animals Article Changes in Acute Phase Proteins in Bitches after Laparoscopic, Midline, and Flank Ovariectomy Using the Same Method for Hemostasis Ayla Del Romero 1,2 , Belén Cuervo 1,2, * , Pau Peláez 1,2 , Laura Miguel 1,2 , Marta Torres 1,2 , Marc Yeste 3,4 , Maria Montserrat Rivera del Alamo 5 , Camila P. Rubio 6 and Mónica Rubio 1,2 1 Bioregenerative Medicine and Applied Surgery Research Group, Department of Animal Medicine and Surgery, University CEU—Cardenal Herrera, CEU Universities, C/Tirant lo Blanc, 7, Alfara del Patriarca, 46115 Valencia, Spain; ayla.delromero@uchceu.es (A.D.R.); pau.pelaez@uchceu.es (P.P.); laura.miguel@uchceu.es (L.M.); marta.torrestorrillas@uchceu.es (M.T.); mrubio@uchceu.es (M.R.) 2 García Cugat Foundation CEU-UCH Chair of Medicine and Regenerative Surgery, University CEU-Cardenal Herrera, CEU Universities, C/Tirant lo Blanc, 7, Alfara del Patriarca, 46115 Valencia, Spain 3 Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, E-17003 Girona, Spain; marc.yeste@udg.edu 4 Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, E-17003 Girona, Spain 5 Unit of Animal Reproduction, Department of Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra (Cerdanyola Del Vallès), Spain; mariamontserrat.Rivera@uab.cat 6 Interdisciplinary Laboratory of Clinical Analysis (Interlab-UMU), Veterinary School, Campus of Excellence Mare Nostrum, University of Murcia, Espinardo, 30100 Murcia, Spain; camila.peres@um.es * Correspondence: belen.cuervo@uchceu.es; Tel.: +34-96-136-9000 (ext. 66017)  Received: 19 October 2020; Accepted: 23 November 2020; Published: 27 November 2020  Simple Summary: Ovariectomy is a common surgical procedure in veterinary medicine, with many techniques involved. The aim of this study is to objectively evaluate the acute phase response by measuring the changes in a panel of acute phase proteins after applying three different ovariectomy techniques. C-reactive protein values showed increases of lower magnitude after laparoscopic ovariectomy compared with other techniques, indicating that this surgical technique induces a reduced inflammatory response and tissue damage. The use of this procedure is in agreement with the current tendency to use minimally invasive procedures for ovariectomy. Abstract: Acute phase proteins (APP) are biomarkers of systemic inflammation, which allow monitoring the evolution of diseases, the response to treatments, and post-operative complications. Ovariectomy (OVE) is frequently performed in veterinary medicine and can be a useful model to evaluate surgical trauma and inflammation in the bitch. The objective was to investigate and compare the acute phase response (APR) after applying three different OVE techniques by measuring serum levels of C-reactive protein (CRP), haptoglobin (Hp), albumin (Alb), and paraoxonase-1 (PON-1). Forty-five intact bitches were included in the study, being randomly distributed into three groups: laparoscopic OVE (L), midline OVE (M), and flank OVE (F). Serum CRP, Hp, Alb, and PON-1 were measured before surgery, 1, 24, 72, and 168 h post-intervention. CRP levels increased significantly 24 h post-surgery in the M and F groups, but no significant variation was observed in the L group at any time of the study period. Hp was significantly higher in group L than in group F 72 h post-surgery. Alb and PON-1 showed no statistical difference among groups or among sampling periods. CRP response suggests that the use of laparoscopic procedures produce lower inflammation compared to open conventional approaches when performing OVE in the bitch. Animals 2020, 10, 2223; doi:10.3390/ani10122223 www.mdpi.com/journal/animals Animals 2020, 10, 2223 2 of 14 Keywords: acute phase proteins; surgical trauma; ovariectomy; laparoscopy 1. Introduction Surgical procedures in veterinary medicine are aimed to help the animal recover its health status or promote the prevention of diseases. Any surgical procedure implies a surgical stress that will induce a response composed of a set of metabolic, hormonal, and inflammatory reactions, which allow the body to adapt to trauma and recover damaged tissues [1,2]. These inflammatory reactions are part of the acute phase response (APR). APR consists of a systemic reaction of the organism to local or systemic disorders in its homeostasis, caused not only by surgical trauma, but also by infection, tissue injury, neoplastic growth, or immunological disorders [3–5]. During the APR, changes in the synthesis and release of acute phase proteins (APP) induced by pro-inflammatory cytokines occur. These proteins are classified into positive APP (if serum concentration increases), or negative APP (if serum concentration decreases) [5,6]. C-reactive protein (CRP) is a major positive APP that has been studied in several inflammatory processes and it is used to monitor the response to treatment in dogs [5,7,8]. CRP can increase up to 95 