Simplified Hypophysectomy Technique for Young Birds

Simplified Hypophysectomy Technique for Young Birds

2356 K. A. HOLLEMAN, B. D. BARNETT AND G. W. WICKER Aroclor 1242 had no effect either on weight gains or livability. Our explanation for this differ...

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K. A. HOLLEMAN, B. D. BARNETT AND G. W. WICKER

Aroclor 1242 had no effect either on weight gains or livability. Our explanation for this difference is that possibly the poults metabolized Aroclor 1242 differently than the chicks. The poult may have the ability to make the PCB less toxic or the chick may convert the PCB to a more toxic form.

REFERENCES Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Flick, D. B., D. Firestone and G. R. Higginbotham, 1972. Studies of the chick edema disease, 9. Response of chicks fed or singly administered synthetic edema-producing compounds. Poultry Sci. 51: 2026-2034.

Flick, D. F., R. G. O'Dell and V. A. Childs, 1965. Studies of the chick edema disease, 3. Similarity of symptoms produced by feeding chlorinated biphenyl. Poultry Sci. 44: 1460-1465. Holleman, K. A., and B. D. Barnett, 1972. Species differences in polychlorinated biphenyls (PCB) response. Poultry Sci. 51: 1870. Koeman, J. H., M. C. Ten Noever De Braux and R. H. DeVos, 1969. Chlorinated biphenyls in fish, mussels and birds from the River Rhine and the Netherlands coastal area. Nature, 221: 1126-1128. McCune, E. L., J. E: Savage and B. L. O'Dell, 1962. Hydropericardium and ascites in chicks fed a chlorinated hydrocarbon. Poultry Sci. 41: 295-299. Rehfield, B. M., R. L. Bradley, Jr. and M. L. Sunde, 1971. Toxicity studies on polychlorinated biphenyls in the chick. 1. Toxicity and symptoms. Poultry Sci. 50: 1090-1096. Steel, T. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Company, Inc., New York, N.Y.

Simplified Hypophysectomy Technique for Young Birds JOHN A. NEGULESCO

Department of Anatomy,

College of Medicine, The Ohio State University, Columbus, Ohio 43210 (Received for publication March 15, 1976)

ABSTRACT The present work introduces a modified oral approach to the pituitary gland of the immature bird with improved localization and visualization of the organ in situ. The hypophysectomy procedure is based on simplified instrumentation readily available or constructed with materials present in most, if not all, laboratories. The technique has the added advantage of minimal exsanguination, virtually no operative trauma, and allows for a rapid post operative animal recovery. POULTRY SCIENCE 55: 2356-2363, 1976

INTRODUCTION

Schooley, 1939; Miller, 1961, 1967), duck (Benoit, 1939; Assenmacher, 1958; TixierVidal and Assenmacher, 1961; Wright et al.,

B

IRDS have been used as experimental

1966; Bradley, 1971), and chicken (Mitchel,

animals in almost every biological field.

1929; Hill and Parkes, 1934; Nalbandov and

Their contribution to embryology, immuno-

Card, 1943; Rothchild, 1948a, b ; Shirley and

logy, physiology and endocrinology competes

Nalbandov,

and

Nalbandov,

closely with rats and mice. Birds as experi-

1961a, b, c; Rosenberg et ai,

1963; Taber

1956;

Opel

mental animals have been hypophysecto-

et al., 1964; Koike et ai, 1964; Opel, 1956a,

mized either partially by removal of either

b; Gibson and Nalbandov, 1966a, b ; King,

the anterior or posterior lobes of the pituitary

1969a, b ; Kudzma et ai,

or totally. Among the more common labora-

and Eglitis, 1975).

tory birds hypophysectomy has been reported for pigeon (Ogata and

Nishimura,

1927;

1973; Negulesco

There are 5 major approaches to the avian hypophysis:

HYPOPHYSECTOMY TECHNIQUE

Temporal (or Bitemporal) of Paulesco (1907). This approach actually requires major neurosurgery and handling of the brain. It does give, however, a good view of the stalk and sellar region. Shrinking one cerebral hemisphere with hypertonic solutions may facilitate this approach. Orbital Approach of Martins (1933). This entails enucleation of the eye and requires breaking of the posterior orbital wall in order to reach the pituitary stalk. Transbuccal Approach of Hill and Parkes (1934). The operator reaches the base of the skull by a ventromedian incision through the lower jaw with the beak closed. The procedure allows for observation of the gland in situ. The operation requires multiple retractors and necessitates the drilling of a fairly large foramen in the posterior portion of the parasphenoid rostrum and basisphenoid bones of the skull (Marshall, 1960).

