Quem sou eu

Minha foto
A Videocirurgia e o videodiagnóstico são recursos de ponta já amplamente empregados na Medicina e que atualmente vêm ganhando espaço na Medicina Veterinária. O VideoVet tem o objetivo de reunir informações sobre a Videocirurgia Veterinária, divulgando documentos, videos e artigos sobre o assunto. Além de informações de curso, eventos, empresas e outras informações a videocirurgia, também sendo uma porta de comunicação entre interessados sobre o tema.

Pesquisar este blog

Artigos e matérias

Esta seção do VIDEOVET está destinada a publicações cientificas e matérias na área de videocirurgia aplicada Medicina Veterinária.





Postado por:
Pedro Paulo Maia Teixeira
Veterinarian
Master Veterinary Surgery
PhD Obstetrics and Animal Reproduction - FCAV / UNESP
ClinicVeterinary Surgery and Obstetrics
p_paulomt@yahoo.com.br






ABSTRACT
Background: Ventricular foreign bodies are common in some species of birds. Depending on the type of the material ingested, the bird can develop ventricular impactions. Diagnosis is made by radiography, palpation (in larger species), endoscopy and exploratory laparotomy. Treatment depends on the nature of the foreign body, and if there is impaction. Endoscopy is a less invasive treatment that can be used in some situations. This paper reports a case of a foreign body in a toucan’s ventriculus, removed by endoscopy.
Case: A young Toco Toucan (Ramphastos toco) was referred to the Veterinary Hospital “Governador Laudo Natel" of  the School of Agricultural and Veterinary Sciences “Júlio de Mesquita Filho”, São Paulo State University (HV/FCAV/UNESP) by the Environmental Police. Survey radiographs indicated a foreign body in the ventriculus. Based on the size of the foreign body, the endoscope removal was the determinate treatment. The patient was fasted for 6 hours and was anesthetized with isoflurane. The endoscope was carefully passed through the esophagus, than the proventriculus and ventriculus. This view was hampered by the large amount of fibrin in gastroesophageal mucus, making it necessary to wash it during the procedure, with 0.9% saline through the operating channel. Was observed hyperemic mucosa throughout the digestive tract and a marked hyperemia on the ventriculus surface. The foreign body was seized using Babcock forceps and gently the whole set was removed, preventing further injury to the gastrointestinal mucosa.  The foreign body was visualized and collected out of the use of the Babcock atraumatic forceps. The overall surgical time was 24 minutes. The patient recovered with no complications of anesthesia and early postoperative period, the surgical recovery was considered excellent.
Discussion: The access allowed visualizes the oral cavity, esophagus (the most difficult access, right of the midline) proventriculus and ventriculus, demonstrating the feasibility of endoscopy in these birds. Nevertheless, the use of the technique in toucans to date, only have been described for sex determination. This report demonstrates the effectiveness of endoscopy and significantly reduced risk of death due to a surgical procedure. Foreign body ingestion is a common indication for endoscopy. If the foreign body is present in the crop, it is easily removed with the help of an endoscope and graspers or by means of an ingluviotomy. If the foreign body has already traveled to the proventriculus or ventriculus, depending on the bird’s length, rigid endoscopy may still be a viable option, even if the access requires more heed and sensibility. The 30 to 45 º incline profess by [4] was conclusive to the success of the procedure, not only to avoid the risk of aspiration of gastric contents, but mostly for better access to the ventriculus, identified by the yellow greenish color of the koilin layer. The use of optics with working channel allowed safer handling of foods and foreign body seized within the gastric chamber.

Foreign body ingestion is a common indication for endoscopy. Orally approach allowed excellent visualization of the ventriculus site, demonstrating the feasibility of the endoscopy in this specie and reducing the death risk due to a surgical procedure to remove the foreign body. The external position at 30 to 45º incline was imperative for procedure success due to the aspiration risk of gastric fluids and the best access to the ventriculus. The use of the endoscope equipment with an operating channel allowed safer manipulation of the foods and the foreign body seized in the gastric chamber.

Keywords: endoscopic, removal, Aglandular stomach, toucan.



Postado por:
Pedro Paulo Maia Teixeira
Veterinarian
Master Veterinary Surgery
PhD Obstetrics and Animal Reproduction - FCAV / UNESP
ClinicVeterinary Surgery and Obstetrics
p_paulomt@yahoo.com.br




Reportagem completa em:  http://www.revistacaesegatos.com.br/pub/curuca/index2/








Postado por:


Pedro Paulo Maia Teixeira
Veterinarian
Master of Veterinary Surgery
PhD in Obstetrics and Animal Reproduction - FCAV / UNESP
ClinicVeterinary Surgery and Obstetrics
 p_paulomt@yahoo.com.br





ABSTRACT
Background: Intraperitoneal adhesions in equines, especially following exploratory celiotomy. Adhesiolysis is the treatment of choice for patients presenting postsurgical adhesions. Laparoscopic approach for adhesiolysis presents several advantageous aspects in human patients. The aim of the current study was to report a case of successful laparoscopic adhesiolysis in a mini pony horse.
Case: A male Shetland Pony, weighing 140 kg, was admitted under complaint of right hind limb trauma and treated surgically for metatarsal fracture reduction. The patient has also had intermittent episodes of colic and was always treated clinically without major complications. The pony had no history of previous abdominal surgery and no episodes of acute abdomen were seen during hospital stay. Three months following ostheosynthesis, an exploratory laparoscopic approach was carried out to assess the possible cause or consequences of the episodes of acute abdomen. The patient was submitted to general anesthesia, positioned in dorsal recumbency and the abdomen was clipped and aseptically prepared for surgery. During the laparoscopic inspection, there were adhesions involving the ventral abdominal wall and a ventral mesogastric segment of duodenum. Laparoscopic adhesiolysis was performed using a two-port approach, by gently breaking the adhesion bands using meticulous traction with a 10-mm laparoscopic atraumatic Babcock forceps. Afterwards, the intestinal loop was rinsed with heparin sodium solution diluted in normal saline. Total surgical time was 58 min. the patient was able to recover without complications. In the early postoperative period, the surgical recovery was considered excellent. No apparent adhesion involving the previously affected intestinal loop was found during the ultrasound exam following 15 days of surgery. Furthermore, the surgical wounds had healed completely, with no complications.
Discussion: In the current case report, the primary cause of the acute abdomen episodes was not determined since the patient had never undergone abdominal surgery. It was hypothesized that an acute inflammation of the duodenal loop that was involved by the adhesion bands may have triggered the adhesiogenesis. Laparoscopy was efficient and presented a short operative time, due to magnification of image and adequate observation of structures surrounded by adhesion bands. Although the use of Babcock forceps is not usually recommended for adhesiolysis in the current literature, it was both effective in manipulating the bowel and performing the adhesiolysis. The heparin solution diluted in normal saline was effective in preventing the recurrence of new adhesions, which was evidenced by ultrasonography following 15 days. The laparoscopic approach usually minimizes the new formation of adhesions as trauma to the peritoneal surfaces is minimized by the use of delicate instruments, as observed in the current study. In addition, laparoscopy reduces the possibility of contact among the peritoneal surfaces and foreign bodies, such as gauze, glove powder and room air particles. Moreover, it maintains the abdominal surfaces in adequate humidity environment.
Keywords: endosurgery, equines, intraperitoneal adhesions.
Descritores: videocirurgia, equinos, aderências intraperitoneais.

INTRODUCTION
Peritoneal adhesions are common complications in horses following exploratory laparotomy, especially regarding small intestine secondary disorders [4,5,7]. Adhesiolysis is the treatment of choice. Moreover, the laparoscopic approach has been reported to present several beneficial effects over the conventional approaches for adhesiolysis in human patients [6]. However, little information is known about the use of minimally invasive endoscopic techniques for adhesiolysis in equine species so far.
Within such context, the aim of this case report was to describe a successful case of minimally invasive laparoscopic adhesiolysis in a mini pony horse.

