Evaluation of the capacity and effective angle of thalamic damage for two commercially available captive bolt tool types on cadaver heads from sows > 200 kg body weight.

This study evaluated the ability of two penetrating captive bolt (PCB) types (PISTOL, INLINE) to reach and disrupt the thalamus when applied in two placements (FRONTAL, BEHIND EAR) to chilled cadaver heads (N=60) from sows > 200 kg. Heads were randomly distributed across six treatments (n=10): FRONTAL-INLINE, FRONTAL-PISTOL, FRONTAL-NO SHOT, BEHIND EAR-INLINE, BEHIND EAR-PISTOL, and BEHIND EAR-NO SHOT. The FRONTAL shot was placed 3.5 cm superior to the optic orbits at the midline; the BEHIND EAR shot was placed directly caudal to the pinna of the ear on the same plane as the eyes and targeting the middle of the opposite eye. For INLINE treatments, a Jarvis PAS-Type C 0.25R Super Heavy Duty PCB with a Long Bolt and 6.0 GR power loads was used. For PISTOL treatments, a Jarvis PAS-Type P 0.25R Pistol PCB with a Long Stunning Rod Nosepiece Assembly and 3.5 GR power loads was used. Heads were split along the bolt with a band saw. Tissue depth measurements are reported as Mean ± SE followed by 97.5% one-sided upper reference limit (URL). Total tissue thickness was less (P < 0.0001) at the FRONTAL (56.31±1.76 mm; URL: 73.17 mm) than the BEHIND EAR placement (95.52±3.30 mm; URL: 126.53 mm). Thalamic depth was less (P < 0.0001) at the FRONTAL (78.31±1.32 mm; URL: 88.19 mm) than the BEHIND EAR placement (111.86±3.22 mm; URL: 135.99 mm). Effective angle was greater (P < 0.0001) at the FRONTAL (4.72±0.20°) than the BEHIND EAR placement (3.22±0.17°). Potential for bolt-brain contact was not different (P = 1.0000) between FRONTAL-INLINE (10/10, 100±0.01%), FRONTAL-PISTOL (10/10, 100±0.01%), BEHIND EAR-INLINE (9/10, 90±9.49%), and BEHIND EAR-PISTOL (10/10, 100±0.01%); brain damage (P = 0.5577) between FRONTAL-INLINE (9/9, 100±0.02%), FRONTAL-PISTOL (10/10, 100±0.02%), BEHIND EAR-INLINE (4/10, 40±15.49%), and BEHIND EAR-PISTOL (1/10, 10±9.49%); potential for bolt-thalamus contact (P = 0.0683) for FRONTAL-INLINE (2/10, 20±12.65%), FRONTAL-PISTOL (8/10, 80±12.65%), BEHIND EAR-INLINE (7/9, 77.78±13.86%), and BEHIND EAR-PISTOL (9/9, 100±0.02%); or thalamic damage (P = 0.8041) for FRONTAL-INLINE (1/10, 10±9.49%), FRONTAL-PISTOL (1/10, 10±9.49%), BEHIND EAR-INLINE (2/8, 25±15.31%), and BEHIND EAR-PISTOL (0/9, 0±0.00%). The FRONTAL placement with an INLINE PCB may present the least risk of failure for the PCB euthanasia of mature sows > 200 kg BW due to less total tissue thickness and thalamic depth, greater effective angle, and prevalent brain damage.

An Experimental Infection Model in Sheep and Goats to Evaluate Salmonella Colonization in Deep Tissue Lymph Nodes and after Carcass Vascular Rinsing with Bacteriophages in Goats.

An animal infection model was evaluated on sheep and goats to confirm which species infected with Salmonella enterica serovar Enteritidis C StR (SE13) would provide a consistent and high frequency of Salmonella colonization in lymph nodes (LNs) without causing undue animal morbidity. Sheep and goats (n = 5) were intradermally inoculated with Salmonella, postincubated for 7 days, and euthanized. Superficial cervical, medial iliac, subiliac, mammary, and popliteal LNs were excised from each carcass. Goat LNs had approximately 53% greater Salmonella level compared to sheep. Also, Salmonella was inconsistently recovered from the sheep LNs. Thus, goats were selected to determine the ability of carcass vascular rinsing (with and without bacteriophages) to reduce Salmonella in infected LNs. Goats with similar characteristics were grouped together before being randomly assigned to 3 postharvest treatments; control (CN, not vascularly rinsed; n = 10), vascularly rinsed with a standard Rinse & Chill® solution (RC; 98.5% water and a blend of saccharides and phosphates; n = 10), or vascularly rinsed with a standard Rinse & Chill® solution plus the addition of bacteriophages (BP; n = 10). Rinse & Chill® system was able to successfully deliver a mean 7.0 log PFU/g to the S. Enteritidis-infected LNs (mean 3.5 log CFU/g). However, neither Rinse & Chill® without bacteriophages nor with bacteriophages caused Salmonella reduction (P > 0.05) compared to the nonrinsed goat carcasses.

