ADRENALECTOMY OF THE CALF AND ITS EFFECTS ON VARIOUS BLOOD CONSTITUENTS

Adrenalectomy of the Calf and Its Effects on Various Blood Constituents1A D R E N A L E C T O M Y O F T H E C A L F A N D I T S E F F E C T S ON V A R I O U S
B L O O D C O N S T I T U E N T S 1
V. L. ESTERGREEN, JR. -~ A~) N. L. VAN DEMARK Department of Dairy Seienee, University of Illinois, Urbana
SUMMARY
Surgical adrenalectomy was successfully performed in calves without suturing the wall of the vena cava, by using a special forceps to facilitate removal of the gland. Main- tenance of adrenalectomized calves was possible under controlled conditions, with the in- tramuscular injection of 25 mg. cortisone acetate or 5 rag. prednisone plus 5 mg. desoxy- corticosterone acetate per 100 lb. body weight, daily or every other day. Calves were also maintained in an apparent ly normal state without steroid therapy by the free-choice feeding of salt, which was consumed at a rate about four times that of the normal calf.
The outward symptoms of adrenal deficiency, manifested during the period when no therapy was given, began with lethargy and weakness in the legs. This was followed by anorexia, extreme apathy, a completely prostrate position, and extreme depression and arrhythmia of the heart beat shortly before death ensued. Loose, foul-smelling feces and a rapid, labored respiration were sometimes observed. The calves survived about six to seven days following withdrawal of maintenance therapy.
In three adrenal-deficient calves there was a decrease in the serum Na :K ratio, a decrease in serum chlorides, and an increase in serum potassium, all of which were highly significant (P less than 0.01). In two of the three animals there was a reduction in serum sodium, total neutrophils, and mature neutrophils, and an increase in RBC and eosino- phils. The influence of adrenalectomy on serum calcium, blood hemoglobin, hematocrit, and neutrophil levels varied considerably between calves. No significant differences were found in WBC, lymphocyte, and monocyte levels following adrenalectomy.
One calf was found to have an adrenal remnant which prevented it from succumbing completely to the effects of adrenalectomy.
One a p p r o a c h to the s t u d y of a d r e n a l g l a n d f u n c t i o n in an a n i m a l is to inves t iga t e the changes which occur in the a n i m a l a f t e r r emova l of the g land . Then, b y s u b s t i t u t i o n t h e r a p y w i t h a d r e n a l s teroids , the levels of these ho rmones needed fo r n o r m a l m a i n t e n a n c e of the a n i m a l can be de t e rmined .
P r i o r to the p r e s e n t s t udy , on ly one ins tance of successfu l su rg i ca l ad re - n a l e c t o m y of a bovine h a d been r e p o r t e d in the l i t e r a t u r e . I n 1957, Ho leombe
(10) r e p o r t e d the a d r e n a l e c t o m y of a bu l l in a s t u d y of the u r i n a r y gluco- cor t i co id excre t ion . Mansson a n d Obel (15) l a t e r r e p o r t e d on the su rg i ca l t echn iques employed . I n 1959, E s t e r g r e e n a n d V a n D e m a r k (6) m a d e a p re - l i m i n a r y r e p o r t on b lood changes in an a d r e n a l e e t o m i z e d calf . Recen t ly , C u p p s et al. (5) r e p o r t e d the successfu l a d r e n a l e e t o m y of two bu l l calves a n d two m a t u r e bulls .
A d r e n a l e c t o m y in the bovine p r e s e n t s some techn ica l p r o b l e m s because of the f i rm a t t a c h m e n t of bo th a d r e n a l s to the wa l l of the p o s t e r i o r vena cava (15,
Received for publication January 13, 1961.
~ The data presented in this paper were taken from a thesis submitted by the senior author to the Graduate College of the University of Illinois in partial fulfillment of the requirements for the Ph.D. degree.
2Present address, Department of Biochemistry, College of Medicine, University of Utah, Salt Luke City, Utah.
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23). These glands have no adrenal vein or a very short one, so special techniques must be devised for their removal without excessive hemorrhage. Cowie and associates (3, 4), working with goats, described special clamps which could be applied between the gland and the wall of the vena cava which would aid in removal of the gland and in sutur ing the defect lef t in the posterior vena cava. Mansson and Obel (15) describe a similar use of long, curved a r te ry forceps for aid in removal of the adrenal gland from a bull.
The present s tudy was under taken in an a t tempt to a) adapt surgical pro- cedures to removal of the adrenal glands of the calf, b) investigate the levels of replacement hormones needed to maintain the adrenalectomized calf, c) s tudy the possibilities of maintaining the adrenaleetomized calf on salt alone, and d) examine the effects of adrenal deficiency on several blood components of the calf.
S U R G I C A L P R O C E D U R E
Young calves were selected for this s tudy because the depth of field in which to work was less, there was less interference from the rumen, and inter- fer ing perirenal fa t deposits were absent as compared with older animals (15). The calves were fasted and kept f rom water for 24 hr. before surgery. Intra- venous sodium pentobarbital (Nembutal) , accompanied by ether as needed dur ing the operation, was the anesthetic used on the first calves. Later, a com- mercial combination of sodium pentobarbital, chloral hydrate, and magnesium sulfate (Equitol) given intravenously proved more effective. A stimulant, amphetamine sulfate (Amphetasul) , was given immediately post-operatively to hasten recovery from the anesthetic.
