EFFECT OF THE UTERUS ON SUBNORMAL LUTEAL FUNCTION IN ANESTROUS BEEF COWS 1

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1 EFFECT OF THE UTERUS ON SUBNORMAL LUTEAL FUNCTION IN ANESTROUS BEEF COWS 1 J. P. Copelin 2, M. F. Smith 2, H. A. Garverick a and R. S. Youngquist 4 University of Missouri, Columbia ABSTRACT The effect of the uterus on luteal lifespan and pattern of secretion of progesterone following early weaning of calves from anestrous beef cows was studied. Calves were weaned from 15 anestrous beef cows 23 to 33 d postpartum, and cows were allotted to a control (sham surgery, n = 8) or a hysterectomy (n = 7) group, with surgery performed at weaning. Cows in the hysterectomy group were injected (ira) with 25 mg prostaglandin F2a (PGF2~) approximately 20 d after first estrus (d 0). The interval from weaning to estrus was longer (P<.05) for the hysterectomy group ( d) than the control group (6.2.5 d). In the control group, the first estrous cycle (8.8.3 d) was shorter (P<.O1) than the second estrous cycle ( d). Following first estrus in the hysterectomy group, cows were not detected in estrus until after injection of PGF2c ~ and did not return to estrus. From d 0 to 5, mean concentrations of plasma progesterone were similar (P>.05) between groups for both estrous cycles; after d 5 of estrous cycle 1, concentrations of plasma progesterone decreased in the control group. Within the hysterectomy group, the pattern of secretion of progesterone from d 0 to 16 was similar after the first and second estrus. Furthermore, there was no difference in the pattern of secretion of progesterone from d 0 to 16 between hysterectomy (first or second estrous cycles) and control (second estrous cycle) groups. These results suggest that by d 6 after first estrus, the uterus has a luteolytic effect on the corpus luteum in cows that have had their calves weaned early. (Key Words: Corpus Luteum, Postpartum Interval, Early Weaning, Progesterone, Uterus.) I ntroduction In cattle, subnormal corpora lutea form spontaneously before the first ovulatory estrus in prepuberal heifers (Berardinelli et al., 1979), postpartum cows (Corah et al., 1974; LaVoie et al., 1981), and following the early weaning of calves from cows (Odde et al., 1980). Similar luteal structures have been induced following early postpartum injection of gonadotropin releasing hormone (GnRH; Lishman et al., 1979), or human chorionic gonadotropin (hcg; Pratt et al., 1982). The function of a short-lived corpus luteum is not clear; however, several studies indicate that a transient increase in progesterone may assist in initiation of normal estrous cycles in anestrous cattle (Gonzalez- Padilla et al., 1975 ; LaVoie et al., 1981 ; Ramirez- Godinez et al., 1981). t Contribution from the Missouri Agr. Exp. Sta. Journal Series No This research supported in part by USDA Grant No. 84-CRSR Dept. of Anita. Sci. 3 Dept. of Dairy Sci. 4 Dept. of Vet. Med. and Surgery. Received September 2, Accepted January 9, Subnormal luteal function may result from 1) inadequate preovulatory follicular development (dizerega and Hodgen, 1981; Ramirez- Godinez et al., 1981); 2) decreased luteotropic stimuli (Kesler et al., 1981;Manns et al., 1983); and(or) 3) premature release of and(or) increased sensitivity to a luteolysin (Pulgisi et al., 1979; Troxel and Kesler, 1984a). A key question is "does a subfunctional corpus luteum have an inherently short lifespan, or does a luteolytic stimulus cause premature luteolysis?" Therefore, the objective of this experiment was to determine the effect of the uterus on luteal lifespan and pattern of secretion of progesterone following early weaning of calves from beef cows. Materials and Methods Fifteen pluriparous beef cows were allotted at parturition by age and breed to a control (sham surgery; n = 8) or a hysterectomy (n = 7) group. All cows were in moderate to good body condition at calving. From parturition until completion of the experiment, cows were observed for estrus twice daily; an androgenized cow was used as an aid to detect estrus. Blood samples were collected via jugular venipuncture from 1506 J. Anita. Sci :

