Effects of Season and Stage of Gestation Hormone Release in Gilts

on

Luteinizing

Craig A. Smith and Glen W. Almond

ABSTRACT This study was designed to examine the effects of two seasons and stage of gestation on luteinizing hormone (LH) release in the gilt. Eleven YorkshireLandrace crossbred gilts were each fitted with an indwelling vena caval cannula. Blood samples were collected at 6 h intervals for six days during early (day 39 to 44) or mid-gestation (day 69 to 74). Serum progesterone, estradiol-17#3 and LH concentrations were determined in samples collected at 6 h intervals. Early and midgestation occurred during August and September in group 1 (n = 6) and during January and February in group 2 gilts (n = 5). To characterize pulsatile LH release, samples were collected at 15 min intervals for 8 h on day 40, 43, 70 and 73 of gestation. Following each 8 h sampling period, gilts were treated intravenously with 0.5 Zg gonadotropinreleasing hormone (GnRH)/kg body weight and blood collected at 10 min intervals for 3 h. Progesterone concentrations decreased (p < 0.01) from 22.1 ± 0.4 ng/mL during early gestation to 18.2 ± 0.4 ng/mL during midgestation. Estradiol-17f3 concentrations increased (p < 0.01) from early to mid-gestation (13.5 ± 0.8 versus 28.4 ± 0.7 pg/mL). Frequency of LH pulses and LH pulse amplitude were higher (p < 0.05) in pregnant gilts during January and February compared to August and September. The exogenous GnRH-induced peak LH concentrations, amplitude of peak LH concentrations, and total amount of LH released were higher (p < 0.01), while the interval from GnRH administration until peak LH concentration was shorter (p < 0.05) in early com-

pared to mid-gestation gilts. The results indicate that endogenous LH release differed between the two seasons. In contrast, LH release in response to exogenous GnRH was affected by stage of gestation, but not by season.

RESUME Le but de cette etude etait d'evaluer les effets saisonniers ainsi que les effets du stade de gestation sur les niveaux d'hormone luteinisante (LH) chez des cochettes. Onze cochettes YorkshireLandrace ont ete catheterisees et des echantillons sanguins provenant de la veine cave ont ete preleves a toutes les six heures pendant six journees consecutives en debut (jour 39-44) et mi-gestation (jour 69-74) et les niveaux de progesterone, de la 17 ,B-estradiol et de LH y ont ete mesures. Chez les six animaux du groupe 1, le debut et la mi-gestation se sont produits durant les mois d'aout et septembre et durant les mois de janvier et fevrier chez les cinq sujets du groupe 2. Afin de verifier le relachement en ondes de la LH, des echantillons sanguins furent recoltes aux 15 minutes pendant une periode de huit heures au jours 40, 43, 70 et 73 de gestation. A la fin de ces periodes de prelevements, les animaux ont regu une injection intraveineuse de l'hormone hypothalamique de liberation de l'hormone luteinisante (GnRH) a la dose de 0.5 dg/kg et des echantillons sanguins ont ete par la suite recueillis a toutes les dix minutes pour une periode de trois heures. Une diminution significative des niveaux de progesterone a ete observee entre le debut et la mi-gestation (22.1 ± 0.4 vs 18.2 ± 0.4 ng/mL) par contre les

niveaux d'estradiol ont significativement augmente (13.5 ± 0.8 vs 28.4 ± 0.7 pg/mL) durant la meme periode. La frequence et l'amplitude des pics de LH etaient superieurs (p < 0.05) en debut d'hiver qu'en fin d'e'te. L'administration de GnRH a provoque (p < 0.05) de plus hauts pics et une augmentation des niveaux seriques de LH, et la periode de latence entre l'injection et le pic de LH a allonge (p < 0.05) avec le nombre de jours de gestation. Ces resultats demontrent que le relachement de LH serait influence par les saisons tandis que la reponse a l'injection de GnRH semblerait plutot affectee par la periode de gestation. (Traduit par Dr Pascal Dubreuil)

INTRODUCTION Although serum concentrations of progesterone (P4) and estradiol-17,B (E2) during gestation have been described (1,2), there is a paucity of data concerning concentrations of the gonadotropic hormones during pregnancy in the pig. Ziecik et al (3) reported that plasma concentrations of luteinizing hormone (LH) during early and mid-pregnancy were comparable to mid-luteal concentrations of LH, but the frequency and amplitude of LH pulses during the second half of gestation were decreased compared to mid-luteal values. Jenkin et al (4) reported that the amount of LH released in ewes in response to a half-maximal dose of gonadotropin releasing hormone (GnRH) was highly correlated with pituitary content of LH. Furthermore, the GnRH-induced LH response in pregnant ewes declined to 35% of

Department of Food Animal and Equine Medicine, North Carolina State University, Raleigh, North Carolina 27606. Present address of Dr. C.A. Smith: School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 53706. Reprint requests to Dr. G.W. Almond. This research was supported by the North Carolina Veterinary Competitive Grants Program. Submitted December 3, 1990.

294

Can J Vet Res 1991; 55: 294-297

anestrous controls by day 56 of pregnancy and decreased further to 14% of anestrous controls by the time of parturition. Similar studies in the pig have not been reported. Although the domestic pig is considered to be capable of year-round reproduction, evidence indicates seasonal influences can adversely influence sow reproduction. Anestrus or delayed return to estrus following weaning is more prevalent during the months of July through September compared to other months of the year (5), and noninfectious abortions are most prevalent in the autumn months (6). The precise endocrine pathogenesis of the anestrous sow or noninfectious abortion remains unknown; however, a recent investigation revealed that the negative feedback effects of E2 on the hypothalamus and pituitary gland contribute to a sow's failure to return to estrus after weaning (7). Therefore, this investigation was designed to identify possible interactions between serum P4 and E2 concentrations and LH release that contribute to noninfectious abortion in the autumn months. Consequently, the present study examined the effects of two seasons and two stages of gestation on serum E2, LH, and P4 concentrations in the gilt.

MATERIALS AND METHODS ANIMAL MANAGEMENT

The experiments followed the guidelines of the "Guide to the Care and Use of Experimental Animals" of the Canadian Council on Animal Care. Eleven primiparous YorkshireLandrace crossbred gilts were used in this study. Following estrous synchronization with oral administration of allyl trenbolone (Hoechst-Roussel, Sommerville, New Jersey) at 16 mg/ gilt/day for 14 days, gilts were bred on the first day of estrus (day 0). Gilts were fed a 14% crude protein cornsoybean meal diet (2.3 kg/gilt/day). During the blood collection phases of the experiment, animals were housed in farrowing crates in an environment controlled room. Photoperiod and temperature were maintained at levels similar to the ambient environment. Outdoor pens were used to maintain

gilts prior to and between blood collection phases. Blood samples were collected during two stages of pregnancy: early (day 39 to 44) and mid-gestation (day 69 to 74). Early and mid-gestation samples were collected in August and September for group 1 gilts (n = 6) and in January and February for group 2 gilts (n = 5). Each gilt was fitted nonsurgically with an indwelling venous cannula one day before each sampling period (day 38 and 68, respectively). The cannula was inserted approximately 10 cm into the anterior vena cava. Blood samples were collected every 6 h during each six day sampling period and at 15 min intervals for 8 h on days 40, 43 and days 70, 73. Following each 8 h frequent sampling period, GnRH (Ceva Laboratories, Overland Park, Kansas) was administered intravenously (0.5 itg/kg body weight) followed by blood sample collection at 10 min intervals for 3 h. Serum P4 and E2 concentrations were determined in samples collected at 6 h intervals, while LH concentrations were quantitated in all samples. ANALYSIS OF HORMONE CONCENTRATIONS

The LH and E2 concentrations were determined using established radioimmunoassay (RIA) techniques (7). The interassay coefficients of variation (CV) for high (4.3 ng/mL) and low (0.9 ng/mL) LH reference sera were 13.8% and 16.807o, respectively (12 assays). The interassay CV for high (140 pg/mL) and low (25 pg/mL) E2 reference sera were 3.8% and 12.807o, respectively (13 assays). The intraassay CV for the LH and E2 assays were less than 5 %o. Progesterone (P4) concentrations were determined using a commercially available RIA kit (Diagnostic Products Corporation, Los Angeles, California). Serum samples of 100 ltL were placed into P4 antibody-coated polypropylene tubes. Each tube received 1.0 mL buffered [1251] P4 followed by a short vortex period and a 3 h incubation. Liquid was decanted, and tubes were allowed to drain, followed by counting on a gamma counter. The percentage of recovery of 0, 0.05, 0.2, 1, 5, 15 and 30 ng P4added to 100 AL of charcoal-stripped serum collected from ovariectomized gilts was 99.0 ±

3.207o (weighted mean ± SEM for 14 replicates). Intra-assay CV was less than 5%o. Interassay CV for reference sera containing high (24 ng/mL) and low (4 ng/mL) concentrations of P4 were 6.60o and 1.4%7o, respectively (three assays). ANALYSES OF DATA

Characteristics of pulsatile LH release during 8 h frequent sampling periods were analyzed as described previously (7). Mean LH concentrations, baseline, frequency of pulses, and pulse amplitude were determined for each 8 h period. The study was conducted as a replicated split-split plot repeated measurement in time design. The effect of sub-plot (day 40 versus 43; day 70 versus 73) was tested and found to be nonsignificant (p > 0.05). Sub-plots were pooled for further analyses. A split-plot-in-time analysis of variance (8) was used to analyze changes in LH, E2, and P4 concentrations in samples collected at 6 h intervals. The statistical model included season, stage of gestation, gilt-withinseason, and two-way interactions. The main effect of season was tested using the gilt-within-season mean square as the error term. When a significant season-by-time interaction was present (p < 0.05), modifications of this model were used to evaluate changes occurring over time within each season (9). Differences in means were compared by Tukey's Studentized range test for multiple comparisons (10). The LH response to GnRH was assessed by determining the maximum concentration of LH released (peak), time of peak relative to GnRH administration, and the difference between the peak LH and the baseline concentration determined from the previous 8 h sampling period. The total amount of LH released was determined by using a trapezoidal technique to calculate the area under the curve (AUC) based on the 19 samples collected during the 3 h interval following GnRH administration (11). The General Linear Model procedure (12) and split-plot analyses of repeated measurements (8) utilized the model described previously. Tukey's Studentized Range test for multiple comparisons was used to compare differences in means. 295

TABLE I. Changes in mean and baseline luteinizing hormone (LH) concentrationsa during 8 h frequent sampling periods at two stages of gestation in gilts during two seasons

Season August/ September January/ February aMean ± SEM b,cMeans within a column F=

Stage of gestation Early Mid Early Mid

with different superscripts are significantly different (p < 0.05)

4 U Gilt 0 Gilt o0k Gilt Gilt ~~~~~~~0

c:n CD CD

3-

E CD

Baseline LH concentration 0.7 + 0.1c 0.3 + 0.1b 0.6 + 0.lc 0.6 + 0.1c

Mean LH concentration 0.9 + 0.lC 0.4 + 0.lb 1.0 + 0.IC 0.9 + 0.IC

2

_

-

J_ rN,

. .

o-

28 28 47 47

Early Mid Early Mid

~

,

DISCUSSION

4-,

'0 120 60 Time After GnRH (min)

0

180

Fig. 1. Comparison of luteinizing hormone (LH) release in response to exogenous gonadotropinreleasing hormone (GnRH) in primiparous gilts at two stages of gestation. Illustrated profiles are for individual gilts representative of their respective groups; Gilt 28 = Group 1 (August-September), Gilt 47 = Group 2 (January-February).

RESULTS Mean steroid hormone concentrations were affected by stage of gestation. Whereas serum E2 concentrations were higher (p < 0.01) in gilts during mid-gestation (28.4 + 0.7 pg/mL) compared to early gestation (13.5 ± 0.8 pg/mL), P4 concentrations were higher (p < 0.01) during early gestation (22.1 ± 0.4 ng/mL) compared to mid-gestation (18.2 + 0.4 ng/mL). Mean daily concentrations of LH exhibited a season-by-stage of gestation interaction. Gilts pregnant during August and September had higher (p < 0.05) mean daily LH concentrations during early gestation (1.6 + 0.03 versus 0.8 ± 0.04 ng/mL), but lower mean daily LH concentrations during mid-gestation (0.34 ± 0.03 versus 0.78 ± 0.04 ng/mL) than their 296

during January and February compared to August and September (1.53 ± 0.09 versus 1.0 ± 0.09 ng/mL, respectively) and was also higher (p < 0.01) during early gestation compared to mid-gestation (1.59 ± 0.09 versus 0.94 ± 0.1 ng/mL, respectively). The release of LH in response to exogenous GnRH was affected by stage of gestation (Fig. 1). The peak LH concentration, amplitude of the peak LH concentration, and total amount of LH released were higher (p < 0.01) during early gestation (3.31 ± 0.11 ng/mL, 2.68 ± 0.12 ng/mL and 377 ± 12 ng/mL/min, respectively) than during mid-gestation (2.17 ± 0.12 ng/mL, 1.71 ± 0.12 ng/mL and 272 ± 12 ng/mL/min, respectively). The interval from administration of GnRH until the peak LH concentration occurred was shorter (p < 0.05) during early compared to mid-gestation (26.8 ± 3.3 versus 37.0 ± 3.4 min, respectively).

counterparts during January and February. The pulsatile LH characteristics were affected by season, stage of pregnancy, and season-by-stage of pregnancy interactions. Whereas mean and baseline LH concentrations were higher (p < 0.05) in early than in mid-gestation during August and September; there were no differences between early and mid-gestation during January and February (Table I). In addition, mean and baseline LH concentrations were greater (p < 0.05) in mid-gestation sows during February than in September. The LH pulse frequency was higher (p < 0.05) in pregnant gilts during January and February compared to August and September (2.35 ± 0.15 versus 1.62 ± 0.14 pulses/8 h, respectively). Amplitude of the LH pulses was higher (p < 0.05) in pregnant gilts

Since the corpus luteum is the principal source of P4 for pregnancy in the pig, a previous report indicated that suppressed P4 concentrations contributed to noninfectious abortions during August to October (13). In contrast, serum P4 concentrations were not affected by season in the present study. Serum concentrations in the present study were lower during midgestation compared to early gestation. In contrast, Robertson and King (1) reported that P4 concentrations were fairly constant throughout gestation until approximately two weeks before parturition. Since E2 treatment decreases plasma P4 concentrations (14,15), the decrease in P4 concentrations in the present study may have been due to the increased fetal-placental production (16) of E2 during midgestation compared to early gestation. The concentrations of E2 observed in the present study were consistent with previous reports (1). Serum E2 concentrations are low (< 15 pg/mL) during early gestation, but increase beginning around day 70 (mid-gestation) and continue to rise until parturition. Frequency and amplitude of LH pulses were higher during January and February compared to August and

September. In addition, mid-gestation ducted in the pig. Assuming that 4. JENKIN G, HEAP RB, SYMNONS DBA. Pituitary responsiveness to synthetic LH-RH gilts had lower mean LH concentra- exogenous GnRH-induced LH release and pituitary LH content at various tions and lower baseline LH concen- reflects pituitary stores of LH, the reproductive stages in the sheep. J Reprod trations during August and September decreased LH response observed with Fertil 1977; 49: 207-214. compared to January and February. advancing gestation in the pig is due, 5. HURTGEN JP, LEMAN AD, CRABO B. Seasonal influence on estrous activity in sows Increased incidence of abortion (6,13) at least in part, to decreased pituitary and gilts. J Am Vet Med Assoc 1980; 176: has been reported during August and concentrations of LH. 119-123. September compared to January and The decreased LH reponse to 6. ALMOND GW, FRIENDSHIP RM, BOSU February. It is conceivable that exogenous GnRH observed in midWTK. Autumn abortions in sows. Can Vet J 1985; 26: 162-163. decreased LH release, as noted in the gestation compared to early gestation 7. ALMOND GW, DIAL GD. Estradiol feedpresent study, contributes to the fail- was similar to the diminished GnRHback inhibition of luteinizing hormone conure of luteal maintenance during early induced LH release observed in ovaricentrations in the anestrous sow. J Anim Sci autumn pregnancies. ectomized sows treated with exogenous 1990; 68: 1077-1086. It has been hypothesized that E2 (7). Since similar serum E2 concen- 8. GILL JL, HAFS HD. Analysis of repeated measurements of animals. J Anim Sci 1971; seasonal patterns of decreased trations were observed in both studies, 33: 331-336. reproductive efficiency are due to an it is likely that the mid-gestation con- 9. STEEL RGD, TORRIE JH. Principles and increased sensitivity of the hypothalamo- centrations of E2 contribute to the Procedures of Statistics. A Biomedical hypophyseal axis to the negative feed- inhibitory effects observed in the presApproach. New York: McGraw-Hill Book Co., 1980: 172-194. back effects of E2. Almond and Dial ent study. Although it has not been 10. SNEDECOR GW, COCHRAN WG. (7) demonstrated that serum concen- demonstrated in the pig, E2 is a potent Statistical Methods. 4th ed. Ames: Iowa trations of E2, similar to those inhibitor of mRNA for a and ,B State University Press, 1980: 233-235. observed during mid-gestation in the subunits of LH in the ewe (19). 11. ALMOND GW, DIAL GD. Sensitivity of the hypothalamo-hypophyseal axis to the present study, suppressed LH release. Therefore, it is tempting to speculate positive feedback effects of estrogen in Thus, the present results support the that the concentrations of E2 present ovariectomized anestrous and diestrous hypothesis of a seasonal increase in at mid-gestation suppress synthesis of Anim Reprod Sci 1990; 22: 243-252. the sensitivity of the hypothalamic- LH. Consequently, the E2 inhibition 12. sows. SAS. SAS User's Guide: Statistics. Cary, hypophyseal axis to the negative feed- of LH secretion indirectly lowers P4 North Carolina: SAS Institute Inc., 1985. back effects of E2. concentrations during mid-gestation. 13. WRATHALL AE, WELLS DE, JONES PC, FOULKES JA. Seasonal variations in In summary, gilts had higher serum It has been demonstrated that E2 serum progesterone levels in pregnant sows. E2 concentrations at mid-gestation will decrease LH release by suppressing Vet Rec 1986; 188: 685-687. GnRH release from the hypothalamus- compared to early gestation. Whereas 14. GUTHRIE HD, REXROAD CE. Endometrial prostaglandin F release in vitro and stalk median eminence (17). In the pulsatile LH release was suppressed plasma 13,14-dihydro-I5-keto-prostaglandin present study, seasonal effects on LH during August and September comF2a in pigs with luteolysis blocked by release were apparent only in the pared to January and February, LH pregnancy, estradiol benzoate or human endogenous release of LH and not in release in response to exogenous chorionic gonadotropin. J Anim Sci 1981; 52: 330-339. the exogenous GnRH-induced release GnRH decreased at mid-gestation ZIECIK A, DOBOSZYNISKA T, DUSZA of LH. Thus, it can be assumed that compared to early gestation, regardless 15. L. Concentrations of LH, prolactin and the increased sensitivity to the negative of season. Evidently, the neuroenprogesterone in early pregnant and LH docrine control of pulsatile release feedback effects of E2 during August oestradiol-treated pigs. Anim Reprod Sci 1986; 10: 215-224. and September were mediated at the is inhibited in pregnant gilts during level of the hypothalamus to decrease August and September, while elevated 16. KENSINGER RS, COLLIER RJ, BAZER FW, KRAELING RR. Effect of number of endogenous GnRH release with mini- serum E2 concentrations during midconceptuses on maternal hormone concenthe and/or gestation suppress synthesis mal inhibitory effects on the anterior trations in the pig. J Anim Sci 1986; 62: pituitary or its ability to respond to release of LH from the anterior 1666-1674. pituitary gland. 17. COX NM, BRITT JH. Effect of estradiol exogenous GnRH. on hypothalamic GnRH and pituitary and The release of LH in response to serum LH and FSH in ovariectomized pigs. REFERENCES exogenous GnRH was decreased in J Anim Sci 1982; 55: 901-908. 18. CROWDER ME, GILLES PA, TAMANINI mid-gestation compared to early gestaROBERTSON HA, KING GJ. Plasma conC, MOSS GE, NETT TM. Pituitary content tion. This phenomenon has not been 1. centrations of progesterone, oestrone, of gonadotropins and GnRH receptors in in the pig, but previously reported oestradiol-17B and of oestrone sulphate in pregnant, post-partum and steroid-treated the pig at implantation, during pregnancy exogenous administration of GnRH ovariectomized ewes. J Anim Sci 1982; 54: and at parturition. J Reprod Fertil 1974; 40: has been demonstrated to result in 1235-1242. 133-141. 19. NILSON JH, NEJEDLIK TM, VIRGIN JB, decreasing release of LH with advanc- 2. ASH RW, HEAP RB. Oestrogen, progesCROWDER ME, NETT TM. Expression of ing gestation in the ewe (4). Furtherterone and corticosteroid concentrations in alpha and luteinizing hormone beta genes in more, the quantity of LH released in of sows peripheral plasma during pregnancy, the ovine anterior pituitary. Estradiol supparturition, lactation and after weaning. J response to a half-maximal dose of presses accumulation of mRNAs for both Endocrinol 1975; 64: 141-154. GnRH has been demonstrated to be alpha subunit and luteinizing hormone beta. ZIECIK A, TILTON JE, WEIGH R, J Biol Chem 1983; 258: 12087-12090. highly correlated to pituitary content 3. WILLIAMS GL. Plasma luteinizing horof LH in the ewe (4,18); however, mone during pregnancy in the pig. Anim Reprod Sci 1983; 5: 213-218. similar studies have not been con297