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Parapharyngeal Approach of Schooley (1939). This represents a modified version of the transbuccal approach described by Hill and Parkes (1934). The incision is made on the ventral surface of the lower jaw (with the beak closed) but slightly sagittal to the median plane. Difficulty is presented by the slightly angular approach to the pituitary gland and by requiring multiple retractors.

Oral Approach of Rothchild (1948). The procedure is performed with the jaws wide open and is considered to induce a minimum amount of operative trauma. It has the advantage that it allows drilling directly under the pituitary gland and thus provides direct exposure of the gland and access to it with good visualization. The present work introduces a modified oral approach to the pituitary gland with improved localization and visualization of the organ. The technique has the added advantage of minimal exsanguination, virtually no oper-

© FIG. 1. The operating table for the hypophysectomy procedure.

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J. A. NEGULESCO

ative trauma, and allows for a rapid post operative animal recovery. MATERIALS AND METHODS The hypophysectomy procedure can be performed with instruments readily available or constructed with materials present in most, if not all, laboratories. The following list represents the basic instrumental requirements for the hypophysectomy procedure: 1. Operating Table. A flat wood or metal surface (Fig. 1) provided with a " C " clamp (Fig. 1A) to immobilize the cephalic region of the animal during the operation. In the present work a " C " clamp was internally lined with foam rubber (Fig. IB) to prevent injury to the temporal and orbital head regions. The superior limb was stretched and affixed with heavy rubber bands to the parallel boards of the operating table for easier access to the wing veins during the procedure. The thoraco-abdominal region was ligated. in mature birds, to the operating table with soft rubber tubing 0.5 cm. in diameter (Fig.

IE) threaded with soft electrical wire. Lower limb fixation, if required for injections and/or to prevent movement, was facilitated by attaching the tibiotarsus and tarsometatarsus to the board with pipe cleaners or soft coated wire to sliding hooks attached to metal runners (Fig. IF).

2. Suction Cannula and Vacuum Source. Pasteur or similar glass pipettes trimmed for the desired suction opening are ideal. A small aperture should be cut with triangular file on the margin of the glass tube or Pasteur pipette to allow digital control of suction. The vacuum source can be provided by a water pump or it may be supplied by a vacuum pump. A trap bottle must be inserted roughly midway between the vacuum source and suction point to allow for collection of blood, mucous, and extracted pituitary glands.

3. Dental Drill. The drill was provided with #6-8 burrs and a foot pedal rheostat for control of off-on activity.

FIG. 2. Cauterization (A) and tissue retraction (B) instruments.

HYPOPHYSECTOMY TECHNIQUE

FIG. 3. Frontal view of the normal palate, the palatal cleft and the transpalatal tissue ridge (arrow) prior to surgery. 4. Cautery Apparatus. Hot needle cauterization was employed to coagulate small bleeders, to burn out tissue remnants after pituitary extraction, and to incise the soft palate and periosteum under the hypophysis. The cautery needle was made from nikon wire surrounded by plastic handles (Fig. 2A). The electrical source was provided by a small transformer (50 Watts, 5 1/2—10 1 /2 volts). It is also possible to use ophthalmological, tonsilar and cutaneous coagulators. 5. Spring Spreaders.

The tissue retractors

FIG. 4. Frontal view of the parasphenoid rostrum (A) and the surgically induced os basisphenoid fenestra (B) post palatal sectioning.

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FIG. 5. Dorsal view of the sella turcica (arrow) of a decorticated animal. were made of coat hanger wire with small, flat, serrated copper plates soldered to the internal surface of the open ends (Fig. 2B). Surgical spreaders and retractors (if available) work just as well. 6. Freeze Coagulator. Free tips of crochet needles surrounded with 2-3 layers of tape were cooled in dry ice and used as freezecoagulators upon excessive bleeding during the hypophysectomy procedure. 7. Dental Probe (Seeker) and Spatula.

A

FIG. 6. Sagittal section through the cephalic region of the bird showing the hypophyseal topography (arrow).

