Linear Mixed Model Procedure Research Articles

Levels of matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, osteopontin, pentraxin-3, and thymic stromal lymphopoietin in crevicular fluid samples from peri-implantitis, periodontitis, and healthy sites.

Periodontitis and peri-implantitis are chronic inflammatory diseases characterized by the destruction of supporting tissues. Despite some similarities, it is essential to understand the differences in how these diseases elicit unique host responses within the oral tissues, including the production of selected matrix metalloproteinases (MMPs) and inflammatory mediators involved in tissue remodelling. The aim of this study was to evaluate the levels of proteolytic enzymes MMP-1, MMP-2, MMP-3, as well as the inflammatory mediators osteopontin (OPN), pentraxin-3 (PTX3), and thymic stromal lymphopoietin (TSLP) in crevicular fluid samples collected from healthy, periodontitis-affected, and peri-implantitis sites. Gingival crevicular fluid (GCF) and peri-implant crevicular fluid (PICF) samples were collected from healthy and diseased teeth and implant sites of 163 patients. The MMP-1, MMP-2, MMP-3, OPN, PTX3, and TSLP levels were determined using commercially available immunoassays. A linear mixed model procedure was adopted for multilevel analyses, using biomarker levels as the outcome variable to compare two types of sites. The diagnostic accuracy of the biomarkers was evaluated by Youden's index to estimate the sensitivity, specificity and the area under curve (AUC). The levels of MMP-1, MMP-2, MMP-3, OPN, and TSLP were higher at sites with periodontitis and peri-implantitis compared to the levels at sites with healthy teeth and healthy implants. No significant differences were observed in the levels of the measured markers between the sites diagnosed with periodontitis and those diagnosed with peri-implantitis. The highest diagnostic potential at implant sites was found for MMP-2 (AUC = 0.74) and TSLP (AUC = 0.72). The highest AUC (0.82) at tooth sites was found for OPN. The findings indicate that the proteolytic enzyme MMP-2 and the cytokine TSLP might be potential biomarkers for both periodontitis and peri-implantitis, whereas the proinflammatory cytokine OPN may serve as a biomarker for periodontitis. Further studies are required to confirm the utility of these biomarkers and explore their potential clinical applications.

PSLBII-28 Effects of dietary protein manipulation on greenhouse gases and ammonia emissions from feedlot beef steers

Abstract Black Angus steers [n = 112; body weight (BW) = 401 ± 3.6 kg] were used in a randomized incomplete block design to evaluate the effects of 3 rumen available protein to microbial crude protein ratios (RAP:MCP) on growth performance, and gaseous emissions from feedlot steers. Steers were blocked by initial BW and randomly assigned to 1 of 3 treatment rations. Diets were fed rations either Deficient (-150 gּ animal-1ּ d-1; DEF), Balanced (0 gּ animal-1ּ d-1; BAL), or Excess (+150 gּ animal-1ּ d-1; EXS) in RAP: MCP. Steers were allocated and housed in cattle pen enclosures (CPE), and treatment diets were delivered daily as a total mixed ration. The present study consisted of two 42-d periods. Refusals were collected weekly and BW every 14 d. Gas measurements were obtained daily in sequential order from all CPE. Data were analyzed with R statistical software (4.2.3). The linear mixed effect model procedure within the “lme4” package was used with CPE as the experimental unit, treatment and week as fixed effects, and block and period as random effects. A contrast coefficient matrix was constructed to test for linear and quadratic effects. Initial, intermediary and final BW were not different for steers receiving the varying RAP:MCP (P ≥ 0.239). A treatment effect (P = 0.004) was observed for mean dry matter intake (DMI) where EXS steers consumed 0.46 kg more DM compared with BAL steers, but DEF steers had similar DMI to those fed EXS and BAL (P = 0.189). Average daily gain (ADG) was only affected by treatment (P = 0.007) on d 0 to 14 where steers receiving DEF gained 0.6 kg more than those fed BAL (P = 0.010), but those fed EXS had similar ADG. Similar to ADG, gain to feed (G:F) was affected by treatment (P = 0.012) only during d 0 to 14. Mean CH4 production from steers fed DEF and EXS were 20% and 13% greater (P = 0.010) compared with those fed BAL, respectively; however, CH4 from EXS vs. DEF steers was not different (P = 0.149). Mean cumulative NH3 emissions increased linearly by treatment with EXS steers emitting up to 52% more NH3 compared with those receiving DEF (P < 0.001). Similarly, EXS steers had increased SO2 emissions by up to 44% compared with those fed DEF (P < 0.001). Emissions of CO2, N2O, and H2S were similar (P > 0.100). The manipulation of RAP:MCP in the diets of feedlot steers can drastically reduce NH3 emissions without affecting growth performance and may be a valuable tool to reduce air pollution from beef production systems.

