Emotive Themes from Tennessee Cattle Producers Regarding Responsible Antibiotic Use.

To improve judicious antimicrobial use (AMU) in food animals in the United States, all feed additives that were medically important antimicrobials were moved from over the counter to Veterinary Feed Directive in 2017. This action required a change in behavior of producers' AMU practices. Because emotions are important aspects of behavior, several behavioral interventions have targeted people's emotions as means of effecting change. Hence, understanding and incorporating the emotional experiences of producers towards current AMU practices can be a starting point to making future behavioral changes that could reduce the emergence of antimicrobial resistance challenge. Between June 2017 and March 2018, seven focus group meetings of Tennessee (TN) beef and dairy cattle producers were conducted to evaluate producers' emotional views regarding responsible AMU in TN cattle. Sixty-two TN cattle producers participated and emotively expressed the following: (1) deep connections to animals in ways that improve animal and public health; (2) pride in their quality of products; (3) distress that consumers misconceive their AMU practices as indiscriminate; and (4) recommended that producers be more transparent about their AMU practices and the public improve their awareness for detecting marketers' deceptive product labels that take advantage of public ignorance. Knowledge of these producers' emotions would help educators target more successful behavioral change campaigns, improving stewardship in AMU practices among producers.

Control of Salmonella Dublin in a bovine dairy herd.

Salmonella enterica serovar Dublin (S. Dublin) was diagnosed in a dairy herd after signs of acute gastroenteritis and sepsis. Two hundred eighty-three Holstein cattle were sampled resulting in 700 observations, and serology for S. Dublin was performed. Holstein cattle sampled were divided by origin and arrival date to determine on-farm exposure. Prevalence estimates were calculated and compared with control measures implemented on the dairy during the outbreak. One group of cows, presumed to be the original carrier animals, had the highest overall seroprevalence (76.5%). Seroprevalence decreased throughout the study, coinciding with testing and management changes. This report documents biosecurity measures that identified S. Dublin after the purchase of subclinical carrier cattle and the steps taken to successfully control herd transmission.

Alterations in pregnancy-associated glycoprotein concentrations of pregnant sheep experimentally infected with bovine viral diarrhea virus.

OBJECTIVE: To compare pregnancy-associated glycoprotein 1 (PAG1) concentrations in maternal (jugular vein) and fetal (uterine vein) circulations and amniotic fluid samples between pregnant ewes that were and were not experimentally infected with bovine viral diarrhea virus (BVDV). ANIMALS: 11 healthy pregnant yearling ewes. PROCEDURES: Before study initiation, all ewes were naïve to BVDV and confirmed pregnant by transabdominal ultrasonography at approximately 60 days of gestation. At 65 days of gestation, ewes were intranasally inoculated with a noncytopathic BVDV type 1b strain (concentration, 107 TCID50/mL; 2 mL/nostril; n = 6) or an equal volume of BVDV-free viral culture medium (control; 5). A blood sample was collected for measurement of PAG1 concentration before inoculation. At 80 days of gestation, each ewe was anesthetized and underwent an ovariohysterectomy. While sheep were anesthetized, blood samples from the jugular and uterine veins and an amniotic fluid sample were collected for measurement of PAG1 concentration. Fetal tissues underwent real-time PCR analysis for BVDV RNA, and placental specimens underwent histologic evaluation and immunohistochemical staining for BVDV antigen. RESULTS: At 80 days of gestation, BVDV RNA in fetal tissues and mild placentitis were detected in 5 of 6 BVDV-inoculated ewes. Mean PAG1 concentrations in the maternal and fetal circulations of BVDV-inoculated ewes were significantly less than those in control ewes. Mean amniotic fluid PAG1 concentration did not differ significantly between the 2 groups. CONCLUSIONS AND CLINICAL RELEVANCE: Concentration of PAG1 in the maternal circulation may be a useful biomarker for determining placental health in sheep after viral infection of the reproductive tract.

Evaluation of the pharmacokinetics and efficacy of transdermal flunixin for pain mitigation following castration in goats.

