Effect of Bovine Interferon-7 on Progesterone Concentrations in. Body Temperature and Plasma Cyclic Dairy Cows

Effect of Bovine Interferon-7 on Progesterone Concentrations in Body Temperature and Plasma Cyclic Dairy Cows M. D. MEYER, P. J. HANSEN, and W. W. THATCHER1 Department of Dairy and Poultry Sciences University of Florida Gainesville 3261 1-0920 M. DROST Large Animal Clinical Sciences College of Veterinary Medicine Gainesville 3261 14136 R. M. ROBERTS Department of Animal Sciences University of Missouri 158 Animal Sciences Research Center Columbia 65211 ABSTRACT We examined the effect of intramuscular injections of recombinant bovine interferon-7 on basal body temperature and plasma concentrations of progesterone in cows. Six mature, nonlactating Holstein cows were synchronized for estrus and assigned randomly to a replicated 3 x 3 Latin square design to receive 0, 1, or 5 mg of recombinant bovine interferon-7 intramuscularly on d 9, 12, or 15 of the estrous cycle. Rectal temperatures were measured, and blood was collected just prior to injection, at hourly intervals for 8 h, and at 10 and 12 h after injection. Treatment with 1 and 5 mg of recombinant bovine interferon-.z caused a rise in body temperature that peaked at 6 h (39.5 f.13"c) and 4 h (40.3 *.13"C), respectively. Analysis of homogeneity of regression for response curves indicated that 5 mg of recombinant bovine interferon-7 decreased plasma progesterone concentrations. This effect was not detected for the 1-mg dose of recombinant bovine interferon-7. Intramuscular administration of 5 mg of recombinant bovine interferon-7 caused a hyperthermic response that was tem- Received December 7, 1994. Accepted February 22, 1995. 'To whom correspondence should be addressed: PO Box 110920. porally associated with the decrease in plasma progesterone. Intramuscular administration of 1 mg of recombinant bovine interieron-.r caused a slight rise in temperature occurring 4 h later than that caused by 5 mg of recombinant bovine interferon-7, and no decrease in plasma progesterone was detected. Recombinant bovine interferon-7, administered at a 1-mg dose, warrants further study to determine whether it will alter survival rate of cattle embryos. (Key words: interferon-7, hyperthermia, progesterone) Abbreviation key: b = bovine, CL = corpus luteum, IFN = interferon, rb = recombinant bovine. INTRODUCTION Interferon (IFN>7 is a unique class of Type I IFN produced by the trophoblast of ruminants and implicated in maintenance of the corpus luteum (CL) during pregnancy (21). Thatcher et al. (23) estimated that 10 to 15% of pregnancies of cows fail because the conceptus produces insufficient IF"-.r to maintain CL lifespan. Accordingly, increased pregnancy rate of cows might be possible if IFN-7 or related molecules were administered at appropriate times during early pregnancy. One related molecule is IFN-a. A recombinant form of bovine (rb) IFN-a has approximately 50% sequence identity of bovine (b) 1995 J Dairy Sci 78:147&1476 1470

INTERFERON-7 AND HYPERTHERMIA 1471 IFN-7 (8) and extends CL lifespan and interestrous interval when administered i.m. (18, 20). However, hyperthermia and a temporal decrease in plasma progesterone concentrations occur upon initial administration of rbifn-a (1, 16, 20). The hyperthermia appears to be dependent on prostaglandin production because a prostaglandin synthesis inhibitor, given concurrently with rbif'n-a, blocks the hyperthermia response (1). Barros et al. (2) reported that rbifn-a decreased fertility in heifers, probably because of the induction of adverse physiological side effects. Therefore, rbifn-a is not a useful agent to enhance cattle fertility. Both rbifn-7 (14) and recombinant ovine IFN- 7 (13) extended CL lifespan without hyperthermic side effects when they were infused into the uterus. Bovine IFN-7 has yet to be tested for its effects on CL lifespan, pyrogenicity, and plasma progesterone when given intramuscularly. The objective of this experiment was to determine whether intramuscular administration of bifn-7 caused physiological effects on progesterone secretion and body temperature that could be inimical to pregnancy. cows MATERIALS AND METHODS Twelve cyclic, nonlactating Holstein cows were synchronized for estrus by placing a previously used CIDR-B progesteronereleasing device (Eazibreed CIDR-B; CHH Agricultural Division, Hamilton, New Zealand) containing approximately 1.14 g of progesterone (24) in the vagina of cows for 7 d. Lutalyse@ (25 mg i.m.; Upjohn Co., Kalamazoo, MI) was