Baselining physiological parameters in three muscles across three equine breeds. What can we learn from the horse?

Mapping-out baseline physiological muscle parameters with their metabolic blueprint across multiple archetype equine breeds, will contribute to better understanding their functionality, even across species. Aims: 1) to map out and compare the baseline fiber type composition, fiber type and mean fiber cross-sectional area (fCSA, mfCSA) and metabolic blueprint of three muscles in 3 different breeds 2) to study possible associations between differences in histomorphological parameters and baseline metabolism. Methods: Muscle biopsies [m. pectoralis (PM), m. vastus lateralis (VL) and m. semitendinosus (ST)] were harvested of 7 untrained Friesians, 12 Standardbred and 4 Warmblood mares. Untargeted metabolomics was performed on the VL and PM of Friesian and Warmblood horses and the VL of Standardbreds using UHPLC/MS/MS and GC/MS. Breed effect on fiber type percentage and fCSA and mfCSA was tested with Kruskal-Wallis. Breeds were compared with Wilcoxon rank-sum test, with Bonferroni correction. Spearman correlation explored the association between the metabolic blueprint and morphometric parameters. Results: The ST was least and the VL most discriminative across breeds. In Standardbreds, a significantly higher proportion of type IIA fibers was represented in PM and VL. Friesians showed a significantly higher representation of type IIX fibers in the PM. No significant differences in fCSA were present across breeds. A significantly larger mfCSA was seen in the VL of Standardbreds. Lipid and nucleotide super pathways were significantly more upregulated in Friesians, with increased activity of short and medium-chain acylcarnitines together with increased abundance of long chain and polyunsaturated fatty acids. Standardbreds showed highly active xenobiotic pathways and high activity of long and very long chain acylcarnitines. Amino acid metabolism was similar across breeds, with branched and aromatic amino acid sub-pathways being highly active in Friesians. Carbohydrate, amino acid and nucleotide super pathways and carnitine metabolism showed higher activity in Warmbloods compared to Standardbreds. Conclusion: Results show important metabolic differences between equine breeds for lipid, amino acid, nucleotide and carbohydrate metabolism and in that order. Mapping the metabolic profile together with morphometric parameters provides trainers, owners and researchers with crucial information to develop future strategies with respect to customized training and dietary regimens to reach full potential in optimal welfare.

Dynamics of training and acute exercise-induced shifts in muscular glucose transporter (GLUT) 4, 8, and 12 expression in locomotion versus posture muscles in healthy horses.

Important changes in glucose transporter (GLUT) expression should be expected if the glucose influx plays a pivotal role in fuelling or connecting metabolic pathways that are upregulated in response to exercise. The aim was to assess GLUT4, 8, and 12 dynamics in response to training and acute exercise. Methods: Sixteen untrained Standardbred mares (3-4 year) performed an incremental SET at the start and end of 8 weeks harness training. M. pectoralis (PM) and M. vastus lateralis (VL) muscle biopsies were taken before and after each SET, allowing for comparing rest and acute samples in untrained (UT) and trained (T) condition using Western Blot for GLUT quantification and Image Pro v.10 for Blot analysis. Data were normalized against GAPDH. Basal GLUT-levels of PM versus VL were analysed with the Wilcoxon matched-pairs signed rank test. The effect of acute exercise or training was assessed using the Friedman test with a post hoc Dunn's. Results: Basal GLUT4 and GLUT12 protein expression were significantly higher in the VL compared to the PM (PGLUT4 = 0.031 and PGLUT12 = 0.002). Training had no effect on basal GLUT4 expression, neither in the VL (p > 0.9999), nor the PM (p > 0.9999). However, acute exercise in trained condition significantly decreased GLUT4 expression in the VL (p = 0.0148). Neither training nor acute exercise significantly changed total GLUT8 protein expression. Training significantly decreased total GLUT12 protein expression in rest biopsies, only visible in the VL (p = 0.0359). This decrease was even more prominent in the VL after acute exercise in trained condition (PVL = 0.0025). Conclusion: The important changes seen in GLUT12 expression downregulation, both in response to training and acute exercise in the horse, the downregulation of GLUT4 expression after acute exercise in trained condition and the lack of differential shifts in GLUT8 expression in any of the studied conditions, questions the importance of glucose as substrate to fuel training and exercise in healthy horses. These findings encourage to further explore alternative fuels for their involvement in equine muscular energetics.

