from That of Cycling Cows and the Effects of Progesterone Pretreatment

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1 BOLOGY OF REPRODUTON 33, (1985) The Postpartum nduced orpus Luteum: Functional Differences from That of ycling ows and the Effects of Progesterone Pretreatment L. M. RUTTER,2 T. D. ARRUTHER, and J. G. MANN Department of Veterinary Physiological ciences University of askatchewan askato on, askatchewan anada 7N OWO ABTRAT Anestrous postpartum (PP) Hereford cows (n=41) were used to compare corpora lutea (L) from gonadotropin-releasing hormone (GnRH)-induced ovulation with L from cycling cows. Postpartum cows were injected i.m. daily with 1 mg progesterone (P4) or oil on Days 25 through 28 PP and then given 2.ig GnRH i.m. on Day 3 PP. orpora lutea were removed from one-half of the PP cows in the oil- and P4-treated groups 6.5 days after GnRH injection, and from the cycling cows 7 days after estrus. ntact PP cows were used to evaluate cycle length. Blood was collected daily from all PP cows from Day 25 PP through luteectomy and on Days 9, 11, and 13 post-gnrh from the oil- and P4-intact cows to determine short (HORT) versus normal (NORM) luteal phases. ycling cows were bled daily from estrus until L removal. NORM PP cows had higher (P<.l) P4 levels than did HORT PP cows from Day 7 through Day 13 post-gnrh, and more (P<.5) P4 -intact cows were NORM compared with oil-intact cows (45.5% vs. 14.3%, respectively). orpora lutea from cycling cows were heavier (P<.5) and had a higher luteinizing hormone (LH) receptor concentration (P<O.5), but L P4 concentration did not differ from PP cows. orpora lutes weight, LH receptor and P4 concentration, and in vitro P4 production were similar in the oil-and P4-treated PP cows. NORM cows had heavier L (P<.5) than HORT cows, although P4 content and LH receptor concentration did not differ. Pretreatment of PP cows with P4 increased the proportion of cows with normal luteal phases after induced ovulation. However, the cause of the short life span of some induced L could not be determined from the functional characteristics measured in this study. NTRODUTON A prolonged interval from parturition to the onset of normal estrous cyclicity is a major cause of decreased reproductive efficiency in the beef cow. ontrolled suckling regimens, gonadotropin-releasing hormone (GnRH) administration, and/or steroid treatment have all been shown to be capable of inducing ovulation in postpartum (PP) anestrous beef cows. However, the luteal phase of the first spontaneous (LaVoie et al., 1981; Manns et al., 1983) or induced (Lishman et al., 1979; Kesler et al., 1981; Walters et al., 1982) estrous cycle is frequently shorter than normal. This first short cycle, which may or may not be preceded by behavioral estrus (chams et al., 1978; Manns et al., 1983), results in elevated serum progesterone (P4) Accepted March 18, Received October 29, These experiments were funded by grants from the Alberta Agricultural Research ouncil, Reprint requests. levels for 3-5 days. The elevated P4 is normally produced by short-lived corpora lutea (L) (Rawlings et al., 198; Williams and Ray, 198; Manns et al., 1983). The reasons for the shortened life span of the first PP L have not been determined, but could include: 1) inadequate levels or pattern of circulating gonadotropins; 2) the presence of luteolytic factor(s); and/or 3) lack of luteinizing hormone (LH) recognition by the developing L. t does not appear that short first luteal phases can be attributed to a lack of circulating LH, because many workers have reported that the amount of LH released before, during, and after the first estrus followed by a short cycle was similar to that released at an estrus followed by a normal cycle (Lewis et al., 1981; Ramirez- Godinez et al., 1982; Manns et al., 1983). However, the secretory pattern of LH release during the formation of the first PP L may influence the functional integrity of the developing uteal tissue. Although Thatcher et al. (198) have reported high levels of 13,14-dihydro-15-ketoprostaglandin F2 a (PGFM) during the early PP 56