Quantification of cooling effects on basic tissue measurements and exposed cross-sectional brain area of cadaver heads from Holstein cows > 30 mo of age.

Penetrating captive bolt (PCB) is the primary method of preslaughter stunning for cattle and is also used for on-farm euthanasia. The objective of this study was to quantify the impact of cooling on the soft tissue thickness, cranial thickness, total tissue thickness, and cross-sectional brain area of cadaver heads collected from mature (> 30 mo of age) dairy cows following the application of a PCB stun in a frontal placement. Hide-on cadaver heads were obtained from culled dairy cows (N = 37) stunned in a frontal location using a handheld PCB device (Jarvis Model PAS-Type C 0.25R Caliber Captive Bolt, Long Bolt) at a commercial slaughter establishment. Following transport to the University of Wisconsin-River Falls, heads were split at midline along the bolt path by a bandsaw and then underwent FRESH, CHILL24, CHILL48, and CHILL72 refrigeration treatments. The FRESH treatment involved images collected immediately after splitting each head, the CHILL24 treatment involved images collected after 24 h of refrigeration, the CHIL48 treatment involved images collected after 48 h of refrigeration, and the CHILL72 treatment involved images collected after 72 h of refrigeration. Measurements of soft tissue thickness, cranial thickness, total tissue thickness, and cross-sectional brain area were recorded for each refrigeration treatment. Soft tissue thickness did not differ caudal to (P = 0.3751) or rostral to (P = 0.2555) the bolt path. Cranial thickness did not differ caudal to (P = 0.9281) or rostral to (P = 0.9051) the bolt path. Total tissue thickness did not differ caudal to (P = 0.9225; FRESH: 24.77 mm, CHILL24: 23.93 mm, CHILL48: 24.27 mm, CHILL72: 42.30, SE: 0.86) or rostral to (P = 0.8931; FRESH: 24.09 mm, CHILL24: 23.99, CHILL48: 24.26, CHILL72: 24.43 mm, SE: 0.79 mm) the bolt path. Cross-sectional brain area was not different (P = 0.0971) between refrigeration treatments (FRESH: 9,829.65 ±â€…163.87 mm2, CHILL24: 10,012.00 ±â€…163.87 mm2, CHILL48: 9,672.43 ±â€…163.87 mm2, CHILL72: 10,235.00 ±â€…166.34 mm2). This study demonstrated that FRESH tissue parameters can be determined from cattle cadaver heads refrigerated for 24, 48, or 72 h.

Use of a Calving Blind That Imitates a Natural Environment.

Many indoor-housed cows isolate at calving when given the opportunity, and calving behaviors vary by blind and pen design. The objectives of this study were to determine if cows preferred calving in a visibly separated (blind) or an open area of a group maternity pen, and if there was a preference for the degree of seclusion provided by the blind (50% vs. 100% coverage). Two calving blinds were provided in a group calving pen, and the amount of visibility through the blinds was created using firehoses secured from the top of a metal frame that lined the entire front of the blind (100%) or with every other hose rolled up (50%). Holstein cows and heifers (n = 79) were enrolled into a dynamic group calving pen 21 ± 3 d before calving. Calving location, the difference in blind use prior to calving compared to a baseline period, and social behaviors were recorded using video observation. There was no difference in the number of cows that calved in or outside of a blind (28 vs. 37 calvings, respectively). Cows were more likely to calve in a blind during the day than at night and as the number of cows in the pen increased. For cows who calved in a blind, there was no preference for calving in the 50% or 100% blind (10 vs. 18, respectively). Providing a varied environment for intensively managed cattle can improve their welfare by allowing cows the opportunity to perform natural behaviors and choice over their environment.

Relationship of tissue dimensions and three captive bolt application sites on cadaver heads from mature swine (Sus scrofa domesticus) <200 kg body weight.

