This research was authorized by the Iranian biomedical research ethics committee [IR.IAU.BABOL.REC.1399.004 (48), IR.IAU.BABOL.REC.1399.015 (49) and IR.IAU.BABOL.REC.1399.093 (50)] and conducted at the Babol branch of Azad University.

In a randomized controlled trial, 18 healthy, large, deep-chested intact female mixed-breed dogs were included. Animals were divided into two equal groups randomly by coin flipping, using a sterile suture sachet (51–53) (NZD) after inducing anesthesia and draping the surgical area. The sample size was evaluated using the software GPower 3.1.9.7. The presence of 9 dogs in each group resulted in a power of 0.9 (Power = 1 − β = 0.9) for TST with effect size d > 1.50 at a significance level of = 0.05.

Shelter dogs with ASA I (the American Society of Anesthesiologists) physical status enrolled in the study (54). The physical exam checked the patient’s heart rate (HR), breathing rate (RR), and rectal temperature (RT). It also checked for internal and external parasites, did a complete blood count, and looked at the Hb, PCV, CRP, and glucose levels in the blood. The study was conducted on bitches in diestrus, based on vaginal smear cytology. Animals with a body condition score between 4 and 6 out of 9 were chosen. Animals under 1 year old, in estrus, pregnant, or lactating, with a weak or no response to painful stimuli (a needlestick in the lower abdomen), with a history of physical or behavioral issues, or with abnormal vaginal secretions were excluded.

Each dog scheduled for surgery on a particular day spent 3 days before and 3 days after the surgery in a separate cage with free access to food and water. Ten days after surgery, the sutures were removed.

The animal’s resting vital parameters (HR, RR, and RT) were recorded before anesthesia. Anesthesia and analgesia were provided by acepromazine (10 mg mL⁻¹, Neurotranq; Alfasan, Woerden, Holland), midazolam (5 mg mL⁻¹, Midazolam; Caspian, Rasht, Iran), pethidine (100 mg 2 mL⁻¹, Petholan; Adeka, İstanbul, Turkey), medetomidine (1,000 mcg mL⁻¹, Dorbene Vet; Syva, León, Spain) and ketamine (50 mg mL⁻¹, Ketamine HCl Inj.; Rotexmedica GmbH, Trittau, Germany), and ketorolac (30 mg mL⁻¹, Ketorolac; Alborz Darou, Tehran, Iran). A 19G catheter was placed aseptically in the cephalic vein for a given lactated Ringer’s solution (250 mL, lactated Ringer’s solution; Shahid Ghazi, Tabriz, Iran) at a rate of 5 mL kg⁻¹ h⁻¹.

The animals were premedicated by acepromazine at 0.02 mg kg⁻¹, midazolam at 0.5 mg kg⁻¹, meperidine at 2 mg kg⁻¹, medetomidine at 20 mcg kg⁻¹, and ketamine at 4 mg kg⁻¹ IM. They received ketorolac at 1 mg kg⁻¹ immediately before surgical asepsis. The maintenance of anesthesia was achieved by administering a consistent anesthetic mixture (ketamine at 4 mg kg⁻¹ and midazolam at 0.27 mg kg⁻¹) at a variable rate of 0.2–0.5 mg kg⁻¹, depending on the ketamine levels present in the mixture. The administration of the anesthetic was monitored through the use of several parameters, including SpO2, ECG, non-invasive blood pressure measured through a blood pressure cuff (size #4) placed proximal to the carpus over the radial artery at five-minute intervals, and respiration. The surgical procedure and manipulation were also taken into account during the administration of anesthesia. The Pm-7000vet, manufactured by Wuhan Zoncare Bio-medical Electronics Co., Ltd., was used to monitor animals. The animals’ cardiorespiratory parameters were monitored until they demonstrated full recovery.

A nociceptive response was defined as a 20% or more rise in heart rate over the base rate, accompanied with an increase in breathing frequency and blood pressure proportionate to a painful surgical procedure (20, 55, 56). Ketamine at 0.5 mg kg⁻¹ was used for rescue analgesia during surgery. The timeline details of measures were provided in Table 1.