2 SUBNORMAL LUTEAL FUNCTION 1507 cows three times weekly from parturition until weaning; concentrations of progesterone in plasma were determined (Cantley et al., 1975) to identify cows that ovulated prior to weaning. Calves were weaned from cows 23 to 33 d following parturition, at which time either sham surgery or hysterectomy was performed. Cows were tranquilized by injecting (ira) 25 mg xylazine and 20 mg acepromazine maleate. Lidocaine hydrochloride (2%) was used as a local anesthetic. The uterus and ovaries were exposed through a mid-ventral incision in both groups using aseptic technique. Ovaries of animals in both control and hysterectomy groups were examined for luteal structures and returned to the body cavity. In the hysterectomy group, the uterus was removed as close as possible to the cervix. Care was taken not to disrupt the ovarian blood supply. In the control group, blood samples were collected daily via jugular venipuncture from Surgery until completion of two estrous cycles. Blood samples were collected daily via jugular venipuncture from cows in the hysterectomy group from surgery until d 24 (estrus = d 0) of the second estrous cycle. Animals that maintained corpora lutea following hysterectomy were injected (ira) with 25 mg prostaglandin F2~ (PGF2~) on approximately d 20 of the first estrous cycle to induce luteolysis. All blood samples were collected into heparinized tubes and immediately placed on ice. Plasma was collected and frozen (-20 C) until analyzed for progesterone (Cantley et al., 1975), luteinizing hormone (LH; Zaied et al., 1980) and estradiol-17/3 (Kesler et al., 1977) by radioimmunoassay. Intra- and inter-assay coefficients of variation for progesterone were 6.1 and 13.1%, and for estradiol-17~ were 4.8 and 2.8%, respectively. Determinations for LH were made in one assay, with an intra-assay coefficient of variation of 5.8%. The data were analyzed by analysis of variance for a split-plot in time design. The general linear model contained effects of treatment, estrous cycle and time. Differences between means were ascertained using the least significant difference procedure (Cochran and Cox, 1957). Interval from weaning to first estrus, and length of estrous cycles 1 and 2 for the control group were analyzed using a Student's t-test (Snedecor and Cochran, 1980). Results Three cows in the control group and one cow in the hysterectomy group ovulated 0 to 3 d before weaning. These cows were included in all data analyses except interval from weaning to estrus. Length of the first estrous cycle for these cows was similar to cows that ovulated after weaning within each treatment group. Cows in the control or hysterectomy groups that did not ovulate before weaning were detected in estrus d or 10.4 t 1.6 d (~ + SE) after weaning (P<.05), respectively. During the 5 d preceding first estrus, concentrations of LH increased (P<.05) similarly for both groups. Pooled mean (~ + SE) concentrations of LH for both groups from d -5 to -1 preceding the first estrus were.9 +.2,.9 +.2,.9 +.1, and ng/ml, respectively. Concentrations of estradiol-1713 also increased (P<.05) similarly for control and hysterectomy groups beginning 3 d before first and second estrus. Pooled mean (~ + SE) concentrations of estradiol-17/3 for both groups from d -3 to -1 prior to the first and secorid estrus were , and pg/ml, respectively U) ~ 2 s a. Estrous Cycle 1 ~4 6 Estrous Cycle f.,,l,$"~'~.& jk Hysterectomy G rou p IE ' s 3 ~ " g2 2 a. 1 "'" I I I I I Day of Estrous Cycle Figure 1. Mean (+ SE) daily concentrations of plasma progesterone in the control and hysterectomy groups for estrous cycles 1 and 2 (estrus = d 0).