Penetrating captive bolt (PCB) is a common method of euthanasia for swine but has not been evaluated for mature swine < 200 kg body weight (BW). The objectives were to determine tissue depth, brain contact plane, and visible brain tissue damage (brain damage[BD]) for the common FRONTAL (F) and alternative TEMPORAL (T) and BEHIND EAR (BE) placements for PCB use on sows and boars weighing < 200 kg. Cadaver heads were obtained from 30 sows and 30 boars (estimated BW, mean ±â€…SD; sows: 165.8 ±â€…22.4 kg; boars: 173.6 ±â€…21.4 kg) from a slaughter establishment after electrical stunning and exsanguination. Heads were cooled at 2 to 4 °C for approximately 64 h. A Jarvis PAS-Type P 0.25R PCB with a Long Stunning Rod Nosepiece Assembly and a 3.5 GR power load was used for all PCB applications at the following placements: F-3.5 cm superior to the optic orbits at midline, T-at the depression posterior to the lateral canthus of the eye within the plane between the lateral canthus and the base of the ear, or BE-directly caudal to the pinna of the ear on the same plane as the eyes and targeting the middle of the opposite eye. For sows, the bolt path was in the brain for 10/10 (100.0%, 95% CI: 69.2% to 100.0%) F, T, and BE heads. In heads that could reliably be assessed for BD, BD was detected in 10/10 (100.0%, 95% CI: 69.2% to 100.0%) F heads, 9/9 (100.0%, 95% CI: 66.4% to 100.0%) T heads, and 0/10 (0.0%, 95% CI: 0.0% to 30.1%) BE heads. For boars, the bolt path was in the plane of the brain for 8/9 (88.9%, 95% CI: 51.8% to 99.7%) F heads, 9/10 (90.0%, 95% CI: 55.5% to 99.7%) T heads, and 11/11 (100.0%, 95% CI: 71.5% to 100.0%) BE heads. In heads that could reliably be assessed for BD, BD was detected in 8/9 (88.9%, 95% CI: 51.7% to 99.7%) F heads, 7/10 (70.0%, 95% CI: 34.8% to 93.3%) T heads, and 4/11 (36.4%, 95% CI: 10.9% to 69.2%) BE heads. Tissue depth was reported as mean ±â€…SE followed by 95% one-sided upper reference limit (URL). For sows, total tissue thickness differed (P < 0.05) between placements (F: 49.41 ±â€…2.74 mm, URL: 70.0 mm; T: 62.83 ±â€…1.83 mm, URL: 76.6 mm; BE: 84.63 ±â€…3.67 mm; URL: 112.3 mm). Total tissue thickness differed (P < 0.05) between placements for boars (F: 54.73 ±â€…3.23 mm, URL: 77.6 mm; T: 70.72 ±â€…3.60 mm, URL: 96.3 mm; BE: 92.81 ±â€…5.50 mm; URL: 135.3 mm). For swine between 120 and 200 kg BW, the F placement may have the greatest likelihood for successful euthanasia due to the least total tissue thickness and may present less risk for failure than the T and BE placements.

Euthanasia is an important procedure to protect animal welfare and cannot be avoided on swine farms. A common method of euthanasia for swine is penetrating captive bolt (PCB), which involves passing a metal bolt through the skull into the brain. This process should result in an immediate loss of consciousness. The use of PCB has been evaluated for market hogs and heavier sows and boars, but not for sows and boars weighing < 200 kg. As pigs mature, the cranial thickness at the common frontal PCB placement increases due to the expansion of sinus cavities. This makes PCB euthanasia more challenging for sows and boars. As a result, alternative PCB application sites, including behind the ear and at a temporal location, have been proposed. This study evaluated frontal, temporal, and behind-ear placements for PCB application to cadaver heads from sows and boars weighing < 200 kg. The frontal placement appeared to be more reliable than the temporal or behind ear placements due to the least tissue for the bolt to travel through to reach the brain, the greatest potential target area, and prevalent brain damage.

Evaluation of tissue depth, captive bolt penetration force and energy, and potential for bolt-thalamus contact in cadaver heads from physically castrated market barrows and immunocastrated boars.

The main objective of this study was to describe tissue thicknesses of cadaver heads from physically castrated market barrows (PC MARKET BARROWS) and immunocastrated boars (IC BOARS) at the frontal penetrating captive bolt (PCB) placement. Other objectives were to describe differences in bolt force and energy requirements to penetrate and describe potential for bolt-thalamus contact. Forty-four heads were obtained from PC MARKET BARROWS (n = 22) and IC BOARS (n = 22) of similar age and size that were rendered insensible with CO2. Mean HCW was 117.32 ±â€…3.52 kg. Snout to poll distance (cm) and maximum deflection distance (cm) were collected in duplicate. Heads were split at midline with a bandsaw and soft tissue and cranial thicknesses were measured with a digital caliper. Images of each cut surface were collected to evaluate the potential for thalamic damage. Tissue samples were retained from each half of each head and a universal tester was used to determine maximum force and energy of bolt penetration. There was no evidence to support a significant difference (P > 0.05) in tissue thicknesses between PC MARKET BARROWS and IC BOARS. Maximum deflection distance (maximum distance from a straight edge that was placed from the tip of the snout to the poll of the head) was not different (P = 0.10) between PC MARKET BARROWS (3.31 ±â€…0.10 cm) and IC BOARS (3.08 ±â€…0.10 cm). There was no evidence to support a difference (P = 0.77) in maximum force between PC MARKET BARROWS (7130.32 ±â€…483.23 N) and IC BOARS (6974.60 ±â€…463.70 N). There was also no evidence to support a difference (P = 0.62) in maximum energy between PC MARKET BARROWS (33.37 ±â€…2.77 J) and IC BOARS (32.04 ±â€…2.50 J). For PC MARKET BARROWS, there was a difference (P = 0.05) between the number of heads where the thalamus was located within the theoretical plane of bolt travel for market placement (21/21) versus mature placement (16/21). For IC BOARS, the number of heads where the thalamus was located within the plane of theoretical bolt path was not different between the two PCB placements (19/21 each). Overall, the data suggest that tissue profiles of PC MARKET BARROWS and IC BOARS do not differ at the frontal PCB placement site and the mechanical tools that are effective for PC MARKET BARROWS should also be effective for IC BOARS.