2 POTPARTUM ORPU LUTEUM 561 period of dairy cows, short estrous cycles are usually detected much later in the PP period of the beef cow. Furthermore, in a study by Manns et al. (1983), short-lived L did not appear to undergo rapid regression with elevated PGFM levels typical of a normal cycle, but rather appeared to simply lose their ability to secrete P4, perhaps because they were no longer responsive to LH stimulation. Kesler et al. (1981) reported a diminished response to LH by luteal cells collected 7 days after GnRH-induced ovulation in PP beef cows compared with luteal cells collected 7 days postestrus in normally cycling cows. These authors also suggested that a lack of LH recognition by the induced L might be the cause of the shortened life span. The life span of induced L can be prolonged by administering exogenous P4 before GnRH treatment or weaning (Ramirez-Godinez et al., 1981; Pratt et al., 1982; heffel et al., 1982). n addition, PP cows have a short P4 rise before the first observed estrus followed by a normal cycle, or exhibit a short cycle if the first PP ovulation is accompanied by estrus (Kiracofe, 198; Ramirez-Godinez et al., 1982). These data indicate that P4 may be involved in the reestablishment of normal estrous cyclicity. Therefore, the objectives of the present study were to: 1) compare functional characteristics of the short-lived L formed after GnRH-induced ovulation in PP cows with L formed after estrus in normally cycling cows; and 2) determine if P4 treatment, attempting to mimic the serum P4 elevation seen with the first short cycle, would alter the life span and functional characteristics of L formed after induced PP ovulation. General MATERAL AND METHOD Forty-one multiparous, spring-calving and six normally cycling, nonlactating Hereford cows were used in this study. All cows were in good to excellent body condition and received diets adequate for lactation and maintenance throughout the experiment. To enhance the success rate of induced PP ovulation, calves were limited to 3 mm suckling once daily from Days 27 through 31 PP (arter et al., 198; Troxel et al., 198; Randel, 1981). alves were allowed to suckle ad libitum at all other times. Postpartum cows were allotted randomly to one of four treatment groups on Day 25 PP, while the nonlctating cows served as the cycling control group. The PP treatments included: 1) P4-luteectomy (n=1o); 2) P4-intact (n=11); 3) oil-luteectomy (n=11); and 4) oil-intact (n=9). Progesterone-treated cows received 1 mg P4 (4-pregnene-3,2-dione; igma hemical Go., t. Louis, MO) in sesame oil i.m. once daily on Days 25 through 28 PP, whereas PP control cows received sesame oil only (Fig. 1). All PP cows received 2 g GnRH (Abbott Laboratories, North hicago, L) i.m. on the afternoon of Day 3 PP to induce ovulation. orpora lutea were obtained from cows in the luteectomy groups by standing mid-flank laparotomy under local anesthesia on Day 37 PP (6.5 days after GnRH). orpora lutes were also obtained from the cycling control cows 7 days after standing estrus had been observed. The P4 -and oil-intact PP cows were used to estimate the proportion of luteectomized PP cows that would have undergone a short or normal luteal phase. Blood samples for serum P4 determination were collected daily via jugular venipuncture from all PP cows on Days 25 through 37 PP. Additional blood samples for P4 analysis were collected from intact PP cows on Days 39, 41, and 43 (Days 9, 11, and 13 post. GnRH injection). Blood samples were also collected daily from the cycling control cows from Days 1 through 7 after estrus. All blood samples were collected at the same time of day and were taken immediately before P4 or oil injection on the days of steroid treatment. Blood samples were allowed to clot at room temperature for approximately 3 mm and centrifuged to harvest the serum, which was stored at -2#{176} until assayed for P4 concentration by radioimmunoassay. One cow in each of the P4-luteectomy and oil-intact groups did not ovulate in response to GnRH injection, as evidenced by lack of luteal tissue at luteectomy or by elevated serum P4 concentrations. One cow in the oil-intact group had elevated serum P4 concentrations indicative of cyclic activity before GnRH injection. Data from these three cows were omitted from analyses. n Vitro L Evaluation The L collected at luteectomy were placed immediately in cold Ham s F-b (GBO, Grand sland, NY) media (containing 25 mm HEPE, igma hemical o., t. Louis, MO) for transport to the laboratory, where they were weighed and processed. One-half of the L were frozen immediately at -7#{176} for later determination of LH receptor concentration and P4 content. The remaining tissue was sliced and dispersed with collagenase (Type 1A; igma hemical o., t. Louis, MO) according to the procedure of immons et al. (1976). Following dispersion and washing with fresh media, free luteal cells were counted and viability was determined by trypan blue exclusion (Patterson, 1979). The large (25-4 m in diameter) and small (1-12 m in diameter) luteal cell populations were also determined for each L. Because we wanted to obtain a representative sample of the in vivo L, no attempt was made to separate the two cell types for incubation, nor did we attempt to determine if all cells included in the culture were capable of steroid secretion. Aliquots of cells were placed in 12 X 75-mm glass test tubes (2, total live cells/tube) and incubated in a Dubnoff metabolic shaker at 37#{176} for 1 h with either, 1, 5, 1, or 5 ng LH (NAMDD-oLH- 24; National Hormone and Pituitary Program, Baltimore, MD) added to the culture media (5 replicates/

