Postbiotic supplements have been used in various livestock species to mitigate the negative effects of stress. This study aimed to determine if similar responses are seen in canine companion animals. We evaluated the effects of a Saccharomyces cerevisiae (S. cerevisiae) postbiotic supplement on inflammatory biomarkers, cortisol, and microbiota dysbiosis index in Labrador Retrievers exposed to exercise and transport stress. Dogs [n = 36; 3.52 ± 0.21 yr old and body weight (BW) = 27.6 ± 2.97 kg] were blocked by sex, age, and BW and assigned to receive 1 of 3 daily top-dressed treatments: 1) 15 g ground corn germ (Control, n = 12); 2) 7.5 g S. cerevisiae yeast postbiotic (Low, n = 12); or 3) 15 g of S. cerevisiae yeast postbiotic (High, n = 12). Dogs were given 1 wk to acclimate to treatments before beginning a 7-wk exercise regimen consisting of twice weekly 4.8 km guided runs. Dogs were exposed to a transportation stressor in wk 8. Fecal samples from d 0 and 56 were analyzed for calprotectin, calgranulin C (S100A12), α1-protease inhibitor, immunoglobulin A, and microbiota dysbiosis index. Blood was collected on d 0 and 63 for serum chemistries and complete blood counts. Blood collection was also performed prior to and 18 to 20 h after first and final runs of the exercise regimen for analysis of inflammatory cytokines. The Four Rivers Kennel (FRK) Gait Inflammation Index score was assessed prior to and 24 hours after the first and last exercise runs with the Gait4Dogs pressure mat system. Saliva cortisol was measured before and after transportation. Blood chemistry did not differ among treatments (P ≥ 0.19), but Control dogs had increased eosinophils (P = 0.01) and tended to have increased white blood cells (P = 0.08) and lymphocytes (P = 0.08) compared with dogs receiving postbiotic. Dogs fed Low dose had elevated monocyte chemoattractant protein-1 (P = 0.01) and maintained Interleukin (IL) -8 after the initial run (P = 0.03). Males in the Low group had increased IL-10 (P = 0.02). Fecal biomarkers and microbiota dysbiosis index score did not differ among treatments by d 56 (P ≥ 0.19). Blautia abundance was decreased in High (P = 0.04). Fusobacterium abundance tended to be reduced in High and increased in Control and Low (P = 0.07). Clostridium hiranonis tended to be greater in Low compared with Control (P = 0.07). The FRK Total Gait Inflammation Index scores decreased over time with acclimation to running (P < 0.01). Saliva cortisol concentrations did not differ among treatments (P ≥ 0.20). The Saccharomyces cerevisiae-based postbiotic tested in this study has potential as a functional ingredient to modulate immune function, improve muscle recovery shortly after exercise and positively change diversity of the gut microbiome in canine companion animal diets.

This study sought to validate the D2Dx rapid immunity test in canines and investigate the age-related changes in the humoral immune system of healthy large breed dogs. Blood plasma with EDTA anticoagulant was collected from healthy Labrador Retrievers (n = 80) housed at Four Rivers Kennel under IACUC protocol FRK-56. Age groups were defined as follows: puppy 0-1.5 yr, adults 1.5-7 yr, senior 7-10, and geriatric 10-14. Immunity biomarkers including c-reactive protein (CRP), interleukin 4 (IL-4), interferon gamma (IFN-γ), complement protein 3 (C3), and immunoglobulin G (IgG) were determined through canine specific enzyme-linked immunosorbent assay (ELISA) kits. The nanoparticle based D2Dx test, which evaluates the cumulative humoral immune response, was also performed. Data were analyzed using a one-way ANOVA between age groups, with a Tukey’s post hoc, and correlations between variables were evaluated through multivariate analysis. The D2Dx score was correlated with body weight, age, IgG, and C3 validating that the test was able to adequately measure the humoral immunity in canines (r ≥ 0.49). D2Dx score was significantly different between age groups (P < 0.01) with puppies having significantly reduced scores compared with all other groups (P < 0.01). C3 was significantly different between age groups (P < 0.01), with puppies significantly less than other life stages (P ≤ 0.03). The C3 of adults averaged significantly greater than seniors and trended greater than geriatric subjects (P ≤ 0.01, 0.07). IgG between ages was also significantly different, with puppies significantly less than other age groups (P < 0.01). There was no significant difference between age groups for IFN-γ, IL-4 ratio, IFN-γ, IL-4, or CRP (P ≥ 0.18). This study showed that the D2Dx Rapid Immunity Test is able to detect changes to humoral immunity in canines. Beyond evaluating the effectiveness of the D2Dx test, this study also provides important information about the under researched topic of canine humoral immunity development and senescence in a robust, healthy cohort of large breed dogs.