Comparison of Shifts in Skeletal Muscle Plasticity Parameters in Horses in Three Different Muscles, in Answer to 8 Weeks of Harness Training.

Training-induced follow-up of multiple muscle plasticity parameters in postural stability vs. locomotion muscles provides an integrative physiological view on shifts in the muscular metabolic machinery. It can be expected that not all muscle plasticity parameters show the same expression time profile across muscles. This knowledge is important to underpin results of metabolomic studies. Twelve non-competing Standardbred mares were subjected to standardized harness training. Muscle biopsies were taken on a non-training day before and after 8 weeks. Shifts in muscle fiber type composition and muscle fiber cross-sectional area (CSA) were compared in the m. pectoralis, the m. vastus lateralis, and the m. semitendinosus. In the m. vastus lateralis, which showed most pronounced training-induced plasticity, two additional muscle plasticity parameters (capillarization and mitochondrial density) were assessed. In the m. semitendinosus, additionally the mean minimum Feret's diameter was assessed. There was a significant difference in baseline profiles. The m. semitendinosus contained less type I and more type IIX fibers compatible with the most pronounced anaerobic profile. Though no baseline fiber type-specific and overall mean CSA differences could be detected, there was a clear post-training decrease in fiber type specific CSA, most pronounced for the m. vastus lateralis, and this was accompanied by a clear increase in capillary supply. No shifts in mitochondrial density were detected. The m. semitendinosus showed a decrease in fiber type specific CSA of type IIAX fibers and a decrease of type I fiber Feret's diameter as well as mean minimum Feret's diameter. The training-induced increased capillary supply in conjunction with a significant decrease in muscle fiber CSA suggests that the muscular machinery models itself toward an optimal smaller individual muscle fiber structure to receive and process fuels that can be swiftly delivered by the circulatory system. These results are interesting in view of the recently identified important fuel candidates such as branched-chain amino acids, aromatic amino acids, and gut microbiome-related xenobiotics, which need a rapid gut-muscle gateway to reach these fibers and are less challenging for the mitochondrial system. More research is needed with that respect. Results also show important differences between muscle groups with respect to baseline and training-specific modulation.

Flexibility of equine bioenergetics and muscle plasticity in response to different types of training: An integrative approach, questioning existing paradigms.

Equine bioenergetics have predominantly been studied focusing on glycogen and fatty acids. Combining omics with conventional techniques allows for an integrative approach to broadly explore and identify important biomolecules. Friesian horses were aquatrained (n = 5) or dry treadmill trained (n = 7) (8 weeks) and monitored for: evolution of muscle diameter in response to aquatraining and dry treadmill training, fiber type composition and fiber cross-sectional area of the M. pectoralis, M. vastus lateralis and M. semitendinosus and untargeted metabolomics of the M. pectoralis and M. vastus lateralis in response to dry treadmill training. Aquatraining was superior to dry treadmill training to increase muscle diameter in the hindquarters, with maximum effect after 4 weeks. After dry treadmill training, the M. pectoralis showed increased muscle diameter, more type I fibers, decreased fiber mean cross sectional area, and an upregulated oxidative metabolic profile: increased ß-oxidation (key metabolites: decreased long chain fatty acids and increased long chain acylcarnitines), TCA activity (intermediates including succinyl-carnitine and 2-methylcitrate), amino acid metabolism (glutamine, aromatic amino acids, serine, urea cycle metabolites such as proline, arginine and ornithine) and xenobiotic metabolism (especially p-cresol glucuronide). The M. vastus lateralis expanded its fast twitch profile, with decreased muscle diameter, type I fibers and an upregulation of glycolytic and pentose phosphate pathway activity, and increased branched-chain and aromatic amino acid metabolism (cis-urocanate, carnosine, homocarnosine, tyrosine, tryptophan, p-cresol-glucuronide, serine, methionine, cysteine, proline and ornithine). Trained Friesians showed increased collagen and elastin turn-over. Results show that branched-chain amino acids, aromatic amino acids and microbiome-derived xenobiotics need further study in horses. They feed the TCA cycle at steps further downstream from acetyl CoA and most likely, they are oxidized in type IIA fibers, the predominant fiber type of the horse. These study results underline the importance of reviewing existing paradigms on equine bioenergetics.

