Proteome and secretome profiling of zinc availability in Cryptococcus neoformans identifies Wos2 as a subtle influencer of fungal virulence determinants.

BACKGROUND: Fungal infections impact over 25% of the global population. For the opportunistic fungal pathogen, Cryptococcus neoformans, infection leads to cryptococcosis. In the presence of the host, disease is enabled by elaboration of sophisticated virulence determinants, including polysaccharide capsule, melanin, thermotolerance, and extracellular enzymes. Conversely, the host protects itself from fungal invasion by regulating and sequestering transition metals (e.g., iron, zinc, copper) important for microbial growth and survival. RESULTS: Here, we explore the intricate relationship between zinc availability and fungal virulence via mass spectrometry-based quantitative proteomics. We observe a core proteome along with a distinct zinc-regulated protein-level signature demonstrating a shift away from transport and ion binding under zinc-replete conditions towards transcription and metal acquisition under zinc-limited conditions. In addition, we revealed a novel connection among zinc availability, thermotolerance, as well as capsule and melanin production through the detection of a Wos2 ortholog in the secretome under replete conditions. CONCLUSIONS: Overall, we provide new biological insight into cellular remodeling at the protein level of C. neoformans under regulated zinc conditions and uncover a novel connection between zinc homeostasis and fungal virulence determinants.

Nonlinear analysis of heart rate variability for evaluating the growing pig stress response to an acute heat episode.

Heart rate variability (HRV) is a proxy measure of autonomic function and can be used as an indicator of swine stress. While traditional linear measures are used to distinguish between stressed and unstressed treatments, inclusion of nonlinear HRV measures that evaluate data structure and organization shows promise for improving HRV interpretation. The objective of this study was to evaluate the inclusion of nonlinear HRV measures in response to an acute heat episode. Twenty 12- to 14-week-old growing pigs were individually housed for 7 days and acclimated to thermoneutral conditions (20.35°C ± 0.01°C; 67.6% ± 0.2% RH) before undergoing one of the two treatments: (1) thermoneutral control (TN; n = 10 pigs) or (2) acute heat stress (HS; n = 10 pigs; 32.6°C ± 0.1°C; 26.2% ± 0.1% RH). In Phase 1 of the experimental procedure (P1; 60 min), pigs underwent a baseline HRV measurement period in thermoneutral conditions before treatment [Phase 2; P2; 60 min once gastrointestinal temperature (Tg) reached 40.6°C], where HS pigs were exposed to heated conditions and TN pigs remained in thermoneutral conditions. After P2, all pigs were moved back to thermoneutral conditions (Phase 3; P3; 60 min). During each phase, Tg data were collected every 5 min and behavioural data were collected to evaluate the amount of time each pig spent in an active posture. Additionally, linear (time and frequency domain) and nonlinear [sample entropy (SampEn), de-trended fluctuation analysis, percentage recurrence, percentage determinism (%DET), mean diagonal line length in a recurrence plot] HRV measures were quantified. Heat stressed pigs exhibited greater Tg (P = 0.002) and spent less time in an active posture compared to TN pigs during P2 (P = 0.0003). Additionally, low frequency to high frequency ratio was greater in HS pigs during P3 compared to TN pigs (P = 0.02). SampEn was reduced in HS pigs during P2 (P = 0.01) and P3 (P = 0.03) compared to TN pigs. Heat stressed pigs exhibited greater %DET during P3 (P = 0.03) and tended to have greater %DET (P = 0.09) during P2 than TN pigs. No differences between treatments were detected for the remaining HRV measures. In conclusion, linear HRV measures were largely unchanged during P2. However, changes to SampEn and %DET suggest increased heat stress as a result of the acute heat episode. Future work should continue to evaluate the benefits of including nonlinear HRV measures in HRV analysis of swine heat stress.

Short communication: Assessment of disbudding pain in dairy calves using nonlinear measures of heart rate variability.

The purpose of this study was to evaluate whether pain-related stress caused by disbudding could be detected using nonlinear measures of heart rate variability (HRV). Twenty-five female Holstein calves (4-7 wk of age) were randomly assigned to 1 of 3 treatments: (1) sham disbud (SHAM; n = 9), (2) disbud with lidocaine-meloxicam pain mitigation (MED; n = 8), or (3) disbud without pain mitigation (NoMED; n = 8). Heart rate variability (sample entropy, percentage determinism, percentage recurrence, or mean length of diagonal lines in a recurrence plot) was recorded on d -1, 0, 1, 3, and 5 relative to the experimental procedure, with disbudding taking place on d 0. The short-term detrended fluctuation analysis scaling exponent was greater in MED calves than in SHAM calves, indicating a greater stress response to the disbudding procedure regardless of pain mitigation. These results indicate that calves in the MED group may have experienced pain-related stress as a result of the disbudding procedure. The remaining nonlinear HRV measures did not differ by treatment. Future research on this topic should address additional potential confounding factors, such as the effect of pain-mitigating drugs on autonomic function or the influence of the autonomic nervous system on wound healing, that may prohibit HRV measurement as an indicator of disbudding pain severity.

Short communication: Effect of age at group housing on behavior, cortisol, health, and leukocyte differential counts of neonatal bull dairy calves.