There are many different kinds of subjective scoring systems, and some of them have been used in veterinary medicine (59). In this study, postoperative pain was measured using the Glasgow composite pain scale short form (GCMPS-SF) (60), the University of Melbourne pain scale (UMPS) (61), and visual analogue scales (VAS) (62). A trained, male examiner (AB) who was single-blinded measured the post-surgical pain (30, 31). He became acquainted with the dogs the day before surgery. Moreover, the similar abdominal closure and the bandage have prevented the procedure from being identified. Multidimensional pain assessments were carried out 1 h before surgery (0) and 6-, 24-, 48-, and 72-h following skin suturing.

Five milliliters of blood were drawn from the lateral saphenous vein after the same pain evaluation times. The samples were kept at the temperature of the operation room for 20–25 min before being transported to the laboratory. The serum separated by centrifuging at 3,000 rpm for 15 min was stored in a microtube at −20°C until the research ended. CRP levels (mg L⁻¹) were determined using the CRP latex agglutination technique using the CRP-LIA kit, Bionik in the laboratory of the faculty (4, 33).

Antimicrobial therapy (63) was started intravenously (IV) during premedication and maintained intramuscularly (IM) every 8 h for 3 days following surgery at 22 mg kg⁻¹ of cefazolin (Exir, Tehran, Iran) in both groups. Ketoprofen (Ketomax; Rooyandarou, Tehran, Iran) at 2 mg kg⁻¹ IM was given to animals having a GCMPS-SF score of 6 or higher out of 24 (45); throughout the examination, the UMPS and VAS ratings were also examined as supplementary criteria for determining a ketoprofen prescription.

The SPSS program (IBM SPSS Statistics for Windows, version 26, IBM Corp., Armonk, NY, United States) was used to look at the data. Along with normality confirmation using the Shapiro–Wilk test (except LigT), the study recruited the suspicious non-normal parametric data (PID, SRT, ShT, ClpT, LigT, and OSPT) after normalization based on the concordance of skewness and kurtosis coupled with stem-and-leaf plots. The duration of surgical steps was analyzed using a T-test. The Pearson correlation coefficient was used to determine the relationship between PIT and TST.

The Friedman test analyzes the progression of pain changes. Using Related-Samples Friedman’s Two-Way ANOVA by Ranks, we compared pain levels between evaluating time intervals throughout each surgical process. Using Kruskal-Wallis H tests on mean, a comparison of pain levels at assessing time intervals between two surgical techniques has been conducted. On the median, Independent-Samples Kruskal-Wallis H followed by Independent-Samples Fisher Exact Sig. (2-sided test, for samples less than 10) has been implemented on the information obtained from three behavioral pain assessment methods.

Variations in CRP were evaluated using repeated measures ANOVA, independent samples t-test, and general linear model-univariate tests.

The correlation between CRP and age, weight, HR, RR, and RT was shown using Pearson’s correlation coefficient. The partial eta squared was used to figure out how CRP and the body condition score (BCS) are related. Spearman’s rho correlation coefficient was used to determine the relation between surgical time intervals and pain (based on GCMPS-SF). The statistical significance level was set at p < 0.05.

There were no significant differences in the distribution of dogs by weight (p = 0.726) or age (p = 0.598) between the two groups. The mean and SD of age, weight, vital parameters (HR, RR, and RT), and BCS are shown in Table 2.

Instrument shank-assisted OHE displayed shorter TSTs than the conventional method (34.70 ± 6.7 and 47.40 ± 9.9 min respectively, p = 0.005). Table 3 provides a comparison of the methods’ time intervals. According to the new method, SRT is identical to ShT. ShT required 74% less time than SRT (p = 0.009), and moreover, LigT improved by 37% (p = 0.005) with the novel approach. Additionally, the OSPT and CT got 26 and 29% shorter, respectively.

Figure 2 shows how time intervals have changed, and Table 3 gives a statistical analysis of the changes. In each compartment of this figure, the difference favors the Instrument shank-assisted method.

According to the comparable correlation pattern (64) between TST and PIT based on ρ = 0.952, p = 0.0001 and ρ = 0.862, p = 0.003 for conventional and instrument shank-assisted OHE, respectively, PIT plays a crucial role in TST during ovariohysterectomy. The correlation analysis of TST and ShT revealed that, from a temporal perspective, ShT alone did not significantly reduce TST (ρ = 0.097, p = 0.804).

During the conventional OHE, a single intraoperative hemorrhage in the ovarian pedicle was managed. The dog was excluded from the study. None of the dogs in either group had problems after surgery.

