An analysis of culture-based methods used for the detection and isolation of Salmonella spp., Escherichia coli, and Enterococcus spp. from surface water: A systematic review.

Identification of methods for the standardized assessment of bacterial pathogens and antimicrobial resistance (AMR) in environmental water can improve the quality of monitoring and data collected, support global surveillance efforts, and enhance the understanding of environmental water sources. We conducted a systematic review to assemble and synthesize available literature that identified methods for assessment of prevalence and abundance of bacterial fecal indicators and pathogens in water for the purposes of monitoring bacterial pathogens and AMR. After screening for quality, 175 unique publications were identified from 15 databases, and data were extracted for analysis. This review identifies the most common and robust methods, and media used to isolate target organisms from surface water sources, summarizes methodological trends, and recognizes knowledge gaps. The information presented in this review will be useful when establishing standardized methods for monitoring bacterial pathogens and AMR in water in the United States and globally.

Interactions between corticotropin releasing factor signaling and prophylactic antibiotics on measures of intestinal function in weaned and transported pigs.

The study objective was to evaluate the interaction between corticotrophin releasing factor (CRF) receptor signaling and prophylactic antibiotic administration on intestinal physiology in newly weaned and transported pigs. Pigs (n = 56; 5.70 ± 1.05 kg) were weaned (20.49 ± 0.64 d), a blood sample was taken, and then pigs were given an intraperitoneal injection of saline (SAL; n = 28 pigs) or a CRF receptor antagonist (CRFA; n = 28 pigs; 30 µg/kg body weight; Astressin B), and then were transported in a livestock trailer for 12 h and 49 min. A second and third intraperitoneal injection was given at 4 h 42 min and 11 h 36 min into the transport process, respectively. Following transport, 4 SAL and 4 CRFA pigs were blood sampled and euthanized. The remaining 48 pigs were individually housed and given dietary antibiotics [AB; n = 12 SAL and 12 CRFA pigs; chlortetracycline (441 ppm) + tiamulin (38.6 ppm)] or no dietary antibiotics (NAB; n = 12 SAL and 12 CRFA pigs) for 14 d post-transport. Blood was collected at 12 h and on d 3, 7, and 14, and then pigs were euthanized on d 7 (n = 24) and d 14 (n = 24) post-weaning and transport. Circulating cortisol was reduced (p = 0.05) in CRFA pigs when compared to SAL pigs post-weaning and transport. On d 7, jejunal villus height and crypt depth was greater overall (p < 0.05) in AB-fed pigs versus NAB-fed pigs. On d 14, ileal crypt depth was reduced (p = 0.02) in CRFA pigs when compared to SAL pigs. Jejunal CRF mRNA abundance tended to be reduced (p = 0.09) on d 7 in CRFA pigs versus SAL pigs. On d 14, jejunal tumor necrosis factor-alpha was reduced (p = 0.01) in AB-fed pigs versus NAB-fed pigs. On d 7, change in glucose short-circuit current tended to be increased (p = 0.07) in CRFA pigs fed the AB diet when compared to CRFA pigs fed the NAB diet. In conclusion, CRFA pigs and pigs fed AB had some similar biological intestinal function measures post-weaning and transport.

A comparison of methods to detect low levels of Salmonella enterica in surface waters to support antimicrobial resistance surveillance efforts performed in multiple laboratories.

Developing effective and sensitive detection methods for antimicrobial resistant Salmonella enterica from surface water is a goal of the National Antimicrobial Resistance Monitoring System (NARMS). There are no specified methods for recovery of S. enterica in surface waters in the U.S. A multi-laboratory evaluation of four methods - bulk water enrichment (BW), vertical Modified Moore Swab (VMMS), modified Standard Method 9260.B2 (SM), and dead-end ultrafiltration (DEUF) - was undertaken to recover S. enterica from surface water. In Phase 1, one-liter volumes of water were collected from the same site on five different dates. Water was shipped and analyzed at four different laboratory locations (A, B, C, and D) for recovery of 1) inoculated fluorescent S. Typhimurium strain (ca. 30 CFU/L) and 2) Salmonella present in the water sampled. At each location, BW, VMMS, or SM recovery was performed on five separate 1 L water samples. Twenty 1 L water samples were subjected to each recovery method, and overall, sixty 1 L samples were assayed for Salmonella. Inoculated, fluorescent Salmonella Typhimurium and environmental Salmonella spp. were recovered from 65 % (39/60) and 45 % (27/60) of water samples, respectively. BW, VMMS, and SM recovered fluorescent S. Typhimurium from 60 %, 60 %, and 75 % of inoculated samples, respectively. Analysis by Chi-squared test determined laboratory location had a significant (p < 0.05) effect on fluorescent S. Typhimurium recovery compared to method or date of water collection. In Phase 2, recovery of inoculated fluorescent S. Typhimurium from 1 L samples by SM and DEUF was compared at laboratory locations B and D. SM and DEUF recovered fluorescent S. Typhimurium from 100 % (20/20) and 95 % (19/20) of inoculated water samples, respectively; laboratory location (p > 0.05) did not affect Salmonella recovery. Uniform laboratory methodology and training should be prioritized in conducting Salmonella recovery from surface water in laboratories.