Agreement of Quantitative and Qualitative Antimicrobial Susceptibility Testing Methodologies: The Case of Enrofloxacin and Avian Pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is the causal agent of colibacillosis, one of the most common bacterial infections in the poultry sector. Antimicrobial susceptibility testing (AST) is essential for rational and prudent antimicrobial therapy. Subsequently, uniformity in test results from the various testing methodologies used in diagnostic laboratories is pivotal. The aim of this study was therefore to evaluate the agreement between different AST methods in determining fluoroquinolone resistance in APEC. Twenty APEC isolates were selected and subjected to four different susceptibility tests: the quantitative microbroth dilution, agar dilution and gradient strip tests, and the qualitative disk diffusion method. The experiments were performed in triplicate. Categorical agreement, essential agreement and different errors were assessed. Moreover, agreement was also evaluated by calculating intraclass correlation coefficients (ICCs) for the quantitative tests and determining the Pearson correlation coefficients for the agreement between the disk diffusion method and the quantitative tests. Categorical agreement and essential agreement when compared with the microbroth technique ranged from 85-95% and 85-100%, respectively. No very major errors (false susceptible) and only one major error (false resistant) and minor errors (results involving an intermediary category) were detected. The calculated ICC values of the three quantitative tests fluctuated around 0.970 (range 0.940-0.988). There was a high negative correlation between the disk diffusion method and the other tests (correlation coefficients ranging from -0.979 to -0.940), indicating a clear inverse relationship between the minimum inhibitory concentration value and the zone diameter of growth inhibition. In conclusion, the overall agreement between the four different testing methodologies was very high. These results confirm the reliability of the disk diffusion and gradient strip test methods as substantiated alternatives, next to the gold standard agar and microbroth dilution, for fluoroquinolone susceptibility testing of APEC isolates.

Unraveling the Contribution of Fluid Therapy to the Development of Augmented Renal Clearance in a Piglet Model.

Augmented renal clearance (ARC) observed in the critically ill pediatric population has received an increased attention over the last years due to its major impact on the disposition and pharmacokinetics of mainly renally excreted drugs. Apart from an important inflammatory trigger, fluid administration has been suggested to contribute to the development of ARC. Therefore, the primary objective of this study was to evaluate the effect of continuous intravenous fluid administration on renal function using a conventional piglet animal model and to quantify the impact of fluid administration on the pharmacokinetics of renally excreted drugs. At baseline, twenty-four piglets (12 treatment/12 control; 7 weeks old, all ♂) received the marker drugs iohexol (64.7 mg/kg body weight (BW)) and para-aminohippuric acid (10 mg/kg BW) to quantify glomerular filtration rate and effective renal plasma flow, respectively. In addition, the hydrophilic antibiotic amikacin (7.5 mg/kg BW) was administered. Following this baseline measurement, the treatment group received fluid therapy as a constant rate infusion of 0.9% saline at 6 mL/kg/h over 36 h. After 24 h of fluid administration, the marker drugs and amikacin were administered again. When comparing both groups, a significant effect of fluid administration on the total body clearances of iohexol (p = 0.032) and amikacin (p = 0.0014) was observed. Clearances of iohexol and amikacin increased with on average 15 and 14%, although large interindividual variability was observed. This led to decreased systemic exposure to amikacin, which was manifested as decrease in area under the plasma concentration-time curve from time 0 h to infinity from 34,807 to 30,804 ng.h/mL. These results suggest that fluid therapy is a key factor involved in the development of ARC and should be taken into account when administering mainly renally excreted drugs. However, further research is necessary to confirm these results in children.

Transverse Maxillary Development in Complete Cleft Patients: Comparison of 2 Treatment Strategies by Longitudinal and Cross-Sectional Model Analysis.