Abstract Age-appropriate diets that meet the unique nutritional needs of aging are in greater demand as senior dogs represent an increasing percentage of the pet population. Studies targeting the specific amino acid requirements of the aging canine are important because they may inform dietary formulations for this specific life stage. In this study, the indicator amino acid oxidation (IAAO) technique was used to determine the minimum requirements for isoleucine (Iso) in young adult and senior Labrador retrievers. After 2-d adaptation to Iso-adequate basal diet (Iso = 0.608% dry matter), dogs underwent individual IAOO studies. In brief, all dogs were randomly fed one of six test diets with varying levels of isoleucine ranging from deficient to sufficient (final Iso content in experimental diets were 0.244, 0.285, 0.326, 0.532, 0.57 and 0.608% dry matter). Test diet was divided into 13 equal meals; at the 5th meal, a tracer amino acid was supplied (a bolus L-[1-13C] phenylalanine based on body weight was first given, followed by [1-13C] Phe doses every 30 min spanning a 4 h period), and breath samples were collected via respiration mask every 30 min. CO2 production was determined via indirect calorimetry and production of 13CO2 at each dose was measured by enrichment of 13C in breath samples via isotope ratio mass spectrometry (IRMS). Results for IRMS data were converted to atom percent excess (APE) and analyzed using segmented linear regression. The Four Rivers mean and population requirements for isoleucine were 898 ± 71 mg/1,000 kcal ME (mean ± 2 SD) for young adults and 885 ± 91 mg/1,000 kcal ME (mean ± 2 SD) in senior dogs, which is slightly greater than the National Research Council’s minimum requirement. As the pet food industry offers more specialized diets for aging canines, updating the amino acid requirements for such animals is increasingly important.

This study examined the effects of varying protein sources on apparent total tract digestibility, inflammatory markers, and fecal microbiota in Labrador Retrievers with historically poor stool quality. Thirty dogs (15 male, 15 female; aged 0.93 to 11.7 yr) with stool quality scores <= 2.5 on a 5-point scale (1 representing liquid stool and 5 representing firm stool) were randomly assigned to 1 of 3 nutritionally complete diets with differing protein sources and similar macronutrient profiles: 1) chicken meal (n = 10); 2) 10% brewer's yeast (n = 10); or 3) 10% torula yeast (n = 10). Another 10 dogs (five male, five female) with normal stool quality (scores ranging from 3 to 4) received diet 1 and served as negative control (NC). All dogs were fed diet 1 for 7 days, then provided their assigned treatment diets from days 7 to 37. Daily stool scores and weekly body weights were recorded. On days 7, 21, and 36, blood serum was analyzed for c-reactive protein (CRP), and feces for calgranulin C (S100A12), alpha(1)-proteinase inhibitor (alpha(1)-PI), calprotectin, and microbiota dysbiosis index. Apparent total tract digestibility was assessed using the indicator method with 2 g titanium dioxide administered via oral capsules. Stool scores were greater in NC (P < 0.01) as designed but not affected by treatment x time interaction (P = 0.64). Body weight was greater (P = 0.01) and CRP lower (P < 0.01) in NC dogs. Dry matter and nitrogen-free extract digestibility did not differ among groups (P >= 0.14). Negative controls had greater fat digestibility compared to BY (94.64 +/- 1.33% vs. 91.65 +/- 1.25%; P = 0.02). The overall effect of treatment was significant for protein digestibility (P = 0.03), but there were no differences in individual post hoc comparisons (P >= 0.07). Treatment did not affect S100A12 or alpha(1)-PI (P >= 0.44). Calprotectin decreased at a greater rate over time in TY (P < 0.01). The dysbiosis index score for BY and TY fluctuated less over time (P = 0.01). Blautia (P = 0.03) and Clostridium hiranonis (P = 0.05) abundances were reduced in BY and TY. Dogs with chronically poor stool quality experienced reduced body weights and increased serum CRP, but TY numerically increased protein digestibility, altered the microbiome, and reduced fecal calprotectin. Torula yeast is a suitable alternative protein source in extruded canine diets, but further research is needed to understand the long-term potential for improving the plane of nutrition and modulating gut health.

Complete uncoated extruded kibble was coated with 5% by weight premium poultry fat (5,000 ppm Naturox, 0.57% FFA; Tyson, Springdale, AR) spiked with 0, 5, 10, 15, or 20 percent free fatty acids (Oleic acid, Millipore Sigma. Burlington, MA), and 3% liquid chicken-based palatant (Tyson, Springdale, AR). Kibbles were then utilized in oral palatability (n = 20 dogs; 10 male, 10 female), aromatic palatability (n = 20 dogs; 10 male, 10 female), and apparent total tract digestibility (ATTG; n = 36 dogs; 18 male, 18 female). Kibble odors were by ultra-fast gas chromatography e-nose (AlphaMOS, Toulouse, France), and SPME GC-MS (MUMC, Columbia, MO). In the aromatic palatability trials, there were no significant differences in first approach (P ≥ 0.21), percent interaction time (P ≥ 0.16), or interaction ratio (P ≥ 0.94). In the oral palatability trials, there were no significant differences in first approach (P ≥ 0.26), first bite (P ≥ 0.50), percent consumed (P ≥ 0.15), or intake ratio (P ≥ 0.59). There was a significant increase in fat digestibility among diets coated with 20% FFA compared with the uncoated diet (P = 0.02), with intermediate diets showing a slight numerical increase but no significant difference. However, there was no significant difference in protein, carbohydrate, or dry matter digestibility (P ≥ 0.06). Principle component analysis of e-nose peaks showed minimal odor changes between samples (DI = -3). SPME GC-MS analysis identified 16 compounds correlated with FFA percentage (r2 ≥ 0.50). When used to coat kibble, poultry fat with up to 20% FFA did not appear to negatively impact palatability or overall odor profile, though it did modify specific volatile compounds.

