genized cow, or other cows, and/or extensive paint mark- ings from a chinball ..... 49:617-621. 8. Cooper DA, Carver DA, Villeneuve P, Silvia WJ, Inskeep EK.
BIOLOGY OF REPRODUCTION 54, 531-537 (1996)
Embryotoxicity of a Regressing Corpus Luteum
in Beef Cows Supplemented with Progestogen' William I. Buford, Nasim Ahmad, F. Neal Schrick, 3 Roy L. Butcher, 4 Paul E. Lewis, and E. Keith Inskeep 2
Division of Animal and Veterinary Sciences, West Virginia University Morgantown, West Virginia 26506- 6108
ABSTRACT Postpartum cows with short-lived corpora lutea produce embryos that arrive at the uterus, but pregnancy rates are low even with exogenous progestogen. Four experiments were conducted to determine whether prostaglandin (PG) F2., known to cause early luteolysis, could have adirect effect on embryonic loss. Exogenous progestogen was injected s.c. twice daily ineach experiment, starting 3 or 4 days after estrus (day of estrus = Day 0). Nonlactating, cycling beef cows were mated and injected i.m. every 8h on Days 4through 7 (experiment 1) or 5 through 8 (experiment 3) with either 15 mg PGF2. or 3 ml saline. In experiment 1, cows in a third group received 1 g flunixin meglumine i.m. every 8 h.Ten of 18 PGF2 -treated cows in experiment 3were luteectomized on Day 5. Pregnancy rates were higher (p < 0.05) in cows given saline or flunixin meglumine (5of 7)than in cows given PGF 2, (1of 5) in experiment 1,and in cows given saline (6of 9) or given PGF 2, and luteectomized (8of 10) than in cows given PGF2. (2of 8) inexperiment 3. Postpartum beef cows, mated at weaninginduced first estrus, received i.m. injections every 8 h on Days 4 through 9 of 3 ml saline or 1g flunixin meglumine (experiment 2); 14 flunixin meglumine-treated cows were luteectomized on Day 7. Pregnancy rates were higher in cows given flunixin meglumine and luteectomized (7 of 14) than in cows given saline (4 of 15) or flunixin meglumine alone (3of 15; p < 0.05). In experiment 4, postpartum cows were luteectomized or sham-operated on Day 5. Pregnancy rates (2of 13 and 2 of 14, respectively) did not differ. Thus, both reduction of endogenous PGF2. and luteectomy were required for embryo survival in postpartum cows with short-lived corpora lutea, whereas luteectomy alone prevented effects of exogenous PGF 2. in cycling cows. INTRODUCTION
toxic effects of PGF2, seemed to be a logical explanation. Indeed, PGF2 . inhibited development of rabbit [91 and rat [10] embryos in vitro. Furthermore, pretreatment of postpartum cows with norgestomet, which produced a normal luteal phase, reduced concentrations of PGF 2 , in uterine flushings on Day 6 compared to concentrations in cows with short luteal phases [7], and the quality of the embryo tended to be negatively correlated to the concentration of PGF2Q in the uterine flushings [7]. Four experiments were conducted to examine effects of PGF2 . and(or) of regressing CL on embryo survival. In two of the experiments, cycling cows treated with PGF2 . were used as a model. The objective of experiment 1 was to determine whether exogenous PGF2,, or flunixin meglumine, an inhibitor of synthesis of PGF2,, affected pregnancy rate directly in cycling cows supplemented with exogenous progestogen. The objectives of experiment 2 were to determine 1) whether pregnancy rate would be improved in postpartum cows that have short luteal phases by suppressing endogenous secretion of PGF2,, with flunixin meglumine during supplementation with exogenous progestogen and 2) whether luteal maintenance per se was important to any effect of flunixin meglumine during supplementation with exogenous progestogen. The objective of experiment 3 was to determine whether luteectomy would abrogate detrimental effects of exogenous PGF2 ,, on pregnancy in cycling cows supplemented with exogenous progestogen. Experiment 4 was conducted to determine whether the regressing