CASE REPORT
The patient, a male Shetland Pony, weighing 140 kg, was admitted at the Large Animal Surgery Division of the Veterinary Teaching Hospital “Governador Laudo Natel" of  the School of Agricultural and Veterinary Sciences “Júlio de Mesquita Filho”, São Paulo State University (HV/FCAV/UNESP), under complaint of right hind limb trauma, and submitted to metatarsal fracture ostheosynthesis. During the anamnesis, it was reported that the patient had been presenting intermittent episodes of acute abdomen and had always been treated clinically without complications. Additionally, there was no record of previous abdominal surgery and no episodes of acute abdominal pain were observed during the hospitalization period.
A diagnostic laparoscopic approach was suggested in order to aid in the diagnosis of the recurrent acute abdomen episodes and possible surgical treatment. The patient was fasted for 12 hours and premedicated using xylazine hydrochloride1 (0.8 mg/kg, IM). Following a 15-min interval, anesthesia was induced using guaifenesin2 (100 mg/kg, IV) and ketamine chloride3 (1 mg/kg, IV) and maintained using halothane4 vaporized in oxygen (100%), to 2 CAM, following tracheal intubation. The abdomens was ventrally clipped and aseptically prepared for surgery. Local anesthetic was injected on the desired laparoscopic portal sites, using 4 mL of lidocaine hydrochloride5.
Patient was placed under supine simple position, without head-down tilt. Two 12-mm trocars were used for abdominal access of a 10-mm, 30º angled laparoscope and a 10-mm atraumatic laparoscopic Babcock forceps. The first port was inserted through the umbilical scar using the open mini-laparotomy technique, which was used for the insertion of the laparoscope. Subsequently, the pneumoperitoneum with sterilized CO2 was created, using intraperitoneal pressure of 8 mmHg and 5 L/min of insufflation flow rate. The second trocar was introduced in the midline, 10 cm caudally to the first port, under laparoscopic assistance.
During general laparoscopic inspection, broad fibrous bands were observed on the ventral mesogastric area, connecting a segment of the duodenum to the ventral abdominal wall (Figure 1A). Adhesiolysis was then performed employing a slight and gentle traction of the adhesion bands proximally to the abdominal wall, using the atraumatic Babcock forceps (Figure 1B). Extreme care was taken not to traumatize the intestinal loop and its mesenteric vessels. No intestinal petechial or bleeding were noted following adhesiolysis (Figure 2). The intestinal loop was finally rinsed using sodium heparin in normal saline (5 IU/mL), in order to prevent adhesion recurrence.

Figure 1. (A) Laparoscopic view of fibrous adhesions involving the abdominal wall (ab) and a loop of the duodenum (du). (B) Blunt adhesiolysis being carried out with a 10-mm atraumatic Babcock forceps.


Figure 2. (A) Laparoscopic view of the abdominal wall (arrow) following adhesiolysis. (B) Duodenum (arrow) free from adhesion bands following blunt dissection. No bleeding was noted.

The pneumoperitoneum was completely drained and the trocars sequentially withdrawn from the abdominal wall. The synthesis of the muscular layer was carried out using an interrupted cross mattress pattern (Sultan pattern), followed by synthesis of the skin with an interrupted cushion pattern (Wolf pattern).
The overall surgical time was 58 minutes. The patient recovered with no complications of anesthesia. In the early postoperative period, the surgical recovery was considered excellent, as only mild pain (< 30) was diagnosed using the visual analogue scale (VAS) for pain assessment (ranging from 0, no pain, to 100, worst pain possible). The patient received flunixin meglumine7 (1.1 mg/kg), ceftiofur8 (2.2 mg/kg) and benzilpenicillin9 (40,000 IU) on the surgery day. The antibiotics were continued for 6 days of coverage.
No adhesion recurrence was noted during the following 15 days after the procedure during the ultrasound scanning. Furthermore, the surgical wounds healed completely, with no complications at any time point.

DISCUSSION
Intraperitoneal adhesions may involve any intestinal segment. Although intestinal anastomosis or enterotomy sites are the main adhesiogenic sites, the hole intestine can be affected [4]. In the current case report, the primary cause was not determined preoperatively since patient had never undergone abdominal surgical procedures. It is believed that an acute inflammation of the intestinal loop involved may have resulted in the formation of adhesions. The formation of adhesions without prior surgical trauma is common in women with uterine inflammatory or degenerative diseases, such as endometriosis [2]. In the current clinical case, the heparin solution diluted in normal saline was effective in preventing the formation of new adhesions following adhesiolysis, as shown during the ultrasonography [1,4,8].
Laparoscopic approach was efficient for the accomplishment of adhesiolysis in a short surgical time. Although Metzenbaum scissors, monopolar dissectors, bipolar coagulation forceps, lasers and ultrasonic shears have been both indicated and used for adhesiolysis in human patients and animals, gentle traction of the adhesion bands using an atraumatic Babcock forceps was effective in both handling the intestinal loop and performing adhesiolysis. This kind of meticulous approach minimizes the formation of new adhesions and the trauma to peritoneal surfaces compared to open surgery. In addition, the contact of intraperitoneal surfaces with foreign bodies is reduced and the tissues are maintained in a closely physiologic humidity and atmosphere-free environment [3]. In the current case report, there was no recurrence of adhesions, no considerable painful and excellent postoperative recovery. Such facts highlight the suitability of the laparoscopic approach in the equine surgical routine.

SOURCES AND MANUFACTURERS
1Rompun® Bayer. SP, Brazil.
2Guaifenesin®, Henrifarma. SP, Brazil.
3Dopalen®, Vetbrands. SP, Brazil.
4Halothane®, Cristalia. SP, Brazil.
5Lidovet®, Bravet, RJ, Brazil.
6Xcel® Ethicon, SP, Brazil.
7Banamine® Injection, Schering-Plough. SP, Brazil.
8Bioxell, Vallé. MG, Brazil.
9Benzapen®. Teuto. SP, Brazil.

Declaration of interest. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

REFERENCES
1      Hillyer M.H. & Wright C.J. 1997. Peritonitis in the horse.  Equine Veterinary Education. 9(3): 136-142.
2      Howard F.M. 2011. Surgical treatment of endometriosis. Obstetics and Gynecology Clinics of North America, 38(4): 677-686.
3      Kavic S.M., Kavic S.M.2002. Adhesions and Adhesiolysis: The Role of Laparoscopy. Journal of the Society of Laparoendscopic Surgeons, (6) 99-109.
4      Kelmer G. 2009. Update on recent advances in equine abdominal surgery. The Veterinary Clinics, Equine Practice, 25:271-282.
5      Leira P.A.T., Martins P., Dércoli T.E., Furini T.F., Berlingieri M.A. 2012. Adesiólise peritoneal trans-cirúrgica seguida de lavagem peritoneal e infusão de solução de carboximetilcelulose 1% (SCMC 1%) através de tubo de silicone implantado na cavidade abdominal. In: XIII Conferência Anual da ABRAVEQ – 2012. (Campinas, SP). p. 984.
6      Li M.Z, Lian L., Xiao L., Wu W., He Y., Song X. 2012. Laparoscopic versus open adhesiolysis in patients with adhesive small bowel obstruction: a systematic review and meta-analysis. American Journal of Surgery. [in Press]
7      Pagliosa G.M., Alves G.E.S. 2004. Fatorespredisponentes das complicaçõesincisionais de laparotomiasmedianasemequinos. Ciência Rural, 34(5):1655-1659.
8      Parsak C.K., SatarS., Akcam T., SatarD., Sunguret I. 2007. Effectiveness of treatment to prevent adhesions after abdominal surgery: an experimental evaluation in rats. Advances in Therapy, 24(4):796-802.