given 1 d prior to removal of the CIDR device. Tailheads were painted with blue paint (Impervo; Benjamin Moore Co., Jacksonville, FL) on d 6, and yellow crayon marker (Paint Stick; LaCo Industries, Chicago, IL) was applied over the paint on d 7 to aid in estrus detection (12). Cows were observed for 30 min twice daily (0700 and 1900 h) to detect estrus from 1 to 4 d after removal of the CIDR-B device. Six cows that exhibited estrus on d 2 after removal of the CIDR-B device were assigned randomly to a replicated 3 x 3 Latin square design (three cows per square; two squares) with repeated measurements over time to receive 0, 1, or 5 mg of rbifn-7 i.m. on d 9, 12, or 15 of the estrous cycle. Each cow received a different sequence of the three doses over the 3 d of treatment, resulting in two cows receiving one of the dosage levels on a given day in each square. Cows were maintained on limited pasture and fed peanut hay and corn silage throughout the experiment at the University of Florida Dairy Research Unit (Hague, FL). Blood sampling was conducted on alternate days from synchronized estrus (d 0) until d 9 for progesterone determinations in plasma. Ten milliliters of blood were collected into heparinized tubes (Vacutainer; Becton- Dickinson, Rutherford, NJ), and plasma was then prepared by centrifugation (at 1200 x g for 30 min at 4 C) three times per week following synchronized estrus. Administration of IFN-T All solutions for intramuscular injection were prepared in buffer (20 mm Tris.HC1 and 150 mm NaCI), ph 8.0, prepared in USP grade water (.25 units of endotoxidml; Baxter Health Care Co., Roundlake, IL).The rbifn-7 (9) was prepared to contain 1 or 5 mg of rbifn-7 in 16 ml of buffer. Additionally, 4 mg of BS (radioimmunoassay grade, fraction V; United States Biochemical Corp., Cleveland, OH) were added to the 1-mg dose so that a total dose of 5 mg of protein was injected for all treatments. Doses of rbifn-7 were based on the amounts of rbifn-a previously shown to cause an increase in rectal temperature or a decrease in plasma progesterone when administered intramuscularly to cattle (1, 2, 16, 20). Control treatment (0 mg of rbim-7) was 5 mg of BSA in buffer. Solutions were sterilized by using a.45-pm filter (Acrodiscm; Gelman Sciences, Ann Arbor, MI) and stored at 4 C until the day of use. Intramuscular injections (16 ml) were split, and one-half the assigned dose of rbifn-7 was given in each side of the cow's hindquarters on d 9, 12, or 15 after synchronized estrus (d 0). Cows were fitted with indwelling jugular catheters on d 9, 12, and 15 (V/9; Bolab Inc., Lake Havasu City, AZ), and blood was collected at 1-h intervals for 8 h and at 10 and 12 h after injection. Plasma was harvested by centrifugation (1200 x g for 30 min at 4 C) and used to measure concentrations of progesterone.

1472 MEYER ET AL. Rectal temperatures were monitored by a digital thermometer (Becton-Dickinson, Franklin Lakes, NJ) and recorded just prior to i.m. injection and at concurrent times of blood sampling. Cows were observed twice daily for estrus as previously described following the last day of i.m. injection of rbifn-7. Progesterone Determination Concentrations of progesterone in plasma were measured by radioimmunoassay (10). A hexane-benzene mixture (2: 1, vol/vol) was used to extract 50, 100, or 200 p1 of plasma. A 1:30,000 antiserum dilution achieved an assay sensitivity of 31.2 pg per tube. Intraassay and interassay coefficients of variation were 13.9 and 8.2%, respectively. Statistical Analysis Changes in plasma progesterone and rectal temperature were analyzed by using the general linear models procedure of SAS (22). The mathematical model included variability from dose (0, 1, or 5 mg of rbifn-.r), cow, day, hour, and residual error. Homogeneity of regression analyses were conducted to evaluate differences among doses over time. Orthogonal contrasts (0 vs. 1 and 5 mg of rbifn-7; 1 vs. 5 mg of rbifn-7) were performed to analyze differences among time trends for doses of rbifn-7. RESULTS Cows receiving 5 mg of rbifn-7 had side effects: trembling in the rear quarters; rapid, shallow breathing; ears laid back; and acute loss of appetite. Onset of these side effects occurred approximately 1 h after injection and lasted from 2 to 6 h. Side effects appeared to be associated temporally with rise in rectal temperature. No side effects were observed in cows treated with 0 or 1 mg of rbifn-7. Day effects were not significant for changes in rectal temperature (Table 1). However, dose x hour trends in rectal