A behavior and physiology-based decision support tool to predict thermal comfort and stress in non-pregnant, mid-gestation, and late-gestation sows.

BACKGROUND: Although thermal indices have been proposed for swine, none to our knowledge differentiate by reproductive stage or predict thermal comfort using behavioral and physiological data. The study objective was to develop a behavior and physiology-based decision support tool to predict thermal comfort and stress in multiparous (3.28 ± 0.81) non-pregnant (n = 11), mid-gestation (n = 13), and late-gestation (n = 12) sows. RESULTS: Regression analyses were performed using PROC MIXED in SAS 9.4 to determine the optimal environmental indicator [dry bulb temperature (TDB) and dew point] of heat stress (HS) in non-pregnant, mid-gestation, and late-gestation sows with respiration rate (RR) and body temperature (TB) successively used as the dependent variable in a cubic function. A linear relationship was observed for skin temperature (TS) indicating that TDB rather than the sow HS response impacted TS and so TS was excluded from further analyses. Reproductive stage was significant for all analyses (P < 0.05). Heat stress thresholds for each reproductive stage were calculated using the inflections points of RR for mild HS and TB for moderate and severe HS. Mild HS inflection points differed for non-pregnant, mid-gestation, and late gestation sows and occurred at 25.5, 25.1, and 24.0 °C, respectively. Moderate HS inflection points differed for non-pregnant, mid-gestation, and late gestation sows and occurred at 28.1, 27.8, and 25.5 °C, respectively. Severe HS inflection points were similar for non-pregnant and mid-gestation sows (32.9 °C) but differed for late-gestation sows (30.8 °C). These data were integrated with previously collected behavioral thermal preference data to estimate the TDB that non-pregnant, mid-gestation, and late-gestation sows found to be cool (TDB < TDB preference range), comfortable (TDB = TDB preference range), and warm (TDB preference range < TDB < mild HS). CONCLUSIONS: The results of this study provide valuable information about thermal comfort and thermal stress thresholds in sows at three reproductive stages. The development of a behavior and physiology-based decision support tool to predict thermal comfort and stress in non-pregnant, mid-gestation, and late-gestation sows is expected to provide swine producers with a more accurate means of managing sow environments.

Characterizing the effect of incrementally increasing dry bulb temperature on linear and nonlinear measures of heart rate variability in nonpregnant, mid-gestation, and late-gestation sows.

Characterizing the sow physiological response to an increased heat load is essential for effective heat stress mitigation. The study objective was to characterize the effects of a 400-min heating episode on sow heart rate variability (HRV) at different reproductive stages. HRV is a commonly used noninvasive proxy measure of autonomic function. Twenty-seven sows were enrolled in the study according to their gestation stage at time of selection: 1) nonpregnant (NP; n = 7), 2) mid-gestation (MID; 57.3 ± 11.8 d gestation; n = 11), and 3) late-gestation (LATE; 98.8 ± 4.9 d gestation; n = 8). The HRV data utilized in the study were collected from each pig as the dry bulb temperature in the room increased incrementally from 19.84 ± 2.15 °C to 35.54 ± 0.43 °C (range: 17.1-37.5 °C) over a 400-min period. After data collection, one 5-min set of continuous heart rate data were identified per pig for each of nine temperature intervals (19-20.99, 21-22.99, 23-24.99, 25-26.99, 27-28.99, 29-30.99, 31-32.99, 33-34.99, and 35-36.99 °C). Mean inter-beat interval length (RR), standard deviation of r-r intervals (SDNN), root mean square of successive differences (RMSSD), high frequency spectral power (HF), sample entropy (SampEn), short-term detrended fluctuation analysis (DFAα1), and three measures (%REC, DET, LMEAN) derived from recurrence quantification analysis were calculated for each data set. All data were analyzed using the PROC GLIMMIX procedure in SAS 9.4. Overall, LATE sows exhibited lower RR than NP sows (P < 0.01). The standard deviation of r-r intervals and RMSSD differed between each group (P < 0.01), with LATE sows exhibiting the lowest SDNN and RMSSD and NP sows exhibiting the greatest SDNN and RMSSD. Late-gestation sows exhibited lower HF than both MID and NP sows (P < 0.0001), greater DFA values than NP sows (P = 0.05), and greater DET compared to MID sows (P = 0.001). Late-gestation also sows exhibited greater %REC and LMEAN compared to MID (P < 0.01) and NP sows (all P < 0.01). In conclusion, LATE sows exhibited indicators of greater autonomic stress throughout the heating period compared to MID and NP sows. However, temperature by treatment interactions were not detected as dry bulb increased. Future studies are needed to fully elucidate the effect of gestational stage and increasing dry bulb temperature on sow HRV.

Pregnant pigs may be at a higher risk of poor physiological outcomes due to heat exposure compared to mature female pigs that are not pregnant. The purpose of this study was to evaluate the stress response of pregnant pigs to increasing environmental temperatures using heart rate variability, a noninvasive measure commonly used to evaluate the physiological stress response. Our findings show that pregnant pigs, particularly those who are closer to giving birth, exhibit greater evidence of physiological stress compared to pigs who are not pregnant. However, we did not find evidence that increasing environmental temperature throughout the experimental period was a primary reason for the increased stress exhibited by pregnant pigs. It is possible that the physiological changes that normally occur during pregnancy may have masked the physiological stress response typically associated with increased heat exposure.