times after surgery, and this increase is related to the degree of tissue injury in dogs. Thus, when different surgical procedures are compared, CRP values correlate with the tissue trauma [9–11]. Changes in the post-operative period report maximum concentrations of CRP after approximately 12–24 h [12] and significantly increased concentrations have been demonstrated for several days after surgery, varying from 7 days [13] to 17 days [14]. Haptoglobin (Hp) is a moderate positive APP involved in host defense responses to infection and inflammation [5]. Increases in Hp concentration have been described after surgical trauma in dogs, as well as other species, such as cats [15–17]. Changes in Hp can be detected at 24 h after a surgical procedure, showing the peak concentration 3–4 days after surgery [5]. Albumin (Alb) is the most abundant protein in the blood and is considered a negative APP [18]. Low Alb is related to poor outcomes in human medicine and it has been used to monitor the effects of different treatments in leishmania in dogs [19]. Paraoxonase-1 (PON-1) is an enzyme associated with high-density lipoproteins (HDL) and considered a negative APP [20]. There are few studies on this inflammatory biomarker in dogs in different medical situations [2,21,22], but its changes after surgery are less described [23]. Surgical neutering is commonly performed in the veterinary practice, as surgical sterilization has several proven benefits, including population control, prevention of some reproductive tract disorders, and elimination of undesirable behaviors associated with hormonal cycling [24,25]. Described techniques for bitch surgical sterilization are ovariectomy (OVE) and ovariohysterectomy (OVH), being both equally effective. OVE has potential advantages as smaller incisions, better viewing of the ovarian vessels and lower risk of surgical complications [26–30]. However, this technique should not be used if there is uterine disease or to prevent uterine neoplasm [26]. The surgical approach to OVE in the bitch has been debated over the last years [31–35]. Midline and flank laparotomies, known as open or conventional OVE, have been the most frequent approaches while laparoscopic OVE has recently gained interest in veterinary practice [36,37]. Many advantages are described for laparoscopic procedures over conventional laparotomies in both human and veterinary medicine: decreased post-operative stress and pain, shorter recovery periods, decreased hospitalization stay, better surgical wound outcome, and improved visualization of the abdominal cavity [38–44]. However, most of these parameters might be affected not only by the intensity of the surgical trauma, but also by some other factors, such as age, health status, breed, or character of the animal [45]. Laparoscopic procedures also have some disadvantages over open conventional procedures: high cost of the equipment, the need of an assistant, higher learning curve, longer surgical time, and the requirement of intra-abdominal carbon-dioxide (CO2 ) insufflation (pneumoperitoneum) [43,46–48]. The latter has been associated Animals 2020, 10, 2223 3 of 14 with several complications, which include visceral pain, local peritonitis, hemodynamic changes, oxidative stress, and inflammation [49–53]. In human medicine, APR after surgical trauma is well described, being this response proportional to the level of tissue injury [54–57]. In veterinary medicine, changes in APP in the post-operative period have also been described, with CRP being one of the most studied biomarkers [10,11,17,58,59]. These inflammatory biomarkers have also been studied in conjunction with the leucogram to monitor the post-operative period in dogs [60,61], as well as after surgical trauma in other species such as cows [62,63]. Specific studies on APR after surgical neutering (OVE or OVH techniques) are described in both dogs and cats [11,16,28,58]. Comparison among several types of surgery of different magnitude shows that APR increases according to the degree of the surgical trauma [10]. Changes in the APP have also been studied in dogs undergoing minimally invasive surgery of different types, showing major increases in the APP than in the open techniques [42]. Some studies compare conventional midline laparotomy with mini-laparotomy techniques or laparoscopic-assisted techniques [15,64], but purely laparoscopic technique is less represented [10,16,65]. Different techniques for hemostasis of the ovarian pedicle (sutures, ties, sealing devices) are used among these studies, which may cause variations in the post-surgical inflammation [37,39,43,44,66]. To the authors’ knowledge, there is no published evidence of the APR in bitches after OVE, comparing midline, flank, and laparoscopic techniques using the same method for hemostasis of the ovarian and uterine pedicles. Therefore, the objective of this study was to evaluate the effect of three different OVE techniques, namely laparoscopic, midline and flank OVE, on the kinetics of two positive APP (CRP and Hp) and two negative APP (Alb and PON-1), and to compare the effects of inflammation by the three procedures in the postoperative period. Knowing this information regarding inflammation could help to better select the technique for OVE in daily practice. 