Introduction

Pain is an unpleasant physical and emotional sensation associated with tissue injury, whether real or perceived (1). In many situations where humans are morally or legally responsible, assessing and treating an animal's pain is crucial for its well-being (2). Analgesia is a vital component of medical therapy, however identifying pain in avian kind leftovers is hard because of species-specific pain presentations (3,4). Inflammation is a protective mechanism developed in higher species to respond to harmful stimuli including microbial infection, tissue injury, and other unpleasant situations (5). Inflammation is a defensive mechanism that restores cellular homeostasis in response to harmful conditions and typically begins within minutes in hosts with a functioning innate immune system (6,7). Inflammation is primarily caused by the innate immune system, which involves mast cells, macrophages, neutrophils, dendritic cells, and lymphocytes (8). Inflammation may cause significant damage to the affected cells and organs, so it is necessary to alleviate this damage through anti-inflammatory medications (9,10). Minocycline is a broad-spectrum semi-synthetic second generation of the tetracycline antibiotic family, in addition to its antimicrobial properties it has a particularly valuable analgesic effect (11). Minocycline has positive benefits in the CNS because of its anti-apoptotic, anti-inflammatory, neuromodulator, and neuroprotective qualities (12). Minocycline has lipophilic solubility, permeability, and nearly complete gastrointestinal (GI) absorption, which allows it to enter the circulatory system, and reaches more target organs, minocycline can more effortlessly pass the blood barrier of the brain (13). The ability of minocycline to cross the blood-brain barrier, a property unique among antibiotics, is key to its distinct characteristics (14). Numerous researches have provided persuasive evidence that both animal models and human participants given minocycline report reduced pain and inflammation (15).

This study was conducted since there was no previous study to evaluate the analgesic and anti-inflammatory effects of minocycline in chicks by determining the lethal and analgesic doses and revealing the safety criteria of the drug in addition to the anti-inflammatory efficacy.

Materials and methods

Ethical approval

The study was permitted by the Committee of Institutional Animal Care and Use, College of Veterinary Medicine, Mosul University, IRAQ. The study adhered to the ARRIVE guidelines for animal research. Ethical approval number UM.VET.2024.070.

Animals and drugs

A total of eighty-six healthy, unsexed one-day-old broiler chicks were used in this study. They were raised seven days before the trial began. The chicks were raised in cages under standard temperature (32-35°C), ventilation, and consistent lighting conditions. The chicks were given the same conventional broiler grower food (drug-free) and unfettered access to water throughout a one-week acclimation period. Before the medicine was administered, water and food were stopped for one hour and six hours, respectively. Minocycline hydrochloride (10⁵ mg/tablet, Hikma Pharmaceuticals, Amman, Jordan) was dissolved in a normal saline to achieve the desired medication concentration, and the volume of administration was 5 mL/kg orally.

Determination of the median lethal dose (LD₅₀) of minocycline

The up-and-down method (16) was used to determine the acute (24-hour) LD₅₀ of minocycline following oral therapy. The experiment was repeated three times to obtain the average median lethal dose. In preliminary experiments conducted on chicks, an initial oral dose of minocycline (1500 mg/kg) was administered to a single subject. Survival (denoted as "O") or mortality (denoted as "X") was recorded 24 hours post-administration. To estimate the median lethal dose (LD₅₀), a sequential dosing protocol was implemented within the range of 1000–1500 mg/kg. Experimental outcomes were analyzed using Dixon’s reference tables in conjunction with the up-and-down method, a statistical approach designed to refine dose escalation and determine the LD₅₀ with minimized animal use. This methodology ensured systematic evaluation of toxicity thresholds while adhering to ethical guidelines for humane experimental design.

Determination of the median analgesic dose (ED₅₀) of minocycline

The following experimental protocol was applied to assess analgesic efficacy of minocycline in chicks: An SRI (UK) electrical stimulation device was utilized to induce standardized nociceptive responses, configured with a maximum output of 10 V, 5 ms pulse width, and 15 Hz continuous current frequency. Electrodes were affixed to the featherless subcutaneous region under the wing (17). Baseline pain thresholds were determined pre-intervention by incrementally increasing stimulus intensity until a reproducible behavioral response (e.g., vocalization or wing flapping) was consistently elicited, as validated by Albadrany et al. (18). Post-administration (1 h), chicks were re-evaluated using their individualized pre-established threshold voltage. The presence or absence of nociceptive behavior was recorded, and subsequent dosing adjustments followed Dixon's up-down sequential method: doses were decreased if analgesia was observed (response abolition, denoted as X) or increased if nociceptive responses persisted (denoted as O). This iterative titration continued until three consecutive chicks exhibited reversal of the initial response trajectory (analgesic to non-analgesic, or vice versa). Final median effective analgesic dose (ED₅₀) values were derived using Dixon's reference tables, with the application of the equation.

