The American alligator (Alligator mississippiensis) is an iconic reptile species of the southeastern United States (1). American alligators are considered a conservation success story as a result of protective federal legislation, the Endangered Species Act of 1973, which was enacted following sharp population declines due to overhunting and habitat loss during the mid-to late twentieth century (1). Today, American alligators and their eggs are collected commercially and raised on farms for meat, leather, and curios (2). Because of their exceptional conservation history, American alligators offer unique opportunities for educational programs within zoos and aquariums (3).

Due to the popularity of maintaining American alligators in zoo, aquaria, and under commercial managed conditions, veterinary medical care and applied animal welfare practices should be considered standard practice for this iconic reptile species. Routine clinical veterinary medical procedures in all reptiles, including crocodilians, may consist of wound cleaning and repair, blood and tissue sampling for diagnostics, diagnostic imaging, and surgical procedures. While there are some published data with respect to sedation and anesthesia of crocodilians, published clinical analgesic data for crocodilians, particularly American alligators, is lacking (4–6). In early reptile analgesia research, young Nile crocodiles (Crocodylus niloticus) developed significantly increased limb withdrawal latencies to a thermal noxious stimulus (i.e., hot plate) for at least 8 h with maximal effect after morphine (0.3 mg/kg and 1.0 mg/kg) was administered intracoelomically (7). In this same crocodile study, pethidine (i.e., meperidine) a short-acting μ-opioid agonist used in human and veterinary medicine, had a similar effect at 1.0 mg/kg intracoelomically, but for less than 3 h (7). However, unlike thermal noxious stimuli used in past crocodilian analgesia research, mechanical noxious stimuli have not been applied for use in crocodilian research even though recent evidence suggests that crocodilians have exquisitely sensitive skin mechanoreceptors in several anatomic areas, referred to as integumentary sensory organs (ISOs) (8). These ISOs were determined to convey a mechanical sensitivity exceeding that of primate fingertips, and were primarily distributed throughout the mandible, maxilla, near eyes and on parts of the fore-and hind limbs (8). Given this mechanosensitivity associated with the head and limbs of crocodilians, it seemed logical to take advantage of these anatomical sites (ISOs) to investigate the efficacy of analgesic drugs before and after exposure to a mechanical noxious stimulus.

Hydromorphone is considered a μ-opioid receptor agonist with some δ-opioid agonist activity, but is primarily considered a semisynthetic ketone derivative of morphine with approximately 10 times the potency (9). Although the exact mechanism of analgesic action is not completely understood, hydromorphone is believed to relieve moderate to severe pain by binding to μ-opioid receptors in the brain and spinal cord (10). While hydromorphone analgesic efficacy has not been evaluated in any crocodilian, it was demonstrated to be provide analgesia in red-eared sliders (Trachemys scripta elegans) using the thermal hind limb withdrawal nociception model at 0.5 mg/kg SC for up to 24 h (11). Additionally, hydromorphone plasma concentrations were considered analgesic, when compared to analgesic concentrations in mammals, for 12–24 h after bearded dragons (Pogona sp.) and red-eared slider turtles were administered either 0.5 or 1.0 mg/kg but, as with morphine, respiratory depression was a side effect at higher doses (12).

With the lack of published data regarding the use of analgesic drugs in alligators, the objective of this study was to evaluate the mechanical antinociceptive efficacy of hydromorphone in American alligators. We hypothesized that hydromorphone would provide antinociception to a mechanical noxious stimulus using von Frey filaments applied to theoretically mechanosensitive anatomic regions in American alligators.

The Texas A&M University–Kingsville IACUC (Protocol Number 2020-06-22) approved this study. Alligators were used in accordance with the Texas Scientific Collecting Permit No. SPR-0620-085.

Hydromorphone lessened the avoidance behavior associated with the application of the von Frey filament mechanosensory stimuli. Drug, sensory location, and von Frey filament diameter interacted (F_{72, 2,878} = 4.03, p < 0.0001; Table 1) with each other in their effects on alligator sensory perception. Differences in sensory perception by alligators occurred between hydromorphone and no drug at filament sizes ≥4.08, 4.17, 4.31, 4.56, and 4.93 grams-force for naris, rostrum, inside of mouth, rostral mandible, and lateral mandible, respectively (Figures 2A–E). Without hydromorphone, increased sensitivity beyond this force level was experienced when filaments reached 4.17, 4.31, 4.56, 4.93 and 5.18 grams-force for these locations, respectively. With administration of hydromorphone, however, sensitivity beyond this force level was experienced at filaments 4.56, 5.07, 5.46, 5.46, and 6.10 grams-force, respectively. Hydromorphone delayed the response by 3, 4, 4, 5, and 5 filament sizes at the naris, rostrum, inside of mouth, rostral mandible, and lateral mandible, respectively (Figures 2A–E). For example, alligator response in the naris occurred initially with the 4.08 grams-force von Frey filament, but on average, hydromorphone delayed the response until the 4.56 grams-force von Frey filament (i.e., 3 filament sizes larger; Figure 2A).