Using the Friedman test, pain score changes (Δ Pain in GCMPS-SF, UMPS, and VAS; Table 3) were statistically significant in both groups. The majority of the time, the data showed that the new method was associated with much less pain (Table 4 and Figures 3–5).

After 24 h, the highest serum CRP levels were observed in both groups. Figure 6 demonstrates that the conventional group’s rate of rise accelerated more rapidly during the initial 6 h following surgery. The slope of the graph is determined to be y = 24.328x − 18.072 for traditional OHE and y = 4.9556x − 1.5667 for instrument shank-assisted OHE within the first 6 h. The traditional group observed a 4.9-fold increase in CRP acceleration on this basis. After 48 h, the Instrument shank-assisted OHE showed a significant decrease to baseline levels (p = 0.032; see Tables 5, 6). In contrast, although CRP levels decreased in the conventional group, they were not significantly different from their peak levels.

The relationship between pain (as measured by GCMPS-SF, UMPS, and VAS), surgical time parameters, CRP, vital signs, age, weight, and BCS has been studied.

OHE, like many other surgical procedures, seems to get more challenging as the size and weight of the animal increase. As body mass increases, the chest sinks deeper, and it becomes more difficult to reach and release the suspensory ligament (11). In the present study, the same higher body weight range enhanced the ovarian exposure challenge, allowing the method to be generalized to various sizes of dogs and cats; however, it may be accompanied by fewer problems in smaller animals.

Insufficient exposure during OHE leads to incorrect technique execution and raises the risk of ovarian remnant syndrome (2). The instrument shank-assisted OHE keeps the ovary outside of the abdomen without an assistant while maintaining the suspensory ligament. In obese and deep-chested dogs, as well as for unskilled surgeons who require a longer incision, the surgical assistant is essential (1, 2). So, a small incision without tearing the ligament is another achievement of the modified method, which led to limited surgical complications including incisional swelling, seroma, infection, delayed healing, ventral body wall dehiscence, self-inflicted trauma, pain (65), and hemorrhaging (66).

During a suspensory ligament release (66), an inexperienced surgeon is more likely to break the blood vessel and induce hemorrhage, which prompts the hurried application of surgical sponges. Stress has a negative impact on the non-technical skills of surgeons (67), leading Rodriguez et al. to conclude that intraoperative hemorrhage from an ovarian pedicle probably increased the retention of surgical sponges in veterinary patients (68). Therefore, removing ligament release from the surgical steps would likely reduce the frequency of hemorrhage and sponge retention; however, more research is required before a conclusion can be reached.

Hilgard’s learning theory suggests that experience is a crucial component of the learning process (69). For unbiased comparisons, the study surgeries must be performed by a surgeon with no or equivalent prior experience in both methods. A minimum of surgeon experience in our study to the level of the veterinary training program according to conventional method seems to impose an inevitable minimal bias. However, the presence of an inexperienced surgeon could be a limitation of the present study. Given the possibility that a surgeon’s lack of expertise might exacerbate surgical stress to the point where the impact of the technique is nullified, a week of training for each method prior to the research allowed the current study to reveal the smallest difference between the procedures. TST has been reported to take between 55 and 130 min for inexperienced surgeons (70, 71). Freeman et al. established an optimal duration of 45 min for inexperienced surgeons after six surgeries (70). In this study, an average of almost 40 min TST demonstrated that the surgeon’s skill is enhanced by training before to the start of main operations, and demonstrating our surgeons’ experience (71) made the findings more realistic (11, 13, 70). Alternatively, previous research on pilots has yielded five levels of skill acquisition, including novice, advanced beginner, competent, proficient, and expert (25); If each 10-min improvement for OHE corresponded to one level of improvement for the surgeon’s expert, then our surgeon’s 12.7 min reduction in surgical duration using the instrument shank-assisted technique would theoretically qualify her as “competent.”

Surgical experience makes sick animals healthier (72). Sir Francis Galton thought that talent was completely innate (73), but experience is a learning growth (25). Yet, from a different perspective, the surgeon’s experience may provide no more than a 25% health improvement (72). On the other hand, OHE is still known as a model of acute pain in research studies. So, when animal pain after a technique is still a problem even after experienced surgeons have used it, it is important to look at the technique itself instead of just how it is taught. Consequently, both correct training and the training of correct techniques are emphasized in the surgical training curriculum, and the new method may contribute to the promotion of the latter, as indicated by the high effect sizes reported in the present study.