The mitigation of pain associated with common management procedures is a rising concern among veterinarians, producers and consumers. Nonsteroidal anti-inflammatory drugs are vital compounds for this purpose due to their cost, convenience, and efficacy. A transdermal formulation of flunixin meglumine (FM) was approved for the treatment of pain in cattle; however, the efficacy has yet to be determined for small ruminants. The current study had two aims: 1) to determine the pharmacokinetics of transdermal flunixin meglumine (TD FM) in bucklings and 2) to evaluate pain mitigation by TD FM following castration. To evaluate pharmacokinetics, 12 male goats (mean age = 6 mo) received 2.2 mg/kg of FM IV (n = 6) or 3.3 mg/kg TD FM (n = 6). Plasma FM concentrations were measured. The mean C max, T max, and harmonic mean half-life for TD FM were 1.09 ± 0.65 µg/mL, 5.50 ± 2.95 h, and 7.16 ± 2.06 h, respectively. To evaluate the efficacy of pain mitigation, 18 goats were randomly assigned to three treatment groups: 1) TD FM and castration (FM CAST) (n = 6); 2) transdermal placebo and castration (PL CAST) (n = 6); and 3) TD FM and sham castration (SHAM) (n = 6). Plasma samples were collected at 0, 12, 24, 36, 48, 72, and 96 h to assess cortisol and prostaglandin E2 (PGE2). Daily dry matter intake (DMI) was recorded and body weight was measured at the beginning and end of the study. Thermography (IRT) images of the scrotum, as well as heart rate (HR), respiratory rate (RR), and rectal temperature, were taken twice daily. Separate mixed analysis of variance models were used to test the effects of treatment, time, and their interaction on mean body temperature, IRT, HR, and RR. Autoregressive covariance structure was utilized to account for repeated measures and individual goat DMI prior to the study was added as a covariate. There were no differences in vital parameters, IRT measurements, cortisol, or PGE2 in animals receiving either TD FM or placebo following castration (P > 0.05). DMI had a treatment by hour interaction and was significantly higher in FM CAST and SHAM groups than the PL CAST group (P = 0.04). Goats in the SHAM group gained weight throughout the study, whereas goats in all other groups lost weight (P = 0.02). Results indicate that TD FM may mitigate pain as demonstrated by increased DMI; however, a single dose may not be sufficient to reduce physiological indicators of pain associated with castration in goats.

A survey of antimicrobial use practices of Tennessee beef producers.

BACKGROUND: Inappropriate antimicrobial use (AMU) is a key modifiable factor that leads to the development of antimicrobial resistance (AMR). The objectives of this study were to determine the following among Tennessee beef cattle producers: (1) the opinions on factors driving AMU (2) opinions on alternatives to antimicrobials, (3) the knowledge and perceptions regarding AMU and AMR, and (4) the preferred avenues for receiving information on prudent AMU. A survey questionnaire was made available to participants both in print and online from January 26, 2018 through May 11, 2018. The questions targeted the producers' demographics and their AMU practices; factors driving producer's choice of antimicrobials; perceptions, opinions and concerns about AMU and AMR in cattle production. Ordinal logistic regression was used to test for associations between the captured demographic information and producers' degree of concern about AMR. RESULTS: Overall, 231 beef producers responded to all or some of the survey questions. More than 60% of the participants mentioned that they kept up-to-date written records on antimicrobial purchases and AMU. Regarding extra-label use, 169 (84.1%) of the 201 respondents did not practice extra-label AMU. Profitability of the beef operation was a key factor influencing the decisions of many producers to use antimicrobials for disease management and prevention on their farms. Of the 228 producers who completed the question on the rating of their degree of concern about AMR, 50 (21.9%) reported that they were very concerned about AMR, 133 (58.3%) were moderately concerned, and 36 (15.8%) reported that they were not concerned about AMR. Nine producers (4%) did not rate their degree of concern about AMR because they were not familiar with what antimicrobial resistance meant. The inferential analyses suggested that younger beef producers were significantly less concerned about AMR when compared to the older ones (P = 0.019). Regarding avenues for receiving information on prudent AMU, no single medium was most preferred by all the respondents. CONCLUSIONS: There is a need to promote the use of written antimicrobial treatment protocols among beef producers in Tennessee. Continued training for beef producers on infection prevention and control and prudent AMU is needed.

Perceptions of Tennessee cattle producers regarding the Veterinary Feed Directive.