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J. A. NEGULESCO

straight blunt tip and curved, sharp, dental probe (Fig. 8A) was used in sectioning and removal of the dural layer of the diaphragma sllae (Figs. 5,6). A fine round tip metal spatula (Fig. 8B) was used for decanting wax [Dentsply, 38° C. (100° F.) or paraffin, Paraplasm 15.5° C. (60° F.)] into the osseous deficiency induced by the extraction of the pituitary gland. The wax or paraffin was warmed with an alcohol lamp. 8. Beak Retractors. The retractors were made of soft wire bent to the desired shape and surrounded with tape to protect the soft tissue of the oral cavity (Fig. 8C). Each retractor was provided with rubber bands or soft wire for attachment to the operating table. A dissecting microscope and light source were used for direct observation of the pituitary gland in situ. A high intensity direct light source or over head flood light is, similarly, required for optimal illumination of the operation field. Anesthesia. Nembutal (sodium pentobarbital, 50 mg./ml.) admistered LP. or I.V. was the general anesthetic of choice in all cases. In the younger birds, at two weeks post hatching, the best results were obtained by using only the I.P. induction method. I.V. was used with the older chicks. Table 1 TABLE 1.—The method, site and amount of sodium pentobarbital (Nembutal) used in induction of animal anesthesia Mean Nem- Method Animal body butal of age weight dosage induc(weeks) (gm.)* ting.)** tion

2

130

4

290

5 10 20

IP IV IV

Site

Rt. leg vein Rt. leg vein Rt. wing vein 6 420 30 IV Rt. leg vein Rt. wing vein *Negulesco, J. A., J. M. Delphia, A. Naumoff and D. L. Clark (1976) **NeguIesco J. A., and J. A. Eglitis (1975).

illustrates the animal age and dosage relationship utilized in inducing 20-30 min. deep anesthesia state (Negulesco and Eglitis, 1975: Negulesco, 1976). Pre-Operative Care and the Operation. The animal, rendered motionless by the anesthetic, was firmly affixed, on its back, to the operating board by immobilizing its legs on the hooks of the metal runners with pipe cleaners. The temporal region was gently but firmly secured into the foam rubber lined " C " clamp. The wings, if required, may be attached to the 3 parallel boards (Fig. IC) with rubber bands. The thoraco-abdominal region was affixed to the operating board with a small rubber tube threaded by a ligation wire. The lower jaw was drawn out as far as possible. Beak retractors were attached to the jaw and via rubber bands they were aff i xed and stabilized to the round head hooks of the operating table (Fig. ID). The upper jaw was, similarly, immobilized and attached via beak retractors to the head region of the operating table. This procedure completely exposed the roof of the oral cavity. The soft palate appeared deeply clefted in the midline except in the posterior 1/3 region where it was interconnected by a tissue ridge (Fig. 3). Probe examination of the palatal cleft revealed a fine midsagittal osseous structure representing the parasphenoid rostrum (Fig. 4A) (Marshall. 1960). The palatal ridge, the mucosa, and the musculature of the soft palate immediately posterior to and above the posterior extent of the rostrum was bisected with hot needle cautery. The hot scalpel procedure prevented blood loss in exposing the periosteum and a small midline bony elevation perpendicular to the posterior attachment of the rostrum. This structure represents the anterior portion of the basisphenoid (Marshall, 1960). A tissue retractor was inserted to spread laterally the two sides of the palatal cleft. The underlying bone region between rostrum and basisphenoid was

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HYPOPHYSECTOMY TECHNIQUE

FIG. 8. Seekers (A), wax spatula (B), and beak retractors (C) for the hypophysectomy procedure. Fio. 7. Post operative dorso-ventral allignment of the sellar region with the surgically induced basisphenoid fenestra. clearly exposed with a few light sweeps of the hot needle cautery. The bone underneath the pituitary was drilled between the posterior portion of the rostrum and the anterior portion of the basisphenoid (Fig. 4B). As soon as the drilling is started the suction and cautery apparatus must be working, and the melted wax ready. The tips of the crochet needles should already have been frozen or freezing on the C 0 2 block. The drilling was started at the posterior attachment of the rostrum keeping the hand piece at roughly 45° (Fig. 7). As the opening through the cancellous bone was gradually rounded, deepened and extended posteriorly into the basisphenoid the hand piece was straightened to a perpendicular position. A 2-3 mm. depth penetration exposed the dural layer underlying the pituitary gland (Fig. 6). Rapid penetration through the basisphenoid (due to impatience) results in large and unnecessary blood loss. The bone should be drilled patiently until the experimenter becomes accustomed to its texture and thickness. Once the dura was exposed the frozen needle was applied around the aperture and over the meninges to prevent blood loss. Gradual freezing of the field, and continuation of the drilling followed by probe

dissection of the dura (blunt and curved seekers) completely exposed the hypophysis. The gland was lifted and removed with probes or by gentle suction. The tissue cleft between the adeno- and neurohypophysis allows the experimenter to be selective in the extraction procedure. Post extraction, the area should be cauterized to eliminate glandular fragments, sponged with a cleansing alcohol solution (60%), and packed with Gelfoam (absorbable gelatin sponge) to prevent bleeding. The osseous deficiency left by the extraction of the gland was filled with melted wax. Surgical closure by suture can be performed on the cut palatal edges but it was found unnecessary since birds were extremely resistant to infection and healed rather well.

