L EVELS of certain hormones fluctuate during the estrous cycle of the cow. Gomes and Erb (1965) reviewed the litera- ture on progesterone levels in corpora ...
PROGESTERONE AND LUTEINIZING HORMONE LEVELS PERIPHERAL BLOOD OF CYCLING BEEF COWS 1
IN
E. A. SPRACUE,2 M. L. HOPWOOD,G. D. NISWENDER AND J. N. WILTBANK Colorado State University, Fort Collins 3 80521, and University o/ Michigan, Ann Arbor 4 48823
L EVELS during
of certain hormones fluctuate the estrous cycle of the cow. Gomes and Erb (1965) reviewed the literature on progesterone levels in corpora lutea of dairy cattle; luteal progesterone was lowest at estrus and gradually increased to peak levels at days 14 to 15. The concentration of progesterone in ovarian venous effluent (Gomes et al., 1963; Dobrowolski, Stupnicka and Domanski, 1968) and in peripheral blood (Plotka et al., 1967; Gupta and Pope, 1968; Stabenfeldt et al., 1968; Shemest, Ayalon and Luidner, 1968) of dairy cattle folIowed the same pattern. Progesterone has not been reported for peripheral blood of beef cows but has been determined in luteal tissue (Niswender et al., 1965). Luteinizing hormone (LH) in the pituitary glands of dairy cattle decreased rapidly at estrus as determined by the ovarian ascorbic acid depletion assay (OAAD) (Rakha and Robertson, 1965). Anderson and McShan (1966), using OAAD, found L H to increase in plasma between 6 and 17 hr. before ovulation. In contrast, Karg et al. (1967) found increasing levels of L H from day 14 to estrus with a high peak prior to ovulation and a secondary peak in some subjects at day 8. In this study progesterone, progestins, corticoids and L H were measured in jugular blood plasma or serum of cycling beef cows.
termined by rectal palpation at 4-hr. intervals starting at the end of estrus. Jugular blood was obtained each day starting on day 16 of the estrous cycle. As soon as estrus was observed a sample was taken every 4 hr. until ovulation occurred and then a daily sample was taken until the start of the next estrus. Two samples of approximately 10 co. were removed at each bleeding. One was allowed to clot to obtain serum for LIt assay and the other was heparinized for steroid assay. The blood was taken immediately to the laboratory for processing where the serum of heparinized blood was frozen and stored until assays could be prepared. Heparinized blood was centrifuged at 20,000g to obtain plasma. Progesterone-414C at 2,000 dpm/ml was added to 5 ml plasma to correct for procedural losses. For progesterone assay the plasma was extracted with 25 ml of n-hexane for 10 hr. at room temperature using gentle swirling on a rotating shaker (Eberbach, Ann Arbor, Michigan 48103). The solvent was removed by pipette and the plasma was washed twice with 5 ml portions of fresh solvent. The extract and washings were combined and evaporated to dryness in vacuo. The extracts were dissolved in absolute methanol and made up to 10 ml with this solvent. All solvents were reagent grade and were used without further purification. Radioactivity in 1 ml portions of the methanol solutions was measured in a Beckman LS-200 scintillation spectrophotometer using a scintillation fluid containing naphthalene 10% and 2,5-diphenyloxazole (PPO) 0.5% dissolved in 1,4-dioxane. Progesterone was determined on 2 ml aliquots of the methanol extracts. The aliquot was evaporated to dryness under Nz at 40C and taken up in a small quantity of the same solvent for applying to Eastman silica gel chromatography sheets. Subsequent procedures were according to Neill et al. (1967). Progestins were determined as suggested by these authors using the protein binding procedure but without purification of the extracted material by chromatography.
Materials and Methods Seven non-lactating beef cows (two Hereford, four Shorthorn, one Angus), previously cycling normally, were checked for estrus every 4 hr. from day 16 of the estrous cycle, until estrus and ovulation occurred and then cows were checked for estrus twice daily until the start of the next estrus. Ovulation was de1 Colorado Agricultural Experiment Station Journal Series No. 1527. Supported in part by Experiment Station Project 57 and American Breeders Service. In cooperation with Regional Research Proiect W-95. In partial fulfillment of the requirements for the M.S. in Animal Science. ~Departments of Animal Science (E.A.S., J.N.W.) and Physiology and Biophysics (M.L.H.). Department of Pathology, University of Michigan (G.D.N.). 99
JOURNAL OF ANIMAL SCIENCE, vol. 33, no. 1, 1971
100
SPRAGUE E T AL.