Pedro Paulo Maia Teixeira
Veterinarian
Master of Veterinary Surgery
PhD in Obstetrics and Animal Reproduction - FCAV / UNESP
ClinicVeterinary Surgery and Obstetrics
Teixeira et al., 2011 - p_paulomt@yahoo.com.br









aUniversidade Estadual Paulista (FCAV/UNESP), Jaboticabal – São Paulo – Brazil.
bUniversidade Federal Rural da Amazônia (CPCOP/ISPA/UFRA), Belém – Pará – Brazil.
* Corresponding author at: Departamento de Medicina Veterinária Preventiva e Reprodução Animal. Universidade Estadual Paulista (FCAV/UNESP), Via de acesso Prof. Paulo Donato Castellane - Vila Industrial CEP: 14884900. Jaboticabal – São Paulo – Brazil.
E-mail address: p_paulomt@yahoo.com.br (P.P.M. Teixeira)




ABSTRACT
Gross and microscopic evaluations of the ovaries of sheep subjected to successive ovum pick-up were performed with the objective of identifying if there was interference in oocyte production and morphology of gonads. Gross evaluation of the internal genital tract was also performed. Eighteen ewes of the Santa Inês breed were randomly distributed into three experimental groups of six animals each; G0, G1 and G9 with 0, 1 and 9 repetitions, respectively. Estrus synchronization was achieved with a short protocol using MAP followed by single dose stimulation with 80 mg of FSHp and 300 IU of eCG (IM). Laparoscopic ovum pick-up was performed 36 hours later, with 7-day intervals. The number of visualized follicles, aspirated follicles and oocytes recovery was recorded. After the last intervention, ovariectomy was performed for evaluation of gross and microscopical appearance and existence of lesions caused by follicular puncture, which were classified as absent (0), mild (1), moderate (2) and severe (3). The number of visualized follicles, aspirated follicles and oocytes recovery were 13.24±2.0, 11.27±3.03 and 5.79±2.3, respectively, with a recovery rate of 51.69%. No statistical difference was found between the nine sessions (p>0.05). We concluded that nine sessions of superovulation and ovum pick-up procedures did not cause ovarian lesions and did not interfere with the production of follicles in ewes of the Santa Inês breed.
Keywords: follicular aspiration, histology, ovary, ewes, laparoscopy.

1. Introduction
The sheep industry has been experiencing a cycle of global growth, especially in developing countries as Brazil (Fonseca, 2005; Simplício et al., 2007). In this context, reproduction biotechnologies are highlighted since they are strong allies in improvement of reproductive efficiency and productivity of herds, ensuring economical viability to be more attractive (Traldi, 2006).
Ovum pick-up, together with the in vitro production of embryos (IVP), a technique of great relevance which was initially used in production of descendents of high genetic value from animals with acquired problems involving reproductive capacity (Bols et al., 1996; Schernthaner et al., 1999) or that would not respond to conventional superovulation treatments (Looney et al., 1994). However, this technique has an embryo production potential and number of gestations over a period of time inferior to that obtained by other techniques (Bousquet et al, 1999). Its application can be extended to prepubertal females (Fry et al., 1998, Majerus et al., 1999), acyclic or in seasonal anestrus, gestating and also, submitted or not to previous hyperstimulation treatments (Berrnardi, 2005), as well as to animals with temporary or irreversible infertility (Baldassare et al., 1996; Kühholzer et al., 1997). This technique can also be applied to other biotechnologies as cloning and transgenic techniques (Basso et al., 2008).
The high efficiency of this technique is related to the possibility of its repeated use with short intervals. It is noteworthy that the majority of studies on identification of viability of repeated ovum pick-up, considering number of oocytes harvested and ovarian lesions promoted by the technique, have been restricted to bovines (Viana et al., 2003; Pieterse et al., 1991; Becker et al., 1996 and Santl et al., 1998). Nevertheless, it is imperative to obtain information on occurrence of histological ovarian alterations and oocyte recovery rate after successive ovum pick-up procedures by laparoscopy in ewes.
Thus, the objective of this study was to perform gross and microscopic evaluations of the ovaries of ewes subjected to successive ovum pick-up procedures and evaluate if there was interference with oocyte production and morphology of gonads, as well as to perform macroscopic observation of the internal genital tract.

2. Material and methods
2.1. Location and characterization of the area
The study was performed at Faculdade de Ciências Agrárias e Veterinárias of Universidade Estadual Paulista “Julio de Mesquita Filho”, located in the municipality of Jaboticabal (Latitude 21º15'17"S and longitude 48º19'20" W of Greenwich, at an altitude of 605 meters), São Paulo state - Brazil.
The present study was conducted following the approval of the Animal Ethics and Welfare Committee of the School of Agrarian and Veterinary Sciences of the São Paulo State University (protocol No 025922-08). The principles of Ethics of the European Commission for experiments involving animals (Directive 83/609EEC) were followed also.

2.2. Animals and treatments
Eighteen adult pluripara ewes of the Santa Ines breed with an average age of 2 years, mean body condition score of 2.9±0.2 (score 1-5, from Jefferies, 1961) and considered eligible after clinical examination (hemogram, total protein, fibrinogen and parasitic control) and ultrasound evaluation of the organs of the reproductive system, were selected for this study. They were kept under intensive system of management, receiving corn silage and balanced feed twice a day, and had access to water and mineral salt ad libitum.
Animals were randomly distributed into three experimental groups (n=6): control (G0), not subjected to the ovum pick-up process; group 1 (G1), subjected to one ovum pick-up session; group 9 (G9), subjected to nine ovum pick-up sessions with 7-day intervals.
For estrus synchronization, animals were treated with intravaginal sponges impregnated with 60 mg of medroxyprogesterone acetate (MAP - Progespon®, Schering-Plough. Brazil) starting at a random day of the estrus cycle (Day 0) and for a total of six days. On Day 5, 37.5 µg of D-cloprostenol (Sincrocio® Orofino S.A, Brazil) and 300 IU of eCG (Novormon®, Schering-Plough, Brazil) were administered intramuscularly.
The ovarian stimulation was performed 12 hours after detection of estrus (confirmed with teaser) and consisted of administration of 80 mg of FSHp (Folltropinâ, Schering-Plough, Brazil) and 300 IU of eCG, and after 36 hours the ovum pick-up procedures were performed by laparoscopy. For the subsequent aspirations, only the ovary stimulation was performed.

2.3. Animal preparation and anesthesia protocol
After 36 hours of food and water fasting, animals were subjected to anesthesia, which was obtained by administration of 0.5 mg/kg of diazepam (IM, Diazepamâ Cristalia., Brazil) and 2 mg/kg of tramadol (IM, Tramal® Cristalia. Brazil) as premedication; subsequently, induction was performed using propofol (IV, Propofol® Cristalia. Brazil) in a dose of 6 mg/kg and anesthesia was maintained with continuous infusion of propofol at a rate of 0.5 mg/kg/min (IV) as well as 1mg/kg bolus and 1mg/kg/min infusion of lidocaine hydrochloride (IV, Lidovetâ, Bravet, Brasil).
Animals underwent tracheal intubation using 8 mm endotracheal tubes with inflatable cuffs. This procedure had the objective of avoiding aspiration of ruminal content and allowing the supply of humidified medical oxygen, under assisted ventilation.
In the surgical field, hair clipping and skin antisepsis with iodine were performed on the abdominal region cranial to the udder, and subsequently, local infiltration anesthesia was performed with 0.4 mL of lidocaine hydrochloride, being 0.2 mL for a subcutaneous administration and 0.2 mL for intramuscular administration.

2.4. Laparoscopy for ovum pick-up
Females were placed in Trendelenburg position. The cutaneous incision was performed with a scalpel, 10 to 15 cm cranial to the udder and 5 cm to the right of the midline to facilitate the introduction of a trocar with insufflation valves (5 mm). Pneumoperinoneum was established with CO2, maintaining intra-abdominal pressure (IAP) at a range of 5 to 8 mmHg and a flow rate of 5 L/min. Subsequently, with video-assisted guidance, the second trocar of 10 mm in diameter was inserted symmetrically to the first and the third of 5 mm was placed on the midline, 20 cm cranial to the udder.
Next, the laparoscope was transferred to the third trocar, obtaining visual field. Afterwards, the atraumatic forceps (Babcock) were introduced through the first and second trocars, which allowed individualization and fixation of ovaries on the mesovarium, always avoiding injuries to any structure (Figure 1A).
Number of follicles measuring 2 to 8 mm was recorded. Next, the aspiration needle was introduced into the cavity, close to the ovary. Puncture was performed by moving the ovaries in different positions with the manipulation atraumatic forceps. The needle was initially placed in parallel position to the ovarian surface, which allowed perforation of the follicles in the extremities, and when that was not possible, the puncture was performed perpendicularly (Figure 1).
Figure 1: Image of the abdominal cavity of a sheep with 10x magnification, note:  fixation of the ovary (circle) with atraumatic forceps (1), and presence of follicles (arrow) (A); needle position (2) for ovum pick-up (B), and aspiration of the follicles (C and D).