The type of operation is a stressor for the surgeon (74). Mental (75) and muscle (76) fatigue can delay an operation. Aside from the fact that the stress was not directly evaluated in our study, time as a major component in calculating surgical stress (74, 77) has been meticulously recorded and analyzed. Controversial is the scenario in which the total surgery time is reduced beyond the time spent on the ovarian pedicle. The technical difference between the two methods was SRT and ShT only had a time difference of 58.58 s in favor of the modified method, but TST was improved by 12.7 min. Non-correlation of these variables with TST indicates they did not directly contribute to the difference in TST, while ovarian exposure caused roughly 63% quicker application of ligatures (4.8 min improvement). The remainder of the improved duration was divided into two parts: (1) 3.1 min from the major procedures in OSPT, which include the separation of the broad ligament on both sides, the second ovary access, uterine arteries ligature placement, and uterine body close and cut; and (2) 4.8 min from CT. The procedures conducted in OSPT and CT were comparable among techniques, although the Instrument shank-assisted OHE required significantly less time. When we were nearing the end of the surgery, or, in other words, when the surgeon had reached extreme fatigue, 4.8 min more time was required to close the abdominal wall using the conventional technique. Therefore, significant time reduction in the mentioned two parts could be related to less fatigue and stress, which was paved through the shorter LigT in the new method. Peeters and Kirpensteijn’s unsuccessful attempt to reduce surgical time by utilizing ovariectomy (OVE) instead of OHE (58) is another example of the surgeon’s mental and physical strain at this time, as they did not eliminate digital strumming. Due to the strong correlation between TST and LigT in the present study, it is evident that the modified instrument shank-assisted technique can reduce the impact of the time required to install the ligatures, which was the primary factor in the significantly longer surgical time when the previous technique was used. Additionally, the small standard deviation (78) in these two portions may indicate the surgeon’s optimal state of stability and fewer technical obstacles during instrument shank-assisted OHE, which may require further investigation.

According to research findings, the surgeon’s stress level may be enhanced due to a lack of familiarity with the members of the surgical team (67). While the present study did not measure the stress level of the surgeon, efforts were made to mitigate concerns regarding the presence of new team members. This was achieved through measures such as facilitating familiarity and collaboration among team members during the pre-study training course as well as maintaining a consistent surgical team.

Surgical supervisor changes complicate student-led surgical procedures (57). This study’s experienced academic surgeon continuously supervised the surgical procedures, eliminating the possibility of this error, as observed in Harris’s study due to a change in supervisor.

The time interval proportion found in this study could be used in general, even though an experienced surgeon could cut the total time needed for surgery. Hence, based on Shivley’s yearly savings of 73.3 h (11), a 26.7% decrease in TST in instrument shank-assisted OHE could save 195.7 h (>2.5 times).

In concluding, as ovarian manipulation and pedicle ligatures were identified as the most essential procedures of OHE in a previous study (79), these findings have been meticulously confirmed in the present study. In the instrument shank-assisted OHE, a more accessible ovarian pedicle facilitated all subsequent steps of the surgery.

Due to the surgical discussion’s focus on inexperienced surgeons, it is reasonable to assume that the significance of this technique will change as the surgeon’s experience grows, whereas the pain-related advantages of this surgical technique will be discussed in a separate chapter, considering all surgeons to be subject to the implementation of this technique.

The current study confirmed that, compared to instrument shank-assisted OHE, digital strumming of the ovarian pedicle during conventional OHE makes ligature placement more challenging, possibly due to unwanted visceral interferences or manipulations, prolongs the duration of surgery, and increases pain. Digital strumming is an unpleasant surgical procedure followed by considerable surgical trauma. Thereby, it is anticipated that modifying traditionally invasive procedures would improve patients’ recoveries and well-being.

Subjective pain scales (44, 80) and, controversially, CRP have been used to measure pain since vital signs are not sensitive enough (81) and animals cannot communicate verbally (47). Several multidimensional structured behavior scales have been adapted for use in veterinary medicine (59), and the multidimensional GCMPS-SF for acute pain has been authorized for use in dogs (45, 60) with more sensitive and consistent results (82). The UMPS (which comprises six categories of physiological data) and VAS (with more flexibility) were used to compensate for the insufficiency of the GCMPS-SF and reduce the secrecy of the pain.