Pregnancy rate in cows obviously will be nil if the corpus luteum (CL) regresses prematurely, because luteal progesterone is essential throughout pregnancy [1, 2]. Ovulation, fertilization, and early embryonic development did not differ in postpartum cows with short (< 14 days) or normal luteal phases [3]. However, replacement therapy with progestogens, at dosages that maintained pregnancy in unilaterally or bilaterally ovariectomized heifers or cows [4, 5], did not maintain pregnancy in cows with short luteal phases [3]. Transfer of good embryos into postpartum cows with short luteal phases on Day 7 [6] (day of estrus = Day 0) and transfer of embryos from postpartum cows with short luteal phases into cycling cows on Day 6 [7] yielded pregnancy rates only half of those obtained when comparable transfers were made into [6] or from [7] postpartum cows with normal luteal phases. Because the short luteal phase is caused by increased secretion of prostaglandin (PG) F2,, on Days 4 through 9 after the induction of ovulation [8], direct embryoAccepted October 16, 1995. Received August 11, 1995. 'This work was supported by USDA Grant 89-37240-4714 and by the Hatch Funds of the West Virginia Agricultural and Forestry Experimental Station, and is published with the approval of the Director as Scientific Paper No. 2524. 2Correspondence. FAX: (304) 293-3740. 3 Current address: Department of Animal Science, University of Tennessee, P.O. Box 1071, Knoxville, TN 37901-1071. 4Professor Emeritus, Department of Obstetrics and Gynecology, West Virginia University, Morgantown, WV 26506.
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CL was directly responsible for low pregnancy rates in postpartum cows supplemented with exogenous progestogen.
MATERIALS AND METHODS General Multiparous beef cows of mixed breeding, including Angus and crosses of Simmental and Hereford with Angus, in moderate body condition [11], were used in these studies. For cows used in experiments 1 and 3, calves had been weaned at 1 mo postpartum, and estrous cycles had been established. In postpartum cows (experiments 2 and 4), calves were weaned at 25-31 days postpartum to induce estrus and ovulation. Reproductive tracts were palpated per rectum for a normal uterus and ovaries in cycling cows, or for normal uterine involution and absence of CL in postpartum cows, before assignment to the experiments. Cows were observed for estrus (Day 0) twice daily with an androgenized teaser cow and penned continuously with one of two Angus bulls of known high fertility. Each teaser cow and each bull was fitted with a chinball marker with different colored ink. Cows were rotated every 12 h between bulls. In postpartum cows, twice daily observation for estrus began at calf removal and continued for 10 days or until the second day after estrus. Cycling cows that had not exhibited estrus during the first four days of observation were synchronized with PGF2,, (Lutalyse; Upjohn Company, Kalamazoo, MI) and estradiol benzoate (Sigma Chemical Co., St. Louis, MO), as described by Peters et al. [12]. Some cows were observed to be mated by the bull; others exhibited estrous activity (attempted mounting by bull, androgenized cow, or other cows, and/or extensive paint markings from a chinball marker) but were not observed to be mated by the bull. Each cow was inseminated artificially with semen of high fertility 12 h after estrous activity or mating was observed in all experiments and again 12 h later in postpartum cows (experiments 2 and 4). Ultrasonographic examination (Aloka 500 [Corometrics Medical Systems, Wallingford, CT] fitted with a 7.5 MHz rectal probe) was conducted at weaning (7 days after the initiation of blood sampling) to determine location of follicles with antral diameters > 5 mm and to evaluate presence or absence of a CL from a previous postpartum ovulation. At one and two days after onset of estrus, cows were scanned to assist in the detection of the first ovulation after weaning. Ovulation was considered not to have occurred before first estrus if no CL was observed by ultrasonography and peripheral concentrations of progesterone were below 1 ng/ mi. Concentrations of estradiol-17P and LH in combination with ultrasonographic records of follicular patterns were used to confirm that ovulation occurred within the normal interval (24-36 h) after observed estrous activity. Ultrasonography was used to determine the presence of CL and