The site of operation was prepared by close clipping with small animal clippers. Jus t before draping, the area was washed with soap and water, dried, and disinfected with iodine and alcohol. Bilateral adrenalectomy was per- formed one side at a time, allowing 2 wk. or more for recovery between opera- tions. The left adrenaleetomy was done first, to avoid the interference of the developing rumen.
The approach to the left gland was made through an 8- to 10-in. oblique incision, s tar t ing close to the first lumbar transverse process and running par- allel to the last rib. Since the r ight adrenal was more anterior, approach was made to it through an incision between the last two ribs on the r ight side, s tart ing as high as possible and keeping the incision close to the anterior adge of the last rib to clear the intercostal blood vessels.
The wDund was spread with an adjustable abdominal re t ractor and on the left side the viscera were packed off with wet toweling and held back with ad- ditional retractors. On the r ight side, a lobe of the liver directly over the gland was careful ly dissected free and retracted, exposing the gland direct ly under the incision.
The gland was freed from surrounding tissue by blunt dissection and all small blood vessels were doubly ligated and cut between ligations. The median border of the gland was careful ly dissected free of the posterior vena cava except for the anterior one-third around the adrenal vein. The gland was ]i]~t~e~
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930 V . L . ESTERGREEN, JR. AND N. L. VAN DEMARK
and a Her r i ck kidney forceps clamped between the gland and the vena cava. The gland was then freed by runn ing a scalpel along the outer edge of the kidney forceps. No sutur ing was done on the vena cava and the clamp was removed a f te r about 5 min. without hemorrhage.
Normal suture and clamping techniques were used for closing the wound. A gauze and collodion dressing was placed over the wound to keep out d i r t and insects. Three hundred thousand I.U. penicillin and 250 mg. dihydrostrepto- mycin were administered in t ramuscular ly immediately post-operat ively and daily for three to five days following. Af te r removal of the second adrenal, steroid substi tution the rapy was begun. Of four calves bi lateral ly adrenal- ectomized, only one (H-3) was lost, p resumably f rom shock, 18 hr. a f t e r surgery.
T R E A T M E N T S AND M ETHODS OF ANALYSIS
Following surgery the first t r ea tment consisted of maintenance of the ani- mal by substitution steroid therapy. The second t rea tment consisted of main- tenance of the animal by supplemental free-choice salt feeding without steroid therapy. The th i rd t rea tment was the no- therapy period, in which the animal was allowed to go into the extreme deficiency state.
Dur ing all the periods of t rea tment the calves were kept in pens in a heated room and subjected to no more stress than was necessary for blood sampling. Two of the calves were fed milk twice daily for the entire period. The thi rd calf was weaned before removal of the second adrenal. A high-quali ty calf s ta r te r grain mix, good-quality hay, and water were kept before the calves at all times.
Blood samples were taken f rom the jugular vein f rom five to 18 times dur ing each t rea tment period on each calf. Results of the analysis on blood com- ponents were compiled for each t rea tment period and only the means are pre- sented in this paper. The serum sodium, potassium, and calcium were deter- mined by means of a Coleman flame photometer, serum chloride by the method of Whi tehorn (24), hemoglobin by a visual comparison test using the Haden- Hausser technique (2), and the hematocri t as the percentage of packed cells following centr i fugat ion at 2,500 r.p.m, for 30 rain. A differential count of the white blood cells was made a f te r staining with Wr igh t ' s stain (2). Total red and white blood cells were counted visual ly on a hemacytometer . Sta- tistical analysis was carr ied out as described by Ostle (18).
RESULTS AND DISCUSSION
In all the calves used in this investigation (ranging in age f rom 3 to 15 wk. at the t ime of surgery) both adrenals were found to be securely encapsulated to the posterior vena cava with a tough fibrous connective tissue. The r ight adrenal was consistently found to be 1 or 2 in. more anter ior than the left and each was found close to the anter ior pole of the kidney. Both adrenals were found slightly anter ior to the location described by Smollich (23) and in some- what differing positions f rom tha t described by Sisson and Grossman (22). Since these authors describe the position in adul t animals, these differences may have been due to age.
ADRENALEOTOMY OF THE CALF 931
The left gland had a very short adrenal vein (1-2 ram.) between the gland and the posterior vena cava. The right gland had no adrenal vein but com- municated directly from the lumen of the gland into the posterior vena cava via a hole in the wall of the blood vessel.
Following early attempts in this experiment at ligating the adrenal vein or suturing the vena caval wall after removal of the gland, a I-Ierrick kidney forceps was used, as described earlier. This forceps, similar to the clamp described and used by Cowie and Tindal (4) on goats, has two bends in the blade, making it easy to insert under a deep gland. On the inside, and running the length of each blade, are longitudinal ridges which crimp the tissue held between the blades. After clamping the vena eava for 5 rain. with this forceps, it was unnecessary to suture the slit in the caval wall. No caval hemorrhaging was noted in the five operations where this clamp was used.