temperature were significant (P <.01). Orthogonal contrasts detected differences among regression curves for rectal temperature of 0 mg versus 1 and 5 mg TABLE 1. Least squares analysis of variance, tests of homogeneity of regression, and orthogonal contrasts of rectal temperature and plasma progesterone responses in cows treated with recombinant bovine interferon-r (rbifn-7). source Dose (D) cow (0 Day (4 Residual 1 Hour' Residual 2 Rectal temperature Plasma progesterone Emor term, df MS df MS residual 2,001 2 9.55 1 5 2.03 5 54.54 1 2.59 2 6.56 1 8 1.10*** 8 61.86*** 2 5 1.52*** 4 9.542 2 180,118 180 4.56 Error term Dose Orthogonal contrast4 Order of Item regression dfz MSZ df MSD~ A B Temperature 5 165.12 10 1,46*** e** *** *** *** Progesterone 4 168 4.56 8 10.53* * 'See tests of homogeneity of regression of fifrh- (rectal temperature) and fourth- (progesterone) order curves. *When individual curves were generated for each treatment group. 3For the difference in residuals between fitting one pooled curve versus fitting an individual curve far each treatment group. %rthogonal contrasts: A = 0 versus 1 and 5 mg of rbifn-7; B = 1 versus 5 mg of rbifn-7. *P <.lo. **P <.05. ***P <.01 Ioumal of Dairy Science Vol. 78, No. 7, 1995

INTERFERON-r AND HYPERTHERMIA 1473 of rbifn-7 (P <.01; Table 1) and 1 versus 5 mg of rbifn-7 (P <.01; Table 1). The 5-mg dose of rbifn-7 caused a rise in temperature from about 2 to 7 h after injection, and peak temperature occurred at 4 h (40.3 f.13"c; Figure 1, panel c). The 1-mg dose of rbifn-7 caused a slight rise in temperature that peaked later than the rise for 5 mg of rbifn-7 (6 vs. 4 h; Figure 1). The duration of the rise in temperature was approximately 6 h, which was similar to that of the group treated with 5 mg of rbifn-7. However, peak response was only 39.5 f.13"c by 6 h after injection (Figure 1). Regression curves were examined for plasma progesterone responses to the different doses of rbifn-7 (Figure 2). Orthogonal contrasts detected differences in regression curves for plasma progesterone responses of 0 mg versus 1 or 5 mg of rbifn-7 (P e.01; Table 1) and 1 versus 5 mg of rbifn-7 (P c.01; Table 1). Examination of regression curves revealed no difference in plasma progesterone between groups treated with 0 or 1 mg of rbifn-7 (Figure 2). In contrast, plasma progesterone concentrations declined for the group treated with 5 mg of rbifn-7; the nadir was reached at 6 h after injection (Figure 2). The decline in plasma progesterone concentrations was associated temporally with the rise in rectal temperatures of the cows in the treatment group that received 5 mg of rbifn-7. Cows were observed for estrus through d 23 of the treatment estrous cycle. The 2 cows receiving a control dose of 0 mg of rbifn-7 on a y = 38.231 -.005x +, 089~~ -,025~3 +, 003~~ 391.4---- 38 37 I e L I I I 42 - r b c 38 y = 38.319 -,061~ +, 158~~ -,029~~ +, 002~~ - 42 41 [y = 38.332-.453x +, 801~~ -,217~3 C +.021x4- h L c = 7.163 +.852x -,055~~ -,014~~ -.001x4 : 6 s '? - E 1 y = 8.752 -,148~ +.199x2 -,014~~ -.OOIx4 b C 37 I i 1 I I I I 0 2 4 6 8 1 0 1 2 Time (h) Figure 1. Effect of recombinant bovine interferon-7 (rbifn-7) on rectal temperature. Cows received 0, 1. or 5 mg of rbifn-s i.m. on d 9, 12, or 15 after estrus, and rectal temperatures were recorded hourly from 0 to 8 h and at 10 and 12 h after injection. Data were pooled over days and shown as least squares means and regression lines for treatment groups receiving doses of 0 mg (a), 1 mg (b). and 5 mg (c) of rbifn-s. 0 - y = 6.441 + 2.710~ -,006~~ - 1.215~~ +.159x3 - I I I J

1474 h4eyer ET AL. d 12 returned to estrus on d 17 and 21 of the estrous cycle. The 4 cows that received 1 or 5 mg of rbifn-7 on d 12 of the estrous cycle had not returned to estrus by d 23. DISCUSSION Results indicate that IFN-7 shares with IFNa the ability to regulate body temperature and to cause an acute decrease in plasma progesterone concentrations. However, results also indicate the possibility of identifying a dose of IFN-7 that has minimal side effects and might therefore be useful for testing the fertilityenhancing effects of IFN-7. Five milligrams of rbifn-7 caused a hyperthermic response that peaked at a rectal temperature of 40.3'C, a magnitude similar to that caused by injection of 1.25 to 20 mg of rbifna (1, 2, 16, 20). The slight rise in rectal temperature caused by injection of 1 mg of rbifn-7 (Figure 1; peak temperature, 39.5-C) did not reach the high temperature (240 C) caused by the 1.25-mg intramuscular injection of