follicles with antral diameters > 5 mm on Days 4 and 11 (experiment 1); Days 7, 10, 14, and 25 (experiment 2); Day 5 (experiment 3); or Days 10, 15, 20, and 25 (experiment 4). Pregnancy was determined in all cows by ultrasonographic visualization of a heartbeat within the embryo at 30 days postmating. Each cow received either 150 mg of progesterone (experiments 1 and 2; Sigma) or 6 mg of flurogestone acetate (experiments 3 and 4; G.D. Searle and Co., Chicago, IL) in corn oil, twice daily (s.c.) from Day 3.5 or 4.5, respectively, until cows were examined for pregnancy. Because flurogestone acetate does not cross-react with the antibody [13] used for assay of progesterone, luteal function in intact cows could be monitored in experiments 3 and 4. Flurogestone acetate was 20-25 times as active as progesterone in the Clauberg test and in blocking ovulation in ewes [14]; therefore, 6 mg of flurogestone acetate was considered equivalent to 150 mg of progesterone. Experiment 1. Effects of PGF2a or Flunixin Meglumine on PregnancyRates in Nonlactating Cows In fall 1992, 12 cows were allotted at random to 3 groups at breeding: saline (n = 4), PGF2 ,, (n = 5), or flunixin meglumine (Schering-Plough Animal Health Corp., Kenilworth, NJ) (n = 3). On Days 4 through 7 after estrus, either 3 ml of saline (i.m.), 15 mg of PGF,, (i.m.), or 1 g of flunixin meglumine (i.m.), respectively, was injected every 8 h. Flunixin meglumine inhibits prostaglandin endoperoxide synthetase, which is reflected by decreased peripheral concentrations of 15-keto 13,14-dihydro-PGF2 ,, (PGFM) for 6-8 h [15-17]. Jugular blood was sampled daily on Days 3 through 11 and then every other day until Day 30. Experiment 2. Effects of Flunixin Meglumine, with or without Luteectomy, on PregnancyRates in Postpartum Cows In spring 1993, 53 cows were allotted at random among three groups: control, flunixin meglumine, or flunixin meglumine and luteectomy. Seven cows were removed because peripheral concentrations of progesterone exceeded 1 ng/ ml before postweaning estrus. Two cows were removed because the LH surge was not synchronous with estrus. Final numbers of cows in each group were 15, 15, and 14, respectively. On Days 4 through 9 after estrus, 1 g of flunixin meglumine was injected (i.m.) every 8 h. Fourteen of the 29 cows that received flunixin meglumine were luteectomized transrectally on Day 7; the CL was removed by gentle pressure around the base of the gland with the thumb and forefinger and dropped into the body cavity. Jugular blood was collected once daily from 18 to 24 days postpartum through Day 7 after estrus. Additional samples were collected once daily in 20 cows on Days 4 through 10 after estrus.
LUTEAL FACTOR(S) IN EMBRYONIC MORTALITY
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TABLE 1. Summary of estrous response and luteal lifespan for cows in experiments 2 and 4. Experiment 2 Variable Cows (n) Interval to estrus (days) from: Parturition Weaning Estrous response (n) Standing Active, but not standing Percentage of cows with CL (mean CL area, mm2 )e on following days after estrus 5 7 10 14 15 20 25 30
Experiment 4
CON a
FMb
FM + LUTd
15
15
14
14
13
34.2 ± 0.7 3.6 + 0.4
33.0 + 0.7 4.5 + 0.5
33.5 ±+0.6 4.8 + 0.4
34.7 + 0.9 3.8 0.6
34.5 ±+0.9 3.9 0.6
10 5
8 7
8 6
100 (321) 93 (299) 87 (286)
100 (330) 100 (318) 100 (290)
100 (307) _f
CON
a
9 5
100 (209)
40 (270) 7 (377)
LUTc
10 3
100 (204)
57 (235) 43 (279) 43 (223) 36 (239) 14 (217)
47 (151) 7 (94)
a Control.
bFlunixin meglumine. cLuteectomy. dFlunixin meglumine plus luteectomy. eMeans based only upon CL remaining visible by ultrasonography at each stage. fLuteal lifespan shortened by luteectomy.