Steroid therapy. Daily intramuscular administration Of approximately 25 rag. cortisone acetate (C.A., Schering Cortogen), or an equivalent amount of prednisone (Schering Meticorten), plus 5 mg. desoxycorticosterone acetate (DOCA, Schering Cortate) per 100 lb. body weight, maintained these adrenal- ectomized calves in an apparently normal condition. Cortisone acetate alone, at the levels given, did not prevent the onset of deficiency symptoms four days after adrenalectomy in Calf H-1. I t was necessary to include injections of DOCA to maintain normal appearance in this calf and, consequently, both steroids were used for maintenance of the other calves in the experiment. This level of steroid substitution therapy is similar to the reports for maintenance of other adrenalectomized ruminants (4, 8, 10).
Although this level of steroid substitution therapy supported these calves under the controlled conditions, it was not adequate for coping with a severe stress. In one instance, the insertion of a venous cannula caused an animal to collapse. A full day was required for recovery following immediate steroid therapy. No information is available on the leveI of substitution therapy needed to support an animal under normal barn conditions or when penned with other animals.
Salt therapy. Free-choice salt feeding successfully maintained these adre- nalectomized calves without substitution steroid therapy for 2 wk. There was every indication that Calves H-1 and G-5 could have been maintained for a much longer period, since no sign of deficiency appeared until at least four days following removal of the supplemental salt. The other calf, H-2, stopped eating salt after five or six days, but until that time no outward symptoms of deficiency were apparent. The high serum levels of Na and C1 exhibited during this salt-feeding period indicate the calf was successfully overcoming the ex- cessive salt loss due to steroid deficiency." The salt consumption of one calf (G-5) was recorded for a ten-day period, and grain consumption determined for five days. During this time an average of 36 g. salt, 0.5 lb. grMn, 9 lb. milk, and some hay were consumed :daily. The total sodium chloride intake during this period was calculated to be 47 g. daily, exclusive of what was provided in the hay. This was more than four times the calculated-Salt con- sumed during the steroid therapy period.
932 V. L. ESTEI~,GI~EEN, JR. AND N. L. VAN D E M A R K
These findings indicate that supplemental salt feeding alone can alleviate the outward manifestations of adrenal deficiency and maintain life in the adrenalectomized calf, as has been found in the adrenal-deficient rat, dog, and man (13, 14, 19). This concurs with the findings of Holcombe (10), who maintained an adrenalectomized bull four days by giving free access to rock salt.
Adrenal insufficiency. The outward manifestations of adrenal insufficiency began with a lethargy and a weakness in the legs, followed by a loss of appetite and extreme apathy. I f no therapy was initiated the calves became almost completely comatose shortly before death. Other symptoms were loose, foul- smelling feces and a rapid, labored respiration before death. Significant was the sudden, extreme drop in hear t rate and an i r regular i ty of the hear t beat. In one calf the heart rate dropped in a few hours f rom a normal of about 100-110 beats per minute to about 50-55 and was very i r regular at the time the calf was prostrate. At this stage, intramuscular steroid therapy was in- effective in restoring the animal to normal. Intravenous infusion of 300-500 ml. of 25% glucose made up in physiological saline was effective in bringing the animal to his feet and alleviating the deficiency symptoms.
Calves H-1 and H-2 exhibited symptoms of adrenal deficiency af ter four days and survived seven and six days, respectively, following withdrawal of maintenance therapy. Death was due to extreme adrenal deficiency in both cases and post-mortem examination revealed no remnant of adrenal tissue at the sites of operation in either animal.
Calf G-5 exhibited signs of deficiency on the fifth day following cessation of maintenance therapy and was in an extremely deficient state by the seventh day. Some recovery occurred on the eighth day and on the tenth day following cessation of maintenance therapy the calf was sacrificed. Histological investi- gations revealed a small nodule of adrenal tissue embedded in the wall of the vena cava at the site of the r ight adrenalectomy.
Changes in blood components. The mean changes in some of the blood con- stituents for each t reatment period are given in Table 1. The effects of adrenal deficiency on the serum electrolytes in these calves, a decrease in Serum Na and C1, and an increase in Serum K, appear to follow closely the effects recorded for rats, dogs, goats, and sheep (3, 4, 8, 13, 14).
Although Serum Na and K levels were near normal dur ing the nondeficient periods in this experiment, there was a significant decrease in the Serum Na : K ratio and in Serum C1 level, and a significant rise in Serum I( in all three calves dur ing adrenal deficiency (see Figure 2). In two of the three calves this was accompanied by a significant decrease in Serum Na. In the th i rd calf, H-2, the Serum Na did not fall significantly, but there was a tremendous increase in Serum K (to 71.8 rag/100 ml.). Thus, the Serum N a : K ratios were similar for all three calves.
Probably the most constant finding in all the calves was the fall in N a : K ratio dur ing the adrenal-deficient period. The mean Na: K ratio range of 13.7-t6.9 for these calves dur ing the periods before bilateral adrenatectomy and dur ing the steroid…