2. Materials and Methods 2.1. Animals Forty-six entire healthy bitches of different breeds were initially included (Table 1). The dogs aged between 5 and 48 months old, and weighed between 2.5 and 51 kg. The study was approved by the Ethics Committee for Animal Experimentation of the University CEU-Cardenal Herrera (2018/VSC/PEA/0195). All animal owners signed a consent form after having been explained the relevant project information. Table 1. Descriptive data of breed for each patient. Dog Breed Dog Breed Dog Breed 1 Crossbreed 17 Shih-Tzu 32 Podenco 2 Crossbreed 18 Mastiff 33 Crossbreed 3 Crossbreed 19 Maltese 34 Crossbreed 4 Crossbreed 20 Crossbreed 35 Yorkshire terrier 5 Weimaraner 21 Podenco 36 Crossbreed 6 Am Staffordshire 22 Am Staffordshire 37 Siberian husky 7 Podenco 23 Beagle 38 Podenco 8 Bull Terrier 24 Yorkshire terrier 39 Crossbreed 9 Crossbreed 25 Jack Russell 40 Crossbreed 10 Spaniel Breton 26 Yorkshire terrier 41 Crossbreed 11 Crossbreed 27 Crossbreed 42 Podenco 12 Crossbreed 28 Crossbreed 43 Podenco 13 Am. Staffordshire 29 Podenco 44 Mastiff 14 Pinscher 30 Podenco 45 Crossbreed 15 Crossbreed 31 Greyhound 46 Crossbreed 16 Crossbreed 32 Dachshund Animals 2020, 10, 2223 4 of 14 Before surgery, dogs underwent a thorough clinical examination to ensure that they were completely healthy. Complete blood cell count and biochemistry profile, thoracic radiographs and electrocardiogram were performed. Uranotest® Quattro (Urano Vet SL, Barcelona, Spain) was used to detect antibodies for Leishmania infantum, Ehrlichia canis and Anaplasma platys, and to detect antigens for Dirofilaria immitis. Only dogs assigned to ASA (American Society of Anesthesiologists) category 1 (normal healthy animals) [67], and with negative results on the Uranotest® were included. As the surgical technique consisted of OVE, any animal in which OVH was recommended for clinical reasons, was excluded. If post-operative complications appeared, the animal was also excluded from the study. 2.2. Study Groups The forty-six bitches were randomly divided into three groups, depending on the surgical technique: 16 animals were located in group L (laparoscopic OVE), 15 animals in group M (midline OVE), and 15 animals in group F (flank OVE). 2.3. Anesthesia and Antibiotic Prophylaxis Anesthetic protocol consisted in premedication with dexmedetomidine (5 µg/Kg, Dexdomitor® , Orion Corporation, Espoo, Finland) and methadone (0.3 mg/Kg, Semfortan® , Eurovet Animal Health BV, Bladel, Holland) intramuscular. Vital signs (respiratory rate, heart rate, blood pressure, and temperature) were taken 15 min after premedication and prior to induction with propofol (2–5 mg/Kg/dose effect, Propofol Lipuro® 1%, Melsungen, Alemania) intravenously (IV). Anesthesia was maintained with sevoflurane (SevoFlo® , Zoetis, Louvain-la-Neuve, Belgium) in 50% oxygen/air mixture. Intermittent positive pressure ventilation (VPPI) was applied to ensure normocapnia and the volume was regulated in order to keep the end-tidal CO2 at normal levels (35–45 mmHg). All animals received IV fluid therapy with Ringer-Lactate solution (5 mL/Kg/h, Braun® , Barcelona, Spain) during the surgical procedure, and a single dose of cefazolin (20 mg/Kg/IV, Cefazolina Normon® , Normon laboratories, Madrid, Spain) before starting the surgeries. Intraoperative monitoring consisted of body temperature, electrocardiogram, capnography, pulse oximetry, noninvasive blood pressure, and oxygen and vapor concentrations. 2.4. Surgical Procedures Three surgical techniques for OVE were applied, laparoscopic (L), midline (M), and flank (F) approach. All techniques were performed by the same surgeon with the help of an assistant. The surgical area was clipped and scrubbed (abdomen for group L and M and flank for group F). In groups M and F, the distance from xiphoid to pubis was measured, to calculate the size of the incision (20% of this length) and standardize the surgical wound. OVE technique was carried out depending on the group. For group M, an incision was made on the abdominal area, caudal to the umbilicus, to approach the linea alba after dissecting the subcutaneous tissue. For group F, a single dorsoventral incision in the skin was made on the left flank of the animal, starting caudal to the midpoint between the last rib and iliac crest. The subcutaneous tissue was dissected and the fibers from the muscle bellies of the external abdominal oblique, internal abdominal oblique and transverse abdominal were opened separately, according to