3 1508 COPELIN ET AL. Within the control group, the first estrous cycle ( d) was shorter (P<.01) than the second estrous cycle ( d; figure 1). All cows subjected to sham surgery had short estrous cycles lasting 8 to 10 d, and all cows that were hysterectomized maintained their corpora lutea until after PGF2~ injection. Following injection of PGF2a, all cows returned to estrus within 48 to 72 h. Following PGF20cinduced estrus and ovulation, cows in the hysterectomy group were not detected in estrus again during the experiment. Circulating concentrations of progesterone from d 0 to 5 were similar between groups for estrous cycles 1 and 2 (figure 1). Within the control group, concentrations of progesterone decreased after d 5 of estrous cycle 1, whereas concentrations of progesterone continued to increase in the hysterectomy group until d 12. Within the hysterectomy group, pattern of secretion of progesterone from d 0 to 16 was similar after the first and second estrus. Furthermore, there was no difference in pattern of secretion of progesterone from d 0 to 16 between hysterectomy (estrous cycles 1 and 2) and control (estrous cycle 2) groups. Both mean concentration and pattern of secretion of LH from d 1 to 5 following the first and second cstrus were similar for both treatment groups. In addition, mean concentration and pattern of secretion of LH from d 1 to 16 was similar for hysterectomy (estrous cycles 1 and 2) and control (estrous cycle 2) groups. Discussw n Because development of the corpus luteum is a continuation of follicular maturation, both intra-follicular and extra-follicular mechanisms have been suggested to influence subsequent luteal function (dizerega and Hodgen, 1981; Garverick and Smith, 1986). During the follicular phase in humans and primates, a decrease in plasma concentrations of follicle stimulating hormone (FSH),or a decrease in the FSH/LH ratio resulted in formation of subnormal corpora lutea (Strott et al., 1970; Wilks et al., 1976). In cattle, the relationship between preovulatory concentrations of FSH and subsequent luteal function is unclear. Preovulatory concentrations of FSH were lower preceding a short vs a normal length estrous cycle in cows from which calves were weaned early (Ramirez- Godinez et al., 1982), or preceding a short vs a normal length estrous cycle in progestogen-pre- treated cows induced to ovulate with hcg (Garcia-Winder et al., 1986). However, administration of pregnant mare serum gonadotropin or FSH to postpartum cows, prior to the induction of ovulation, did not enhance subsequent luteal function (Lishman et al., 1979; Sheffel et al., 1982). Regardless, it is unlikely that inadequate follicular growth resulted in subnormal luteal function in this study, because preovulatory concentrations of estradiol-173 were similar for both treatment groups. Furthermore, the pattern of secretion of progesterone was similar for the first estrous cycle in the hysterectomy group and the second estrous cycle (normal luteal phase) of the control: group. Subnormal luteal function might be due to inadequate luteotropic stimuli. Luteinizing hormone is necessary for normal luteal development and function in cattle (Hansel and Convey, 1983). Therefore, decreased concentrations of LH, or an inability to respond to LH might decrease luteai lifespan. However, in the present study, plasma concentrations of LH were similar regardless of treatment or estrous cycle. In addition, other investigators have reported no differences in the mean concentration, pulse frequency, or pulse amplitude of LH during the postovulatory period of a short vs a normal estrous cycle (Ramirez-Godinez et al., 1982; Garcia-Winder et al., 1986). In the presence of adequate concentrations of LH, subfunctional luteal tissue may not be responsive to a luteotropic stimulus. In cattle (Kesler et al., 1981) and sheep (McNeilly et al., 1981), subnormal corpora lutea that formed following gonadotropin-induced ovulations secreted less progesterone in vitro in response to LH compared with normal, spontaneously formed corpora lutea. Conversely, Rutter et al. (1985) and O'Shea et al. (1984) found no difference in the ability of corpora lutea anticipated to have short or normal lifespans to secrete progesterone in vitro in response to LH stimulation in cattle and sheep, respectively. Following early weaning of calves from anestrous cows there was no difference in luteal weight, LH receptor concentrations (occupied and unoccupied), concentrations of plasma and luteal progesterone and adenylate cyclase activity for corpora lutea anticipated to have short or normal lifespans (Smith et al., 1986). Thus, subnormal luteal function was not due to an inability of the luteal tissue to bind LH or synthesize cyclic AMP. Because the first luteal tissue formed in the hysterectomy group secreted