To determine the effect of age at grouping on behavior, health, and production of dairy bull calves, 90 Holstein-Friesian bull calves were housed in individual pens until moved to 1 of 3 treatments. Calves were housed in groups of 3 calves at 3 d old (GH3), 7 d old (GH7), or 14 d old (GH14) until 7 wk of age. Ten groups of 3 calves for each treatment were used, with 5 pens/treatment in each of 2 replications (10 pens/treatment, 3 treatments, 3 calves/treatment; 90 calves total). Direct behavioral observations using instantaneous scan sampling every 10 min were conducted twice per week for 7 wk. At the same times, video data were recorded for continuous observations at feeding time to observe the overall activity of group-housed calves. Hip height, heart girth, and health scores were recorded weekly and body weight was recorded at the start and end of the study. Calves in GH3 spent more time playing and but more time cross-sucking and displacing other calves from milk bottles. Calves engaged in social interaction as early as 3 d of age, and social interactions between 3 to 6 wk of age increased markedly. Calves housed in GH14 vocalized more than did calves in GH7 and GH3. No difference was found between treatments in growth performance. Calf fecal, cough, and nasal and ocular discharge scores, differential leukocyte counts, and plasma cortisol concentrations were not affected by age at grouping. However, during the first week of grouping, when calves were moved from individual pens to group pens, some calves were unable to find their milk bottles and required guidance. In conclusion, these data show no adverse effects on health or performance and some benefits on social behavior for early (d 3) grouping of calves.

Feed withdrawal and transportation effects on Salmonella enterica levels in market-weight pigs.

Feed withdrawal and transport commonly occur together in pigs. Objectives of this study were to determine if these preslaughter stressors, feed withdrawal and transportation, affect the levels of , stress hormone concentrations, and immune functions in infected market pigs. A 2 × 2 factorial analysis of a randomized complete block design with feed withdrawal and transport as fixed effects was used. Sixty market-weight pigs were individually inoculated with serovar Typhimurium. The experiment was replicated 3 times (blocking factor) with 20 pigs per replicate. Three days after inoculation, the pigs were randomly assigned to 1 of 4 treatments (5 pigs per treatment in each/replicate), including 1) control (Control; or no stress), 2) feed withdrawal for 12 h (FW), 3) transportation for 2 h (T), and 4) feed withdrawal for 12 h followed by transportation for 2 h (FWT). Feed withdrawal by itself or followed by transportation caused an increase of levels in ileal contents ( < 0.05), whereas only FWT caused an increase of levels in cecal contents ( < 0.05). Rectal contents (feces) consistently contained very low levels of , with no difference among treatments ( > 0.10). Cortisol increased in pigs from all 3 stress treatments ( < 0.001), with T and FWT pigs having greater concentrations than Control pigs ( < 0.05), although total white blood cell counts were lower for FWT pigs compared with Controls ( > 0.03). Each granulocyte percentage (neutrophil, eosinophils, and basophils) increased ( < 0.05) following transport but was attenuated ( > 0.05) by feed withdrawal with transport. Lymphocytes were suppressed ( < 0.05) by all stressors, and the greatest suppression occurred when pigs were transported (T and FWT). However, monocytes were suppressed ( < 0.05) compared with Controls only by FWT. Expression of IL-1 (produced by monocytes/macrophages) from the spleen cells increased ( < 0.05) with FW compared with Controls, whereas its receptor antagonist was suppressed by FWT ( < 0.05). It is concluded that some typical preslaughter practices, such as feed withdrawal and transportation, lead to greater intestinal levels and gut-associated lymphoid tissue markers of inflammation in market pigs and, consequently, to an increased food safety risk.

Evaluating the behavior, growth performance, immune parameters, and intestinal morphology of weaned piglets after simulated transport and heat stress when antibiotics are eliminated from the diet or replaced with L-glutamine.

Study objectives were to evaluate the effects of post-weaning transport during heat stress (HS) and thermoneutral (TN) conditions when dietary antibiotics are removed or replaced with a nutraceutical. Sixty mixed sex piglets from 10 sows ( = 6 piglets/sow) were weaned (18.8 ± 0.8 d of age) and then herded up ramps into 1 of 2 simulated transport trailers in either TN (28.8 ± 0.2°C) or HS (cyclical 32 to 37°C) conditions where they remained for 12 h. During the 12 h of simulated transport, fans were used to simulate air movement through the trailer, feed and water were withheld, and rectal temperature (T) was measured hourly. Following the 12 h simulated transport, piglets were unloaded from the trailer, weighed, and then housed individually in TN conditions [28.5 ± 0.1°C; 29.1 ± 0.1% relative humidity (RH)] and assigned to 1 of 3 dietary treatments balanced by weaning weight, sex, sow, and transport environment. Treatments were dietary antibiotics [A; = 20 piglets; 5.5 ± 0.2 kg BW; chlortetracycline (400 g/ton) + tiamulin (35 g/ton)], no dietary antibiotics (NA; = 20 piglets; 5.6 ± 0.2 kg BW), or 0.20% L-glutamine (GLU; = 20 piglets; 5.6 ± 0.2 kg BW) fed for 14 d. During the diet treatment period, feed intake (FI), BW, and behaviors were monitored daily. At the conclusion of the diet treatment period, all piglets were euthanized and intestinal samples were collected for histology. The T and post-transport BW loss were increased in HS (40.7°C and 0.43 kg, respectively) compared to TN-exposed (39.2°C and 0.27 kg, respectively) piglets during simulated transport. Throughout the 14 d dietary treatment phase, FI was greater overall ( < 0.01; 60.3%) in GLU compared to A and NA pigs, and tended to be greater (37.7%) in A compared to NA pigs. BW was greater overall ( < 0.01; 8.7%) in GLU and A compared to NA pigs, but no differences were detected between A and GLU pigs. Lying behavior was greater ( = 0.05; 11.7%) in NA compared to A and GLU piglets in the first 2 d following simulated transport. The villus height to crypt depth ratio was greater ( < 0.05) in the duodenum (12.1%) and jejunum (12.8%) for A and GLU compared to NA pigs, and greater in the ileum (15.6%) for GLU compared to A and NA pigs. In summary, withholding dietary antibiotics after weaning and transport can negatively affect piglet productivity and measures of intestinal morphology compared to dietary antibiotic administration and L-glutamine provision.