PSVI-19 Abundance of some cecal bacteria in Lohmann LSL lite pullets and hens fed omega-3 fatty acids and yeast bioactives in low or high spacing allowance

Abstract Nutritional optimizations and rearing environment that meets welfare standards are prerequisite in the performance and health of laying hens. Rearing practices influence gastrointestinal development, microbiota, and immune function. However, little is known on the implications of rearing spacing allowance (SA) and feed ingredients on intestinal microbiota. We determined the abundance of selected cecal bacteria in pullets and hens in response to rearing SA and dietary omega-3 fatty acids (N-3 FA) and yeast bioactive (YB) from placement to 17 wk of age (woa). One-day old chicks (n = 2,632) were placed in cages in 2 SA of 24 cages each: High SA (HSA, 348 cm2/bird; ~50 birds/cage) and Low SA (LSA, 284 cm2/bird; ~68 birds/cage). Birds were fed corn and soybean-based diets (C), C + 3% co-extruded full fat flaxseed and pulse mixture (N-3 FA) and C + 0.05% YB processed with β-1,3-glucan hydrolase (YB). Birds were allocated to diets within SA according to 2 x 3 factorial design (n = 8 cage/treatment) and fed ad libitum. At 4 and 16 woa, 1 and 2 pullets/treatment respectively, were necropsied for ceca digesta from each cage and independently analyzed. At 18 woa, hens were placed in enriched cages based on rearing treatment identities in egg production facility and fed a commercial diet. At 35 woa, 18 hens per treatment were necropsied for ceca digesta samples and analyzed independently. Samples were analyzed for viable bacterial counts for E. coli, Enterococcus and Lactobacillus. Statistical analysis was performed by general linear mixed model procedures of SAS with diets, SA, sampling age and associated interactions as fixed factors, difference with P < 0.05 was considered significant. Comparison of log-transformed means (CFU/g) showed that diets and SA had no significant effect on cecal E. coli counts at 4, 8 and 35 woa (P > 0.05; Table 1). However, at 16 woa the least E. coli counts was found in LSA (P < 0.0001), with N-3-FA-fed and YB-fed pullets having fewer numbers (P = 0.007) than control-fed pullets. There was no interaction effects between diet and density for E. coli in all sampling wk (P >0.05). No significant effect of SA or diets were observed at any sampling week for Enterococcus counts, except LSA had a tendency at 8 woa (P = 0.078). At 8 woa, the cecal Lactobacillus counts (P = 0.009) were less in LSA relative to HSA pullets. In conclusion, feeding of N3-FA or YB during rearing influenced cecal microbiota. Supplementation of YB seemed to promote growth of beneficial Lactobacillus, which is associated with strengthened immunity.

489 Antibiotic Resistance of cecal Escherichia coli isolates from Lohmann LSL lite pullets and laying hens fed omega-3 fatty acids or yeast bioactives, reared at different spacing allowance

Abstract Poultry diets provide components that alter gut microbiota composition. We investigated antimicrobial resistance (AMR) phenotypes of E. coli isolates from ceca digesta of pullets and hens reared at different spacing allowance (SA) and dietary source of omega-3 fatty acids (N-3 FA) and yeast bioactives (YB) from placement to 17 wk of age (woa). A total of 2,632-day old chicks were placed in cages at two SA (24 cages each): High SA (HSA, 348 cm2/bird; ~50 birds/cage) and Low SA (LSA, 284 cm2/bird; ~68 birds/cage). Birds were fed corn and soybean-based diets (C), C + 3% co-extruded full fat flaxseed and pulse mixture (N-3 FA) and C + 0.05% YB processed with β-1,3-glucan hydrolase. At 4, 16 and 35 woa, ceca samples were collected to isolate E. coli using Chromocult agar. Their susceptibility to 14 antibiotics were determined by automated Sensititre system according to CLSI and CIPARS guidelines to determine their antimicrobial susceptibility profile. Number of antibiotics resistant E. coli isolates were analyzed using general linear mixed model procedure of SAS with diets, SA, sampling age and associated interactions as fixed factors. Cochran-Mantel-Haenszel test was used to determine the relationship between these treatments, and differences between treatments were considered significant at P < 0.05. Out of 428 presumptive E. coli isolated, 153 of 428 isolates (35.7%) were resistant to one antimicrobial class but no resistance against ciprofloxacin, azithromycin and nalidixic acid were detected. The prevalence of antimicrobial resistant E. coli decreased as the birds aged from wk 4–35 (P < 0.05). At wk 4 (P = 0.002) and 16 (P = 0.011), the least prevalence of ampicillin (AMP) resistance was in ceca digesta from N-3 FA-fed birds. At 35 woa, no AMP and cefoxitin (FOX) resistance were detected in birds fed YB (Table 2). At 16 woa, the prevalence of streptomycin resistance was greater in N-3 FA-fed than in YB-fed birds (P = 0.02), which was less than control-fed birds. Of the 428 isolates, 59 (13.8%) were resistant to more than two antimicrobial classes, with most from TIO-FOX-AMP-TET and STR-SULF-TET. At wk-4, SA had a significant effect on multiple resistance spectrum with LSA having the lowest MDR E. coli (P = 0.02) mainly from N-3 FA-fed pullets. This study showed an overall low AMR prevalence which decreases with age of the bird. This study is the first to report that feed additives (N-3 FA or YB) influence the AMR profile of cecal E. coli in pullets and layers. Prevalence of AMP resistance was least in N-3 FA-fed birds, whereas least STEP- and SULF resistance was observed in YB-fed birds. Our next study would help understand the molecular and evolutionary aspect of this resistant E. coli and their potential to cause colibacillosis.

Effect of the Presence of Emergency Departments With 300 or More Hospital Beds in Health Service Areas on 30-Day Mortality in Korea: A Nationwide Retrospective Cross-sectional Study.

