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The use of fine needle fibre endoscopy in fish for in vivo examination of visceral organs, with special reference to ovarian evaluation
Aquaculture, 40 (1984) 255-259 Elsevier Science Publishers B.V., Amsterdam -Printed
255 in The Netherlands
Technical Note THE USE OF FINE NEEDLE FIBRE ENDOSCOPY IN FISH FOR IN VIVO EXAMINATION OF VISCERAL ORGANS, WITH SPECIAL REFERENCE TO OVARIAN EVALUATION
R.D. MOCCIA, E.J. WILKIE, K.R. MUNKI’M’RICK and W.D. THOMPSON International (Canada)
Aquaculture
Developments
Company,
P.O. Box 434, Erin, Ont. NOB ITO
(Accepted 16 February 1984)
ABSTRACT Moccia, R.D., Wilkie, E.J., Munkittrick, K.R. and Thompson, W.D., 1984. The use of fine needle fibre endoscopy in fish for in vivo examination of visceral organs, with special reference to ovarian evaluation. Aquaculture, 40: 255-259. Endoscopic techniques for visceral organ and ovarian examination of rainbow trout (Salmo gairdneri) are described. Internal organs can be readily identified and qualitatively assessed., It is possibIe to distinguish sex, and in the female, state of maturity can be identified, as well as some morphological characteristics of individual ova. Repeated use of the initial entry hole for the endoscope is recommended. Gross healing of the surgical incision is 70% complete in 7-10 days, without signs of inflammation or other internal damage. The advantages of fine needle fibre endoscopy are discussed.
INTRODUCTION
Endoscopy is the internal, visual inspection of any cavity of the body by means of a specialized scope (an endoscope). During the development of the field of endoscopy in human medicine, a variety of endoscopes have become available, varying from simple, hollow tubes to sophisticated forms of flexible-optic, video camera display units. Endoscopes have simplified examinations and surgical procedures involving the gas&o-intestinal tract, peritoneal cavity, urogenital systems and joints. In most cases, endoscopic procedures take less time, and patients experience less trauma and quicker recoveries, when compared with surgical procedures utilizing conventional techniques. In recent years, many types of investigations involving teleosts have been hindered by an inability to examine live fish internally. For example, a major stumbling block in the successful hormonal manipulation of fish has been the inability to assess gonadal state accurately (Lam, 1982). In some species it is sufficient to examine external features of female fish which suggest readiness to spawn, such as the presence of a “soft swollen abdomen” or a “pink vent”. Less reliable methods include the measurement of maximum
0044-8486/84/$03.00
o 1984 Elsevier Science Publishers B.V.
256
circumference of the fish, or the determination of condition factors, ponderal indices or gonadosomatic indices (for review see Harvey and Hoar, ‘1979; Davy and Chouinard, 1981; Lam, 1982). There have been several attempts to sample ovarian tissue from intact fish, but most techniques are traumatic to the ovaries, or to the entire fish, and may require a functional oviduct to gain access to the gonadal tissue. Preliminary testing has shown that the endoscope is superior to most other available methods for organ observation, as well as allowing a greater differentiation of gonadal maturation stages. Fine needle fibre endoscopy minimizes the stress imposed on a fish during an internal examination, and eliminates unnecessary sacrifice of experimental fish. This technique can be applied to studies requiring sexual differentiation, gonadal observation or qualitative assessment, examination of visceral organs, clinical assessment of disease, or the examination of preserved, rare or expensive specimens. This report describes the development of endoscopic techniques used on female rainbow trout (Sulmo guirdneri) for the assessment of ovarian maturation. METHODS
Endoscopic equipment The endoscope used is an Olympus SEC1711D Selfoscope with a 2.0 mm exterior trocar tube. The endoscope has an outer sleeve diameter of 1.7 mm and a working length of 105 mm (total length = 215 mm). The optical system gives a 55” field of view in air (forward viewing), 5” offset from straight line of sight, and a depth of field from 1 mm to infinity. The light source consists of an Olympus ILK-4 cold light supply with a 150 W halogen lamp, transmitting light to the endoscope via a flexible, fibre optic cord. The photographic system consists of an OM-1 mounting adapter connected to an Olympus OM-2, 35 mm camera body. Endoscopy procedure Wherever possible, standard aseptic techniques should be practiced when handling equipment and fish. Before examination, fish are anaesthetized in 1 : 12 000 tricaine methanesulphonate (MS 222) and placed upside down in a plexiglass surgery table (see Ferguson et al., 1982, for details). Recirculated, oxygenated water contaming MS 222 (1:12 000) is continuously perfused over the gills through 6.4 mm I.D. surgical tubing. Anaesthetic bath water should be replaced on a regular basis to prevent contamination. Fish specimens are ready for examination when they cease reaction to tactile stimulation, and lose the ocular righting response. With the exception of the surgical area, fish are draped in wet tissues to maintain skin moisture
257
Trocor
tube
I Inserted through peritoneum
IEndoscope Fig. 1. Schematic of endoscopic equipment.