Testing the feasibility of using a conveyor belt to load weanling and nursery pigs for transportation.

Transportation is known to be a multi-faceted stressor, with the process of loading being one of the most significant factors impacting the stress to which animals are exposed. This project was designed to determine if using a conveyor to load pigs into the top deck of a simulated straight deck trailer could lower the stress to which pigs and handlers are exposed. Pigs were assigned to either a Control group that were herded up a stationary conveyor ramp into a top deck trailer (2.5 m above the ground); or Conveyor group which were herded onto a mobile conveyor into a top deck trailer. The conveyor was 7.6 m long, 0.9 m wide and rose to 2.5 m high at a 16° slope, and moved 11.3 m/min. Two age groups were tested; Weanling pigs which were moved in groups of 20 (n = 14 groups/treatment) and Nursery pigs which were moved in groups of 10 (n = 15 groups/treatment). Behavior was recorded during loading, including slips and falls, vocalizations, assists, and time to load. Heart rate of 2 sentinel pigs/group and the handler were recorded during loading, and body temperature of the handler after loading. Pigs were held in the simulated trailer for 30 min while heart rate was recorded. After which, they were unloaded and held in a holding pen for an additional 30 min while heart rate was recorded. There were no treatment differences for slips or falls (P < 0.90). Vocalizations were too few to analyze. Both Weanling (2.8 ± 0.7) and Nursery (1.6 ± 0.5) Conveyor pigs needed to be assisted onto the conveyor more than Weanling (1.2 ± 0.4) and Nursery (0.3 ± 0.1) Control pigs (P < 0.06). There was no difference in total loading time between the treatments for any age group (P < 0.15), with Weanling and Nursery pigs loading in 50 to 45 s, respectively. There were no treatment differences for heart rate variability measures (P > 0.10). However, loading increased heart rate of Nursery pigs (204.9 ± 5.7 bpm, P < 0.005), but not Weanling pigs (172.1 ± 9.0 bpm). Nursery pigs had a greater ratio of low frequency to high frequency power during loading (P < 0.02) compared to other phases of the procedure in both Control and Conveyor groups. Heart rate (93.9 ± 1.9 bpm) and body temperature (31.1 ± 0.3°C, eye temperature) of the handler was not affected by treatment (P < 0.26). Based on behavior and physiology, the pigs had similar experiences in both treatments. This study shows that it is feasible to use a conveyor to load pigs, but it may not be advantageous.

Including dietary fiber and resistant starch to increase satiety and reduce aggression in gestating sows.

Aggression during mixing of pregnant sows impacts sow welfare and productivity. The aim of this study was to increase satiety and reduce aggression by including dietary fiber and fermentable carbohydrates. Sows were housed in individual stalls 7 to 14 d after breeding (moving day was considered d 0 of treatment) and were fed (at 0700 h) with a CONTROL (corn-soybean meal based with no additional fiber sources), RSTARCH (10.8% resistant starch), BEETPULP (27.2% sugar beet pulp), SOYHULLS (19.1% soybean hulls), or INCSOY (14.05% soybean hulls) for 21 d (5 sows/diet × 5 diets × 8 replications = 200 sows). The CONTROL diet was targeted to contain 185 g(d∙sow) NDF and the other diets were targeted to contain 350 g(d∙sow) NDF. The INCSOY diet was fed at 2.2 kg/(d∙sow) and the other diets were fed at 2 kg(d∙sow). On d 22, sows were mixed in groups of 5 (at 1200 h). Behaviors in stalls (on d 1, 7, 14, and 21) and after mixing (d 22 and 23), heart rate (on d 1, 7, 14, and 21), blood metabolites (on d 2, 8, 15, 22, and 25), and the effects of diets on production were collected and analyzed. Sows stood more ( < 0.01) and rested less ( < 0.001) over time irrespective of the diet. Sows on BEETPULP stood more ( < 0.01) and sows on SOYHULLS rested more ( < 0.01). Sham chewing increased over days irrespective of the diet. Chewing behavior (bar and feeder) increased with days on diet ( < 0.001) and was lowest in sows on the SOYHULLS diet ( = 0.045). When mixed, biting frequency in the first hour was highest for sows on the CONTROL diet (236.5 ± 62.6) and lowest for sows on the RSTARCH diet (90.5 ± 30.5). Skin lesions increased ( < 0.001) 24 h after mixing sows irrespective of diet. Blood urea nitrogen (BUN) concentration was lowest in sows fed BEETPULP and SOYHULLS ( < 0.001). Serum glucose concentration was highest in sows fed RSTARCH and BEETPULP ( = 0.04), but there was no day effect ( = 0.62) or diet × day interaction ( = 0.60). The NEFA was greatest in sows fed RSTARCH, BEETPULP, and SOYHULLS ( < 0.001). Lactate ( < 0.001) and BUN concentrations were greatest on d 2 but dropped and remained constant after d 8. Average heart rate was lowest for sows on SOYHULLS and INCSOY compared with sows on the other diets ( = 0.03). Number of piglets born and average weaning weight were not affected by diets ( > 0.05). Average birth weight was lowest in the INCSOY diet ( = 0.02). This study demonstrates that RSTARCH and SOYHULLS can improve the welfare of sows by reducing aggression and increasing satiety in limit-fed pregnant sows without affecting production.