Once inserted in the follicle, the needle was carefully moved to ensure that all the content was aspirated. Vacuum pressure was adjusted for a maximum of 50 mmHg.
A single lumen (same internal diameter) aspiration system was used, which was composed of a 16 G needle with a short bevel connected to a 50 cm length cannula, connected to a silicone cork (Handle Cook®, Ribeirão Preto-SP, Brazil) which were connected to the collection tube (50 mL). Vacuum was produced by an aspiration pump (Nevoni, Brazil) connected to a silicone hose also connected to the collection tube. Previous to the oocytes aspiration, a washout was performed with the harvesting medium, with approximately 2 mL of the medium left for the end of the procedure to receive the oocytes.
Ovaries were washed with 10 mL of 0.9% NaCl solution to remove surface clots, minimizing formation of adherences. Skin suture was performed using horizontal "U-shaped" suture pattern. Next, the surgical wound was cleaned with povidone iodine and a repellent/healing ointment was used around the wound.
Females were placed in a clean and calm environment and were observed until standing in all four legs. On the subsequent interventions, incisions were made laterally to the first.

2.5. Postoperative evaluation
All animals were weighed weekly and after the aspirations, were evaluated on the first 24 hours for pain manifestation taking into account the curvature of the back, difficulty moving and decreased appetite (Mellor and Stafford, 2004). Grades were established using these parameters allowing for classification of this symptom, and for each of them a score of 0 to 2 was determined (0 – absent; 1 – moderate; 2 – intense) characterizing the intensity of painful discomfort. The sum of the values referring to each of the parameters was used as indicative of manifestation of pain, which could reach a maximum grade of 6 (0 to 2 – absent to mild; 2 to 4 – mild to moderate and 4 to 6 – moderate to intense).

2.6. Analysis of visualized follicles, aspirated follicles and oocytes recovery
The number of visualized follicles, aspirated follicles and oocytes recovery was recorded and the averages of these values were analyzed comparatively between sessions.
In a laboratory, the aspirated liquid was carefully deposited in petri dishes and taken for observation in stereomicroscope with 40X magnification. Once located, oocytes were transferred to another dish containing 300 to 500 mL of washing medium and were classified according to their quality in agreement with Hewitt and England (1997).  

2.7. Gross evaluation of the ovaries, oviducts and uterus
These structures were observed by laparoscopy and evaluated macroscopically to confirm the presence or absence of adherences and fibrosis, especially on the ovaries and other abdominal structures.
Seven days after the last aspiration, ovariectomy was performed by laparoscopy for gross and microscopic evaluation of the ovaries. Macroscopically, the presence of scarring lesions and consistency of the gonad were evaluated and quantified in a score of 0 to 2 (0 - characteristic consistency; 1 - moderate hardening and 2 - fibrous tissue consistency).

2.8. Histological processing and evaluation of ovaries
Histological sections of 7 mm thickness were obtained from four fragments per ovary. Slides were mounted and stained with hematoxylin-eosin and Masson’s trichrome.
The injury severity was analyzed from the formation of scar tissue, inflammatory cell infiltration and presence of luteal tissue scattered in the ovarian stroma, according to Viana et al. (2003), and hemorrhagic follicles or partially luteinized according to Gibbons et al. (1994).
Slides were analyzed per fields and a score of the inflammatory process was established and classified as: negative (0), absent or low occurrence (1), medium occurrence (2) and high occurrence (3).
2.9. Statistical analysis
Data obtained from visualized follicles, aspirated follicles, quantity and quality of oocytes harvested per session, data referring to gross analysis, and weekly body weight were expressed in averages ± standard deviation and were submitted to analysis of variance (ANOVA) and Tukey’s test (P<0.05). Histological analysis and score of pain were submitted to analysis of chi-square, using GraphPad Prisma 4 statistics program.

3. Results
3.1. Trans and postoperative evaluations
The 36h fasting, besides avoiding reflux of ruminal contents, facilitated, together with a pneumoperitoneum of 5 mmHg and 5L/min, an adequate visualization of the abdominal cavity which had its image transmitted to video with 10X magnification, allowing for a surgical time of 26.75 ± 9.6 min.
The bleeding observed from follicular punctures was incipient and the washout of the ovaries promoted removal of clots, avoiding formation of adherences in all animals.
The weight of the animals did not vary along evaluations (p>0.05), the ewes exhibited good anesthetic recovery and they would stand in four legs without difficulties in average within 20 minutes. At painful discomfort evaluation 90% of animals presented score zero and 10% score 1.
.  
3.2. Ovarian stimulation and oocyte production
Most ewes (72,2%) showed estrus 36 hours after removal of progestogens (5,5% at 12 hours, 11,1% at 24 hours, 72,2% at 36 hours and 11,1% at 42 hours). For the others sessions of G9 there was no necessity of estrus observation, although follicular waves were estimulated, which were observed through laparoscopic view at ovarian aspiration moment.   
The average number of visualized follicles, aspirated follicles and oocytes recovery was 13.24±2.0, 11.27±3.03 and 5.79±2.3, respectively, with a recovery rate of 51.69% (aspirated follicles/oocytes recovery), and no variation was observed between the sessions (p>0.05), as seen in figure 2. In some sessions, animals exhibited corpora lutea (CL), with 0.46±0.84 CL/animal/session.
Figure 2: Graphical representation of number of visualized follicles (VF), aspirated follicles (AF) and recovered oocytes (RO) over time in weeks. No significant difference was found between sessions (p>0.05).


There was also no difference in oocyte quality along the observations (p>0.05) and the frequency distribution of grade classification of oocytes can be seen in figure 3.
Figure 3: Graphical representation of qualitative classification of oocytes during the nine weeks of the study. No significant difference was found between sessions (p>0.05).

3.3. Gross and microscopic evaluation of the ovaries
At laparoscopy, no lesions were observed on the internal reproductive system (uterine body, horns and oviduct) and similarly, no lesions were seen in the ovaries during the surgical procedures and after ovariectomy (score zero).
No significant lesions were found in the histological evaluation comparing G0, G1 and G9 (p>0.05). Only two animals of G1 exhibited small areas of scarring tissue and mononuclear infiltrate, possibly on the locations of follicular punctures (score zero for all groups (G0 (control), G1 and G9), can be seen in figure 4.
Figure 4: Photomicrograph of ovary sections of sheep subjected to ovum pick-up, stained with hematoxylin and eosin (HE) and Masson's trichrome (MT). Tissue integrity can be seen in all images; images A, B, C and D correspond to G1, images E, F, G and H to G9. Note unaltered germinal epithelium on the cortical region (arrows) and ovarian follicles in various stages of development, primordial follicles (1), primary (2), secondary (3) and tertiary (4) preovulatory follicles.