Disparities in emergency care accessibility exist between health service areas (HSAs). There is limited evidence on whether the presence of an emergency department (ED) that exceeds a certain hospital bed capacity is associated with emergency patient outcomes at the regional level. The objective of this study was to evaluate the effect of HSAs with or without of regional or local emergency centers with 300 or more hospital beds (EC300 or nEC300, respectively) by comparing the 30-day mortality of patients with severe emergency diseases (SEDs) admitted to the hospital through the ED. The study retrospectively evaluated data from the National Health Information Database (NHID) of the National Health Insurance Service (NHIS) Claims database and enrolled patients who were admitted from the ED for SEDs. SEDs were defined using ICD-10 (International Classification of Diseases 10th Revision) codes for 28 disease categories with high severity, and 56 HSAs were designated as published by the NHIS. We performed hierarchical logistic regression analysis using multilevel models with the generalized linear mixed model (GLIMMIX) procedure to evaluate whether EC300 was associated with the 30-day mortality of SED patients, adjusting for patient-level, prehospital-level, hospital-level, and HSA-level variables. In total, 662 478 patients were analyzed, of whom 54 839 (8.3%) died within 30 days after hospital discharge. Of the 56 HSAs, 46 (82.1%) were included in the EC300 group. After adjustment for patient-level, prehospital-level, hospital-level, and HSA-level variables, nEC300 was significantly associated with increased 30-day mortality in SED patients (adjusted odds ratio [AOR]: 1.33, 95% CI: 1.137-1.153). In addition, patients who visited EDs with fewer annual SED admissions were associated with higher 30-day mortality. nEC300 had a greater risk of 30-day mortality in patients treated with SEDs than EC300. The results indicate that not only the number of EDs in each HSA is important for ensuring adequate patient outcomes but also the presence of EDs with adequate receiving capacity.

Little Owl Aggression and Territory in Urban and Rural Landscapes.

Urbanization is a major land use change across the globe with vast effects on wildlife. In this paper, we studied (1) the territorial displays of Little Owls in urban and rural landscapes, analyzing also (2) the size and habitat composition of the territories, and (3) the factors affecting territory size in both landscapes. To do that, we used t-tests, Principal Components Analysis, and General Linear mixed model procedures. The territory size was smaller in urban than in rural landscapes. Urban territories of Little Owls are characterized by a lower cover of grassland, tall crops, short crops, gardens, and orchards, as well as a higher cover of built-up areas than territories in rural landscapes. Territory size in rural landscapes was negatively correlated with seasonal progress and positively correlated with altitude. The rate of territorial displays was similar between urban and rural territories; however, birds differentially utilized various structures. In urban territories, birds mostly used buildings, whereas in rural territories, birds used electric pylons and trees. The compositional differences between territories in the two landscapes may have important consequences for other behavior types and possibly reproductive output in this species.

PSV-29 Plasma Citrulline Is Strongly Correlated with Jejunum and Ileum Citrulline Concentrations in Pigs at Weaning

Abstract Pigs at weaning are abruptly exposed to numerous stressors that negatively affect gut health. While the term gut health lacks a clear definition, it is best measured by its key functions, including nutrient digestion, absorption, and metabolism, mucosal immunity, and barrier function. The gut is the main endogenous source of citrulline, a potential biomarker of gut function. The objective of this study was to determine the relationship between plasma and intestinal concentrations of citrulline and citrulline precursors in pigs. Pigs (initial BW = 7,233 ± 683 g) were either weaned at 21 d age and euthanized at 23 d age (W; n = 9) or were not weaned, remained with the sow, and euthanized at 23 d age (NW; n = 9). At euthanasia, blood, jejunum, and ileum tissue was collected and snap-frozen in liquid nitrogen; segments of mid jejunum and distal ileum were collected for morphological analysis. Plasma and tissue glutamine (Gln), glutamate (Glu), proline (Pro), ornithine (Orn), and citrulline (Cit) concentrations were determined by reverse-phase HPLC following pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Data were analyzed with the generalized linear mixed model and correlation procedures of SAS (version 9.4). Pearson correlation coefficients between 0.50 and 0.69 were considered as moderate, between 0.70 and 0.89 as strong, and between 0.90 to 1 as very strong. Jejunum villus height was reduced in W compared with NW pigs (370 versus 246 ± 18 µm; P < 0.001), whereas ileum villus height was not different between NW and W pigs (257 versus 215 ± 27 µm; P = 0.27). Among precursors for gut Cit synthesis, plasma Pro was strongly correlated with jejunum Pro (r = 0.83; P < 0.001) and very strongly correlated with ileum Pro (r = 0.91; P < 0.001). Plasma Glu was moderately correlated with ileum Glu (r = 0.58; P = 0.014), but not with jejunum Glu (r = 0.18, P = 0.48), whereas plasma Gln was moderately correlated with ileum Gln (r = 0.57; P = 0.016) and strongly correlated with jejunum Gln (r = 0.80; P < 0.001). Plasma Orn was strongly correlated with both jejunum Orn (r = 0.73; P < 0.001) and ileum Orn (r = 0.78; P < 0.001). Plasma Cit was strongly correlated with jejunum Cit (r = 0.77; P < 0.001) and very strongly correlated with ileum Cit (r = 0.95; P < 0.001). Plasma Cit can be used to estimate gut Cit content and may be predictive of gut function in nursery pigs undergoing weaning stress.