Abstract Methionine and cysteine are the principal sulfur-containing amino acids that are incorporated into proteins, although homocysteine and taurine also have important physiological roles in canines. As limited studies have been conducted for total sulfur-containing amino acids (TSAA) requirements across the canine life cycle, our objective was to determine the minimum requirements (MR) for TSAA at 4 life stages using the indicator amino acid oxidation (IAAO) technique. Labrador Retrievers (n = 24) were used in feeding studies to evaluate the changing requirements of TSAA as dogs mature: 6 puppies aged 10 to 14 wk, 6 puppies >14 wk to 9 mos, 6 adults 2 to 5 yr (yr), and 6 seniors > 8yr. Following a 2-d adaptation to a TSAA-adequate basal diet (TSAA = 1.12% dry matter), dogs underwent individual IAAO studies. In brief, all dogs were randomly fed one of six test diets with varying levels of TSAA ranging from deficient to sufficient (final TSAA content in experimental diets were 0.41, 0.46, 0.51, 0.98, 1.05, and 1.12% dry matter, with indispensable amino acids formulated at 1.6x NRC values). The test diet was divided into 13 equal meals; at the 5th meal, a tracer amino acid was included (a bolus L-[1-13C] phenylalanine based on body weight was first given, followed by [1-13C] Phe doses every 30 spanning 4 h). Breath samples were collected via a respiration mask every 30 min. Total CO2 production was determined via indirect calorimetry and production of 13CO2 at each dose was measured by enrichment of 13C in breath samples via isotope ratio mass spectrometry (IRMS). Results for IRMS data were converted to atom percent excess (APE) and analyzed using segmented linear regression. The Four Rivers mean and population requirements for TSAA were estimated to be 1.55 ± 0.069 g/1,000 kcal (mean ± 2 SD) for puppies 10 to 14 wk, 1.53 ± 0.21 g/1,000 kcal (mean ± 2 SD) for puppies 14 wk to 9 mo, 1.4 ± 0.30 g/1000 kcal (mean ± 2 SD) for adults 2 to 5 yr, and 1.27 ± 0.23 g/1,000 kcal (mean ± 2 SD) for seniors. These results may contribute to the formulation of diets that meet the age-appropriate requirements of TSAA for large-breed dogs at all life stages.

Methionine is a limiting amino acid in canine diets, but few requirement studies have been conducted across life stages in large-breed dogs. The objective of this study was to determine the minimum requirement (MR) for methionine (Met) in Labrador retrievers at 4 life stages using the indicator amino acid (AA) oxidation (IAAO) technique. In total, 24 dogs were used: 6 puppies aged 10 to 14 wk, 6 puppies aged 14 wk to 9 mo, 6 adults aged 2 to 5 yr, and 6 seniors aged over 8 yr in feeding studies to evaluate the changing requirements of methionine (Met) as canines age. After 2-d adaptation to Met-adequate basal diet (Met = 0.56% dry matter), dogs underwent individual IAAO studies. In brief, all dogs were randomly fed one of six test diets with varying levels of Met ranging from deficient to sufficient (final Met content in experimental diets was 0.1982, 0.2386, 0.279, 0.49, 0.525, 0.56% dry matter, with indispensable amino acids formulated at 1.6x NRC values. Test diet was divided into 13 equal meals; at the 5th meal, a tracer amino acid was supplied (a bolus L-[1-13C]phenylalanine based on body weight was first given, followed by [1-13C]Phe doses every 30 min spanning a 4-h period), and breath samples were collected via respiration mask every 30 min. Total production of 13CO2 during isotopic steady state was determined by enrichment of 13CO2 in breath samples and total CO2 production measured via indirect calorimetry and isotope ratio mass spectrometry (IRMS). Results for IRMS were converted to atom percent excess (APE) and analyzed using segmented linear regression. The Four Rivers mean and population requirements for Met were estimated to be 0.84 ± 0.05 g/1000 kcal (mean ± 2SD) for puppies 10 to 14 wk, 0.78 ± 0.16 g/1,000kcal (mean ± 2SD) for puppies 14 wk to -9 mo, 0.68 ± 0.19 g/1,000kcal (mean ± 2SD) for adults 2 to 5 yr, and 0.62 ± 0.17 g/1,000kcal (mean ± 2SD) for seniors. This research provides important information to assist in accurate formulation of diets that contain appropriate methionine for large breed dogs at all life stages.