4 SUBNORMAL LUTEAL FUNCTION 1509 concentrations of progesterone that were similar to a normal luteal phase, it was unlikely that the tissue had an impaired ability to respond to LH. Another mechanism that may be associated with subnormal luteal function is a premature release of and(or) an increased sensitivity to a luteolysin. Results of the present study demonstrate that the first corpus luteum formed after early weaning does not have an inherently short lifespan. Instead, presence of the uterus results in premature luteolysis. While luteolysis (decrease in concentrations of plasma progesterone, occurred after d 5 following first estrus in the control group, concentrations of progesterone in the hysterectomy group continued to increase from d 5 to 12 post-estrus. Similar results were reported following formation of the first luteal tissue in prepuberal lambs, anestrous ewes, and postpartum cows. The first luteal tissue formed in prepuberal lambs had a reduced lifespan; however, hysterectomy resulted in sustained secretion of progesterone (Keisler et al., 1983). In anestrous ewes, induction of ovulation with GnRH was followed by formation of a corpus luteum with a short lifespan; however, hysterectomy resulted in maintenance of luteal lifespan as determined by elevated concentrations of progesterone for 21 d (Southee et al., 1985). In cattle induced to ovulate with GnRH (short cycle), intrauterine administration of indomethacin (prostaglandin synthetase inhibitor) prolonged luteal lifespan (Troxel and Kesler, 1984b). Therefore, removal of the uterus or inhibition of prostaglandin synthesis (cyclooxygenase pathway) prevented subnormal corpora lutea from undergoing premature luteolysis. Increased sensitivity of corpora lutea to PGF2a has been reported for hysterectomized cows (Stellflug et al., 1975; Hansel and Fortune, 1978) and prepuberal pigs (Pulgisi et al., 1979). However, subnormal luteal function in anestrous cows induced to ovulate with GnRH was not due to an increased sensitivity of the corpora lutea to PGF2a (Copelin et al., 1986). Alternatively, reduced luteal lifespan may be due to an increased or premature release of PGF2a. Plasma concentrations of the PGF2a metabolite [ 13,14 dihydro-15-keto-prostaglandin F2a (PGFM)] were elevated during the early postpartum period of dairy cows (Thatcher et al., 1980) and ewes (Fredriksson, 1985). In the ewe, PGFM levels decreased substantially during the first 3 d postpartum but remained elevated for approximately 3 wk (Fredriksson, 1985). However, in the cow, concentrations of PGFM declined by d 15 postpartum (Lindell et al., 1982). Thus, sustained elevated concentrations of PGFM do not appear to be a major factor in subnormal luteal function in cattle. Elevated postpartum concentrations of PGFM are probably involved in uterine involution (Kiracofe, 1980). A premature release of PGF2a following the first luteal tissue formation may be associated with subnormal luteal function. Peak release of PGFM occurred later in postpartum cows having a normal compared with a short estrous cycle (d 16 vs d 10, respectively; Troxel and Kesler, 1984a). However, it is unclear from the preceding study whether or not peak release of PGFM preceded the decline in progesterone. Alternatively, Garcia-Winder et al. (1986) found no evidence for an effect of progestogen pretreatment on plasma PGFM concentrations in anestrous cows. In progestogen-pretreated postpartum ewes, concentrations of PGF2a were decreased on d 7 after estrus; however, luteal lifespan was not prolonged by progestogen pretreatment (Lewis et al., 1981). The mechanism associated with the timing of PGF2a release following the first luteal tissue formation in postpartum cows is unknown. Although PGF2a is a well-known uterine luteolysin, other possible luteolysins should not be ignored. Prostacyclin (PGI 2) stimulates progesterone synthesis and secretion in vitro and in vivo (Milvae and Hansel, 1980), and 5-hydroxyeicosatetraenoic acid (5-HETE), which is a product of the 5-1ipoxygenase pathway of arachidonic acid metabolism, inhibits PGI2 synthesis in dispersed bovine luteal cells in vitro (Milvae, 1986). In cattle, intrauterine infusion of an inhibitor of the lipoxygenase pathway [nordihydroguaiaretic acid (NDGA)] on d 14 through 18 of the estrous cycle delayed luteolysis (Milvae, 1986). Thus, 5-HETE may be a uterine luteolysin that deserves further investigation. In summary, by d 6 after the first estrus, the uterus has a luteolytic effect on the corpus luteum in cows that have had their calves weaned early. Literature Cited Berardinelli, J. G., R. A. Dailey, R. L. Butcher and E. K. Inskeep Source of progesterone prior to puberty in beef heifers. J. Anim. Sci. 49:1276. Cantley, T. C., H. A. Garverick, C. J. Bierschwal, C. E. Martin and R. S. Youngquist Hormonal response of dairy cows with ovarian cysts to

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