Acute phase cytokines, TAC1, and toll-like receptor4 mRNA expression and health associated with group size in veal calves.

Chronic stressors are a major health and well-being issue in animals. Immune status of animals under chronic stress is compromised, thus reducing disease resistance and compromising well-being of the animal. The objective of this study was to determine the influence of group size of veal calves on immune status and leukocyte mRNA expression of acute phase cytokines, toll-like receptor 4 (TLR4) and tachykinin 1 (TAC1) over a five-month finishing period. Holstein bull calves (n=168), 44±3 days of age were assigned to one of three treatments; 2, 4, or 8 calves/pen (pen space allowance of 1.82m(2)/calf). Jugular blood samples were collected at the day of grouping and then monthly for 4 months. The differential leukocyte counts were determined and mRNA was extracted from the leukocytes. Reverse transcription-qPCR was used to measure the gene expression of interleukin-1 (IL-1ß), IL-1 receptor antagonist (IL-1Ra), tumor necrosis factor (TNF-α), TLR4, and TAC1 in leukocytes. Health was evaluated before grouping and monthly for 4 months. On the 1st month after grouping, veal calves that were housed in groups of 8 have greater expression of IL-1ß mRNA than calves housed in groups of 4 or 2 (treatment×month, P=0.04). Also at 1 month, groups of 8 had greater TAC1 expression (P<0.05) than calves housed in groups of 4 or 2. However, the expression of IL-1Ra, TNF-α, and TLR4 were not influenced by group size. In the first month of the trial, calves in groups of 8 coughed more (P<0.05) than calves in groups of 2 and coughed more than calves in groups of 4 and 2 during the 2nd month (treatment×month, P=0.03). Calves housed in groups of 8 tended to have greater neutrophil percentage (P=0.09), neutrophil to lymphocyte ratio (P=0.06), and had lower lymphocyte percentage (P=0.06) than those housed in groups of 4 or 2. In conclusion, the number of veal calves in a group, given the same space during the finishing period did not alter IL-1Ra, TNF-α, and TLR4 mRNA expression. However, housing of calves in groups of 8 was associated with greater expression of IL-1ß and TAC1 mRNA in peripheral blood leukocytes, and coughing during the first 2 months after grouping. Therefore, housing of veal calves in larger groups may lead to greater susceptibility to respiratory disease and stress.

Serotonin's role in piglet mortality and thriftiness.

Improving piglet survivability rates is of high priority for swine production as well as for piglet well-being. Dysfunction in the serotonin (5-HT) system has been associated with growth deficiencies, infant mortalities, or failure to thrive in human infants. The aim of this research was to determine if a relationship exists between infant mortality and failure to thrive (or unthriftiness), and umbilical 5-HT concentration in piglets. Umbilical blood was collected from a total of 60 piglets from 15 litters for analysis of 5-HT and tryptophan (Trp; the AA precursor to 5-HT) concentrations. Behavior was scan sampled for the first 2 days after birth. Brain samples were also taken at 8 h after birth from healthy and unthrifty piglets (n = 4/group). The raphe nucleus was dissected out and analyzed for 5-HT and dopamine concentrations as well as their major metabolites 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA), respectively. Data were analyzed by ANOVA. Piglets that died within 48 h of birth (n = 14) had significantly lower umbilical blood 5-HT concentrations at the time of their birth compared to their healthy counterparts (n = 46, P = 0.003). However, no difference in Trp was detected (P 0.38). Time spent under the heat lamp and sleeping were positively correlated with umbilical 5-HT levels (P = 0.004 and P = 0.02, respectively), while inactivity had a negative correlation with 5-HT levels (P = 0.04). In the raphe nucleus, the center for brain 5-HT biosynthesis, unthrifty piglets had a greater concentration of 5-HIAA (P = 0.02) and a trend for higher concentrations of 5-HT (P = 0.07) compared with healthy piglets. Dopamine levels did not differ between thrifty and unthrifty piglets (P = 0.45); however, its metabolite HVA tended to be greater in unthrifty piglets (P = 0.05). Our results show evidence of serotonergic dysfunction, at both the central and peripheral levels, accompanying early piglet mortalities. These data suggest a possible route for intervention, via the 5-HT system, to improve piglet survivability. However, further research is required to validate this hypothesis.

Group space allowance has little effect on sow health, productivity, or welfare in a free-access stall system.