Postbiotics are emerging as potential functional ingredients for companion animal diets. This study aimed to determine if a Saccharomyces cerevisiae-based postbiotic can alter cytokine and stress responses to exercise and transport stress in adult Labrador Retrievers. Dogs received 15 g ground corn germ (Control, n = 12), 7.5 g postbiotic (Low, n = 12), or 15 g postbiotic (High, n = 12), daily for 63 days. Exercise was twice weekly for 7 weeks, and a single transport per dog occurred in week 8. Fecal inflammatory biomarkers, serum chemistries, and complete blood counts were assessed at the beginning and end of the study. Serum cytokines were quantified before and 18–20 h after the first and last exercise runs. Gait analysis was assessed before and 24 h after the first and final runs. Saliva cortisol was measured before and after transportation. Treatment did not affect blood chemistries, gait, fecal biomarkers, or saliva cortisol (p ≥ 0.19). Eosinophils increased slightly in Controls (p = 0.01), though remained below 0.80 × 109 cells/L. Most cytokines were unaffected by treatment (p ≥ 0.15), but there were minor changes in circulating monocyte chemoattractant protein-1 (p = 0.01) and IL-8 over time at the initial run (p = 0.03) and IL-10 in males (p = 0.02) in the Low dose dogs. The High dose decreased Blautia (p = 0.04) slightly and tended to decrease Fusobacterium abundances (p = 0.07). The Low dose tended to increase Clostridium hiranonis (p = 0.07) slightly. The tested S. cerevisiae postbiotic produced small changes in immune function and gut microbial species in dogs.

Nutritional requirements vary across the life stages of canines, and the consumer demand for advanced diets that meet the age-appropriate needs of their pets has steadily increased. Availability of veterinary care and high-quality nutrition has led to a longer average lifespan in companion animals, and although senior dogs represent a significant proportion of the pet population, few studies have targeted the specific amino acid requirements of the aging canine. In this study, the indicator amino acid oxidation (IAAO) technique was used to compare the tryptophan (Trp) requirements of young adult and senior Labrador retrievers. After 2-day adaptation to Trp-adequate basal diet (Trp = 0.256% dry matter), dogs underwent individual IAOO studies. In brief, all dogs were randomly fed one of six test diets with varying levels of tryptophan ranging from deficient to sufficient (final Trp content in experimental diets was 0.05, 0.1, 0.15, 0.224, 0.25 and 0.256% dry matter). Test diet was divided into 13 equal meals; at the 5th meal, a tracer amino acid was supplied (a bolus L-[1-13C] Phe based on body weight was first given, followed by [1-13C] Phe doses every thirty minutes spanning a four-hour period), and breath samples were collected via respiration mask every 30 minutes. Total production of 13CO2 during isotopic steady state was determined by enrichment of 13CO2 in breath samples and total CO2 production measured via indirect calorimetry and isotope ratio mass spectrometry (IRMS). Results for IRMS were converted to atom percent excess (APE) and analyzed using segmented linear regression. The Four Rivers mean and population requirements for tryptophan were 377.7 ± 31.9 mg/1,000 kcal ME (mean ± 2SD) in the young adults and 362.9 ± 31.5 mg/1,000 kcal ME for senior dogs, which are greater than the currently recommended amounts by NRC and AAFCO. As the pet food industry offers more specialized diets for aging canines, updating the amino acid requirements for such animals is increasingly important.

PURPOSE:Staff management is the most cited ART/IVF laboratory inspection deficiency. Small ART/IVF clinics may be challenged to perform these activities by low staff volume; similarly, large ART/IVF networks may be challenged by high staff volume and large datasets. Here, we sought to investigate the performance of an automated, digital platform solution to manage this necessary task.METHODS:The ART Compass (ARTC) digital staff management platform was used to assess the clinical decision-making of ART laboratory staff. The survey modules presented standardized instructions to technologists and measured inter- and intra-technologist variability for subjective "clinical decision-making" type questions. Internal and external comparisons were achieved by providing technologists two answers: (1) a comparison to their own lab director and (2) to the most popular response collectively provided by all lab director level accounts. The platform is hosted on HIPAA compliant Amazon web servers, accessible via web browser and mobile applications for iOS (Apple) and Android mobile devices.RESULTS:Here, we investigated the performance of a digital staff management platform for single embryologist IVF practices and for three IVF lab networks (sites A, B, C) from 2020 to 2022. Embryology dish preparation survey results show variance among respondents in the following: PPE use, media volume, timing of oil overlay, and timing of moving prepared dishes to incubators. Surveying the perceived Gardner score and terms in use for early blastocysts reveals a lack of standardization of terminology and fair to poor agreement. We observed moderate inter-technologist agreement for ICM and TE grade (0.47 and 0.52, respectively). Lastly, the clinical decision of choice to freeze or discard an embryo revealed that agreement to freeze was highest for the top-quality embryos, and that some embryos can be highly contested, evenly split between choice to freeze or discard.CONCLUSIONS:We conclude that a digital platform is a novel and effective tool to automate, routinely monitor, and assure quality for staff-related parameters in ART and IVF laboratories. Use of a digital platform can increase regulatory compliance and provide actionable insight for quality assurance in both single embryologist practices and for large networks. Furthermore, clinical decision-making can be augmented with artificial intelligence integration.