Since January 1, 2017, the United States Food and Drug Administration (FDA) has fully implemented the Veterinary Feed Directive (VFD) final rule aimed at facilitating the judicious use of medically important antimicrobials in food-producing animals. The objective of this study was to identify the common perceptions of Tennessee (TN) cattle producers regarding the VFD. We used a combination of focus groups and survey questionnaires to explore TN cattle producers' perceptions regarding the VFD. Preliminary findings from seven focus groups of 62 producers were used in the development of the questionnaire sent both online and in-print to rest of cattle producers in TN. The beef focus group participants perceived the VFD: to be a top-down policy; to have led to unregulated access to in-feed antimicrobials; a regulation that has limited the producers' ability to prevent disease and leading to economic losses; to negatively affect small producers; and to be affected by challenges related to prescription writing and disposal of un-used medicines. The dairy focus group participants perceived the VFD as unnecessary and burdensome, to have affected small producers, and introduced additional costs. Among the survey questionnaire respondents, 35 (15.4%) beef producers and 6 (13.6%) dairy producers respectively were not familiar at all with the VFD. Forty-eight (21.1%) beef producers and 11 (25%) dairy producers were slightly familiar with VFD. Gender was significantly associated (P = 0.02) with the beef producers' belief in the usefulness of the VFD. Similarly, for dairy producers, herd size was significantly associated (P = 0.002) with their perceptions regarding the usefulness of the VFD. The findings of this study could inform future VFD policy review processes. More awareness regarding the VFD and its benefits is needed among both beef and dairy producers in TN.

Drivers, alternatives, knowledge, and perceptions towards antimicrobial use among Tennessee beef cattle producers: a qualitative study.

BACKGROUND: In recent years, there has been an increased awareness of antimicrobial resistance in both animals and humans, which has triggered concerns over non-judicious antimicrobial use. In the United States, antimicrobial use in food-producing animals for growth promotion or improved feed efficiency is perceived as non-judicious. To facilitate judicious antimicrobial use, the United States Food and Drug Administration implemented the Veterinary Feed Directive, effective from January 1, 2017. Interventions, such as the VFD, designed to ensure the judicious use of antimicrobials among cattle producers may be more effective if the factors that inform and influence producer AMU practices are addressed. The specific objectives of this study were to determine the following among Tennessee beef cattle producers: (1) the most common drivers for using antimicrobials, (2) the perceived alternatives to antimicrobials, (3) the knowledge and perceptions regarding antimicrobial resistance, and (4) the preferred avenues for receiving information on prudent antimicrobial use. A total of 5 focus group meetings with beef producers were conducted in East, Middle, and West Tennessee. Each focus group was video recorded and thematic analysis was performed using NVivo. RESULTS: The factors that producers considered to drive antimicrobial use were the type of cattle operation, disease and animal welfare, economic factors, veterinarian consultation, producer's experience and peer support, Veterinary Feed Directive, and perceived drug efficacy. Vaccination, proper nutrition, and other good management practices were considered alternatives to antimicrobial use. To encourage vaccine use among small producers, participants suggested packaging vaccines into smaller quantities. Antimicrobial resistance was perceived to be a problem affecting animal and public health. Participants suggested additional education for cattle producers on the prudent use of antimicrobials as a measure for improving antimicrobial use. The veterinarian, producer associations and meetings, and county extension agents emerged as trusted avenues for channeling information on prudent antimicrobial use to cattle producers. CONCLUSIONS: Several factors drive antimicrobial use among cattle producers in Tennessee. Participants generally perceived their antimicrobial use to be discreet and only when necessary. More awareness of drivers for the development of antimicrobial resistance and continuing education on prudent antimicrobial use is needed for Tennessee beef producers.

Drivers of Antimicrobial Use Practices among Tennessee Dairy Cattle Producers.