4. Discussion
A surgical time was similar to the 35 minutes described by Cordeiro (2006) and 18 to 20 minutes by Wieczorek et al. (2010) in ovum pick-up procedures in goats and sheep, respectively. Duarte et al. (2009) described 23 minutes for liver biopsies in rams and Bleul et al. (2005) described 120 to 150 minutes for ovariectomies in bovines. With these citations we can suggest that the laparoscopic technique favors the execution of different procedures with very convenient operative time, particularly in small ruminants. However, it should be noted that the surgeon skills are important for obtainment of this result, fact that was also described by those authors.
The surgical technique used allowed for excellent postoperative recovery of animals, which corroborates with the fact that no painful discomfort was observed, which was evaluated by loss of appetite, difficulty moving and curvature of the back, as described by Mellor & Stafford (2004). Curiously, this type of observation considered important was not mentioned as a specific criterion by any of the authors reported above; probably due to obtainment of favorable recovery. Therefore, this condition allows one to infer about the superiority of this surgical procedure over conventional ovum pick-up methods.
The interval of seven days used between surgical interventions in animals of G9 was adequate and sufficient, which allowed the obtainment of oocytes without compromising the clinical condition of the animals; which was evidenced by maintenance of body condition score and behavior of animals. Stangl et al. (1999) did not perform clinical and behavioral observations, but reported that the weekly interval was sufficient and no alteration was seen in oocyte production in 10 repetitions of laparoscopic ovum pick-up in sheep.
The synchronization and ovarian stimulation protocols were correct for most of the animals showed estrus 36 hours after removal of MAP implants. Thereby, ovarian stimulation using eCG and FSHp induced a new follicular wave, exhibiting a satisfactory number of follicles/sheep. Even though in some cases corpora lutea were seen, which result from follicles that ovulated before the aspiration, these losses were not significant (p>0.05).
The average number of follicles/sheep founded in this study was close to the values found by Baldassare and Karatzas (2004) of 13.4 follicles/sheep and Basso et al. (2008) of 14.3 follicles/sheep. The oocyte recovery rate is within the interval of 40 to 90% (Cognié et al., 2004; Morton et al., 2005; Rodriguez et al., 2006; Cox e Alfaro, 2007; Gibbons et al., 2007). 
However, Abdullah et al. (2008), when comparing the best time for ovarian stimulation in goats, observed that ovum pick-up was performed 60 and 72 hours after stimulation using FSH and hCG,  showed superior results than performed 36 hours after stimulation, both in quantity and quality of oocytes recovery.
In several sessions of the present study, follicles were close to the minimum size required for aspiration (2 mm); therefore, we could establish a hypothesis that such losses can be resolved by studying other hormonal protocols with the aim of promoting better ovarian stimulation, which will result in more viable follicles (from 2 to 8 mm) at the moment of ovum pick-up.
The absence of variation in quantity and quality of aspirated oocytes throughout the successive sessions of ovum pick-up is a favorable factor for animal production because the animals will have a higher reproductive performance when using LOPU for IVP. Cordeiro (2006) reported reduction in oocytes production, both quantitative and qualitatively, along consecutive sessions when studying LOPU in goats. Even though it was not the object of this study, authors as Roy et al. (1999), Drion et al. (2001) and De Ruigh et al. (2000) mentioned the hypothesis of formation of antibodies, such as anti-eCG and anti-FSH, after prolonged use of exogenous hormones, which would compromise the obtainment of oocytes. However, our results stand in contrast to those mentioned by these authors, which allows the questioning of these reports and we propose the development of further research to clarify this fact.
The absence of gross lesions (adherences, fibroses, and others) was probably due to the fact that laparoscopy is a minimally invasive procedure, not perforating the other organs of the reproductive system, as happens when using ultrasound-guided ovum pick-up in bovines (Viana et al, 2003). Histological evaluation similarly did not reveal presence of lesions in the ovaries and we consider this is due to punctures being performed in specific locations, since ovarian follicles are located on the surface of the ovary, on the cortical region.
In ultrasound-guided ovum pick-up there is a relation between number of aspirated follicles and the occurrence of ovary adherences; however, there was no reduction in number of oocytes recovery (Viana et al., 2003). Neither formation of fibrous cords around the organ, nor the presence of hemorrhagic or partially luteinized follicles were seen, differently than what has been described for cows aspirated by ultrasound-guided (Gibbons et al., 1994).
None of those findings were found in the present study, which is justified by the fact that ultrasound-guided OPU is a more traumatic procedure since there is no way of puncturing only the surface of the ovarian follicle, as well as due to manual manipulation of the ovary which can cause lesions on the surface of the gonad.
There is no information regarding ovarian lesions caused by follicle aspirations in small ruminants. However, Cordeiro (2006) reported the occurrence of adherences in reproductive organs of 15% of the goats which underwent ovum pick-up, even with the use of heparinized solution, and Stangl et al. (1999) reported unilateral and bilateral adherences on ovaries of sheep subjected to ten sessions of ovum pick-up with weekly intervals.
Such consequences did not occur in this study even without the use of substances such as heparinized solutions, used by Cordeiro (2006) and Baldassare and Karatzas (2004), and carboxymethilcellulose used by Ewoldt etal. (2004). This can be justified by the care taken on genital tract manipulation and the removal of clots from the ovarian surface using 0.9% NaCl solution.
It is possible that the absence of histological alterations in the ovaries results from punctures being performed only on the surface of the ovarian follicle as well as from the physiological recovery capacity of the ovary. Physiologically, the corpus hemorrhagicum, porterior corpus luteum from the ovulated follicle, is totally eliminated by apoptosis (Silva et al., 2010). This mechanism occurs by action of immunological cells (macrophages and T lymphocytes) which produce cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor (TNF-α) and interleukin- β (ILβ-1), which can modify the syntheses of progesterone and prostaglandins by luteum cells, causing direct cytotoxic effects in which the dead luteum cells are phagocyted by macrophages (Miyamoto, 1996; Pate, 1996). This phenomenum can also occur in the punctured follicles, so there is no scarring tissue seven days after the ovum pick-up procedure.


5. Conclusions
The repetition process of ovum pick-up did not cause painful discomfort to the animals which could limit the technique; the interval between ovum pick-up sessions allowed adequate recovery time for the animals.
Also, the repetition of ovum pick-up sessions did not alter the number of visualized and aspirated follicles, or the quantity of oocytes recovery. Probably the quality of oocytes was not affected.
Besides, the surgical sequences for the ovum pick-up procedures did not cause macro or microscopic lesions in any of the structures evaluated, particularly the ovaries. Therefore, consecutive ovum pick-up procedures in nine weekly sessions were feasible and show that the technique has a great potential for assisted reproduction optimization in this animal species.

Acknowledgements
The authors gratefully thank FAPESP for support in the study.