Retrospective studies have suggested that gonadectomies, such as the spays and neuters common in companion animal populations, may be correlated with the development of autoimmune conditions later in the life of an animale. This study investigated the potential influences of gonadectomy on peripheral blood lymphocyte populations through flow cytometry. Twenty senior Labrador Retrievers (10 males and 10 females) were randomly selected from Four Rivers Kennel’s existing research colony according to their intact or altered status. Groups were balanced by age and body weight (average 11.4 y/o and 64.5 kg) as both have been previously shown to alter lymphocyte populations. A single blood collection was performed and stained with canine cross-reactive conjugated antibodies CD3-FITC, CD4-Superbright 600, CD8-Superbright 702, and a fixable lime viability stain (Invitrogen, Waltham MA). Samples were processed through a whole blood based lyse no-wash method and analyzed on an Attune NXT Flow Cytometer equipped with blue and violet lasers (ThermoFisher Waltham, MA). Measurement of cell populations was completed on a defined volume of each sample, and all subsets were gated to live, singlet lymphocytes. Statistical significance was determined through one-way ANOVAs on JMP Pro 16 (Cary, NC). There were no significant differences in CD4+/CD8+ ratio between groups (P = 0.56), or total lymphocyte populations (P = 0.34). Additionally, there were no significant differences between groups regarding CD3+ MFI (P = 0.25), CD8+ MFI (P = 0.70), or CD4+ MFI (P = 0.43). Overall, in age and body weight balanced senior Labrador Retrievers, spay or neuter status did not significantly influence T-cell populations. This suggests that previous gonadectomy does not influence the adaptive immune system as an animal ages. Further research is needed to clarify the relationship between gonadectomy and the later development of autoimmune disorders.

Samples of rendered chicken meal (CM) and chicken by-product meal (CB) were obtained from a commercial plant with samples representing various storage time points and a range of peroxide values from < 10 (1), 20-50 (2), 50-100 (3), 100-200 (4). Samples were utilized in a novel aromatic palatability trial with 20 working Labrador Retrievers (n = 10 males and n = 10 females). Aggregated interaction time showed the most pronounced difference between the lowest PV sample and the highest PV for both CB and CM (P < 0.0001, 0.001). Both CB1 and CM1 were significantly different from all other samples (P = 0.05). Samples were also sent to a third party for HERACLES Neo flash gas chromatography electronic nose analysis (e-nose). Analysis of e-nose chromatograms showed good discrimination between samples (DI = 97) with the largest spatial differences between the low and high PV samples. Chromatogram peaks were tentatively identified according to their Kovats index and the AlphaChemBase database. Component 1 (X-axis) accounted for 99.4% of the difference while component 2 (Y-axis) showed 0.54% of the difference. The strongest odor, as determined by the highest area peak was hexanal which produces a fatty and fishy smell. When evaluated in a bivariate model hexanal vs PV was well correlated on a 2P curve (R2= 0.986, P < 0.0001). However, hexanal was less correlated with aromatic palatability (R2 = 0.616. P = 0.09). Therefore, a primary least squares model was created to better display the relationship between palatability and oxidation. Utilizing two factors, the PLSR model explained 72% of the variation in the e-nose peak area data (X) and 90% of the variation in the PV*palatability (Y). Of the 61 aromatic compounds identified, the model identified 45 compounds with a VIP over 0.8. Both PV and palatability were well-fitted by the model as indicated by their position between the outer two ellipses (R2 = 75% to 100%). In the model, Hexanal had a VIP of 1.1 suggesting it was an important component, although other compounds had significantly better VIP scores and are likely to better predict palatability. Therefore, these data show aromatic hexanal is an accurate predictor of the oxidation of a product, although it may not provide the full picture regarding palatability.