338 A Survey of Mycotoxin Occurrence in Us Corn and Corn By-Product Feed Ingredients from 2018 to 2022 Harvest Years

Abstract The aim of this survey was to compare the occurrence and contamination levels of six major mycotoxin categories found in corn and corn by-product feed ingredients harvested in the United States from 2018 through 2022. Mycotoxins are secondary fungal metabolites that can negatively impact animal health and performance and have been routinely found in a variety of feedstuffs including, but not limited to, cereal grains, by-products, silages, dry hays and pasture grasses. Mycotoxin categories evaluated in this survey included: aflatoxins (AFLA), type A trichothecenes (A-Trich), type B trichothecenes (B-Trich), fumonisins (FUM), zearalenone (ZEN), and ochratoxin A (OTA). This study includes data from corn samples labeled as 2022 harvest and corn b-product feed ingredients submitted from October 2022 through March 2023 and were analyzed for 17 different metabolites via the Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) method. Samples that tested above the limit of detection for each mycotoxin group were analyzed using the generalized linear mixed model procedure of SAS (SAS 9.4, Cary, NC) using harvest year as a fixed effect. The effect of harvest year on average contamination level for each mycotoxin is presented in Table 1. For 2022 harvest year, 260 corn samples and 66 corn by-product samples have been evaluated to date. Mean B-Trich, ZEN, FUM and AFLA levels (parts per billion, ppb) for corn grain samples were similar in 2022 as previous years (P > 0.05). However, for corn grain samples, FUM prevalence and concentration were numerically greater in 2022 than in 2021 and 2020. In the 66 samples of corn by-products, FUM was found in all the samples (100%) and B-Trich were found in the majority of the samples (97%). Mean concentration of FUM in 2022 corn by-product samples was greater (P < 0.001) compared with 2021. Preliminary results of this survey suggest mycotoxin occurrence and contamination remains similar to previous harvest years except for greater mean FUM concentration levels in corn and corn by-products feed ingredients.

Resting pattern and preferences when lying down at night in a semi-captive population of African elephants

Despite recent improvements in semi-captive African elephant habitats and husbandry, night-time behaviour patterns are still not well documented in this species. Hence, this study investigated resting behaviour when lying down, using data recorded nine African elephants maintained in semi-captivity in South Africa. A total of 111 nights were recorded over a 33-month period (2015–2018). Closed circuit television cameras were used to record the elephant’s behaviour from 17:00–07:00. Three elephants were individually penned in a boma at night while the rest of the herd could move freely between the boma and an outdoor camp. Time laying down and standing up, location, resting side, head direction, sawdust quality, weather conditions, and number of elephants present in the boma were recorded. Data were analysed using linear mixed model and Chi-square test procedures. On average ( ± SE), the elephants slept laying down for 65.64 ± 0.79 mins (1–267 mins). They laid down 3.22 ± 0.05 times per night (0−8), with an average bout length of 69.79 ± 2.65 min and switching side at a rate of 51.03%. No difference in resting duration when laying down was observed between sex, age category, resting side, head direction, whether they were penned or not, and location (P > 0.05). Elephants rested longer laying down between 01:00 and 04:00 (P < 0.0001), with a peak between 01:00 and 02:00. They also rested longer when the weather was clear as compared to overcast and rainy conditions (P < 0.0001). An intermediate freshness of sawdust also resulted in longer resting duration (P < 0.001). Furthermore, elephants laid down more when they were unpenned (P < 0.05); when the sawdust was at an intermediate freshness (P < 0.001); when the number of elephants in the boma was at an intermediate number of 4 (P < 0.001); when the weather was overcast (P < 0.001), and when the ambient temperature was mild (P < 0.001). They were observed laying down more often between 23:00 and 03:00 (P < 0.001), with mature adults preferring the areas opposite to the main entrance of the boma while the younger elephants preferred areas closer to the main entrance (P < 0.0001). Finally, high individual variations were observed in resting duration, number of bouts and average bout length. Further studies are therefore needed to investigate how preferences, personality traits, life-history and external factors associated with eco-tourism can affect the quality of resting laying down in captive African elephants.

How to observe the principle of concurrent control in an arm-based meta-analysis using SAS procedures GLIMMIX and BGLIMM.

Network meta-analysis is a popular method to synthesize the information obtained in a systematic review of studies (e.g., randomized clinical trials) involving subsets of multiple treatments of interest. The dominant method of analysis employs within-study information on treatment contrasts and integrates this over a network of studies. One advantage of this approach is that all inference is protected by within-study randomization. By contrast, arm-based analyses have been criticized in the past because they may also recover inter-study information when studies are modeled as random, which is the dominant practice, hence violating the principle of concurrent control, requiring treated individuals to only be compared directly with randomized controls. This issue arises regardless of whether analysis is implemented within a frequentist or a Bayesian framework. Here, we argue that recovery of inter-study information can be prevented in an arm-based analysis by adding a fixed study main effect. This simple device means that it is possible to honor the principle of concurrent control in a two-way analysis-of-variance approach that is very easy to implement using generalized linear mixed model procedures and hence may be particularly welcome to those not well versed in the more intricate coding required for a contrast-based analysis.

Effects of dietary macronutrient profile on apparent total tract macronutrient digestibility and fecal microbiota, fermentative metabolites, and bile acids of female dogs after spay surgery