OBJECTIVE: This study aimed to compare differences in the maxillary transverse dimension following cleft repair using 2 protocols involving a 2-staged palatal closure with similar timing but different techniques. DESIGN: Retrospective study. Two matched study samples, each containing 10 patients. SETTING: Cleft center A is a university hospital. Cleft Center B is a general teaching hospital. PATIENTS: Patients with cleft lip alveolus and palate (CLAP) were matched by cleft type, gender, age, and palatal morphology at the age of 1 month. Differences in the transverse dimension were measured at 4 ages in 2 matched groups (A and B from center A and B), each including 5 patients with unilateral CLAP (UCLAP) and 5 patients with bilateral CLAP (BCLAP). INTERVENTIONS: Orthodontic/orthopedic expansion treatment began at 8 and 5 years of age at centers A and B, respectively. RESULTS: The mean intercanine distance decreased by 8 mm (standard deviation [SD] = 1.6; P = .030) in group A patients with UCLAP between 1 and 5 years (SD = 1.6; P = .030) and by 6.3 mm (SD = 3.2; P = .016) in group B patients with BCLAP. The mean intermolar distance increased by 4 mm (SD = 3.4; P = .076) in patients with BCLAP. A trend toward improved canine positioning was observed in patients with BCLAP by 12 years of age, with improvement occurring between 4 and 12 years. CONCLUSION: The type of palatal closure (inducing scar tissue at the bony level) and the late installment of orthodontic/orthopedic expansion probably reduced the intercanine width in bilateral complete clefts.

Impact of transportation duration on stress responses in day-old chicks from young and old breeders.

The aim of this study was to assess the interaction effect of transportation duration and parental age on physiological stress responses and quality in day-old chicks. After hatch, 3240 chicks from either 29-week old (young) or 60-week old (old) broiler breeders were transported for 1.5h (short) or 11h (long). Thereafter, 228 chicks were assessed for quality and blood plasma was assayed for corticosterone (CORT), lactate, glucose, and thiobarbituric acid-reactive substances (TBARS, increased concentrations can indicate oxidative stress). No interaction effects of parental age and transportation duration were found (all P>0.10). Chicks from young breeders showed higher CORT levels (P=0.007) and were of higher quality (Tona method, P<0.001) than those from old breeders. After long transportation, chicks showed increased CORT (P<0.001) and lower TBARS levels (P<0.001) compared to after short transportation. No evidence was provided that long transportation differently affected the quality or stress responses of chicks from breeder flocks of two ages.

Broiler chickens dead on arrival: associated risk factors and welfare indicators.

The pre-slaughter phase of the production process has potentially important animal welfare implications, including mortality (recorded as "Dead on Arrivals"; DOA: ). To reduce DOA%, specific risk factors need to be identified. DOA% can also be used as a first and quick screening of pre-slaughter broiler welfare under commercial conditions. The aim of the present study was to identify risk factors for DOA% in commercial broiler flocks and to find associations between DOA% and animal-based indicators of broiler welfare during the pre-slaughter phase. Eighty-one transported flocks to five slaughter plants were assessed. Farmers provided information on the flock and observers gathered data on pre-slaughter factors. DOA% was recorded by slaughter plant personnel. The association between risk factors and DOA% was tested using linear mixed models, with slaughter plant as the random effect. Mean (± SE) DOA% was 0.30 ± 0.05% and median was 0.19% (range 0.04 to 3.34%). Two risk factors for DOA% were identified, both related to flock health status during the production phase. Higher DOA% was found when farmers did not check chick quality upon arrival (P = 0.011), although one extreme DOA% caused this significant effect. In addition, on-farm mortality during production (including selective culling) was negatively associated with DOA% (P = 0.011), potentially due to the selective culling on farm. Further research is needed on the impact of on-farm health status on welfare during the pre-slaughter phase, as the current study provided some evidence that on-farm factors are linked to mortality during the pre-slaughter phase. No specific characteristics of the pre-slaughter phase could be identified as risk factor for increased mortality. DOA% was positively associated with five welfare indicators, including indicators of thermal stress, which indicates the potential to use DOA% as a quick, cost-efficient screening tool for those welfare aspects.