The results of pain assessments vary based on the experience and knowledge of the veterinarians, which are affected by age, gender, and time since graduation (31, 83). It was anticipated that using a trained, blinded assessor (30), a wound dressing, and three distinct pain scales would reduce the influence of qualitative variable bias in the current study. On the other hand, demographic data and dog acclimation before surgery suggest that individual pain tolerance, species, age, body condition, and environmental factors that can change or mask pain intensity are not confounding variables.

Since severe pain after surgery is often underestimated (84), it needs to be measured in a new surgical procedure (85). The perception of postoperative pain is dependent not only on surgical duration and technique (86), but also on analgesic type (87), dose, multimodality (88–90), the use of preventative analgesia (91), route of administration, and the pharmacokinetics of medications (92). Pain can result in delayed wound healing and surgical site infection (93), and bandages may not be adequate for preventing suture line contamination (94). So, surgeons prefer to modify surgical procedures to reduce postoperative pain (47). This was one of the most important goals of instrument shank-assisted OHE, which had an effect size d of >1.27, > 1.32, and > 1.77 and a power of 0.811, 0.836, and 0.968 at T6, T24, and T48, respectively, based on provided GCMPS-SF pain measurements.

Sampling time is essential for accurately determining pain on time. In this study, sampling times were adjusted according to four theoretical elements: (1) the clinical duration of action of the analgesic agent (meperidine, medetomidine, and ketorolac), which may provide enough pain relief; (2) the plasma half-life of the anesthetic selected for premedication, induction, and maintenance of anesthesia (acepromazine, midazolam, and ketamine), which may change the responses given for pain evaluation; (3) the minimum estimated duration for pain onset, peak, and subsidence; and (4) the pathophysiology of CRP turnover. By giving dogs nonsteroidal anti-inflammatory drugs (NSAIDs) before surgery to help with pain after surgery (95, 96), it was thought that ketorolac might be enough for the first few hours after surgery. However, the results have only shown that this is true for the new technique. It was anticipated that the pharmacologic effects of the long-half-life (t_½) drugs acepromazine (97), ketorolac (98, 99), and ketamine (nor-ketamine) (100) with residual effects of 7.1, 4.5 (or 10 based on relevant reference), and 6.2 h, respectively, would be felt from premedication until endotracheal extubation. Because sedatives, analgesics, and injectable dissociative anesthetics can change responses like facial expression, salivation, mydriasis, and cardiorespiratory parameters, which were evaluated in the GCMPS-SF, UMPS, and VAS, early pain assessment (<6 h) was not considered in this study.

The requirement of rescue analgesia may be regarded as a reliable indicator of the surgical technique’s incompetence. According to a previous report in humans (101), after hip and knee arthroplasty, ketoprofen had the same analgesic effect as extradural morphine. Mathews et al. find that ketoprofen has a comparable impact to meloxicam and conclude that it could be a useful way for controlling postoperative pain (102). Similar to carprofen, meloxicam, and tolfenamic acid, ketoprofen produced excellent postoperative analgesia in cats, but with a lesser effect on tenderness (103). In the meantime, more recent studies indicate the administration of NSAIDs is superior to opioids due to faster recovery of normal functions and greater satisfaction with postoperative well-being (104). Nevertheless, these findings should be taken with care when applied to OHE in veterinary medicine. In the present study, animals that got rescue analgesia were not excluded, and 8 dogs in the conventional group who received rescue analgesia at 13 evaluation times were included for analysis. If getting rescue analgesics improved outcomes, the dogs in the first group were unable to demonstrate superior outcomes despite receiving frequent pain treatment. The number of animals administered rescue analgesics may be a reflection of the severity of surgical trauma and the invasiveness of the conventional technique. Receiving rescue analgesics in 1 dog out of 2 evaluation times can be attributed to greater well-being using the modified method and can be interpreted in two aspects: first, a standard protocol of analgesics is still recommended after surgery, and second, it may be useful in shelter or stray dogs that may not receive proper follow-up treatment, for example.