3 562 RUTTER ET AL. Daily P4 nject ions (1 mg GnRH (2?) Luteectomy T,V Parturition FG. 1. Outline of experimental protocol. x, days when jugular blood samples were collected for P4 determination alves One. 29 uckled Daily DAY #{149} 35 POTPARTUM DAY POT-ETR(J ntst Postpartum ows dose of Lii). Following incubation, cells were placed in a 7#{176}water bath for 2 mm to kill the cells and to denature any steroid enzyme systems present. The tubes were then centrifuged at 35 rpm for 1 mm and the supernatants were decanted and stored at -2#{176} for later determination of P4 concentration. Progesterone Radio immunoassay All P4 assays utilized a specific antibody raised against 4-pregnen-1 lcs-ol-3,2-dione hemisuccinate/ bovine serum albumin (rabbit #1) according to the procedure of Niswender (1973). The antibody was used at a 1:1, dilution in phosphate-buffered saline/o.1% gelatin (PB-gel). ross-reactivity calculated at 5% binding was: 1% with progesterone; 16% each with 5a- and 5j3-pregnan-3,2-dione; 1.9% with corticosterone; less than.3% with / - and L -pregnen-3-ol-2-one;.14% with cholesterol;.8% with testosterone; and less than.5% with androstenedione, cortisol, estradiol-17, estrone, androsterone, and 5a-pregnan-3,2a-diol. Parallelism was determined with three different volumes of serum from five cows assayed over a 4-fold range of concentrations. Mean P4 concentrations obtained were 3.7, 3.54, and 3.95 ng/ml, respectively. n addition, recovery of 5 and 1 ng of cold P4 added to pooled cow sera was 17.% and 14.8%, respectively. Progesterone standards were prepared by drying 1 ml of a stock solution in ethanol (1 ng P4/ml) under forced air and then adding 1 ml of PB-gel to give 1 ng P4/ml. The standard solution in buffer was then shaken gently overnight at 37#{176}on a Dubnoff metabolic shaker to ensure that the P4 went into solution. erial dilutions of the stock standard were made in buffer and ranged from 1 to 1 pg/2 M lodinated P4 (progesterone.11glucuronide.[lssl iodotyr amine; Amersham, Oakville, Ontario, anada) served as the tracer. After a 24-h incubation period at 4#{176}, the P4-antibody complex was precipitated with 1 ml propylene glycol (2% in PB) and centrifugation at 3 rpm for 2 mm. Total binding of 25-P4 in the absence of cold hormone was 3 7.8%, with nonspecific 7 #{149}t binding of 8.1%. ntra- and interassay coefficients of variation, based on pooled PP cow sera assayed in quadruplicate with each standard curve, were 11.9% and 6.3%, respectively. era (2 M) were extracted twice with petroleum ether, dried under forced air, brought up to 1 ml with PB-gel, and then incubated at 37#{176} in a Dubnoff metabolic shaker for 2 h to ensure that P4 dissolved in the buffer. All tubes were cooled to 4#{176}before addition of antibody and tracer. Extraction of 3H-P4 ([1,2,6,7,21-s H(N)J progesterone; New England Nuclear, Boston, MA) from duplicate pools of serum was used to estimate recovery and averaged 81.3%. erum P4 concentrations were corrected for extraction procedure losses. Dispersed luteal cell supernatants were diluted 1:2 in PB-gel immediately before P4 assay and all samples were assayed in one assay in duplicate 1-Ml aliquots without extraction. An aliquot (1 M) of the crude L homogenate (see below) was extracted twice with petroleum ether, dried under forced air, and then diluted to 1 ml with PB-gel. An additional 1:2 dilution of the extracted L homogenate was assayed in duplicate O-M aliquots. All L homogenates were analyzed in one assay. Luteinizing Hormone Receptor Assay ndividual, weighed L halves were thawed, minced with fine scissors, and suspended in 1 ml homogenization buffer (1 mm Na2 EDTA, 25 mm Tris-H, 27% sucrose; ph 7.4). After homogenization for 2-3 s with a Polytron homogenizer, 5 M1 of the crude homogenate were stored at -7#{176} for later determination of L P4 content. The remaining L homogenate was further reduced in a cold, ground glass, Ten Broeck-type homogenizer. Homogenates were then transferred into weighed 25 X 1-mm plastic centrifuge tubes and centrifuged at 4#{176}and 11, X g for 2 mm. The supernatants were discarded and the tubes drained and weighed to determine wet weight of membrane. The L membrane pellet was resuspended in buffer to give a concentration of 2 mg/m and was stored at -7#{176} until analyzed