and temperature. The anatomical site selected for laparotomy is located approximately 2.5 cm anterior to the pelvic girdle and l-2 cm lateral to the midline. This site allows easy access to the ovaries, while reducing the possibility of traumatizing vital organs. The surgical area is then dried, disinfected with 70% ethanol, and a 12 gauge cutting needle is inserted anteriorly at approximately a 45” angle. The needle is withdrawn and replaced by a trocar tube containing a triangular tipped trocar (or obturator). The trocar tube is 11 cm long and 2 mm in diameter. The tipped trocar is removed and replaced by the endoscope (Fig. 1). An injection port on the trocar tube can be attached to an air pump set at low pressure to induce slight dilation of the body wall, facilitating manipulation of the endoscope within the peritoneal cavity. A camera attachment for the endoscope is available, permitting photographic recording (Fig. 2). The endoscope is carefully manipulated dorsally and cranially along the peritoneum, until the swim bladder is located, and the bladder followed anteriorly until either the liver or ovary are detected. Generally the liver and ovaries are located adjacent to each other, and in females approaching maturity, the distended ovary is easily located and recognized, Once the examination is completed (5-10 min), the endoscope and trocar tube are withdrawn, and any excess air remaining in the body cavity is purged by gentle abdominal pressure. The area around the incision is again disinfected, dried and manually appositioned, while a small drop of tissue adhesive (methyl cyanoacrylate) is applied to close the incision. The adhesive is air-dried for 20-30 s before the fish is removed from the surgery table and revived immediately in fresh water.
258
Fig. 2. Endoscopy technique: specimen on surgery table received recirculated, oxygenated water containing tricaine methanesulphonate (1 : 12 000). The endoscope is inserted through an incision anterior to the pelvic girdle and lateral to the midline. A camera records observations.
DISCUSSION
Spleen, liver, swim bladder, visceral fat, intestinal tract and gonadal tissue can be identified after a minimal amount of practice. The manipulation of the endoscope is easily controlled to minimize trauma to the fish, and necropsy of specimens reveals no evidence of internal damage. Postanaesthesia revival time varies between 1 and 3 min, and once fish are alert and free from the effects of anaesthesia, normal behaviour resumes. Within a week, the entry incisions are well healed, and remain infection-free without prophylactic antibiotic treatment. Within 3 weeks, significant healing progresses, and there may be a slight depigmentation of the skin surrounding the incision in some fish. Fish subjected to multiple examinations with the endoscope exhibit a tendency to mild formation of abdominal fibrous adhesions, although these appear inconsequential. Reuse of the initial entry incision is accomplished by reinserting the trocar tube and triangular tipped trocar through the previous incision. This enables easier entrance to the body cavity, and is preferred over the formation of a second incision. No abnormalities have been detected in the healing of reopened incisions, even in fish which have undergone more than 20 examinations in less than 4 months.
