be limited, at least under the present study conditions.

Valine Treatment Enhances Antimicrobial Component Production in Mammary Epithelial Cells and the Milk of Lactating Goats Without Influencing the Tight Junction Barrier.

The production of antimicrobial components and the formation of less-permeable tight junctions (TJs) are important in the defense system of lactating mammary glands and for safe dairy production. Valine is a branched-chain amino acid that is actively consumed in the mammary glands and promotes the production of major milk components like ß-casein; additionally, branched-chain amino acids stimulate antimicrobial component production in the intestines. Therefore, we hypothesized that valine strengthens the mammary gland defense system without influencing milk production. We investigated the effects of valine in vitro using cultured mammary epithelial cells (MECs) and in vivo using the mammary glands of lactating Tokara goats. Valine treatment at 4 mM increased the secretion of S100A7 and lactoferrin as well as the intracellular concentration of ß-defensin 1 and cathelicidin 7 in cultured MECs. In addition, an intravenous injection of valine increased S100A7 levels in the milk of Tokara goats without influencing milk yield and milk components (i.e., fat, protein, lactose, and solids). In contrast, valine treatment did not affect TJ barrier function either in vitro or in vivo. These findings indicate that valine enhances antimicrobial component production without influencing milk production and TJ barrier function in lactating mammary glands; thus, valine contributes to safe dairy production.

Effects of ozonized glycerin on inflammation of mammary glands induced by intramammary lipopolysaccharide infusion in goats.

Although ozone shows antimicrobial activity against mastitis-causing pathogens in ruminants, its anti-inflammatory effect on mammary glands remains unclear. The aim of this study was to examine the anti-inflammatory effects of ozonized glycerin (OG) on experimentally induced inflammation in the mammary glands of six Shiba and two Tokara lactating goats. We infused 1 µg lipopolysaccharide (LPS) into all udders on day -1. On day 0, post LPS infusion, OG (ozone group), and glycerin (control group) were infused into the right and left sides of the udders, respectively. Milk samples were collected once daily from days -1 to 7. The somatic cell count and lactoperoxidase (LPO) activity, along with the interleukin-1ß (IL-1ß), tumor necrosis factor-α, IL-8, IL-10, lactoferrin, and sodium ion concentrations in milk were measured. IL-8, IL-10, and lactoferrin levels after LPS infusion in the ozone group were significantly lower than those in the control group, and the LPO activity tended to be lower than that observed in the control group. This study showed that OG has anti-inflammatory potential against LPS-induced inflammation in the mammary glands.

Effect of 6-n-propyl-2-thiouracil or dexamethasone administration on the responses of antimicrobial components in goat milk to intramammary lipopolysaccharide infusion.

Heat stress impacts the immune system of dairy animals by altering the hypothalamic-pituitary-adrenal axis and thyroid function, leading to conditions such as hypothyroidism and hypercortisolism. This study aimed to elucidate the effect of hypothyroidism and hypercortisolism on the response of mammary innate immune function to inflammation caused by Escherichia coli in dairy goats. To induce hypothyroidism and hypercortisolism, we administered 6-n-propyl-2-thiouracil (PTU; for 21 days) and dexamethasone (DEX; for 5 days), respectively, to six goats each; six goats without treatment were used as the control group. After treatment, lipopolysaccharide (LPS) from E. coli O111 was infused into the mammary gland. Somatic cell counts (SCC) and levels of lactoferrin (LF), S100A7, immunoglobulin A (IgA), and interleukin-8 (IL-8) in milk until 7 days after LPS infusion were measured. An increase in SCC after LPS infusion was inhibited in both PTU and DEX groups, and an increase in LF after LPS infusion was inhibited in PTU group, compared with that in the control group. The results of the present study suggest that the recruitment of neutrophils and LF production decreased under hypothyroidism or hypercortisolism, which may be one of the causes underlying increased incidence of mastitis in dairy animals under heat stress conditions.

Differences in antimicrobial components between bacterial culture-positive and culture-negative bovine clinical mastitis milk.

A bacterial culture of milk is the most common test to determine the presence of mastitis-causing pathogens, which informs appropriate treatment. However, a certain proportion of clinical mastitis milk shows no growth of any mastitis-causing pathogens. We hypothesized that bacterial culture-negative clinical mastitis milk is associated with the activity of antimicrobial components contained in the milk. In this study, the differences in antimicrobial components (lactoferrin, transferrin, lysozyme, lactoperoxidase, and lingual antimicrobial peptide [LAP]) between bacterial culture-positive and culture-negative bovine clinical mastitis milk were investigated using Holstein cows. Our results showed that 37 out of 71 samples of clinical mastitis milk had negative bacterial cultures. The LAP concentration in bacterial culture-negative milk was lower than that in positive milk (31.95 ± 1.64 nM vs. 42.85 ± 4.01 nM). In contrast, the lysozyme concentration in bacterial culture-negative milk was higher than that in positive milk (0.76 ± 0.15 µg/ml vs. 0.42 ± 0.06 µg/ml). In conclusion, the concentration of antimicrobial components was different between bacterial culture-positive and culture-negative bovine clinical mastitis milk, which suggests that antimicrobial components are related to bacterial culture results.