At 6 h postoperatively, eight out of nine dogs treated with the conventional method received rescue analgesia. This demonstrates at least two important points: (1) ketorolac provides inadequate postoperative analgesia for the conventional OHE performed by an inexperienced surgeon, although it has been used in humans to control moderate to severe post-operative pain, and it may be effective in dogs (105), as effective as flunixin, and more effective than butorphanol or a low dose of oxymorphone (106), by affecting opioid receptors centrally with comparable efficacy to morphine (99), and (2) the current modification has decreased postoperative pain to the point that ketorolac could control it, so that none of the dogs in the second group required T6 rescue analgesia.

Our “competent” surgeon has made the surgery quick and competitive in terms of time (see “Surgical perspective”). Since the new method causes less pain and this surgery is still done to create a model of acute pain for research, it is safe to assume that this surgical method is not limited to a certain group of surgeons, no matter how much experience they have, and that it is better to make it more general.

In addition to the psychological burden experienced by the surgeon, contemporary approaches have been developed to assess the degree of pain and surgical stress imposed on the patient. While certain methods, including pupillometry, surgical pleth index (SPI), skin conductance, cardiovascular, and cardiorespiratory indices, require further advancements in sensor technology and interpretation algorithms to investigate animal responses to anesthesia and surgery, their applicability has not yet been confirmed. In the field of veterinary medicine, additional quantitative techniques have been introduced for animal assessment. These include the parasympathetic tone activity index (PTA index), which analyzes heart rate variability, and the bispectral index (BIS), which analyzes electroencephalography, with the potential for animal interpretation. Despite ongoing debates regarding the universal implementation of their use in all treatment procedures and drug protocols (107), the accessibility of these remedies may not always be assured. It is worth noting that the application of PTA as a means of assessing pain in conscious animals within the field of veterinary medicine is challenging. This is primarily due to the presence of unwanted movements by the animal, which directly impact heart rate variability. Consequently, alternative models capable of detecting these fluctuations should be employed (20, 32, 107, 108). The use of more recent medications, and therefore the recording of the quantity of anesthetics used (55) and the vital parameters throughout the operation, seems to be a large issue that would need review in separate research, other than that this technology was not available for the current study.

A number of different physiological parameters, such as plasma vasopressin, urine noradrenaline, and creatinine concentrations, have been suggested to assess the degree of irritation and pain caused by a surgical method. It appears that documenting additional facts and aiding in the final assessment of the effectiveness of the presented technique may be accomplished by comparing the changes in these parameters during anesthesia between the two methods. The conventional OHE involves applying extremely stretching stress to digitally strumming the suspensory ligament, while the new method involves keeping this ligament under tension all the way through the ligature placement process. Further research is warranted to compare the two methods from this perspective, as acute noxious stimuli during stretching of the pedicles can increase systolic blood pressure, heart rate, plasma vasopressin concentration, and urinary noradrenaline/creatinine ratio (55).

One of the initial observable events following surgery is the elevation in temperature and inflammation of the surgical site, attributed to enhanced blood circulation to the area where surgery was performed. Infrared thermography (IRT) is a proficient technique for assessing thermal variations in problematic areas, as it measures the surface temperature of the skin through thermographic maps. This technique may be used to recognize the localized changes in blood supply and localized increases in temperature that occur in response to stress. The assessment of the efficacy of local anesthesia through the analysis of alterations in surface blood circulation linked to sympathetic activity is among the additional functionalities of IRT (109). However, it has been observed that this technique has not demonstrated sufficient effectiveness in dogs (110). The present study suggests that while the implementation of IRT for evaluating pain and inflammation in the surgical approach area was effective, practical limitations arose due to the bandage covering the surgical site. Conversely, the thermographic assessment of regions where the suspensory ligament has been torn, situated on the roof of the abdominal cavity, may not be deemed reliable in theory. This is due to the fact that the heating of the dermis surface is directly linked to the local dermal microcirculation, which is under the control of the ANS. Nevertheless, the non-invasive nature of this method of evaluation may be an appealing subject for further research.

Pupil shape has been a key indicator for neurological assessment for over a century (111), and automated pupillometers have become increasingly important due to the difficulty of detecting the “reactive pupil” characteristic (112). In addition, assessing pupil reactivity using a pupillometer offers an objective, rather than subjective, evaluation of the neurological examination. Automated pupillometers can distinguish between canine conscious and anesthetic pupillary light reflex (PLR) and continuously assess an animal before, during, and after anesthesia (113). It seems that PLR devices could be useful in research like the current one, provided that the assessments are standardized.