Characterizing the postnatal hypothalamic-pituitary-adrenal axis response of in utero heat stressed pigs at 10 and 15 weeks of age.

In utero heat stress alters postnatal physiological and behavioral stress responses in pigs. However, the mechanisms underlying these alterations have not been determined. The study objective was to characterize the postnatal hypothalamic-pituitary-adrenal axis response of in utero heat-stressed pigs. Pigs were subjected to a dexamethasone suppression test followed by a corticotrophin releasing hormone challenge at 10 and 15 weeks of age. Following the challenge, hypothalamic, pituitary, and adrenal tissues were collected from all pigs for mRNA abundance analyses. At 10 weeks of age, in utero heat-stressed pigs had a reduced (P < 0.05) cortisol response to the corticotrophin releasing hormone challenge versus controls. Additionally, the cortisol response tended to be greater overall (P < 0.10) in 15 versus 10-week-old pigs in response to the dexamethasone suppression test. The cortisol response tended to be reduced overall (P < 0.10) in 15 versus 10-week-old pigs in response to the corticotrophin releasing hormone challenge. Hypothalamic corticotropin releasing hormone mRNA abundance tended to be greater (P < 0.10) in in utero heat-stressed versus control pigs at 15-weeks of age. In summary, in utero heat stress altered some aspects of the hypothalamic-pituitary-adrenal axis related to corticotropin releasing hormone signaling, and age influenced this response.

Thermoregulatory and physiological responses of nonpregnant, mid-gestation, and late-gestation sows exposed to incrementally increasing dry bulb temperature.

Gestating sows may be more susceptible to increasing dry bulb temperatures (TDB) due to greater metabolic heat production and increased body mass, especially as gestation advances. However, there are few studies on the thermoregulatory and physiological responses of sows at differing gestation stages exposed to gradually increasing temperatures. The study objective was to determine the thermoregulatory and physiological responses of nonpregnant (n = 12; parity 3.27 ± 0.86), mid-gestation (59.7 ± 9.6 d pregnant, n = 12; parity 3.25 ± 0.83), and late-gestation (99.0 ± 4.8 d pregnant, n = 12; parity 3.33 ± 0.75) sows exposed to increasing TDB. Prior to the experiment (5.0 ± 0.7 d), jugular catheters were placed in all sows. During the experiment, the TDB was increased incrementally by 2.45 ± 0.43 °C every 60 min from 19.84 ± 2.15 to 35.54 ± 0.43 °C over 400 min, and relative humidity was recorded at 40.49 ± 18.57%. Respiration rate (RR), heart rate (HR), skin temperature, and vaginal temperature were measured, and blood samples were obtained via the jugular catheter every 20 min. Data were analyzed using PROC MIXED in SAS 9.4. RR increased at a lower TDB (P < 0.01) in late-gestation sows compared with mid-gestation and nonpregnant sows, but no differences were detected between mid-gestation and nonpregnant sows. Overall, late-gestation sows had greater RR (P < 0.01; 23 ± 2 breaths per min [brpm]) compared with mid-gestation (16 ± 2 brpm) and nonpregnant (15 ± 2 brpm) sows. Late-gestation sows had an overall greater HR (P < 0.01; 84 ± 5 beats per min [bpm]) than mid-gestation (76 ± 5 bpm) and nonpregnant (69 ± 5 bpm) sows. Late-gestation sows had overall reduced bicarbonate and total carbon dioxide levels (P = 0.02; 23.89 ± 1.97 and 25.41 ± 2.07 mmol/L, respectively) compared with mid-gestation (27.03 ± 1.97 and 28.58 ± 2.07 mmol/L, respectively) and nonpregnant (26.08 ± 1.97 and 27.58 ± 2.07 mmol/L, respectively) sows. Moreover, late-gestation sows had overall greater nitric oxide levels (P < 0.01; 248.82 ± 34.54 µM) compared with mid-gestation (110.47 ± 34.54 µM) and nonpregnant (41.55 ± 34.54 µM) sows. In summary, late-gestation sows appear to be more sensitive to increasing TDB as indicated by thermoregulatory and physiological responses when compared with mid-gestation or nonpregnant sows. The results from this study provide valuable information regarding thermoregulatory thresholds of sows at differing gestation stages.

Replacing dietary antibiotics with 0.20% l-glutamine in swine nursery diets: impact on intestinal physiology and the microbiome following weaning and transport.

