Mastitis, inflammation of the udder, is one of the most common diseases hampering milk yield of dairy cows. Methionine (Met) and arginine (Arg) are key nutrients with potential to regulate inflammation and oxidative stress. The aim of this study was to evaluate the effect of increased supply of Met and Arg on mRNA and protein abundance associated with innate immune response and redox balance during lipopolysaccharide (LPS) stimulation in primary bovine mammary epithelial cells (BMEC). Primary BMEC (n = 4 replicates per treatment) were pre-incubated for 12 h in media with the following amino acid combinations: ideal profile of amino acids (control; Con), increased Met supply (incMet), increased Arg supply (incArg), and increased supply of Met and Arg (incMetArg). Subsequently, cells were challenged with or without LPS (1 µg/mL) and incubated for 6 h. Data were analyzed as a 2 × 2 × 2 factorial using the MIXED procedure of SAS 9.4 (SAS Institute Inc., Cary, NC). The downregulation of SLC36A1 and SLC7A1 mRNA abundance induced by LPS was attenuated in the incArg cultures. Although challenge with LPS led to lower abundance of proteins related to the antioxidant response (NFE2L2, NQO1, GPX1), lower levels of ATG7, and lower mRNA abundance of GPX3, we found little effect in cultures with incMet or incArg. Cultures with incMet, incArg, or incMetArg led to attenuation of the upregulation of SOD2 and NOS2 induced by LPS. Abundance of phosphorylated p65 (RELA) was greater after LPS stimulation, but the response was attenuated in cultures with incMet. The greater ratio of pRELA to total RELA in responses to LPS was also attenuated in cultures with incMetArg. The greater mRNA abundance of the proinflammatory cytokine IL1B induced by LPS was attenuated in cultures with incMet, and the same trend induced by LPS on CXCL2 was also alleviated in cultures with incArg. Overall, the data suggest that greater supply of Met and Arg alleviated the proinflammatory responses triggered by LPS through controlling the abundance of proinflammatory cytokines and chemokines and activity of NF-κB. Little benefit on oxidative stress induced by LPS challenge in BMEC was detected with greater supply of Met and Arg.

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Body condition alters glutathione and nuclear factor erythroid 2-like 2 (NFE2L2)-related antioxidant network abundance in subcutaneous adipose tissue of periparturient Holstein cows. By Liang et al. We explored periparturient s.c. adipose tissue (SAT) antioxidant mechanisms in cows with high and low body condition score (BCS) in late-prepartum. Although overall activation of the antioxidant transcription regulator NFE2L2 was lower and reactive oxygen species concentrations were greater in SAT from high BCS cows, the greater protein abundance of glutathione S-transferase mu 1 associated with glutathione (an antioxidant) metabolism in those cows underscored the importance of antioxidant mechanisms at the tissue level.

SummaryThe success rate in treatment against Staphylococcus aureus mastitis with antibacterial drugs is marginal. Low antibacterial drug concentrations in the mammary tissue is part of the reason for this failure. Enrofloxacin has only been administered on few occasions through the intramammary route i.e., combined with gentamicin in an undisclosed vehicle or dose. A new solvate form of enrofloxacin (enrofloxacin hydrochloride-dihydrate (enro-C)) prepared as 1.5% suspension pH 5.5 was tested in a field mastitis outbreak due to coagulase-negative S. aureus (CNS) in F1 (Holstein/Zebu) cows aged 5-8 years destined to be culled. Enro-C was administered intramammarily daily (300 mg/infected quarter) for 8 days both to healthy and mastitic cows; milk and serum enro-C concentrations were determined on days 1 and 8. Maximum serum concentration (C MAX ) values were similar in both groups (9.4 to 10.7 µg/mL). The area under the moment curve (AUMC) for enrofloxacin increased only by 11% in healthy cows and by 10% in infected cows from day 1 to day 8, a fact that suggests little accumulation with this dose regime. Peak concentrations in milk ranged from 18.5 to 19.8 µg/mL on day 1 and from 20.2 to 22 µg/mL on day 8. Cure rates on day 21 after treatment were 75% (12 out of 16 cows) or 69.4% (26 out of 36 glands). Somatic cell counts and California mastitis test showed a positive trend in cured animals. Uniquely high milk enrofloxacin and ciprofloxacin concentrations were obtained after intramammary administration of enro-C. These concentrations seem effective for treating CNS mastitis. The feasibility of incorporating this experimental pharmaceutical preparation of enrofloxacin is discussed.

