MATERIALS AND METHODS

Drug and chemicals: Tulathromycin (TLM) (100 mg mL^–1) was supplied as an injectable solution (Draxxin®) by animal health division Pfizer company, Cairo, Egypt. The LDH kits (Biosystems S.A. Costa Barva 30, Barcelona, Spain), AST and ALT kits (Spinreact, S.A./S.A.U. Ctra Santa Coloma, Spain), creatinine kits (Diamond) and cortisol kits (the ADVIA Centaur and ADVIA Centaur Xp Assay Manual).

Animals and housing: Twenty seven apparently healthy adult albino male rats (Average weight 160-200 g) were obtained from breeding unit of Helwan farm of laboratory animals (Helwan, Egypt). Animals were group housed (3/cage) in (47×25×21) cm dimension. Rats were kept for 2 week prior to the onset of the study in department of Pharmacology, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef, Egypt. Animals within each social group (i.e., homecage) received the same experimental treatment throughout the experiment. Three replicates were used for each group.

Rats were maintained at 21±2°C temperature, 45±5% humidity, with a 12:12 h light: dark cycle (07.00-19.00 h), fed a commercially prepared pellet diet and watered ad libitum throughout the study. Bedding was changed twice per week. Cages, feeders and water bottles were washed twice weekly.

Experimental design: After two weeks of acclimatization, rats were equally divided into three groups (9 rats, each 3/cage). Rats in the control group were subcutaneously (S/C) injected with 0.1 mL normal saline once weekly for three weeks, while those in the second group (G1) were S/C injected with TLM in a dose of (2.5 mg kg^–1 b.wt.) (Er and Yazar, 2010) twice with 15 day interval and the third group (G2) were S/C injected with TLM (2.5 mg kg^–1 b.wt.) for three successive doses (once weekly).

The study was conducted in accordance with the principles and guidelines of the Canadian Council on Animal Care and approved by department of Animal care and Welfare, Faculty of Veterinary Medicine, Beni-Suef University.

Marking of experimental animals: Rats were marked using three different stains whereas, each rat had a fixed color during the experiment to facilitate tracking of its behavior throughout the experiment. The non toxic stains were smeared on various parts of the animal body. Re-marking of rats was necessary at interval according to disappearance of the stain.

Behavioral data collection and ethogram: Behavior of each three rats (in same cage) was videotaped for 30 min session three days weekly, three times per day (once in the morning 8:30", anther at noon the 12:00" and the last at afternoon time 3:30") using digital video camera (Sony, Japan). Three videos for each group were collected for behavior analysis.

Thereafter, rat’s behavior was analyzed using focal observation (Lehner, 1996). In all groups, rats behaviors including, self-grooming (licking and nibbling the fur, washing face with forelimbs, licking ears, head in front of the snout with forelimbs), feeding, comfort (Rest and sleep) and locomotor (walking) behaviors were analyzed. Frequency and duration/min of each behavior pattern were calculated.

Moreover, aggressive behavior of the rats was analyzed using zero-one score method (Lehner, 1996). Rats aggression was scored as 0 when aggressive bouts duration was less than 30 sec (Miczek and De Bore, 2004) while, that was prolonged for more than 30 min was scored as 1.

Blood sampling: After the end of injection period, blood was collected from the retro-orbital venous plexus (Poole, 1987), using clean microcapillary tubes. Collected blood was kept in a clean screw-capped bottle and incubated at 37°C until clotting, centrifuged at 3000 rpm for 15 min to obtain serum which stored at -20°C till use.

Cortisol and biochemical assay: Serum cortisol was assayed using direct chemiluminescent technology (Chodosh and Daniels, 1993). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured colourimetrically, serum creatinine activity was measured by thecolourimetric kinetic method and lactate dehydrogenase (LDH) was measured spectrophotometerically according to the method described by Yang (2001). Serum cortisol level was measured according to the ADVIA Centaur Xp Assay Manual.

Statistical analysis: All statistical analyses were performed using Advanced Models SPSS version 20.0. Data were analyzed by One-Way ANOVA. Post hoc analysis was carried out using Dunnett’s multiple comparison test. The p-values less than 0.05 were considered significant.