259
A qualitative ovarian index based on an estimation of ovum diameter, internal egg morphology, and the amount of vascularization in the ovary has been developed. This indexing allows the estimation of ovulation within a 2-3 week period (Moccia et al., in prep.). The cost of the endoscope and related surgical equipment is under $3 000 (Cdn.). This may restrict the application of the technique, despite the advantages direct visual observation of visceral organs may offer. A relatively inexpensive otoscope was used by Fijan (1975) to observe ova in situ and has been used for sexing largemouth bass (Micropterus sahoides) (Driscoll, 1969). However, the otoscope requires entry of the gonad through an oviduct, provides a very small field of view, and identification of sex and gamete development may be difficult unless the gonads are close to maturity (Fijan, 1975). The endoscope requires no oviduct or other natural portal of entry into the body cavity, the field of view is large, and the unit is easily adapted to photographic equipment for recording results. Further work to refine the technique is currently in progress and the equipment shows promise for a wide variety of applications. ACKNOWLEDGEMENTS
This work was financially supported by the Industrial Research Assistance Program, National Research Council of Canada, and Shamrock Springs Trout Farm Ltd. (Erin, Ont.). The authors gratefully acknowledge Dr. R.W. Jackson (Toronto Western Hospital, Toronto, Ont.) for his suggestions regarding the medical applications of endoscopic techniques.
REFERENCES Davy, F.B. and Chouinard, A., 1981. Induced fish breeding in Southeast Asia: a report of a workshop held in Singapore, 25-28 November 1980. IDRC-1’78e, Ottawa, Ont., 48 DriI%I, D.P., 1969. Sexing the largemouth bass with an otoscope. Prog. Fish Cult., 31: 183-184. Ferguson, H.W., Claxton, M.J., Moccia, R.D. and Wilkie, E.J., 1982. The quantitative clearance of bacteria from the bloodstream of rainbow trout (Salmo gairdneri). Vet. Pathol., 19: 687-699. Fijan, N., 1975. Induced spawning, larval rearing and nursery operations - Silurus glanis. Workshop on Controlled Reproduction of Cultivated Fishes, Hamburg, 21-25 May 1973. EIFAC/T25, pp. 130-138. Harvey, B.J. and Hoar, W.S., 1979. The theory and practice of induced breeding in fish. IDRC-TS2le, Ottawa, Ont., 48 pp. Lam, T.J., 1982. Applications of endocrinology to fish culture. Can. J. Fish. Aquat. Sci., 38: 111-137.
255 in The Netherlands
Technical Note THE USE OF FINE NEEDLE FIBRE ENDOSCOPY IN FISH FOR IN VIVO EXAMINATION OF VISCERAL ORGANS, WITH SPECIAL REFERENCE TO OVARIAN EVALUATION
R.D. MOCCIA, E.J. WILKIE, K.R. MUNKI’M’RICK and W.D. THOMPSON International (Canada)
Aquaculture
Developments
Company,
P.O. Box 434, Erin, Ont. NOB ITO
(Accepted 16 February 1984)
ABSTRACT Moccia, R.D., Wilkie, E.J., Munkittrick, K.R. and Thompson, W.D., 1984. The use of fine needle fibre endoscopy in fish for in vivo examination of visceral organs, with special reference to ovarian evaluation. Aquaculture, 40: 255-259. Endoscopic techniques for visceral organ and ovarian examination of rainbow trout (Salmo gairdneri) are described. Internal organs can be readily identified and qualitatively assessed., It is possibIe to distinguish sex, and in the female, state of maturity can be identified, as well as some morphological characteristics of individual ova. Repeated use of the initial entry hole for the endoscope is recommended. Gross healing of the surgical incision is 70% complete in 7-10 days, without signs of inflammation or other internal damage. The advantages of fine needle fibre endoscopy are discussed.
INTRODUCTION
Endoscopy is the internal, visual inspection of any cavity of the body by means of a specialized scope (an endoscope). During the development of the field of endoscopy in human medicine, a variety of endoscopes have become available, varying from simple, hollow tubes to sophisticated forms of flexible-optic, video camera display units. Endoscopes have simplified examinations and surgical procedures involving the gas&o-intestinal tract, peritoneal cavity, urogenital systems and joints. In most cases, endoscopic procedures take less time, and patients experience less trauma and quicker recoveries, when compared with surgical procedures utilizing conventional techniques. In recent years, many types of investigations involving teleosts have been hindered by an inability to examine live fish internally. For example, a major stumbling block in the successful hormonal manipulation of fish has been the inability to assess gonadal state accurately (Lam, 1982). In some species it is sufficient to examine external features of female fish which suggest readiness to spawn, such as the presence of a “soft swollen abdomen” or a “pink vent”. Less reliable methods include the measurement of maximum
0044-8486/84/$03.00
o 1984 Elsevier Science Publishers B.V.
