Introduction
Castration involves the removal or destruction of the testes, epididymis, and a portion of each spermatic cord to make the testis nonfunctional in-situ [1,2]. It is a common practice carried out in goats in several countries, to reduce aggressive behavior and improve meat quality [3]. Other purposes include preventing unwanted mating, mounting activities, and injuries accompanied by or for testicular or inguinal pathology [4].
Several castration techniques exist and the use of each method depends on the animals’ species, the anatomy of the testes, the age of the animals, the purpose for use, whether the testis is normal or diseased, and convenience [4,3]. The conventional techniques, which include physical methods, which result in removal, irreversible damage, or destruction of the testis such as the application of elastrator bands or rubber rings, use of Burdizzo clamp, and surgical removal (by open, closed, or modified closed techniques) [5,3]. These techniques are usually associated with stress, pain, and inflammatory responses, and the inflammatory responses usually result in the production of pro-inflammatory cytokines [6].
One of the major cytokines synthesized after routine surgery is interleukin-6 (IL-6) and this can stimulate acute phase protein synthesis in the liver and can affect lymphocyte function and hematopoiesis [7]. IL-6 is a cytokine comprising 184 amino acids, produced by various cell types and expressed during periods of cellular stress, including inflammation, infection, wound healing, and cancer. During these states, IL-6 levels may escalate significantly, potentially aiding in orchestrating the response to tissue homeostasis dysregulation [8]. This multifunctional regulator plays a role in inflammation, immune response, and fibrosis.
IL-6 can be released due to tissue injury or an inflammatory stimulus and can act at the site and systemically to give way to different responses in the body [9]. IL-6 levels are elevated after surgery, trauma, and certain sicknesses. The magnitude of the IL-6 increase aligns with the degree of tissue trauma/injury or severity [9]. The role of inflammation in addressing surgical injury is significant. It serves as a defense mechanism aimed at repairing damaged tissue and restoring homeostasis under stress, as proposed by Medzhitov [10]. Nevertheless, if unregulated, this response can result in pathological conditions and chronic inflammation, as highlighted by Medzhitov [10]. Pro-inflammatory cytokines are important mediators of inflammatory responses and were reported to be involved in cellular activities that included proliferation, differentiation, and apoptosis [11]. Physical methods of castration tend to generate some pain levels irrespective of the castration methods utilized. There is a lack of information on the effects of orchidectomy, Burdizzo castration, and in-situ spermatic cord ligation on IL-6 responses in red Sokoto bucks. Thus, in this study, the effect of orchidectomy, Burdizzo castration, and in-situ spermatic cord ligation on IL-6 responses in red Sokoto bucks was evaluated.
Materials and Methods
Location of the study
The study was conducted in Zaria Kaduna State, Nigeria. The surgical procedures were performed in the Large Animal Surgery Unit, Department of Veterinary Surgery and Radiology, Ahmadu Bello University (A.B.U.), Zaria.
Ethical approval
Approval for this study was obtained from the A.B.U. Committee on Animal Care and Use (ABUCAUC/2022/047), A.B.U., Zaria.
Experimental animals
A total of 16 Sokoto red bucks, 6 months to 1 year old and weighing between 11 and 12 kg, were used for the study. The bucks were purchased from the livestock market in Giwa local Government area of Kaduna State and kept in the small ruminant pens of the Department of Veterinary Surgery and Radiology, A.B.U., Zaria. Before the arrival of the bucks, the pens were cleaned, disinfected, and treated with insecticide. The bucks were stabilized and acclimatized for 2 weeks, during which routine examination was carried out for any sign of illness. Also, clinical evaluations were conducted on each animal to determine their baseline data. The animals were fed with groundnut hay, bean husks, and maize offal twice a day, water was provided ad libitum.
Grouping of animals
The animals were randomly allocated into four groups (A, B, C, and D) each comprising four bucks as follows: A (Burdizzo castration), B (in-situ spermatic cord ligation), C (orchidectomy), and D (control).
Experimental design
The bucks were randomly assigned into four groups (A, B, C, and D) of four animals each. Bucks in group A were castrated using Burdizzo, B by in situ spermatic cord ligation, and C by orchidectomy, while D was not castrated. Blood (3 ml) was collected from each buck via jugular venipuncture pre-castration, immediate (0 hour), 4, 8, 12, 16, 20, 24-, 32-, 48-, and 72-hours post-castration (hpc), dispensed into labeled plain tubes and allowed to clot. The serum was harvested after that and used for IL-6 assay.
Determination of serum IL-6 level
Serum IL-6 was determined using a Goat IL-6 ELISA kit (KTE 50039) obtained from Abbkine, Incorporated China by following the manufacturer’s instructions as follows.