Sensory perception differed significantly among locations for alligators without hydromorphone (F_4,2,527 > 2.59, p < 0.0352) as well as with hydromorphone (F_4,2,527 > 4.69, p < 0.0352), although these differences depended on filament size (Figures 3A,B). Alligators displayed a response to von Frey filaments beginning at 4.08 and 4.95 grams-force without and with hydromorphone, respectively. In general, nares were the most sensitive location, followed by inside the mouth, rostral mandible, rostrum, and lateral mandible (Figures 3A,B). It is worth noting that alligators reacted to the largest von Frey filament (i.e., 6.65 grams-force) at each location >60% of the applications, but that level of response was only observed in the nares after alligators were administered hydromorphone.

Areas under the curve represent sensory perception by alligators (Figures 2A–E), which, without hydromorphone, ranged from 196.6 to 58.2 units for naris to lateral mandible, respectively. However, with hydromorphone, the area under the curve decreased. For example, for nares, the area was 74.1, which means that hydromorphone decreased sensory perception by 62.3%. Similar calculations for lateral mandible, rostrum, rostral mandible, and inside of mouth are 92.2, 89.4, 80.3, and 69.6%, respectively.

Blindfolding and von Frey filament also interacted (F_18,2,878 = 4.33, p < 0.0001) in their effects on sensory perceptions by alligators (Table 1 and Figure 4). Blindfolding reduced sensory perception at filament ≥4.31 grams-force. Area-under-the-curve comparisons showed that blindfolding decreased sensory perception by 24.8%.

No obvious visible signs of sedation, such as lethargy, droopy eyelids, frequent blinking of eyes, unsteady movements, or slower breathing and heart rate, were apparent, nor were adverse reactions to hydromorphone apparent, such as vomiting, lateral shaking of head common in nausea, or excretion. Alligators remained alert and their body temperatures (x̄ = 31.5 ± 0.8) did not change (F_3,256 = 0.22, p = 0.88) with the addition of hydromorphone or fluctuate (F_21,256 = 0.56, p = 0.94) during the experiment.

Hydromorphone reduced and delayed the perception of mechanical noxious stimuli in American alligators using von Frey filaments. Hydromorphone inactivates metabolites that have been associated with neuroexcitatory states within the central and peripheral nervous system by acting primarily as a receptor agonist at μ-and δ-opioid receptors to elicit analgesia (22). Morphine (0.8 mg/kg) given intramuscularly has been used on saltwater crocodiles [Crocodylus porosus; (7)]. However, morphine has several known adverse side effects, such as respiratory depression and vomiting (10). For example, in red-eared slider turtles, morphine administered subcutaneously caused profound respiratory depression (23). Although hydromorphone is chemically similar to morphine, its structural differences impact the route of metabolism, which may help resolve unwanted side effects (24), albeit the majority of published side effects have been documented in humans (24). In red-eared slider turtles, hydromorphone was antinociceptive using a thermal noxious stimulus model (11). Therefore, use of hydromorphone as an analgesic may be preferable in other reptile species, including crocodilians.

Alligators in managed care are susceptible to traumatic wounds (e.g., bite wounds, lacerations, burns), as well as primary and secondary infections associated with a variety of microorganisms (25). Potential injuries and illness to free-ranging alligators include exposure to toxic chemicals and pesticides (26–28), oral injuries from debris in water (i.e., fish hooks, wooden sticks, etc.), tooth abscesses (29), and skin wounds and lesions from trauma and infection (29). Therefore, the use of analgesic drugs is warranted in alligator management practices and is an important component of standard veterinary medical care for reptiles (30). However, information on the use of analgesics in crocodilians is extremely limited. Published laboratory-based studies in crocodilians demonstrated that morphine, a μ-opioid agonist, attenuates the limb withdrawal behavior after exposure to a thermal noxious stimulus (7). The results of our study support, and build upon these data, such that hydromorphone, also a μ-opioid agonist, diminished the response to a mechanical noxious stimulus in alligators by >60%.

Crocodilians seem to have exquisitely sensitive mechanoreceptors, also referred to as integumentary sensory organs or ISOs, now renamed dome pressure receptors [i.e., DPRs; (31)] on specific anatomical regions of their bodies, particularly the head in alligators and caiman, but are found in nearly every scale of the body surface in other crocodilian species (8). It was hypothesized that these DPRs function as part of a complex mechanosensory system and are adaptive to several ecologically relevant behaviors, including detection of water movements from predators and prey, contact with prey items, and fine oral tactile discrimination (8). These data suggest that mechanoreception may be more salient for crocodilians than thermoreception and the sensitivity of crocodilian DPRs is equivalent to the sensitivity of a primate fingertip, based on the use of very fine diameter von Frey filaments (8). We tested multiple cephalic locations to verify that analgesic effects were centralized and not local effects. While most crocodilians have DPRs on virtually all cranial and postcranial scales, DPRs in alligators are only found on the cranial region (31). Therefore, we tested the analgesic effects of hydromorphone at multiple sites that were deemed sensitive to alligators. Although alligator responses differed by location, hydromorphone delayed and reduced the response to von Frey filaments at each cephalic location. The density of DPRs differs throughout the head of American alligators, which may explain the varying responses observed in this study (32). However, the results of our study differ from that of Leitch and Catania (8), which indicated that stimulation of the nares would elicit a reduced response as compared to stimulation of the rostral mandible.