RESULTS

Statistical analysis of serum cortisol level and biochemical data showed significant difference between injected rats and control.

Table 1 illustrates significant decrease (p = 0.035 and p = 0.028) in serum cortisol level of rats treated with two and three repeated doses of TLM (2.5 mg kg^–1 b.wt.), respectively, in relation to control group. Serum cortisol level was increased to 1.4 and 1.5 times that of control in rats in group 1 and 2, respectively. Moreover, liver and kidney functions in rats treated with two and three repeated doses of TLM (2.5 mg kg^–1 b.wt.) showed no significant alterations in ALT, AST, LDH and creatinine levels in serum compared to control group.

Rats behavior alterations in response to repeated S/C injection of TLM in a dose of (2.5 mg kg^–1 b.wt.) are demonstrated in Fig. 1. Self grooming behavior of rats in group 1 (injected with two repeated doses) was performed for longer (p = 0.042) time than control rats. However, rats in both groups (injected with two and three doses) showed shorter (p = 0.04 in G1 and p = 0.02 in G2) (Fig. 1a) duration of self grooming performance than those of control (Fig. 1b) at the third week of TLM injection.

Feeding of rats in treated groups was not significantly different than those in control (Fig. 1c and d). Similarly, comfort (Fig. 1e and f) and locomotor behaviors (Fig. 1g and h) of rats repeatedly injected with TLM did not differ from control.

The aggression bouts observed per social group (i.e., each cage of 3) are illustrated in Table 2. Aggression bouts were scored as 1 in rats injected for two times while, those injected with three doses of TLM and in control group were scored as 0.

Table 1:	Effect of repeated subcutaneous administration of tulathromycin; TLM (2.5 mg kg–1 b.wt.) on serum cortisol level, ALT, AST, LDH and creatinine levels of rats
Control: Injected with 0.1 mL saline, Group 1: Injected with 2 doses, 2.5 mg kg–1 b.wt., of TLM with one week interval and Group 2: Injected with 3 doses, 2.5 mg kg–1 b.wt., of TLM with one week interval (Mean±SD), n = 5. *Significance at p<0.05 according to post hoc analysis, Dunnett’s multiple comparison test, ALT: Alanine aminotransferase, AST: Aspartate aminotransferase and LDH: Lactate dehydrogenase

Fig. 1(a-h):

Effect of tulathromycin (TLM) on rats (a) Self grooming frequency, (b) Self grooming duration/min, (c) Feeding frequency, (d) Feeding duration/min, (e) Comfort frequency, (f) Comfort duration/min of rats, (g) Locomotor frequency and (h) Locomotor duration/min in control rats (injected with 0.1 mL saline) group 1 (injected with 2 doses, 2.5 mg kg–1 b.wt., of TLM with (15 days interval) and group 2 (injected with 3 doses; 2.5 mg kg–1 b.wt., of TLM with one week interval) at first, second and third weeks of TLM injection. All values are the Mean±SD (n = 9). *Significant difference between the exposure group and corresponding control (white bars) according to post hoc analysis, Dunnett’s multiple comparison test at p<0.05

Table 2:	Effect of tulathromycin on aggressive behavior of rats
Control: injected with 0.1 mL saline, group 1: Injected with 2 doses, 2.5 mg kg–1 b.wt., of TLM with one week interval and Group 2: Injected with 3 doses, 2.5 mg kg–1 b.wt., of TLM with one week interval, Rats aggression was scored as 0 when aggressive bouts duration was less than 30 sec while, that was prolonged for more than 30 min was scored as 1, (n = 3 social groups; each 3 rats), TLM: Tulathromycin

DISCUSSION

Overall, this study revealed the stressful effect of TLM on rat (Rattus norvigious albinus) expressed by marked increase in serum cortisol level and behavioral alterations. The increased cortisol level in serum of TLM (2.5 mg kg^–1 b.wt.) injected (S/C) rats suggested stressful effect of repeated (two and three) injection of the drug. Similar results were recorded in individuals exposed to stress (Retana-Marquez et al., 2003). On the other hand, macrloids caused no significant alterations in serum cortisol level in the treated patients (Kanoh and Rubin, 2010). Therefore, the reported increase in serum cortisol might be as a result of severe pain and irritation of TLM injection (Giguere, 2013).