Referências
Abdullah R.B., Liow S.L.; Rahman A.N.M.A., Chan W.K.; Wan-Khadijah W.E.S.C. N.G. 2008. Prolonging the interval from ovarian hyperstimulation to laparoscopic ovum pick-up improves oocyte yield, quality, and developmental competence in goats. Theriogenology, 70 765–771.
Baldassare H., Karatzas C.N., 2004. Advances assited reproduction technologies (ART) in goats. Anim Reprod Sci,  82 (82) 255-266.
Basso A.C., Martins J.F.P., Ferreira, C.R., Ereno A., Tannura, J.,Tabet A., Figueiredo C.L., De Oliveira P.C., Pontes J.H.F. 2008. Biotecnologia da Reprodução na Espécie Ovina: Produção in vitro de Embriões Ovinos: Aspectos da Técnica de Aspiração Folicular e do Tratamento Hormonal de Doadoras. O Embrião, 38 (10) 8-11.
Becker F., Kanitz W., Nürnberg G., Kurth J., Spitschak M. 1996. Comparison of repeated transvaginal ovum pick up in heifers by ultrasonographic and endoscopic instruments. Theriogenology, 46 999-1007.
Bernardi, M.L. 2005. In vitro production of ovine embryos, Acta Sci Vet, 33(1) 1- 16.
Bleul U., Hollenstein K., Kähn W. 2005. Laparoscopic ovariectomy in standing cows. Anim Reprod Scie, 90 193–200.
Bols P.E.J., Van Soom A., Vanroose G., De Kruif A. 1996. Transvaginal ovum pick-up in infertile Belgian Blue donor cows: preliminary results. Theriogenology, 45 359.
Bousquet D., Twagiramungu H., Morin N., Brisson C., Carboneau G., Durocher J. 1999. In vitro embryo production in the cow: an effective alternative to the convencional embryo production approach. Theriogenology, 51 59-70.
Cordeiro M.F. Avaliação da laparoscopia na aspiração folicular em fêmeas caprinas pré-púberes e adultas com ou sem estimulação ovariana hormonal. 59f. Tese (Doutorado) – Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista, Jaboticabal. 2006.
Cognié Y., Poulin N., Locatelli Y. 2004. State of the art production, conservation and transfer of in vitro produced embryos in small ruminants. Reprod Fert Develop, 16, 437-445.
Cox J.F., Alfaro V. 2007. In vitro fertilization and development of OPU derived goat and sheep oocytes. Reprod Domes Anim, 42 83-87.
De Ruigh L., Mullaart E., Van De Wagtendonk, Leeuw A.M. 2000. The effect of FSH stimulation prior to ovum pick-up on oocyte and embryo yield. Theriogenology, 53 (1) 429.
Drion P.V., Furtoss V., Baril G., Manfredi E., Bouvier F., Pougnard J.L., Bernelas D., Caugnon P., Macnamara E.M., Remy B., Sulon J., Beckers J.F., Bodin L., Lebouef B. 2001. Four year of induction/synchronization of estrus in dairy goats: effect on the evolution of eCG biding rate in relation with parameters of reproduction. Reprod Nutr Dev, 41 401-412.
Duarte A.L.L., Cattelan J.W., Bezerra M.B., Vicente W.R.R., Cordeiro M.F. 2009. Biópsia hepática com agulha tru-cut guiada por videolaparoscopia em caprinos. Arq Bras Med Vet Zootec, 61 (1) 12-19.
Ewoldt J.M., Anderson D.E., Hardy J., Weisbrode S.E. 2004. Evaluation of a Sheep Laparoscopic Uterine Trauma Model and Repeat Laparoscopy for Evaluation of Adhesion Formation and Prevention with Sodium Carboxymethylcellulose. Vet Surg, 33 668-672.
Fonseca JF. 2005. Estratégias para o controle do ciclo estral e superovulação em ovinos e caprinos. In: Congresso Brasileiro de Reprodução Animal, 16, 2005, Anais... CBRA.
Fry R.C., Simpson T.L., Squires T.J. 1998. Ultrasonically guided transvaginal oocyte recovery from calves treated with or without GnRH. Theriogenology, 49 1077-1082.
Gibbons J.R., Beal W.E., Krisher R.L., Faber E.G., Pearson R.E., Gwazdauskas F.C. 1994. Effect of once versus twice-weekly transvaginal follicular aspiration on bovine oocyte recovery and embryo development. Theriogenology, 42 405-419.
Gibbons A., Pereyra Bonnet F., Cueto M.  2007. A procedure for maximizing oocyte harvest for in vitro embryo production in small ruminants. Reprod Dom Anim, 42 423-426.
Hewitt D. A., England G. C. W. 1997. Effect of preovulatory endocrine events upon maturation of oocytes of domestic bitches. J Reprod Ferty, 83-91. Supplement 51.
Jefferies, B. C. Body condition scoring and its use in management. Tasmanian J Agricult, v. 32, p. 19-21, 1961.
Kühholzer B., Müller S., Treuer A., Seregi J., Besenfelder U., Brem G. 1997. Repeated endoscopic ovum pick-up in hormonally untreated ewes: a new technique. Theriogenology, 48 545-550.
Looney C.R., Lindsey B.R., Gonseth C.L., Johnson D.L. 1994. Commercial aspects of oocyte retrieval and in vitro fertilization (IVF) for embryo production in problem cows. Theriogenology, 41 67-72.
Majerus V., De Roove R., Etienne D., Kaidi S., Massip A., Dessy F., Donnay I. 1999. Embryo production by ovum pick up in unstimulated calves before and after puberty. Theriogenology, 52 1169-1179.
Mellor D.J. and Stafford K.J. 2004. Physiological and Behavioural Assessment of Pain in
Ruminants: Principles and Caveats. 32 (1) 267–271.
Miyamoto A.  1996. Intraluteal mechanisms involved in prostaglandin F2a induced luteolysis in Ewe. J Reprod Develop, 49 61-63.
Morton K.M., De Graaf S.P., Campbell A., Tomkins L.M., Chis Maxwell W.M., Evans G. 2005. Repeat Ovum Pick-up and In Vitro Embryo Production from Adult Ewes with and without FSH Treatment. Reprod Dom Anim, 40 422–428.
Pate, J.L. 1996. Intercellular communication in the bovine corpus luteum. Theriogenology, 45 1381–1397.
Pieterse M.C., Vos P.L.A.M., Kruip T.A.M., Wurth Y.A., Beneden TH. H., Willemse A.H., Taverne M.A.M. 1991.Transvaginal ultrasound guided follicular aspiration of bovine oocytes. Theriogenology, 35 19-24.
Rodríguez C., Anel L., Alvarez M., Anel E., Boixo J.C., Chamorro C.A.,  Paz P. 2006. Ovum Pick-up in Sheep: a Comparison between Different Aspiration Devices for Optimal Oocyte Retrieval. Reprod Dom Anim, 41 106–113.
Roy F., Maurel M.C., Combes B., Vaiman D., Cribiu E.P., Lantier I., Pobel T., Deletang F., Combarnous Y., Guillou F. 1999. The negative effect of repeated equine chorionic gonadotropin treatment on subsequent fertility in Alpine goats is due to a humoral immune response involving the major histocompatibility comples. Biol Reprod, 60 805-813.
Santl B., Wenigerkind H., Schernthaner W., Mödl J., Stojkovic M., Prelle K., Holtz W., Brem G., Wolf E. 1998. Comparison of ultrasound-guided vs laparoscopic transvaginal ovum pick-up (OPU) in Simmental heifers. Theriogenology, 50 89-100.
Schernthaner W., Wenigerkind H., Stojkovic M., Palma G.A., Modl J., Wolf E., Brem G. 1999. Pregnancy rate after ultrasound-guided follicle aspiration in nonlactating cows from different breeds. J Vet Med, 46 33-37.
Silva A.S.L., Teixeira P.P.M., Vicente W.R.R. 2010. Mecanismos fisiológicos e bioquímicos da luteólise: revisão de literatura.  Rev Cien Eletr Med Vet. 8 (15).
Simplício A.A.V., Freitas V.J.F., Fonseca J.F. 2007. Biotechniques of reproduction as techniques of reproductive management in sheep. Rev Bras Repro Anim, 31 (2) 234-246.
Stangl M., Kühholzer B., Besenfelder U., Brem G. 1999. Repeated endoscopic ovum pick-up in sheep. Theriogenology, 52 709-716.
Traldi A.S. 2006. Biotecnicas aplicadas em reprodução de pequenos ruminantes. III Feinco.
Viana J.H.M., Nascimento A.A., Pinheiro N.N.L., Ferreira A.M., Camargo L.S.A., Sá W.F., Marques Júnior A.P. 2003. Caracterização de seqüelas subseqüentes à punção folicular em bovinos. Pesq Vet Bras 23 (3)119-124.
Wieczorek J., Kosenyuk Y., Cegła M., Ryńska B. 2010. A new concept in laparoscopic ovum pick-up (OPU) in sheep – efficiency of method and morphology of recovered oocytes. Ann Anim Sci, 10 (1) 39–48.













Postado por:
Pedro Paulo Maia Teixeira
Médico Veterinário
Mestre em Cirurgia Veterinária
Doutorando em Medicina Veterinária (Reprodução Animal)


Abstract
The aim of the study was to compare the use of open ovariectomy, to the video-assisted laparoscopic approach or total laparoscopic ovariectomy in Santa Ines ewes. Surgical time and body weight gain/loss were recorded and post-surgical pain assessed using a behavioral scale. Laparotomy involved a longer surgical time (75±29.5 min.), than the video-assisted (37.5±13.04 min.; p<0.05) or total laparoscopic approach (27.5±2.89; p<0.01). Behavioral pain recorded score was higher for the laparotomy ovariectomy (5.6±0.5), compared to the video-assisted (0.3±0.5) and laparoscopic approaches (0.3±0.5) (p<0.0001). No significant differences were recorded regarding body weight gain/loss during the first 30 days post-surgery, between the techniques. The video-assisted laparoscopic and total laparoscopic techniques of ovariectomy showed a tendency to have more advantages than the use of laparotomy as such. Less surgical trauma, a shorter surgical time, minimal post-surgical stress and better surgical recovery being highlighted as the main advantages of the endoscopic approaches in sheep.
Keywords: Sheep, ovariectomy, video-assisted, laparoscopy, laparotomy.