We previously reported increases in lean mass and lean:fat ratio in senior Labrador Retrievers fed a commercial whey protein supplement after a 26-week trial with exercise regimen. Further work on this project examined the impacts of this supplement on immunity and inflammation. Thirty-six senior (>7 yr) Labrador Retrievers received a basal diet meeting AAFCO crude protein (CP) requirements (45g/1,000 kcal, 18% CP on DM basis) and were randomly assigned to one of three supplements: 1) an additional 22.5 g CP/1,000 kcal (27% CP on DM basis) sourced from hydrolyzed pea protein isolate (Pea); 2) additional 22.5 g CP/1,000 kcal (27% CP on DM basis) sourced from whey protein (Whey; Hilmar Protelyze Pet; Hilmar, CA) or 3) poultry fat and sucrose to equalize calories but provide no additional CP (Control). All dogs participated in an exercise regimen consisting of twice weekly 2.2 km runs. Blood samples were collected via jugular venipuncture at week 0 (baseline) and week 26 (end of trial) for analysis of cytokines (IL-2, IL-6, IL-8, IL-10, IL-12, NGF-β, IFN-γ, TNF-α, MCP-1, VEGFA, and SCF) and immune cell proliferation. Cytokines were measured with Luminex MAGPIX analyzer. Peripheral blood mononuclear cells were isolated from whole blood, plated, challenged with pokeweed mitogen. Proliferation was evaluated with a 5-bromo-2’-deoxyuridine proliferation assay kit. Gait analysis was evaluated at weeks 0 (baseline), 4, 8, 16 and 26 using a Gait4Dogs pressure walkway (CIR Systems, Inc; Franklin, NJ). Gait data were processed to generate a FRK Total Gait Inflammation Index, calculated by the sum of the differences from the ideal scores for gait lameness score, total pressure index, step/stride ratio, and hind reach. Data were analyzed using one-way ANOVA with Tukey’s post-hoc adjustments. We observed increases in IL-12 (P = 0.004), MCP-1 (P = 0.04), VEGFA (P = 0.05), and SCF (P = 0.04) in Control vs Whey at week 26, with Pea intermediate. Spontaneous immune cell proliferation was less in both Pea and Whey compared with Control (P < 0.01). Proliferation after challenge was less in Whey compared with Pea (P = 0.05), with Control intermediate. The FRK Total Gait Inflammation Index significantly increased after exercise in Control and Pea dogs, but not Whey (P < 0.01). Scores of Control dogs remained increased compared with Whey dogs from weeks 4 to 26 (P < 0.01). Dietary supplementation with whey protein at 1.5 x AAFCO minimum proved beneficial for Labrador Retrievers by maintaining more lean mass with improved lean/fat ratio, reduced inflammatory biomarkers, immune cell proliferation, and FRK Total Gait Inflammation Index after an exercise regimen when compared with seniors only consuming a 18% protein basal diet. Supplemental whey protein may better maintain senior dogs experiencing chronic inflammation from aging or disease.

Four Rivers Kennel examined the effects of varying protein sources on digestibility, inflammation, and gut microbiota in Labrador Retrievers with historically poor stool quality. Thirty dogs (n = 15 males and n= 15 females; ages 3 to 10 years) with stool quality scores ≤ 2.5 on a 5 point scale were randomly assigned to 1 of 3 diets meeting AAFCO requirements with differing protein sources and similar nutrient profiles: 1) chicken meal (PC); 2) 10% brewer’s yeast (BY); and 3) 10% torula yeast (TY; SylPro, Arbiom, Durham, NC). Another 10 dogs (n = 5 males and n = 5 females) with normal stool quality (score ≥ 3) received diet 1 and served as negative control (NC). After 7 days acclimation to diet 1, dogs began treatments. Daily stool scores and weekly body weights were recorded. On days 7, 21, and 36, blood serum was analyzed for C-reactive protein (CRP), and feces for S100A12, alpha-1 proteinase inhibitor (a1PI), calprotectin, and microbiota dysbiosis index (MDI). Apparent total tract digestibility was assessed using the indicator method with 2 g titanium dioxide administered orally via gel capsules. Statistical analysis was performed by repeated measures model in SAS for stool scores, body weight, biomarkers, and microbiota or by one-way ANOVA in JMP for digestibility coefficients. Stool scores were greater in NC (P < 0.01), changed daily in all groups (P < 0.01) without noticeable overall trends, and were not affected by treatment*day interaction (P = 0.64). Body weight was greater (P = 0.01) and CRP less (P < 0.01) in NC dogs than poor stool dogs. Dry matter and carbohydrate digestibility did not differ between groups (P ≥ 0.14). NC dogs had greater fat digestibility compared with BY dogs (94.64 ± 1.33 % vs 91.65 ± 1.25%; P = 0.02). Protein digestibility was reduced in PC (58.91 ± 4.68%) and BY (58.26 ± 4.42%) compared with NC (75.52 ± 5.01 %), but TY diet improved protein digestibility to levels similar to NC (72.18 ± 5.01%, P = 0.03). There were no treatment effects on S100A12 or a1PI (P ≥ 0.44). Calprotectin decreased at a greater rate overtime in TY dogs (P < 0.01). There was a treatment*day interaction for MDI; BY and TY fluctuated less overtime (P = 0.01). Blautia (P = 0.03) and Clostridium hiranonis (P = 0.05) abundances were reduced in BY and TY. Dogs never exhibited significant dysbiosis (MDI < 2), and bacterial abundances remained within normal ranges. Dogs with chronically poor stool quality experience reduced body weights, increased serum CRP, and altered nutrient digestibility, but TY helped improve protein and fat digestibility, sustain microbiome, and reduce fecal calprotectin. Feeding torula yeast long-term may help improve plane of nutrition to better maintain weight and reduce inflammation in dogs with chronically poor stool quality.