Previous research demonstrates that supplementing 0.20% l-glutamine (GLN) in the diets of newly weaned and transported pigs improves growth rate to a similar extent as providing dietary antibiotics (AB). However, research comparing the effects of GLN vs. AB on intestinal physiology and the microbiome is limited. Therefore, the study objective was to compare the effects of supplementing nursery diets with GLN, AB, or no dietary antibiotics (NA) on intestinal physiology and the microbiome of pigs in a production environment following weaning and transport. Mixed-sex piglets (N = 480; 5.62 ± 0.06 kg body weight [BW]) were weaned (18.4 ± 0.2 d of age) and transported for 12 h in central Indiana, for two replicates, during the summer of 2016 and the spring of 2017. Pens were blocked by BW and allotted to one of the three dietary treatments (n = 10 pens/dietary treatment/replicate [8 pigs/pen]): AB (chlortetracycline [441 ppm] + tiamulin [38.6 ppm]), GLN (0.20% as-fed), or NA fed for 14 d. From day 14 to 34, pigs were fed common AB-free diets in two phases. On day 33, villus height:crypt depth tended to be increased (P = 0.07; 7.0%) in GLN and AB pigs vs. NA pigs. On day 33, glucagon-like peptide 2 (GLP-2) mRNA abundance was decreased (P = 0.01; 50.3%) in GLN and NA pigs vs. AB pigs. Crypt depth was increased overall on day 33 (P = 0.01; 16.2%) during the spring replicate compared with the summer replicate. Villus height:crypt depth was reduced (P = 0.01; 9.6%) during the spring replicate compared with the summer replicate on day 33. On day 13, tumor necrosis factor-alpha and occludin mRNA abundance was increased (P ≤ 0.04; 45.9% and 106.5%, respectively) and zonula occludens-1 mRNA abundance tended to be greater (P = 0.10; 19.2%) in the spring replicate compared with the summer replicate. In addition, AB pigs had increased (P = 0.01; 101.3%) GLP-2 mRNA abundance compared with GLN and NA pigs. Microbiome analysis indicated that on day 13, dietary treatment altered the microbiota community structure (P = 0.03). Specifically, the AB pigs tended to be distinct from both the NA and GLN pigs (P = 0.08), and Lactobacillus was increased nearly 2-fold in AB compared with NA pigs (q = 0.04) and GLN pigs (q = 0.22). In conclusion, GLN supplementation tended to improve some morphological markers of intestinal health similarly to AB pigs, while the microbiome composition in GLN pigs was more similar to NA pigs than AB pigs.

Effects of Feed Removal during Acute Heat Stress on the Cytokine Response and Short-Term Growth Performance in Finishing Pigs.

The study objective was to evaluate the effects of feed removal during acute heat stress (HS) on the cytokine response and its short-term effect on growth performance in finishing pigs. Thirty-two pigs (93.29 ± 3.14 kg initial body weight; 50% barrows and 50% gilts) were subjected to thermoneutral (TN; 23.47 ± 0.10 °C; n = 16 pigs) or HS (cycling of 25 to 36 °C; n = 16 pigs) conditions for 24 h. Within each temperature treatment, 50% of the pigs were provided with feed (AF; n = 8 pigs/temperature treatment) and 50% of the pigs had no feed access (NF; n = 8 pigs/temperature treatment). Following the 24 h temperature and feeding treatment (TF) period, all pigs had ad libitum access to feed and water and were maintained under TN conditions for 6 d. During the first 12 h of the TF period, gastrointestinal (TGI) and skin (Tsk) temperatures were recorded every 30 min. Serum cytokines were determined at 0, 4, 8, 12, and 24 h during the TF period and on Days 3 and 6 of the post-TF period. Average daily gain (ADG) and average daily feed intake were measured on Days 1, 3, and 6 of the post-TF period. Behavioral data were collected from Days 1 to 6 of the post-TF period. Heat stress increased (p < 0.02) the TGI and Tsk. During the post-TF period, interleukin-1α was greater (p < 0.01) in HS + NF compared to HS + AF and TN + NF pigs. From Days 1 to 2 of the post-TF period, the ADG was reduced (p < 0.01) in TN + AF compared to HS + AF, HS + NF, and TN + NF pigs. In conclusion, feed removal during an acute HS challenge did not reduce the cytokine response or improve short-term growth performance in finishing pigs.

In utero heat stress alters the postnatal innate immune response of pigs.

The effects of in utero heat stress (IUHS) range from decreased growth performance to altered behavior, but the long-term impact of IUHS on postnatal innate immune function in pigs is unknown. Therefore, the study objective was to determine the effects of early gestation IUHS on the immune, metabolic, and stress response of pigs subjected to an 8 hr lipopolysaccharide (LPS) challenge during postnatal life. Twenty-four pregnant gilts were exposed to thermoneutral (TN; n = 12; 17.5 ± 2.1 °C) or heat stress (HS; n = 12; cyclic 26 to 36 °C) conditions from days 6 to 59 of gestation, and then TN conditions (20.9 ± 2.3 °C) from day 60 of gestation to farrowing. At 12 wk of age, 16 IUHS and 16 in utero thermoneutral (IUTN) pigs were selected, balanced by sex and given an intravenous injection of LPS (2 µg/kg BW mixed with sterile saline [SAL] and injected at 2 µL/kg BW) or SAL (2 µL/kg BW). Body temperature was monitored every 30 min, and blood was obtained at 0, 1, 2, 3, 4, 6, and 8 hr following the LPS challenge. Blood samples were analyzed for glucose, insulin, non-esterified fatty acids (NEFA), cortisol, and cytokine concentrations. In addition, white blood cell counts were determined at 0 and 4 hr. Hour 0 data were used as covariates. Body temperature was increased (P < 0.01) in LPS (40.88 ± 0.08 °C) vs. SAL (39.83 ± 0.08 °C) pigs. Eosinophils tended to be decreased overall (P = 0.09; 43.9%) in IUHS vs. IUTN pigs. Glucose concentrations were reduced overall (P = 0.05; 5.9%) in IUHS vs. IUTN pigs. The NEFA concentrations tended to be greater (P = 0.07; 143.4%) in IUHS-LPS pigs compared with all other treatments, and IUTN-LPS pigs tended to have greater (127.4%) circulating NEFA concentrations compared with IUTN-SAL and IUHS-SAL pigs. Cortisol was increased (P = 0.04) in IUHS-LPS compared with IUTN-LPS pigs at 3 hr (21.5%) and 4 hr (64.3%). At 1 hr, tumor necrosis factor α was increased (P = 0.01; 115.1%) in IUHS-LPS compared with IUTN-LPS pigs. Overall, interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) were greater (P < 0.04; 281.3% and 297.8%, respectively) in IUHS-LPS pigs compared with all other treatments, and IUTN-LPS pigs had increased IL-1ß and IL-6 concentrations compared with IUTN-SAL and IUHS-SAL pigs. In summary, IUHS altered the postnatal cytokine, metabolic, and physiological stress response of pigs during postnatal life, which may have negative implications toward the innate immune response of IUHS pigs to pathogens.