Experiment 3. Effects of PGF2Q and PGF2 , plus Luteectomy on Pregnancy Rates in Non-Lactating Cows In fall 1993, 27 cows were assigned at random among three groups: control (n = 9), PGF2Q (n = 8), or PGF 2 . plus luteectomy (n = 10). On Days 5 through 8 after estrus, either 3 ml of saline or 15 mg of PGF2 . was injected (i.m.) every 8 h. On Day 5, 10 cows that received PGF 2a were luteectomized transrectally, as described for experiment 2. Jugular blood was sampled 30 min and 3 h after initiation of treatment on Day 5, every 8 h on Days 5 through 8, and every 5 days from Day 10 until Day 30. Experiment 4. Effects of Luteectomy on PregnancyRates in Postpartum Cows In spring 1994, 48 cows were allotted at random between two groups: 1) no treatment or 2) luteectomy. Fourteen cows were removed because peripheral concentrations of progesterone exceeded 1 ng/ml before postweaning estrus. Seven other cows were removed because the LH surge was not synchronous with estrus. Transvaginal surgery [18] was performed on each cow on Day 5 to handle the ovaries (sham operation; n = 14) or to perform luteectomy (n = 13). Jugular blood was sampled once daily beginning 1824 days postpartum and continuing through Day 5 after estrus. Additional samples were collected once every 5 days on Days 10 through 30 after estrus. RIAs Blood samples were allowed to clot at 4C, and serum was collected after centrifugation at 1800 X g for 20 min in
experiment 1; plasma was collected after centrifugation of blood samples containing sodium citrate in experiment 2, and containing heparin in experiments 3 and 4. Plasma was collected in experiments 2 and 3, and samples were placed immediately on ice, in order to quantify PGFM without a contribution from the clotting process [19]. All samples were stored at - 20 0C until RIAs were conducted to quantify progesterone [20], PGFM [21], oxytocin [8, 22], estradiol-17 [23, 24], and LH [25]. Standard for LH was NIH-LH-B9. Intraand interassay coefficients of variation, respectively, were progesterone, 10.9% and 19.4%; PGFM, 4.3% and 10.4%; oxytocin, 4.6% (one assay); estradiol-17fi, 12.0% and 7.3%; and LH, 10.7% (one assay). Sensitivities of the assays were as follows: progesterone, 20 pg/tube (100 pl); PGFM, 34 pg/ ml (50 1l); oxytocin, 2.8 pg/tube (200 gl); estradiol-173, 0.5 pg/tube (1 ml); and LH, 0.025 ng/tube (100 aI).
StatisticalAnalysis
Data from all 39 nonlactating, cycling cows were analyzed (experiments 1 and 3). Of the 103 postpartum cows assigned to the studies, data were utilized from 71 cows (69%; 44 in experiment 2 and 27 in experiment 4) that ovulated within 10 days after weaning and showed sufficient estrous activity for insemination (naturally and/or artificially) at an appropriate time relative to ovulation. Pregnancy rates in each experiment were compared among groups by chi-square analysis using orthogonal contrasts. Changes in concentrations of oxytocin (experiment 3) and in mean concentrations of PGFM (experiment 3 and 20
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FIG. 2. Mean concentrations of PGFM in jugular plasma at 0 time (open bars), 30 min (black bars), 3 h (dotted bars), and 8 h (gray bars) after initiation of treatment in experiment 3. CON, controls (n = 9); PGF, PGF2,, (n = 8); PGF + LUT, PGF,,2 plus luteectomy (n = 10). Treatments within time period or time periods within treatment differ (p < 0.05) when letters above bars are different.