Proper production of steroid hormones, such as progesterone (P4), is essential for maintenance of pregnancy. Provision of gluconeogenic substrates from the diet may alter postabsorptive energy supplies and associated hormone signaling in a manner supportive of elevating P4 concentrations. Crossbred Angus heifers (n=27) were balanced for pre-trial bodyweight (BW) and body condition score (BCS), then assigned to isoenergetic diets leveraging starch (ES) or fat (EF) as the primary source of energy (45.7% starch vs 11.5% fat). The heifers were placed on the assigned diets 8 days prior to the initiation of estrous synchronization and continued the diets through the second pregnancy diagnosis, 52 days after the initial insemination. On day 28 post-breeding, pregnancy status was diagnosed via ultrasonography. Measurements of follicle size and number were collected via rectal palpation and ultrasonography on days −10, −8, −4, and day 0 relative to breeding. Blood samples were collected on days −10, 1 through 7, 10, 14, 18, 21, 24, and 28 to analyze plasma for P4 concentrations and placental-associated glycoprotein (PAG) concentrations. Data were analyzed using a linear mixed effects model with a fixed effect for treatment and a random effect for pen. Observations collected over time were analyzed using a repeated measures approach with fixed effects for treatment, time, initial condition, and a random effect for pen. Interactions between treatment and pregnancy status were also tested for PAG and P4 concentrations. No treatment differences were observed in final BCS ( P= 0.12) and total dry matter intake (DMI; P= 0.51), though heifers on the ES treatment tended to reach an increased BW ( P= 0.10). Although heifers fed ES diets had numerically higher conception rates compared to heifers fed EF diets (71.4% vs. 66.7%), there was no statistically significant difference. Furthermore, there were no significant differences observed among diets in terms of pregnancy outcomes, follicle size, or number. A treatment by pregnancy status interaction was detected for both PAG and P4 concentrations. These results suggest that short-term dietary starch supplementation provided around the time of breeding can result in shifts to reproductive hormone abundance that may be conducive for pregnancy establishment.

Cattle temperament significantly impacts production traits such as reproduction. The objective of this study was to assess the effects of temperament on pregnancy rates to fixed-timed artificial insemination (TAI) in Bos taurus beef heifers. A total of 297 Angus influenced heifers from 3 different locations were evaluated for temperament based on chute score and exit velocity on the first day of the estrus synchronization protocol (d-9) and classified by temperament type based on temperament score (calm ≤ 3 < excitable). Pregnancy status was determined by transrectal ultrasonography approximately 40 d after TAI. Hair from the tail switch was collected at d-9 and at d0 (TAI) for cumulative cortisol evaluation. A subset of 43 heifers from location 3 had blood samples collected at all handling events and evaluated for cortisol concentrations. Overall, 71% of heifers were classified as calm whereas 29% as excitable. Pregnancy rates to TAI were reduced (P = 0.042) in excitable heifers compared to calm heifers (36% vs. 55%, respectively). Mean concentration of cortisol in the hair was reduced over time (P < 0.001) from d-9 (3.5 ± 0.3 pg/mg) to d0 (1.74 ± 0.3 pg/mg) in all heifers, regardless of temperament. In addition, excitable heifers had increased circulating concentrations of cortisol when compared to calm heifers in all handling events (P = 0.015). More interestingly, despite of temperament, concentrations of circulating cortisol were reduced as the protocol events progressed, with greater cortisol concentrations on d-9, intermediate on d-3, and lesser on d0 (P = 0.031). Therefore, the present study demonstrates that heifer temperament has negative effects in pregnancy rates to TAI programs. However, according to blood cortisol concentrations, no chronic stress response was detected due to cattle handling for the TAI protocol. In fact, blood cortisol concentration was reduced between the initiation and completion of the protocol, indicating that heifers were acclimated to handing. These results support the adoption of acclimation protocols and proper cattle handling as a strategy to increase fertility of heifers that are exposed to TAI.

This study evaluated the effect of an injectable multi-mineral complex supplementation on grazing beef cows on subsequent offspring birth weight (BW) and adjusted weaning weight (AWW). Experiment 1 included 1,128 Angus crossbred multiparous suckled cows, from 9 locations in Virginia, that were randomly assigned to one of two treatments: 1) One dose of an injectable trace mineral (6 mL; Multimin® 90, Multimin USA, Ft. Collins, CO) at 71.4±16.8 d of lactation (n = 560); or 2) a negative control with no trace mineral injection (n = 568). In experiment 2, 986 Angus crossbred multiparous suckled cows, at 7 locations in Virginia, were randomly assigned to one of two treatments: 1) Two doses of an injectable trace mineral (6 mL; Multimin® 90) at 118.3±17 days before calving and at 67.7±17.4 d of lactation (n = 494); or 2) negative control with no trace mineral injection (n = 492). All cows had ad libitum access to forage and water, and received additional mineral supplementation in form of mineral blocks. Calves were weaned at approximately 7 months of age, and 205-day AWW was calculated based on birth day, age of weaning and age of dam. No differences between treatments were detected for BW (33.7±1.01 kg; P = 0.50) and AWW (212.2±5.0 kg; P = 0.25) when only one dose of injectable trace mineral was administered to the dam at early lactation. Similarly, when two doses of injectable trace mineral were administrated BW (35.6±1.1 kg; P = 0.70) and AWW (259.4±6.1 kg; P = 0.83) did not differ between treatments. In conclusion, one or two doses of injectable trace mineral administrated at late gestation and/or during lactation had no effect on offspring BW and AWW.

