GONADOTROPIN RESPONSE BY POSTPARTUM MARES TO GONADOTROPIN-RELEASING HORMONE
Colorado State University, Fort Collins 80523 ABSTRACT
We hypothesized that the LH response to GnRH would be greater as the interval from foaling increases, whereas the FSH response would decrease, and that corpus luteum function after the first ovulation would be similar to that after the second ovulation. At parturition, mares were assigned to receive GnRH (2 p&g) intravenously on 1) d 3 postpartum (n = 6); 2) d 6 postpartum (n = 6); 3) d 1 of first postpartum estrus (foal estrus) and again on d 1 of second postpartum estrus (n = 8). Blood was collected through an indwelling cannula at -2, -1 and 0 h relative to GnRH stimulation (basal concentrations) and at .25, .5, .75, 1.0, 1.5, 2.0,2.5,3.0, 3.5 and 4.0 h post-GnRH. Samples were assayed for concentrations of LH and FSH. Basal concentrations of LH were lower (P < .05) for mares given GnRH on d 3 postpartum than for mares on d 1 of foal estrus. A rise in concentrations of LH was noted within 30 min in all groups, but the response to GnRH on d 1 of the first estrus was less (P c .05) than on d 1 of second postpartum estrus. As the interval from parturition increased, the amount of LH secreted in response to GnRH increased. The maximum response to GnRH was greater (Pc .05) during d 1 of the first estrus than on d 3 or 6 postpartum and was greater on d 1 of cycle 2 than on d 1 of cycle 1. In contrast, the amount of PSH secreted in response to GnRH was similar (P > .05) for all days. Pituitary concentrations of LH presumably were depressed in early postpartum mares, but pituitary concentrations of LH were quickly restored. (Key Words: LH, FSH, GnRH, Parturition, Mares.) J. Anim. Sci. 1990. 68:2r130-2435
lntroductlon
Postpartum anestrous in the ewe and cow has been associated with insufficient concentrations of LH in serum and in the anterior pituitary gland (Nett et al., 1987, 1988). Apparently, the high concentrations of estrogens in the peripheral circulation during late gestation are associated with inhibition of LH synthesis in the anterior pituitary (Moss et al., 1980, 1981). In sheep, the amount of LH secreted after GnRH challenge was correlated highly with concentration of LH in the anterior pituitary gland (Crowder et al., 1982). Likewise, the content of LH in the anterior pituitary
lAnim. Reprod. Lab. *Supported in part by Colorado State Exp. Sta. Received June 12,1989. Accepted November 17,1989.
gland of mares was correlated positively with the amount of LH released in response to a maximally stimulatory dose of GnRH (Silvia et al., 1987). Unlike species such as the ewe and cow, the mare resumes normal ovarian cyclicity soon after parturition (Ginther, 1979). Even though concentrations of estrone and estradiol decline during the last third of gestation (Nett et al., 1973), concentrations still are adequate to suppress synthesis of LH and inhibit accumulation of LH in the anterior pituitary gland. Nett et al. (1989) challenged mares with a maximally stimulatory dose of GnRH on d 240 and 320 of gestation, d 3 postpartum and (in a separate group) on d 2 or 3 of estrus. Basal concentrations and total quantity of LH released after GriRH was similar (P> .05) on d 240 and 320 of gestation and d 3 postpartum, but release was greater by d 3 of the first postpartum estrus. Release of FSH after GnRH
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L. A. Harrison, E. L. Squires'J, T. M. Nett and A. 0. McKinnon
RESPONSE BY POSTPARTUM MARES TO GnRH
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decreased continually (P < .05) from d 240 of every other day during first and second periods of diestrus and were assayed for progesterone gestation to d 3 of estrus. The present study was conducted to test (Niswender, 1973). Data from one mare in Group 3 (no. 779) these hypotheses: 1) LH response to GnRH is were excluded from all analyses. This mare greater as the interval from parturition increases, whereas the FSH response to GnRH had a high baseline concentration of LH during decreases; and 2) corpus luteum function after the first postpartum period of estrus (127.0 ng/ the first ovulation is similar to that after the ml vs 41.6 ng/ml for all other mares in this group) and elevated baseline concentrations of second ovulation. FSH during the first (70.2 ng/ml vs 12.5 ng/ ml, all others) and second (92.2 ng/ml vs 14.6 Materials and Methods ng/d all others) postpartum cycle. Twenty lactating embryo transfer recipient Three characteristics of LH and FSH were mares that foaled in May, June and July were established from the GnRH-challenge: baseused in this study. At parturition, mares were line, maximum response and area under the assigned randomly to one of three groups: 1) responsive curve. Baseline concentration of GnRH challenge on d 3 postpartum (n = 6); 2) each gonadotropin was calculated by averaging GnRH challenge on d 6 postpartum (n = 6); values from the three pre-injection samples. and 3) GnRH challenge on d 1 of the first Maximum response was identified as the postpartum period of estrus (foal estrus) and greatest concentration of hormone achieved again on d 1 of the second postpartum period after injection minus the baseline. k e a under of estrus (n = 8). Mares were administered a the response w e was determined from maximally stimulatory dose of onRH3 (2 pg/ samples collected between 0 and 4 h after kg) in .9% saline intravenously (Nett et al., GnRH using the SAS program (1987); data 1988). All mares were maintained on pasture, were adjusted for baseline concentrations. fed 1.5 kg grain per day and had ad libitum Differences in these characteristics among the access to alfalfa hay. Mares were exposed to a three groups (d 3, 6 and d 1, cycle 1) were stallion each day for detection of estrus. determined by analysis of variance. When Palpation and ultrasonography of the genital significant group effects were observed, means tract per rectum was initiated on d 2 postpar- for each group were compared by Tukey's tum and continued daily throughout the study. Honestly Significant Differences test (Steel Mares were separated from their foals during and Tome, 1980). T-tests were used to teasing. determine difference in LH and FSH values for For collection of blood, mares and foals mares at d 1, cycle 1 versus d 1, cycle 2. were housed in 3- x 15-m pens with access to Concentrations of progesterone on d 1,3,5,7, water. A 16-gauge indwelling cannula was 9, 11 and 13 of each diestrous period were placed in the jugular vein of each mare and the averaged and mcans were compared by t-test. first blood sample was taken at 0730. Blood samples were collected at -2, -1 and 0 h Results relative to GnRH stimulation to determine basal concentrations, then at .25, .5, .75, 1.0, hior to treatment, basal concentrations of 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 h after GnRH. LH were lower (P < .OS) for mares given Twenty milliliters of blood was collected for GnRH on d 3 postpartum than for those each sample, placed in centrifuge tubes and administered GnRH on d 1 of foal estrus stored at room temperature for approximately Vable 1). Concentration of LH in mares on d 20 min, then at 5'C overnight. Samples were 6 of cycle 1 were similar to values in the other centrifuged for 20 min, and serum was groups (Figure 1). Although a rise in concenharvested and frozen at -20'C until it was trations of LH was noted within 30 min in all quantified for concentrations of LH (Nett et groups, the response to GnRH treatment on d 3 al., 1975) and FSH (Nett et al., 1979) by RIA. or 6 postpartum was only modest. In contrast, Additional blood samples were collected daily a marked release of LH was detected after during first and second periods of estrus and treatment on d 1 of the second postpartum estrus, whereas treatment with GnRH on d 1 of foal estrus resulted in a lower response than that on d 1 of cycle 2 Figure 1). This indicates h, MSMO, sigma chemical CO.,st ~ o u i s MO. ,
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HARRISON ET AL. TABLE 1. RESPONSE TO GnRH STIMULATION' Parameter
GrOUDb
LH
m e , units FSH
Day 3 Day 6 Day 1,cycle 1 Day 1,cycle2
79.2 f 87.0 f 297.2 f 595.2 f
97.8' 54.8' 103.5' 235.1d
Maximum response, nglml FSH
LH
16.4 f 202 f 76.5 f 122.6 f
43.8 f 30.5' 13.7 f 11.4' 12.0 f 25.4' 23.2 f 20.1'
14.8' 21.5 f 9.4 1O.P 12.4 f 9.5 28.gd 12.5 f 8.8 24Se 14.6 f 4.4
Baseline, ng/ml FSH
LH 10.0 f 21.1 f 41.6 f 45.8 f
8.5' 13.@ 22.od 16.8d
28.1 24.2 20.9 21.2
f f f f
4.5' 10.V 8.V 5.3'
'Mean f SD. b a y 3 = 3 d p o s t p m day 6 = 6 dpstpartum; day 1, cycle 1 = d 1 of firstpostpartumestrus; day 1, cycle2 = d 1 of second postpartum estrus. '*dMeanswithin column with different superscriptsare different (P< .05).
that as the interval from parturition increased, the amount of LH secreted in response to GnRH increased. However, the areas under the response curve for mares on d 3 and 6 postpartum and d 1 of the first estrus were similar (P > .05). Similarly, the maximum response of LH to GnRH for mares challenged during the first postpartum estrus was greater (P < .OS) than for mares challenged on either d 3 or 6 postpartum. The maximum response of LH to GnRH also increased from d 1, cycle 1 to d 1, cycle 2. Concentrations of FSH prior to GnRH treatment were similar (P > .05) among all groups (Figure 2). In addition, the response to GnRH as measured by total area under the curve, response area under the curve and maximum response were similar (P > .05) among all groups. However, the greatest numerical response to GnRH was noted in mares treated on d 3 postpartum; FSH re-
sponse to GnRH in the other three groups was minimal. Duration of estrus for the first and second postpartum estrous periods was similar (P > .05) at 4.9 f 1.8 and 4.4 f 1.1 d, respectively. Durations of the first and second diestrous periods also were similar (P > .05) at 13.6 f 2.0 and 13.1 f 3.0 d, respectively. Concentrations of progesterone during both diestrous periods are presented in Figure 3. Values were similar (P> .05) for the two diestrous periods (overall means being 2.9 f .8 and 2.8 f .6 for cycle 1 and 2, respectively). Peak progesterone during the first and second postpartum cycles was 3.8 f 2.1 and 4.0 f 1.5 ng/ml, respectivelY
-
Dlscusslon
Silvia et al. (1987) reported a correlation of .82 between pituitary concentration of LH and
50-
150130.-
--
110-
A 1st h e a t 2nd h e a t
-- 40..
E 90-
-
70--
3
50.-
A 2nd heat
E
\
rn
\
p
0 Day 3
5
30-
I v, LL
20-
30IO--
04 - 3 - 2 - 1
0
I 2 TIME ( h )
3
4
I
IO.
5
Figure 1. Concentrations of LH in postpartum mares before and after administration (time 0) of GnRH.
Figure 2. Concenhations of FSH in postpartum mares before and after administrationof GnRH.
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Area under
RESPONSE BY POSTPARTUM MARES TO GnRH
ov
Figure 3. Progesteroneprofiie during estrous cycle 1 and 2 postpartum.
baseline serum LH concentration. In addition, the correlation coefficient for area under the c w e in response to GnRH and pituitary concentrations of LH was .72. Assuming this finding holds true for postpartum mares as it does for postpartum ewes (Moss et al., 1980), then results of the present study and a previous study (Nett et al., 1989) suggest that pituitary concentrations of LH were depressed in pregnant mares and early postpartum mares. However, in contrast to the beef cow and ewe, pituitary concentrations of LH were replenished quickly after parturition even when mares were nursing foals. Baseline concentrations of LH were greater (P< .05) during the first estrous period in both lactating and nonlactating mares compared with d 240 or 320 of gestation or d 3 and 6 after parturition. Maximum LH response to GnRH and area under the response curve increased as the interval from parturition increased. This apparent early replenishment of pituitary content of LH probably is responsible for the mare’s ability to exhibit a postpartum cycle within 9 to 11 d after pamuition, in contrast to the longer periods of postpartum anestrus noted in sheep and beef cattle. Postpartum anestrus in the ewe and beef cow has been associated with insufficient concentration of LH, in the anterior pituitary gland and serum, to stimulate follicular maturation and owlation. Very high concentrations of estrogen (Camegie and Robertson, 1978) and progestin (Stabenfeldt et al., 1972) are present in both ewes and cows until pamuition (Moss et al., 1980, 1981). Therefore, regulation of the rate of LH synthesis determines postpartum repre
ductive activity. Nilson et al. (1983) demonstrated a reduction in the amount of mRNA of the subunits of LH in pituitary glands of ovariectomized ewes treated with estradiol. This was associated with a reduction in pituitary content of LH. A similar decrease in mRNA for LH also has been demonstrated during gestation in the ewe (Wise et al., 1986); this decline was suggested as the cause for the decrease in pituitary stores of LH. Thus, gene expression for subunits of LH was suppressed by high semm concentrations of estradiol during gestation. During the early postpartum period of ewes, concentration of mRNA for both alpha and beta subunits increased rapidly, followed by a slow increase in pituitary content of LH during the postpartum period (Wise et al., 1986). This slow increase in pituitary content of LH in the cow and ewe appears to be responsible for the delay in return to reproductive activity. In contrast, rapid restoration of LH in the pituitary gland of the mare allows for an early return to cyclicity. Pituitary receptors for GnRH and estradiol were elevated by d 15 in postpartum suckled beef cows (Nett et al., 1988). These researchers suggested that increased receptors for GnRH and estradiol and increased sensitivity of the anterior pituitary gland to these hormones lead to an increased rate of synthesis of LH by d 30 after parturition. If these findipgs are true in the mare, then possibly there is an earlier and greater elevation of these receptors in the mare, which allows for a much more rapid restoration of LH. Endocrinology of late pregnancy is unique in the mare compared with other species. Progesterone concentrations are increased (Holtan et al., 1975) and estrogen concentrations are decreased (Nett et al. 1973) during the last month of pregnancy. This is the opposite of the cow (Henricks et al., 1972) and ewe (Stabenfeldt et al., 1972). Because of the presence of progesterone throughout gestation, concentrations of estrogens are still adequate to suppress synthesis of LH during late gestation (Nett et al., 1989). Garcia and Ginther (1978) reported that, in ovariectomized mares, a combination of progesterone and estradiol resulted in a greater suppression of LH secretion than progesterone did alone. There are several similarities between postpartum mares and seasonally anestrous mares. Silvia et al. (1986) demonstrated that pituitary content and baseline concentrations of LH
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ov
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HARRISON ET AL.
or second postpartum estrous cycle, negative feedback of estradiol (Gana et al., 1986) or inhibm (Miller et al., 1981) from the preovulatory follicle reduces secretion of FSH in response to GnRH stimulation. Because there is no follicular activity in the late pregnant mare, synthesis and secretion of FSH remain high. Implications
Pituitary concentration of luteinizing hormone was low initially after parturition but increased very rapidly during the first month after foaling. This rapid change in pituitary luteinizing hormone allows the mare to exhibit estrus and ovulate earlier in the postpartum period than other farm species. Llterature Clted Camegie, S. A. audH. A. Robertson. 1978. Conjugated and anconjugated eseogens in fetal and maternal fluids of the pregnant ewe. A possible role of estrone sulfate during pregaancy. Biol. Repmd. 19202. Crowder. M. E., P. A. Gilles,C. Tamanid, G. E. Moss and T. M.Nett. 1982. pituitary content of gonadotropins and GnRH-receptors in pregnant, postpartum and steroidh a t e d ovx ewes. J. Anim. Sci. 541235. Garcia, M.C. and 0.J. M e r . 1978.Regulation of plasma LH by estradiol and progesterone in ovariectomized mares. Biol. Reprod. 19447. Gama, F., Jr., D. L. ll~ompson, Jr., R. L. St. George and D. D. French. 1986. Androgen and estradiol effects on gonadotropin secretion and response to GnRH in ovariectomized pony mares. J. Anim. Sci. 621654. Gin&, 0. J. 1979. Reproductive biology of the mare basic and applied aspects. Published by author, Dept. Vet Sci., Univ. of Wisconsin, Madison Hemiclcs,D. M,, J. F.Dickey,J. R. Hill and W. E. Johnston. 1972. Plasma estrogen and pmgestmne levels after mating,during late pregnaracy and postpartumin cows. Endocrinology W1336. Hines,K. K.,E.P.Fitzgdd and R G. b y . 1987.Effect of palsatilegon&jotrophinreleaseonmeanserumLHand PSH in periparturient mares. J. Reprod. Fertil. Suppl. 35:635. H o l m D. W., T. M. Nett and V. L. Estergreen. 1975. Plasma progestinsin pregnaat postpartum and cycling mares. J. Anim. Sci. 40:251. M e , C.€LG. and M. J. Evans. 1978. FSH and LH concentrations precedingpostpartum ovulationin the mare. NZ. Vet. J. 26310. Miller, K. F., J. A. Wesson and 0. J. Ginther. 1981. Interaction of estradiol and a nonsteroidal folliculiu fluid substance in the regulation of gonadotropin secretion in the mare. Biol. Reprod. 24954. Moss, G. E.,T.E.Adams. G. D. Niswender and T. M.Nett. 1980. Effect of @tion and suckliug on concentrations of pituitary gollladohopins, hypothalamic GnRH and Pituitary responsiveness to GnRH in ewes. J. Anim. Sci. 50496. Moss, G. E., M.E. Crowder and T. M.Nett. 1981. GnRHReceptor Interaction. VI. Effect of progesterone and
Downloaded from https://academic.oup.com/jas/article-abstract/68/8/2430/4704885 by Iowa State University user on 17 January 2019
were low in the mare in winter anestrus as well as during the early transition period. Replenishment of pituitary stores of LH were necessary before the initial ovulation of the breeding season. In the present study, pituitary content of LH changed rapidly between d 6 and d 1 of the first estrus (d 7.5 f 1.7). Although peak response and area under the curve after a challenge with GnRH were lower for mares at the first estrus than at the second postpartum estrus, apparently sufficient LH was present to promote follicular development and development of a normal corpus luteum. Others (Irvine and Evans, 1978) have reported that peripheral concentrations of LH were similar during the first postpartum estrus and the second postpartum cycle. "he basal concentrations of LH during the first postpartum estrus (41.6 ng/ml) and second postpartum estrus (45.8 nglml) were similar to those reported previously for normal cycling mares (59 ng/d, Nett et al., 1987). This is interesting in light of the fact that the LH response to GnRH was lower during the frst estrus than during the second postpartum cycle. The reduced pituitary content of LH, noted in the present study, was not attributed to season because our experiment was conducted during the middle of the breeding season (June and July). Unlike the release of LH in response to GnRH, the amount of FSH secreted in response to GnRH tended to decrease from d 3 to d 6 postpartum. Response of FSH to GnRH for mares challenged on d 6 or during the first or second postpartum estrous cycle was minimal and similar to that reported previously by Silvia et al. (1987) in normal cycling mares. Hines et al. (1987) also reported a reciprocal relationship between LH and FSH concentrations in periparturient mares. At parturition, FSH concentrations were high and LH concentrations were low; however, by d 4 there was a reversal of this relationship. Thus, pituitary stores of FSH are not decreased in the pregnant mare during the last trimester, early postpartum or transition period (January to March). This is similar to what has been reported in the postpartum ewe and cow (Moss et al., 1980, 1981; Walters et al., 1982). Apparently, synthesis in the pituitary gland and secretion from the pituitary is not regulated in the same manner for FSH and LH. As the mare develops follicular activity, either during the transition period or during the first
RESPONSE BY POSTPARTUM MARES TO GnRH
luteinizinghormone of beta-genesin the ovine anterior pituitary. J. Biol. Chem. 25812087. Niswender, G. D. 1973.Influence of the site of conjugation on the specificity of antibodies to progesterone. Stmids 22:413. SAS. 1987.SASISTATGuide for PersonalComputers.SAS Inst., Inc., Cary, NC. Sihria, P.J., E. L.Squires and T. M. Nett. 1986.Changes in the hypoulalamic hypophyseal axis of mares associated with seasonal reproductive recrudescence. Biol. Reprod. 35:897. Silvia, P.J., E.L. Squires and T. M. Nett. 1987. pituitary responsiveness of mares challenged with GnRH at various stages of the transition into the braeding season. J. Anim. Sci. 64:790. Stabenfeldt, G. H., M. h o s t and C. E. b t i . 1972. Peripheral plasma progesterone lewels in the ewe daring pregnancy and parhxition. Endocrinology 90: 144. Steel, RGD. and Torrie, J. H. 1980. Principles and Roceduresof Statistics: A BiometricalApproach (2nd Ed.). McoraW-Hill Book Co., New York. Walters, D. L., C. C. Kaltenbach, T. G. Duna and R. R. Short. 1982.Pituitary and ovarian function in postparhm beef cows. I. Effect of suckling on serum and follicular fluid hormones and follicular gonadotropin receptors. Biol. Reprod. 26:W. Wise, M.E.,J. D. Glassand T. M. Nett. 1986.Changesin the concentrationof hypothalamic and hypophyseal receptors for estradiol m pregnant and postpartum ewes. J. Anim. Sci. 62:1021.
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estradiol on hypophyseal receptors for GnRH, and serum and hypophyseal concentrations of gonadotropins in ovariectomizedewes. Biol. Reprod. 25:938. Nett, T. M., D. Cermak, T. Braden, J. Manns and G. D. Niswender. 1988.Receptors for GnRH and estradiol and pituitary contmt of gonadotropins in beef cows. E. Changes during postpartum period. Domest. Anim. Endocrinol. 581. Nett, T. M,D. W. Holtan and V. L. Esterpen. 1973. Plasma estrogensin pregnant and postpartum mares.J. Anim. Sci. 37:962. Nett, T. M.,D. W. Holm and V.L. Estergreen. 1975.Serum levels of oestrogens, luteinizinghormone and prolactin inpOSt-partammanS. J. Reprod. Fertil. SUpPl. 23~201. Nett, T. M., B. W. pickett and E.L.Squires. 1979.Effects of equimate (ICI-81008)on levels of luteinizing hormone, folliclsstimulating honnone and progesterone during the estrous cycle of the mare. J. Anim. Sci. 48: 69. Nett, T. M.,C. F. Shoemaker and E. L. Squires. 1987. GnRH-stimulated release of LH during pregnancy and after parturition. J. Reprod. Pertil. Suppl. 35:729. Nett, T. M., C. F. Shoemaker and E. L. Squires. 1989. Changes in serum conccntT(Ltiom of luteinizing hormone and folliclastimulating hormone following injection of gonadotropin-releasing hormone during pregnancy and after paaaritiOn in mares. I. Anim. Sci. 6 71330. Nilson, J. H.,M.T. NejedWc, J. B. Virgin,M. E. Crowder and T. M. Nett. 1983.Expression of alpha-subunitand
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Colorado State University, Fort Collins 80523 ABSTRACT
We hypothesized that the LH response to GnRH would be greater as the interval from foaling increases, whereas the FSH response would decrease, and that corpus luteum function after the first ovulation would be similar to that after the second ovulation. At parturition, mares were assigned to receive GnRH (2 p&g) intravenously on 1) d 3 postpartum (n = 6); 2) d 6 postpartum (n = 6); 3) d 1 of first postpartum estrus (foal estrus) and again on d 1 of second postpartum estrus (n = 8). Blood was collected through an indwelling cannula at -2, -1 and 0 h relative to GnRH stimulation (basal concentrations) and at .25, .5, .75, 1.0, 1.5, 2.0,2.5,3.0, 3.5 and 4.0 h post-GnRH. Samples were assayed for concentrations of LH and FSH. Basal concentrations of LH were lower (P < .05) for mares given GnRH on d 3 postpartum than for mares on d 1 of foal estrus. A rise in concentrations of LH was noted within 30 min in all groups, but the response to GnRH on d 1 of the first estrus was less (P c .05) than on d 1 of second postpartum estrus. As the interval from parturition increased, the amount of LH secreted in response to GnRH increased. The maximum response to GnRH was greater (Pc .05) during d 1 of the first estrus than on d 3 or 6 postpartum and was greater on d 1 of cycle 2 than on d 1 of cycle 1. In contrast, the amount of PSH secreted in response to GnRH was similar (P > .05) for all days. Pituitary concentrations of LH presumably were depressed in early postpartum mares, but pituitary concentrations of LH were quickly restored. (Key Words: LH, FSH, GnRH, Parturition, Mares.) J. Anim. Sci. 1990. 68:2r130-2435
lntroductlon
Postpartum anestrous in the ewe and cow has been associated with insufficient concentrations of LH in serum and in the anterior pituitary gland (Nett et al., 1987, 1988). Apparently, the high concentrations of estrogens in the peripheral circulation during late gestation are associated with inhibition of LH synthesis in the anterior pituitary (Moss et al., 1980, 1981). In sheep, the amount of LH secreted after GnRH challenge was correlated highly with concentration of LH in the anterior pituitary gland (Crowder et al., 1982). Likewise, the content of LH in the anterior pituitary
lAnim. Reprod. Lab. *Supported in part by Colorado State Exp. Sta. Received June 12,1989. Accepted November 17,1989.
gland of mares was correlated positively with the amount of LH released in response to a maximally stimulatory dose of GnRH (Silvia et al., 1987). Unlike species such as the ewe and cow, the mare resumes normal ovarian cyclicity soon after parturition (Ginther, 1979). Even though concentrations of estrone and estradiol decline during the last third of gestation (Nett et al., 1973), concentrations still are adequate to suppress synthesis of LH and inhibit accumulation of LH in the anterior pituitary gland. Nett et al. (1989) challenged mares with a maximally stimulatory dose of GnRH on d 240 and 320 of gestation, d 3 postpartum and (in a separate group) on d 2 or 3 of estrus. Basal concentrations and total quantity of LH released after GriRH was similar (P> .05) on d 240 and 320 of gestation and d 3 postpartum, but release was greater by d 3 of the first postpartum estrus. Release of FSH after GnRH
2430
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L. A. Harrison, E. L. Squires'J, T. M. Nett and A. 0. McKinnon
RESPONSE BY POSTPARTUM MARES TO GnRH
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decreased continually (P < .05) from d 240 of every other day during first and second periods of diestrus and were assayed for progesterone gestation to d 3 of estrus. The present study was conducted to test (Niswender, 1973). Data from one mare in Group 3 (no. 779) these hypotheses: 1) LH response to GnRH is were excluded from all analyses. This mare greater as the interval from parturition increases, whereas the FSH response to GnRH had a high baseline concentration of LH during decreases; and 2) corpus luteum function after the first postpartum period of estrus (127.0 ng/ the first ovulation is similar to that after the ml vs 41.6 ng/ml for all other mares in this group) and elevated baseline concentrations of second ovulation. FSH during the first (70.2 ng/ml vs 12.5 ng/ ml, all others) and second (92.2 ng/ml vs 14.6 Materials and Methods ng/d all others) postpartum cycle. Twenty lactating embryo transfer recipient Three characteristics of LH and FSH were mares that foaled in May, June and July were established from the GnRH-challenge: baseused in this study. At parturition, mares were line, maximum response and area under the assigned randomly to one of three groups: 1) responsive curve. Baseline concentration of GnRH challenge on d 3 postpartum (n = 6); 2) each gonadotropin was calculated by averaging GnRH challenge on d 6 postpartum (n = 6); values from the three pre-injection samples. and 3) GnRH challenge on d 1 of the first Maximum response was identified as the postpartum period of estrus (foal estrus) and greatest concentration of hormone achieved again on d 1 of the second postpartum period after injection minus the baseline. k e a under of estrus (n = 8). Mares were administered a the response w e was determined from maximally stimulatory dose of onRH3 (2 pg/ samples collected between 0 and 4 h after kg) in .9% saline intravenously (Nett et al., GnRH using the SAS program (1987); data 1988). All mares were maintained on pasture, were adjusted for baseline concentrations. fed 1.5 kg grain per day and had ad libitum Differences in these characteristics among the access to alfalfa hay. Mares were exposed to a three groups (d 3, 6 and d 1, cycle 1) were stallion each day for detection of estrus. determined by analysis of variance. When Palpation and ultrasonography of the genital significant group effects were observed, means tract per rectum was initiated on d 2 postpar- for each group were compared by Tukey's tum and continued daily throughout the study. Honestly Significant Differences test (Steel Mares were separated from their foals during and Tome, 1980). T-tests were used to teasing. determine difference in LH and FSH values for For collection of blood, mares and foals mares at d 1, cycle 1 versus d 1, cycle 2. were housed in 3- x 15-m pens with access to Concentrations of progesterone on d 1,3,5,7, water. A 16-gauge indwelling cannula was 9, 11 and 13 of each diestrous period were placed in the jugular vein of each mare and the averaged and mcans were compared by t-test. first blood sample was taken at 0730. Blood samples were collected at -2, -1 and 0 h Results relative to GnRH stimulation to determine basal concentrations, then at .25, .5, .75, 1.0, hior to treatment, basal concentrations of 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 h after GnRH. LH were lower (P < .OS) for mares given Twenty milliliters of blood was collected for GnRH on d 3 postpartum than for those each sample, placed in centrifuge tubes and administered GnRH on d 1 of foal estrus stored at room temperature for approximately Vable 1). Concentration of LH in mares on d 20 min, then at 5'C overnight. Samples were 6 of cycle 1 were similar to values in the other centrifuged for 20 min, and serum was groups (Figure 1). Although a rise in concenharvested and frozen at -20'C until it was trations of LH was noted within 30 min in all quantified for concentrations of LH (Nett et groups, the response to GnRH treatment on d 3 al., 1975) and FSH (Nett et al., 1979) by RIA. or 6 postpartum was only modest. In contrast, Additional blood samples were collected daily a marked release of LH was detected after during first and second periods of estrus and treatment on d 1 of the second postpartum estrus, whereas treatment with GnRH on d 1 of foal estrus resulted in a lower response than that on d 1 of cycle 2 Figure 1). This indicates h, MSMO, sigma chemical CO.,st ~ o u i s MO. ,
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HARRISON ET AL. TABLE 1. RESPONSE TO GnRH STIMULATION' Parameter
GrOUDb
LH
m e , units FSH
Day 3 Day 6 Day 1,cycle 1 Day 1,cycle2
79.2 f 87.0 f 297.2 f 595.2 f
97.8' 54.8' 103.5' 235.1d
Maximum response, nglml FSH
LH
16.4 f 202 f 76.5 f 122.6 f
43.8 f 30.5' 13.7 f 11.4' 12.0 f 25.4' 23.2 f 20.1'
14.8' 21.5 f 9.4 1O.P 12.4 f 9.5 28.gd 12.5 f 8.8 24Se 14.6 f 4.4
Baseline, ng/ml FSH
LH 10.0 f 21.1 f 41.6 f 45.8 f
8.5' 13.@ 22.od 16.8d
28.1 24.2 20.9 21.2
f f f f
4.5' 10.V 8.V 5.3'
'Mean f SD. b a y 3 = 3 d p o s t p m day 6 = 6 dpstpartum; day 1, cycle 1 = d 1 of firstpostpartumestrus; day 1, cycle2 = d 1 of second postpartum estrus. '*dMeanswithin column with different superscriptsare different (P< .05).
that as the interval from parturition increased, the amount of LH secreted in response to GnRH increased. However, the areas under the response curve for mares on d 3 and 6 postpartum and d 1 of the first estrus were similar (P > .05). Similarly, the maximum response of LH to GnRH for mares challenged during the first postpartum estrus was greater (P < .OS) than for mares challenged on either d 3 or 6 postpartum. The maximum response of LH to GnRH also increased from d 1, cycle 1 to d 1, cycle 2. Concentrations of FSH prior to GnRH treatment were similar (P > .05) among all groups (Figure 2). In addition, the response to GnRH as measured by total area under the curve, response area under the curve and maximum response were similar (P > .05) among all groups. However, the greatest numerical response to GnRH was noted in mares treated on d 3 postpartum; FSH re-
sponse to GnRH in the other three groups was minimal. Duration of estrus for the first and second postpartum estrous periods was similar (P > .05) at 4.9 f 1.8 and 4.4 f 1.1 d, respectively. Durations of the first and second diestrous periods also were similar (P > .05) at 13.6 f 2.0 and 13.1 f 3.0 d, respectively. Concentrations of progesterone during both diestrous periods are presented in Figure 3. Values were similar (P> .05) for the two diestrous periods (overall means being 2.9 f .8 and 2.8 f .6 for cycle 1 and 2, respectively). Peak progesterone during the first and second postpartum cycles was 3.8 f 2.1 and 4.0 f 1.5 ng/ml, respectivelY
-
Dlscusslon
Silvia et al. (1987) reported a correlation of .82 between pituitary concentration of LH and
50-
150130.-
--
110-
A 1st h e a t 2nd h e a t
-- 40..
E 90-
-
70--
3
50.-
A 2nd heat
E
\
rn
\
p
0 Day 3
5
30-
I v, LL
20-
30IO--
04 - 3 - 2 - 1
0
I 2 TIME ( h )
3
4
I
IO.
5
Figure 1. Concentrations of LH in postpartum mares before and after administration (time 0) of GnRH.
Figure 2. Concenhations of FSH in postpartum mares before and after administrationof GnRH.
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Area under
RESPONSE BY POSTPARTUM MARES TO GnRH
ov
Figure 3. Progesteroneprofiie during estrous cycle 1 and 2 postpartum.
baseline serum LH concentration. In addition, the correlation coefficient for area under the c w e in response to GnRH and pituitary concentrations of LH was .72. Assuming this finding holds true for postpartum mares as it does for postpartum ewes (Moss et al., 1980), then results of the present study and a previous study (Nett et al., 1989) suggest that pituitary concentrations of LH were depressed in pregnant mares and early postpartum mares. However, in contrast to the beef cow and ewe, pituitary concentrations of LH were replenished quickly after parturition even when mares were nursing foals. Baseline concentrations of LH were greater (P< .05) during the first estrous period in both lactating and nonlactating mares compared with d 240 or 320 of gestation or d 3 and 6 after parturition. Maximum LH response to GnRH and area under the response curve increased as the interval from parturition increased. This apparent early replenishment of pituitary content of LH probably is responsible for the mare’s ability to exhibit a postpartum cycle within 9 to 11 d after pamuition, in contrast to the longer periods of postpartum anestrus noted in sheep and beef cattle. Postpartum anestrus in the ewe and beef cow has been associated with insufficient concentration of LH, in the anterior pituitary gland and serum, to stimulate follicular maturation and owlation. Very high concentrations of estrogen (Camegie and Robertson, 1978) and progestin (Stabenfeldt et al., 1972) are present in both ewes and cows until pamuition (Moss et al., 1980, 1981). Therefore, regulation of the rate of LH synthesis determines postpartum repre
ductive activity. Nilson et al. (1983) demonstrated a reduction in the amount of mRNA of the subunits of LH in pituitary glands of ovariectomized ewes treated with estradiol. This was associated with a reduction in pituitary content of LH. A similar decrease in mRNA for LH also has been demonstrated during gestation in the ewe (Wise et al., 1986); this decline was suggested as the cause for the decrease in pituitary stores of LH. Thus, gene expression for subunits of LH was suppressed by high semm concentrations of estradiol during gestation. During the early postpartum period of ewes, concentration of mRNA for both alpha and beta subunits increased rapidly, followed by a slow increase in pituitary content of LH during the postpartum period (Wise et al., 1986). This slow increase in pituitary content of LH in the cow and ewe appears to be responsible for the delay in return to reproductive activity. In contrast, rapid restoration of LH in the pituitary gland of the mare allows for an early return to cyclicity. Pituitary receptors for GnRH and estradiol were elevated by d 15 in postpartum suckled beef cows (Nett et al., 1988). These researchers suggested that increased receptors for GnRH and estradiol and increased sensitivity of the anterior pituitary gland to these hormones lead to an increased rate of synthesis of LH by d 30 after parturition. If these findipgs are true in the mare, then possibly there is an earlier and greater elevation of these receptors in the mare, which allows for a much more rapid restoration of LH. Endocrinology of late pregnancy is unique in the mare compared with other species. Progesterone concentrations are increased (Holtan et al., 1975) and estrogen concentrations are decreased (Nett et al. 1973) during the last month of pregnancy. This is the opposite of the cow (Henricks et al., 1972) and ewe (Stabenfeldt et al., 1972). Because of the presence of progesterone throughout gestation, concentrations of estrogens are still adequate to suppress synthesis of LH during late gestation (Nett et al., 1989). Garcia and Ginther (1978) reported that, in ovariectomized mares, a combination of progesterone and estradiol resulted in a greater suppression of LH secretion than progesterone did alone. There are several similarities between postpartum mares and seasonally anestrous mares. Silvia et al. (1986) demonstrated that pituitary content and baseline concentrations of LH
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ov
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HARRISON ET AL.
or second postpartum estrous cycle, negative feedback of estradiol (Gana et al., 1986) or inhibm (Miller et al., 1981) from the preovulatory follicle reduces secretion of FSH in response to GnRH stimulation. Because there is no follicular activity in the late pregnant mare, synthesis and secretion of FSH remain high. Implications
Pituitary concentration of luteinizing hormone was low initially after parturition but increased very rapidly during the first month after foaling. This rapid change in pituitary luteinizing hormone allows the mare to exhibit estrus and ovulate earlier in the postpartum period than other farm species. Llterature Clted Camegie, S. A. audH. A. Robertson. 1978. Conjugated and anconjugated eseogens in fetal and maternal fluids of the pregnant ewe. A possible role of estrone sulfate during pregaancy. Biol. Repmd. 19202. Crowder. M. E., P. A. Gilles,C. Tamanid, G. E. Moss and T. M.Nett. 1982. pituitary content of gonadotropins and GnRH-receptors in pregnant, postpartum and steroidh a t e d ovx ewes. J. Anim. Sci. 541235. Garcia, M.C. and 0.J. M e r . 1978.Regulation of plasma LH by estradiol and progesterone in ovariectomized mares. Biol. Reprod. 19447. Gama, F., Jr., D. L. ll~ompson, Jr., R. L. St. George and D. D. French. 1986. Androgen and estradiol effects on gonadotropin secretion and response to GnRH in ovariectomized pony mares. J. Anim. Sci. 621654. Gin&, 0. J. 1979. Reproductive biology of the mare basic and applied aspects. Published by author, Dept. Vet Sci., Univ. of Wisconsin, Madison Hemiclcs,D. M,, J. F.Dickey,J. R. Hill and W. E. Johnston. 1972. Plasma estrogen and pmgestmne levels after mating,during late pregnaracy and postpartumin cows. Endocrinology W1336. Hines,K. K.,E.P.Fitzgdd and R G. b y . 1987.Effect of palsatilegon&jotrophinreleaseonmeanserumLHand PSH in periparturient mares. J. Reprod. Fertil. Suppl. 35:635. H o l m D. W., T. M. Nett and V. L. Estergreen. 1975. Plasma progestinsin pregnaat postpartum and cycling mares. J. Anim. Sci. 40:251. M e , C.€LG. and M. J. Evans. 1978. FSH and LH concentrations precedingpostpartum ovulationin the mare. NZ. Vet. J. 26310. Miller, K. F., J. A. Wesson and 0. J. Ginther. 1981. Interaction of estradiol and a nonsteroidal folliculiu fluid substance in the regulation of gonadotropin secretion in the mare. Biol. Reprod. 24954. Moss, G. E.,T.E.Adams. G. D. Niswender and T. M.Nett. 1980. Effect of @tion and suckliug on concentrations of pituitary gollladohopins, hypothalamic GnRH and Pituitary responsiveness to GnRH in ewes. J. Anim. Sci. 50496. Moss, G. E., M.E. Crowder and T. M.Nett. 1981. GnRHReceptor Interaction. VI. Effect of progesterone and
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were low in the mare in winter anestrus as well as during the early transition period. Replenishment of pituitary stores of LH were necessary before the initial ovulation of the breeding season. In the present study, pituitary content of LH changed rapidly between d 6 and d 1 of the first estrus (d 7.5 f 1.7). Although peak response and area under the curve after a challenge with GnRH were lower for mares at the first estrus than at the second postpartum estrus, apparently sufficient LH was present to promote follicular development and development of a normal corpus luteum. Others (Irvine and Evans, 1978) have reported that peripheral concentrations of LH were similar during the first postpartum estrus and the second postpartum cycle. "he basal concentrations of LH during the first postpartum estrus (41.6 ng/ml) and second postpartum estrus (45.8 nglml) were similar to those reported previously for normal cycling mares (59 ng/d, Nett et al., 1987). This is interesting in light of the fact that the LH response to GnRH was lower during the frst estrus than during the second postpartum cycle. The reduced pituitary content of LH, noted in the present study, was not attributed to season because our experiment was conducted during the middle of the breeding season (June and July). Unlike the release of LH in response to GnRH, the amount of FSH secreted in response to GnRH tended to decrease from d 3 to d 6 postpartum. Response of FSH to GnRH for mares challenged on d 6 or during the first or second postpartum estrous cycle was minimal and similar to that reported previously by Silvia et al. (1987) in normal cycling mares. Hines et al. (1987) also reported a reciprocal relationship between LH and FSH concentrations in periparturient mares. At parturition, FSH concentrations were high and LH concentrations were low; however, by d 4 there was a reversal of this relationship. Thus, pituitary stores of FSH are not decreased in the pregnant mare during the last trimester, early postpartum or transition period (January to March). This is similar to what has been reported in the postpartum ewe and cow (Moss et al., 1980, 1981; Walters et al., 1982). Apparently, synthesis in the pituitary gland and secretion from the pituitary is not regulated in the same manner for FSH and LH. As the mare develops follicular activity, either during the transition period or during the first
RESPONSE BY POSTPARTUM MARES TO GnRH
luteinizinghormone of beta-genesin the ovine anterior pituitary. J. Biol. Chem. 25812087. Niswender, G. D. 1973.Influence of the site of conjugation on the specificity of antibodies to progesterone. Stmids 22:413. SAS. 1987.SASISTATGuide for PersonalComputers.SAS Inst., Inc., Cary, NC. Sihria, P.J., E. L.Squires and T. M. Nett. 1986.Changes in the hypoulalamic hypophyseal axis of mares associated with seasonal reproductive recrudescence. Biol. Reprod. 35:897. Silvia, P.J., E.L. Squires and T. M. Nett. 1987. pituitary responsiveness of mares challenged with GnRH at various stages of the transition into the braeding season. J. Anim. Sci. 64:790. Stabenfeldt, G. H., M. h o s t and C. E. b t i . 1972. Peripheral plasma progesterone lewels in the ewe daring pregnancy and parhxition. Endocrinology 90: 144. Steel, RGD. and Torrie, J. H. 1980. Principles and Roceduresof Statistics: A BiometricalApproach (2nd Ed.). McoraW-Hill Book Co., New York. Walters, D. L., C. C. Kaltenbach, T. G. Duna and R. R. Short. 1982.Pituitary and ovarian function in postparhm beef cows. I. Effect of suckling on serum and follicular fluid hormones and follicular gonadotropin receptors. Biol. Reprod. 26:W. Wise, M.E.,J. D. Glassand T. M. Nett. 1986.Changesin the concentrationof hypothalamic and hypophyseal receptors for estradiol m pregnant and postpartum ewes. J. Anim. Sci. 62:1021.
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estradiol on hypophyseal receptors for GnRH, and serum and hypophyseal concentrations of gonadotropins in ovariectomizedewes. Biol. Reprod. 25:938. Nett, T. M., D. Cermak, T. Braden, J. Manns and G. D. Niswender. 1988.Receptors for GnRH and estradiol and pituitary contmt of gonadotropins in beef cows. E. Changes during postpartum period. Domest. Anim. Endocrinol. 581. Nett, T. M,D. W. Holtan and V. L. Esterpen. 1973. Plasma estrogensin pregnant and postpartum mares.J. Anim. Sci. 37:962. Nett, T. M.,D. W. Holm and V.L. Estergreen. 1975.Serum levels of oestrogens, luteinizinghormone and prolactin inpOSt-partammanS. J. Reprod. Fertil. SUpPl. 23~201. Nett, T. M., B. W. pickett and E.L.Squires. 1979.Effects of equimate (ICI-81008)on levels of luteinizing hormone, folliclsstimulating honnone and progesterone during the estrous cycle of the mare. J. Anim. Sci. 48: 69. Nett, T. M.,C. F. Shoemaker and E. L. Squires. 1987. GnRH-stimulated release of LH during pregnancy and after parturition. J. Reprod. Pertil. Suppl. 35:729. Nett, T. M., C. F. Shoemaker and E. L. Squires. 1989. Changes in serum conccntT(Ltiom of luteinizing hormone and folliclastimulating hormone following injection of gonadotropin-releasing hormone during pregnancy and after paaaritiOn in mares. I. Anim. Sci. 6 71330. Nilson, J. H.,M.T. NejedWc, J. B. Virgin,M. E. Crowder and T. M. Nett. 1983.Expression of alpha-subunitand
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