Obesity and estrogen reduction are known to affect the gut microbiota and gut microbial-derived metabolites in some species, but limited information is available in dogs. The aim of this study was to determine the effects of dietary macronutrient profile on apparent total tract macronutrient digestibility, fecal microbiota, and fecal metabolites of adult female dogs after spay surgery. Twenty-eight adult intact female beagles (age: 3.02 ± 0.71 yr, BW: 10.28 ± 0.77 kg; BCS: 4.98 ± 0.57) were used. After a 5-wk baseline phase (week 0), 24 dogs were spayed and randomly allotted to one of three experimental diets (n = 8 per group): 1) control (CO) containing moderate protein and fiber (COSP), 2) high-protein, high-fiber (HPHF), or 3) high-protein, high-fiber plus omega-3 and medium-chain fatty acids (HPHFO). Four dogs were sham-operated and fed CO (COSH). All dogs were fed to maintain BW for 12 wk after spay and then allowed to consume twice that amount for 12 wk. Fecal samples were collected at weeks 0, 12, and 24 for digestibility, microbiota, and metabolite analysis. All data were analyzed using repeated measures and linear mixed models procedure of SAS 9.4, with results reported as a change from baseline. Apparent organic matter and energy digestibilities had greater decreases in HPHF and HPHFO than COSH and COSP. Increases in fecal acetate, total short-chain fatty acids, and secondary bile acids were greater and decreases in primary bile acids were greater in HPHF and HPHFO. Principal coordinates analysis of weighted UniFrac distances revealed that HPHF and HPHFO clustered together and separated from COSH and COSP at weeks 12 and 24, with relative abundances of Faecalibacterium, Romboutsia, and Fusobacterium increasing to a greater extent and Catenibacterium, Bifidobacterium, Prevotella 9, Eubacterium, and Megamonas decreasing to a greater extent in HPHF or HPHFO. Our results suggest that high-protein, high-fiber diets alter nutrient and energy digestibilities, fecal metabolite concentrations, and fecal gut microbiota, but spay surgery had minor effects. Future research is needed to investigate how food intake, nutrient profile, and changes in hormone production influence gut microbiota and metabolites of dogs individually and how this knowledge may be used to manage spayed pets.

Voluntary daily fluctuation in dry matter intake is associated to feedlot performance, feeding behavior and rumen morphometrics in beef cattle

• Cattle from low-DMI fluctuation group improved feedlot performance. • High-DMI fluctuation pens took longer to eat and ruminate a kg of both DM and NDF. • High-DMI fluctuation negatively impacted blood gas profile and rumenitis scores. The degree of day-to-day fluctuation in voluntary DMI may influence feeding and rumination behavior, with potential impacts on feedlot performance. The purpose of this study was to evaluate the effect of voluntary fluctuation of DMI by pens of cattle given free-choice access to feed. These responses were measured in a dataset compiled from ten feedlot studies from 2006 to 2015. The data set included a total of 956 16-mo-old Bos indicus bulls (343.60 ± 44.15 kg) randomly allocated to a total of 238 pens (4 bulls per pen); cattle were fed high concentrate feedlot diets for an average of 106-d (minimum: 84-d; maximum: 140-d), and slaughtered at a commercial abattoir. To balance statistically for differences in animal background, type, and condition, for each year within treatments, pens were allocated into one of two groups: below or above the mean DMI fluctuation. The low- and the high- DMI fluctuation groups had day-to-day fluctuation in DMI of 5.18% and 6.22%, respectively ( P < 0.01) or 0.45 vs. 0.55 kg/day (P < 0.01). Data were analyzed using a generalized linear mixed model procedure with the statistical software R. Cattle within the low-DMI fluctuation group had 4.2% greater (1.48 vs. 1.42 kg/d; P < 0.01) ADG and 3.7% greater (2.26 vs. 2.18 kg/d; P < 0.001) DMI expressed as a percentage of mean BW; but no significant effect was detected for G:F ratio ( P = 0.91). Animals from the low-DMI fluctuation group had greater ( P ≤ 0.01) daily increase in LM area and 12 th rib fat as well as heavier HCW (266 vs. 263 kg; P = 0.04). Cattle from low-DMI fluctuation group consumed more DMI per meal ( P < 0.001) and had greater NDF intake ( P = 0.01) than cattle from the high-DMI fluctuation group. In addition, animals from high-DMI fluctuation pens took longer to consume and ruminate each kg of diet DM ( P < 0.01) and NDF ( P < 0.01) compared to cattle from low-DMI fluctuation group. A effect ( P = 0.04) was detected for blood base excess (BEB) with low-DMI fluctuation pens having a greater concentration of BEB. Moreover, the low-DMI fluctuation group tended to have ( P = 0.07) lower rumenitis scores. Although causal effects cannot be differentiated from casual relationships in this study, the fact that reduced daily DMI fluctuation was related to greater ADG and carcass weight makes this measurement relevant as an index of performance, and efforts to reduce DMI variability are warranted. Reducing day-to-day fluctuations in DMI should improve cattle health and performance.

Effects of seeding rate and hybrid relative maturity on yield, nutrient composition, ruminal in vitro neutral detergent fiber digestibility, and predicted milk yield of dairy cows in whole-plant corn forage in subtropical conditions

We aimed to evaluate the effects of season (SEA), relative maturity (RM), and seeding rate (SR) on yield, nutrient composition, ruminal in vitro NDF digestibility at 30 h, and predicted milk yield in whole-plant corn forage grown in subtropical regions. During the summer of 2016 and spring of 2017, 3 hybrids varying in RM [118 (RM118); 124 (RM124); and 130 (RM130) d] were planted at 2 different SR [63,000 (63K) or 73,000 (73K) plants/ha] in a randomized complete block design with 4 blocks. Data were analyzed as a 2 (SEA) × 3 (hybrid RM) × 2 (SR) factorial using linear mixed model procedures, where hybrid RM, SR, SEA, and all their interactions were fixed effects, and block was the sole random effect. A 3-way interaction was observed for DM yield, which was greatest (18.0 Mg/ha) for RM130 planted at 63K for summer and RM130 planted at 73K (27.0 Mg/ha) for spring. Later RM hybrids had lower starch concentration and yield, but both were also influenced by season. The NDF concentration was lowest for hybrid RM118. The NDF and TDN yield were both greater for the spring season compared with the summer, but both were influenced by SR. Predicted milk yield per megagram of forage (kg/Mg) and starch yield were both lowest when planted at 63K for hybrid RM130. Greater NDF and lower undigested NDF concentrations were observed in 63K compared with 73K for SR. Seeding rate can be used to increase DM yield, but yield is also affected by hybrid RM and season. Hybrid RM can also be used to increase DM yield with later RM or improve nutrient composition with an earlier RM hybrid by increasing starch concentration and decreasing NDF concentration. Additionally, the greater starch concentration combined with the greater DM yield observed for treatments in the spring season suggest producers should focus their efforts on growing the bulk of corn silage during the spring. Last, different levels of SR and different hybrid RM combinations can be used during different seasons to maximize corn silage productivity.