256
circumference of the fish, or the determination of condition factors, ponderal indices or gonadosomatic indices (for review see Harvey and Hoar, ‘1979; Davy and Chouinard, 1981; Lam, 1982). There have been several attempts to sample ovarian tissue from intact fish, but most techniques are traumatic to the ovaries, or to the entire fish, and may require a functional oviduct to gain access to the gonadal tissue. Preliminary testing has shown that the endoscope is superior to most other available methods for organ observation, as well as allowing a greater differentiation of gonadal maturation stages. Fine needle fibre endoscopy minimizes the stress imposed on a fish during an internal examination, and eliminates unnecessary sacrifice of experimental fish. This technique can be applied to studies requiring sexual differentiation, gonadal observation or qualitative assessment, examination of visceral organs, clinical assessment of disease, or the examination of preserved, rare or expensive specimens. This report describes the development of endoscopic techniques used on female rainbow trout (Sulmo guirdneri) for the assessment of ovarian maturation. METHODS
Endoscopic equipment The endoscope used is an Olympus SEC1711D Selfoscope with a 2.0 mm exterior trocar tube. The endoscope has an outer sleeve diameter of 1.7 mm and a working length of 105 mm (total length = 215 mm). The optical system gives a 55” field of view in air (forward viewing), 5” offset from straight line of sight, and a depth of field from 1 mm to infinity. The light source consists of an Olympus ILK-4 cold light supply with a 150 W halogen lamp, transmitting light to the endoscope via a flexible, fibre optic cord. The photographic system consists of an OM-1 mounting adapter connected to an Olympus OM-2, 35 mm camera body. Endoscopy procedure Wherever possible, standard aseptic techniques should be practiced when handling equipment and fish. Before examination, fish are anaesthetized in 1 : 12 000 tricaine methanesulphonate (MS 222) and placed upside down in a plexiglass surgery table (see Ferguson et al., 1982, for details). Recirculated, oxygenated water contaming MS 222 (1:12 000) is continuously perfused over the gills through 6.4 mm I.D. surgical tubing. Anaesthetic bath water should be replaced on a regular basis to prevent contamination. Fish specimens are ready for examination when they cease reaction to tactile stimulation, and lose the ocular righting response. With the exception of the surgical area, fish are draped in wet tissues to maintain skin moisture
257
Trocor
tube
I Inserted through peritoneum
IEndoscope Fig. 1. Schematic of endoscopic equipment.
and temperature. The anatomical site selected for laparotomy is located approximately 2.5 cm anterior to the pelvic girdle and l-2 cm lateral to the midline. This site allows easy access to the ovaries, while reducing the possibility of traumatizing vital organs. The surgical area is then dried, disinfected with 70% ethanol, and a 12 gauge cutting needle is inserted anteriorly at approximately a 45” angle. The needle is withdrawn and replaced by a trocar tube containing a triangular tipped trocar (or obturator). The trocar tube is 11 cm long and 2 mm in diameter. The tipped trocar is removed and replaced by the endoscope (Fig. 1). An injection port on the trocar tube can be attached to an air pump set at low pressure to induce slight dilation of the body wall, facilitating manipulation of the endoscope within the peritoneal cavity. A camera attachment for the endoscope is available, permitting photographic recording (Fig. 2). The endoscope is carefully manipulated dorsally and cranially along the peritoneum, until the swim bladder is located, and the bladder followed anteriorly until either the liver or ovary are detected. Generally the liver and ovaries are located adjacent to each other, and in females approaching maturity, the distended ovary is easily located and recognized, Once the examination is completed (5-10 min), the endoscope and trocar tube are withdrawn, and any excess air remaining in the body cavity is purged by gentle abdominal pressure. The area around the incision is again disinfected, dried and manually appositioned, while a small drop of tissue adhesive (methyl cyanoacrylate) is applied to close the incision. The adhesive is air-dried for 20-30 s before the fish is removed from the surgery table and revived immediately in fresh water.