The worldwide production of in vitro-produced embryos in livestock species continues to grow. The current gold standard for selecting quality oocytes and embryos is morphologic assessment, yet this method is subjective and varies based on experience. There is a need for a non-invasive, objective method of selecting viable oocytes and embryos. The aim of this study was to determine if ooplasm area, diameter including zona pellucida (ZP), and ZP thickness of artificially activated oocytes and in vitro fertilized (IVF) zygotes are indicative of development success in vitro and correlated with embryo quality, as assessed by total blastomere number. Diameter affected the probability of development to the blastocyst stage in activated oocytes on day 7 (P < 0.01) and day 8 (P < 0.001), and had a tendency to affect IVF zygotes on day 8 (P = 0.08). Zona pellucida thickness affected the probability of development on day 7 (P < 0.01) and day 8 (P < 0.001) in activated oocytes, and day 8 for IVF zygotes (P < 0.05). An interaction between ZP thickness and diameter was observed on days 7 and 8 (P < 0.05) in IVF zygotes. Area did not significantly affect the probability of development, but was positively correlated with blastomere number on day 8 for IVF zygotes (P = 0.01, conditional R2 = 0.09). Physical parameters of bovine zygotes have the potential for use as a non-invasive, objective selection method. Upon further development, methods used in this study could be integrated into embryo production systems to improve IVF success.

The use of in vitro produced embryos in dairy and beef cattle has increased in recent years, but compromised post-transfer pregnancy success prevents producers from capturing all the benefits this technology can provide. This study explored whether supplementing interleukin-6 (IL6) during in vitro embryo development influences post-transfer development of the embryo-proper, fetus and placenta during early gestation in cattle. Slaughterhouse-derived cumulus oocyte complexes underwent IVM (day -1) and IVF (day 0). On day 5 post-fertilization, embryos were treated with either 0 (CONT) or 100 ng/mL recombinant bovine IL6. No difference in blastocyst formation was detected on day 7.5 post-fertilization, but an increase (P < 0.05) in inner cell mass cell numbers and tendency for increased (P = 0.08) trophectoderm cell numbers were detected in IL6-treated blastocysts. A subset of the blastocysts was loaded individually into transfer straws, and embryo transfer (ET) was completed using estrous cycle stage-matched, nonlactating commercial beef and dairy cows. A subset of cows from each group underwent timed artificial insemination (TAI). Pregnancy rates were similar among all three treatment groups at day 28 and 70. No differences in crown-rump length (CRL), crown nose length (CNL), abdominal diameter (AD), or placental fluid volume (PFV) were detected between TAI and ET-IL6 groups. Reductions (P < 0.05) in CRL and AD were detected at day 56 and a tendency for a reduction (P = 0.08) in PFV was detected on day 35 when comparing the ET-CONT group with the TAI group. Reductions (P < 0.05) in CRL and PFV on day 28 and CNL and AD on day 56 as well as a tendency for a reduction (P = 0.08) in PFV on day 35 were detected when contrasting ET-CONT with ET-IL6. Circulating plasma pregnancy-associated glycoprotein concentrations were similar among all treatment groups. In summary, IL6 treatment to IVP embryos before ET produced pregnancies that more closely resembled TAI-generated pregnancies than pregnancies generated using conventionally cultured embryos. These findings failed to find any adverse effects of IL6 supplementation on early development of the embryo-proper and fetus or on placental activity. Rather, these observations suggest that IL6 treatment may normalize the developmental trajectory of the embryo-proper and fetus for in vitro produced embryos.

The objective of this study was to assess differences in reproductive performance of natural service and artificial insemination (AI) sired beef females based on pregnancy outcomes, age at first calving, and calving interval. Data were sourced from 8,938 cows sired by AI bulls and 3,320 cows sired by natural service bulls between 2010 and 2017. All cows were in a commercial Angus herd with 17 management units located throughout Virginia and represented spring and fall calving seasons. All calves were born to dams managed with estrus synchronization. Pregnancy was analyzed with generalized linear mixed models and other reproductive measures with linear mixed models in R. Six models were evaluated with the dependent variables of pregnancy status at the first diagnosis, pregnancy status at the second diagnosis, pregnancy type (AI or natural service) at the first diagnosis, pregnancy type at the second diagnosis, calving interval, and age at first calving. Independent variables differed by model but included sire type of the female (AI or natural service), prebreeding measures of age, weight, and body condition score, postpartum interval, sex of the calf nursing the cow, and management group. No differences were observed between AI- and natural service-sired females based on pregnancy status at first and second pregnancy diagnosis (P > 0.05). Sire type was only found to be significant for age at first calving (P < 0.05) with AI-sired females being 26.6 +/- 1.6 d older at their first calving, which was expected because AI-sired females were born early in the calving season making them older at breeding. Surprisingly, age and body condition score were not significant predictors of pregnancy (P > 0.05). Body weight at breeding was not significant for pregnancy (P > 0.05) but was significant for age at first calving (P < 0.05). These data suggested that lighter heifers calved earlier which contradicts our original hypothesis. Overall, commercial Angus females sired by AI or natural service bulls had similar reproductive performance. Factors that were commonly associated with reproductive success were not significant in this commercial Angus herd managed with estrus synchronization. Given the size of these data, the importance of body condition, age, and weight should be reassessed in modern genetics and management practices.