Broken tails in Holstein dairy cattle: A cross-sectional study.

Dairy cows are regularly handled when moved to the milking parlor and during other routine procedures. Low-stress handling methods are important in avoiding negative welfare states for dairy cattle. Tail twisting is used by some handlers to prompt cattle movement. However, when used inappropriately with excessive force, tail twisting can lead to a broken tail. The aim of this cross-sectional study was to determine cow-level factors that may be associated with the prevalence of broken tails in dairy cattle. A subset of 229 Holstein dairy cows (68 primiparous and 161 multiparous) at a single dairy were assessed for broken tails from the larger herd (N = 1,356). Tails were visually assessed for the presence of fractures by a single trained observer. A tail was classified as unfractured if it laid straight when at rest and as fractured if there were deviations in the tail when at rest. Poisson regression models were used to identify associations between cow-level characteristics and broken tails and compute adjusted prevalence ratios (PR). The prevalence of broken tails was 45.8% (105/229) at the time of assessment. Multiparous cows had a greater prevalence of broken tails than primiparous cows [PR = 1.70; 95% confidence interval (CI): 1.11-2.59]. The prevalence of broken tails was also greater for cows treated for mastitis ≥2 times than cows treated once for mastitis (PR = 1.84; 95% CI: 1.08-3.13) and cows never treated for mastitis (PR = 1.36; 95% CI: 1.02-1.82). Results from this study indicated that the longer a cow was present on the farm and the more times she was treated for mastitis, the more likely she was to experience a broken tail. These findings suggest that the relationship between dairy cow handling, health, and welfare is a multifactorial issue.

Assessment of United States Department of Agriculture Food Safety Inspection Service Humane Handling Enforcement Actions: 2018-2020.

Federally inspected slaughter establishments in the United States must adhere to the Humane Methods of Slaughter Act and regulations that enforce it. Failure to comply with this law results in a Humane Handling Enforcement Action (HHEA) issued by the United States Department of Agriculture Food Safety Inspection Service (USDA FSIS). The objective of this study was to systematically analyze and describe HHEAs issued between 2018 and 2020. Enforcement action notification letters were accessed from the USDA FSIS website and date, location, regulatory action, reason for noncompliance, species, and follow up action for each HHEA was recorded. Summary statistics (proportions and percentages) were calculated for the entire population dataset. Between 2018 and 2020, FSIS issued 293 HHEAs; 109 in 2018, 85 in 2019, and 99 in 2020. The majority of HHEAs (64.16%; 188 of 293) were related to the mechanical stunning of bovine (39.93%; 117 of 293) and porcine (24.23%; 71 of 293) species. The majority (50.23%; 107 of 213) of causative reasons for mechanical stun failure across all species were not clearly described; however, of those that were, most (39.12%; 68 of 213) were related to the placement of mechanical stuns. Addressing these issues through improved training and research would help to reduce the total number of HHEAs. Additional detail in reporting the events that result in HHEAs from USDA FSIS would aid in guiding corrective actions on an industry-wide scale.

Female Athletes and the Menstrual Cycle in Team Sports: Current State of Play and Considerations for Future Research.

Over the past ten years there has been a dramatic rise in female sport participation and accompanying female professional national leagues across multiple sports, yet research has not followed suit. Although there are known variations between female and male physiology, training protocols in female sport are predominantly underpinned by research undertaken in male athletes. The hormonal variability experienced by women across the menstrual cycle, as well as the menstrual cycle variability between women, may contribute to the complexity of conducting rigorous physiological studies, leading to a paucity of robust sports-specific research that can be confidently applied to female athletes. Moreover, barriers exist in female sport that potentially limit the ability to conduct research, including the lack of full-time programs and limited resources. Recently, there has been increased interest in the potential effects of fluctuations in the female sex hormones, progesterone and oestrogen, on sport performance across different phases of the menstrual cycle. However, current research evaluating the menstrual cycle and physical performance (such as strength, speed, aerobic fitness, and athletes' perception of their performance) have shown inconsistent results. Additionally, methodological design across studies has shown little consistency, making it difficult to draw firm conclusions, which potentially prevents female athletes optimising their physical and sporting performance. It further impacts coaches and sports science researchers in their ability to provide appropriate training recommendations and educational opportunities. It is important to progress in female athlete research with an understanding of how the unique physiology of female athletes may influence their ability to physically perform in their respective sport, which requires representation in sports science research. This paper will provide an overview on current evidence and limitations within menstrual cycle research and provide considerations and directions for future research in this space within team sports.

History and best practices of captive bolt euthanasia for swine.