Nonjudicious antimicrobial use (AMU) and inadequate antimicrobial stewardship are known modifiable factors driving the occurrence of antimicrobial resistance (AMR). A mixed methods approach using a combination of focus groups and survey questionnaires was used to explore the AMU practices of Tennessee (TN) dairy cattle producers. Specifically, the objectives of the study were to determine the following: (1) the most common drivers for using antimicrobials, (2) perceived alternatives to antimicrobials, (3) knowledge of and perceptions regarding AMR, (4) and the appropriate avenues for receiving information on prudent AMU. Two focus groups were conducted, one in July 2017 and the other in March 2018. The questionnaire was simultaneously made available to participants both in print form and online from January 26, 2018, through May 11, 2018. Twenty-three dairy producers participated in the focus groups and 45 responded to the survey. Eight (18.6%) producers never used bacterial culture and sensitivity testing (C/S) to select antimicrobials, more than half (25 producers (58.1%)) sometimes used C/S, four (9.3%) used C/S about half the time, five (11.6%) most of the time, and one (2.3%) always used C/S. The most common drivers for using antimicrobials were disease and animal welfare, pathogen surveillance, economic factors, veterinarian recommendation, producer's experience and judgment, drug attributes, and the Veterinary Feed Directive. Good management practices, vaccination, use of immunomodulatory products, and use of appropriate technology for early disease detection were considered alternatives to AMU. Four (9.1%) dairy producers were very concerned about AMR, 27 (61.4%) moderately concerned, and 10 (22.7%) not concerned. The veterinarian was considered to be a trusted source of information on prudent AMU. Use of C/S test results for antimicrobial selection is widespread among TN dairy producers. There is a need to popularize/promote selective dry cow therapy among TN dairy producers.

Passive protection against anthrax in mice with plasma derived from horses hyper-immunized against Bacillus anthracis Sterne strain.

In this study, equine source polyclonal anti-Bacillus anthracis immunoglobulins were generated and utilized to demonstrate passive protection of mice in a lethal challenge assay. Four horses were hyper-immunized with B. anthracis Sterne strain for approximately one year. The geometric mean anti-PA titer in the horses at maximal response following immunization was 1:77,936 (Log2 mean titer 16.25, SEM ± 0.25 95% CI [15.5 -17.0]). The geometric mean neutralizing titer at maximal response was 1:128 (Log2 mean titer 7, SEM ± 0.0, 95% CI 7). Treatment with hyper-immune plasma or purified immunoglobulins was successful in passively protecting A/J mice from a lethal B. anthracis Sterne strain challenge. The treatment of mice with hyper-immune plasma at time 0 h and 24 h post-infection had no effect on survival, but did significantly increase mean time to death (p < 0.0001). Mice treated with purified immunoglobulins at time 0 h and 24 h post-infection in showed significant increase in survival rate (p < 0.001). Bacterial loads in lung, liver and spleen tissue were also assessed and were not significantly different in mice treated with hyper-immune plasma from placebo treated control mice. Mice treated with purified antibodies demonstrated mean colony forming units/gram tissue fourfold less than mice receiving placebo treatment (p < 0.0001). Immunotherapeutics harvested from horses immunized against B. anthracis Sterne strain represent a rapidly induced, inexpensive and effective expansion to the arsenal of treatments against anthrax.

Behavioral and physiologic changes in Holstein steers experimentally infected with Mannheimia haemolytica.

OBJECTIVE To evaluate changes in behavior and surfactant protein (SP) A and D concentrations in serum and bronchoalveolar lavage fluid (BALF) samples of calves experimentally infected with Mannheimia haemolytica. ANIMALS Twelve 4- to 5-month-old Holstein steers. PROCEDURES Calves were divided into 2 treatment groups and instrumented with a data logger to collect behavioral data. After 10 days of acclimation, calves were experimentally inoculated with 3 × 109 CFUs to 5 × 109 CFUs of M haemolytica suspended in approximately 5 mL of PBS solution (infected calves; n = 6) or 5 mL of PBS solution without M haemolytica (control calves; 6) through a catheter into the right accessory lung lobe. Calves were clinically evaluated twice daily for 7 days after inoculation. Blood and BALF samples were collected from all calves at predetermined times for determination of serum and BALF SP-A and SP-D concentrations. Serum and BALF concentrations of SP-A and SP-D and behavioral data were evaluated over time and between treatment groups. RESULTS Compared with control calves, infected calves spent more time lying in general and more time lying on the right side during the 24 hours and 6 days after inoculation, respectively. Mean rectal temperature for infected calves (41.3°C) was significantly greater than that for control calves (39.2°C) 12 hours after inoculation. Mean respiratory rate for infected calves (52.5 breaths/min) was significantly greater than that for control calves (45.4 breaths/min) throughout the observation period. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated continuous behavioral monitoring may improve detection of calves with respiratory tract disease.

Diagnosis of lumbosacral diskospondylosis in a bucking bull assisted by high-definition thermal and nuclear scintigraphic imaging.