their fiber direction as described in the literature [32]. In both groups, after identifying the ovaries, a vessel sealing device LigaSure™ (Medtronic [formerly Covidien], Minneapolis, MN, USA) was used for the sealing of the suspensory ligament, the ovarian artery and vein, and the uterine artery and vein, just caudally to the ovaries so the uterus was not excised. For group L, a two cannulas laparoscopic technique [68] was used, so each animal had two incisions (1 cm cranial and 1 cm caudal to the umbilicus) that were the same size of the cannulas (5 mm). Pneumoperitoneum was stablished by insufflating CO2 to a pressure of 7 mm Hg. The patient was rotated into right lateral oblique recumbency for removing the left ovary and subsequently into left lateral oblique recumbency for removing the right ovary. A circular 0-size needle was used during Animals 2020, 10, 2223 5 of 14 each procedure to transabdominally stabilize the ovary against the abdominal wall. Ovaries were removed through the cranial 5-mm cannula after sealing the ovarian vessels using the LigaSure™ (Medtronic [formerly Covidien], Minneapolis, MN, USA) sealing device. In group M, linea alba was closed using a simple continuous pattern, and in group F, muscles were sutured in two layers with the transverse and internal oblique muscle together, and the external oblique muscle as a second layer, as described in the literature [33,69]. Subcutaneous tissue was sutured with a simple continuous pattern, and skin was closed with an intradermic pattern. For all of the layers, absorbable monofilament glyconate suture was used (Monosyn® , B. Braun VetCare SA, Barcelona, Spain). For group L, the same type of suture was placed to close the abdominal wall at each cannula site, using a cruciate pattern. Subcutaneous tissue was closed using a cruciate pattern and surgical glue was used to seal the skin. No complications related to anesthesia or intraoperative hemorrhage were observed during surgery in any case. Post-operative care consisted of hospitalization during the first 24 h and analgesia receiving methadone (0.2 mg/kg/6 h Semfortan® , Eurovet Animal Health BV, Bladel, Holland) (IM). Only one dog experienced post-operative complications (a dog in group L that licked at the wound site), and it was excluded from the study 2.5. Blood Sampling Blood samples (3 mL) were taken by jugular venipuncture just before ovariectomy (B) and at 1, 24, 72, and 168 h post-surgery. Blood was allowed to clot at room temperature for 30 min and centrifuged (3000 rpm, 10 min). The obtained serum was separated and kept in Eppendorf microtubes (Eppendorf, Hamburg, Germany) at −80 ◦ C until analysis. Serum concentration of CRP was measured using a human immunoturbidimetric test (CRP OSR 6147 Olympus Life and Material Science Europe GmbH, Lismeehan, O’Callaghan’s Mills, Co., Clare, Ireland). A colorimetric method (kit haptoglobin TrideltaPhase range SAA kit, Tridelta Development Ltd., Co., Kildare, Ireland) was used to measure Hp concentration. Albumin was determined using a Bromocresol green reagent (Albumin OSR 6102 Olympus Life and Material Science Europe GmbH Lismeehan). Finally, an automated spectrophotometric assay was used to determine PON-1 concentration (Sigma-Aldrich, St. Louis, MO, USA). All techniques had been previously validated for use in dogs [70–72]. All analyses were performed within 6 months since the sampling procedure. 2.6. Statistical Analysis Data were analyzed through a statistical package (IBM SPSS for Windows, Ver. 25.0; Armonk, NY, USA). First, data were checked through Shapiro–Wilk and Levine tests for normal distribution and homogeneity of variances, respectively. When distribution of data was not normal and/or variances √ √ were not homogenous, they were transformed through arcsine x or x. Following this, a linear mixed model followed by post-hoc Sidak test was run per variable (changes in concentration of albumin and PON-1 in serum, age, and weight of animals and time of surgery). In this model, the between-subjects factor was the surgery treatment (laparoscopic ovariectomy, midline ovariectomy, and flank ovariectomy) and the within-subjects factor was the time of evaluation (before surgery, 1, 24, 72, and 168 h post-intervention). When, even after linear transformation, data did not match with parametric assumptions (changes in the concentrations of CRP and Hp in serum), Friedman and Wilcoxon tests were used as non-parametric alternatives. Correlation between variables were calculated with Pearson coefficient when data fit with parametric assumptions (changes in concentration of albumin and PON-1 in serum, and with Spearman coefficient when distribution was not normal and/or variances were not homogeneous (changes in the concentrations of CRP and Hp in serum). In all statistical analyses, the level of significance was set at p ≤ 0.05. Data are shown as mean ± standard