The definition of animal welfare includes how an animal dies. As such, euthanasia is intrinsically linked to animal welfare, and ensuring a good death through effective, safe, and validated practices is a critical piece of promoting positive animal welfare. The objective of this review is to provide a better understanding of the literature on the euthanasia of swine via penetrating captive bolt (PCB) and nonpenetrating captive bolt (NPCB), as well as a history of captive bolt use, and indicators of sensibility and insensibility. To do this, we performed a systematic review that included 30 peer-reviewed articles and 17 other publications. NPCB devices have been validated as an effective single-step euthanasia method for neonatal and preweaning swine, as well as a two-step euthanasia method for nursery swine. PCB devices have been validated as an effective euthanasia method for nursery and market swine up to 120 kg, but further investigation is required for the use of captive bolt devices on mature breeding sows and boars.

Relationship of tissue dimensions and three captive bolt placements on cadaver heads from mature swine (Sus scrofa domesticus) > 200 kg body weight.

Three penetrating captive bolt (PCB) placements were tested on cadaver heads from swine with estimated body weight (BW) >200 kg (sows = 232.9 ± 4.1 kg; boars = 229.3 ± 2.6 kg). The objectives were to determine tissue depth, cross-sectional brain area, visible brain damage (BD), regions of BD, and bolt-brain contact; and determine relationships between external head dimensions and tissue depth at each placement. A Jarvis PAS-Type P 0.25R PCB with a Long Stunning Rod Nosepiece Assembly and 3.5 g power loads was used at the following placements on heads from 111 sows and 46 boars after storage at 2 to 4 °C for ~62 h before treatment: FRONTAL (F)-3.5 cm superior to the optic orbits at midline, TEMPORAL (T)-at the depression posterior to the lateral canthus of the eye within the plane between the lateral canthus and the base of the ear, or BEHIND EAR (BE)-directly caudal to the pinna of the ear on the same plane as the eyes and targeting the middle of the opposite eye. For sows, the bolt path was in the plane of the brain for 42/42 (100%, 95% confidence interval [CI]: 91.6% to 100.0%) F heads, 39/40 (97.5%, 95% CI: 86.8% to 99.9%) T heads, and 34/39 (87.5%, 95% CI: 72.6% to 95.7%) BE heads; for the heads that could reliably be assessed for BD damage was detected in 25/26 (96.2%, 95% CI: 80.4% to 99.9%) F heads, 24/35 (68.6%, 95% CI: 50.7% to 83.2%) T heads, and 5/40 (12.5%, 95% CI: 4.2% to 26.8%) BE heads. For boars, the bolt path was in the plane of the brain for 17/17 (100.0%, 95% CI: 80.5% to 100.0%) F heads, 18/18 (100.0%, 95% CI: 81.5% to 100.0%) T heads, and 14/14 (100.0%, 95% CI: 76.8% to 100.0%) BE heads; damage was detected in 11/12 (91.7%, 95% CI: 61.5% to 99.8%) F heads, 2/15 (13.3%, 95% CI: 1.7% to 40.5%) T heads, and 7/14 (50.0%, 95% CI: 23.0% to 77.0%) BE heads. Tissue depth was reported as mean ± standard error followed by 95% one-sided upper reference limit (URL). For sows, total tissue thickness was different (P < 0.05) between placements (F: 52.7 ± 1.0 mm, URL: 64.1 mm; T: 69.8 ± 1.4 mm, URL: 83.9 mm; BE: 89.3 ± 1.5 mm, URL: 103.4 mm). In boars, total tissue thickness was different (P < 0.05) between placements (F: 41.2 ± 2.1 mm, URL: 56.3 mm; T: 73.2 ± 1.5 mm, URL: 83.4 mm; BE: 90.9 ± 3.5 mm, URL: 113.5 mm). For swine > 200 kg BW, F placement may be more effective than T or BE due to less soft tissue thickness, which may reduce concussive force. The brain was within the plane of bolt travel for 100% of F heads with BD for 96.2% and 91.7% of F sow and boar heads, respectively.

Efficacy of oral meloxicam as primary pain mitigation following caustic paste disbudding of three day old Holstein calves1.

The purpose of this study was to investigate the effects of perioperative administration of oral meloxicam prior to and following the application of caustic paste to disbud neonatal dairy calves. Sixty-one 3-4-d-old Holstein heifer calves were randomly assigned to one of four treatment groups of 15-16 calves. The treatment groups were: 1) M1, caustic paste disbudding and oral meloxicam (45 mg) with a placebo 24 h later; 2) M2, treatment M1 followed by a second 45-mg dose of meloxicam 24 h later instead of placebo; 3) CONTROL, treatment M1 with placebo in place of meloxicam; and 4) SHAM, sham disbudding with placebo in place of meloxicam. Infrared thermography was used to quantify eye and horn bud temperatures. Pressure algometry was used to measure Mechanical nociceptive threshold (MNT) surrounding the horn bud. Average daily gain and body weight (BW) were obtained by weighing each animal throughout the study and calculating the changes over time. Plasma was collected and analyzed for cortisol and substance P concentrations. Substance P and cortisol decreased in all animals over time, regardless of treatment. Mean plasma substance P concentration across all time points was greater (P < 0.05) in the SHAM group than M1 or M2 but not different (P > 0.05) than the CONTROL group. The MNT and ocular temperatures decreased over time across all treatments (P < 0.05). Mean BW increased over time across all treatments (P < 0.05). A significant interaction (P < 0.05) between treatment and sampling time was observed at 12 h following treatment application for both mean horn bud temperature and the ratio between horn bud and ocular temperature. Overall, the results of this study suggest that meloxicam administration at a dose of 45 mg per animal may have limited influence as the primary modulator of pain and inflammatory response in calves that have been disbudded with caustic paste at 3 d of age.