Reagent preparation
All reagents were brought to room temperature before use and the wash buffer was diluted with distilled water at 1:30 (96T).
Standard
150 μl of standard diluent was pipette into each tube. The stock solution was used to produce a 2-fold dilution series. Each tube was mixed thoroughly before the next transfer. The undiluted standard serves as the high standard and the standard diluent serves as the zero standard.
Assay procedure
After the standard was prepared all the standards and samples were added in duplicate to the microplate. A diluted standard of 50 μl was added to the standard well. A sample diluent of 40 μl was added to the testing sample well and another sample of 10 μl was added to the testing sample well and nothing was added into the blank well.
The microplate was covered with a plate cover and incubated for 45 minutes in an incubator at 37°C. Each well was aspirated and washed, repeating the process four times for a total of five washes, 1–3 minutes per time. It was also washed by filling each well with wash buffer (250 μl) using a manifold dispenser. Complete removal of liquid at each step was done to enhance good performance. After the last wash was done, the remaining wash buffer was removed by decanting and the plate was inverted and blotted against clean paper towels. A conjugate detection antibody of 50 μl was added to each well, except the blank well. It was covered with a plate covered and incubated for 30 minutes at 37°C. The aspiration/wash process was repeated five times as mentioned earlier. Chromogen solution A 50 μl and chromogen solution B 50 μl was added to each well, it was gently mixed and incubated for 15 minutes at 37°C. It was protected from light. 50 μl stop solution was added to each well and the color in the wells changed from blue to yellow. The optical density was read at 450 nm using a microtiter plate reader within 15 minutes.
Data analysis
Graph pad prism version 5.0 (San Diego, California, USA) was used for the analysis. Data were presented using charts as the mean and standard error of the mean (mean ± SEM), subjected to one-way analysis of variance with repeated measures with Tukey’s posthoc test. The value of p ≤ 0.05 was considered significant.
Experimental procedures
Burdizzo castration method
This was performed using the Burdizzo castrator (Agri Health castrator; Agri Health Ltd, Ireland) as described by Olaifa and Opara [12]. After proper restraint of each buck, the hind limbs were spread apart and the scrotal area was exposed to the surgeon. Castration was achieved by applying the Burdizzo laterally onto the scrotal neck. The first finger and thumb were used to hold the cord laterally in the scrotal neck, while the second hand slowly directed the position of the jaws until they were about 8–10 mm apart to grip the skin and cord firmly. The surgeon ordered and maintained rapid closure for 15–30 seconds while ensuring that the cord was properly crushed (Fig. 1).
Figure 1.
Photograph of a red Sokoto buck undergoing Burdizzo castration. Note the Burdizzo castrator crushing the spermatic cord in the scrotum (Blue arrow).
Figure 2.
Photograph of the scrotum of a red Sokoto buck undergoing in-situ spermatic cord ligation (see blue arrow).
In-situ spermatic cord ligation procedure
In-situ spermatic cord ligation was carried out following the aseptic preparation of the skin enveloping the spermatic cord [13]. Each buck was restrained on the surgical table in lateral recumbency, and local anesthesia was achieved through a linear subcutaneous infiltration of 1 ml of 2% Lidocaine HCl (Afirst Life Science Ltd, India) on each lateral aspect of the scrotum. Non-absorbable suture material (Nylon size 2/0), (Anhui Kangning Industries, China) was used for a double external trans-fixing ligation of the entire spermatic cord, 2 cm apart. The procedure was repeated on the other cord (Fig. 2).
Orchidectomy
Orchidectomy was performed by modifying the procedure described by Malbrue and Zorilla [14]. The scrotal area was shaved, and scrubbed with soap and water, and chlorhexidine (Saro Life Care Limited, Nigeria) was applied to disinfect. The buck was sedated intramuscularly with 0.05 mg/kg Xylazine (Bioveta, Komenskeho, Czech Republic). A linear subcutaneous infiltration of 2% lidocaine HCl (A First Life Science Ltd, India) was achieved at 4 mg/kg on the lateral aspect of the scrotal sac and was used to achieve local anesthesia. Each goat was restrained on the surgical cradle in dorsal recumbency. The scrotum was then grasped, and a vertical incision through skin and fascia at the lateral part of the scrotum was made. This allowed the exteriorization of one of the testicles which were stripped off the vaginal tunic with a gauze sponge. The spermatic cord was ligated in three places with an absorbable suture ligature (Chromic catgut size 2/0, Anhui Kangning Industries, China), which was covered by a vaginal tunic. The spermatic cord was cut 1 cm below the ligature and the stump was checked for bleeding. Similarly, the opposite testicle was removed (Fig. 3).