PregnancyRates
FIG. 1. Pregnancy rates in cycling (top; experiments 1 and 3)and postpartum (bottom; experiments 2 and 4)cows at 30 days after induced estrus. CON, controls; FM, flunixin meglumine; PGF, PGF2,; LUT, luteectomy; PGF + LUT, combined treatment; FM + LUT, combined treatment. PGF2,-treated cows in experiments 1 and 3 are models for control cows in experiments 2 and 4. Treatments differ (p < 0.05) within experiment when letters above bars are different.
In cycling cows, pregnancy rates at Day 30 were reduced by treatment with PGF,, (1 of 5; 20%) compared to saline or flunixin meglumine (5 of 7; 71%; p < 0.05) in experiment 1. This result was confirmed in experiment 3: pregnancy rates at Day 30 were reduced in cows treated with PGF2 , (2 of 8; 25%) compared to cows treated with saline (6 of 9; 67%; p < 0.05). Luteectomy of cows treated with PGF2 ,, restored pregnancy rate (8 of 10; 80%; p < 0.05 compared to PGF2 , alone; experiment 3). Data for both experiments are summarized in Figure 1 (top).
cows in experiment 2) were compared by analysis of variance for a split-plot design, with treatment in the main plot and time in the subplot, by the general linear models procedure of the Statistical Analysis System [26].
RESULTS
Proportions of postpartum cows that exhibited standing estrus (63% overall in experiments 2 and 4) did not differ among treatments (Table 1). Average luteal life span did not differ in cows treated with flunixin meglumine or saline, as indicated by ultrasonographic records of presence and area of CL (Table 1) in experiment 2 or by ultrasonography and progesterone in plasma in experiment 4. The interval from parturition or weaning until estrus, and degree of estrous response, did not differ among treatments for either experiment 2 or 4 (Table 1).
FIG. 3. Effect of PGF2 . on mean change from 0 time in concentrations of oxytocin (OT) in jugular plasma at 30 min (black bars) and 3 h (gray bars) after initiation of treatment in experiment 3. CON, controls; PGF, PGF2,; PGF + LUT, PGF2~ plus luteectomy. Means differ (p < 0.05) within time period when letters above bars are different.
LUTEAL FACTOR(S) IN EMBRYONIC MORTALITY Pregnancy rates at Day 30 in experiment 2 did not differ between cows treated with saline (4 of 15; 27%) and all cows treated with flunixin meglumine (10 of 29; 34%; p > 0.1). However, pregnancy rates in cows treated with flunixin meglumine were increased by luteectomy (7 of 14 or 50% in luteectomized cows compared to 3 of 15 or 20% in intact cows; p < 0.05). CL were maintained in all 4 cows treated with saline and in 2 of 3 intact cows treated with flunixin meglumine that remained pregnant. In the third cow treated with flunixin meglumine, in which the CL had regressed before Day 25 after estrus, the embryo was alive on Day 30, but died by Day 33. In experiment 4, pregnancy rates at Day 30 did not differ in control cows (2 of 14; 14%) and luteectomized cows (2 of 13; 15%; p > 0.1; Fig. 1, bottom). The two pregnant control cows maintained their CL. Overall, in cows with short luteal phases, pregnancy was maintained in 0 of 23 control cows and 1 of 12 cows treated with flunixin meglumine. Concentrations of Hormones In experiment 2, mean concentrations of PGFM did not differ among treatments in the 20 cows sampled 8 h after previous injection on Days 4 through 10 (overall mean = 132 + 18 pg/ml; p > 0.05). In experiment 3, as expected, mean concentrations of PGFM at 30 min, 3 h, and 8 h after the initial injection of PGF2a were higher in treated cows than in cows that received saline (p < 0.05; Fig. 2). At 30 min after initial treatment with PGF2Q, concentrations of PGFM were higher in luteectomized cows than in cows that remained intact (p < 0.05; Fig. 2). By 8 h after injection of PGF2,, or saline, both initially (Fig. 2) and on Days 5 through 8, mean concentrations of PGFM remained higher in cows treated with PGF2 . than in saline-treated cows (p < 0.05; Fig. 2), but were not affected by luteectomy. Concentrations of oxytocin differed among