Evaluation of a novel surgical procedure extends beyond surgical parameters and postoperative discomfort. Surgical invasiveness may be assessed separately. Based on past research, the invasiveness of the surgical procedure may be related to postoperative pain. Consequently, the question of surgical trauma severity is a separate topic that will be addressed further in “CRP perspective.”

In this study, the traditional OHE method was changed in a way that reduced the amount of trauma. With the new technique, the amount of surgery-related trauma had a smoother pattern and went back to normal almost 30% faster. These results predicted a shorter period of recovery time subsequent to instrument shank-assisted OHE.

The postoperative acute-phase response develops faster in dogs compared to humans. Tissue damage and pain after invasive surgery are associated with a rise in blood acute-phase proteins, primarily CRP, which may be a valuable diagnostic biological marker of early postoperative complications (114, 115). The CRP, a sensitive biomarker of infection (116), inflammation, and tissue damage (46), is more sensitive than serum cortisol (81) in detecting surgical trauma (13) and can assess various surgical procedures in dogs (33, 117), peaking 24 h postoperatively (42). The short half-life of canine CRP (19 h) makes it a useful marker for identifying the intensity of mild clinical stressors (33, 118, 119) whose effects dissipate more rapidly.

It has been stated earlier that the slope of changes is a reliable predictor variable for the expected peak (13). A five-fold smoother slope in the elevation of CRP concentration generated a milder peak after instrument shank-assisted ovariohysterectomy, so its return level to the base value showed a significant reduction at T48 compared to that of the animals in the opposite group. Thus, it is suggested that future research on the slope of the post-OHE CRP increase would also be planned to reduce study duration and be used for designing and scheduling postoperative analgesia protocols and lengths. As CRP is elevated approximately 6 h after a single stimulation (119), the present data revealed that there is no clinical necessity for sampling before 6 h after surgery. Moreover, because serum concentration peaks between 24 and 48 h in dogs (42, 115, 119), and CRP has a short half-life (33, 118, 119), the last measurement time of 72 h after surgery was appropriate for CRP return.

It has been imagined that OVE can satisfy surgeons’ hopes by reducing the consequences of OHE. Moldal et al. did not find any differences in the levels of CRP, glucose, or iron in the blood between them. Therefore, the greatest trauma in OHE occurs during the surgeon’s manipulation of the ovarian pedicle (86). Thereby, the instrument shank-assisted method’s unique characteristic can be considered an advantage. By reducing manipulation of the ovarian pedicle through eliminating digital strumming, the surgical trauma has also been reduced to a level close to the lowest expected minimum. Experienced surgeons have the advantage of avoiding unnecessary organ manipulation (26, 27), which causes minimal surgical trauma and postoperative serum CRP concentration. Hence, the present modified technique, which entails less organ touch, presents surgical quality closer to that of an experienced surgeon.

It is not without merit to state that CRP concentrations seem to be a decent predictor of how invasive an operation is (120), despite some contradictory reports, such as that there is no correlation between the length of surgery and CRP, despite its rise (121), or that CRP concentrations may not be significant in the diagnosis of a disease (118). In the present study, the return to baseline after a moderate rise in CRP levels utilizing the instrument shank-assisted OHE, as contrasted to the conventional group’s high CRP levels, suggests that the instrument shank-assisted OHE could be concluded as having a minor invasive nature.

The risk of infection is increased when abnormal CRP responses are seen 5 or 7 days following surgery (116). A progressive decrease in serum CRP content in both groups could indicate the absence of infection. It is not unlikely that a procedure requiring fundamentally less organ manipulation would result in improved recovery, but ultimate healing was not the focus of this study.

Finally, the pattern of CRP changes followed the pain charts without correlation, according to the data. So, at least when the surgery is minor, there may not be a statistical correlation between changes in the CRP and pain. This may be because of the wide range of its reported changes. In this instance, the CRP profile may be able to anticipate pain patterns, but it cannot be used to make statistical conclusions.

The CRP changes showed that OVE and other similar procedures cannot be the final solution to animal comfort, and modification of the surgical technique on the more severe parts of the surgery is necessary, whether it is the method proposed in the current study or other solutions that may be introduced later. Although the graphs of CRP and pain changes appear similar, establishing a definitive relationship or statistically significant correlation between them requires further research.