4 POTPARTUM ORPU LUTEUM 563 for LH receptor content. Mid-luteal phase bovine L obtained from the local slaughterhouse were processed identically to provide standard luteal membrane tissue. Human chorionic gonadotropin (hg) was used to determine lutes! cell membrane LH receptor concentrations. Highly purified hg (2 Mg; anfield R- 119, 116 lu/mg) was radioiodinated with 1 mi Na 2 (Amersham orp., Oakville, Ontario, anada) using 2 pg lactoperoxidase and 1 ng H23 in a 2-mm incubation at room temperature (Morrison and Bayse, 197). Labeled hg was isolated on a G-25 ephadex column and specific activity was determined by self-displacement (Diekman et al., 1978). Experimental L membranes were assayed in duplicate at 5 mg and 1 mg tissue by a standard curve procedure (Nett et al., 1981). The standard curve was generated using a standard membrane preparation at concentrations of -3 mg/tube. Nonspecific binding was assessed for each tube by the addition of 1 g cold hg. tatistical teectomy. Analyses erum P4 concentrations were analyzed by multiple analyses of variance (teel and Torrie, 196) with treatment and/or class and time as main effects. n vitro P4 release was analyzed by split-plot analysis of variance with treatment or class and dose of LH as main effects (Gill, 1978). Where indicated, differences between group or dose means were tested using a multiple range test. orpora lutes characteristics, including cell viability, small and large cell populations, tissue P4 content, and LB receptor concentration, were analyzed by one-way analysis of variance with treatment or class as main effects. u-square analysis was used to determine P4 pretreatment effects on the life span of the induced L. REULT As anticipated, daily injections of 1 mg P4 caused elevated (P<.5) serum P4 concentrations in the PP P4 -treated group compared to the PP oil-treated controls (Fig. 2). irculating P4 concentrations did not return to basal levels in the P4-treated cows as rapidly as we expected once injections stopped (Day 28 PP). nstead, the P4 -treated cows continued to show higher serum P4 concentrations than did the oiltreated cows (P<.5) for 4 days after cessation of injections, which also meant that the P4- treated cows had elevated P4 levels at the time of GnRH injection. Endogenous P4 concentrations did not begin to rise until 5 days after GnRH in the P4-treated cows compared to 3 days in the oil-treated cows (P<.5). No differences were observed in serum P4 concentrations between the oil- and P4-treated cows from Days 5 through 13 after GnRH. However, the cycling control cows exhibited higher serum P4 levels (P<.5) than did either the oil- or P4-treated postpartum cows on the day of lu- Based on serum P4 patterns found in the intact PP cows and in the cycling control cows, PP females were further divided into one of two classifications (Table 1): 1) the short cycle (HORT) group (n=2), which contained any PP cow whose serum P4 concentration started declining on or before 6-7 days after GnRH injection; and 2) the normal cycle (NORM) group (n=18), which contained any PP cow whose serum P4 concentration did not show a decline through at least 13 days after GnRH. ows undergoing luteectomy 6.5 days after GnRH were considered to be NORM if serum P4 concentrations were still rising. ows classified as NORM had higher (P<.1) serum P4 concentrations on the day of luteectomy (2.8 ±.3 ng/ml) than did those cows classified as HORT (1.5 ±.2 ng/ml). However, the cycling control cows had higher (P<.5) serum P4 concentrations (5.6 ±.9 ng/ml) on this day than did the HORT or NORM PP cows. The P4-intact PP cows had fewer (P<.5) short luteal phases after GnRH-induced ovulation than did oil-intact cows (Table 1). However, examination of the serum P4 data from the intact cows revealed that we could not predict accurately whether cycles would have been 6 5 P4 GnRH -6 5 DAY RELATVE TO GnRH (OR ETRU) FG. 2. Mean (± EM) serum P4 concentrations in oil-treated PP cows (.-.), P4-treazed PP cows (.----.), and cycling control cows (...). Postpartum cows received once-daily injections of 1 mg P4 (P4 -treated) or vehicle (oil-treated) on Days -6, -5, -4, and -3 (indicated by arrows) relative to injection of 2 Mg GnRH., significant differences (P<O.5) between the groups.