1. Introduction
Laparoscopy is a minimal invasive surgical technique and with the use of a trans-abdominal endoscope, it is possible to visually observe the organs within the abdominal cavity (Bouré, 2005). The laparoscopic approach also provides a better success rate in small ruminants, and can be performed several times in the same recipient if necessary, with minimal surgical trauma (Graff et al., 1999; Baldassarre et al., 2002; Cordeiro, 2006). Many different laparoscopic techniques have thus been applied in livestock for e.g. liver biopsy (Chiesa et al., 2009; Duarte et al., 2009), cistotomy (Franz et al., 2006), ruminoscopy (Franz et al., 2009) and ovariectomy (Bleul et al., 2006).
Ovariectomy (removal of the ovaries) results in many advantages for livestock, such as easier handling, the suppression of heat behavior in beef animals for the maintenance of male and female cattle in the same environment (Garber et al, 1990), faster weight gain and the production of a high quality carcass (Silva et al., 2006). This technique has then also been employed for the recovery of the gonads from animals of high genetic and productive performance, and for other reproductive techniques (Padula et al., 2002).
In the modern livestock breeding industry, surgical procedures are generally aimed at providing minimal stress. Painful procedures may decrease the productive performance and thus impair the body weight gain, while animal welfare remains a major concern in surgical procedures, especially in the beef industry (Fitzpatrick, et al., 2006; Luna, 2008).
The aim of the present study was to compare the surgical techniques, post-operative pain and complications of ovariectomy performed by laparotomy, the video-assisted approach and a total laparoscopic approach in Santa Ines ewes.

2. Material and Methods
2.1. Animals and experimental design
The present study was conducted following the approval of the Animal Ethics and Welfare Committee of the School of Agrarian and Veterinary Sciences of the São Paulo State University (protocol No. 025988-08). The Principles of Ethics of the European Commission for experiments involving animals (Directive 86/609EEC) were followed also. Eighteen adult female Santa Ines ewes were randomly allocated to one out of the three surgical techniques of ovariectomy. Ovariectomy was either performed by laparotomy (n=6), the video-assisted laparoscopic approach (n=6) or the full laparoscopic approach (n=6).
2.2. Anesthetic protocol and pre-operative care
All ewes were fasted for 36h prior to each surgical procedure. The animals were premedicated using diazepam (0.5mg/kg, IM - Diazepamâ Cristalia., Brazil), and tramadol (2mg/kg, IM - Tramal® Cristalia. Brazil) and anesthesia was induced using a mixture of propofol (6 mg/kg, IV - Proporfol® Cristalia. Brazil) and lidocaine chloride (1mg/kg, IV).

Anesthesia was maintained with propofol (0.5mg/kg/min., IV) and lidocaine-chloride (1 mg/kg/min, iv) at a constant rate of infusion. After induction, the animals were intubated using a cuffed 8 mm tracheal tubes, to provide 100% oxygen. The sheep were positioned and kept in dorsal recumbency for laparotomy and in the Trendelenburg position for video-assisted and normal laparoscopic ovariectomy. Local anesthesia with lidocaine chloride (2 ml) was administered at the portal sites prior to skin incision in the laparoscopic procedures.

2.3. Laparotomic ovariectomy

The ovariectomy was accomplished following a 10 cm prepubic median incision laparotomic for adequate exposure of the uterus and ovaries. Due to the difficulty in finding the internal genital tract, assisted vaginal palpation was required in some cases. Once the ovaries had been exposed, a transfixation ligature around each ovarian pedicle was performed and the ovary excised. Following final abdominal inspection, the muscular layer was sutured with continuous simple pattern interspersed with simple interrupted knots. The skin was sutured in a simple interrupted pattern.

2.4. Video-assisted laparoscopic ovariectomy
            The video-assisted ovariectomy was performed using three portals. Firstly, a 6 mm trocar was introduced into the linea alba, 30 cm cranial to the udder, using the Hasson technique, for insertion of a 5 mm rigid endoscope (Vilos et al., 2007). The pneumoperitoneum was established under 5 L/min. CO2 flow rate and the intra-abdominal pressure maintained between 5 and 8 mmHg. One 6 mm and one 11 mm instrument port was positioned bilaterally using the laparoscopic-assisted technique, 10 cm cranial to the udder and 5 cm lateral to the linea alba. A 10 mm Babcock atraumatic forceps and a 5 mm atraumatic grasping forceps was then introduced through the 11 mm and 6 mm instrument ports, respectively. The uterus, fallopian tubes and ovaries were manipulated and the ovaries then grasped and exteriorized from the abdominal cavity, via the 11 mm trocar. The ligature of the ovarian pedicle was carried out in the same manner as for the laparotomy technique. The same technique was used for the opposite ovary. After resection of both ovaries, the CO2 pneumoperitoneum was adequately drained. Skin sutures were performed at the portal sites, using the Wolf interrupted pattern with 2-0 nylon. The muscular layer was not sutured.


2.5. Laparoscopic ovariectomy
The laparoscopic ovariectomy was carried out using a two-port technique. One 11 mm port was placed on the midline, 10 cm cranial to the udder, using the Hasson technique (Bouré, 2005). A 10 mm rigid endoscope with a working channel for instruments of up to 5.5 mm was inserted and the initial visual abdominal inspections performed. The pneumoperitoneum was located in the same fashion as in the video-assisted technique. A second 6 mm trocar was placed on the left side, using the laparoscopic-assisted technique, 5 cm from the linea alba and 5 cm from the 11 mm port. A 42 cm in length Babcock forceps was introduced through the working channel of the endoscope. A second 33 cm in length Babcock forceps was inserted through the 6 mm trocar, in order to manipulate the ovaries. The second forceps was used to expose the ovarian pedicle. Following adequate ovarian pedicle exposure, the first forceps was replaced by a bipolar forceps with a simultaneously coagulation and cutting function (Lina Tripol PowerBlade® – WEM & VIVAMED – Ribeirão Preto – SP – Brazil). The left pedicle was cauterized and the ovary was excised and exteriorized through the 11 mm port, along with the trocar. The 11 mm trocar was then reinserted through the abdominal incision. The same procedure was performed for the opposite ovary. The trocars were withdrawn from the abdominal cavity and the pneumoperitoneum drained. The incisions were closed in the same fashion as for the video-assisted technique.

During the early post-operative period, all animals received a single dose of a long acting oxytetracycline (20 mg/Kg, IM).

2.6. Trans and post-surgical assessment
Surgical time and trans-surgical complications were recorded for the groups and post surgical pain also assessed , using a behavioral scale proposed in the present study. This consisted of a kyphosis score for difficulty in movement and spontaneous feed intake score. The score ranged from 0 to 3 for each variable evaluated and the final score was then the sum of the scores of each variable. The maximum pain score ranged from 0 to 9. All animals were weighed weekly, for a 30 day post treatment period and the weight loss/gain compared between the groups.

2.7. Statistical analysis
Surgical time, post-surgical pain score and body weight loss/gain were recorded as the mean (±SD). An analysis of variance (ANOVA) was used and the comparison between the groups were performed using the Tukey test. A confidence level of P<0.05 was considered to be significant. Trans and post-operative complications were assessed descriptively.

3. Results
The ovaries were generally difficult to locate during laparotomy. Exposure of the genital tract was poor and intense traction was required to withdraw the ovaries from the abdominal cavity. However, visualization and manipulation of the genital tract were usually easy to perform during the two endoscopic techniques.
Surgical time was longer for laparotomy (75.8 ± 29.5 min; mean ± SD) than following laparoscopy (27.5 ± 2.9 min; P<0.01) and video-assisted laparoscopy (40.0 ± 13.0 min; P<0.05). The difference in surgical time between laparoscopy and video-assisted laparoscopy was not significant (Fig. 1). Behavioral pain score (Fig. 2) was significantly higher for laparotomy (5.6 ± 0.5; P<0.001), than following laparoscopy (0.3 ± 0.5) or video-assisted laparoscopy (0.3 ± 0.5). No significant differences were recorded in live body weight changes between the treatments during the first 30 day post-operative period.