rbifn-a in a previous study (16). Intramuscular injection of 5 mg of rbifn-7 decreased plasma progesterone concentrations and was associated temporally with the increase in rectal temperature. An initial rise in plasma progesterone was seen in the group treated with 5 mg of rbifn-7. The reason for this is not known. Treatment with 1 mg of rbifn-7 did not alter plasma progesterone from those of controls (Figure 2). In contrast, earlier studies (1, 16) indicated that intramuscular injections of rbifn-a of as low as 1.25 mg depressed plasma progesterone. Both rbifn-7 (18) and rbifn-a (2, 18, 19, 20) extended CL lifespan and attenuated oxytocin-induced secretion of uterine PGF2,. The similarity of effects caused by 5 mg of rbifn-7 and varying doses of rbifn-a indicated that these IFN probably act through the same mechanisms to cause pyrogenic responses and decreases in plasma progesterone. Barros et al. (1) reported that inhibiting prostaglandin production with a prostaglandin synthesis inhibitor blocked the hyperthermic effect of rbifn-a. Production of E series prostaglandins in the hypothalamus apparently plays a role in the pyrogenic effect of rbifn-a (4, 15). Bovine IFN-7 and rbifn-a decrease prostaglandin E2 secretion by bovine en- dometrial explants (3) and endometrial epithelial cells (5). Perhaps rbifn-a and bifn- 7 regulate prostaglandin secretion differently in the uterus than in the hypothalamus. Decreases in plasma progesterone caused by rbifn-a appear to be mediated through a decrease in LH secretion from the anterior pituitary (1). However, higher doses of rbifn-7 are likely needed to cause the same magnitude of effects on body temperature and progesterone as rbifn- a. The diminished side effects of rbifn-.r relative to rbifn-a might be considered surprising because both bind to bovine endometrial receptor with almost identical affinities (11). However, different human IFN-a exhibit quite different pyrogenic effects on patients (6), and numerous examples exist in which a particular biological activity (e.g., antiproliferative potency) is poorly matched with receptor-binding affinity (6, 7). In addition, there are good reasons to think that the receptor system consists of one or more accessory polypeptides as well as a primary binding component (17). The varied cellular responses to different IFN subtypes probably depend upon which accessory polypeptides associate with the activated IFN receptor. This study was not designed to test the effect of rbifn-7 on the interestrous interval, but the 2 cows that received 0 mg of rbifn-.r on d 12 had estrous cycle lengths of 17 and 21 d, and cows that received either 1 or 5 mg of rbifn-7 on d 12 had estrous cycle lengths >23 d. The two cows that received 0 mg of rbifn-7 on d 12 received either 1 or 5 mg of rbifn-7 on d 9 or 15 of the estrous cycle. These results suggest that d 10 to 14 of early pregnancy may be critical if exogenous bifn-7 is to be administered i.m. for amplifying the process leading to maintaining the CL. Pregnancy rate was decreased in heifers when treated with 20 mg of rbifn-cy intramuscularly once daily from d 14 to 17 of the estrous cycle or 40 mg of rbifn-a intramuscularly on d 13 of the estrous cycle (2). Hyperthermic effects caused by rbifn-a decreased with repeated exposure (16, 20), but attempts to improve fertility by administering gradually increasing doses of rbifn-a to heifers failed and were associated with decreased pregnancy rates (2). Results from the present study indicated that the 1-mg dose of rbifn-7 does not

INTERFERON-7 AND HYPERTHERMIA 1475 have the same side effects as does the higher 5-mg dose of rbifn-7 or the reported detrimental effects of rbifn-a. In summary, the present study indicates that rbifn-7, administered at a 1-mg dose, does not cause the detrimental effects of hyperthermia and decreased plasma progesterone observed with a 5-mg dose of rbifn-7. Intramuscular administration of rbifn-.r in cattle will likely extend the CL lifespan and interestrous interval because intrauterine injection of rbifn-7 (14) and intramuscular injection of rbifn-a (2, 18, 19, 20) extend these reproductive responses. Therefore, a 1-mg dose of recombinant bim-7 administered intramuscularly warrants further testing for its effects on CL lifespan, interestrous interval, and potential ability to alter embryo survival of cattle. ACKNOWLEDGMENTS Funding was provided by USDA Grant Number 89-37240-4583 and National Institutes of Health Grant Number HD21896. 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