Quantification of cooling effects on basic tissue measurements and exposed cross-sectional brain area of cadaver heads from market pigs.

The objective of this project was to determine the impact of cooling on the soft tissue thickness, cranial thickness, and cross-sectional brain area of cadaver heads from market pigs. Documenting the effect of cooling on tissue dimensions of swine heads is valuable and important for future investigations of physical stunning and euthanasia methods that use cadaver heads. Scalded and dehaired cadaver heads with intact jowls were sourced from market pigs stunned with CO2 gas. After transport to the data collection location, a penetrating captive bolt (PCB) shot (Jarvis Model PAS-Type P 0.25R Caliber Captive Bolt Pistol with Medium Rod Assembly and Blue Powder Cartridges) was applied in the frontal position. Following PCB application, each head (n = 36) underwent an UNCHILLED treatment followed by CHILLED treatment. The UNCHILLED treatment involved images collected immediately after splitting each head along the bolt path, and the CHILLED treatment involved images of the same heads after storage in a walk-in cooler for 24 h at 2 to 4°C. All measurements for each treatment were collected from images of the heads on the plane of the bolt path immediately prior to and immediately after the refrigeration treatment. Measurements were performed by two observers. Across all measurements, mean interobserver coefficient of variation was 11.3 ± 0.6%. The soft tissue caudal to the bolt path was different (P = 0.0120) between treatments (CHILLED: 6.4 ± 0.2 mm; UNCHILLED: 7.2 ± 0.2 mm). The soft tissue thickness rostral to the bolt path was different (P = 0.0378) between treatments (CHILLED: 5.5 ± 0.2 mm; UNCHILLED: 6.1 ± 0.2 mm). Cranial thickness caudal to the bolt path was not different (P = 0.8659; CHILLED: 18.1 ± 0.6 mm; UNCHILLED: 18.3 ± 0.6 mm), nor was there a significant difference (P = 0.2593) in cranial thickness rostral to the bolt path between treatments (CHILLED: 16.2 ± 0.6 mm; UNCHILLED: 15.2 ± 0.6 mm). Cross-sectional brain area did not differ (P = 0.0737; CHILLED: 3633.4 ± 44.1 mm; UNCHILLED: 3519.9 ± 44.1 mm). A correction factor of 1.12 was determined from this study for cases where estimation of UNCHILLED soft tissue thickness from CHILLED soft tissue thickness is necessary.

Inspiratory flow-resistive breathing, respiratory muscle-induced systemic oxidative stress, and diaphragm fatigue in healthy humans.

We questioned whether the respiratory muscles of humans contribute to systemic oxidative stress following inspiratory flow-resistive breathing, whether the amount of oxidative stress is influenced by the level of resistive load, and whether the amount of oxidative stress is related to the degree of diaphragm fatigue incurred. Eight young and healthy participants attended the laboratory for four visits on separate days. During the first visit, height, body mass, lung function, and maximal inspiratory mouth and transdiaphragmatic pressure (Pdimax) were assessed. During visits 2-4, participants undertook inspiratory flow-resistive breathing with either no resistance (control) or resistive loads equivalent to 50 and 70% of their Pdimax (Pdimax50% and Pdimax70%) for 30 min. Participants undertook one resistive load per visit, and the order in which they undertook the loads was randomized. Inspiratory muscle pressures were higher (P < 0.05) during the 5th and Final min of Pdimax50% and Pdimax70% compared with control. Plasma F2-isoprostanes increased (P < 0.05) following inspiratory flow-resistive breathing at Pdimax70%. There were no increases in plasma protein carbonyls or total antioxidant capacity. Furthermore, although we evidenced small reductions in transdiapragmaic twitch pressures (PdiTW) after inspiratory flow-resistive breathing at Pdimax50% and Pdimax70%, this was not related to the increase in plasma F2-isoprostanes. Our novel data suggest that it is only when sufficiently strenuous that inspiratory flow-resistive breathing in humans elicits systemic oxidative stress evidenced by elevated plasma F2-isoprostanes, and based on our data, this is not related to a reduction in PdiTW.NEW & NOTEWORTHY We examined whether the respiratory muscles of humans contribute to systemic oxidative stress following inspiratory flow-resistive breathing, whether the amount of oxidative stress is influenced by the level of resistive load, and whether the amount of oxidative stress is related to the degree of diaphragm fatigue incurred. It is only when sufficiently strenuous that inspiratory flow-resistive breathing elevates plasma F2-isoprostanes, and our novel data show that this is not related to a reduction in transdiaphragmatic twitch pressure.