groups at 0 time (injection of PGF2,, or saline) on Day 5 in experiment 3 (p < 0.05). Therefore, data for 30 min and 3 h were analyzed as changes from 0 time (Fig. 3). Thirty minutes after the initial injection of PGF2,,, oxytocin secretion had increased in intact cows (PGF 2a versus saline; p < 0.05), but not in luteectomized cows (PGF2,, plus luteectomy versus saline; p > 0.05). Changes in oxytocin from 0 time did not differ among groups at 3 h after treatment. DISCUSSION Frequent injections of PGF 2, on Days 4 through 7 or 5 through 8 in mated cycling cows (experiments 1 and 3) caused regression of the CL as expected [27, 28]. Despite replacement therapy with progestogen, treatment with PGF2, reduced pregnancy rate to 23% (versus 69% in controls), a value similar to that obtained in untreated postpartum cows after early weaning (21%) in experiments 2 and 4. Flunixin meglumine exerted no detrimental effects on
535
embryo survival in cycling cows (experiment 1). Embryonic survival was not improved when postpartum cows were treated with flunixin meglumine (experiment 2). Although life span of the CL was not extended, ultrasonography was not performed frequently enough to detect a small increase in persistence of a CL, so detection of luteal lifespan was not precise. Furthermore, progesterone was not measured after Day 7 or 10, and serum progesterone concentrations reflected both endogenous and exogenous progesterone. Also, by 8 h after each treatment with flunixin meglumine, values for PGFM were not different from those of controls. Although PGFM is not a sensitive indicator of PGF2, [8], the 1-g dose of flunixin meglumine was expected to reduce PGF2,, for only 6 to 8 h [15], so small periodic rises in PGF2,, at 8-h intervals or an increase in PGF20 after the termination of treatment [29] could have caused the CL to regress in the cows treated with flunixin meglumine. Pregnancy rate in postpartum cows expected to have short luteal phases was increased significantly when luteectomy was used in conjunction with flunixin meglumine and exogenous progestogen (experiment 2). In cycling cows supplemented with exogenous progestogen, luteectomy with PGF2,, produced a significant increase in pregnancy rate (80%) over treatment with PGF2,, alone (25%; experiment 3). Fertility was restored by luteectomy (control fertility was 670/o), even though concentrations of PGFM were significantly higher in luteectomized cows than in other treatment groups for at least 8 h after surgery. Hence, experiment 4 was conducted to determine whether an embryotoxic factor from the regressing CL was the only direct cause of the lower fertility in postpartum cows with short luteal phases. Pregnancy rates were not increased by luteectomy alone. Therefore, both premature secretion of PGF2,, and a factor released by the regressing CL may contribute to embryonic death in the postpartum cow with a short luteal phase. Two possible mechanisms for involvement of both the regressing CL and uterine PGF2 a in embryonic death can be considered. First, the entire embryotoxic effect may be due to PGF2 a. Endogenous secretion of PGF2 a would be more continuous in postpartum cows [8] than the pattern produced by injections of PGF2a every 8 h in the cycling cow, and the CL could be an important secondary source of PGF2Q [30]. Thus, removal of the CL and treatment with flunixin meglumine may reduce PGF2a more than either alone. Such an effect was not detected by measuring PGFM, but PGFM is a relatively insensitive indicator of secretion of PGF 2 ~ [8]. If the role of the CL is indirect via release of luteal oxytocin [31-33], which in turn released more uterine PGF 2, [32-34], removal of the CL in cycling cows should yield a less continuous pattern of exposure to PGF 2,,. Luteectomy of cycling cows prevented the rise in oxytocin in response to injections of PGF 2. but increased concentrations of PGF2 . in cycling cows temporarily, as was observed after manip-
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BUFORD ET AL.