Evaluation and mitigation of the effects of in utero heat stress on piglet growth performance, postabsorptive metabolism, and stress response following weaning and transport.

In utero heat stress (IUHS) increases the energy requirements of pigs during postnatal life, and this may compound weaning and transport stress. The study objective was to evaluate and mitigate the negative effects of IUHS following weaning and transport through the provision of a nutrient-dense (ND) nursery diet formulated to meet the greater energy requirements of IUHS pigs during the first 14 d postweaning and transport. Twenty-four pregnant gilts were exposed to thermoneutral (TN; n = 12; 17.5 ± 2.1 °C) or heat stress (HS; n = 12; cycling 26 to 36 °C) conditions for the first half of gestation (day 6 to 59) and then TN conditions (20.9 ± 2.3 °C) until farrowing. Nine TN gilts and 12 HS gilts produced litters. At weaning (16.2 ± 0.4 d), mixed-sex piglets (N = 160; 4.78 ± 0.15 kg body weight [BW]) were transported (loading + transport + unloading) for 11 h 40 min. Following transport, piglets were blocked into pens (n = 4 pigs/pen) by in utero and dietary treatments: in utero thermoneutral (IUTN) + control (C) diet (n = 10 pens), IUTN + ND (n = 10 pens), IUHS + C (n = 10 pens), and IUHS + ND (n = 10 pens). Treatment diets were fed from day 1 to 14 postweaning and transport (period 1), and the C diet was fed to all pigs from day 14 to 35 postweaning and transport (period 2). Production measures were taken in 7 d intervals to calculate average daily gain (ADG), average daily feed intake (ADFI), average daily net energy intake (ADEI), gain:feed, and gain:net energy intake. Blood samples were collected prior to transport, following transport, and on days 2, 7, 14, 28, and 35 postweaning and transport to analyze cortisol, glucose, insulin, and nonesterified fatty acids. Behavior was assessed through video-recording on days 3, 5, 8, 11, and 13 postweaning and transport. In period 1, ADG was reduced (P = 0.04; 20.0 g/d) in IUHS vs. IUTN pigs. Pigs fed ND diets had reduced ADFI (P = 0.02; 9.3%) compared with C diet-fed pigs during period 1, which resulted in similar ADEI (P = 0.23; 1,115 ± 35 kcal/d). During transport, cortisol was decreased (P = 0.03; 25.8%) in IUHS vs. IUTN pigs. On day 2, glucose was decreased (P = 0.01; 13.8%) in IUHS vs. IUTN pigs. No in utero treatment-related behavior differences were observed but lying was reduced (P = 0.03; 6.5%) and standing was increased (P = 0.04; 14.1%) in ND vs. C pigs overall. In summary, IUHS reduced growth performance in pigs following weaning and transport, and providing an ND diet did not rescue the lost performance.

Replacing dietary antibiotics with 0.20% l-glutamine and synbiotics following weaning and transport in pigs.