CASE DESCRIPTION An 8-year-old Brahman-cross bull was evaluated for left hind limb lameness of 2 months' duration. The lameness was first noticed during a rodeo bucking performance, immediately after the bull appeared to land inappropriately on the affected limb. CLINICAL FINDINGS Physical examination findings revealed left hind limb lameness, ataxia, and left-sided epaxial muscle atrophy. Palpation per rectum along the lumbar portion of the vertebral column revealed evidence of exostosis of the ventral aspect. High-definition infrared thermal imaging revealed a pattern of reduced skin temperature in the area of the left lumbar and gluteal regions suggestive of a disruption in the sympathetic control of peripheral blood flow. Nuclear scintigraphy revealed a focal area of increased radioisotope uptake on the left ventrolateral aspect of the L2-3 intervertebral joint. A presumptive diagnosis of ventrolateral vertebral spondylosis resulting in spinal nerve impingement was made. TREATMENT AND OUTCOME 200 mg of methylprednisolone was epidurally injected at the site of the lesion, and treatment with polysulfated glycosaminoglycans was initiated (500 mg, IM, every 4 days for 7 treatments, then monthly thereafter). The lameness and ataxia observed in the left hind limb resolved within 1 week after treatment began. Subsequently, the bull was discharged from the hospital and was used successfully for semen collection and live-cover breeding. CLINICAL RELEVANCE Use of thermography for the bull of this report provided additional insight into neurovascular physiologic function that classical imaging modalities are unable to provide and, when combined with nuclear scintigraphy, aided in identifying the most critical lesion in a complex clinical case.

Comparison of penetrating and nonpenetrating captive bolt methods in horned goats.

OBJECTIVE To use MRI and CT to compare the amount of tissue damage (soft tissue and bone) to the heads of goats after administration of a nonpenetrating or penetrating captive bolt. ANIMALS Cadavers of twelve 1- to 5-year-old mixed-breed goats that had been euthanized with an overdose of pentobarbital as part of an unrelated study. PROCEDURES Cadavers were randomly assigned to receive a nonpenetrating (n = 6) or penetrating (6) captive bolt. The head of 1 cadaver was imaged via CT and MRI. The muzzle of a device designed to administer either a penetrating or nonpenetrating captive bolt was then placed flush on the dorsal midline of each head at the level of the external occipital protuberance (poll) and aimed downward toward the cranialmost portion of the intermandibular space, and the assigned bolt was administered. Heads were removed, and CT and MRI of each head were performed. After imaging, each skull was transected along the sagittal plane to permit gross evaluation of central nervous tissue and obtain digital photographic images. In addition, 1 head that received a nonpenetrating captive bolt was further evaluated via blunt dissection and removal of adnexa from the external surface of the calvarium. RESULTS MRI, CT, and dissection of skulls revealed severe skeletal and soft tissue damage after impact with the penetrating and nonpenetrating captive bolts. CONCLUSIONS AND CLINICAL RELEVANCE The nonpenetrating captive bolt appeared to cause damage similar to that of the penetrating captive bolt in the cranium and soft tissues of the head in caprine cadavers. This damage suggested that administration of a nonpenetrating captive bolt as described here may be an acceptable method of euthanasia in goats.

Impact of the source and serial passaging of goat mesenchymal stem cells on osteogenic differentiation potential: implications for bone tissue engineering.

BACKGROUND: Adult mesenchymal stem cells (MSCs) can be conveniently sampled from bone marrow, peripheral blood, muscle, adipose and connective tissue, harvested from various species, including, rodents, dogs, cats, horses, sheep, goats and human beings. The MSCs isolated from adult tissues vary in their morphological and functional properties. These variations are further complicated when cells are expanded by passaging in culture. These differences and changes in MSCs must be considered prior to their application in the clinic or in a basic research study. Goats are commonly used as animal models for bone tissue engineering to test the potential of stem cells for bone regeneration. As a result, goat MSCs isolated from bone marrow or adipose tissue should be evaluated using in vitro assays, prior to their application in a tissue engineering project. RESULTS: In this study, we compared the stem cell properties of MSCs isolated from goat bone marrow and adipose tissue. We used quantitative and qualitative assays with a focus on osteogenesis, including, colony forming unit, rate of cell proliferation, tri-lineage differentiation and expression profiling of key signal transduction proteins to compare MSCs from low and high passages. Primary cultures generated from each source displayed the stem cell characteristics, with variations in their osteogenic potentials. Most importantly, low passaged bone marrow MSCs displayed a significantly higher and superior osteogenic potential, and hence, will be the preferred choice for bone tissue engineering in future in vivo experiments. In the bone marrow MSCs, this process is potentially mediated by the p38 MAPK pathway. On the other hand, osteogenic differentiation in the adipose tissue MSCs may involve the p44/42 MAPK pathway. CONCLUSIONS: Based on these data, we can conclude that bone marrow and fat-derived MSCs undergo osteogenesis via two distinct signaling pathways. Even though the bone marrow MSCs are the preferred source for bone tissue engineering, the adipose tissue MSCs are an attractive alternative source and undergo osteo-differentiation differently from the bone marrow MSCs and hence, might require a cell-based enhancer/inducer to improve their osteogenic regenerative capacity.