DISCUSSION The success of the procedure depends on the amount of time spnt by the experimenter in acquiring the technique. Dissection of animal heads obtained from colleagues working with the animal in related experiments or fields was a plus before the onset of the operations. One should not be discouraged with a high animal mortality in the early phases since no researcher is born with unearned surgical skills. Patience and continuous practice can, eventually, lead to almost

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100% animal survival. Care of the animal, although minimal, should be most strictly enforced. The animals, if provided by supply companies and not raised on the premises, should be allowed to adjust to the new environment for at least 48 hours before the onset of the operation. All heads should be checked and preferably sectioned at the end of each experimental procedure to ensure the success of the operation. REFERENCES Assenmacher, I., 1958. Recherches sur le controle hypothalamique de la function gonadotrope prehypophysaire chcz le canard. Arch. Anat. Micr. Morph. Exp. 47: 447-572. Benoit, J., 1937. Facteurs externes et internes de l'activite sexuelle. II. Etude du mecanisme de la stimulation par la lumiere de l'activite testiculaire chez le canard domestique. Role de I'hypophyse. Bull. Biol. 71: 393-437. Bradley, E. L., and W. N. Holmes, 1971. The effects of hypophysectomy on adrenocortical function in the duck. J. Endocrinol. 49: 437-457. Brown, N. L., J. D. Bayle, C. G. Scanes and B. K. Follett, 1975. Chicken gonadotropins: Their effects on the testes of immature and hypophysectomized Japanese quail. Cell Tissue Res. 156:499-520. Gibson, W. R., and A. V. Nalbandov, 1966a. Lipid mobilization in obese hypophysectomized cockerels. Amer. J. Physiol. 211: 1345-1351. Gibson, W. R., and A. V. Nalbandov, 1966b. Lipolysis and lipogenesis in liver and adipose tissue of hypophysectomized cockerels. Amer. J. Physiol. 211: 1352-1356. Hill, R. T., and A. S. Parkes., 1934. Hypophysectomy of birds. Proc. Roy. Soc. B. 115: 402-409. King, D. B., 1969a. Effect of hypophysectomy on the radioactive phosphorus uptake of chick adrenals. Poultry Sci. 48: 459-464. King, D. B., 1969b. Effect of hypophysectomy of young cockerels with particular reference to body growth, liver weight and liver glycogen level. Gen. Comp. Endocr. 12: 242-255. Koike, T. I., A. V. Nalbandov, M. K. Dimick and Y. Matsumura, 1964. Blood glucose levels of fasted hypophysectomized, depancreatized and normal chickens. Endocrinology, 74: 944-948. Kudzma, D. J., P. M. Hegstad and R. E. Stoll, 1973. The chick as a laboratory model for the study of estrogen-induced hyperlipemia. Metabolism, 22: 423-434.

Marshall, A. J., 1960. The skeleton of birds In: Biology and Comparative Physiology of Birds. Academic Press, New York and London, P. 241-300. Martins, T., 1933. Technique de l'hypophysectomie chez les oiseaux. Compt. Rend. Soc. Biol. 14: 837-839. Miller, R. A., 1961. Hypertrophic adrenals and their responses to stress after lesions in the median eminence of totally hypophysectomized pigeons. Acta Endocr. (Kbh), 37: 565-576. Miller, R. A., 1967. Regional responses of interrenal tissue and of chromaffin tissue to hypophysectomy and stress in pigeons. Acta Endocr. (Kbh), 55: 108-118. Mitchell, J. B., 1929. Experimental studies of the bird hypophysis. I. Effects of hypophysectomy in the Brown Leghorn fowl. Physiol, Zool. 2: 411-437. Nalbandov, A. V., and L. E. Card, 1943. Effect of hypophysectomy on growing chicks. J. Exptl. Zool. 94: 387-409. Negulesco, J. A., 1976. Accumulative effects of two weeks exposure to 2 G hypergravity state and estrogen treatment upon intact and fractured radii of youngfemale birds. Part II. Aviat. Space Environ. Med., (in press). Negulesco, J. A., J. M. Delphia, A. Naumoff and D. L. Clark, 1976. Somatic and hepato-cardiac mass adjustments of young female birds subjected to estrogen treatment and 29 hypergravity state. Unpublished data. Negulesco, J. A., and J. A. Eglitis, 1975. Effect of hypophysectomy and estrogen treatment on long bone fracture healing of young domestic fowls. Ohio J. Sci. 75: 217-222. Ogata, D., and H. Nishimura, 1927. New (orbital) method for extirpation of the hypophysis of the pigeon. Endocrinology, 11: 457-460. Opel, H., 1965a. Oviposition in chickens after removal of posterior lobe of the pituitary by an improved method. Endocrinology, 76: 673-677. Opel, H., 1965b. Failure of neurohypophysectomy to reduce egg shell thickness in chickens. Poultry Sci. 44: 1135-1136. Opel, H., 1969. Transbuccal hypophysectomy in the Japanese quail. Poultry Sci. 48: 722-728. Opel, H., and A. V. Nalbandov, 1961a. Onset of follicular atresia following hypophysectomy of the laying hen. Proc. Soc. Exp. Biol. Med. 107: 233-235. Opel, H., and A. V. Nalbandov, 1961b. Follicular growth and ovulation in hypophysectomized hens. Endocrinology, 69: 1016-1028. Opel, H., and A. V. Nalbandov, 1961c. Ovulability of ovarian follicles in the hypophysectomized hen. Endocrinology, 69: 1029-1035. Paulesco, N. C , 1907. Contribution a la morphologie