Dietary antibiotic use has been limited in swine production due to concerns regarding antibiotic resistance. However, this may negatively impact the health, productivity, and welfare of pigs. Therefore, the study objective was to determine if combining dietary synbiotics and 0.20% l-glutamine would improve pig growth performance and intestinal health following weaning and transport when compared with traditionally used dietary antibiotics. Because previous research indicates that l-glutamine improves swine growth performance and synbiotics reduce enterogenic bacteria, it was hypothesized that supplementing diets with 0.20% l-glutamine (GLN) and synbiotics (SYN; 3 strains of Lactobacillus [1.2 × 10^9 cfu/g of strain/pig/d] + ß-glucan [0.01 g/pig/d] + fructooligosaccharide [0.01 g/pig/d]) would have an additive effect and improve pig performance and intestinal health over that of dietary antibiotics. Mixed-sex pigs (N = 226; 5.86 ± 0.11 kg body weight [BW]) were weaned (19.4 ± 0.2 d of age) and transported for 12 h in central Indiana. Pigs were blocked by BW and allotted to one of two dietary treatments (5 to 6 pigs per pen): antibiotics (positive control [PC]; chlortetracycline [441 ppm] + tiamulin [38.5 ppm]), no antibiotics (negative control [NC]), GLN, SYN, or the NC diet with both the GLN and SYN additives (GLN + SYN) fed for 14 d. From day 14 post-weaning to the end of the grow-finish period, all pigs were provided common antibiotic-free diets. Data were analyzed using PROC GLIMMIX and PROC MIXED in SAS 9.4. Overall, haptoglobin was greater (P = 0.03; 216%) in NC pigs compared with PC pigs. On day 13, GLN and PC pigs tended to have reduced (P = 0.07; 75.2% and 67.3%, respectively) haptoglobin compared with NC pigs. On day 34, the jejunal goblet cell count per villi and per millimeter tended to be greater (P < 0.08; 71.4% and 62.9%, respectively) in SYN pigs compared with all other dietary treatments. Overall, jejunal mucosa tumor necrosis factor-alpha (TNFα) gene expression tended to be greater (P = 0.09; 40.0%) in NC pigs compared with PC pigs on day 34. On day 34, jejunal mucosa TNFα gene expression tended to be greater (P = 0.09; 33.3%, 41.2%, and 60.0%, respectively) in GLN pigs compared with SYN, GLN + SYN, and PC pigs. Although it was determined that some metrics of pig health were improved by the addition of GLN and SYN (i.e., haptoglobin and goblet cell count), overall, there were very few differences detected between dietary treatments and this may be related to the stress load incurred by the pigs.

Large-Scale Phenotyping of Livestock Welfare in Commercial Production Systems: A New Frontier in Animal Breeding.

Genomic breeding programs have been paramount in improving the rates of genetic progress of productive efficiency traits in livestock. Such improvement has been accompanied by the intensification of production systems, use of a wider range of precision technologies in routine management practices, and high-throughput phenotyping. Simultaneously, a greater public awareness of animal welfare has influenced livestock producers to place more emphasis on welfare relative to production traits. Therefore, management practices and breeding technologies in livestock have been developed in recent years to enhance animal welfare. In particular, genomic selection can be used to improve livestock social behavior, resilience to disease and other stress factors, and ease habituation to production system changes. The main requirements for including novel behavioral and welfare traits in genomic breeding schemes are: (1) to identify traits that represent the biological mechanisms of the industry breeding goals; (2) the availability of individual phenotypic records measured on a large number of animals (ideally with genomic information); (3) the derived traits are heritable, biologically meaningful, repeatable, and (ideally) not highly correlated with other traits already included in the selection indexes; and (4) genomic information is available for a large number of individuals (or genetically close individuals) with phenotypic records. In this review, we (1) describe a potential route for development of novel welfare indicator traits (using ideal phenotypes) for both genetic and genomic selection schemes; (2) summarize key indicator variables of livestock behavior and welfare, including a detailed assessment of thermal stress in livestock; (3) describe the primary statistical and bioinformatic methods available for large-scale data analyses of animal welfare; and (4) identify major advancements, challenges, and opportunities to generate high-throughput and large-scale datasets to enable genetic and genomic selection for improved welfare in livestock. A wide variety of novel welfare indicator traits can be derived from information captured by modern technology such as sensors, automatic feeding systems, milking robots, activity monitors, video cameras, and indirect biomarkers at the cellular and physiological levels. The development of novel traits coupled with genomic selection schemes for improved welfare in livestock can be feasible and optimized based on recently developed (or developing) technologies. Efficient implementation of genetic and genomic selection for improved animal welfare also requires the integration of a multitude of scientific fields such as cell and molecular biology, neuroscience, immunology, stress physiology, computer science, engineering, quantitative genomics, and bioinformatics.

The anorexigenic effect of neuropeptide K in chicks involves the paraventricular nucleus and arcuate nucleus of the hypothalamus.

Neuropeptide K (NPK) induces satiety in birds and mammals. We demonstrated that in birds this effect was associated with the hypothalamus, but beyond this little is known in any species regarding the central mechanism of action. Thus, this study was designed to identify hypothalamic molecular mechanisms associated with the food intake-inhibiting effects of NPK in chicks. In Experiment 1, intracerebroventricular (ICV) injection of 1.0 and 3.0 nmol of NPK reduced food intake and we identified an effective dose for microinjection. In Experiment 2, food intake was reduced when NPK was microinjected into the PVN. In Experiment 3, whole hypothalamus was collected from chicks at 1 h post-ICV NPK injection. The abundance of corticotropin-releasing factor (CRF) and agouti-related peptide (AgRP) mRNA was reduced in NPK-injected chicks. In Experiment 4, within the isolated paraventricular nucleus (PVN) there was less CRF mRNA, and within the arcuate nucleus (ARC) there was less AgRP mRNA, in NPK- than vehicle-treated chicks at 1 h post-injection. We conclude that there are first order neurons for NPK that reside within the PVN, and the anorexigenic effect of NPK is associated with a decrease in AgRP in the ARC.