Relationship of captive bolt stunning location with basic tissue measurements and exposed cross-sectional brain area in cadaver heads from market pigs.

The objective of this study was to contrast the soft tissue thickness, cranial thickness, total tissue thickness, cross-sectional brain area, and bolt-brain contact from the common frontal application of captive bolt euthanasia with the alternative location behind the ear in cadaver swine heads. Twenty-three cadaver heads from pigs that were approximately 136 kg and 6 mo of age were collected from a regional slaughter establishment following CO2 stunning and assigned to either the FRONTAL (n = 11) or the CAUDAL TO PINNA (n = 12) application of the captive bolt. The soft tissue thickness was different (P < 0.0001) between the 2 applications (FRONTAL: 8.3 ± 3.4 mm; CAUDAL TO PINNA: 56.5 ± 3.4 mm). The cranial thickness was different (P < 0.0001) between the applications (FRONTAL: 23.4 ± 2.9 mm; CAUDAL TO PINNA: 26.5 ± 2.9 mm). There was also a difference (P < 0.0001) in the total tissue thickness between the 2 applications (FRONTAL: 31.7 ± 3.8 mm; CAUDAL TO PINNA: 73.4 ± 3.8 mm). Cross-sectional area was calculated from images collected immediately after the heads were cut along the plane of bolt travel by bandsaw and was different (P = 0.0028) between the 2 applications (FRONTAL: 25.2 ± 1.3 cm2; CAUDAL TO PINNA: 18.9 ± 1.3 cm2). Bolt-brain contact was also assessed from the images, and a difference (P = 0.0360) between the 2 applications (FRONTAL: 100 ± 10.5%; CAUDAL TO PINNA: 66.7 ± 10.5%) was identified. The results of this study suggest that the FRONTAL application may provide a bolt path with less tissue to travel through when compared with the CAUDAL TO PINNA application for pigs of the approximate age and weight of those in this study. Ultimately, the FRONTAL location may present less risk for the captive bolt euthanasia of swine at market weight at this time. Additional refinement of the CAUDAL TO PINNA procedure and modification to the captive bolt device to penetrate to a suitable depth to ensure brain damage is recommended.

Parkinson disease-associated mutation R1441H in LRRK2 prolongs the "active state" of its GTPase domain.

Mutation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD). A disease-causing point mutation R1441H/G/C in the GTPase domain of LRRK2 leads to overactivation of its kinase domain. However, the mechanism by which this mutation alters the normal function of its GTPase domain [Ras of complex proteins (Roc)] remains unclear. Here, we report the effects of R1441H mutation (RocR1441H) on the structure and activity of Roc. We show that Roc forms a stable monomeric conformation in solution that is catalytically active, thus demonstrating that LRRK2 is a bona fide self-contained GTPase. We further show that the R1441H mutation causes a twofold reduction in GTPase activity without affecting the structure, thermal stability, and GDP-binding affinity of Roc. However, the mutation causes a twofold increase in GTP-binding affinity of Roc, thus suggesting that the PD-causing mutation R1441H traps Roc in a more persistently activated state by increasing its affinity for GTP and, at the same time, compromising its GTP hydrolysis.

Threshold levels of ABL tyrosine kinase inhibitors retained in chronic myeloid leukemia cells determine their commitment to apoptosis.

The imatinib paradigm in chronic myelogenous leukemia (CML) established continuous BCR-ABL inhibition as a design principle for ABL tyrosine kinase inhibitors (TKI). However, clinical responses seen in patients treated with the ABL TKI dasatinib despite its much shorter plasma half-life and the apparent rapid restoration of BCR-ABL signaling activity following once-daily dosing suggested acute, potent inhibition of kinase activity may be sufficient to irrevocably commit CML cells to apoptosis. To determine the specific requirements for ABL TKI-induced CML cell death for a panel of clinically important ABL TKIs (imatinib, nilotinib, dasatinib, ponatinib, and DCC-2036), we interrogated response of CML cell lines and primary CML cells following acute drug exposure using intracellular fluorescence-activated cell sorting and immunoblot analyses of BCR-ABL signaling, apoptosis measurements, liquid chromatography/tandem mass spectrometry of intracellular drug levels, and biochemical TKI dissociation studies. Importantly, significant intracellular TKI stores were detected following drug washout, levels of which tracked with onset of apoptosis and incomplete return of BCR-ABL signaling, particularly pSTAT5, to baseline. Among TKIs tested, ponatinib showed the most robust capacity for apoptotic commitment showing sustained suppression of BCR-ABL signaling even at low intracellular levels following extensive washout, consistent with high-affinity binding and slow dissociation from ABL kinase. Together, our findings suggest commitment of CML cells to apoptosis requires protracted incomplete restoration of BCR-ABL signaling mediated by intracellular retention of TKIs above a quantifiable threshold. These studies refine our understanding of apoptotic commitment in CML cells and highlight parameters important to design of therapeutic kinase inhibitors for CML and other malignancies.

