Medical Research Institute Iowa State University Ames, Iowa 50010 USA


By W. C.


R. E. Strohbehn and P. A. Larson

ABSTRACT Studies were conducted to determine the effect on corpus luteum (CL) function of ACTH or hydrocortisone administered by ovarian perfusion on day 11 of the oestrous cycle or by carotid infusion on days 2\p=n-\9 of the oestrous cycle. No significant local effects on CL function were observed during the ovarian perfusion study. Continuous carotid infusion of ACTH or hydrocortisone on days 2\p=n-\9 resulted in a decreased slope of the progesterone increase during the infusion period. However, no difference in plasma progesterone level on days 9\p=n-\11between control and treated cycles was detected and there was no difference between treated and control CL at day 11 with regard to CL weight or progesterone content. The data suggest a possible direct effect of hydrocortisone on either the hypothalamus or pituitary which results in decreased progesterone secretion but not CL development. No residual effect was observed.

Observations on the depressed reproductive function found in overpopulation studies in animals and the possible role of the adrenal gland have been re¬ ported by Christian et al. (1965) and Andrews (1970), among others. Treatment with ACTH has been reported to block ovulation in the rat (Hagino et al.

1969), pig (Liptrap 1970; Schilling & von Rechenberg 1973), cow (Liptrap & McNally 1976), and baboon (Hagino 1972). Furthermore, highly potent syn¬ thetic corticoids were demonstrated to be capable of blocking ovulation e.g., 158

dexamethasone in the rat (Hagino et al. 1969) and triamcinolone acetonide in the baboon (Hagino 1972), while natural corticoids such as hydrocortisone or corticosterone had no effect in these same studies. Schilling 8c von Rechen¬ berg (1973) also reported that dexamethasone was effective in altering (de¬ laying) ovulation time in pigs. Brunner et al. (1969) were able to reduce corpus luteum (CL) size in heifers by daily treatment with a dosage of 100 IU long-acting ACTH. Wagner et al. (1972) also reported a decrease in midcycle plasma progesterone levels in heifers treated with 100 IU ACTH daily. The present study was undertaken to deter¬ mine the effect of ACTH or hydrocortisone succinate treatment on CL function using (1) acute ovarian perfusion studies at cycle day 10 or 11 and (2) con¬ tinuous carotid infusion of these same compounds during days 2-9 of the oestrous cycle of heifers.


Eighteen virgin Holstein heifers exhibiting normal oestrous cycles were used for this study. They were housed in stanchions in a heated building and fed a diet of alfalfa hay and 14% protein concentrate mix according to recommended levels. All animals had access to trace minerals in an exercise lot. Except during periods of actual in¬ fusion, all animals were observed twice daily for oestrus in an exercise lot.






Twelve animals were used for this study on day 10 or 11 of the oestrous cycle (day 0 day of standing oestrus). Each animal was used for a treatment perfusion (ACTH or hydrocortisone) and a saline control perfusion. The sequence of treatment vs control was arranged so that three animals in each group received the treatment (ACTH or HS) during the first treatment cycle and three the control or saline treat¬ ment. One normal oestrous cycle was allowed to occur between the first and second =



Surgical procedures. All feed was withheld for 24 h prior to surgery. Premedication consisting of atropine and chlorpromazine was given 1 h prior to surgery. In addition all animals in this experiment received 2.5 mg flumethasone im prior to surgery. Thiopental sodium was given iv to induce anaesthesia and allow placement of an -

endotracheal tube. The animals were then maintained under anaesthesia with halothane. The animals were restrained on a surgery table tilted at a 25° angle to permit better access to the ovarian stalk. A flank incision was made just anterior to the hind leg extending ventrally from the tuber coxa for approximately 30 cm. The ovarian vein and artery were dissected free. Approximately 10 cm proximal to the ovary a vinyl cannula was placed in the artery directed toward the ovary and secured with double silk ligatures. The ovarian vein was then cannulated with silastic tubing inserted toward the ovary and secured with double silk ligatures. A vinyl cannula was also placed in the jugular vein. Hydrocortisone succinate (HS) (Upjohn Co.) or ACTH (Porcine, Sigma Chem. Co.) was used as the treatment perfusion. ACTH and HS solutions were prepared in 0.9 °/o (w/v) NaCl solution containing 0.1 "la (w/v) gelatin


and 400 U heparin/ml. This saline solution was also used for the control infusion and pre- and post-treatment infusion periods. The ovarian perfusion lasted 120 min and consisted of a 30 min saline period, 60 min treatment (ACTH or HS) period and a subsequent 30 min saline period. Control perfusion consisted of a 120 min saline perfusion. An infusion pump was used to deliver the solutions at a rate of 0.68 ml/min. A treated ovary received either 30.6 IU ACTH or 8.16 fig HS during the 60 min treatment period. Blood samples were collected at 5 min intervals from the jugular vein and an aliquot of the pooled 5 min effluent from the ovarian vein was saved for hormone analysis. Virtually all ovarian venous effluent was collected in order to avoid having any of the infused material enter the general circulation. During the perfusion and sampling period, all animals received sterile 0.9 "la (w/v) saline iv at a rate sufficient to equal the rate of blood removal. At the end of the perfusion period the cannulation system was verified by perfusion with a small amount of India ink. The data were discarded from any animal in which darkening of the venous effluent did not occur with this procedure. The ovary was then removed, the CL dissected free, weighed and a portion frozen in 95 "la ethanol for subsequent assay.