Background We aimed to characterize the protective effects and the molecular mechanisms of action of a Saccharomyces cerevisiae fermentation product (NTK) in response to a mastitis challenge. Eighteen mid-lactation multiparous Holstein cows (n = 9/group) were fed the control diet (CON) or CON supplemented with 19 g/d NTK for 45 d (phase 1, P1) and then infected in the right rear quarter with 2500 CFU of Streptococcus uberis (phase 2, P2). After 36-h, mammary gland and liver biopsies were collected and antibiotic treatment started until the end of P2 (9 d post challenge). Cows were then followed until day 75 (phase 3, P3). Milk yield (MY) and dry matter intake (DMI) were recorded daily. Milk samples for somatic cell score were collected, and rectal and udder temperature, heart and respiration rate were recorded during the challenge period (P2) together with blood samples for metabolite and immune function analyses. Data were analyzed by phase using the PROC MIXED procedure in SAS. Biopsies were used for transcriptomic analysis via RNA-sequencing, followed by pathway analysis. Results DMI and MY were not affected by diet in P1, but an interaction with time was recorded in P2 indicating a better recovery from the challenge in NTK compared with CON. NTK reduced rectal temperature, somatic cell score, and temperature of the infected quarter during the challenge. Transcriptome data supported these findings, as NTK supplementation upregulated mammary genes related to immune cell antibacterial function (e.g., CATHL4, NOS2), epithelial tissue protection (e.g. IL17C), and anti-inflammatory activity (e.g., ATF3, BAG3, IER3, G-CSF, GRO1, ZFAND2A). Pathway analysis indicated upregulation of tumor necrosis factor α, heat shock protein response, and p21 related pathways in the response to mastitis in NTK cows. Other pathways for detoxification and cytoprotection functions along with the tight junction pathway were also upregulated in NTK-fed cows. Conclusions Overall, results highlighted molecular networks involved in the protective effect of NTK prophylactic supplementation on udder health during a subclinical mastitic event.

OBJECTIVE To determine the concentration of tilmicosin in mammary gland secretions of dairy cows following administration of an experimental preparation once or twice during the dry period (45-day period immediately prior to calving during which cows are not milked) and to evaluate its efficacy for the treatment of cows with intramammary infections (IMIs) caused by Staphylococcus aureus at dry off (cessation of milking; first day of dry period), compared with that of an intramammary infusion of ceftiofur. ANIMALS 172 cows. PROCEDURES Milk samples were collected for microbiological culture 5 days before dry off and at calving and 15 and 30 days after calving. Cows with Staphylococcus IMIs were randomly assigned to receive an experimental preparation of tilmicosin (20 mg/kg, SC) once at dry off (n = 58) or at dry off and again 20 days later (56) or receive a long-acting intramammary preparation of ceftiofur (500 mg/mammary gland; 56) at dry off. Mammary gland secretions were collected from 5 cows in the tilmicosin-treated groups every 5 days after dry off until calving for determination of tilmicosin concentration. RESULTS Mean maximum concentration of tilmicosin in mammary gland secretions ranged from 14.4 to 20.9 μg/mL after the first dose and was 17.1 μg/mL after the second dose. The bacteriologic cure rate was 100% for all 3 treatments. Tilmicosin was detectable for 0 and 18 days after calving in the milk of cows treated with 1 and 2 doses of tilmicosin, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Administration of an experimental preparation of tilmicosin (20 mg/kg, SC) once to dairy cows at dry off might be useful for the treatment of S aureus IMIs.

Choline chloride is used to provide choline in dog foods; however, in other domestic species, it has been replaced with a polyherbal containing phosphatidylcholine. A polyherbal containing Achyrantes aspera, Trachyspermum ammi, Citrullus colocynthis, Andrographis paniculata, and Azadirachta indica was evaluated in adult dogs through body weight changes, subcutaneous fat thickness, blood metabolites, and gene expression. Forty dogs (4.6 ± 1.6 years old) who were individually housed in concrete kennels were randomly assigned to the following treatments: unsupplemented diet (377 mg choline/kg), choline chloride (3850 mg/kg equivalent to 2000 mg choline/kg diet), and polyherbal (200, 400, and 800 mg/kg) for 60 days. Blood samples were collected on day 59 for biochemistry, biometry, and gene expression analysis through microarray assays. Intake, final body weight, and weight changes were similar for the two choline sources. Feed intake variation among dogs (p = 0.01) and dorsal fat (p = 0.03) showed a quadratic response to herbal choline. Dogs that received the polyherbal diet had reduced blood cholesterol levels (Quadratic, p = 0.02). The gene ontology analysis indicated that 15 biological processes were modified (p ≤ 0.05) with implications for preventing cardiovascular and metabolic diseases, cancer prevention, inflammatory and immune response, and behavior and cognitive process. According to these results that were observed in a 60 day trial, the polyherbal form could replace choline chloride in dog diets at a concentration of 400 mg/kg.

Peripartal cows mobilize not only body fat but also body protein to satisfy their energy requirements. The objective of this study was to determine the effect of prepartum BCS on blood biomarkers related to energy and nitrogen metabolism, and mRNA and protein abundance associated with AA metabolism and insulin signaling in subcutaneous adipose tissue (SAT) in peripartal cows. Twenty-two multiparous Holstein cows were retrospectively classified into a high BCS (HBCS; n = 11, BCS ≥ 3.5) or normal BCS (NBCS; n = 11, BCS ≤ 3.17) group at d 28 before expected parturition. Cows were fed the same diet as a total mixed ration before parturition and were fed the same lactation diet postpartum. Blood samples collected at −10, 7, 15, and 30 d relative to parturition were used for analyses of biomarkers associated with energy and nitrogen metabolism. Biopsies of SAT harvested at −15, 7, and 30 d relative to parturition were used for mRNA (real time-PCR) and protein abundance (Western blotting) assays. Data were subjected to ANOVA using the MIXED procedure of SAS (v. 9.4; SAS Institute Inc., Cary, NC), with P ≤ 0.05 being the threshold for significance. Cows in HBCS had greater overall plasma nonesterified fatty acid concentrations, due to marked increases at 7 and 15 d postpartum. This response was similar (BCS × Day effect) to protein abundance of phosphorylated (p) protein kinase B (p-AKT), the insulin-induced glucose transporter (SLC2A4), and the sodium-coupled neutral AA transporter (SLC38A1). Abundance of these proteins was lower at −15 d compared with NBCS cows, and either increased (SLC2A4, SLC38A1) or did not change (p-AKT) at 7 d postpartum in HBCS. Unlike protein abundance, however, overall mRNA abundances of the high-affinity cationic (SLC7A1), proton-coupled (SLC36A1), and sodium-coupled amino acid transporters (SLC38A2) were greater in HBCS than NBCS cows, due to upregulation in the postpartum phase. Those responses were similar to protein abundance of p-mTOR, which increased (BCS × Day effect) at 7 d in HBCS compared with NBCS cows. mRNA abundance of argininosuccinate lyase (ASL) and arginase 1 (ARG1) also was greater overall in HBCS cows. Together, these responses suggested impaired insulin signaling, coupled with greater postpartum AA transport rate and urea cycle activity in SAT of HBCS cows. An in vitro study using adipocyte and macrophage cocultures stimulated with various concentrations of fatty acids could provide some insights into the role of immune cells in modulating adipose tissue immunometabolic status, including insulin resistance and AA metabolism.