error of the mean (SEM). Animals 2020, 10, x 6 of 15 Animals 2020, 10, 2223 6 of 14 3. Results Animals 2020, 10, x 6 of 15 Dogs aged 5 to 48 months (15.7 ± 12.1) and weighted 2.5 to 51 kg (13.7 ± 9.4). The group did not 3. Results 3. Results differ with regards to age (p = 0.58) or body weight (p = 1.00). The total surgical time was 26 to 52.4 Dogs min Dogs ±aged (37.1 aged 55 to 13.8). to 48 months months Surgical 48 (15.7 time(15.7 ±± 12.1) 12.1)Land for group and weighted wasweighted 39.7 to 52.52.5min 2.5 to 51 to 51(46 kg±(13.7 kg (13.7 3.2,);±±for 9.4). The group group 9.4). The group M wasdiddid not 26.8 notto differ 40 min differ with (33.2 with regards ± 3.2),toto regards andage age for =group (p(p =0.58) 0.58) F or or was body body 26.1weight (p =(p to 38.5 weight min= 1.00). The±The (32.3 1.00). total 3.1). total surgical Figure surgical 1time time waswas represents to26 26 the to min 52.4 surgical 52.4 min time (37.1 per ± 13.8). group. Surgical time for group L was 39.7 to 52.5 min (46 ± 3.2,); for (37.1 ± 13.8). Surgical time for group L was 39.7 to 52.5 min (46 ± 3.2,); for group M was 26.8 to 40 min group M was 26.8 to 40 min (33.2 ± 3.2), and for group F was 26.1 to 38.5 min (32.3 ± 3.1). Figure 1 (33.2 ± 3.2), and for group F was 26.1 to 38.5 min (32.3 ± 3.1). Figure 1 represents the surgical time represents the surgical timegroup. per per group. Figure 1. Surgical time in minutes per study group. L: laparoscopic, M: midline, F: flank. * p < 0.05: Statistically Figure significant 1. Surgical difference time in minutesamong groups. per study ° Outlier group. values. M: midline, F: flank. * p < 0.05: L: laparoscopic, Statistically significant ◦ Outlier values. Figure 1. Surgical timedifference in minutesamong groups. per study group. L: laparoscopic, M: midline, F: flank. * p < 0.05: 3.1. CRP and Hpsignificant difference among groups. ° Outlier values. Statistically 3.1. CRP and Hp CRP values peaked at 24 h in all groups, although it was only statistically significant compared 3.1. CRP and Hp peaked at 24 h in all groups, although it was only statistically significant compared withCRP values baseline value in groups M (p = 0.001) and F (p = 0.001). At this time point, CRP concentration in with groupbaseline CRP value values L was in groups peaked 24Mh (p at lower significantly = all0.001) inthan groups, in groups M=(p0.001). andalthough F (p wasAtonly =it0.001) thisstatistically and time F (p =point, CRP concentration significant 0.028). Figure in 2 compared represents group with inLfold wasvalues. CRP baseline significantly lower value in groups than M (p in groups = 0.001) and FM (p(p = 0.001) = 0.001). Atand (p =point, thisFtime 0.028).CRP Figure 2 represents concentration in CRP in fold values. group L was significantly lower than in groups M (p = 0.001) and F (p = 0.028). Figure 2 represents CRP in fold values. Figure 2. Fold C-reactive protein (CRP) concentration along the experimental period. L: laparoscopic ovariectomy (OVE), Figure 2. Fold M: midline C-reactive proteinOVE, (CRP)and F: flank OVE. concentration Different along letters (a, b) the experimental indicate period. differences L: laparoscopic between groups. B: baseline. * p < 0.05: statistically significant difference compared to baseline. ovariectomy (OVE), M: midline OVE, and F: flank OVE. Different letters (a, b) indicate differences between Figure groups. 2. Fold B: baseline. C-reactive * p <(CRP) protein 0.05: statistically concentration significant along thedifference compared experimental toL: period. baseline. laparoscopic ovariectomy (OVE), M: midline OVE, and F: flank OVE. Different letters (a, b) indicate differences between groups. B: baseline. * p < 0.05: statistically significant difference compared to baseline. Animals 2020, 10, x 7 of 15 Hp Animals concentration 2020, 10, x for group L was significantly increased above baseline at 24 h (p = 0.034) and 7 of 15 72 h (p = 0.001), showing the highest levels 72 h after the surgery. In groups M and F, Hp Animals 2020, 10, 2223 7 of 14 concentrations did notfor Hp concentration increase group significantly compared L was significantly to basal increased values. above When baseline at surgical 24 h (p =procedures 0.034) and were 72 h compared, (p = 0.001),statistically showing the significant highestdifferences levels 72 between L and h after the F groups surgery. In atgroups 72 h were observed M and F, Hp (p = 0.026), with concentrations L group did not showing increase higher levels significantly of Hp compared than to F group. basal Figure values. When3 represents surgical Hp concentration for group L was significantly increased above baseline at 24 h (p = 0.034) and Hp in proceduresfold values. were compared, 72 h (p statistically = 0.001), showing the significant differences highest levels 72 h afterbetween L and the surgery. In Fgroups groupsMatand72 F, h Hp were observed (p concentrations =did 0.026), not increase significantly compared to basal values. When surgical procedures wereHp with L group showing higher levels of Hp than F group. Figure 3 represents in fold compared, values. statistically significant differences between L and F groups at 72 h were observed (p = 0.026), with L group showing higher levels of Hp than F group. Figure 3 represents Hp in fold values. Figure 3. Fold Hp concentration along the experimental period. L: laparoscopic OVE, M: midline OVE, Figureand F: flank 3. Fold Hp OVE. Differentalong concentration letters the(a,experimental b) indicate differences between groups. period. L: laparoscopic OVE, B: M:baseline, *p< midline OVE, 0.05: Figurestatistically and F: flank 3. FoldOVE.significant Hp Differentdifference letters concentration (a, compared along b)the indicate to baseline. differences experimental between period. groups. B: baseline, L: laparoscopic p < 0.05: OVE, M:* midline statistically OVE, and F: significant flank OVE.difference Different compared letters (a, b) to indicate baseline.differences between groups. B: baseline, * p < 3.2. Alb 0.05:and PON-1 significant difference compared to baseline. statistically 3.2. Alb and PON-1 Albumin and PON-1 showed no significant differences among the sampling times in each 3.2. Alb and PON-1 Albumin and PON-1 showed surgical procedure, nor among thenosurgical significant differences procedures. among Figure the sampling 4 represents times Alb in each in fold surgical values and procedure, Figure nor 5 representsamong the surgical PON-1 in procedures. fold values. Figure 4 represents Alb in fold values and Figure 5 Albumin and PON-1 showed no significant differences among the sampling times in each represents PON-1 in fold values. surgical procedure, nor among the surgical procedures. Figure 4 represents Alb in fold values and Figure 5 represents PON-1 in fold values. Figure 4. Fold albumin (Alb) concentration along the experimental period. L: laparoscopic OVE, M: midline OVE, and F: flank OVE. B: baseline. Animals 2020, 10, x 8 of 15 Figure 4. Fold albumin (Alb) concentration along the experimental period. L: laparoscopic OVE, M: Animalsmidline 2020, 10, OVE, 2223 and F: flank OVE. B: baseline. 8 of 14 Figure 5. Fold paraoxonase-1 (PON-1) concentration along the experimental period. L: laparoscopic OVE, Figure M: 5. Fold midline OVE,paraoxonase-1 (PON-1) and F: flank OVE. concentration along the experimental period. L: laparoscopic B: baseline. OVE, M: midline OVE, and F: flank OVE. B: baseline. 4. Discussion 4. Discussion Surgical neutering is a widely used method of contraception in veterinary medicine, and many bitchesSurgical undergo major abdominal neutering is a widely surgery for elective used method OVE yearly around of contraception the world. in veterinary The traumatic medicine, and many surgical bitchesinjury undergo results in the major release ofsurgery abdominal pro-inflammatory for electivecytokines OVE yearly by local monocytes around the world.and macrophages The traumatic and stimulates surgical injuryacute inflammation results and synthesis in the release of APP [7]. According of pro-inflammatory cytokinesto by the local authors’ knowledge, monocytes and this is the firstand macrophages study in evaluating stimulates the APR in and acute inflammation bitches after OVE synthesis of APP by[7]. comparing Accordingthree to thedifferent authors’ surgical knowledge,approaches, this is thelaparotomy, first study midline and flank. in evaluating the Studying the APR APR in bitches after after OVEOVE byusing these three comparing three different differenttechniques objectively evaluates surgical approaches, laparotomy, the degree midline of the andsurgical trauma and flank. Studying thepermits APR afterto compare OVE usingthe post-operative inflammatory these three different techniquesresponse. objectively evaluates the degree of the surgical trauma and permits CRP peaked to compare 24 h after surgery the post-operative in groups inflammatory M and F, showing that a moderate inflammatory response. responseCRP peaked occurs after24these h after twosurgery in groupswhich open techniques, M and F,agreement is in showing that withathat moderate previouslyinflammatory observed inresponse occurs bitches after OVHafter[58,61,73]. these twoInopen techniques, contrast, CRP levels which in is in agreement group L did not with undergothat significant previously observed changes over in the bitches after OVHperiod. post-operative [58,61,73].TheIn contrast, present dataCRP levels with coincides in group a study L did not undergo confirming that significant CRP allows to changes over degrees differentiate the post-operative of inflammation period. Thepost-operative in the present data period coincides withdifferent of three a study confirming soft-tissue that CRP surgical allows toEven procedures. differentiate though the degrees