5 564 RUTTER ET AL. TABLE 1. Treatment effect on the incidence of short cycles following GnRH-induced ovulation in postpartum beef cows. Treatment group n % horts (n) % Normals (n) P4-Luteectomy P4-lntact (2) 55(6)b 78 (7) 45(5) Total 2 4 (8)c 6 (12) Oi-Luteectomy Oil-ntact (6) 86(6)b 45 (5) 14(1) Total (12)c 33 (6) athe basis for classifying cows as short or normal is given in the Results section. b4 -intact cows showed a lower (P<.5) incidence of short first luteal phases than did the oil-intact controls. All P4-treated cows (combined intact and luteectomized) showed a lower (P<O.1) incidence of short first luteal phases than did all oil-treated control cows. short or normal at the time of L removal (6.5 days after GnRH; Days 37 PP) in the luteectomized p cows. Based on the above definition, four of six P4-intact and two of six oil-intact cows would have been classified as having normal cycles at Day 37, but actually had short cycles. However, based on results with the P4- intact versus oil-intact cows, we expected a significantly higher proportion of normal cycles in the P4 -treated luteectomized group. n addition, based on our definition of a short cycle, all L classified as HORT were from cows with declining serum P4 concentrations at the time of luteectomy. Therefore, we compared L that were certain to have a shortened life span with L that had a high probability of having a normal life span. The point to be emphasized is the minor variation around the mean L characteristics measured between the cows classified as HORT versus those classified as NORM, suggesting that the postpartum L were similar 6.5 days after GnRH regardless of whether they were of definitely short or predominantly normal duration. TABLE 2. Mean (t EM) L characteristics in postpartum and cycling beef cows. Treatment group n L weight (g) There was no difference in L weight, P4 concentration, or LH receptor concentration between the P4- and oil-treated PP cows (Table 2). However, the cycling control cows had a greater L weight and LH receptor concentration (P<.5) than did either HORT or NORM PP cows. NORM PP cows had heavier L (P<.5) than did HORT PP cows, although LH receptor concentration did not differ between these two groups. orpora lutea P4 concentration did not differ between the cycling control, NORM PP, or HORT PP cows. ince cycling control L were significantly heavier than the PP-induced L, total P4 content/l was also greater (P<.5) in the cycling cows (data not shown). None of the dispersed L characteristics (Table 3) differed between either the oil- versus L P4 concentration (Mg/g) L LH Receptor concentration (fmol/mg L) Oi-Luteectomy ± ± ±.1 P4-Luteectomy ± ± ±.1 hort Normal ycling cycle cycle controls ± ± 3b 4.6 ± O ± ± ± ± O.la.65 t.l 1.23 ± a,b,cmeans in the same column with different superscripts differ (P<.5).

6 POTPARTUM ORPU LUTEUM 565 ii,fl t.4 OsOe4 P4-uteectomy PP cows or the HORT versus NORM PP cows. However, the cycling control cows had a higher (P<.1) percentage of live dispersed cells, and a greater number of small cells/g of L (P<.5), large cells/g of L (P<.5), and total cells/g of L (P<.5) 4.4 V ii V 56 u u 4 (4-4 V. tc-. u - V 2. t-.r. O -- N r4.,-. Or- -- Nr r-. -4N,nW Nln N * N N* AN ** H t.utos #{149} vvv. 4 5, 5, V U u 5, ci, v, EEE than did either the HORT or NORM PP cows. The small-large luteal cell ratio did not differ among groups. n vitro LH stimulation increased (P<.1) P4 production by dispersed luteal cells relative to unstimulated cells ( ng/ml LH) in all groups. Progesterone responses to 1, 5, 1, or 5 ng/ml LH did not differ (P>.15;.82 for full model,.22 for individual main effects); therefore, a mean stimulated release was calculated (Table 4). n vitro P4 production did not differ between luteal cells from oil- and P4-treated PP cows or between those from PP and cycling cows (P>.1). Luteal cells from HORT PP cows tended to have greater basal and mean stimulated P4 release (P<.15) than did those of either NORM PP or cycling control cows. However, there was no difference observed between groups when mean LH-stimulated P4 release was expressed as a percentage of basal P4 production. There were no treatment or class by LH dose interactions (P>.1). DUON Data from this study show that induction of ovulation in PP cows with exogenous GnRH resulted in an approximately 5% incidence of short luteal phases. imilarly, Walters et al. (1982) reported a 55% incidence of short cycles after pulsatile GnRH injection and a 64% incidence after weaning in PP beef cows. Furthermore, a significant reduction in the incidence of short luteal phases with P4-treated cows is consistent with other reports of lengthened luteal life span with a progestogen pretreatment (Pratt et al., 1982; heffel et al., 1982; Troxel and Kesler, 1983). irculating P4 levels (Odde et al., 198; Ramirez-Godinez et al., 1982; Manns et al., 1983), L weights (Kesler et al., 1981; picer et a!., 1981), and L P4 concentrations (mith et al., 1984) during a u o. En 4 4 u - 2#{176} U - (4(4 UJ nn Os.. utzl. #{176}. u U t;#{149}t; 2.. > 2.. UUU ct. U short versus a normal luteal phase are in close agreement with the values reported in this study. The P4 treatment was designed to mimic the brief elevation in serum P4 concentrations observed before a normal cycle in PP cows. t was expected that serum P4 concentrations would decline rapidly after cessation of treat-