These findings showed that both two and three S/C repeated injections of TLM (2.5 mg kg^–1 b.wt.) caused no significant changes in liver and kidney functions. Serum ALT, AST, LDH and creatinine activities were within normal limits. These results were agreeable with Yazar et al. (2004) reported no alterations in serum creatinine level after treatment with macrolid antibiotics. On the other hand, CVMP (2002) found that TLM caused slight increase in liver enzymes (ALT and AST) in rats and dogs. In additions, Er et al. (2011) recorded an increase in serum creatinine of rabbits treated with TLM. The different data in kidney and liver enzymes in response to macrolids treatment may be due to different doses and treated animals species. Thus, the used TLM dose in this study was safe and bearable for liver and kidney of rats.

The observed data indicated that duration of self grooming behavior showed opposite responses to repeated TLM (2.5 mg kg^–1 b.wt.) S/C injection. Grooming duration was increased after two successive injections one week in between. However, rats groomed during short duration after the third injection one week later. Similar result was observed in rats injected two doses 15 days in between. In early reports, both results were observed. Smolinsky et al. (2009) reported that stress increase grooming behavior of rat. Meanwhile, Kalueff and Tuohimaa (2004) recorded less grooming activity in mice exposed to strong stress. These alterations in grooming performance of rats may be attributed to the increased level of cortisol (Zwiers et al., 1981). Thus, grooming behavior may be useful tool for assessing stressful impacts of repeated subcutaneous TLM injection.

Grooming is critical behavior for health and survival of rats (Borchelt, 1980). Hence, repeated injection of TLM drugs may disturb this behavior and consequently threaten animals health.

This study indicated that feeding, comfort and locomotor behaviors of rats subcutenously injected with TLM (2.5 mg kg^–1 b.wt.) were not significantly altered. This is in agreement with Silveira et al. (2005) who observed no effect of stress on feeding of rats. On the contrary, it was reported that stress may decrease feeding (Haque et al., 2013). Moreover, macrolids (roxithromycin) decreased locomotor behavior and prolonged sleeping period (Deshmukh and Tamboli, 2013). Hence, the used dose did not affect feeding, locomotor and feeding behavior of rat.

The increased aggression bouts of rats that were S/C injected with TLM (2.5 mg kg^–1 b.wt.) for two times may be argued to increased level of serum cortisol (Guimont, 2009). Similarly, aggressive behavior was increased in response to stress (Sgoifo et al., 1996). Moreover, aggression of the rats three times injected was not marked despite of high serum cortisol level. This data is in agreement with Wood et al. (2003) who reported that acute stress did not alter aggressive behavior of rats. These findings support the concept of wide inter-individual aggression differentiation in coping style (Sgoifo et al., 1996) during response to stress. Thus, repeated injection of TLM may result in social disruption due to increase of aggression.

It is worth noting that, the group of rats performed long time grooming showed normal aggression bouts duration and vice versa. It is hypothesize that rats display one pattern only of motor activities (i.e., grooming or aggression) as an active coping strategy in attempting to deal with or escape from pain (Sgoifo et al., 1996).

Further studies are needed to investigate the irreversibility of these effects of the tested drug on the experimental animals.

CONCLUSION

The response of rats to repeated subcutaneous injection of TLM in a dose of (2.5 mg kg^–1 b.wt.) expressed in high serum cortisol level and increase behaviors of anxiety (self grooming and aggression) suggested its stressful effect on animals. Moreover, prolonged suffering and consequent biochemical and behavioral disruption might threaten animal health. Therefore, repeated subcutaneous injection of TLM is not recommended.

ACKNOWLEDGMENT

We acknowledge professor doctor Hosney Hafeez Emeash head of Animal Hygiene Management and Zoonoses department for directing us during behavioral analysis.