Effects of complete replacement of corn flour with sorghum flour in dairy cows fed Parmigiano Reggiano dry hay-based ration

The aim of this research was to evaluate of the productive responses of cows fed a dry hay based total mixed ration (TMR) in which sorghum (SOR) or corn (COR) meal (8 kg/h/d) were the main source of starch. The study involved two dairy herds located in the Parmigiano Reggiano PDO area, for a total of 1,400 cows (30% primiparous and 70% pluriparous). Each herd was fed alternatively SOR or COR TMR for 4 periods of 3 weeks: two weeks of adaptation and one week of data collection. Total milk production and composition, cheesemaking properties, fatty acid content and cheese yield were measured. In addition, within each herd, a random subgroup of 50 cows was selected for individual milk production, composition and cheesemaking properties analysis. Fibre digestibility was evaluated on faecal samples collected in 15 cows randomly selected in each subgroup. Data were analysed by a linear mixed model procedure with diet, herd, days in milk, parity and their interactions as fixed effects and cow as random effect. Individual milk production increase in SOR (32.43 vs. 31.34 kg, for SOR and COR, respectively; p<.0001) however, bulk milk and cheese yield did not show differences. Milk urea content was higher in SOR (27.38 vs. 22.79 mg/dL, for SOR and COR, respectively; p < .05). In this study the complete replacement of corn with finely ground sorghum meal in dairy cow diets in the Parmigiano Reggiano region did not result in negative effects on cows’ productivity, cheese making properties and production. Highlights Sorghum meal can be used as a substitute of corn without negative effect on herd productivity, milk quality and cheese yield. Replacement of corn meal with sorghum meal in the dairy cows’ ration permit to decrease the soy content of the ration, thus improving sustainability and costs of feeding. Utilising sorghum meal could increase the amount of crop produced in the geographical area of the farm, contributing to increase its economic sustainability.

6 Immunological aspects of ovarian follicle ovulation and corpus luteum formation in cattle

Ovarian follicle ovulation and subsequent luteinization have been described as a controlled inflammatory event, comprising tissue damage and repair. To elaborate this further in cattle, the contribution of immune cells to dominant follicle luteinization, ovulation, and corpus luteum formation was investigated. Ovulation in beef heifers was synchronized using an 8-day progesterone-based synchronization program. Heifers were slaughtered at a local abattoir at 5 timepoints (T): (T1) 24h before ovulation (n=10); (T2) 2h before ovulation (n=9); (T3) 6h after ovulation (n=10); (T4) 24h after ovulation (n=10); (T5) 72h after ovulation (n=10), and ovarian tissue was collected and returned to the laboratory on ice. Follicular fluid, theca, granulosa, and corpus luteum (CL) tissues were recovered by dissection and processed for analysis. The concentrations of a panel of cytokines were measured using an antibody-conjugated magnetic bead immunoassay. The abundance of T-lymphocytes, mast cells, neutrophils, eosinophils, monocytes, macrophages, and dendritic cells was determined by immunohistochemistry. The mRNA relative abundance of candidate genes, including angiogenic growth factors, adhesion factors, chemokines and cytokines, was determined by quantitative real-time PCR analysis. The resulting datasets were analysed using the linear mixed model procedure of SAS and data are presented as least squares means; reported differences were deemed significant at P≤0.05. The cytokines IFNy, IP-10, IL-10, IL-36RA, MCP-1, MIP-1a, MIP-1b, and VEGF-A were detected in follicular fluid. The concentrations (pg mL−1) of IL-10 and VEGF-A were significantly higher in T1 follicular fluid samples compared with T2 (7.70 vs. 0.86 and 2193.33 vs. 293.93, respectively). Although dendritic cells were the most abundant cells in bovine ovulatory follicular and early corpus luteum tissue at all time points (P&amp;lt;0.05), their numbers peaked in ovulatory (T2) thecal tissue (261.5 cells/mm2). The greatest number of neutrophils was identified in thecal tissue at T1 (45/mm2); thereafter, their numbers declined to 1.1/mm2 in CL tissue by T5. Similarly, the numbers of T-lymphocytes, mast cells, monocytes, and macrophages declined in CL tissue at T4 and T5. Candidate gene mRNA expression profiles appeared to be time- and tissue-specific; for example, IFNA was highest in the preovulatory granulosa tissue (T1), IL8 was highest in peri-ovulatory thecal tissue (T2), VEGFA and MMP9 were highest in the early CL tissue (T4 and T5), MMP1, TIMP1, and VCAM1 expression was highest in theca, granulosa, and CL tissue collected on or after ovulation (T2, T4, T5), expression of the prostaglandin-related genes PTGES and PTGS2 was lowest in CL tissue, and that of PTGIS was highest. The current findings support the hypothesis that ovulation in heifers is characterised by an initial proinflammatory cascade followed by a dramatic switch to tissue repair, growth, and remodelling, all occurring within a 72-h period and commencing with the LH surge. Our results highlight the roles of neutrophils, dendritic cells, and macrophages as the key actors in this process.