Impact of the separate pre-slaughter stages on broiler chicken welfare.

Before broilers are slaughtered, several parties are involved in catching, loading, transportation and lairage. During these pre-slaughter activities, broilers are exposed to a variety of stressors. A detailed understanding of how broiler welfare is impaired during each stage could help the responsible parties to make targeted improvements. The aim of this study was to identify welfare problems occurring during the consecutive stages of commercial broiler transportation and to identify risk factors associated with the identified welfare problems. Commercial Belgian transports (n = 81) were assessed in spring (n = 14), summer (n = 33), autumn (n = 10), and winter (n = 24), and potential risk factors were recorded by the observer. Animal-based welfare indicators were scored before the start of the pre-slaughter phase as well as after the catching, transport and lairage, and slaughter stages to assess the impact of each stage. The most frequently observed welfare impairments were vent and thigh lesions, panting, wing fractures, and bruising on wings and breasts. Our results show that the impact of the pre-slaughter phase on broiler welfare is multifaceted. The overall pre-slaughter phase resulted in a mean weight decrease of 5.3%, a prevalence of 1.4% in leg bruising, and 3.7% in breast or wing bruising. Wing fractures occurred mainly during the catching stage: Prevalence increased from 0.1% to 1.9% (P = 0.003). A welfare comparison before and after transportation and lairage revealed that plumage had become more soiled (P = 0.003), body temperature decreased by 0.7°C (P < 0.001), huddling prevalence increased by 0.5% (P = 0.008), prevalence of birds with splayed legs increased by 0.08% (P = 0.008), prevalence of supine birds decreased by 0.05% (P = 0.003), and 0.1% fewer birds with wings stuck in the crates (P = 0.010) were observed. Risk factor analyses revealed that carefully choosing the catching crew, minimizing thermal stress, reducing duration of transportation, and worker training are promising actions that may improve broiler welfare during the pre-slaughter phase.

Effect of post-hatch transportation duration and parental age on broiler chicken quality, welfare, and productivity.

Broiler chicks are transported to production sites within one to 2 d post-hatch. Possible effects of this transportation are poorly understood and could vary among chicks from breeder flocks of different ages. The aim of the present study was to investigate the effects of transportation duration and parental flock age on chick welfare, productivity, and quality. After hatch in a commercial hatchery, 1,620 mixed-sex chicks from 29-wk old (young) and 1,620 chicks from 60-wk old (old) breeders were subjected to transportation of 1.5 h or 11 h duration. After transportation, 2,800 chicks were divided among 100 pens, with each pen containing 28 chicks from one transportation crate (2 or 3 pens per crate). From the remaining chicks, on average 6 chicks (min 4, max 8) per crate (n = 228) were randomly selected and assessed for chick quality, weighed, and culled for yolk sac weighing (one d). Chicks that had not been assigned to pens or were not used for post-transportation measurements, were removed from the experiment (n = 212). Mortality, ADG, BW, and feed conversion ( FC: ) of the experimental chicks were recorded until 41 d. Meat quality was measured for breast fillets (n = 47). No interaction effect of parental age and transportation duration was found for any variables. BW and yolk sac weight at one d were lower for chicks transported 11 h than 1.5 h and for chicks from young versus old breeders. The effect of parental flock age on BW persisted until slaughter. Additionally, parental age positively affected ADG until slaughter. Chick quality was lower in chicks from old versus young breeders. Chick quality and productivity were not affected by transportation duration. Mortality and meat quality were not affected by either parental age or transportation duration. To conclude, no long-term detrimental effects were found from long post-hatch transportation in chicks from young or old parent flocks. Based on these results, we suggest that 11 h post-hatch transportations under similar conditions do not impose long-term welfare or productivity risks.

In vitro selenium accessibility in pet foods is affected by diet composition and type.