Free-access stalls allow sows to choose the protection of a stall or use of a shared group space. This study investigated the effect of group space width, 0.91 (SS), 2.13 (IS), and 3.05 (LS) m, on the health, production, behavior, and welfare of gestating sows. Nine replications of 21 (N = 189) gestating sows were used. At gestational d 35.4 ± 2.3, the pregnant sows were distributed into 3 pens of 7 sows, where they remained until 104.6 ± 3.5 d. Each treatment pen had 7 free-access stalls and a group space that together provided 1.93 (SS), 2.68 (IS), or 3.24 (LS) m(2)/sow. Baseline measurements were obtained before mixing. Back fat depth, BW, BCS, and lameness were measured monthly, and skin lesions were scored weekly. Blood was collected monthly for hematological, immunological, and cortisol analyses. Sow behavior was video recorded continuously during the initial 4 d of treatment and 24 h every other week thereafter. Behavior was analyzed for location, posture, pen investigation, social contact, and aggression. Skin response to the mitogen concanavalin A (Con A) was tested at mean gestational d 106. Litter characteristics including size and weight were collected at birth and weaning. The data were analyzed using a mixed model. Multiple comparisons were adjusted with the Tukey-Kramer and Bejamini-Hochberg methods. Group space allowance had no effect on any measure of sow health, physiology, or production (P ≥ 0.10). Sows in the SS, IS, and LS pens spent 77.88% ± 3.88%, 66.02% ± 3.87%, and 63.64% ± 3.91%, respectively, of their time in the free-access stalls (P = 0.12). However, SS sows used the group space less than IS and LS sows (P = 0.01). Overall, pen investigatory behavior was not affected by group space allowance (P = 0.91). Sows in the LS pens spent more time in a social group than SS sows (P = 0.02), whereas sows in IS pens were intermediate to, but not different from, the other treatments (P ≥ 0.10). The size of the social groups was also affected by the group space allowance (P = 0.03), with SS sows forming smaller groups than LS sows; again, IS sows were intermediate to, but not different from, the other treatments. Although the group space allowance had no measurable impact on the health, physiology, or productivity of the sows, the lower group space use and social contact of the SS sows reduced the behavioral diversity benefits of group housing and may indicate an avoidance of social stressors or a lack of physical comfort in the smallest pens.

Postnatal piglet husbandry practices and well-being: the effects of alternative techniques delivered in combination.

After birth, piglets undergo procedures likely to cause stress. The aim of this study was to evaluate stress responses evoked by 2 combinations (More Stressful [all a] or Less Stressful [all a] or More Stressful [all b]) of alternative methods for performing the following processing procedures: 1) teeth resection (TR) ­ [a] clip vs. [b] grind; 2) identification (ID) ­ [a] ear tag vs. [b] ear notch; 3)iron administration (FE) ­ [a] inject vs. [b] oral; 4) castration (CA) ­ [a] cords cut vs. [b] cords torn; 5) taildocking (TD) ­ [a] cold clip vs. [b] hot clip [corrected]. Ten litters of eight 2- and 3-d-old piglets were assigned to each procedure. Within each litter 1 male and 1 female piglet was assigned to 1 of 4 possible procedures: the 2 combinations, sham procedures, and sham procedures plus blood sampling. Blood was collected before processing and at 45 min, 4 h, 48 h, 1 wk, and 2 wk afterward and assayed for cortisol and ß-endorphin concentrations. Procedures were videotaped and analyzed to evaluate the time taken to perform the procedure and the number of squeals, grunts, and escape attempts. Vocalizations were analyzed to determine mean and peak frequencies and duration. Piglets were weighed before the procedure and at 24 h, 48 h, 1 wk, and 2 wk afterward. Identification, tail docking, and castration lesions were scored on a 0 to 5 scale at 24 h, 1 wk, and 2 wk postprocedure. Both combinations of methods took longer to carry out than sham procedures and resulted in more squeals, grunts, and escape attempts during the procedures and higher peak frequencies of vocalizations compared with the control treatments (P < 0.05). Cortisol concentrations 45 min after processing were also higher in the 2 combination treatments than in the sham treatments (P < 0.05). Comparing between procedure treatments, the More Stressful combination of methods took longer to carry out, resulted in higher ß-endorphin concentrations at 1 wk, had higher peak frequency of vocalizations, and increased ear (P < 0.05) and tail wound (P < 0.1) lesion scores at 1 wk than the Less Stressful combination. Growth during d 2 to 7 postprocedure was lower in More Stressful piglets than control piglets (P < 0.05) but by 2 wk, growth was unaffected. Using measures of behavior, physiology, and productivity, the More Stressful combination of procedures decreased welfare relative to the Less Stressful combination; however, both combinations decreased welfare relative to controls. The time taken to carry out the procedure would appear to be an important factor in the strength of the stress response.

Growth and reproductive development of male piglets are more vulnerable to midgestation maternal stress than that of female piglets.

In many mammalian species, prenatal stress masculinizes female and feminizes male offspring impairing their reproductive capacity. Regrouping gestating sows is a common, stressful production practice, but its impact on the developing pigs of the sow is not fully known. This study examined the effects of regrouping gestating sows and the administration of exogenous glucocorticoids on the growth and external reproductive morphology of pigs. At 37.2 ± 0.26 d of gestation, 6 cohorts of 18 sows (N = 108) were placed in 1 of 3 treatments: socially stable (Stable), hydrocortisone acetate (HCA), or mixed (Mixed). The HCA sows were administered 70 mg HCA, a synthetic glucocorticoid, twice daily during the 21 d experimental period. Each Mixed sow was penned with 2 companion sows (Companion) and regrouped on d 7 and 14 with 2 different Companion sows in a new pen. Stable and HCA sows were penned in treatment groups of 3 sows. Sow social rank was assessed weekly during feeding. After the 21 d experimental period, all sows were housed in gestation stalls for the duration of pregnancy. During the 21 d, Companion sows gained more weight than HCA and Mixed sows (P < 0.05) with Stable sows intermediate. High ranked sows gained more weight than middle and low ranked sows (P < 0.05). Mixed sows had greater head lesion scores than Stable and HCA sows (P < 0.05) with Companion sows intermediate. Head lesions increased with lower social rank (P < 0.001). Sow treatment did not affect farrowing rate, litter size, or sex ratio (P > 0.10). Social rank also had no effect on farrowing rate (P > 0.10), but affected total litter size (P = 0.03). High ranked sows bore and weaned more live females than low ranked sows (P < 0.05), in part due to differential preweaning mortality among female pigs (P = 0.01). Only male pigs were affected by sow treatment. Preweaning mortality was higher among male pigs from HCA than from Mixed sows (P = 0.04) with other treatments intermediate. Despite no weight differences in the preweaning period, at 160 d of age males from HCA sows weighed more than males from Stable sows (P = 0.01) with other treatments intermediate. Males born to Companion sows had longer relative anogenital distances, a marker of fetal testosterone exposure, than males from Mixed sows (P = 0.03) with other treatments intermediate. The prenatal environment affected the pigs in a sex-specific manner altering the growth and reproductive morphology of the males more than that of the females.