4. Discussion
The approach to the ovaries was found to be difficult during the laparotomic procedure, due to the presence of extensive gastrointestinal viscera and the caudal topography of the genital tract of the ewe. Vaginal palpation performed may be useful during this surgical step in laparotomic ovariectomy. In the video-assisted and laparoscopic ovariectomy no difficulties were experienced in locating the ovaries. The Trendelenburg position, pneumoperitoneum and amplification of the field of view with the aid of a rigid endoscope and camera system played a crucial role during the endoscopic procedures. This thus resulted in shorter surgical times when the endoscopic approaches were used. Several studies have indicated that laparoscopic surgical procedures usually imply short surgical times in livestock e.g. ovariectomy in cows (Bleul et al., 2005), liver biopsies in sheep (Duarte et al., 2009) and ovarian follicular aspiration in goats (Cordeiro, 2006) and sheep (Teixeira et al., 2010b).
In the present study for endoscopic evaluations, an intra-abdominal CO2 pressure of 5 mmHg provided a satisfactory surgical field. Furthermore, no visceral injury was observed during trocar insertion and surgical handling. However, other studies have indicated that a higher intra-abdominal pressure is required to obtain a good visual surgical field and to avoid visceral injury during trocar insertion (Tabet et al., 2005; Cordeiro, 2006; Duarte et al., 2009, Teixeira et al., 2010a). It is strongly believed that higher intra-peritoneal pressure is not necessary to access the reproductive tract of sheep when the Trendelenburg position is used. High intra-abdominal pressure may lead to respiratory depression, acidosis and an increase in EtCO2 in animals under spontaneous breathing (Uemura et al., 2004).
Generally both laparoscopic techniques were less invasive than the open surgical procedure, and resulted in lower pain scores. The performance of the laparotomy technique plays an important role in post-operative pain. While this technique required a 10 cm-long median incision, the video-assisted required two punctures of 6 mm and one of 11 mm, and the general laparoscopic assisted ovariectomy required one puncture of 6 mm and one of 11 mm. This fact was also observed when conventional and laparoscopic approaches for cistotomy (Franz et al., 2009) and ovariectomy (Bleul et al., 2005) were compared in cows.
The endoscopic approaches proved to be less painful than laparotomy. Even though no changes regarding body weight gain/loss were recorded between the groups during the 30 days of post-operative assessment, the sheep subjected to the video-assisted and laparoscopic techniques generally showed better post-surgical recovery, compared to those submitted to the open procedure. Moreover, the behavioral pain score used in the present study identified variations in post-surgical pain between the groups, satisfactorily. Furthermore, this study recorded physiologic changes similar to those stated in other studies regarding the pain and welfare of ruminants (Mellor & Stafford, 2004).
Regarding the body weight loss/gain, all ovariectomy techniques assessed in the present study may be acceptable for use in sheep, with no difference being detected between the groups. Meirelles et al. (2007) observed a negative effect on body weight gain in cows submitted to a vaginal accessed colpotomy. Although Silva et al. (2006) did not observe any body weight gain changes, a better carcass quality was reported to be obtained with ovariectomy accessed through the flank in Nelore heifers.

5. Conclusion
The endoscopic techniques showed advantages above the laparotomy procedure of ovariectomy in sheep, which resulted in minimal invasiveness, less post-operative pain, a shorter surgical time and a faster recovery time.

Acknowledgements
The authors gratefully thank FAPESP for financial support in the study.

References
Baldassarre, H., Wang, B., Kafidi, N., Keefer, C.L., Lazaris, A., Karatzas, C.N., 2002. Advances in the production and propagation of transgenic goats using laparoscopic ovum pick-up and in vitro embryo production technologies. Theriogenology 57 (1), 275-284.
Bleul, U., Hollenstein, K., Kähn, W., 2005. Laparoscopic ovariectomy in standing cows. Anim. Reprod. Sci. 90, 193-200.
Bouré, L., 2005. General principles of laparoscopy. Vet. Clin. Food Anim. 21, 227-249.
Chiesa, O.A., Von Bredow, J., Smith, M., Thomas, M., 2009. One-port video-assisted laparoscopic kidney biopsy in standing steers. Res. Vet. Sci. 87, 133-134.
Cordeiro, M.F., 2006. Laparoscopic assessment of ovum pick-up in prepubertal and adult ewes, with or without ovarian hormonal stimulation. Tesis (PhD Fellowship of Veterinary Medicine), School of Agrarian and Veterinary Sciences, São Paulo State University, Jaboticabal, São Paulo, Brazil. 59pp.
Duarte, A.L.L., Cattelan, J.W., Bezerra, M.B., Vicente, W.R.R., Cordeiro, M.F., 2009. Laparoscopic-assisted hepatic biopsy with tru-cut needle in caprines. Arq. Bras. Med. Vet. Zootec. 61 (1), 12-19.
Fitzpatrick, J., Scott, M., Nolan, A., 2006. Assessment of pain and welfare in sheep. Small Rumin. Res. 62, 55-61.
Franz, S., Dadak, A.M., Schoffmann, G., Khol, J.L., Baumgartner, W., Dupre, G., 2009. Laparoscopic-assisted cystotomy: an experimental study in male sheep. Vet. Med. 54 (8), 367-373.
Garber, M.J., Roeder, R.A., Combs, J.J., Eldridge, L., Miller, J.C., Hinman, D.D., Ney, J.J., 1990. Efficacy of vaginal spaying and anabolic implants on growth and carcass characteristics in beef heifers. J. Anim. Sci. 68, 1469-1475.
Graff, K.J., Meintjes, M., Dyer, V.W., Paul, J.B., Denniston, R.S., Ziomek, C.A., Godke, R.A., 1999. Transvaginal ultrasound-guided oocyte retrieval 50 following FSH simulation of domestic goats. Theriogenology 51 (6), 1099-1119.
Luna, S.P.L., 2008. Pain, sentience and animal welfare. Ciênc. Vet Tróp. 11 (Suppl.), 17-21.
Meirelles, C., Bueno Junior, C.F., Kozicki, L.E., Weiss, R.R., Segui, M.S., 2007. Evaluation of the body weight gain in heifers ovariectomyzed with the trans-vaginal technique. Rev. Acad. 5 (3), 303-307.
Mellor, D.J., Stafford, K.J., 2004. Physiological and behavioral assessment of pain in ruminants: principles and caveats. ATLA. 32 (Suppl 1), 267-271.
Padula, A.M., Borman, J.M., Wright, P.J., Macmillan, K.L., 2002. Restoration of LH output and 17beta-oestradiol responsiveness in acutely ovariectomised holstein dairy cows pre-treated with a GnRH agonist (deslorelin) for 10 days. Anim. Reprod. Sci. 70, 49-63.
Silva, L.A.F., Pales, A.P., Fioravanti, M.C.S., Pádua, J.T., Silva, O.C., Dos Santos, K.J.G., 2006. Latex ring applied on the ovarian pedicle of Nelore heifers. Acta. Sci. Anim. Sci. 28 (1), 97-103.
Tabet, A.F., Silva, L.C., Shinhorini, I.L., 2005. Comparisson between two techniques of laparoscopic-assisted renal biopsy in equines. Braz. J. Vet. Res. Anim. Sci. 42, 150-156.
Teixeira, P.P.M., Oliveira, M.E.F., Padilha, L.C., da Silva, A.S.L., Motheo, T.F., Bandarra, M.B., Vasconcelos, R.O., Vicente, W.R.R., 2010a. Ovarian histology in stimulated ewes submitted to follicular aspiration – a pilot study. In: 14th Annual Conference of the European Society for Domestic Animal Reproduction. Reprod. Dom. Anim. 45 (Suppl.), 40-41.
Teixeira, P.P.M., Padilha, L.C., Motheo, T.F., Oliveira, M.E.F., Silva, A.S.L., Barros, F.F.P.C., Coutinho, L.N., Vicente, W.R.R., 2010b. Follicular development and oocyte recovery evaluation in Santa Ines ewes submitted to sequential follicular aspirations In: 24th Annual Conference of the Brazilian Society for Embryo Technology, Porto de Galinhas, CE, Brazil. Acta. Sci. Vet. 38 (Suppl.), 768-768.
Uemura, K., McClaine, R.J., de la Fuente, S.G., Manson, R.J., Campbell, K.A., McClaine, D.J., White, W.D., Stamler, J.S., Eubanks, W.S., Reynolds, J.D., 2004. Maternal insufflation during the second trimester equivalent produces hypercapnia, acidosis, and prolonged hypoxia in fetal sheep. Anesthesiol. 101 (6), 1332-1338.
Vilos, G.A., Ternamian, A., Dempster, J., Laberge, P.Y., 2007. Laparoscopic entry: a review of techniques, technologies, and complications. J. Obstet. Gynaecol. Can. 29 (5), 433-465.



Nenhum comentário:

Postar um comentário