main of inflammation differences betweenin the post-operative groups for that study period wereof atthree 12 hdifferent soft-tissue[10]. post-intervention surgical procedures. In human Even athough medicine, smaller theelevation main differences in CRP was between groups detected for after that study were laparoscopic at 12 h post-intervention[10]. hysterectomy compared with the open In human abdominal medicine, procedurea smaller [74]. Inelevation veterinaryinmedicine, CRP was detected several after laparoscopic different laparoscopic hysterectomy procedures have compared showed with lowerthe open abdominal concentrations of CRP procedure compared[74]. withIn veterinary open surgical medicine, techniques several different [13,15,60], whichlaparoscopic procedures is in agreement with the have showed present lower concentrations of results. CRPAscompared it has been with open surgical previously stated, techniques Hp can increase [13,15,60], from which 24 h onisafter in agreement surgery, reachingwith the thepresent peak results. 3–4 days later. [5] In accordance with this, peak level for Hp in the present study was reached 72 h (3 days)Aspost-intervention. it has been previously At thisstated, Hp can time point, Hpincrease levels infrom group 24Lhwere on after highersurgery, than inreaching groups M the peak and F. Hp3–4strongly days later. [5] In blinds freeaccordance hemoglobin with this, peak resulting level in the for HpHP-hemoglobin complex in the present study was reached [15,75,76]. Thus, ree72Hph (3 indays) serum post-intervention. may decrease on Atsituations this time point, Hp levelsor of hemorrhage in hemolysis, group L were higherthat making than in groups M and determination by F. Hp strongly hemoglobin blindsassays binding free hemoglobin resulting[3,5,77–79]. may be unreliable in the complex In M HP-hemoglobin and F groups, [15,75,76]. Thus, free a higher degree of Hp in serum hemolysis may decrease immediately afteron situations surgery wouldof hemorrhage be expectedor duehemolysis, to larger making that determination tissue injury, whereas in the by hemoglobin L-group, binding the better assays viewing may of the be unreliable abdominal cavity[3,5,77–79]. and vesselsIn M and implies F manipulation less groups, a higher degree of the tissues.of Ashemolysis previous immediately studies have after surgery reported, this would imply be expected due to largeristissue that a hemorrhage injury, whereas less probable in the [80]. Surgical L-group, the hemorrhage or better viewingblood intraoperative of thelossabdominal cavity and can be quantified vessels using impliesand colorimetric lessgravimetric manipulation of the methods. tissues. As However, theprevious blood loss studies was nothave reported, measured in this nonewould of theimply that being surgeries, a hemorrhage a limitation is less probable of the [80]. study [81]. Animals 2020, 10, 2223 9 of 14 Another fact that must be elicited is that bitches in group L were subjected to the effect of the pneumoperitoneum. The insufflation of CO2 into the abdominal cavity has numerous effects both local and systemic, mainly an ischemia-reperfusion injury which is caused by decreased splenic blood flow and organ ischemia due to oxidative stress [82,83]. Visceral pain and discomfort are reported in human medicine after laparoscopic procedures [84] and it has been proved that the morphologic integrity of the peritoneum is transiently altered after CO2 pneumoperitoneum in mice [85]. This phenomenon is less studied in veterinary medicine, but could explain the results of Hp obtained in group L. A study in rats subjected to pneumoperitoneum showed an increase of inflammatory cytokines and histopathological changes in lung tissue, with an oxidative status that correlated with the degree of intra-abdominal pressure [53]. Further studies on the effect of the pneumoperitoneum in dogs are needed, so its role in post-operative pain could be better understood. Alb levels did not change among the sampling times neither among surgical procedures. This APP is mainly affected by chronic status rather than acute damage [86], so maybe the possible variations had not occurred yet. On the other hand, being a marker for chronic damage would explain the scarce literature regarding Alb after surgical trauma in dogs. In addition, none of the dogs included in the study reported surgical post-operative complications, being another possible reason for Alb levels to remain stable over the complete study period. PON-1 did not undergo significant changes during the post-operative period in none of the studied groups. This is in accordance with a study where PON-1 levels, 4 days after surgery, were not different from basal values [23]. The lack of variations could be explained by the fact that OVE procedure does not produce enough oxidative stimulus. Even though, a trend for lower levels of PON-1 were