Anorexigenic effects of mesotocin in chicks are genetic background-dependent and are associated with changes in the paraventricular nucleus and lateral hypothalamus.

Mesotocin (MT) decreases food intake and induces hyperthermia in chicks although hypothalamic mechanisms are unknown. The purpose of this study was thus to investigate effects of receptor antagonists and MT on feeding behavior and hypothalamic physiology. Intracerebroventricular injection of 2.5 nmol into broiler chicks was associated with decreased food intake for 180 min and water intake from 60 to 180 min. Cloacal temperatures were elevated in chicks injected with 0.156 and 0.625 nmol at 30 and 60 min, and up to 180 min in those injected with 2.5 nmol. MT also increased temperatures and decreased food and water intake in chicks from lines selected for low (LWS) or high (HWS) body weight with a higher dose threshold but longer food intake response in HWS chicks. An oxytocin receptor antagonist prevented MT-mediated changes in food intake but not water intake or temperature. Yohimbine, an α2-adrenergic receptor antagonist, did not affect food intake, temperature, or MT-mediated effects. MT increased c-Fos immunoreactivity in the paraventricular nucleus (PVN) and lateral hypothalamus (LH). Hypothalamic agouti-related peptide, corticotropin-releasing factor receptor sub-type 1, and melanocortin receptor 3 mRNAs increased in response to MT. There was increased MT mRNA in the LH and L-aromatic amino acid decarboxylase mRNA in the PVN of MT-injected chicks. In conclusion, MT induced anorexia and hyperthermia and reduced water intake. MT was associated with activation of the PVN and LH and differences in the mRNA abundance of some appetite-associated factors, thus implicating these nuclei and several signaling pathways in the effects observed.

Central injection of oxytocin reduces food intake and affects hypothalamic and adipose tissue gene expression in chickens.

Oxytocin (OT) is a well-characterized neurotransmitter that participates in a wide range of physiological processes including the inhibition of food intake. The avian ortholog, mesotocin (MT), differs from OT by a single amino acid. Little is known regarding the function of OT in regulating energy balance in birds; thus, this study was designed to determine the effects of central OT injection on food intake and adipose tissue physiology in chicks. At 4-d post-hatch, broiler chicks were fasted for 3 h and injected intracerebroventricularly with 0 (vehicle), 0.63, 2.5, 5.0, or 10 nmol OT. Oxytocin decreased food and water intake during the entire 180-min observation period. The reduction in water intake was likely not prandial because chicks that were food restricted after OT injection also drank less. There was increased c-Fos immunoreactivity in several appetite-associated hypothalamic nuclei in OT-injected chicks at 1 h, including the arcuate (ARC), dorsomedial nucleus (DMN), lateral hypothalamus (LH), paraventricular nucleus (PVN), and ventromedial hypothalamus (VMH). OT treatment was associated with reduced hypothalamic corticotropin-releasing factor (CRF) mRNA and increased cloacal temperature at 1 h post-injection. We then investigated appetite- and adipose tissue-associated effects of OT in chicks from lines that have undergone long-term selection for either low (LWS) or high (HWS) juvenile body weight. Central injection of OT decreased food intake in both lines with the magnitude of response greater in the HWS than LWS chicks. Adipose tissue abundance of fatty acid-binding protein 4, monoglyceride lipase (MGLL), MT, and perilipin-1 mRNA was greater in LWS than HWS chicks. Lipoprotein lipase, MGLL, and MT mRNAs increased in response to OT injection in LWS but not HWS chicks. In conclusion, central injection of OT induced anorexia, reduced water intake, increased body temperature, and was associated with activation of the ARC, DMN, LH, PVN, and VMH in the hypothalamus. The effects on appetite and body temperature may involve CRF signaling in the hypothalamus and lipolysis in the adipose tissue, respectively. There were differences in the appetite, and adipose tissue response to OT in body weight-selected lines of chicks supports that MT plays a role in energy balance regulation in chickens.

Fasting and refeeding induce differential changes in hypothalamic mRNA abundance of appetite-associated factors in 7 day-old Japanese quail, Coturnix japonica.

There is little information regarding effects of fasting on feeding behavior and hypothalamic physiology in young Japanese quail. The aim was thus to measure food intake and hypothalamic mRNA in response to fasting and refeeding. Five d-old quail ate little during the dark cycle. Food intake was greatest during the first 2 h of the light cycle. Six day-old quail fasted for 6 h ate the most during the first 15 min of refeeding. In 7 d-old quail, 3 h of fasting up-regulated hypothalamic neuropeptide Y (NPY), NPY receptor sub-type 2 (NPYR2), agouti-related peptide (AgRP), orexin receptor 2 (ORXR2), melanocortin receptors 3 and 4 (MC3R and MC4R, respectively), and neuropeptide S (NPS) and decreased corticotropin-releasing factor receptor sub-type 1 (CRFR1) mRNA. Quail fasted for 3 h and refed for 1 h had greater NPY, AgRP, POMC, and MC3R but less CRFR1 mRNA than fed quail. Quail fasted for 6 h expressed more NPY, NPYR1, NPYR2, and MC3R and less ORXR2, prolactin releasing peptide (PrRP), cocaine- and amphetamine-regulated transcript (CART), and calcitonin (CAL) mRNA than fed quail. Quail fasted for 6 h and refed for 1 h expressed more NPY, NPYR1, NPYR2, AgRP, MC3R, MC4R, and NPS and less galanin, ORXR2, PrRP, CART, and CAL mRNA than fed birds. Hence, fasting induced changes in hypothalamic mRNA, with the largest changes occurring in genes associated with NPY and melanocortin signaling. Most genes remained elevated or downregulated after refeeding, suggesting that there was a time lag for transcription to respond to compensatory feeding.