Repeated mixing and isolation: measuring chronic, intermittent stress in Holstein calves.

Objectives of this study were to determine the physiological effects of psychological stress applied to dairy calves and to test if molasses consumption could be used to validate that a stressed condition was achieved. Twenty male calves (3 wk old) received jugular catheters and were randomly assigned to control (CTR; n = 4 pens of 1 calf per pen) or social stress treatments (STR; n = 4 pens of 4 calves per pen). The STR treatment included 5 cycles of 24-h isolation followed by regrouping with unfamiliar animals for 48 h (over 15 d). An ACTH challenge (0.1 IU/kg of body weight) was used to determine adrenal fatigue. Peak and total cortisol concentrations were greater for STR calves until the ACTH challenge. After the ACTH challenge, CTR calf cortisol increased and STR calf cortisol continued to decrease, suggesting adrenal fatigue. The number of calves that became positive for fecal shedding of Salmonella after the acute stress of being moved and the number of calves that were positive after the move decreased with each move. Fifty-six percent of STR calves changed from negative to positive for shedding after the first move compared with 18.75% of STR calves remaining negative after the third move. Difference in fecal shedding of Enterobacteriaceae from samples taken before and after moving calves on d 6 was less than that on d 2, 3, and 5. Leukocyte counts were not different, but trends for day effects were detected for neutrophil and monocyte percentages. Molasses consumption was greater for STR calves on d 2 and 11, as was total consumption. Latency to lie after eating also increased as the study progressed; STR calves required more time to lie after eating on d 12 than on d 3, and latency to lie was greater for STR than CTR on d 4, 8, 12, and 14. The STR calves also stood more than the CTR calves in the 4-h afternoon period on d 4, 5, 7, and 14. However, during the 4-h morning observations on d 14 (ACTH challenge), CTR calves stood more than STR calves. This model induced chronic stress, as characterized by adrenal fatigue, which was confirmed by molasses consumption and behavior changes. Therefore, molasses consumption could be used to confirm social stress in experimental models.

Effect of group size on behavior, health, production, and welfare of veal calves.

The objective of this study was to investigate the effect of group size on behavior, growth, health, and welfare of veal calves. Holstein-Friesian bull calves (n=168; 44±3 d of age) were assigned randomly to 1 of 3 treatments of group housing with 2, 4, or 8 calves per pen. The pens used for housing were 3 by 1.20 m (2 calves per pen), 3 by 2.40 m (4 calves per pen), and 3 by 4.80 m (8 calves per pen), supplying a total pen space allowance of 1.82 m2/calf, regardless of pen size. Behavior was recorded from video data throughout the day from 0700 to 1900 h during a single day each month for 5 mo using scan sampling every 5 min within 30-min observation sessions. On d 0, 1, 5, 14, 42, and 70 after grouping, continuous focal sampling around feeding time (30-min intervals before, during, and after feeding) focused on oral and aggressive behaviors. Calves housed in large groups (4 or 8 calves per pen) showed more (P≤0.001) conspecific contact, walking, and standing and less (P<0.001) manipulation of objects, self-licking, and lying when compared to calves housed in small groups (2 calves per pen). Group size had no effect on play behavior (P=0.11) throughout the experiment. During feeding times group size had no (P≥0.07) effect on any behavioral patterns except for duration of conspecific contact (P<0.01). Aggression at feeding time was not (P>0.23) affected by treatment. Group size treatments were similar for hip height change (P=0.41) and heart girth change (P=0.18) over the duration of the experiment; however, both hip height and heart girth increased (P=0.001) with calf age. During mo 1, calves in groups of 8 or 4 coughed more than calves in groups of 2 whereas calves in groups of 8 coughed more than calves in groups of 4 or 2 in mo 2 (treatment×month, P=0.03). Furthermore, during mo 4, calves in groups of 8 had less nasal discharge than calves in groups of 2 or 4 (treatment×month, P=0.02). Ocular discharge, ears, and fecal scores did not differ (P≥0.05) among treatments. Plasma cortisol was not (P≥0.37) affected by group size. The number of veal calves in a group when given the same space did not affect production and physiological indicators of welfare but had a transient effect on health during the 5-mo finishing period. If increased play and social contact and decreased aggression are considered as primary indicators of welfare, group size did not alter calf welfare.

Effects of early serotonin programming on behavior and central monoamine concentrations in an avian model.