Determination of drug safety indices

Subsequently, by determining the median lethal dose and the median effective analgesic dose in previous experiments and by referring to some of the equations approved in pharmacology, drug safety indices can be determined. The equations used were as follows: Therapeutic Index =LD₅₀/ED₅₀, Standard Safety Margin =LD₁/ED₉₉ and Therapeutic Ratio = LD₂₅/ ED₇₅ (19).

Determination of the analgesia of minocycline over time

Eighteen chicks were divided into three groups of sex chicks per each. The first group received normal saline (5 ml/kg) orally, and the second and third groups received minocycline 150 and 300 mg/kg orally, respectively, which represent the ED100 and ED200 obtained from previous experiments. At 0, 1, 2, 4, and 8 h after treatment, we recorded the lowest voltage produced an aversive pain reaction in each chick. The analgesic response of minocycline was evaluated by statistically testing the rise in voltage in a separately group.

Determination of the analgesic and anti-inflammatory activity of minocycline in formalin test

Formalin test (20) was used to measure the analgesic and anti-inflammatory effects of minocycline. Eighteen chicks were divided into three groups of sex chicks per each. The control group received normal saline (5 ml/kg) orally, while the second and third groups received minocycline 150 and 300 mg/kg orally, respectively. One hour after receiving therapy, the chicks experienced pain and inflammation immediately after formalin injection (0.05 ml,0.1%) in the paw of the right foot, and within 3 minutes of formalin administration the onset (latency to first foot lift) and frequency (total number of lifts within 3 minutes) the right foot were recorded (21). The anti-inflammatory activity of minocycline was assessed by measuring foot thickness (mm) previously and one hour later formalin injection using a digital caliper (22). The anti-inflammatory activity was calculated as follows (in percentages): The anti-inflammatory activity % = [V_control – V _test / V_control] ×100

Determination of the anti-inflammatory activity of minocycline in cotton pellet granuloma

The cotton pellet granuloma test, validated in avian models for chronic inflammation assessment, was selected to evaluate granulomatous tissue formation over seven days (23). Three groups received a daily dose of minocycline (0, 150, and 300 mg/kg) for seven days after a sterile cotton pellet weighing 10±1 mg was implanted under the skin into the thigh area of chick anesthetized with propofol (80 mg/kg, i.p). On day eight, the chicks were slaughtered, the pellets were carefully removed and the weight of the wet cotton pellets was measured. To determine the dry cotton pellet weight the pellets were dried in an oven at 40°C for 12 hours. The percentage of inhibition was calculated by estimating the mean weight of the granuloma tissue that had grown around each pellet (24). Percentage of inhibition % = [Control –Treated/ Control] ×100

Statistical analysis

Data are shown as the mean ±SEM for each group of six animals. One-way ANOVA and the LSD post hoc tests were used to compare between groups (25).

Results

In chicks, the median lethal dose of minocycline administered orally over 24 hour was 1278 mg/kg (Table 1). Stillness, closing eyes, gasping, wing drooping, convulsions, and death were among the signs of poisoning that appeared within two hours of the drug administration.

Table 1: Median lethal dose (LD₅₀) of minocycline

^a X- death; O-survival. Xf: last dose administered in the trial. K: Tabular value. d: fixed decrease and increase in dose.

The median analgesic dose of minocycline administered orally was 73.841 mg/kg (Table 2). The drug safety indices of minocycline including the therapeutic index, standard safety margin, and therapeutic ratio were 17.5, 0.17, and 5.83 respectively.

Table 2: Median effective analgesic dose (ED50) of minocycline

^a X- analgesia; O-none analgesia. Xf: last dose administered in the trial. K: Tabular value. d: fixed decrease and increase in dose.

Minocycline at 150 and 300 mg/kg, orally produced significant analgesia in chicks in a dose-dependent manner. Analgesia was observed after one hour of treatment and the peak of analgesia observed at two hours then began to decrease during the following hours and continued for more than eight hours after treatment (Table 3).

Table 3: Analgesic effect of minocycline over time in chicks

Mean±SE (6 chicks/group). The values in each column are significantly different when followed by various superscript letters (P≤0.05).

In the formalin test, the pain resulting from the formalin injection in the paw of the right foot was reduced by oral minocycline at 150 and 300 mg/kg. This was evident from a significant increase in the onset of foot elevations and a significant decrease in the frequency of foot elevations in comparison with control (Table 4). Anti-inflammatory efficacy was evident from a notable reduction in foot thickness in comparison with a control group and the anti-inflammatory activity ratios were 43 and 66 % separately (Table 4).

Table 4: Anti-inflammatory and analgesic activity of minocycline in formalin test

Mean±SE (6 chicks/group). The values in each column are significantly different when followed by various superscript letters (P≤0.05).

In the cotton pellet granuloma test, a substantial reduction in the weight of wet and dry cotton seeds relative to the control group with inhibition percentages of 30 and 44%, respectively, indicated that oral administration of minocycline at 150 and 300 mg/kg inhibited the development of chronic inflammation (Table 5).