ulation of the reproductive tract for 2 min in postpartum cows [35]. Increased secretion of PGF 2. in postpartum cows with short luteal phases was not necessarily associated with increased secretion of luteal oxytocin. Peter et al. [36] found increased concentrations of oxytocin in association with increased concentrations of PGFM in dairy cows with short luteal phases, but Cooper et al. [8] did not find consistent increases in oxytocin in association with increases in PGF2,, in plasma from the inferior vena cava of beef cows. Luteectomy alone (experiment 4) may have increased concentrations of PGF2 a considerably more than luteectomy during treatment with flunixin meglumine (experiment 2) in postpartum cows, thus overcoming the beneficial effect of removing the CL (as a source of either oxytocin or PGF2 ,). Second, the CL may produce an embryotoxin other than PGF2 , and the two may act together to reduce embryonic survival. Oxytocin could be one candidate. Cycling cows with an intact CL released oxytocin after the initial injection of PGF 2a (experiment 3) and had lower pregnancy rates than those that were luteectomized, in which oxytocin remained at preinjection concentrations. Exogenous oxytocin injected into cows for the first 14 days after mating resulted in maintenance of embryos to Day 15 of pregnancy in only 42% compared to 74% of control cows [371. In the remaining cows treated with oxytocin, either the CL had regressed or the CL was present without the presence of an embryo [37]. Treatment with oxytocin for the first 12-14 days of pregnancy in ewes reduced pregnancy rates, but in some cases of pregnancy loss, CL function was maintained [381. None of the animals in these studies received supplemental progestogen, so it was not possible to determine whether embryonic loss or regression of the CL occurred first in the termination of pregnancy. Embryos in early stages of development (Days 6 through 7 after estrus, when the morula develops into the blastocyst) are more susceptible to mortality [39, 40]. While oxytocin is released rapidly from the CL in response to PGF2 . [41-44], it also is released from the CL of pregnancy (ewe, Days 16-17 [45]; cow, Days 14-18, [46]). Therefore, if oxytocin is embryotoxic, it may be so during only a limited period. An embryotoxic factor other than oxytocin could be released during luteal regression. Glycoproteins of the class II major histocompatibility gene complex are found on macrophages [47, 48], and increased infiltration of macrophages was observed during luteal regression [49-51]. Macrophages and neutrophilic granulocytes are rich sources of the pleiotropic cytokines, tumor necrosis factor alpha (TNFa) and interleukin-1 [481, which stimulate production of PGF2a [52] by bovine luteal cells [53] but do not affect steroidogenesis [54]. TNFa, an inflammatory cytokine, caused fetal death in pregnant mice [55], was at its highest concentration during luteal regression in the rabbit [50], and increased in the bovine CL during regression [56]. Therefore, a variety of secretory products of immune cells [52, 57]
could be secreted or cause luteal cells to release other products during the regression of the CL. Apoptosis, a mechanism of controlled cell death that involves structural changes different from classical coagulative necrosis [58], has been implicated as a mechanism underlying regression of the CL in ewes [591 and cows [60]. One [61, 62] or two [63] tissue inhibitors of metalloproteinases were identified during luteolysis. Products associated with these factors could influence embryonic survival during shortened luteal phases or periods of reduced luteal function in the postpartum cow [64]. In conclusion, multiple injections of PGF2,, early in the luteal phase reduced pregnancy rates in cycling cows despite supplementation with progestogen. Thus the cycling cow treated with PGF2a showed promise as a model for the postpartum cow with a short luteal phase. However, both suppression of PGF2,, and removal of the regressing CL were required for embryonic survival in the early postpartum cow with a short luteal phase, whereas luteectomy alone effectively restored fertility in the cycling cow treated with PGF2,,. Further research is needed to better understand the mechanisms that destroy an embryo in a cow with a short luteal phase after the first postpartum ovulation. ACKNOWLEDGMENTS The authors thank Dr. G.D. Niswender, Colorado State University, Fort Collins, CO, for antiserum to LH; Dr. L.E. Reichert, Jr., Albany Medical College, Albany, NY, for purified LH; Dr. E.C. Townsend for assistance in statistical analysis; Dr. John Chenault of The Upjohn Company for prostaglandin F2 (Lutalyse); and Larry Whetsell and Jill Houghton for conduct of the RAs.
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