Screening for novel LRRK2 inhibitors using a high-throughput TR-FRET cellular assay for LRRK2 Ser935 phosphorylation.

BACKGROUND: Mutations in the leucine-rich repeat kinase-2 (LRRK2) have been linked to Parkinson's disease. Recent studies show that inhibition of LRRK2 kinase activity decreased the level of phosphorylation at its own Ser910 and Ser935, indicating that these sites are prime targets for cellular readouts of LRRK2 inhibition. METHODOLOGY/PRINCIPAL FINDINGS: Using Time-Resolved Förster Resonance Energy Transfer (TR-FRET) technology, we developed a high-throughput cellular assay for monitoring LRRK2 phosphorylation at Ser935. LRRK2-Green Fluorescence Protein (GFP) fusions were expressed in cells via BacMam. Phosphorylation at Ser935 in these cells is detected using a terbium labeled anti-phospho-Ser935 antibody that generates a TR-FRET signal between terbium and GFP. LRRK2 wild-type and G2019S are constitutively phosphorylated at Ser935 in cells as measured by TR-FRET. The phosphorylation level is reduced for the R1441C mutant and little could be detected for the kinase-dead mutant D1994A. The TR-FRET cellular assay was further validated using reported LRRK2 inhibitors including LRRK2-IN-1 and our results confirmed that inhibition of LRRK2 can reduce the phosphorylation level at Ser935. To demonstrate the utility of this assay for screening, we profiled a small library of 1120 compounds. Three known LRRK2 inhibitors were identified and 16 hits were followed up in the TR-FRET and a cytotoxicity assay. Interestingly, out of the top 16 hits, five are known inhibitors of IκB phosphorylation, two CHK1 and two CDC25 inhibitors. Thirteen hits were further tested in a biochemical LRRK2 kinase activity assay and Western blot analysis for their effects on the phosphorylation of Ser910, Ser935, Ser955 and Ser973. CONCLUSIONS/SIGNIFICANCE: We developed a TR-FRET cellular assay for LRRK2 Ser935 phosphorylation that can be applied to the screening for LRRK2 inhibitors. We report for the first time that several compounds such as IKK16, CHK1 inhibitors and GW441756 can inhibit LRRK2 Ser935 phosphorylation in cells and LRRK2 kinase activity in vitro.

Pharmacological inhibition of LRRK2 cellular phosphorylation sites provides insight into LRRK2 biology.

Mutations in LRRK2 (leucine-rich repeat kinase 2) have been linked to inherited forms of PD (Parkinson's disease). Substantial pre-clinical research and drug discovery efforts have focused on LRRK2 with the hope that small-molecule inhibitors of the enzyme may be valuable for the treatment or prevention of the onset of PD. The pathway to develop therapeutic or neuroprotective agents based on LRRK2 function (i.e. kinase activity) has been facilitated by the development of both biochemical and cell-based assays for LRRK2. LRRK2 is phosphorylated on Ser910, Ser935, Ser955 and Ser973 in the N-terminal domain of the enzyme, and these sites of phosphorylation are likely to be regulated by upstream enzymes in an LRRK2 kinase-activity-dependent manner. Knowledge of these phosphorylation sites and their regulation can be adapted to high-throughput-screening-amenable platforms. The present review describes the utilization of LRRK2 phosphorylation as indicators of enzyme inhibition, as well as how such assays can be used to deconvolute the pathways in which LRRK2 plays a role.

Functional characterization of an isoform-selective inhibitor of PI3K-p110ß as a potential anticancer agent.

UNLABELLED: Genetic approaches have shown that the p110ß isoform of class Ia phosphatidylinositol-3-kinase (PI3K) is essential for the growth of PTEN-null tumors. Thus, it is desirable to develop p110ß-specific inhibitors for cancer therapy. Using a panel of PI3K isoform-specific cellular assays, we screened a collection of compounds possessing activities against kinases in the PI3K superfamily and identified a potent and selective p110ß inhibitor: KIN-193. We show that KIN-193 is efficacious specifically in blocking AKT signaling and tumor growth that are dependent on p110ß activation or PTEN loss. Broad profiling across a panel of 422 human tumor cell lines shows that the PTEN mutation status of cancer cells strongly correlates with their response to KIN-193. Together, our data provide the first pharmacologic evidence that PTEN-deficient tumors are dependent on p110ß in animals and suggest that KIN-193 can be pursued as a drug to treat tumors that are dependent on p110ß while sparing other PI3K isoforms. SIGNIFICANCE: We report the first functional characterization of a p110ß-selective inhibitor, KIN-193, that is efficacious as an antitumor agent in mice. We show that this class of inhibitor holds great promise as a pharmacologic agent that could be used to address the potential therapeutic benefit of treating p110ß-dependent PTEN-deficient human tumors.