Table 5: Anti-inflammatory activity of minocycline in cotton pellet granuloma

Mean±SE (6 chicks/group). The values in each column are significantly different when followed by various superscript letters (P≤0.05).

Discussion

Chicks were used in our study due to their economic importance in veterinary science and their long history as a model in biological research. Chicks were used as a model for analgesic (26), anti-inflammatory (27), anesthetic (28), neurobehavioral (29), and toxicological studies (30). Our study revealed the analgesic effect of minocycline by determining the median effective analgesic dose, drug safety indices, and degree of safety. The rapidity of analgesia was observed within the first hour with peak analgesia occurring within two hours, and analgesia continuing for more than eight consecutive hours. Results revealed the anti-inflammatory effects in acute (formalin test) and chronic (cotton pellet granuloma) inflammation. The results confirm the possibility of using minocycline to relieve pain and treat inflammation of both acute and chronic types, in addition to using it as an antibacterial agent.

Minocycline’s analgesic and anti-inflammatory properties, alongside its antibacterial efficacy, are mediated through modulation of ion channels, NMDA receptor inhibition, and enhancement of endogenous opioid pathways. Several studies have highlighted the extraordinary capacity of minocycline to efficiently modify numerous ion channels that are intricately engaged in the complex process of pain signaling. Minocycline has the extraordinary capacity to delicately and precisely disrupt the important functions of NMDA receptors, which are widely recognized for their critical involvement in both the formation and maintenance of pain. Minocycline strongly inhibits the activity of voltage-gated calcium channels, causing a significant decrease in neuronal excitability and reducing pain transmission (31-34). Minocycline efficiently modulated the endogenous opioid pathway, which is important for pain management. It has remarkable potential to dramatically augment the release of endogenous opioids, such as endorphins and enkephalins, thereby magnifying their binding capacity to opioid receptors in the peripheral and central nervous systems (35). The strong analgesic effect mediated by these opioid systems reinforces the reputation of minocycline as a vital candidate for pain management, showing its excellent pain-relieving qualities (36).

Minocycline is an effective COX-2 inhibitor in various cellular models. Further experiments have revealed that minocycline inhibits COX-2 in activated cells and monocytes in a concentration-dependent manner (37). COX-2 is triggered by inflammatory processes and creates prostaglandins that sensitize nociceptors, induce fever, and promote inflammation through vasodilation and an increase in vascular permeability (38). So, inhibiting the COX-2 enzyme will reduce pain sensitivity and inflammatory reaction.

The potential analgesic effect of minocycline in both the spinal cord and the brain is linked to the activation then control of glial cells including astrocytes and microglia. It has a strong and selective inhibitory action on microglia rather than astrocytes or neurons, and has neuroprotective and anti-inflammatory effects on the central nervous system (39). Minocycline inhibits microglial activation, leading to reduced pain hypersensitivity in rat models. Extensive research has clearly shown that it can efficiently stop the ongoing activation of microglial cells, thus greatly lowering the release of pro-inflammatory cytokines like IL-1β, IL-6, TNF-α, and many other inflammatory mediators in both the spinal cord and the brain (40).

Inflammation is important in the host's defense against infections and injuries, but it also plays a vital role in the pathophysiology of many chronic illnesses (41). Inflammation is defined by enhanced pro-inflammatory cytokine production, leukocyte recruitment, and systemic and local control of leukocyte responses (42). Thus, interactions between inflammatory mediators and immune cells regulate various elements of both acute and chronic inflammation processes (43). The formalin test is a widely used model of nociception, involving both acute peripheral inflammation and central sensitization (44). The cotton pellet granuloma test is a commonly used model of chronic inflammatory reactions (45). In both tests, minocycline significantly inhibited the rate of inflammation compared within the control group.

Minocycline decreases inflammation by modulating inflammatory cytokine release and inflammatory cell chemotaxis. Minocycline reduces pro-inflammatory cytokines like TNF alpha, IL-6, IL-1β, PGE2, and PLA2, while increasing anti-inflammatory cytokines like IL-10 (46). Minocycline has potential benefits as an anti-inflammatory medication with fewer side effects, including liver damage, weight gain, hyperglycemia, and osteoporosis, making it use an alternative to corticosteroids (47). Minocycline can influence inflammatory processes, defend against neuronal damage, modulate ion channels, limit the synthesis of pain mediators, and improve the endogenous opioid system, making it an attractive candidate for pain treatment (48).

Conclusion

We conclude that minocycline displays an analgesic effect on acute pain induced by electrical stimulation, with high safety and sustained efficacy. Its anti-inflammatory action in both acute and chronic inflammation underscores its possible as a safe therapeutic agent for pain and inflammation management in poultry. These findings highlight minocycline’s dual therapeutic benefits, offering a valuable unconventional for veterinary applications with fewer adverse effects compared to traditional anti-inflammatory agents.

Acknowledgments

The authors of this paper would like to express their gratitude to the deanery of the College of Veterinary Medicine, University of Mosul, for providing the necessary facilities to finish the research.

Conflicts of Interest

None declared.