HYPOPHYSECTOMY TECHNIQUE

de 1'hypophyse du cerveau. J. Med. Int. (Paris), XI: 6-8. Rosenberg, L. L., M. K. Dimick and G. Laroche. 1963. Thyroid function in chickens and rats. Effect of iodine content of the diet and hypophysectomy on iodine metabolism in White Leghorn cockerels and Long-Evans rats. Endocrinology, 72: 749-758. Rothchild, I. V., 1948a. A simplified technique for hypophysectomy of the domestic fowl. Endocrinology, 43: 293-297. Rothchild, I. V., 1948b. Notes on survival and body weight changes of adult hens following hypophysectomy. Endocrinology, 43: 298-305. Schooley, J. P., 1939. Technic for hypophysectomy of pigeons. Endocrinology, 25: 372-378.

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Shirley, H. V., and A. V. Nalbandov, 1956. Effects of transecting hypophyseal stalks in laying hens. 58: 694-700. Taber, E., J. S. Knight, C. Ayers and J. I. Fishburn, 1964. Some of the factors controlling growth and differentiation of the right gonad in female domestic fowl. Gen. Comp. Endocr. 4: 343-352. Tixier-Vidal, A., and I. Assenmacher, 1961. Influence of pre-hypophysectomy on thyroid function in the male duck. C. R. Acad. Sci. (Paris), 252: 1215-1217. Wright, A., J. G. Phillips and D. P. Huang, 1966. The effect of adenohypophysectomy on the extrarenal and renal excretion of saline-loaded duck (Ansa platyrhunchos). J. Endocr. 36: 249-256.

Selection for Increased Semen Yield in the Turkey K A R L E . NESTOR

Department of Poultry Science, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691 (Received for publication March 16, 1976)

ABSTRACT Selection was effective in greatly increasing semen yield in the turkey. After five generations of selection semen yield in the selected line was more than double that of the randombred control line from which the semen line was developed. The realized heritability of semen yield was .35 ± .20. There was no consistent change in sperm concentration or frequency of bent sperm associated with the increased yield. Egg production was increased in the semen line. The increase occurred in the first generation of selection and was maintained throughout the remaining generations of selection. Body weight during the growing period exhibited an initial increase in the semen line but declined to randombred control levels in later generations. There was no consistent change in percent fertility, percent hatchability of fertile eggs or number of poults produced per hen which was associated with the genetic increases in semen yield. POULTRY SCIENCE 55: 2363-2369, 1976

INTRODUCTION

T

HE production of a large quantity of high quality semen is important to turkey hatching egg producers since almost all turkey hens are artificially inseminated. A genetic increase in semen production would result in a fewer number of males being required to inseminate a given group of females and, therefore, is an economically important trait.

Approved for publication as Journal Article No. 28-76 of the Ohio Agricultural Research and Development Center, Wooster, Ohio 44691.

In chickens, Siegel (1963a) obtained a heritability estimate of .14 for semen volume in the first four generations of selection in lines selected for high and low body weight at eight weeks of age. In White Plymouth Rocks, Soller et al. (1965) estimated the heritability of semen quantity to be 0.41. Carson et al. (1955) reported that the heritability of semen volume produced by turkey males housed under natural lighting conditions was high, averaging 0.86 based on sire plus dam components of variance. Since, to the knowledge of the present author, there were no reported heribability estimates of