258
Fig. 2. Endoscopy technique: specimen on surgery table received recirculated, oxygenated water containing tricaine methanesulphonate (1 : 12 000). The endoscope is inserted through an incision anterior to the pelvic girdle and lateral to the midline. A camera records observations.
DISCUSSION
Spleen, liver, swim bladder, visceral fat, intestinal tract and gonadal tissue can be identified after a minimal amount of practice. The manipulation of the endoscope is easily controlled to minimize trauma to the fish, and necropsy of specimens reveals no evidence of internal damage. Postanaesthesia revival time varies between 1 and 3 min, and once fish are alert and free from the effects of anaesthesia, normal behaviour resumes. Within a week, the entry incisions are well healed, and remain infection-free without prophylactic antibiotic treatment. Within 3 weeks, significant healing progresses, and there may be a slight depigmentation of the skin surrounding the incision in some fish. Fish subjected to multiple examinations with the endoscope exhibit a tendency to mild formation of abdominal fibrous adhesions, although these appear inconsequential. Reuse of the initial entry incision is accomplished by reinserting the trocar tube and triangular tipped trocar through the previous incision. This enables easier entrance to the body cavity, and is preferred over the formation of a second incision. No abnormalities have been detected in the healing of reopened incisions, even in fish which have undergone more than 20 examinations in less than 4 months.
259
A qualitative ovarian index based on an estimation of ovum diameter, internal egg morphology, and the amount of vascularization in the ovary has been developed. This indexing allows the estimation of ovulation within a 2-3 week period (Moccia et al., in prep.). The cost of the endoscope and related surgical equipment is under $3 000 (Cdn.). This may restrict the application of the technique, despite the advantages direct visual observation of visceral organs may offer. A relatively inexpensive otoscope was used by Fijan (1975) to observe ova in situ and has been used for sexing largemouth bass (Micropterus sahoides) (Driscoll, 1969). However, the otoscope requires entry of the gonad through an oviduct, provides a very small field of view, and identification of sex and gamete development may be difficult unless the gonads are close to maturity (Fijan, 1975). The endoscope requires no oviduct or other natural portal of entry into the body cavity, the field of view is large, and the unit is easily adapted to photographic equipment for recording results. Further work to refine the technique is currently in progress and the equipment shows promise for a wide variety of applications. ACKNOWLEDGEMENTS
This work was financially supported by the Industrial Research Assistance Program, National Research Council of Canada, and Shamrock Springs Trout Farm Ltd. (Erin, Ont.). The authors gratefully acknowledge Dr. R.W. Jackson (Toronto Western Hospital, Toronto, Ont.) for his suggestions regarding the medical applications of endoscopic techniques.
REFERENCES Davy, F.B. and Chouinard, A., 1981. Induced fish breeding in Southeast Asia: a report of a workshop held in Singapore, 25-28 November 1980. IDRC-1’78e, Ottawa, Ont., 48 DriI%I, D.P., 1969. Sexing the largemouth bass with an otoscope. Prog. Fish Cult., 31: 183-184. Ferguson, H.W., Claxton, M.J., Moccia, R.D. and Wilkie, E.J., 1982. The quantitative clearance of bacteria from the bloodstream of rainbow trout (Salmo gairdneri). Vet. Pathol., 19: 687-699. Fijan, N., 1975. Induced spawning, larval rearing and nursery operations - Silurus glanis. Workshop on Controlled Reproduction of Cultivated Fishes, Hamburg, 21-25 May 1973. EIFAC/T25, pp. 130-138. Harvey, B.J. and Hoar, W.S., 1979. The theory and practice of induced breeding in fish. IDRC-TS2le, Ottawa, Ont., 48 pp. Lam, T.J., 1982. Applications of endocrinology to fish culture. Can. J. Fish. Aquat. Sci., 38: 111-137.