Serotonin (5-HT) acts as a neurogenic compound in the developing brain; however serotonin altering drugs such as SSRIs are often prescribed to pregnant and lactating mothers. Early agonism of 5-HT receptors could alter the development of serotonergic circuitry, altering neurotransmission and behaviors mediated by 5-HT signaling, including memory, fear and aggression. This study was designed to investigate the effects of early serotonin agonism on later behaviors. An extremely aggressive White leghorn strain (15I5) was used in the study. The chicks were injected with 5-MT (a serotonin agonist) at 2.5mg/kg (low dose), 10mg/kg (high dose) or saline (control) on the day of hatch and a second dose 24h later (n=9/sex/trt). Chicks' fear response and memory were tested at 2 weeks of age. In the fear test, chicks were subjected to a social isolation test for 20min, time to first vocalization and numbers of vocalizations were recorded. In the memory test, chicks were placed in a running wheel and presented with an imprinted object (white box with a red light) and a similar shaped novel object (blue box with a white light), respectively. The distance traveled in the wheel toward each object was measured. At 10 weeks of age birds were tested for aggression and concentrations of catecholamines were determined from the raphe nucleus and hypothalamus by HPLC (n=12). Expression of 5-HT1A and 5-HT1B receptor genes were measured by RT-PCR. Both high and low dose chicks tended to have shorter latency to first vocalization and a greater number of vocalizations compared with control chicks. Memory test showed that chicks from all groups traveled a similar distance toward a familiar object. However, control chicks walked the least toward a novel object, low dose chicks tended to walk further, and high dose chicks walked significantly further for a novel object. In aggression tests, both high and low dose males exhibited greater frequency of aggressive behaviors compared to controls, while no difference in aggression was evident in the females. Norepinephrine concentrations were also reduced in the low dose birds in the hypothalamus and in the raphe nucleus. Serotonin concentrations tended to be lower only in the both hypothalamus and raphe nucleus of the low dose birds. 5-HT1A expression was greatest in the hypothalamus and raphe nucleus of low dose birds. The agonism of the serotonin system during neural development of birds genetically predisposed to aggression alters both the dopaminergic and serotonergic systems further increasing their aggressiveness.

Effects of rubber flooring during the first 2 lactations on production, locomotion, hoof health, immune functions, and stress.

Some housing systems on dairy farms can result in long-term chronic pain. The effects of acute pain on immunity have been explored, but chronic pain's influence on immune responses is still poorly understood. Therefore, the objective of this research was to determine chronic effects of flooring on immune responses and production in freestall housing for dairy cows. Thirty heifers were studied from before calving as first-calf heifers until d 180 of their second lactation. Treatments were rubber (Kraiburg; Agromatic Inc., Fond du Lac, WI) flooring or concrete with diamond grooves in a freestall barn, each in 2 quadrants of the barn. Heifers entered the treatments after calving, so the system was dynamic and each cow was considered an experimental unit. At the end of the first lactation, cows were housed in a bedded pack barn with pasture access until calving was imminent. At that time, they returned to their assigned treatment, but not necessarily into the same quadrant. Production, reproduction, cortisol, acute-phase proteins, and health data were recorded throughout lactation 1, locomotion was scored weekly, and hoof scoring and care was conducted on d 60 and 180 of lactations 1 and 2, and quantitative real-time-PCR of blood leukocytes was analyzed in mid lactation of lactation 1. Mature-equivalent milk fat, milk protein, and protein percentages during the first lactation were greater for cows on the rubber flooring. Hoof and leg therapy treatments per cow were fewer for rubber floor-housed cows. Locomotion scores were less for cows housed on rubber during the second lactation. White blood cell counts were less for cows housed on rubber, and caused by greater lymphocyte counts for cows housed on concrete. The possibility of chronic inflammation was substantiated by less IL-1ß and more IL-1 receptor antagonists for cows housed on rubber at d 150 in the second lactation. Cortisol and acute-phase proteins did not differ between the treatments. Interferon-γ, IL-12, the modulator of tissue reconstruction (B-cell-transforming growth factor 1), and pain-modulating neurokinin (tachykinin 1) were not different at d 105. These data show indicators of chronic inflammation for cows housed on the concrete flooring compared with those housed on rubber. Implications for the use of rubber flooring in freestall barns are broader than just lameness and may affect many aspects of cow physiology and production.

Gas alternatives to carbon dioxide for euthanasia: a piglet perspective.