Carotid infusion

350-400 kg which had previously exhibited two normal oestrous cycles were used for this study. Animals were prepared by implanting catheters on the day of oestrus (day 0) and were then infused with the appropriate materials be¬ ginning at 0900 on day 2 and continuing through 0900 of day 9 of the oestrous cycle. Each animal was used for two or three infusion trials and one normal oestrous cycle occurred between infusion periods. The catheter placements were performed under general anaesthesia (halothane). Silastic tubing (Dow-Corning) (i.d. 1.016 mm, o.d. 2.032 mm) was inserted in the carotid artery and the jugular vein. Each catheter was then passed under the skin to the dorsum of the shoulder region. When not in use for infusion or blood sampling procedures, the cannulas were kept filled with sterile saline containing 400 U heparin/ml. Blood samples were collected from the jugular cannula at 0900, 1200 and 1600 each day from day 2 through day 11 of the oestrous cycle. The heparinized blood samples were chilled, centrifuged at 4°C, the plasma removed and stored at -20°C until assayed. The CL was removed per vagina at 0915 on day 11 for progesterone analysis. The treatments applied were either ACTH, hydrocortisone succinate (HS) or saline. The diluent solution used was 0.9 "la NaCl solution containing 0.1 "la (W/V) gelatin and 45 U heparin/ml. This served as the saline control infusion as well as the vehicle for the hormonal treatment. The experiment was designed to allow each animal to receive all three treatments (HS, ACTH and saline) with the precise sequence rando¬

Six heifers

weighing -

mized between animals. The sequence of ACTH, HS and saline infusions was ar¬ ranged so that each treatment would occur in position 1, 2 or 3 in an equal number of animals (2). The dosage of HS was 1 mg/h throughout the infusion and of ACTH was 10 IU/h (2 animals), 4 lU/h (2 animals) or 1 IU/h (1 animal). All infusion so¬ lutions were maintained at 5°C and delivered to the animals using a peristaltic pump.



Using procedures previously described, competitive protein-binding systems were used to measure plasma progesterone (Wagner et al. 1972) and corticoids (Wagner 160

Se Oxenreider 1972). Adrenalectomized dog plasma was used for the source of corticoid-binding globulin (CBG) for both assays. The progesterone procedure included thin-layer chromatogram purification of the petroleum ether extract prior to the assay itself. In the corticoid system, the plasma was first washed with hexane to remove progestins, followed by méthylène chloride extraction and assay. The corticoid assay was demonstrated to measure the hydrocortisone succinate used in these studies. Corpus luteum progesterone was determined by the method of Seifart Se Hansel (1968) using hot ethanol extraction, thin layer chromatography and quantification in a spectrophotometer.




Good blood flow was maintained to all ovarian perfusions as evidenced by normal pink colour of the ovarian tissues and the similar venous flow rates of about 15 ml/min observed in all preparations. Two animals were discarded from this experiment in the ACTH group because of failure to demonstrate a patent perfusion system. This was probably due to failure of the arterial cannula in both cases. Since there is collateral circulation from the uterine artery to the ovarian artery distal to the position of the cannula, this cannulation does not severely affect the blood flow to the ovary (Lamond & Drost

1974). Jugular corticoid values demonstrated a gradual increase during the per¬ fusion period from approximately 150 ng to 250 ng/ml plasma in all of the

Table 1.

Effect of local





CL function.

Progesterone Expt.

A-ACTH A-Saline

B-Hydrocortisone B-Saline II


Hydrocortisone Saline

No. Observations




4 4 6 6

5.66 5.47 3.96 4.53

5 5 5

4.83 + 0.61 4.95 ± 0.85 5.72 ± 0.48

*Mean ± SE

161 Acta endocr. 85, 1

±0.51* + 0.87 ± 0.37

± 0.48





± 1.9 ± 9.8 ± 8.2

348.7 ± 39.2 361.1 ±111.8 133.9 ± 22.5 190.6 ± 50.2

49.1 ± 9.8 37.7 ± 4.8 35.7 ± 8.5

262.0 ± 34.1 176.8 ± 32.0 175.7 ± 44.9

61.2 63.3 33.9 39.6

± 4.7

& control). Jugular progesterone values remained rather constant in all animals at 4.5-5.0 ng/ml. Concentrations of corticoids ranged from 250 to 350 ng/ml in ovarian vein plasma except for the ACTH perfused ovaries which ranged between 300 and 500 ng/ml. This was not a significant difference. Ovarian vein proges¬ terone in saline and HS perfused ovaries averaged about 2400 ng/ml but was higher in the ACTH perfused ovaries (2000 to 4000 ng/ml). This difference was significant but is probably caused by the LH contamination of the ACTH material (50 ng LH/IU ACTH) as determined by RIA procedures. Data on CL size and progesterone content are given in Table 1. There was no treatment effect observed between groups within each experiment. We have no explanation for the difference in progesterone content between groups A and B in Expt. I. animals


Experiment 11 Due to problems with maintenance of catheters following CL removal, only three animals received heifer received saline and ACTH,


and abdominal adhesions all three treatments. One received saline and HS and one re-








Jugular plasma corticoids during continuous infusion of ACTH via the carotid artery at three dose levels (10 IU/h .-., 4 IU/h .-., 1 IU/h .). Day 0 of the oestrous cycle was day of standing oestrus. Two animals received the 10 IU dosage, two received the 4 IU dosage and one animal received the 1 IU dosage. 162









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Effect of local or parenteral application of ACTH or hydrocortisone on bovine corpus luteum function.

Veterinary Medical Research Institute Iowa State University Ames, Iowa 50010 USA EFFECT OF LOCAL OR PARENTERAL APPLICATION OF ACTH OR HYDROCORTISONE...
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