Corticoid assay was carried out o n the plasma by the competitive protein-binding procedure of Murphy (1967). I t was necessary to use 0.05 ml of plasma for the bovine in place of 0.01 ml used by Murphy for the human being. Luteinizing hormone (LH) was determined on the serum samples, which were frozen until used in the procedure of Niswender et al. (1969). Statistical procedures were according to Li (1964). R e s u l t s and Discussion
Luteinizing Hormone. The levels of L H in the seven beef cows are shown in figure i. Four of the animals had cycles and three of these (2,001, 5,002, 6,504) had very distinct L H peaks. The fourth cycled in 13 days and L H levels were not distinctly elevated. For the second cycle the L H peak was only apparent for cow 5,002. I t is probable that the second peaks were not detected because of the infrequency of sampling (every 24 hr.) during the second estrus. These data show that L H peak and onset of estrus are coinci-
dent and that ovulation occurred from 0 to 52 hr. (avg 24 hr.) thereafter. Two of the seven cows showed no estrus or ovulation while being bled while a third showed estrus but no ovulation during this period and no marked changes in L H were detected in the blood from these three animals. Progesteron'e. Of the four cows that cycled, progesterone was declining at estrus (figure 2) and reached a low point 8 to 20 hr. after the L H peak. Table 1 summarizes the events during the estrous cycle. Progesterone levels increased from days 2 to 3 and declined on day 4 except for cow 6,504. The concentration of progesterone in plasma was highest at 8 to 11 days after estrus and, except for cow 6,503, declined gradually until the next estrus. Progesterone concentrations were not much higher in two of the noncycling cows (4,502, 1,001) in comparison with the cows cycling regularly, but cow 6,505 (showing estrus but not ovulating) had the highest concentrations with great fluctuation during estrus. Cow 2,001 (cycling) went into the first estrus after a delayed, 30 day cycle. Blood sampling was discontinued for 2 days
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P R O G E S T E R O N E AND L U T E I N I Z I N G H O R M O N E S
101
T A B L E 1. T I M E R E L A T I O N OF E V E N T S D U R I N G T H E ESTROUS CYCLE Hours Cow number
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after which, the onset of estrus was observed and blood was again drawn. The blood drawn before estrus was higher in progesterone than any collected during the second luteal phase. We cannot account for some of the changes in progesterone levels in the anestrous phase. Corticoids. The corticoids, corticosterone and cortisol have been identified in the plasma of dairy cattle, with cortisol found at an average of 2.4 times more than corticosterone (Estergreen and Venkataseshu, 1967). Plasma levels of corticoids in the beef cows in the present study were high during estrus (figure 3). In cow 2,001, however, the highCORTICOID$
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est levels were in the phase prior to the first estrus and while this estrus was delayed. Levels for the four cycling cows averaged 49.8 ng/ml through the cycle while the three cows that failed to ovulate averaged 61.4 ng/ml corticoids in the plasma. I t is not conclusive that the elevated corficoid levels in cows failing to cycle are responsible for this behavior, but it is highly suggestive that the frequency or method of drawing blood contributed to the failure of three of the seven cows to cycle with the fourth (2,001) returning to estrus when bleeding was discontinued. In order to avoid this type of response in the future a method of bleeding which will cause less stress should be used. Corpora lutea were not retained in the noncycling cows. Dobrowolski el al. (1968) observed failure to cycle in two of 11 cows with cannulas in the ovarian vein and bled every 6 hours. Composite LH, progesterone and corficoid values of the four cycling cows are graphically shown in figure 4. With respect to the progesterone levels at estrus the values for protein binding estimation in beef cows compare favorably with those in dairy cows of Shemesh et al. (1968), Gupta and Pope (1968) and Stabenfeldt et al. (1968). These workers used gas chromatography, the latter two groups in addition used electron capture detection. For peak levels of progesterone our values agree with those of Shemesh et al. (1968) but are approximately one-half those of the other groups. These differences may be due to the smaller amount of luteal tissue of the beef cow (Niswender et al., 1965). The progesterone levels in this study are less than one-fifth those reported by Plotka et al., (1967), who used a double isotope dilution procedure to determine progesterone in dairy cattle. L H levels in the beef cow are peaked sharply at estrus. No other peaks were observed throughout the cycle, although blood was obtained at 24-hr. intervals after ovula-
102
SPRAGUE E T AL.