7 566 RUTTER ET AL. TABLE 4. n vitro luteal cell response to LU stimulation (mean ± EM) in postpartum and cycling beef 5.a Treatment group n Basal P4 release Mean stimulated releaseb % P4 increase above basal Oil-Luteecromy ± ± P4-Luteectomy ± ± HORT PP ± ± 1O NORM PP ± ± ycling control 6 8. ± ± ap4 production is expressed as ng/2, total live cells. b4 production by luteal cells stimulated with either 1, 5, 1, or 5 ng/ml of LH added to the culture media was similar (P>.1). Therefore, a mean stimulated release was calculated for each cow as the mean P4 production stimulated by 1, 5, 1, and 5 ng/ml LH added to the culture media. timulated P4 release was greater (P<.1) than basal (+ ng LH) for all groups tested with no group X dose of LH interaction (P>.1). Basal and mean stimulated P4 release tended to be greater in HORT (P<.15) compared to NORM or cycling control, but no differences (P>.1) were detected when P4 release was expressed as a percentage of basal production. ment (Manns et al., 1983) and would therefore be at basal levels at the time of GnRH injection. However, P4-treated cows exhibited higher P4 concentrations at the time of GnRH injection and also showed a delay in the time from GnRH to serum P4 concentrations above 1 ng/ml compared with oil-injected cows. With a single 25-mg i.m. injection of progesterone, Williams and Ray (198) reported increased serum concentrations within 6 h that persisted above controls for an average of 66 h. The P4-treated cows in the present experiment received four times this dose for a period of 4 days, and this prolonged treatment may have delayed the decline in serum concentrations to basal levels. n support of this contention, Azzazi et al. (1984) found P4 concentrations of approximately 1.5 ng/ml at the time of GnRH injection 48 h after one subcutaneous injection of 15 mg P4 in dairy cows. Unfortunately, these workers did not report any data on ovulatory response or subsequent L function. n normally cycling cows, ovulation takes place within 24-3 h after the LH surge (Foote, 1974). n our P4-treated cows, the first rise in serum P4 concentrations after GnRH occurred very synchronously within the treatment group. Therefore, it seems likely that the P4 -treated cows responded to 2 pg GnRH with an ovulatory surge of LH and unlikely that they then had an endogenous LH surge 48 h after GnRH injection. t is important to recognize that even if the P4 -treated cows did have delayed ovulation due to the high serum P4 concentrations at the time of GnRH injection, there was no difference in L characteristics measured between the oil- and P4-treated PP cows even though the latter L may have been 2 days younger. Despite its enhancing effect on subsequent luteal life span, P4 pretreatment did not appear to have a specific positive effect on the functional characteristics measured in the induced corpora lutea. Luteinizing hormone receptor concentrations reported herein are similar to the results of harpe (1984) for early PP L analyzed in our laboratory. These receptor concentrations, using frozen membrane preparations, are lower than values reported for fresh membranes (Rao et a!., 1979; picer et al., 1981). Progesterone pretreatment did not appear to affect the number of LH receptors on the induced L, nor were the receptor concentrations different between PP cows undergoing a short luteal phase and those cows assumed to be undergoing a normal luteal phase. imilarly, mith et al. (1984) found no difference in the number of unoccupied LH receptors in Day 5 L from beef cows at the first and third estrus following early weaning. n the present study, however, all PP cows had significantly fewer LH receptors in the early L than did normally cycling cows. Although the PP L had fewer LH receptors than did cycling cows, both basal and LH-stimulated in vitro P4 production was similar. This is in contrast to the study by Kesler et al. (1981),