Appetite-associated responses to central neuropeptide Y injection in quail.

The appetite-associated effects of neuropeptide Y (NPY) have been extensively studied in mammalian models. Less knowledge exists for other vertebrate species including birds. The aim of this study was to determine the effects of central injection of NPY on feeding behavior and hypothalamic physiology in 7 day-old Japanese quail (Coturnix japonica). During the light cycle, intracerebroventricular injection of 1.9 pmol, 0.5, and 1.0 nmol doses of NPY did not affect food intake, 0.031 to 0.13 nmol increased food intake, and 2.0 nmol NPY decreased food intake, in comparison to vehicle injection. Multiple doses of NPY stimulated water intake, but when food was not available, water intake was not affected. When injected during the dark cycle, NPY did not influence food intake. NPY-injected chicks had more c-Fos immunoreactive cells in the arcuate nucleus of the hypothalamus (ARC) and greater hypothalamic agouti-related peptide and neuropeptide Y receptors 1 and 2 (NPYR1 and NPYR2, respectively) mRNA than vehicle-injected chicks. Within the ventromedial hypothalamus, NPY-treated chicks expressed less NPYR1 mRNA, within the dorsomedial hypothalamus less NPY mRNA, and in the ARC greater NPYR2 mRNA than vehicle-injected chicks. Lastly, quail injected with NPY increased feeding pecks, escape attempts, and time spent preening, while locomotion, the number of steps, and time spent perching decreased compared to chicks injected with the vehicle. Results demonstrate that NPY stimulates food intake in quail, consistent with mammals and other avian species, but with some unique responses at the molecular level that are not documented in other species.

Dietary macronutrient composition affects hypothalamic appetite regulation in chicks.

OBJECTIVE: The objective was to determine the effects of high-protein and high-fat diets, and fasting and refeeding, on appetite regulation in chicks. METHODS: Day of hatch chicks were fed one of four diets: basal, high protein (25% crude protein), and 15 and 30% high fat (15 and 30% metabolizable energy derived from soybean oil, respectively), and assigned to one of three treatments at 4 days: (1) access to feed, (2) 3 hours of fasting, or (3) fasting followed by 1 hour of refeeding. The hypothalamus was collected, total RNA isolated, and mRNA abundance measured. RESULTS: Food intake was reduced in chicks fed the high-protein and high-fat diets. Agouti-related peptide, neuropeptide Y (NPY), NPY receptors 1, 2, and 5, melanocortin receptors 3 and 4 (MC3R and 4R, respectively), mesotocin, corticotropin-releasing factor (CRF), and CRF receptor sub-type 2 (CRFR2) mRNAs were greatest in chicks that consumed the basal diet. Refeeding was associated with increased MC3R mRNA in the high-protein diet group. CRFR2 mRNA was increased by fasting and refeeding in chicks that consumed the high-protein diet. DISCUSSION: Food intake and hypothalamic gene expression of some important appetite-associated factors were reduced in chicks fed the high-protein or high-fat diets. Fasting and refeeding accentuated several differences and results suggest that the CRF and melanocortin pathways are involved.

Dietary macronutrient composition and central neuropeptide Y injection affect dietary preference and hypothalamic gene expression in chicks.

OBJECTIVE: The objective of this study was to determine the influence of dietary macronutrient composition on central NPY's orexigenic effect in chicks. METHODS: Day-of-hatch chicks were fed one of three diets (3000 kcal ME/kg) ad libitum from hatch: high carbohydrate (HC), high fat (HF; 30% ME derived from soybean oil), and high protein (HP; 25 vs. 22% CP). In Experiment 1, chicks received intracerebroventricular injections of 0 (vehicle), 0.2, or 2.0 nmol NPY on day 4 and food intake was recorded for 6 hours. In Experiment 2, chicks were given all three diets before and after injection. In Experiment 3, hypothalamus was collected at 1-hour post-injection for gene expression analysis. RESULTS: The HC diet-fed chicks responded with a greater increase, while the chicks fed the HF diet had a lower threshold response in food intake to NPY. Neuropeptide Y dose-dependently increased food intake in chicks fed the HC and HP diets. Chicks administered 0.2 nmol NPY preferred the HC and HP diets over the HF diet. Relative quantities of hypothalamic NPYR1 and MC4R mRNA were reduced by NPY in chicks that consumed the HP and HC diets, respectively. DISCUSSION: Consumption of the HC diet was associated with the most robust NPY-induced increase in food intake. Injection of NPY accentuated differences among dietary groups in hypothalamic gene expression of several appetite-associated factors, results suggesting that the NPY/agouti-related peptide and melanocortin pathways are associated with some of the diet- and NPY-induced differences observed in this study.