Butaphosphan and Cyanocobalamin Supplementation in Semen Extender on Chilled Boar Sperm Quality and Life Span.

The objective of the present study was to determine the effect of butaphosphan and cyanocobalamin supplementation in semen extender on chilled boar sperm quality and life span. A total of 35 ejaculates of boar semen were included. The semen was diluted with Beltsville thawing solution extender supplemented with different concentrations of butaphosphan and cyanocobalamin [0 (control), 0.1, 0.2, 0.3, 0.4, and 0.5%] in the diluted semen. The semen samples were evaluated using a computer-assisted sperm analysis system to determine sperm motility and sperm kinetic parameters (i.e., the curvilinear velocity, VCL; straight line velocity, VSL; average path velocity, VAP; linearity, LIN; straightness, STR; amplitude of lateral head, ALH; wobble, WOB; and beat cross frequency, BCF). Additionally, sperm viability, acrosome integrity, mitochondrial activity, and plasma membrane integrity were evaluated after 4 (day 0), 72 (day 3), 120 (day 5), and 168 (day 7) h of storage using SYBR-14–ethidium homodimer-1 (EthD-1), EthD-1, JC-1, and the short hypo-osmotic swelling test, respectively. The analyses were carried out by using the general linear mixed model (MIXED) procedure of SAS. The statistical models for each data set included group, day after storage, and interaction between group and day after storage. The boar was included as a random effect. On day 0 after storage, progressive motility, VCL, VSL, VAP, and plasma membrane integrity of boar sperm in 0.3% of butaphosphan and cyanocobalamin supplementation were greater than those in the 0.4 and 0.5% groups (P < 0.05). On day 3 after storage, total motility and progressive motility, VCL, VSL, VAP, LIN, WOB, BCF, and plasma membrane integrity in 0.3% of butaphosphan and cyanocobalamin supplementation were significantly greater than those in the control group (P < 0.05). The total motility and progressive motility, VAP, and WOB in 0.3% of butaphosphan and cyanocobalamin supplementation were greater than those in the control group on day 5 after storage (P < 0.05). No effects of butaphosphan and cyanocobalamin supplementation on acrosome integrity and mitochondria activity were found on days 3, 5, and 7 after storage. However, the motility and progressive motility and the values for all sperm kinetic parameters except ALH in 0.3% of butaphosphan and cyanocobalamin supplementation were greater than those in the control group on day 7 after storage (P < 0.05). In conclusion, 0.3% of butaphosphan and cyanocobalamin supplementation in semen extender improved sperm motility, sperm activity, morphology, and life span in chilled boar sperm.

PSVII-28 Program Chair Poster Pick: Effects of SMAD6 and SUGCT genes on Litter traits in pigs

Abstract One of the main goals in the swine industry is a finding of significant SNPs associated with Litter traits. Therefore, SNPs localized in SMAD6 (g.164674664G &amp;gt;A) and SUGCT (g.53672799A &amp;gt;G) genes are of great interest. The purpose of this work is optimizing the SNPs genotyping for real-time PCR technology and detection additive and dominant effects on Litter Traits. The research was conducted on Large White breed sows (n = 400). Total number born (TNB), Total number born alive (NBA), Litter weight, piglets born alive (BALWT) were estimated. Associative analysis was performed using the Linear mixed model (LMM) procedure in the R (coded additive effect 0, 1, 2 and dominant effect 0, 1, 0). For the Real-time PCR genotyping we used the TaqMan method, based on the 5`nuclease activity of Taq polymerase, which detaches nucleotides from oligonucleotide probes hybridized with DNA. Two TaqMan probes with different polymorphic sites are needed. These probes have different fluorescent dyes at the 5’ end and fluorescence dampers at the 3’ end. The primers for SNP_g.164674664G &amp;gt;A genotyping: R6G-TGGTCCCCGGGACAGGC-BHQ-1; FAM-TGGTTCCCGGGACAGGC-BHQ-1; F5’-CAAGGTCAGACAAAGGGACTGC-3’; R5’-ACTGTATGTAAAGCATGGTGTAAGC-3’ The primers for SNP_g.53672799A &amp;gt;G genotyping: FAM-CTTCAGCATCTGAACAGAACA-BHQ-1 R6G-CTTCAGCGTCTGAACAGAACA-BHQ-1 F5’-GGAACTGCTATCTGTTTCATTTCTT-3’ R5’-GGAGCTCAAATCCCAGTATTGC-3’. The additive and dominant effects of SNP_g.164674664G &amp;gt;A on Litter weight are determined. Litter weight were 1.8 (12.6%, P &amp;lt; 0.05) and 1.1 (7.7%, P &amp;lt; 0.05) higher in sows of SNP_g.164674664_AA and SNP_g.164674664_AG genotypes respectively. The negative dominant effect SNP_g.53672799_AG is found for TNB and BALWT (Table). Homozygous sows comparing to heterozygous, have higher TNB and BALWT by 0.7 (4.7%, P ≤ 0.05) and 1.0 (7.4%, P ≤ 0.05), respectively. The reported study was funded by RFBR according to the research project № 18-016-00050-A.