8 POTPARTUM ORPU LUTEUM 567 who reported higher basal and LH-stimulated P4 production in Day 7 postestrus cycling L compared with Day 7 post-gnrh-induced L. Based on their results, these authors suggested that the short life span of the first PP L might have been due to a lack of LH recognition by the induced L. However, in view of the decreasing serum P4 concentrations reported by those investigators, the Day 7 post-gnrh L were probably already in the process of regression, and the lower response to LH stimulation may have been the result of the early regression and not the causative factor. n the current study, the L were removed 6.5 days after GnRH, or approximately 5.5 days after ovulation, considered to be a critical time period in the development of a normal or short cycle. That is, L were removed after the time when a L theoretically becomes responsive to LH stimulation (Day 4 postovulation; Kesler et al., 1981) but before the onset of premature regression (Day 6-7 postovulation). Although the L classified as HORT, based on declining serum P4 concentrations at the time of luteectomy, were subfunctional in terms of systemic P4 levels, in vitro basal and LH-stimulated P4 release appeared to be at least equal to or greater than that of PP L classified as NORM or the cycling L. As a percentage increase in P4 above basal, there was no detectable difference between the L from PP and cycling cows. n terms of dispersed cell numbers, normally cycling cows had a greater number of both small and large cells compared with the PP cows, although the small-large cell ratio did not differ between groups. ell viability was lowest in cows with short cycles, again perhaps indicative of early regression in these PP L, but there was no detectable difference in cell number or type between any of the PP groups. To our knowledge, no other studies have reported actual luteal cell types in the short-cycling PP cow, although Manns et al. (1983) indicated a possible difference in the percentage of large cells in the young PP-induced L. As Fitz et al. (1982) have pointed out, the large luteal cell is responsible for basal P4 release and carries the PGF receptor. During normal L function, there is a progressive increase in the number of large cells throughout the cycle. Although we were unable to show a significant difference in the number of large luteal cells in the L known to be undergoing premature regression, the dispersed luteal cell preparation from these L appeared to release more basal P4 (P<.15) into the incubation media than either normal PP L or cycling L. Therefore, there may have been an alteration in the luteal cell population such that more large cells were present at an earlier stage in the life span of the L. Alternatively, the affinity of the large cell receptor for PGF may have increased, as Rao et al. (1979) demonstrated during the onset of luteal regression in the normally cycling cow. n either case, these changes would effectively increase the L s susceptibility to PGF-induced regression. n conclusion, this study has shown clearly that the postpartum-induced L has both LH receptors and the ability to respond to LH stimulation in vitro. These results indicate that a lack of LH recognition by the short-lived L is not the causative factor in early luteal regression. AKNOWLEDGMENT The authors would like to express their sincere appreciation of the expert technical assistance provided by L. M. larke, P. J. Lewing, and. Deptuck. REFERENE Azzazi, F., Krause, G. F. and Garverick, H. A. (1984). Alteration of the GnRH-induced LU release by steroids in postpartum dairy cattle. J. Anim. ci. 57: arter, M. L., Dierschke, D. L., Rutledge, J. J. and Hauser, E. R. (198). Effect of gonadotropinreleasing hormone and calf removal on pituitaryovarian function and reproductive performance in postpartum beef cows. J. Anim. ci. 51: Diekman, M. A., O allaghan, P., Nets, T. M. and Niswender, G. D. (1978). Validation of methods and quantification of luteal receptors for LU throughout the estrous cycle and early pregnancy in ewes. Biol. Reprod. 19: Fits, T. A., Mayan, M. H., awyer, H. R. and Niswender, G. D. (1982). haracterization of two steroidogenic cell types in the ovine corpus luteum. Biol. Reprod Foote, W. D. (1974). Reproductive cycles. attle. n: Reproduction in Farm Animals (E..E. Hafez, ed.). Lea and Febiger, Philadelphia, p Gill, J. L. (1978). Design and Analysis of Experiments in the Animal and Medical ciences (Vol. 1 & 2). owa tate University Press, Ames, A. esler, D. J.,Weston, P. G., Pimentel,. A., Troxel, T. R., Vincent, D. L. and Hixon, J. E. (1981). Diminution of the in vitro response to luteinizing hormone by corpora lutea induced by gonadotropin releasing hormone treatment of postpartum suckled beef cows. J. Anim. ci. 53: Kiracofe, G. H. (198). Uterine involution: its role in regulating postpartum intervals. J. Anim. d.