seen in group M and F compared to group L at 24, 72, and 168 h. It has been seen that surgical inflammatory response is affected by surgical time, thus, the differences in surgical time may have an impact in the results [57]. There is no agreement in terms of the duration of laparoscopic and laparotomic OVE. Some studies report shorter surgical times in laparoscopic compared to laparotomic procedures, but the latter were done using ligatures instead of sealing devices for the pedicles and that could bias the result [39]. Other studies report longer surgical times in laparoscopic OVE likely due to the learning curve associated to this technique [37]. In this study, laparoscopic OVE took longer than midline and flank OVE. However, CRP levels were lower in the post-operative time for L group. No correlation between surgical time and APP has already been described in previous studies [42,87,88]. The use of the LigaSure™ (Medtronic, [formerly Covidien], Minneapolis, MN, USA) sealing device in the open OVE groups could explain the shorter time described, as these conventional procedures are usually performed using ligatures rather than vessel sealing devices. The surgeon’s experience is also important as it is intimate related to the length of the surgery and to the trauma caused to the tissues [89]. In this project, the experience of the surgeon, the small size of the wound and the use of the vessel sealing device in the open technique, could justify the results regarding surgical time. Reference intervals have been established for some species [90,91], and also some interbreed variation of APP and other biomarkers appear in the literature [92,93]. There were 15 different breeds represented in the study and no statistical difference related to breed has been observed. In addition, to avoid a possible breed or individual effect, values were corrected by basal value for each APP (fold values). Likewise, no significant differences have been described in APP in terms of age and sex [94]. Minimizing tissue trauma and successive stress response is an important goal of surgery [89]. Currently, many surgical procedures in veterinary medicine are performed by laparoscopic procedures, and laparoscopic OVE is commonly preferred by pet owners when deciding to neuter their pet [47,68]. The results from the present study show that open OVE by midline and flank approaches produce a moderate and similar post-surgical inflammation based on the CRP values measured, being these values higher than those observed after laparoscopic OVE. Animals 2020, 10, 2223 10 of 14 5. Conclusions On the basis of the findings in this study, the authors conclude that concentrations of CRP in serum of bitches when performing laparoscopic OVE are lower than when midline and flank approaches are used. However, Hp concentrations may need further studies to understand the possible influence of blood loss on this parameter. Thus, based on the results of CRP changes, laparoscopy would be a favorable technique over conventional open approaches for OVE in the bitch. Author Contributions: Conceptualization, B.C., A.D.R. and M.R.; methodology, B.C., A.D.R. and M.R.; software, M.Y. and M.M.R.d.A.; validation, C.P.R.; formal analysis, A.D.R., M.Y. and M.M.R.d.A.; investigation, A.D.R., B.C., P.P., M.T., L.M. and C.P.R., resources, B.C., A.D.R., C.P.R. and M.R.; data curation, A.D.R., M.Y. and M.M.R.d.A.; writing—original draft preparation, A.D.R.; writing—review and editing, A.D.R., B.C., M.T., M.M.R.d.A., C.P.R. and M.R.; visualization, A.D.R., M.Y., and M.M.R.d.A.; supervision, B.C. and M.R.; project administration, B.C. and M.R.; funding acquisition, M.R. All authors have read and agreed to the published version of the manuscript. Funding: This project received financial support from the García Cugat Foundation CEU-UCH Chair of Medicine and Regenerative Surgery. Acknowledgments: The authors would like to thank the García Cugat Foundation for its funding support, the veterinary teaching hospital of the University CEU-Cardenal Herrera for its facilities support, the Interdisciplinary Laboratory of Clinical Analysis (Interlab-UMU) in Murcia for the processing of the samples and Urano Vet SL (Barcelona, Spain) for donating the Uranotest® Quattro. Conflicts of Interest: The authors declare no conflict of interest. References 1. Jacobsen, S.; Nielsen, J.V.; Kjelgaard-Hansen, M.; Toelboell, T.; Fjeldborg, J.; Halling-Thomsen, M.; Martinussen, T.; Thoefner, M.B. Acute phase response to surgery of varying intensity in horses: A preliminary study. Vet. Surg. 2009, 38, 762–769. [CrossRef] [PubMed] 2. Tvarijonaviciute, A.; García-Martínez, J.D.; Caldin, M.; Martínez-Subiela, S.; Tecles, F.; Pastor, J.; Ceron, J.J. Serum paraoxonase 1 (PON1) activity in acute pancreatitis of dogs. J. Small Anim. Pract. 2015, 56, 67–71. [CrossRef] [PubMed] 3. 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