Graphene supports in vitro proliferation and osteogenic differentiation of goat adult mesenchymal stem cells: potential for bone tissue engineering.

Current treatments for bone loss injuries involve autologous and allogenic bone grafts, metal alloys and ceramics. Although these therapies have proved useful, they suffer from inherent challenges, and hence, an adequate bone replacement therapy has not yet been found. We hypothesize that graphene may be a useful nanoscaffold for mesenchymal stem cells and will promote proliferation and differentiation into bone progenitor cells. In this study, we evaluate graphene, a biocompatible inert nanomaterial, for its effect on in vitro growth and differentiation of goat adult mesenchymal stem cells. Cell proliferation and differentiation are compared between polystyrene-coated tissue culture plates and graphene-coated plates. Graphitic materials are cytocompatible and support cell adhesion and proliferation. Importantly, cells seeded on to oxidized graphene films undergo osteogenic differentiation in fetal bovine serum-containing medium without the addition of any glucocorticoid or specific growth factors. These findings support graphene's potential to act as an osteoinducer and a vehicle to deliver mesenchymal stem cells, and suggest that the combination of graphene and goat mesenchymal stem cells provides a promising construct for bone tissue engineering.

Sound intensity and noise evaluation in a critical care unit.

BACKGROUND: Sound levels in intensive care units can be high. Unfortunately, high levels of sound tend to result in poor sleep quality, which leads to slower healing, poorer immune response, and decreased cognitive function. OBJECTIVES: To measure sound levels to which patients in intensive care units are typically exposed. METHODS: Peak sound pressure levels of alarms on medical devices set at different output levels were measured. Additionally, ambient sound pressure levels for durations of 10 to 24 hours were measured on 12 occasions in patients' rooms in the intensive care unit. RESULTS: Peak levels of equipment alarms measured inside a patient's room were high, and increased as the setting of the alarm level increased. The levels of these alarms when measured in an adjacent room did not increase with alarm output level. Mean sound levels inside the patient's room were generally less than 45 dB(A), but peak levels were often greater than 85 dB(C). Closing the door of the adjacent room did not decrease these peak levels. Peak and mean levels did not differ systematically during 24 hours of measurement. CONCLUSIONS: High-intensity equipment alarms disturb patients' sleep but are critical in a medical emergency. However, nurses should not assume that raising the alarm output level will ensure that the alarm is audible from an adjacent room. Ambient noise measurements indicate high peak levels during both day and night.

Effect of probe tube insertion depth on spectral measures of speech.

This study investigated how depth variations in the tip of the probe tube affected spectral measures of speech recorded in the external ear canal. Consonant-vowel nonsense syllables were recorded with a probe tube microphone system in 10 adult participants with normal middle ear function. Recordings were made with the probe tube tip placed 1 mm, 5 mm, and 10 mm beyond the medial tip of a custom earmold. The effect of probe depth was evaluated on spectral levels (one-third octave and one- twelfth octave band). Extending the probe tube 10 mm past the medial tip of the earmold gave the most accurate results, with relatively lower sound levels for either the 1-mm or 5-mm insertion depth. In general, the effect of insertion depth was minimal at frequencies below 3 to 4 kHz, although this varied with the specific phoneme and the width of the analysis bands. The authors found no significant difference between 1- and 5-mm insertion depths, suggesting that as long as the tip of the probe tube is sufficiently close to the tympanic membrane to capture the highest frequency of interest, it makes little difference if it is less than 5 mm beyond the earmold tip.