Se bioavailability in commercial pet foods has been shown to be highly variable. The aim of the present study was to identify dietary factors associated with in vitro accessibility of Se (Se Aiv) in pet foods. Se Aiv is defined as the percentage of Se from the diet that is potentially available for absorption after in vitro digestion. Sixty-two diets (dog, n 52; cat, n 10) were in vitro enzymatically digested: fifty-four of them were commercially available (kibble, n 20; pellet, n 8; canned, n 17; raw meat, n 6; steamed meat, n 3) and eight were unprocessed (kibble, n 4; canned, n 4) from the same batch as the corresponding processed diets. The present investigation examined if Se Aiv was affected by diet type, dietary protein, methionine, cysteine, lysine and Se content, DM, organic matter and crude protein (CP) digestibility. Se Aiv differed significantly among diet types (P< 0·001). Canned and steamed meat diets had a lower Se Aiv than pelleted and raw meat diets. Se Aiv correlated positively with CP digestibility in extruded diets (kibbles, n 19; r 0·540, P =0·017) and negatively in canned diets (n 16; r - 0·611, P =0·012). Moreover, the canning process (n 4) decreased Se Aiv (P =0·001), whereas extrusion (n 4) revealed no effect on Se Aiv (P =0·297). These differences in Se Aiv between diet types warrant quantification of diet type effects on in vivo Se bioavailability.

Prepubertal gonadectomy in cats: different injectable anaesthetic combinations and comparison with gonadectomy at traditional age.

Anaesthetic and analgesic effects of three different injectable anaesthetic combinations for prepubertal gonadectomy (PPG) in cats were studied. One anaesthetic protocol was compared with a similar one for gonadectomy at traditional age (TAG). Kittens were randomly assigned to PPG or TAG. For PPG, three different protocols were compared: (1) intramuscular (IM) administration of 60 µg/kg dexmedetomidine plus 20 µg/kg buprenorphine followed by an IM injection of the anaesthetic agent (20 mg/kg ketamine) (DB-IM protocol); (2) oral transmucosal (OTM) administration of 80 µg/kg dexmedetomidine plus 20 µg/kg buprenorphine followed by an IM injection of 20 mg/kg ketamine combined with 20 µg/kg dexmedetomidine (DB-OTM protocol); (3) IM injection of a 40 µg/kg medetomidine-20 µg/kg buprenorphine-20 mg/kg ketamine combination (MBK-IM protocol). For TAG, a DB-IM protocol was used, but with different doses for dexmedetomidine (40 µg/kg) and ketamine (5 mg/kg). All cats (PPG and TAG) received a non-steroidal anti-inflammatory before surgery. Anaesthetic and analgesic effects were assessed pre- and postoperatively (until 6 h). Cumulative logit, linear and logistic regression models were used for statistical analysis. Compared with the DB-OTM protocol, the DB-IM and MBK-IM protocols provided better anaesthesia with fewer adverse effects in PPG cats. Postoperative pain was not significantly different between anaesthetic protocols. PPG and TAG cats anaesthetised with the two DB-IM protocols differed significantly only for sedation and pain scores, but sedation and pain scores were generally low. Although there were no anaesthesia-related mortalities in the present study and all anaesthetic protocols for PPG in cats provided a surgical plane of anaesthesia and analgesia up to 6 h postoperatively, our findings were in favour of the intramuscular (DB-IM and MBK-IM) protocols.

Investigating clustering in interval-censored udder quarter infection times in dairy cows using a gamma frailty model.

Udder infections in dairy cows are observed at udder quarter level. Therefore, the best strategy to study infection dynamics of particular bacteria causing mastitis is to follow up and model individual udder quarter infection times. Udder quarter infection times, however, are not independent as they are clustered within a cow and herds. Another challenge in modelling infection times is that the exact infection time is unknown; it is only known that the infection has taken place in the interval between the last negative and the first positive sample. We applied a technique based on the gamma frailty model which handles the clustering and interval censoring simultaneously. Parameter estimates can be obtained analytically and their variance is obtained by the inverse of the hessian matrix. The proposed technique was applied to udder quarter infection times for Corynebacterium bovis, Staphylococcus aureus and Streptococcus uberis. Multiparous cows were more likely to get infected earlier in lactation with C. bovis or S. uberis than primiparous cows. The times to infection of all three bacteria were highly clustered at cow level and the results of a stratified model on a subset of herds suggested a high clustering on herd level for C. bovis and S. uberis.