We recognize that our study has limitations because pain is perceptual and multidimensional and is affected by numerous interactive factors; thus, making accurate measurement difficult at best (33). Genetic strain, age, sex, and reproductive status of animals, stocking density, pen size, and environment can influence the perception of pain (34). Hence, this is the reason why we elected to use same litter, wild-collected eggs, and captive-raised females within same style of pens and similar animal densities until they were nearly 5-years-old. Such confounding variables were experimentally held constant.

There are also limitations to analgesiometry models used in pain research (35, 36). Laboratory-based analgesiometry models cannot completely replicate multidimensional, pathologic pain states. Most of the laboratory-based analgesiometry models rely on the application of thermal (e.g., Hargreaves apparatus), mechanical (e.g., von Frey filaments, toe pinch, hypodermic needle insertion) or chemical (acetic acid test, subcutaneous capsaicin) noxious stimuli and administering a variety of analgesic drugs in order to measure changes (a diminution) in a behavioral, typically a withdrawal, response to the drugs. While these models can certainly cause brief pain or discomfort, they cannot be directly compared to experimental models using surgical or visceral pain. In addition to the experimental model issues, some of the opioid drugs commonly used in pain research, may cause sedation such that a diminution in behavioral responses might be misinterpreted as analgesia, but may realistically be better attributed to sedation.

While the use of Von Frey filaments has never been applied to crocodilians to our knowledge, this method is widely used in mammalian pain/nociceptive research as a mechanical noxious stimulus. We recognize the following limitations associated with using Von Frey filaments in any animal or human subject. Importantly, Von Frey filaments only measure mechanical sensitivity and cannot replicate the multidimensional aspects of pain; in other words, application may not be clinically relevant. When applying the filaments, there is an inherent subjectivity in measuring the response of each subject, such that the observer must interpret the presence of a withdrawal response, which potentially adds variability to the data. For this study, we used one observer in order to decrease interobserver variability and maintain consistency in how the fibers were applied. Data variability also may be associated with application to different anatomical sites on an animal, and the force of filaments can be inconsistent due to variability in application pressure or overuse (damage) to the filaments. The Von Frey filaments used in this study were brand new and the anatomical sites chosen were based on the presence of the most sensitive mechanical nociceptors on crocodilians. Similarly, some individuals may respond more robustly than others due to a variety of methodologic variables (e.g., presence of observer may affect the subject’s response or there may be a lack of subjects’ being appropriately conditioned to the experimental setup). The alligator subjects in this study were conditioned to the experimental environments and blindfolded in order to decrease the effects associated with observer presence. Finally, if the mechanical noxious threshold exceeds the maximum force of the available filament diameters or, alternatively, falls below the lowest possible force of the smallest diameter fibers, data measurements may be less accurate. Withdrawal responses from the alligators in this study were clear and unambiguous.

As had been stated in past studies, anthropomorphism may not be the best way to assess pain, but it is a starting point (37). Although behavior always cannot be treated as evidence of a noxious experience in all animals, there are cases where the degree of deviation in behavior is equal to the degree of pain (38). Thus, we offer our study as a starting point to assess analgesic efficacy of hydromorphone in crocodilians.

Some results presented here are intuitive. For example, increased reactions by alligators to increasing grams-force of von Frey filaments and to the visual stimuli of an approaching filament was expected. However, it was necessary to incorporate these effects into the experimental design to document the antinociceptive effect of hydromorphone in the study subjects. Use of blindfolds is a common practice during capture and restraint in wildlife management to keep animals tranquil (39–41). Our results with alligators were similar to that observed in green iguanas (Iguana iguana), in that blindfolds appeared to have a calming effect on the alligators’ responses as well as minimize the presence of the observer (42).

While we did not use pulmonary plethysmography to accurately measure ventilation in our subjects, alligators in our study did not appear to experience respiratory depression based on observational measuring rate of respiration, nor did the alligators experience hypothermia, both of which can contribute to deleterious consequences after administration of anesthetics and analgesics in reptiles (5). Although we do note that ventilation efficiency cannot be assessed by respiratory rate alone, as it is possible for rates to remain constant even with a significant decrease in tidal volume. However, our data suggest that hydromorphone is a potentially effective analgesic choice with negligible, clinically observable respiratory depression in American alligators.