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tion and other L H surges may have been undetected. Schams and Karg (1969a) found peaks at day 8 or 9 of the cycle and at 15 to 22 hr. prior to ovulation in dairy cattle. In view of the short half life of exogenous L H in the cow also revealed by these workers (Schams and Karg, 1969b), it is obvious that more frequent sampling may have revealed other significant changes in L H levels. Summary Progesterone, progestins, L H and corticoid concentrations were determined in peripheral plasma or serum from seven cows. During the trial three of the animals previously cycling regularly, failed to cycle. One of the seven returned to estrus when daily blood sampling was discontinued. Two of these four cows failed to show estrus or ovulate. The fourth cow, from which blood was drawn at the beginning of estrus failed to ovulate. The four cows that showed estrus ovulated at 0 to 52
hr. from estrus and were bled at 4-hr. intervals during estrus to ovulation and daily thereafter to the next estrus period. LH, determined by radioimmunoassay, peaked sharply coincident with the onset of estrus, rising from nearly zero to between 2.5 and 61 ng/ml. A second, less recognizable peak was again observed at the second estrus. Progesterone, progestins and corticoids were determined by competitive protein binding procedures. Progesterone levels were lowest 20 hr. after detection of estrus, peaked briefly between days 2 to 3, and fell on day 4 to a level similar to that observed at 20 hours. The concentration rose gradually to remain above 2.50 ng/ml between days 6 and 12 with peaks on days 9 and 12 and declined thereafter to the next estrus. Progestin levels were significantly lower than progesterone values ( P < .005) indicating interference with the competitive protein binding procedure~ Corticoid levels were higher during estrus and averaged higher in animals failing to show normal cyclic behavior.
PROGESTERONE Literature
AND
LUTEINIZING
Cited
Anderson, R. R. and W. It. McShan. 1966. Lutenizing hormone levels in pig, cow, rat plasma during the estrous cycle. Endocrinol. 78:976. Dobrowolski, W., E. Stupnicka and E. Domanski. 1968. Progesterone levels in ovarian venous blood during the oestrus cycle of the cow. J. Reprod. and Fertil. 15:409. Estergreen, V. L. and G. K. Venkataseshu. 1967. Positive identification of corticosterone and cortisol in jugular plasma of dairy cattle. Steroids. 10:83. Gomes, W. R. and R. E. Erb. 1965. Progesterone in bovine reproduction: a review. J. Dairy Sci. 48: 314. Gomes, W. R., V. L. Estergreen, Jr., O. L. Frost and R. E . Erb. 1963. Progestin levels in jugular and ovarian venous blood, corpora lutea and ovaries of the nonpregnant bovine. J. Dairy Sci. 46:553. Gupta, S. K. and G. S. Pope. 1968. Variation in the level of progesterone in the systemic plasma of the cow. J. Endocrinol. 40:XII. (Abstr.). Karg, H., D. Aust and S. Bohn. 1967. Versuche zur bestimmung des Luteinisierungshormons (LH) im blut yon Kuhen unter berucksichtigung des zyklus. Suchthyg. 2: 55. Li, C. R. 1964. Statistical Inference I. Edwards Brothers Inc. Ann Arbor, Mich. Murphy, B. E. P. 1967. Some studies of the protein binding of steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive proteinbinding radioassay. J. Clin. Endocrinol. Met. 27: 973. Neill, ~I. D., E. D. Johanssen, J. K. Datta and E. Knobil. 1967. Relationship between the plasma
HORMONES
103
levels of luteinizing hormone and progesterone during the normal menstrual cycle. J. Clin. Endocrinol. Met. 27 : 1167. Niswender, G. D., C. C. Kaltenbach, R. P. Shumway, J. N. Wiltbank and D. R. Zimmerman. 1965. Alteration of ovarian activity in cycling beef heifers with small daily injections of estradiol. J. Anita. Sci. 24:986. Niswender, G. D., L. E. Reichert, Jr., A. R. Midgley, Jr. and A. V. Nalbandov. 1969. Radioimmunoassay for bovine and ovine lutenizing hormone. Endocrinol. 84:1166. Plotka, E. D., R. E. Erb, C. J. Callahan and W. R. Gomes. 1967. Levels of progesterone in peripheral blood plasma during the estrous cycle of the bovine. J. Dairy Sci. 50:1158. Rakha, A. M. and H. A. Robertson. 1965. Changes in levels of follicle-stimulating hormone and lutenizing hormone in the bovine pituitary gland at ovulation. J. Endocrinol. 31:245. Schams, D. and H. Karg. 1969a. Radioummunologische LH-Bestimmung im Blutserum vom Rind unter besonderer Berucksichtigung des Brunstzyklus. Acta Endocrinol. 61 : 96. Schams, D. and H. Karg. 1969b. Zeitlicher Verlauf und analytische Erfassbarkeit des endogen bzw. exogen erhoten Blutstiegels an Luteinisierungshormon beim Rind. Suchthyg. 4:61. Shemesh, M., N. Ayalon and H. R. Lindner. 1968. Early effect of conceptus on plasma progesterone levels in the cow. J. Reprod. and Fertil. 15:161. Stabenfeldt, G. H., E. L. Akins, J. A. Holt, L. E. McDonald and M. C. Morrissette. 1968. Comparative levels of progesterone in the peripheral blood of cow, gilt and ewe during the estrous cycle. Fed. Proc. 27:689.