The search for alternative methods to euthanize piglets is critical to address public concern that current methods are not optimal. Scientific evidence supports that blunt force trauma is humane when performed correctly, but most people find it visually difficult to accept. The use of CO2 is often recommended; at the same time, it is criticized as being aversive to pigs. This research sought to 1) identify a method of scientifically determining if piglets find a gas aversive, using an approach-avoidance test, which relies on the perspective of the piglet, and 2) test different gas mixtures to determine if they are effective and humane for neonatal piglet euthanasia. Pigs were allowed to walk freely between 1 chamber filled with air and another chamber either gradually filled with gas mixtures (Exp. 1) or prefilled with gas mixtures (Exp. 2). Experiment 1 tested CO2 (90%) and air (10%), N2O (60%) and CO2 (30%), Ar (60%) and CO2 (30%), and N2 (60%) and CO2 (30%). Because piglets had to be removed when they started to flail, the test was shortest (P < 0.01) for the pigs in the CO2 treatment compared with pigs in the N2O/CO2, Ar/CO2, and N2/CO2 treatments, 3.1 ± 0.2, 8.5 ± 0.6, 9.6 ± 0.4, and 9.9 ± 0.1 min, respectively. Nonetheless, all gas mixtures adversely affected the pigs, causing the pigs to leave the test chamber. In Exp. 2, piglets were allowed to enter a chamber prefilled with N2/CO2 or N2O/CO2 (both 60/30%). Pigs exposed to the prefill chambers started to flail in fewer than 20 s, much faster in comparison with the gradual fill method, which supports that this method was more aversive. In Exp. 3, piglets were euthanized using a 2-step procedure. Pigs were first placed in a gradual fill chamber with 1 of 4 gas mixtures: 90% CO2, N2/CO2, N2O/CO2, or N2O/O2 (the last 3 mixtures at 60/30%) followed by placement into a 90% CO2 prefill chamber when the pigs started to flail or were anesthetized. All 3 gas treatments that contained CO2 killed pigs more quickly than N2O/O2 (P < 0.05). However, N2O/O2 was the only treatment that anesthetized the pigs instead of causing squeals or flailing although requiring about 12 min longer. Although longer, a 2-step procedure in which pigs are anesthetized with a mixture of N2O and O2 before being euthanized by immersion in CO2 may prove to be more humane than CO2 alone.

The effects of R-salbutamol on growth, carcass measures, and health of finishing pigs.

A pure form of salbutamol has the potential to deliver positive production benefits to the swine industry. The aim of this experiment was to determine the effects of salbutamol on growth, carcass measures, and health of finishing pigs. The study used 192 pigs (89 ± 1 kg BW) housed in groups of 6 in 32 pens and assigned to 1 of 4 treatments: 1) control (CTL), 0 mg/kg salbutamol; 2) 2R, control diet with 2 mg/kg of the pure R-enantiomer of salbutamol; 3) 4R, control diet with 4 mg/kg of pure R-salbutamol; or 4) 8RS, control diet with 8 mg/kg of a 50:50 mixture of the R- and S-enantiomers. All diets were offered ad libitum for 4 wk. All pigs were weighed and pen feed intakes were recorded weekly. At slaughter, individual HCW and measurements of the 10th-rib loin muscle area (LMA), color, marbling, firmness, and back fat, last lumbar, and midline back fat depths were collected. Data were analyzed using Proc GLM of SAS, with pen as the experimental unit. Overall, 2R and 4R pigs had greater ADG than CTL pigs (P < 0.05) and, at slaughter, were heavier than CTL pigs (P < 0.01). Overall, 8RS pigs had decreased ADFI (P < 0.05), and CTL pigs had poorer G:F (P < 0.001) than the other 3 treatments. All salbutamol-fed pigs had 5 to 6 kg greater HCW (P < 0.001), 2% to 3% increased carcass yield (P < 0.001), 5.6 cm(2) larger LMA (P < 0.01), 3 to 4 mm less 10th-rib back fat (P < 0.01), and 2 mm less lumbar back fat (P < 0.05) than CTL pigs. However, control pigs had greater loin muscle color scores (P < 0.05) and marbling scores (P < 0.001) than all salbutamol-treated pigs. Taken together, these data indicate that as little as 2 mg/kg R-salbutamol has a positive effect on pig growth and carcass composition. However, the effects of salbutamol on meat quality require further research.

Nitrous oxide by itself is insufficient to relieve pain due to castration in piglets.

Surgical castration is performed on most male piglets in the United States. However, castration is painful and analgesics have been considered to relieve pain. Inhalant gases with analgesic properties allow for a fast induction, have short-term and reversible effects, and are a needle-free option. Nitrous oxide (N(2)O; "laughing gas") has been widely used in human surgery and dental offices as an analgesic, sedative, and anxiolytic drug, yet N(2)O has not been thoroughly investigated for use in farm animals. We hypothesized that the analgesic effect of N(2)O could reduce the pain experienced by piglets during or immediately after castration. Twenty-four male piglets, from 12 litters, were castrated at 3 d of age. One piglet received N(2)O and a littermate received air as a control. After 150 s of exposure to the gas, castration was performed while the piglet remained exposed to the gas. Agitation scores and total vocalization length were recorded during castration. Behavioral observations were continued for 3 d postcastration by using a 5-min scan-sampling method for 4 h the first morning and for 2-h periods in the morning and afternoon of each day thereafter. Body weight gain was measured on the day before castration, at 3 d postcastration, and at weaning. Data were analyzed using a mixed model in SAS (Cary, NC). Nitrous oxide successfully induced anesthesia in all N(2)O piglets, as validated by a skin pinch test and the loss of the palpebral reflex. Total vocalization length was shorter in piglets receiving N(2)O during the induction phase (P = 0.003) but was not different during castration itself because piglets receiving N(2)O awoke and vocalized as much as control piglets (P = 0.87). Agitation scores during the whole procedure were reduced in piglets receiving N(2)O in both frequency (P = 0.005) and intensity (P = 0.026). For 2 h after castration, piglets receiving N(2)O displayed less huddling behavior than did control piglets (P = 0.01). Over the 3 d, piglets receiving N(2)O performed more tail wagging (P = 0.02) and tended to show fewer sleep spasms (P = 0.06) than did control piglets. Piglets given N(2)O tended to have a reduced growth rate compared with control piglets at 3 d postcastration and at weaning (P = 0.05 and P = 0.06, respectively). Nitrous oxide was effective in inducing anesthesia in neonatal piglets during handling. Nonetheless, its analgesic effects appeared insufficient in preventing castration-induced pain.