Results
There was no significant (p > 0.05) difference in the IL-6 concentration in all groups of goats’ pre-castration (Fig. 4). Post-castration, there was a significant (p < 0.05) increase in the IL-6 concentration in all castrated goats from 4 hpc and reaching peak levels at 20 hpc with the highest recorded in goats castrated by orchidectomy (18.06 ± 0.75 pg/ml; 94.61 ± 1.74 pg/ml). From 24 hpc, there was a significant (p < 0.05) decrease in IL-6 concentration up to 72 hpc (Fig. 1).
Discussion
IL-6 is initiated in response to any infections and tissue-level injuries, thus contributing majorly to the host defense mechanism [15]. In this study, the serum IL-6 level started increasing from 4 hpc and peaked at 20 hours in all castrated goats with the highest level in those castrated by orchidectomy. Yoo et al. [16] reported increased IL-6 mRNA levels on day 1 after castration using Newberry knives and a Henderson castrating tool in Korean cattle bull calves. Also, it increased leukocyte mRNA levels of IL-6 genes at 6 hpc in the Korean cattle bull calves were reported by Yoo et al. [17]. Ji and Young [18] reported that IL-6 increased 10~40 fold at 30 minutes after the end of the operation on patients and at 24 hours after the end of the operation. Furthermore, in a cohort study of three patients, the levels of IL-6 surged following surgical trauma, peaking 24 hours post-surgery, as observed by Nishimoto et al. [19]. These initial investigations suggested that IL-6 might contribute to the outcomes associated with surgery-induced inflammation, as indicated by Zhang et al. [20]. The increased IL-6 level might be due to the activation of pro-inflammatory cytokine in most animals resulting from tissue damage leading to the release of a cytokine such as IL-6 which tends to increase in virtually all inflammatory conditions [21]. In the inflammatory state, IL-6 was reported to increase in proportion to the duration and severity of the condition [7]. This finding is consistent with the report of Randeep et al. [9]. Randeep et al. [9] stated that Systemic IL-6 concentrations can increase as a result of surgical procedures carried out. The degree of increase correlates with the magnitude of tissue injury and therefore varies by surgical approaches. In addition, a diminished cytokine response is often observed in laparoscopic surgery, as this is believed to correlate with less tissue injury, and consequently less immunosuppression, and therefore faster recovery [22]. Kashiwabara et al. [23] reported postoperative serum IL-6 elevations in patients who went through surgery that were proportional to the magnitude of the surgical stress they were exposed to.
Figure 3.
Photograph of a red Sokoto buck undergoing closed castration (Orchidectomy) (see blue arrow).
Figure 4.
Changes in IL-6 levels of red Sokoto bucks before and after bilateral castration using the Burdizzo method, in situ spermatic cord ligation, and orchidectomy. Values with different alphabets at the same time differ significantly at p < 0.05.
Since castration is a minimally invasive procedure, the higher IL-6 level in goats castrated by orchidectomy suggests that the damages induced were prolonged and severe compared to those induced by in-situ spermatic cord ligation and Burdizzo castration. Surgical procedures with severe tissue trauma may trigger high secretion of IL-6 and stimulate high pain intensity after the surgical procedure [24]. Increasing evidence suggests that many of the systemic responses that occur after injury and infection are related to the elaboration of cytokines by the host [18].
According to the gathered evidence, IL-6 emerges as a dependable indicator of injury severity during the acute inflammatory response in surgical, traumatic, and critical care scenarios. Predictably, instances with minimal tissue trauma correspond to a reduced IL-6 response. In situations where the inflammatory response is localized, IL-6’s relevance, particularly its paracrine effects, can be assessed through tissue immunochemistry. Moreover, systemic IL-6 concentrations not only serve as indicators of disease severity but also facilitate patient stratification for therapeutic interventions, as evidenced by significant sepsis trials [9].
Conclusion
The three castration methods, orchidectomy, Burdizzo castration, and in-situ spermatic cord ligation, elicited IL-6 responses but the response was highest with orchidectomy, followed by in-situ spermatic cord ligation Burdizzo castration provoked the least inflammatory response in the neutered bucks. This indicates that there was increased inflammation by the castration methods, thus suggesting that IL-6 can be used to determine inflammatory responses for bucks after castration. This is the first study to demonstrate a relationship between orchidectomy, Burdizzo castration in-situ spermatic cord ligation, and serum IL-6 levels in the red Sokoto bucks.
Acknowledgment
The authors wish to acknowledge the technical assistance rendered by Mr. Yau Hamza and Mr Lawal Musa to make this work a success.
Conflict of interest
The authors have no conflict of interest to declare.
Author contribution
NTO: Carried out conceptualization, design of the experiment, performance of the surgical procedure, and writing of the original draft.
OO: Was involved in the writing of the methodology, performance of the surgical procedure, and data analysis.
TAJ: Was also involved in writing the methodology, editing, and proofreading.