Free Calcium Concentration, Calpain-2 Activity, and Final Product Tenderness of Electrically Stimulated Beef

The objective of this study was to evaluate the effect of timing of electrical stimulation on free calcium concentration, calpain-2 activity, Warner-Bratzler shear force (WBSF), and consumer sensory analysis. Twenty-three beef steers were harvested and stimulated (S) using extra-low voltage or not stimulated (NS), at exsanguination and/or 1 h postmortem, resulting in 4 different stimulation treatments: NS-NS, NS-S, S-NS, or S-S. Samples were removed from the longissimus lumborum (LL) and semimembranosus (SM) for free calcium and calpain-2 analysis on days 1, 4, and 14 postmortem. WBSF and sensory analysis steaks were removed on day 4 and frozen (4 d) or aged to 14 d postmortem. Data were analyzed using the mixed model or generalized linear mixed model procedure of SAS (SAS Institute, Inc., Cary, NC), with significance determined at P < 0.05. There was a tendency for an aging-period-by-stimulation-treatment interaction for LL free calcium concentration (P = 0.05), and there was a significant difference between aging periods (P < 0.01). No difference was observed in free calcium concentration in the SM between stimulation treatments (P = 0.44); aging, however, significantly increased SM free calcium concentration (P < 0.01). Stimulation did not impact native calpain-2 activity in the LL (P = 0.71) or SM (P = 0.89). Stimulation treatment did not improve tenderness values for WBSF analysis for the LL (P = 0.69) or SM (P = 0.61) or consumer sensory analysis in the LL (P = 0.56) or SM (P = 0.36). A longer aging period tended to increase calpain-2 activity in the SM (P = 0.08), improve WBSF in the LL (P = 0.09), and significantly improve consumer tenderness scores in the SM (P < 0.01). In conclusion, the timing of electrical stimulation utilized in the current study tended to influence free calcium concentration in the LL but did not influence calpain-2 activity or beef tenderness. Aging, however, improved tenderness.

Antimicrobial activity as a potential factor influencing the predominance of Bacillus subtilis within the constitutive microflora of a whey reverse osmosis membrane biofilm

Current cleaning and sanitation protocols may not be adequately effective in cleaning separation membranes and can result in the formation of resilient multispecies biofilms. The matured biofilms may result in a bacterial predominance with resilient strains on membranes with a prolonged use. In our previous study, we isolated organisms such as Bacillus subtilis, Bacillus licheniformis, Exiguobacterium aurantiacum, and Acinetobacter radioresistens from an 18-mo-old reverse osmosis membrane. The competitive exclusion studies revealed the predominance of B. subtilis within the membrane biofilm microflora. This study investigated the antimicrobial activity of the B. subtilis isolate as a potential cause of its predominance. The culture isolate was propagated in tryptic soy broth at 37°C, and microfiltered to prepare cell-free extracts (CFE) at 8-, 10-, 12-, 14-, 16-, and 18-h intervals. The CFE were freeze-dried and suspended in minimum quantities of HPLC-grade water to prepare concentrated solutions. The antimicrobial activities of CFE were tested using the agar-well assay against the biofilm constitutive microflora. The experiments were conducted in triplicates and means were compared for significant differences using a general linear mixed model procedure. The results indicated the highest antimicrobial activity of 12-h CFE of B. subtilis against other constitutive microflora such as Exiguobacterium sp., E. auranticum, and A. radioresistens, with average inhibition zone sizes of 16.5 ± 0.00, 16.25 ± 0.66, and 20.6 ± 0.00 mm, respectively. Upon treatment with proteinase K, the CFE completely lost its antimicrobial activity, establishing it to be a proteinaceous compound. The AA profiling revealed the total crude protein in CFE to be 51% (wt/wt), with its major constituent as glutamic acid (11.30% wt/wt). The freeze-dried CFE was thermally stable on exposure to the common temperature used for sanitizer applications (23.8°C for 5 and 10 min) and over a pH range of 3.0 to 6.3. The study helped us understand the role of the antimicrobial compound produced by B. subtilis as a potential cause of its predominance within the biofilm constitutive microflora.

The effect of diet-induced obesity and pasture on blood pressure and serum cortisol in Standardbred mares.

Obesity is associated with insulin resistance, vascular dysfunction and altered cortisol metabolism both in humans and in horses. Evaluate the effect of weight gain induced by a haylage diet low in nonstructural carbohydrates (NSC) on insulin sensitivity, blood pressure and serum cortisol concentrations. In vivo experiment. Nine adult Standardbred mares fed a fat supplemented haylage diet at 250% of the horses' daily metabolisable energy requirements for 22weeks. Horses were then turned out on pasture for 4weeks. Insulin sensitivity (SICLAMP ) was measured before and after weight gain and after 4weeks of pasture using the euglycemic hyperinsulinaemic clamp (EHC) method. Body condition score (BCS), blood pressure and serum cortisol were monitored throughout the study. All data were analysed using the linear mixed model procedure. Values of P<0.05 were considered as statistically different. All horses became obese during the weight gain period (BCS>7). Mean arterial blood pressure (MAP) increased during the weight gain period and was significantly higher than initial values at the end of the weight gain period (78±3mm Hg vs 92±3mmHg). MAP remained increased on pasture (93±3mmHg). SICLAMP was unaffected by weight gain 0.9±0.1 vs 1.0±0.1 ([mg/kg/min×103 ]/[µIU/mL×mmol/L])) but improved after pasture (1.6±0.1 ([mg/kg/min×103 ]/ [mU/L]). Serum cortisol concentrations increased during the weight gain period (80±9nmol/L vs 112±9nmol/L) and remained increased during pasture. Limited number of horses and no control group. Obesity was associated with a linear increase in blood pressure and an increase in serum cortisol that was not associated with insulin sensitivity.