9 568 RUTFER ET AL. 51: LaVoie, V., Han, D. K., Foster, D. B. and Moody, E. L. (1981). uckling effect on estrus and blood plasma progesterone in postpartum beef cows. J. Anim. d. 52: Lewis, G.., Lishman, A. W., Butcher, R. L.. Dailey, R. A. and nskeep, E. K. (1981). Factors affecting function of induced corpora lutea in postpartum anestrous ewes. J. Anim. ci. 52: Lishman, A. W., Allison,.M.J., Fogwell, R. L., Butcher, R. L. and lnskeep, E. K. (1979). Foilicular development and function of induced corpora lutea in underfed postpartum anestrous beef cows. J. Anim. ci. 48: Manns, J. G., Humphrey, W. D., Flood, P. F., Mapletoft, R. J., Rawlings, N. and heng, K. W. (1983). Endocrine profiles and functional characteristics of corpora lutes following onset of postpartum ovarian activity in beef cows. an. J. Anim. ci. 63: Morrison, M. and Bayse, G.. (197). atalysis of iodination by lactoperoxidase. Biochemistry 9: Nett, T. M., rowder, M. E., Moss, G. E. and Duello, T. M. (1981). GnRH-receptor interaction. V. Down-regulation of pituitary receptors for GnRH in ovariectomized ewes by infusion of homologous hormone. Biol. Reprod. 24: Niswender, G. D. (1973). nfluence of the site of conjugation on the specificity of antibodies to progesterone. teroids 22: Odde, K. G., Ward, H.., Kiracofe, G. H., McKee, R. M. and Kirtok, R. J. (198). hort estrous cycles and associated serum progesterone levels in beef cows. Theriogenology 14: Patterson, M. K., Jr. (1979). Measurement of growth and viability of cells in culture. Methods Enzymol. 58: Pratt, B. R., Berardinelli, J. G., tevens, L. P. and lnskeep, E. K. (1982). nduced corpora lutea in the postpartum beef cow.. omparison of gonadotropin releasing hormone and human chorionic gonadotropin and effects of porgestogen and estrogen. J. Anim. ci. 54: Ramirez-Godinez, J. A., Kiracofe, G. H., McKee, R. M., challes, R. R. and Kittok, R. J. (1981). Reducing the incidence of short estrous cycles in beef cows with norgestomet. Theriogenology 15 : Ramirez-Godinez, J. A., Kiracofe, G. H., challes, R. R. and Niswender, G. D. (1982). Endocrine patterns in the postpartum beef cow associated with weaning: a comparison of the short and subsequent normal cycles. J. Anim. ci. 55: Randel, R. D. (1981). Effect of once-daily suckling on postpartum interval and cow-half performance of first-calf Brahman X Hereford heifers. J. Anim. ci. 53: Rao,. V., Estergreen, V. L., arman, F. R., Jr. and Moss, G. E. (1979). Receptors for gonadotrophin and prostaglandin F3 in bovine corpora lutea of early, mid and late luteal phase. Acta Endocrinol. 91: Rawlings, N.., Weir, L., Toddy, B., Manns, J. and Hyland, J. H. (198). ome endocrine changes associated with the post-partum period of the suckling beef cow. J. Reprod. Fertil. 6: chams,., challenberger, E., Menzer,., tangi, J., Zottmeier, K., Hoffman, B. and Karg, H. (1978). Profiles of LH, FH and progesterone in postpartum dairy cows and their relationship to the commencement of cyclic function. Theriogenology 1: harpe, P. H. (1984). Reproductive Function in Beef ows and Ewes during the Postpartum Period. Ph.D. Thesis, University of askatchewan, askatoon, askatchewan anada. heffel,. E., Pratt, B. R., Ferrell, W. L. and nskeep, E. K. (1982). nduced corpora lutes in the postpartum beef cow.. Effects of treatment with progestogen and gonadotropins. J. Anim. ci. 54: immons, K. R., affrey, J. L., Phillips, J. L., Abel, J. H. and Niswender, G. D. (1976). A simple method for preparing suspensions of luteal cells. Proc. oc. Exp. Biol. Med. 152: mith, M. F., Garverick, H. A., Youngquist, R.. and Zahler, W. L. (1984). LH receptor concentrations, adenylate cyclase and phosphodiesterase activity in bovine corpora lutes: comparison of short and normal cycles. Biol. Reprod. 3(uppl. 1):88. (Abstr.). picer, L. J., reland, J. J. and Roche, J. F. (1981). hanges in serum LU, progesterone, and specific binding of -hg to luteal cells during regression and development of bovine corpora lutea. Biol. Reprod. 25: teel, R.G.D. and Torrie, J. H. (196). Principles and Procedures of tatistics. McGraw-Hill Book o., New York, NY. Thatcher, W. W., Wilcox,. J., ollier, R. J., Eley, D. and Head, H. (198). Bovine conceptus-maternal interaction during pre- and post-partum periods. J. Dairy ci. 63: Troxel, T. R. and Kesler, D. J. (1983). Progestin pretreatment enhances the function of corpora lutea induced by gonadotropin releasing hormone treatment in postpartum suckled beef cows. J. Anim. ci. 57(uppl. 1):17. (Abstr.). Troxel, T. R., Kesler,. J., Noble, R.. and arlin,. E. (198). Ovulation and reproductive hormones following steroid pretreatment, calf removal and GnRH in postpartum suckled beef cows. J. Anim. ci. 51: Walters, D. L., hort, R. E., onvey, E. M., taigmiller, R. B., Dunn, T. G. and Kaltenbach,.. (1982). Pituitary and ovarian function in postpartum beef cows. ll. nduction of estrus, ovulation and luteal function with intermittent small-dose injection of GnRH. Biol. Reprod. 26: Williams, G. L. and Ray, D. E. (198). Hormonal and reproductive profiles of early postpartum beef heifers after prolactin suppression of steroidinduced luteal function. J. Anim. ci. 5: