Treatment of bovine respiratory disease with a single administration of tulathromycin and ketoprofen.

BACKGROUND: The therapeutic strategy of bovine respiratory disease (BRD) often involves a combination of an antibiotic with an anti-inflammatory agent. Aim of this study was to evaluate the clinical effect of a new combination product containing tulathromycin and ketoprofen for the treatment of naturally occurring BRD. METHODS: Two hundred and eighty animals were randomized upon diagnosis of BRD. One hundred forty animals each were treated once subcutaneously with tulathromycin-ketoprofen or tulathromycin. Rectal temperature of each animal was measured at 1, 2, 4, 6, 8, 10, 12 and 24 h post-treatment. Individual respiration and depression scores were determined at 6 h post-treatment. Daily rectal temperature, respiration and depression scores were recorded from day 2 to 14 and on day 21. RESULTS: The tulathromycin-ketoprofen and tulathromycin treatment group demonstrated a treatment success rate of 94.2% and 95.0%, respectively and a relapse rate of 3.8% and 4.0%, respectively. Tulathromycin-ketoprofen demonstrated superior pyrexia control compared to tulathromycin within the first 24 h following treatment. Tulathromycin-ketoprofen-treated animals demonstrated faster improvement of their clinical symptoms (respiration and depression score). CONCLUSION: Efficacy of tulathromycin-ketoprofen for the treatment of BRD was non-inferior to tulathromycin. The combination product clearly exhibited more pronounced fever control than tulathromycin which is considered beneficial for animal welfare.

Co-formulation of ketoprofen with tulathromycin alters pharmacokinetic and pharmacodynamic profile of ketoprofen in cattle.

The current studies aimed to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) profile and to establish a PK-PD model for ketoprofen in a new fixed combination product containing tulathromycin (2.5 mg/kg) and ketoprofen (3 mg/kg) to treat bovine respiratory disease associated with pyrexia in cattle. Firstly, the effect of different ketoprofen doses as mono-substance (1, 3, and 6 mg/kg subcutaneous) on lipopolysaccharide-induced fever was evaluated which indicated that rectal temperature reduction lasted longer in the calves receiving 3 and 6 mg/kg ketoprofen. Secondly, the PK profile of the combination product was compared with mono-substance products (3 mg/kg subcutaneous and intramuscular). The PK profile of ketoprofen in the combination product was characterized by longer t1/2 , lower Cmax and increased AUC in comparison with mono-substance products. Due to prolonged ketoprofen exposure in the combination product, the pyrexia reducing effect of the combination product lasted longer in a second lipopolysaccharide challenge study in comparison with mono-substance products. Finally, a PK-PD model for the anti-pyretic effect of ketoprofen was developed based on the data from the different studies. The PK-PD model eliminated the need for additional animal experiments and indicated that a 3 mg/kg ketoprofen dose in the combination product provided optimal efficacy.

A Genome-Wide Association Study for Calving Interval in Holstein Dairy Cows Using Weighted Single-Step Genomic BLUP Approach.

The aim of the present study was to identify genomic region(s) associated with the length of the calving interval in primiparous (n = 6866) and multiparous (n = 5071) Holstein cows. The single nucleotide polymorphism (SNP) solutions were estimated using a weighted single-step genomic best linear unbiased prediction (WssGBLUP) approach and imputed high-density panel (777 k) genotypes. The effects of markers and the genomic estimated breeding values (GEBV) of the animals were obtained by five iterations of WssGBLUP. The results showed that the accuracies of GEBVs with WssGBLUP improved by +5.4 to +5.7, (primiparous cows) and +9.4 to +9.7 (multiparous cows) percent points over accuracies from the pedigree-based BLUP. The most accurate genomic evaluation was provided at the second iteration of WssGBLUP, which was used to identify associated genomic regions using a windows-based GWAS procedure. The proportion of additive genetic variance explained by windows of 50 consecutive SNPs (with an average of 165 Kb) was calculated and the region(s) that accounted for equal to or more than 0.20% of the total additive genetic variance were used to search for candidate genes. Three windows of 50 consecutive SNPs (BTA3, BTA6, and BTA7) were identified to be associated with the length of the calving interval in primi- and multiparous cows, while the window with the highest percentage of explained genetic variance was located on BTA3 position 49.42 to 49.52 Mb. There were five genes including ARHGAP29, SEC24D, METTL14, SLC36A2, and SLC36A3 inside the windows associated with the length of the calving interval. The biological process terms including alanine transport, L-alanine transport, proline transport, and glycine transport were identified as the most important terms enriched by the genes inside the identified windows.

Lipolysis modulates the biosynthesis of inflammatory lipid mediators derived from linoleic acid in adipose tissue of periparturient dairy cows.

Oxidized linoleic acid metabolites (OXLAM) are products of adipocyte lipolysis with the potential to modulate adipose tissue (AT) lipid metabolism and inflammation. In periparturient cows, linoleic acid is preferentially mobilized from AT during lipolysis by hormone-sensitive lipase (HSL) compared with other polyunsaturated fatty acids. Enzymatic and nonenzymatic reactions generate OXLAM from linoleic acid. Among OXLAM, 9-, 10-, and 12-hydroxy-octadecadienoic acids (HODE) are associated with pro-inflammatory responses, whereas 9- and 13-oxo-octadecadienoic acids (oxoODE) and 13-HODE can facilitate inflammation resolution and promote lipogenesis. This study evaluated the effect of HSL activity on OXLAM biosynthesis using subcutaneous AT explants collected from multiparous dairy cows at 10 d before and again at 10 and 24 d after calving. Explants were treated for 3 h without or with the ß-adrenergic agonist isoproterenol (ISO; 1 µM; MilliporeSigma, Burlington, MA) to induce HSL activity. The contribution of HSL to OXLAM biosynthesis was determined by inhibiting its activity with CAY10499 (2 µM; Cayman Chemical, Ann Arbor, MI). After treatments, media and explants were collected for lipidomic analysis using HPLC-tandem mass spectroscopy. Results indicated that ISO increased the biosynthesis of 9-, 12-, and 13-HODE and 9-oxoODE, and this effect was reduced at 24 d after calving. Inhibiting HSL activity partially reversed ISO effects on HODE and 9-oxoODE. Our ex vivo model demonstrated for the first time a direct effect of HSL activity on the biosynthesis of OXLAM in AT, especially at 10 d before and 10 d after calving. The biosynthesis of anti-inflammatory OXLAM is limited during the first weeks after parturition and may promote AT inflammation and lipolytic responses to negative energy balance. These results indicate that HSL activity releases linoleic acid for OXLAM biosynthesis in concentrations of a magnitude that may bypass the need for the activation of phospholipases linked with the inflammatory cascade and thus supports, in part, lipolysis-driven inflammation within AT of periparturient cows.

Genome-wide association for milk production and lactation curve parameters in Holstein dairy cows.

The aim of this study was to identify genomic regions associated with 305-day milk yield and lactation curve parameters on primiparous (n = 9,910) and multiparous (n = 11,158) Holstein cows. The SNP solutions were estimated using a weighted single-step genomic BLUP approach and imputed high-density panel (777k) genotypes. The proportion of genetic variance explained by windows of 50 consecutive SNP (with an average of 165 Kb) was calculated, and regions that accounted for more than 0.50% of the variance were used to search for candidate genes. Estimated heritabilities were 0.37, 0.34, 0.17, 0.12, 0.30 and 0.19, respectively, for 305-day milk yield, peak yield, peak time, ramp, scale and decay for primiparous cows. Genetic correlations of 305-day milk yield with peak yield, peak time, ramp, scale and decay in primiparous cows were 0.99, 0.63, 0.20, 0.97 and -0.52, respectively. The results identified three windows on BTA14 associated with 305-day milk yield and the parameters of lactation curve in primi- and multiparous cows. Previously proposed candidate genes for milk yield supported by this work include GRINA, CYHR1, FOXH1, TONSL, PPP1R16A, ARHGAP39, MAF1, OPLAH and MROH1, whereas newly identified candidate genes are MIR2308, ZNF7, ZNF34, SLURP1, MAFA and KIFC2 (BTA14). The protein lipidation biological process term, which plays a key role in controlling protein localization and function, was identified as the most important term enriched by the identified genes.

Impact of uterine macrophage phenotype on placental retention in dairy cows.

Reproductive diseases affect 25% of dairy cows in the US and often develop from retention of the placenta. It is well established that expulsion of the placenta is a highly regulated inflammatory process, but the mechanisms by which dysregulation of uterine immune responses impair this process are poorly understood. In healthy non-ruminants, pro-inflammatory M1 macrophages are predominant in uterine tissue after parturition. However, macrophage phenotype in the postpartum bovine uterus is unknown. Our study compared macrophage phenotypes in the uterine caruncles of multiparous dairy cows that during the first day postpartum either retained (RET, n = 5) or had normal expulsion (NOR, n = 5) of placenta. Immune cells were sorted magnetically from the caruncular endometrial cell fraction using the CD172a marker and monocyte/macrophage population was characterized using flow cytometry. Transcriptional and protein expression studies were performed on uterine caruncles. Compared to NOR, RET samples showed a lower CD14+/CD16+ expression (P < 0.05) in caruncle monocyte/macrophage population. As opposed to NOR, RET further demonstrated greater expression of anti-inflammatory M2 macrophage associated genes CD206, C-type lectin domain family 7 member A (CLEC7A), and RNASE6. In addition, caruncles from RET showed decreased signal transducer and activator of transcription 3 (STAT3) activation, an important promoter of proteolytic activity, compared to NOR. Our studies demonstrate that there is an overall lower number of macrophage populations in the caruncle of cows with RET placenta and these are polarized towards M2 phenotype. Excessive accumulation of M2 macrophages may lead to reduced trafficking of immune cells into the caruncle thus impairing the inflammatory, phagocytic and proteolytic processes that lead to placental expulsion.

Isolation and Characterization of Functionally Active Extracellular Vesicles from Culture Medium Conditioned by Bovine Embryos In Vitro.

Extracellular vesicles (EVs) play a possible role in cell⁻cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrepTM density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25⁻230 nm with an average concentration of 236.5 ± 1.27 × 108 particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group.

The contribution of hormone sensitive lipase to adipose tissue lipolysis and its regulation by insulin in periparturient dairy cows.

Hormone sensitive lipase (HSL) activation is part of the metabolic adaptations to the negative energy balance common to the mammalian periparturient period. This study determined HSL contribution to adipose tissue (AT) lipolysis and how insulin regulates its activity in periparturient dairy cows. Subcutaneous AT (SCAT) samples were collected at 11 d prepartum (dry) and 11 (fresh) and 24 d (lactation) postpartum. Basal and stimulated lipolysis (ISO) responses were determined using explant cultures. HSL contribution to lipolysis was assessed using an HSL inhibitor (CAY). Basal lipolysis was higher in SCAT at dry compared with fresh. CAY inhibited basal lipolysis negligibly at dry, but at fresh and lactation it reduced basal lipolysis by 36.1 ± 4.51% and 43.1 ± 4.83%, respectively. Insulin inhibited lipolysis more pronouncedly in dry compared to fresh. Results demonstrate that HSL contribution to basal lipolysis is negligible prepartum. However, HSL is a major driver of SCAT lipolytic responses postpartum. Lower basal lipolysis postpartum suggests that reduced lipogenesis is an important contributor to fatty acid release from SCAT. Loss of adipocyte sensitivity to the antilipolytic action of insulin develops in the early lactation period and supports a state of insulin resistance in AT of cows during the first month postpartum.

Short communication: Effects of body fat mobilization on macrophage infiltration in adipose tissue of early lactation dairy cows.

Intense lipolysis triggers an inflammatory response within adipose tissue characterized by adipose tissue macrophage (ATM) infiltration; however, the mechanisms triggering this process are poorly characterized in transition dairy cows. The aim of this study was to determine the association between ATM infiltration and body fat mobilization in the transition period, markers of excessive lipolysis, and adipose tissue expression of genes related to chemotactic and inflammatory responses. Subcutaneous adipose tissue samples were taken from the tailhead of 9 multiparous Holstein cows, 27 ± 2.2 d (far-off) and 10 ± 1.5 d (close-up) before and 9 ± 0.3 d after calving (fresh). Blood samples were collected by coccygeal venipuncture 2 h before adipose sample collections. Body condition score (BCS) was assessed independently by 3 experienced technicians at every time point. Based on BCS loss intensity between the close-up and fresh period, cows were divided into 2 groups: low BCS loss (LBCSL, change in BCS <0.25 units, n = 5) and high BCS loss (HBCSL, change in BCS >0.25 units, n = 4). Although none of the LBCSL cows had a health event, all cows in the HBCSL group suffered from one or more clinical disorder (retained placenta, milk fever, or ketosis) in the transition period. The number of ATM was determined by immunohistochemistry, and expression of selected chemotactic and inflammatory genes was determined by reverse-transcription quantitative real-time PCR in subcutaneous adipose tissue samples. The proportion of ATM in subcutaneous adipose tissue increased in HBCSL during the postpartum period. The proportion of ATM was not associated with serum ß-hydroxybutyrate or free fatty acid concentrations on the day of adipose tissue collection. The ATM infiltration in the fresh period was associated with local expression of the chemotactic genes, C-C motif chemokine ligand 22 (CCL22), osteopontin (SPP1), and the receptor for SPP1, cluster of differentiation 44 (CD44). This supports a potential chemotactic role of CCL22 and SPP1 for ATM in bovine adipose tissue. None of the genes encoding pro- or anti-inflammatory mediators, tumor necrosis factor (TNF), IL6, and IL10 were associated with the proportion of ATM. Our results indicate that ATM infiltration of subcutaneous adipose tissue is associated with body fat mobilization in early-lactation dairy cows and supports a role for ATM in the adaptation of adipose tissues to the metabolic challenges of the transition period.

Symposium review: Modulating adipose tissue lipolysis and remodeling to improve immune function during the transition period and early lactation of dairy cows.

Despite major advances in our understanding of transition and early lactation cow physiology and the use of advanced dietary, medical, and management tools, at least half of early lactation cows are reported to develop disease and over half of cow deaths occur during the first week of lactation. Excessive lipolysis, usually measured as plasma concentrations of free fatty acids (FFA), is a major risk factor for the development of displaced abomasum, ketosis, fatty liver, and metritis, and may also lead to poor lactation performance. Lipolysis triggers adipose tissue (AT) remodeling that is characterized by enhanced humoral and cell-mediated inflammatory responses and changes in its distribution of cellular populations and extracellular matrix composition. Uncontrolled AT inflammation could perpetuate lipolysis, as we have observed in cows with displaced abomasum, especially in those animals with genetic predisposition for excessive lipolysis responses. Efficient transition cow management ensures a moderate rate of lipolysis that is rapidly reduced as lactation progresses. Limiting FFA release from AT benefits immune function as several FFA are known to promote dysregulation of inflammation. Adequate formulation of pre- and postpartum diet reduces the intensity of AT lipolysis. Additionally, supplementation with niacin, monensin, and rumen-protected methyl donors (choline and methionine) during the transition period is reported to minimize FFA release into systemic circulation. Targeted supplementation of energy sources during early lactation improves energy balance and increases insulin concentration, which limits AT lipolytic responses. This review elaborates on the mechanisms by which uncontrolled lipolysis triggers inflammatory disorders. Details on current nutritional and pharmacological interventions that aid the modulation of FFA release from AT and their effect on immune function are provided. Understanding the inherent characteristics of AT biology in transition and early lactation cows will reduce disease incidence and improve lactation performance.

Insulin resistance in dairy cows.

Glucose is the molecule that drives milk production, and insulin plays a pivotal role in the glucose metabolism of dairy cows. The effect of insulin on the glucose metabolism is regulated by the secretion of insulin by the pancreas and the insulin sensitivity of the skeletal muscles, the adipose tissue, and the liver. Insulin resistance may develop as part of physiologic (pregnancy and lactation) and pathologic processes, which may manifest as decreased insulin sensitivity or decreased insulin responsiveness. A good knowledge of the normal physiology of insulin is needed to measure the in vivo insulin resistance of dairy cows.